Journal Pre-proof

Performance of microbial induced carbonate precipitation for

immobilizing Cd in water and soil

Dinghua Peng (Conceptualization) (Methodology) (Supervision)

(Data curation) (Investigation) (Writing - original draft) (Writing -
review and editing), Suyu Qiao (Investigation) (Methodology)
(Software) (Supervision), Yao Luo (Investigation) (Visualization),
Hang Ma (Data curation) (Investigation), Lei Zhang (Data curation)
(Software), Siyu Hou (Writing - review and editing), Bin Wu (Writing
- review and editing), Heng Xu (Conceptualization) (Funding
acquisition) (Project administration)

PII: S0304-3894(20)31105-5
DOI: https://doi.org/10.1016/j.jhazmat.2020.123116
Reference: HAZMAT 123116

To appear in: Journal of Hazardous Materials

Received Date: 4 November 2019

Revised Date: 23 April 2020
Accepted Date: 1 June 2020

Please cite this article as: Peng D, Qiao S, Luo Y, Ma H, Zhang L, Hou S, Wu B, Xu H,
Performance of microbial induced carbonate precipitation for immobilizing Cd in water and
soil, Journal of Hazardous Materials (2020),
doi: https://doi.org/10.1016/j.jhazmat.2020.123116
This is a PDF file of an article that has undergone enhancements after acceptance, such as
the addition of a cover page and metadata, and formatting for readability, but it is not yet the
definitive version of record. This version will undergo additional copyediting, typesetting and
review before it is published in its final form, but we are providing this version to give early
visibility of the article. Please note that, during the production process, errors may be
discovered which could affect the content, and all legal disclaimers that apply to the journal
pertain.

© 2020 Published by Elsevier.

 Performance of microbial induced carbonate precipitation for immobilizing Cd

in water and soil

Dinghua Peng1, Suyu Qiao1, Yao Luo1, Hang Ma1, Lei Zhang1, Siyu Hou1, Bin Wu1

Heng Xu1*

of
1
Key Laboratory of Bio-Resource and Eco-Evironment of Ministry of Education,
College of Life Sciences, Sichuan University, Chengdu 610065, P.R.China

ro
1* Corresponding author: xuheng64@sina.com (H.Xu)
-p
Tel: +86 28 85414644; Fax: +86 28 85418262
re
lP
na

Graphical Abstract
ur
Jo

1
 of
ro
Highlights
-p
re
 A novel Cd-resistant ureolytic bacteria was isolated and identified as Enterobacter

sp.
lP

 The highest Cd remove rate in solution reached 99.50% within 7 days by MICP


na

Feasibility of replacing urea with oyster shell waste in the MICP process was

evaluated.
ur

 Oyster shell waste can alleviate the adverse effect of MICP on soil microecology
Jo

2
Abstract

Microbial induced carbonate precipitation (MICP) is known as a significant

process for remediating heavy metals contaminated environment. In this study, a novel

Cd-resistant ureolytic bacteria was isolated and identified as Enterobacter sp. Its

performances for immobilizing Cd in solution and soil were systematically discussed

at different treatment conditions. Results showed that initial pH and Cd concentration

of
were important parameters to influence Cd removal rate. The maximal Cd removal rate

ro
in solution reached 99.50% within 7 days by MICP. The precipitation produced in Cd

removal process were characterized by X-ray diffraction, scanning electron microscopy

-p
and energy dispersive spectrometer to understand the removal mechanism. Analyses
re
showed that Cd removal mechanism of CJW-1 was predominately via biominerals

including calcites and vaterites to absorb Cd2+. Cd immobilization tests demonstrated

lP

that the highest Cd-immobilization rate in soil could reach 56.10%. Although all
na

treatments contribute to soil pH, fertility, and enzyme activities improvement, oyster

shell wastes (OS) had a better effect on soil cation exchange capacity. All treatments
ur

had negative effects on soil respiration and bacterial community, but OS can alleviate

such adverse influence. Our results emphasized that Cd-resistant ureolytic bacteria
Jo

strain CJW-1 combined with OS had excellent ability and reuse value to remediate Cd-

contaminated environment.

Keywords: Cadmium; Ureolytic bacteria; Oyster shell wastes; Bioremediation; Soil

3
microecology

1. Introduction

More and more pathways including mining, smelting, atmospheric precipitation,

high-throughput input of agricultural chemicals caused seriously environmental

problem of heavy metals including of cadmium (Cd) pollution [1-3]. The Chinese Soil

Pollution Bulletin in 2014 showed that 7.0% of soils exceeded the standard of Cd. Many

of
crops such as rice may absorb excess Cd in Cd contaminated water and soil, which

ro
could threaten the health of humans and other organisms through the food chain [4].

Therefore, an effective way to remediate Cd polluted water and soil, especially for
-p
remediating paddy soil, is urgently needed nowadays. Which is important significant to
re
ensure food security and promote economic development.

Microbial induced carbonate precipitation (MICP) has been widely considered to

lP

be an emerging in-situ remediation technology for heavy metals (HMs) pollution in the
na

environment[5, 6]. This process happens through the ureolytic pathway conducted by

necessary metabolic genes present in microbes. Urea in the pathway was hydrolyzed
ur

into NH4+ and CO2 by urease secreted by ureolytic bacteria. During the hydrolysis

process, CO32- was released, causing Ca2+ to precipitation in the form of CaCO3[7]. It
Jo

can be further converted to other HMs precipitation or adsorb other HMs to cause co-

precipitation. This principle and process can be expressed as follow chemical equation

according to previous study[8]:

(NH2)2CO+H2O → 2NH3 + CO2 (1)

4
2NH3 +2H2O → 2NH4+ +2OH− (2)

CO2+ 2OH− → HCO3– + OH− → CO32–+ H2O (3)

Ca2+ + CO32– → CaCO3(↓) + M (HMs) → MCO3 (↓) (4)

Previous study showed that the soluble-exchangeable Cu in soil decreased from

45.54 mg kg-1 to 1.55 mg kg-1 [9]. The MICP process improved immobilization of Cr

and Pb in soil that the immobilization rates reached to 95.31% and 94.69%, respectively

[6]. Moreover, the MICP process could also improve the biochemical quality of sandy

of
soils [10-12]. Although MICP has been extensively studied on the HMs immobilization

ro
and sand soil equality improvement. While the ability to remediate Cd simultaneously

in solution and soil using Enterobacter sp. strain has not yet been fully elucidated.
-p
Especially for the effects of the MICP process on soil physicochemical properties and
re
microecological environment are rarely reported.

Urea is the key substance for ureolytic bacteria to form carbonate precipitation and
lP

immobilize HMs by MICP process. However, many studies have suggested that large
na

amounts of urea added to HMs contaminated soil will make soil acidification,

salinization and inhibit soil microbial biomass[13-15]. That hinder the application of
ur

MICP to remediate HMs including Cd contaminated paddy soil. Therefore, it is

particularly important to find a substance comparable to urea and expand the

Jo

application value of MICP. Oyster shell wastes (OS), containing rich in calcium sources

including CaCO3, CaO and amino acids, are considered a natural waste product that

were randomly discarded in fields or landfilled. This has caused a series of urgent

environmental problems in China and Korea [16, 17]. Some studies have proved that

5
OS can immobilize heavy metals in soil [17, 18]. Considering the need for calcium

sources in the MICP process and the natural wastes is rich in nutrients such as N\C and

amino acids [6]. So, we believe that the combination of ureolytic bacteria and OS may

have a better immobilization effect on Cd in soil than the single treatment, which is

comparable to the cooperative effect of urea and ureolytic bacteria. Furthermore,

similar study to remediate Cd contaminated paddy soil using the combination of

ureolytic bacteria and OS has not been reported so far.

of
Accordingly, the purposes of our study were to 1) isolate and identify a novel Cd-

ro
resistant bacteria that secret urease; 2) evaluate the ability of the bacteria to remove Cd

in water by MICP process; 3) compare the Cd immobilization effect of the ureolytic

-p
bacteria combined urea with OS on flooded paddy soil; 4) reveal the influences of the
re
ureolytic bacteria combined urea with OS on soil physicochemical properties, fertility

and bacterial community.

lP

2. Materials and Methods

na

2.1 Paddy soil and oyster shells wastes (OS)

Slightly contaminated soil obtained from a paddy field located in Mianzhu City,
ur

Sichuan Province, China (E 104.220515, N 31.265996). The collected soil was air-dried,

grinded, passed through a 2 mm sieve to remove large debris and get homogeneous soil
Jo

particles. Then it was mixed with prepared CdCl2.2H2O solution to make the total Cd

in soil was about 20 mg.kg-1.

The urea and OS presented in this experiment were purchased from Chengdu

Kelong Corp (China). The properties of soil, urea and OS are shown in Table S1.

6
2.2 Isolation and identification of ureolytic bacteria

Cd-resistant bacterial strain possessing the ability to secret urease was isolated

from the sediment samples at the bottom of an acid wastewater ditch in a pyrite mining

area. The area was severely damaged and contaminated with heavy metals in XingWen

City, Sichuan Province, China (E 105.096674, N 28.17121). The method of screening

target strains was similar to previous study[19]. In brief, a moderate amount of sediment

samples was added in Luria Broth (LB) liquid medium containing different Cd

of
concentration (20-200 mg L-1). The well-grown strains under high Cd concentration

ro
were further separated and purified by streak plate method. To verify whether the Cd-

resistant strains have the ability to produce urease, the strains were cultivated in
-p
medium that used urea as the sole nitrogen source and phenol red has been used as an
re
indicator. The resistant strain that rapidly reddened the above identification medium

and has the highest urease activity in liquid medium was selected and named CJW-1.
lP

Subsequently, the growth rate of bacterial cells in LB medium with or without urea was
na

measured by turbidimetry. Finally, strain CJW-1 was identified by 16S rDNA sequence

analysis and the blast program in GenBank at NCBI server was used to match the
ur

obtained gene sequences [20].

2.3 Performance of strain CJW-1 in Cd contaminated solution

Jo

A set of experiments were carried out in liquid medium to determine the ability of

strain CJW-1 to produce urease and remove Cd by MICP process. Briefly, 100 mL LB

medium containing different Cd concentrations (20, 40, 60, 80, 100 mg/L) and initial

pH values (from 4 to 9) respectively were added in 250 mL erlenmeyer flasks and

7
incubated in a constant temperature oscillator (37 °C, 150 rpm). These mediums were

supplemented with 2% of urea, 40 mM CaCl2 and 1% of bacterial cell suspension

(OD600 = 0.5), which were named MICP group [6]. Control groups were just added

with 1% of bacterial cell suspension (OD600=0.5). Cultured samples were harvested at

7th day to measure pH, urease activity and Cd concentration in solution.

The precipitates, bio-mediated by CJW-1 strain in LB medium, were gathered at

7th day, which were characterized by X-ray diffraction (XRD), scanning electron

of
microscope (SEM, JSM-7500F) and energy dispersive spectrometer (EDS,

ro
EMPYREAN).

2.4 Cd immobilization tests

-p
Eight treatments including CK (control group), T1 (urea), T2 (OS), T3 (CJW-1),
re
T4 (urea + OS), T5 (CJW-1+ urea), T6 (CJW-1+ OS), T7 (CJW-1+urea + OS) were

used in the whole incubation experiment, which were conducted in 1000 ml plastic
lP

beaker containing 0.5 kg of Cd contaminated soil. Considering the previous study and
na

the need of microbial induced carbonate precipitation [9, 21, 22], 2% of OS and 50 mL

of bacteria suspension (∼109 CFU mL-1) and 1% of urea were added in corresponding
ur

treated groups, respectively. Meanwhile, the same amount of sterilized saline was

added in the control groups. All treatments including three replicates were immersed in
Jo

water 1-2 cm above the surface of the soil in the whole experiment to maintain the

native environment of the paddy soil. Experiment was conducted at room temperature

(30±5℃) and soil properties including bacterial community were measured after 40

days of cultivation.

8
2.5 Soil Cd fractions

Tessier’ method was used to analyze the Cd fractions in contaminated soil, which

was named soluble-exchangeable, carbonate-bound, Fe-Mn oxides bound, organic-

matter bound, and residual fractions according sequential extraction procedure [23].

The concentration of Cd fractions was analyzed by flame atomic absorption

spectrometry (FAAS, VARIAN SpectrAA-220Fs) according previous study [24].

2.6 Soil physicochemical properties and fertility

of
Some indexes involving soil pH values, Cation exchange capacity (CEC),

ro
available phosphorus (OSP), available potassium (OSK), alkali hydrolyzed nitrogen

(ALN) and organic matter (OM) were determined to evaluate the influence of MICP on

soil physicochemical properties and fertility.

-p
re
Soil pH values were measured in a suspension with a water to soil ratio of 2.5. Soil

CEC was determined by Hexaamminecobalt Chloride Solution Extraction-

lP

Spectrophotometric Method (HJ 889-2017). The OSP was extracted with sodium
na

bicarbonate solution (pH=8.5) and measured by spectrophotometrically at 880 nm, the

method of alkali solution diffusion by the diffusion plate was used to measure the ALN
ur

according to previous study [24]. The OSK was measured as previous method [25]. The

OM was analyzed by potassium dichromate volumetric method [26].

Jo

2.7 Soil Microecology

Soil samples were collected and determined to discuss the effect of MICP on soil

respiration, enzymes activities and bacterial community after remediated at day 40.

Soil respiration was measured according to Jia et al[27], and represented by mg

9
CO2 kg-1 soil h-1. Soil urease and dehydrogenase activities were determined by

spectrophotometry at 578 nm and 492 nm, respectively [28]. Urease activity was

expressed as the amount of NH4+-N (mg) produced per gram of soil per 24 h.

Dehydrogenase activity was presented as the production (mg) of TPF per g soil per 24

h. According to our previous research [29], soil bacterial DNA was extracted using a

OMG Soil DNA Kit (Vazyme Biotech Co.,Ltd.) in accordance with the manufacturer’s

instructions and amplified for further bacterial community analysis. The bacterial

of
community was analyzed on the Illumina MiSeq platform, which was conducted by

ro
Majorbio Bio-Pharm Technology Co., Ltd (Shanghai, China). The final results were

analyzed on the free online platform of Majorbio I-Sanger Cloud Platform (www.i-

sanger.com).
-p
re
2.8 Data analysis

Mean and standard deviation values of three replications were calculated in this
lP

study. SPSS 21.0 and Microsoft Excel 2016 were used to analysis experiment data.
na

3. Result and discussion

3.1 Characterization of isolated strain and its immobilized ability on Cd in solution

ur

3.1.1 Isolation and identification of strain CJW-1

Two Cd-resistant bacteria strains that could grow at 100 mg L-1 Cd stress were
Jo

isolated and screened on a urease selective medium, which was used for the qualitative

detection of urease. When the color of above medium turned pink, it proved urease was

produced [7]. One strain, named CJW-1, was selected for further study for its ability to

make the medium intense pink quickly (Figure S1) and have the highest urease

10
activities between two Cd-resistant bacteria strains (Table S2). Subsequently, the

growth rate of strain CJW-1 was plotted as Figure S2.

The sequence of the strain CJW-1 was analyzed using 16S rDNA sequencing and

compared in NCBI GenBank, which was identified as Enterobacter sp. Moreover, its

16S rDNA sequence was saved in the NCBI database with accession number

MN420673.1.

3.1.2 Cd removal in solution

of
Removing efficiency was directly influenced by the initial HMs concentration in

ro
living environment of microorganisms. The effects of different Cd concentration on

urease activity and Cd removal rate were studied when CJW-1 were cultivated in LB
-p
medium with pH=7 for 7 days. Results showed that the urease activity (Figure 1a) and
re
Cd removal rate (Figure 1b) in the process of MICP were always greater than that of

control group. And the highest urease activity and the maximum Cd removal rate
lP

occurred simultaneously at initial Cd concentration of 20 mg/L, reaching 2.17 and 98.80,

na

respectively. Subsequently, the urease activity and Cd removal rate gradually slowed

down with the increase of Cd concentration, which probably due to strain CJW-1
ur

growth was inhibited by higher Cd concentration. Previous studies have showed that

bacterial growth was influenced due to higher concentration HMs, which have higher
Jo

toxicity for bacterial intracellular substances such as protein and nucleic acid [30].

Moreover, the trend of Cd removal rate was always corresponds with urease activity,

indicated the immobilization of strain CJW-1 to Cd was mainly bio-mediated by urease

driving [31]. On the other hand, Cd was also removed slightly in the control group,

11
which may due to bacteria cell adsorption and the abiotic precipitation by Cd and CO32-

under alkaline conditions [6, 32]. Although the strain CJW-1 was very sensitive on the

changes of HMs concentration, and the removal rate was decreased by 64.86%, 51.83%,

40.95% and 10.82% at higher Cd concentration of 40, 60, 80, 100 mg/L, respectively,

its performance in low concentration of Cd stress was very excellent than other reports

[33, 34]. Hence, CJW-1 induced carbonate precipitation could be considered as an

effective way to remediate Cd pollution in water.

of
To judge the influence of pH on urease activity and Cd removal rate, some

ro
experiments were conducted with the diverse pH values varying from 4.0 to 9.0 at initial

Cd concentrations of 20 mg/L. As Figure 1d presented, urease activity was increased

-p
firstly and then decreased with the improvement of pH in solution. The highest urease
re
activity was both observed at pH=5.0 in control group and MICP, indicating strain CJW-

1 had a better ability of biological adaptability and rapid proliferation in relatively

lP

acidic environment, which may due to the strain was isolated from acidic pyrite slag.
na

However, the urease activity was the lowest (less than 0.1 OD values) at pH=4, which

might because the acidity condition restrained bacterial growth and metabolic activities
ur

[30]. On the other hand, the highest Cd removal rate in MICP process was occurred at

pH=6, reached to 99.50% (Figure 1e), which was not consistent with the pH, that may
Jo

be resulted by initial pH in solution. Generally, carbonate precipitates including CdCO3

were hard to form directly at low pH values. Because the CO32-, produced by the process

of ureolytic, was directly resolve to CO2. Moreover, final pH in solution was also

measured under different initial Cd concentration and pH as presented in Figure 1c and

12
Figure 1f. Results showed the final pH in the control group and MICP was increased

compared to initial pH, suggesting CJW-1 has great potential in immobilization of HMs.

3.1.3 Characteristics of precipitation

The precipitates (Figure S3), produced by strain CJW-1 in Cd removal process,

was collected and characterized by SEM, EDS and XRD. SEM picture (Figure 2a)

showed that the precipitates were roughly and very loose in shape, and there was no

apparently smooth crystal structure. This was not consistent with the crystal shape of

of
calcite precipitation induced by other report [35]. Which may due to the fact that

ro
complex carbonates containing Cd2+ is more difficult to crystallize than single calcite.

Furthermore, the elemental composition of precipitates was analyzed by EDS. Results

-p
indicated that large amounts of Cd appeared on the precipitated surface which also
re
contained a lot of C, O, Ca (Figure 2b).

XRD was conducted to further identify the composition of precipitates. The XRD
lP

pattern analysis clearly showed the main forms of precipitation. It was confirmed strain
na

CJW-1 hydrolyzes urea to produce CO32-, because vaterites and calcites were clearly

identified under XRD spectra (Figure 2c). However, no corresponding significant peaks
ur

proven CdCO3 were found in the XRD pattern. Which was similar to previous research

[36]. Which may due to Cd2+ concentration in solution was too low to form stable
Jo

CdCO3 crystals. On the other hand, it can also be further inferred that the removal

mechanism of Cd2+ was more likely to be an adsorption based on the results of XRD,

SEM and EDS. In other word, these calcites and vaterites induced by strain CJW-1

adsorb Cd2+ to make them co-precipitate, which was consistent with previous study

13
[37]. Moreover, these results can also prove that strain CJW-1 induces carbonate

precipitation during Cd removal in our study. Therefore, our results demonstrated that

Cd removal mechanism of strain CJW-1 was predominately via biominerals including

calcites and vaterites to absorb Cd2+. Which make the Cd2+ in solution were converted

into insoluble carbonate precipitation by Cd-resistant ureolytic bacteria strain CJW-1

through MICP process to achieve lower soluble Cd concentration and toxicity of

solution.

of
3.2 Cd fraction in soil

ro
The fractions of Cd were the standard to estimate its mobility and toxicity in

environment. Tessier’s method in this study was widely used to reflect the effects of
-p
MICP on soil Cd fractions in flooded environment [23]. Figure 3 showed the results of
re
Cd fraction. Compared to CK, soluble-exchangeable Cd fraction in all treatments was

decreased by 15.30-56.10%, and the contents carbonated-Cd were increased by 75.90-

lP

253.80%. The fractions including Fe-Mn oxide-Cd, organic-Cd and residual-Cd were
na

similar between treatment groups and CK. Meanwhile, the contents of organic-Cd and

residual-Cd in soil were less than 2%, which was similar previous research [38]. It
ur

suggested that Cd was relatively active in flooded soil, and the toxicity to

microorganisms or plants was relatively high [39, 40].

Jo

MICP is a natural bio-mediated process that has been used to remediate heavy

metals pollution in environment, and its ability to immobilize Cd in soil also proved

again according our results [41, 42]. The content of exchangeable Cd was remarkably

decreased in the cultivation with strain CJW-1 compared with noncultivation groups,

14
especially for the CJW-1 + urea + OS complex in the T7 group, indicating the important

role of CJW-1 in the process of MICP. According to the content changes between

exchangeable-Cd and carbonated-Cd, it could be inferred that the main style to decrease

HMs toxicity and mobility was covert soluble-exchangeable to carbonated bound

during the process of MICP. This phenomenon of fraction difference was also found in

previous studies [7, 9]. Moreover, our results also suggested that OS was an effective

material to immobilize heavy metal including Cd [16]. Meanwhile, it was proved that

of
there was no significant difference between the Cd immobilized rate by strain CJW-1

ro
combined with oyster shell wastes (OS) and that by strain CJW-1 combined with urea.

The result was probably duo to the following reasons. Firstly, OS had many substances
-p
containing the same functional groups as urea, such as amino acids, which could also
re
be utilized by urease. Secondly, calcium sources containing CaCO3 and Ca(OH)2 were

provided for the ureolytic bacteria due to add OS to the soil, which made MICP process
lP

to occur faster. And it may also occur that the slightly soluble CaCO3 was directly
na

converted into water-insoluble CdCO3 in flooded soil. Therefore, our results also

proved that OS could be combined with ureolytic bacteria, similar to urea, to achieve
ur

similar Cd immobilized effect in Cd contaminated paddy soil.

3.3 Effects of MICP on soil phychemical properties

Jo

Soil pH and CEC were measured to judge the effects of MICP on soil

physicochemical properties. As Figure 4, soil pH values in all treatments were

significantly increased by 0.21 - 0.78 units, the highest and lowest pH values appeared

in the T4 and T3 groups, respectively. The pH values were lower in the cultivation with

15
strain CJW-1 compared with non-cultivation groups, which maybe the urea and OS

were utilized by CJW-1, resulting the effects of them on soil pH slightly weaken.

However, contrary to the trend of soil pH values, the soil CEC in the cultivation of

strain CJW-1 was significantly decreased by 4.35-7.95% of CK, but soil CEC was

improved most with the single OS in the T2 groups.

Soil pH values is an important parameter influencing Cd transfer behavior in soil.

Normally, the combination ability between H+ and metal-binding ligands was

of
strengthened with pH decreased in soil, the heavy metal adsorption capacity decreased,

ro
and the bioavailability and mobility of heavy metals in the soil increased [43]. Our

results suggested that urea and OS had higher improvement on soil pH values than
-p
strain CJW-1. And the process of MICP had a slight influence on soil pH values than
re
the noncultivation groups, indicating that microorganisms were more gentleer than the

simple chemical recuperate agent in the soil pH improvement. Soil pH values increased
lP

that maybe because the production of NH4+ originated from the process of MICP
na

including strain CJW-1 and indigenous microorganism decompose urea or OS [7].

Unbelievable as it was, CEC values in our study, representing the ability of the soil to
ur

maintain nutrients and moisture, significantly decreased in the cultivation strain CJW-

1 and urea groups. This is contrary to what others have reported [9], which may because
Jo

our experiment was carried out in a flooded environment. And the content of OM was

decreased compared CK, which resulted in the production of large amounts of water-

insoluble carbonate compounds during the process of MICP and reduce CEC values in

our results. However, OS slightly increased soil CEC that was consistent with previous

16
research [21], which maybe because it contains many Ca2+.

3.4 Effects of MICP on soil fertility

Soil fertility could directly affect the future function of soil after remediation.

Therefore, the suitability of MICP for remediating paddy soil was judged by measuring

these indicates including OSP, OSK, ALN, OM. As Figure 5 presented, the content of

ALN, OSP and OSK, except that OSK in T2 group decreased slightly, were increased

to 60.71-305.16%, 29.70-57.46% and 6.99-35.28% respectively in all treatments

of
compared with CK. Urea hydrolysis promoted the increase of soil organic or inorganic

ro
nitrogen, the content of ALN were significantly different between the urea-containing

and urea-free groups in this experiment, which was consistent with previous study [44].
-p
In addition, the combination of strain CJW-1 with OS and urea promoted soil fertility
re
compared to single treatment, especially for OSK. The highest concentration for ALN

and OSK were both shown in the T7 treatment but OSP in the T6 treatment, which were
lP

305.16%, 35.28% and 57.46% higher than CK. These results indicated that
na

Enterobacter strain CJW-1, as a urease-producing strain, could not only induce

carbonate precipitation to immobilize heavy metal in the soil, but also improve soil
ur

ALN, OSP and OSK [45, 46]. Generally, increased soil pH can increase the availability

of OM, accelerating the utilization of OM by soil bacteria [47]. So, the content of OM
Jo

in all treatments were decreased except for T1 group in our study, which may due to the

addition of OS and strain CJW-1, which stimulated the metabolism and number of soil

microorganisms and accelerated the consumption of soil OM.

3.5 Soil microecology analysis

17
 Soil microecology after remediation were evaluated by determining soil

respiration and enzyme activities.

As shown Figure 6a, soil respiration intensity was decreased 9.89-39.56%

compared with CK after remediation. Which suggested correlative treatments have

adverse effect on soil microorganism. The influence may be due to soil hardening and

salinization to make some microbial nutrients decreased. Because many process

including urea hydrolysis, OS added and biomineralization accelerate soil hardening

of
and salinization [15, 21, 48]. Moreover, OM contents in almost all treated groups

ro
(Figure 5d) decreased in our study, which could also explain microbial nutrients

reduced leads to soil respiration intensity decreased. However, whether OS was added
-p
alone or in combination with strain CJW-1, the soil respiration intensity was higher than
re
that of the corresponding urea treated groups. This may be because OS itself contains

abundant microorganisms compared with urea, the addition of OS increased the

lP

diversity of soil microorganisms, resulting in soil respiration intensity increased.

na

Therefore, our results suggested that it was more suitable to remediate Cd-contaminated

paddy soil by OS combined with ureolytic bacteria, compare to urea.

ur

Urease activities (Figure 6b) were greatly intensified with the cultivation of strain

CJW-1 compared with the noncultivation groups, which was remarkably raised by
Jo

81.42-306.44% of CK, except for the T2 group. Furthermore, the activity of urease was

promoted most with the CJW-1+urea+OS complex in the T7 group, reached to 306.44%

higher than CK. Compared with the Cd fraction (Figure 3), we found the highest urease

activity and carbonate bound-Cd, but the least exchangeable-Cd content was appeared

18
simultaneously in T7 group. Which further suggested that the passivation of Cd was

mainly caused by soil microbes including CJW-1, which produce urease to decompose

urea or OS, resulting Cd to form carbonate precipitation. Previous studies had also

indicated that urease, a key enzyme in the process of MICP [9], not only affects the

immobilization effect of heavy metal in soil, but also closely related to the geochemical

process of N cycle [49]. Meanwhile, urease activity was also increased with the

noncultivation of strain CJW-1 groups compared to CK, indicated urea and OS could

of
stimulate soil microorganisms to secret urease. Which would rapidly increase urease

ro
activity in soil, which was similar to the results of previous study [9]. Simultaneously,

increased urease activity will further accelerate the process of MICP mediated by
-p
indigenous microorganisms using urea and OS, resulting in Cd being immobilized.
re
The trend of dehydrogenase activities (Figure 6b) was generally consistent with

urease activities. In addition, dehydrogenase activities in the single OS treated group

lP

were increased by 23.60-431.04% of CK. The cultivation of strain CJW-1 significantly

na

enhanced the dehydrogenase activities compared with the noncultivation groups.

Highest dehydrogenase activities were also shown in the T7 group, reached to 431.04%
ur

higher than CK. Although dehydrogenase is not essential in the process of MICP, it has

always been regarded as an important indicator to evaluate the soil microbial activation
Jo

[50] and the degree of heavy metal pollution [51]. There may be two reasons for the

changes of dehydrogenase activities in this study. The first was that soil pH increase as

mentioned above, leads to dehydrogenase activities increased. Another reason was that

the addition of OS inhibited the active of bacteria secreting dehydrogenase, and

19
decreased soil dehydrogenase activity.

3.6 Soil bacterial community

A total of 285646 available sequences in all treated soil samples were obtained to

analysis bacterial community. Some indices including Ace、Chao 1, Shannon and

Simpson were used to evaluate soil bacterial diversity and richness [52], as shown in

Table S3. The coverage values greater than 99%, which suggested our results were

accurate and scientific [9]. Compared to CK, all treated groups have lower Ace、Chao

of
1 and Shannon, and higher Simpson indices values. Which suggested soil bacterial

ro
diversity and richness were decreased after remediation [53]. Moreover, OTUs index

was down more than 29% compared with CK. The lowest Ace, Chao 1 and Shannon
-p
index, and the highest Simpson index were determined in T5. These results further
re
demonstrated that ureolytic bacteria CJW-1 combined with OS or urea have adverse

influence on soil microorganisms, but OS could reduce the negative influence. Which
lP

was consistent with previous study about MICP [9].

na

In order to further reflect soil bacterial community and strain CJW-1 colonization,

the relative abundance of soil bacteria on genus level were calculated and analyzed by
ur

platform (https://cloud.majorbio.com). The genus whose abundance ratio were less than

2% in all samples were classified as others (Figure 7). Results showed that relative
Jo

abundance of some bacteria have significant changes. For example, some bacterial

abundance had decreased such as Rhodanobacter, Flavisolibacter and

norank_o__Subgroup_7 and so on, but some including Paenisporosarcina and

Pseudomonas had increased after remediated by different treatments. Which

20
demonstrated soil microorganisms were very sensitive to environmental change, these

microbes may perhaps serve as indicators. Many studies suggested luminous bacteria

was often used as environmental indicators because it’s sensitive to the degree of

environmental pollution [54, 55]. Therefore, the microorganisms mentioned above may

be very sensitive to soil salinization and hardening due to urea hydrolysis, OS added

and biomineralization. Moreover, the abundance of others in T5, T6 and T7 groups

decreased to different degrees, and it was the lowest in T5 groups. Which further

of
suggested ureolytic bacteria CJW-1 combined with OS or urea have adverse influence

ro
on bacterial abundance, but OS could reduce the negative influence. This is consistent

with some results mentioned above. Furthermore, Enterobacter sp. was determined in
-p
the T3 (18.61%), T5 (0.32%), T6 (38.62) and T7 groups (26.03%) at the end of the
re
experiment. The presence of Enterobacter sp. probably proved that strain CJW-1 was

successfully colonized in soil during remediation[56]. This result also showed that urea
lP

was more unfavorable for strain CJW-1 colonization than OS. Therefore, the analysis
na

of microbial community may further prove that OS combined with ureolytic bacteria

has a better long-term remediation effect in heavy metal contaminated soil compared
ur

with urea.

4. Conclusion
Jo

The results manifested that Cd removal rate was closely related to urease activity.

The highest Cd removal rate reached to 99.50% when initial Cd concentration equal to

20 mg/L and pH=7 in solution. Cd immobilization tests demonstrated that the highest

Cd immobilizing rate in soil could reach to 56.10% in the group with strain CJW-1-

21
urea-OS within 40 days. However, there was no significant difference between the Cd

immobilizing rate by strain CJW-1 combined with oyster shell wastes (OS) and that by

strain CJW-1 combined with urea. Soil pH values and soil fertility (except for soil OM

and CEC) were significantly increased in the groups containing CJW-1 coupled with

OS or urea. Furthermore, all treatments had negative impacts on soil microecology

indicators including soil respiration and bacterial community after remediation, but OS

can alleviate such adverse influence. Therefore, our results provided some valuable

of
information for comprehensive understand for MICP process. On the one hand, the

ro
ability of environment remediation by MICP was strongly influenced by pH and the

concentration of hazardous substances in environment. On the other hand, soil fertility

-p
indicators including N, P, K were improved by MICP process, but it had negative
re
influence on soil microecology including bacterial community. This study will provide

theoretical basis for further enhancing remediation ability and application values of
lP

MICP in the further.

na

Credit Author statement

ur

None
Jo

Acknowledgements

This study was financially supported by the National Key Research and

Development Program (2018YFC1802605), the Key Research and Development

Program of Sichuan Province (2017SZ0181, 2018NZ0008) and the Science and

22
Technology Project of Sichuan Province (2019JDRC0092, 2018RZ0110) ， the

Fundamental Research Funds for the Central Universities (SCU2019D013). The

authors also wish to thank Professor Guanglei Cheng and Hui Wang from Sichuan

University for the technical assistance.

References

of
[1] F. Zang, S. Wang, Z. Nan, J. Ma, Y. Li, Q. Zhang, Y. Chen, Immobilization of Cu,

ro
Zn, Cd and Pb in mine drainage stream sediment using Chinese loess, Chemosphere,

181 (2017) 83-91.

-p
[2] P. Liao, B. Li, L. Xie, X. Bai, H. Qiao, Q. Li, B. Yang, C. Liu, Immobilization of
re
Cr(VI) on engineered silicate nanoparticles: Microscopic mechanisms and site energy

distribution, Journal of Hazardous Materials, 383 (2020).

lP

[3] P. Xu, M. Chen, G. Zeng, D. Huang, C. Lai, Z. Wang, M. Yan, Z. Huang, X. Gong,
na

B. Song, T. Li, A. Duan, Effects of multi-walled carbon nanotubes on metal

transformation and natural organic matters in riverine sediment, Journal of Hazardous

ur

Materials, 374 (2019) 459-468.

[4] Y. Xia, H. Liu, Y. Guo, Z. Liu, W. Jiao, Immobilization of heavy metals in

Jo

contaminated soils by modified hydrochar: Efficiency, risk assessment and potential

mechanisms, Science of the Total Environment, 685 (2019) 1201-1208.

[5] J. Zhang, D. Kumari, C. Fang, V. Achal, Combining the microbial calcite

precipitation process with biochar in order to improve nickel remediation, Appl.

23
Geochem., 103 (2019) 68-71.

[6] X. Qian, C. Fang, M. Huang, V. Achal, Characterization of fungal-mediated

carbonate precipitation in the biomineralization of chromate and lead from an aqueous

solution and soil, Journal of Cleaner Production, 164 (2017) 198-208.

[7] X. Zhu, W. Li, L. Zhan, M. Huang, Q. Zhang, V. Achal, The large-scale process of

microbial carbonate precipitation for nickel remediation from an industrial soil,

Environ Pollut, 219 (2016) 149-155.

of
[8] N.J. Jiang, R. Liu, Y.J. Du, Y.Z. Bi, Microbial induced carbonate precipitation for

ro
immobilizing Pb contaminants: Toxic effects on bacterial activity and immobilization

efficiency, Sci Total Environ, 672 (2019) 722-731.

-p
[9] X. Chen, V. Achal, Biostimulation of carbonate precipitation process in soil for
re
copper immobilization, Journal of Hazardous Materials, 368 (2019) 705-713.

[10] X.H. Sun, L.C. Miao, R.F. Chen, Effects of Different Clay's Percentages on
lP

Improvement of Sand-Clay Mixtures with Microbially Induced Calcite Precipitation,

na

Geomicrobiol. J., 36 (2019) 810-818.

[11] Y.F. Gao, X.Y. Tang, J. Chu, J. He, Microbially Induced Calcite Precipitation for
ur

Seepage Control in Sandy Soil, Geomicrobiol. J., 36 (2019) 366-375.

[12] J.T. Dejong, K. Soga, E. Kavazanjian, S. Burns, L.A. Van Paassen, A. Al Qabany,
Jo

A. Aydilek, S.S. Bang, M. Burbank, L.F. Caslake, C.Y. Chen, X. Cheng, J. Chu, S.

Ciurli, A. Esnault-Filet, S. Fauriel, N. Hamdan, T. Hata, Y. Inagaki, S. Jefferis, M. Kuo,

L. Laloui, J. Larrahondo, D.A.C. Manning, B. Martinez, B.M. Montoya, D.C. Nelson,

A. Palomino, P. Renforth, J.C. Santamarina, E.A. Seagren, B. Tanyu, M. Tsesarsky, T.

24
Weaver, Biogeochemical processes and geotechnical applications: Progress,

opportunities and challenges, Geotechnique, 63 (2013) 287-301.

[13] D. Tian, S. Niu, A global analysis of soil acidification caused by nitrogen addition,

Environmental Research Letters, 10 (2015) 024019.

[14] D. Geisseler, K.M. Scow, Long-term effects of mineral fertilizers on soil

microorganisms – A review, Soil Biology and Biochemistry, 75 (2014) 54-63.

[15] M. Wang, W.U. Shijun, Y. Yang, F. Chen, Microbial Induced Carbonate

of
Precipitation and Its Application for Immobilization of Heavy Metals: a Review,

ro
Research of Environmental Sciences, (2018).

[16] Y.S. Ok, J.E. Lim, D.H. Moon, Stabilization of Pb and Cd contaminated soils and
-p
soil quality improvements using waste oyster shells, Environ Geochem Health, 33
re
(2011) 83-91.

[17] Y. Chen, J. Xu, Z. Lv, R. Xie, L. Huang, J. Jiang, Impacts of biochar and oyster
lP

shells waste on the immobilization of arsenic in highly contaminated soils, J Environ

na

Manage, 217 (2018) 646-653.

[18] N. Seco-Reigosa, A. Bermúdez-Couso, B. Garrido-Rodríguez, M. Arias-Estévez,

ur

M.J. Fernández-Sanjurjo, E. Álvarez-Rodríguez, A. Núñez-Delgado, As(V) retention

on soils and forest by-products and other waste materials, Environmental Science and
Jo

Pollution Research, 20 (2013) 6574-6583.

[19] N. Tirry, N. Tahri Joutey, H. Sayel, A. Kouchou, W. Bahafid, M. Asri, N. El

Ghachtouli, Screening of plant growth promoting traits in heavy metals resistant

bacteria: Prospects in phytoremediation, J Genet Eng Biotechnol, 16 (2018) 613-619.

25
[20] M. Wu, J. Deng, J. Li, Y. Li, J. Li, H. Xu, Simultaneous biological-photocatalytic

treatment with strain CDS-8 and TiO2 for chlorothalonil removal from liquid and soil,

Journal of Hazardous Materials, 320 (2016) 612-619.

[21] Y. Chen, J. Xu, Z. Lv, R. xie, L. Huang, J. Jiang, Impacts of biochar and oyster

shells waste on the immobilization of arsenic in highly contaminated soils, Journal of

Environmental Management, 217 (2018) 646-653.

[22] K. Xiao, Y. Li, Y. Sun, R. Liu, J. Li, Y. Zhao, H. Xu, Remediation Performance

of
and Mechanism of Heavy Metals by a Bottom Up Activation and Extraction System

ro
Using Multiple Biochemical Materials, ACS Applied Materials & Interfaces, 9 (2017)

30448-30457.
-p
[23] A. Tessier, P.G.C. Campbell, M. Bisson, Sequential extraction procedure for the
re
speciation of particulate trace metals, Analytical Chemistry, 51 (2002) 844-851.

[24] Y. Wang, Y. Xu, D. Li, B. Tang, S. Man, Y. Jia, H. Xu, Vermicompost and biochar
lP

as bio-conditioners to immobilize heavy metal and improve soil fertility on cadmium

na

contaminated soil under acid rain stress, Sci Total Environ, 621 (2018) 1057-1065.

[25] L. Guangming, Z. Xuechen, W. Xiuping, S. Hongbo, Y. Jingsong, W. Xiangping,

ur

Soil enzymes as indicators of saline soil fertility under various soil amendments,

Agriculture, Ecosystems & Environment, 237 (2017) 274-279.

Jo

[26] D.W. Nelson, L.E. Sommers, D.L. Sparks, A.L. Page, P.A. Helmke, R.H. Loeppert,

P.N. Soltanpour, M.A. Tabatabai, C.T. Johnston, M.E. Sumner, Total carbon, organic

carbon, and organic matter, Methods of Soil Analysis, 9 (1996) 961-1010.

[27] Z. Jia, D. Jing, N. Chen, W. Shi, H. Xu, Bioremediation of cadmium-dichlorophen

26
co-contaminated soil by spent Lentinus edodes substrate and its effects on microbial

activity and biochemical properties of soil, Journal of Soils & Sediments, 17 (2016) 1-

11.

[28] P. Yu, X. Tang, A. Zhang, G. Fan, S. Liu, Responses of soil specific enzyme

activities to short-term land use conversions in a salt-affected region, northeastern

China, Science of The Total Environment, 687 (2019) 939-945.

[29] D. Peng, B. Wu, H. Tan, S. Hou, M. Liu, H. Tang, J. Yu, H. Xu, Effect of multiple

of
iron-based nanoparticles on availability of lead and iron, and micro-ecology in lead

ro
contaminated soil, Chemosphere, 228 44-53.

[30] M. Wu, Y. Li, J. Li, Y. Wang, H. Xu, Y. Zhao, Bioreduction of hexavalent

-p
chromium using a novel strain CRB-7 immobilized on multiple materials, J Hazard
re
Mater, 368 (2019) 412-420.

[31] S.P. Chaparro-Acuña, M.L. Becerra-Jiménez, J.J. Martínez-Zambrano, H.A.

lP

Rojas-Sarmiento, Soil bacteria that precipitate calcium carbonate: mechanism and

na

applications of the process, Acta Agronómica, 67 (2018) 277-288.

[32] A. Choińska-Pulit, J. Sobolczyk-Bednarek, W. Łaba, Optimization of copper, lead

ur

and cadmium biosorption onto newly isolated bacterium using a Box-Behnken design,

Ecotoxicology & Environmental Safety, 149 (2018) 275.

Jo

[33] W. Yuan, J. Cheng, H. Huang, S. Xiong, J. Gao, J. Zhang, S. Feng, Optimization

of cadmium biosorption by Shewanella putrefaciens using a Box-Behnken design,

Ecotoxicol Environ Saf, 175 (2019) 138-147.

[34] J.F. Su, L. Xue, T.L. Huang, Z. Wang, J.X. Wang, Kinetic analysis of denitrification

27
coupled with Cd(II) removal by Cupriavidus sp. CC1 and its removal mechanism,

Research in Microbiology, 170 (2019) 214-221.

[35] X. Qian, C. Fang, M. Huang, V. Achal, Characterization of fungal-mediated

carbonate precipitation in the biomineralization of chromate and lead from an aqueous

solution and soil, Journal of Cleaner Production, 164 198-208.

[36] M.J. Harbottle, Toxicity effects on metal sequestration by microbially-induced

carbonate precipitation, Journal of Hazardous Materials, 314 237-248.

of
[37] A. Rouff, R. Reeder, N. Fisher, Pb (II) Sorption with Calcite: A Radiotracer Study,

ro
Aquatic Geochemistry, 8 203-228.

[38] B. Wu, G. Cheng, K. Jiao, W. Shi, C. Wang, H. Xu, Mycoextraction by Clitocybe

-p
maxima combined with metal immobilization by biochar and activated carbon in an
re
aged soil, Sci Total Environ, 562 (2016) 732-739.

[39] N. Köleli, S. Eker, I. Cakmak, Effect of zinc fertilization on cadmium toxicity in

lP

durum and bread wheat grown in zinc-deficient soil, Environmental Pollution, 131
na

(2004) 453-459.

[40] G.J. Wagner, Accumulation of Cadmium in Crop Plants and Its Consequences to
ur

Human Health, in: D.L. Sparks (Ed.) Advances in Agronomy, Academic Press, 1993,

pp. 173-212.
Jo

[41] C.H. Kang, S.J. Oh, Y.J. Shin, S.H. Han, I.H. Nam, J.S. So, Bioremediation of lead

by ureolytic bacteria isolated from soil at abandoned metal mines in South Korea,

Ecological Engineering, 74 (2015) 402-407.

[42] Y. Zhao, J. Yao, Z. Yuan, T. Wang, Y. Zhang, F. Wang, Bioremediation of Cd by

28
strain GZ-22 isolated from mine soil based on biosorption and microbially induced

carbonate precipitation, Environ Sci Pollut Res Int, 24 (2017) 372-380.

[43] J.F. Peng, Y.H. Song, P. Yuan, X.Y. Cui, G.L. Qiu, The remediation of heavy metals

contaminated sediment, J Hazard Mater, 161 (2009) 633-640.

[44] H. Ding, X. Zheng, J. Zhang, Y. Zhang, J. Yu, D. Chen, Influence of chlorothalonil

and carbendazim fungicides on the transformation processes of urea nitrogen and

related microbial populations in soil, Environmental Science and Pollution Research,

of
(2019).

ro
[45] Z. Kong, Z. Wu, B.R. Glick, S. He, C. Huang, L. Wu, Co-occurrence patterns of

microbial communities affected by inoculants of plant growth-promoting bacteria

-p
during phytoremediation of heavy metal-contaminated soils, Ecotoxicology and
re
Environmental Safety, 183 (2019) 109504.

[46] D. Bulgarelli, K. Schlaeppi, S. Spaepen, L.v.T.E. Ver, P. Schulze-Lefert, Structure

lP

and functions of the bacterial microbiota of plants, Annual Review of Plant Biology, 64
na

(2012) 807-838.

[47] A. Silva-Sánchez, M. Soares, J. Rousk, Testing the dependence of microbial

ur

growth and carbon use efficiency on nitrogen availability, pH, and organic matter

quality, Soil Biology and Biochemistry, 134 (2019) 25-35.

Jo

[48] C.X. Qian, Q. Pan, R. Wang, Cementation of sand grains based on carbonate

precipitation induced by microorganism, Science China Technological Sciences, 178-

186.

[49] Y. Wang, X. Y, L. D, T. B, M. S, J. Y, X. H, Vermicompost and biochar as bio-

29
conditioners to immobilize heavy metal and improve soil fertility on cadmium

contaminated soil under acid rain stress, Science of the Total Environment, 621 (2017)

1057.

[50] R.G. Burns, International Conference: Enzymes in the Environment: Activity,

Ecology and Applications, Soil Biology & Biochemistry, 32 (2000) 1815-1815.

[51] M. Perez-Mateos, S. Gonzalez-Carcedo, Effect of silver, cadmium and lead on soil

enzyme activity, Revue Decologie Et De Biologie Du Sol, (1987).

of
[52] D. Peng, B. Wu, H. Tan, S. Hou, M. Liu, H. Tang, J. Yu, H. Xu, Effect of multiple

ro
iron-based nanoparticles on availability of lead and iron, and micro-ecology in lead

contaminated soil, Chemosphere, 228 (2019) 44-53.

-p
[53] H.H. Kong, S. Conlan, E.A. Grice, C.B. Deming, J. Davis, A.C. Young, G.G.
re
Bouffard, R.W. Blakesley, P.R. Murray, E.D. Green, Topographical and Temporal

Diversity of the Human Skin Microbiome, 324 1190-1192.

lP

[54] S. Belkin, A Panel of Stress-Responsive Luminous Bacteria for Monitoring

na

Wastewater Toxicity, Methods in Molecular Biology, 102 247-258.

[55] A. Götzl, H. Malissa, W. Riepe, A rapid toxicological test method with luminous
ur

bacteria for laboratory and on﹕ite assessment of wastes and contaminated soils, Field

Analytical Chemistry & Technology, 3 (1999) 329-337.

Jo

[56] K. Xiao, Y. Li, Y. Sun, R. Liu, J. Li, Y. Zhao, H. Xu, Remediation Performance

and Mechanism of Heavy Metals by a Bottom Up Activation and Extraction System

Using Multiple Biochemical Materials, ACS Appl Mater Interfaces, 9 (2017) 30448-

30457.

30
 Figure captions

Figure 1. Influence of initial Cd concentration on urease activity (a), Cd removal rate

(b) and finial pH (c) at day 7 when initial pH values equal to 7. Influence of initial pH

on urease activity (d), Cd removal rate (e) and finial pH (f) at day 7 when initial Cd

concentration equal to 20 mg/L. Bacterial dose added is 1% cell suspension (OD600 =

0.5) during bioremediation with and without MICP treatment.

Figure 2. SEM images of precipitation (a), EDS spectrum of precipitation (b) and XRD

of
spectrum of precipitation (c)

ro
Figure 3. The fractions of Cd and Cd immobilized rate in soil among different

treatments.
-p
Figure 4. Soil pH values and CEC in treatments at day 40. Error bars represent the
re
standard deviation of three sampled pots. The same column with different letters

indicated significant (P<0.05) according to the LSD test (n =3) differences among
lP

different treatments

Figure 5. Soil fertility in treatments at day 40. Error bars represent the standard
na

deviation of three sampled pots. The same column with different letters indicated
significant (P<0.05) according to the LSD test (n =3) differences among different
ur

treatments
Figure 6. Soil respiration (a) and enzymes activities (b) in treatments at day 40. Error
Jo

bars represent the standard deviation of three sampled pots. The same column with
different letters indicated significant (P<0.05) according to the LSD test (n =3)
differences among different treatments.
Figure 7. Soil bacteria community analysis at genus level in treatments after 40 days
of incubation

31
 of
ro
-p
re
Figure 1 Influence of initial Cd concentration on urease activity (a), Cd removal rate (b)
lP

and finial pH (c) at day 7 when initial pH values equal to 7. Influence of initial pH on
urease activity (d), Cd removal rate (e) and finial pH (f) at day 7 when initial Cd
concentration equal to 20 mg/L. Bacterial dose added is 1% cell suspension (OD600 =
na

0.5) during bioremediation with and without MICP treatment

ur
Jo

32
a b

of
c

ro
-p
re
lP
na

Figure 2 SEM images of precipitation (a), EDS spectrum of precipitation (b) and
XRD spectrum of precipitation (c)
ur
Jo

33
 of
ro
Figure 3 The fractions of Cd and Cd immobilized rate in soil among different
treatments.
-p
re
lP
na
ur
Jo

Figure 4 Soil pH values and CEC in treatments at day 40. Error bars represent the
standard deviation of three sampled pots. The same column with different letters
indicated significant (P<0.05) according to the LSD test (n =3) differences among
different treatments

34
 of
ro
-p
Figure 5 Soil fertility in treatments at day 40. Error bars represent the standard deviation
of three sampled pots. The same column with different letters indicated significant
re
(P<0.05) according to the LSD test (n =3) differences among different treatments
lP
na
ur
Jo

35
a

of
ro
b -p
re
lP
na
ur
Jo

Figure 6 Soil respiration (a) and enzymes activities (b) in treatments at day 40. Error
bars represent the standard deviation of three sampled pots. The same column with
different letters indicated significant (P<0.05) according to the LSD test (n =3)
differences among different treatments.

36
 of
ro
Figure 7 Soil bacteria community analysis at genus level in treatments after 40 days of
incubation
-p
re
lP
na
ur
Jo

37