Ecotoxicology and Environmental Safety 192 (2020) 110294

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Ecotoxicology and Environmental Safety

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

Characteristics and in situ remediation effects of heavy metal immobilizing T

bacteria on cadmium and nickel co-contaminated soil
Ying Wanga,1, Yao Luoa,1, Guoquan Zenga, Xudong Wua, Bin Wua, Xue Lib,∗∗, Heng Xua,∗
a
Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610065, Sichuan, PR China
b
Chongqing University of Technology, Chongqing, 400054, China

A R T I C LE I N FO A B S T R A C T

Keywords: Cadmium (Cd) and nickel (Ni) in soil have caused serious environmental problems and increased healthy risks to
Heavy metal humans and biota, it is vital important and necessary to develop effective methods to resolve the combined
Bacteria contaminated problems. In this study, strains L5 and L6 with good heavy metal resistant and immobilizing
Characteristic capacities were isolated from Cd and Ni contaminated soil. Bacterial characteristic experiment illustrated that
Immobilization
many functional groups (-OH, –NH2 and –COO et al.) were distributed on the surface of L5 and L6. Under the
Soil
stress of heavy metals, bacterial appearances were distorted. The pot experiment indicated that the con-
centrations of HOAc-extractable Cd and Ni in soil reduced 6.26–15.33% and 13.31–19.53% with the inoculation
of L5 and L6. In addition, the immobilization rates on Cd and Ni improved 61.27–128.50% and 23.69–39.66%
with re-inoculation of strains L5 and L6 at 30 days, respectively. After inoculation of strains L5 and L6 for 60
days, the activities of FDA hydrolysis, acid phosphatase, urease, invertase and dehydrogenase in soil increased
obviously. Furthermore, bacterial diversity indexes and community structure of soil were also improved. Thus,
given the beneficial remediation effects of the isolated strains, L5 and L6 have great potentials for heavy metals
contaminated soil remediation.

1. Introduction
Due to rapid development of industrialization and urbanization,
numerous heavy metals have been discharged into soil (Wei and Yang,
2010). Especially in southwest of China, cadmium (Cd) and the rich
mineral source Nickel (Ni) are the main contaminants in soil (Sun et al.,
2014; Yuan et al., 2012). The environmental quality standard requires
that the contents of Cd and Ni in soil should not exceed 0.6 and 60 mg/
kg, respectively, when the soil pH > 7.5 (Ministry of Natural
Resources of the People's Republic of China, 2014). In soil, Cd and Ni
are able to transfer from soil to plant and finally accumulate into
human body through food chain (Mclaughlin et al., 1999). Ad-
ditionally, high concentrations of Cd and Ni could cause serious dis-
eases, such as age-adjusted prostate, breast cancer and other respiratory
problems (Pan et al., 2010; Satarug et al., 2004). Thus, it is vital im-
portant and necessary to develop effective methods to resolve the
combined contaminated problems.
So far, heavy metal contaminated soil could be remediated through
biological, physical, chemical and combined methods (Guo et al., 2016;
Wang et al., 2017a; Wu et al., 2016). However, not all heavy metal
contaminated soil can be remediated through plant or other physical
extraction methods, especially the soil was heavily contaminated (Liu
et al., 2018). Compared to the extraction method, immobilization
method could be carried out simultaneously with normal agricultural
production through inhibiting the availability of heavy metals in soil
and therefore reducing the environmental toxicity and the amount of
heavy metals that adsorbed by plants (Wang et al., 2014). In previous
study, owing to the excellent immobilization effect, chemical im-
mobilization agents were applied to immobilize heavy metals in soil.
However, the evident side effects of the chemical immobilization such
as soil structure destruction and soil fertility reduction could not be
ignored (Yao et al., 2012). Compared with chemical agents, bacteria
such as Stenotrophomonas sp., Bacillus subtilis and Escherichia coli possess
advantages like large comparative area, fast growing speed and abun-
dant functional groups on the surface were reported to have the capa-
city to adsorb or precipitate heavy metals in soil without destroying soil
structure or soil fertility (Kiran et al., 2017; Krishnaswamy and Wilson,
2000; Lu et al., 2006; Wang et al., 2014). Therefore, microorganisms

∗
Corresponding author. Sichuan University, No.24 South Section 1, Yihuan Road, Chengdu, 610065, China.
∗∗
Corresponding author.
E-mail addresses: leexcoco217@163.com (X. Li), xuheng64@sina.com (H. Xu).
1
The first two authors contributed equally to this work and should be considered co-first authors.

https://doi.org/10.1016/j.ecoenv.2020.110294
Received 26 August 2019; Received in revised form 14 January 2020; Accepted 1 February 2020
Available online 07 February 2020
0147-6513/ © 2020 Published by Elsevier Inc.
Y. Wang, et al. Ecotoxicology and Environmental Safety 192 (2020) 110294

Table 1 characterize the isolated strains; (3) investigate individual and asso-
Design of immobilization pot experiment. ciative remediation effects of the isolated indigenous strains on Cd and
Treatment Strain Inoculation frequency Ni co-contaminated soil with different inoculation frequencies; (4) de-
termine the influence of the indigenous strains on soil ecology.
Control None None
T1 L5 Once
T2 L6 Once 2. Materials and methods
T3 L5 and L6 Once
T4 L5 Twice 2.1. Materials
T5 L6 Twice
T6 L5 and L6 Twice
CdCl2·2.5H2O, NiCl2 ·6H2O and other reagents were purchased from
Kelong Chemical Reagent (China). All of the chemicals in our experi-
have great potentials for heavy metal contaminated soil remediation. ment are analytical grade.
In previous researches, bacteria were mainly applied to the con-
taminated situation that differed to the isolated sites (Ahemad, 2014; 2.2. The isolation of Cd and Ni adsorption and resistant bacteria
Emenike et al., 2017). Owing to the competition between indigenous
and exogenous bacteria, it was difficult for exogenous bacteria to co- Cd and Ni resistant strains were isolated from Cd and Ni con-
lonize and maintain the immobilization effects in the contaminated soil. taminated soil in Sichuan University, Chengdu, China (30°38′3″N,
On the contrary, indigenous bacteria could adapt to the environment 104°5′19″W). The basic properties of soil samples are presented in
better than other exogenous microorganisms (Li et al., 2016). Table S1. The brief isolation process was: 1 g fresh soil was collected
Therefore, strains that isolated from the heavy metals contaminated and shaken with 9 mL sterilized water under 37 °C and 120 rpm for 1 h.
soil have been applied to the original contaminated soil for in situ re- After shaking, the mixture was diluted by the sterilized water and
mediation in the present study. The objectives of this study are to: (1) spread uniformly on Luria-Bertani (LB) culture medium (composition:
isolate bacteria resistant to high Cd and Ni concentrations; (2) yeast extract 5, peptone tryptone 10, and NaCl 10 g/L) with 20 mg/L Cd

Fig. 1. Evolutionary relationships of Strain L2, Strain L5, Strain L6 and Strain L9. The numbers at the branch are bootstrap values.

2
Y. Wang, et al.
Fig. 2. SEM images of strains L5 and L6 before and after heavy metal adsorption: A: strain L5 in the absence of Cd and Ni; B: strain L5 in the presence of Cd and Ni; C:
strain L6 in the absence of Cd and Ni; D: strain L6 in the presence of Cd and Ni.
and 100 mg/L of Ni (the concentrations were close to the contaminants
in soil), respectively (Netherlands, 2008). Then, the culture medium
was cultivated at 37 °C for 72 h. Through this preliminary isolation
process, Cd and Ni resistant bacteria were isolated by selecting the
individual bacterial colonies.
The isolation of Cd and Ni adsorption bacteria was carried out as the
modified method of Jiang et al. (2015). The isolated Cd and Ni resistant
strains from the previous experiment were inoculated into LB liquid
culture medium without any heavy metal addition and cultivated in the
shaking table for 24 h at 37 °C, 160 rpm. Then, the mixture was cen-
trifuged at the speed of 3000 rpm for 5 min to pour out the supernatant
and collect strains. In order to remove impurities, strains were washed
by 10 mM phosphate buffered saline (pH = 7.0). Afterwards, strains
were cultivated into LB liquid culture medium for 48 h, which con-
tained 20 mg/L Cd and 100 mg/L of Ni, respectively. Then, the bac-
terial cells were harvested by centrifugation (3000 rpm for 10 min), and
washed with sterilized deionized water. To analyze heavy metal con-
tents in strains, 0.2 g of strains were digested in a microwave with the
mixture of HNO3/HCl/HClO4 (3:2:3, v/v/v) under medium high,
medium and low temperatures for 3 min of each. The contents of heavy
metals in strains were detected by flame atomic absorption spectro-
metry (FAAS: Varian, SpectrAA 220FS) (Sungur et al., 2015).
The heavy metal adsorption ability of strains on Cd and Ni were
defined as follows:
The heavy metal adsorption ability of strains = Cs /Cp
In the equation, Cs (mg/L) and Cp (mg/L) represented the con-
centrations of heavy metals in strains and LB liquid culture medium,
respectively. Therefore, the higher value equation represented the
higher adsorption efficiency of the strains. From this adsorption pro-
cess, dominant strains have the abilities to adsorb both Cd and Ni were
isolated. Afterwards, the minimum inhibitory concentration (MIC) of
selected strains were conducted according to Manasi et al. (2016).
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Ecotoxicology and Environmental Safety 192 (2020) 110294
2.3. Characteristics of the isolated dominant bacteria
The isolated dominant strains were identified through 16 S rDNA
sequence analysis according to the previous method (Ge et al., 2011).
Afterwards, the obtained 16 S rDNA sequences were operated by BLAST
program in NCBI and analyzed by sequence alignment in GenBank
database. Subsequently, the phylogenetic trees of strains were con-
structed using the reference sequences obtained from GenBank by the
MEGA 7 program.
Besides, scanning electron microscope (SEM) and Energy Dispersive
Spectrometer (EDS) (JSM-5900LV, Japan) were employed to monitor
the surface characteristics and elements distribution of heavy metal on
the isolated strains before and after heavy metal adsorption (Ma et al.,
2013a). Inductively Coupled Plasma Mass Spectrometry (ICP-MS, Per-
kinElemer NexION 350, USA) was also applied to conform the ab-
sorption capacity of strains on heavy metal. In order to determine the
functional groups on the surface of the isolated strains, Fourier Trans-
form Infrared Spectrometer (FTIR) examination (FTIR-NEXUS 670,
USA) was also subjected.
2.4. Heavy metals immobilization experiment
2.4.1. Soil preparation and pot experiment design
To verify the immobilization capacity of the isolated strains, the
isolated strains were inoculated into the original contaminated soil. All
soil samples were air-dried and sieved through 2-mm sieve to wipe out
extraneous matters in soil. The experiment was carried out in plastic
pots (pot size: 9 cm height and 10 cm base) with 0.5 kg of the sieved
soil. The basic soil properties were shown as Table S1.
After comparing the heavy metal resistant and adsorption capacities
of isolated strains, two dominant strains (L5 and L6) were selected
accordingly. The specific experiment design in pot experiment was
presented as Table 1. 25 mL of the isolated strain suspensions
(108 CFU/mL) and sterile deionized water were sprayed into the
Y. Wang, et al.
Fig. 3. Energy spectrum (EDS) and Inductively Coupled Plasma Mass
Spectrometry (ICP-MS) analysis of strains L5 and L6 after Cd and Ni adsorption.
A: EDS analysis for strain L5; B: EDS analysis for strain L6; C: ICP-MS analysis
for the Ni absorbed in strains L5 and L6.
treatment groups and control group, respectively. Afterwards, soil was
mixed up to ensure strains were uniformly distributed. During the ex-
periment period, sterile deionized water was sprayed into soil to make
the soil water holding capacity steady (13.06 ± 0.13%). In addition,
after inoculation the strains for 30 days, in T4 (inoculated with L5), T5
(inoculated with L6) and T6 (combined inoculated with L5 and L6), the
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Ecotoxicology and Environmental Safety 192 (2020) 110294
same amounts of the isolated strains were re-inoculated into soil to
detect the impacts of re-inoculation on soil properties. To monitor the
dynamic changes of soil physical-chemical indicators and bio-available
heavy metal contents in soil, soil samples were determined every 15
days for 4 times during the pot experiment.
2.4.2. Bio-availabilities of Cd and Ni in soil samples
The bio-available Cd and Ni contents in soil samples were de-
termined by FAAS (VARIAN, SpectrAA 220FS). 0.11 mol/L HOAc so-
lution was thought to remove predominately water-soluble, exchange-
able and carbonate-bound metals in soil (Lu et al., 2007). Thus, the
determination of HOAc-extractable Cd and Ni was taken as an index to
predict the bio-availabilities of heavy metal in soil (M. Pueyo et al.,
2003). In specific, HOAc-extractable heavy metals were detected as the
following: 1 g of air-dried soil sample and 40 mL of 0.11 mol/L HOAc
was shaken at 25 °C, 250 rpm for 16 h, and centrifuged at the speed of
3000 rpm for 20 min to collect the supernatant for the HOAc-ex-
tractable heavy metal content analysis.
2.4.3. Soil enzyme activity determination
Soil samples were collected at the end of the pot experiment in each
treatment to assay soil enzyme activities. Activities of fluorescein dia-
cetate (FDA) hydrolysis, acid phosphatase, dehydrogenase, urease and
invertase were determined according to Tabatabai and Bremner (1969),
Adam and Duncan (2001), Chen et al. (2010), Gu et al. (2009) and Zhan
et al. (2010). The activity of FDA hydrolysis was spectro-
photometrically determined at 490 nm and presented by the amount of
fluorescein (μg) produced per hour and per gram of soil (g−1 h−1). The
activities of acid phosphatase and invertase were spectro-
photometrically determined at 410 nm and 508 nm, and presented as
the production of p-nitrophenol (pNP) and microgram glucose per gram
soil per 24 h, repsectively. Dehydrogenase activity was measured at
spectrophotometrically at 492 nm and presented as the production of
triphenylformazan (TPF) per gram soil per hour. In addition, the ac-
tivity of urease was spectrophotometrically measured at 578 nm and
presented as microgram NH4+ per gram soil per hour.
2.4.4. Bacterial community analysis in soil
The DNA of bacteria in soil sample was extracted from 5 g soil
through soil DNA extraction kit (Omega Bio-tek Inc., USA). Afterwards,
universal primers 338 F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806 R
(5′-GGACTACHVGGGTWTCTAAT-3′) were applied to amplify 16 S
rDNA (Mori et al., 2014; Xu et al., 2016). The analysis of bacterial
community was performed by Majorbio Bio-Pharm Technology Co., Ltd
(Shanghai, China) through the system of Illumina MiSeq. To improve
the quality of analysis results, filtering treatment was conducted on the
original data to get the optimal sequence, and the optimal sequence was
classified into different operational taxonomic units (OTUs). In addi-
tion, the analysis of biological information was carried on through
Usearch (version 7.1) with the similarity of 97% in OTU, and systematic
classification at multiple levels was carried out basing on the biological
information through ribosomal database project (RDP) (Wang et al.,
2007). On account of OTU cluster analysis results, various diversity
indexes of OTU were analyzed. In view of taxonomic information,
statistics of community structure could be analyzed on various classi-
fication levels (Jiang et al., 2013). Therefore, the community of bacteria
in soil with different treatments could be summarized.
2.5. Data analysis
In this experiment three replications were set in each treatment. All
results were presented by value ± standard deviation. Statistical sig-
nificance was evaluated by SPSS 21.0 with ANOVA, and means were
compared using least significant differences (LSD) calculated at a sig-
nificance level of P < 0.05 among different treatments. All figures
were performed using Origin V8.0 (USA) software and Photoshop CS
Y. Wang, et al.
Fig. 4. FTIR spectrograms of strains in the absence and in the presence of Cd and Ni. A: strain L5; B: strain L6.
Fig. 5. The contents of HOAc-extractable heavy metals in soil with different bacteria inoculations and frequencies. Error bars represent the standard deviation of
three sampled pots.
(USA). Phylogenetic tree of bacteria was exported by MEGA 7 (S.
Kumar et al., 2016).
3. Results and discussion
3.1. Isolation of Cd and Ni adsorption and resistant bacteria
In this experiment, 22 strains were isolated from Cd and Ni con-
taminated soil. Through re-isolation experiment, strains L2, L3, L4, L5,
L6, L7, L9 and L11 with better removal effects on heavy metals were
obtained (Table S2). Specifically, strains L2, L5, L6 and L9 performed
Cd adsorption capability with 20.80 ± 0.72%, 21.72 ± 0.12%,
22.72 ± 0.48% and 21.80 ± 0.72% adsorption rates in LB liquid
culture medium solution, respectively. Meanwhile, strains L2 and L6
had good performances on Ni removal with 56.67 ± 1.56% and
74.11 ± 3.01% adsorption rates in LB liquid culture medium solution,
respectively. Compared with other heavy metal adsorption bacteria, the
isolated strains showed better adsorption capacities on heavy metals in
liquid. In the research of Kumar et al., (2010), bacteria Bacillus sp.
Could respectively reduce 48% and 56% of Ni and Cu in solution, but
showed very weak capacity on Cd. Actinomyces sp., Streptomyces sp. And
Bacillus sp. Were able to accumulate about 5 mg/L and 10 mg/L of Cd
and Ni in liquid as the concentration of heavy metals were 1 g/L
(Karakagh et al., 2012). Therefore, with the relatively higher adsorp-
tion effects on both Cd and Ni, strains L2, L5, L6 and L9 were selected to
conduct the following experiments.
3.2. Identification of Cd and Ni adsorption bacteria
16 S rDNA sequences of L2, L5, L6 and L9 were shown in the sup-
plementary information and the phylogenetic tree of the isolated strains
was shown as Fig. 1, which indicated that strains L2, L6 and L9 be-
longed to Bacillus sp. And L5 belonged to Paenibacillus sp.. Many strains
belonging to Bacillus sp. And Paenibacillus sp. have been reported that
they could remediate heavy metal contaminated soil through absorbing
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Ecotoxicology and Environmental Safety 192 (2020) 110294
and precipitating process (Jiang et al., 2009; Rau et al., 2009). With
polysaccharide distributing on bacterial surface, Paenibacillus sp. Could
immobilize heavy metals including lead (Pb) cobalt (Co), zinc (Zn),
copper (Cu) as well as Cd and Ni through chemical reactions in soil
(Morillo et al., 2006; Morillo Pérez et al., 2008; Prado et al., 2005).
Thus, taking the heavy metal adsorption capacity and species of the
isolated strains into consideration, strains L5 and L6 were selected to
conduct the following experiment. Moreover, MIC of strains L5 and L6
toward Cd were 100 mg/L and 500 mg/L, respectively. Meanwhile, MIC
of strains L5 and L6 toward Ni were 250 mg/L and 500 mg/L, respec-
tively.
3.3. Characteristics of the isolated bacteria
3.3.1. Surface characteristics of the isolated bacteria
The surface characteristics of strains L5 and L6 were presented in
Fig. 2. It could be concluded that L5 were spherical particles with a
smooth surface before heavy metal addition (Fig. 2A). After heavy
metal adsorption (Fig. 2B), the appearance of strain L5 was distorted
and became rough. In addition, the bacteria shapes changed from el-
liptical circular to short rod and rod transition. It was reported that
heavy metals could induce various morphological changes in cells,
which might be the survival mechanisms of cells or the manifestation of
heavy metal poisoning (Nepple et al., 1999; Wu et al., 2019b). Similar
to the present experiment, Chakravarty et al. (2007) and Antony et al.
(2010) reported that strains survived from heavy metal threat were
beneficial from the cell elongation and the reduction of cell surface–-
volume ratio, which decreased the binding sites of cell surface with
heavy metals. Besides, the morphology of strain L6 became irregular,
depressed and wrinkled after the adsorption of Cd and Ni (Fig. 2D),
which might be caused by the heavy metal poisonings and was con-
sistent with the research of Tan et al. (2019). Furthermore, under the
threat of heavy metals, both strains L5 and L6 gathered together and
grew in clusters, which was benefit to resist adverse environmental
impacts and bacteria synergies (Y.M. Hua et al., 2011).
Y. Wang, et al.
Fig. 6. The activities of soil enzymes with different bacteria inoculations and frequencies during the remediation process. Error bars represent the standard deviation
of three sampled pots (A: FDA; B: Invertase; C: Acid phosphatase; D: Urease; E: Dehydrogenase).
3.3.2. The distribution of elements and functional groups on immobilizing
bacteria
The energy spectrum results of strains L5 and L6 after Cd and Ni
adsorption were shown in Fig. 3. As shown in Fig. 3A and B, Cd was
observed on both strains L5 and L6 surfaces with the treatment of Cd.
However, Ni was not observed either on the surface of L5 or L6. As we
known, Ni was an important component of enzymes, such as hydro-
genase, methylcoenzyme M reductase, urease and carbon monoxide
dehydrogenase in microorganism (Anke et al., 1984; Hausinger, 1994).
Thus, Ni might have gotten into the internal of strains and been
exploited. The content of Ni in strains was determined by ICP-MS and
presented in Fig. 3C, which demonstrated that various signal changes
were obtained with the addition of different concentrations of the strain
digestion solutions and conformed that Ni was absorbed into both
strains L5 and L6.
The FTIR adsorption spectra of strains L5 and L6 were shown in
Fig. 4. For strains L5 and L6, a wide absorption peak was observed
between 3100 and 3500 cm−1, and the peak at 3305 cm−1 and
3294 cm−1 indicated the vibration of –OH and –NH, respectively (Luo
et al., 2011), which played important roles in heavy metal adsorbing
and were abundant in cell polysaccharide, fatty acid and protein (Ma
et al., 2013b). The remaining band around 2927 cm−1 belonged to the
asymmetric stretch of alkane (C–H), which reflected the information of
water based lipid molecules on the strain surface (Jin, 2000). The
characteristic peak of protein was observed at 1654 cm−1 and reflected
the information of –NH2 (Géraldine Sarret et al., 1998). Thus, com-
parison of the FTIR spectra of strains before and after heavy metal
adsorbing, the absorption peak intensity of strains indicated Cd and Ni
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Ecotoxicology and Environmental Safety 192 (2020) 110294
might be adsorbed on hydroxide (-OH), alkane (-CH), amide (-NH2) and
carboxyl (-COOH) functional groups on the strains.
3.4. Alteration of HOAc-extractable heavy metal contents in soil
The dynamic content changes of soil HOAc-extractable Cd and Ni
with immobilizing bacterial inoculation during 60 days were presented
in Fig. 5. With bacterial inoculation, the contents of HOAc-extractable
heavy metals decreased significantly (Table S3). After 30 days of bac-
terial inoculation, the contents of HOAc-extractable heavy metals
tended to be stable in T1, T2 and T3 treatments. However, after re-
inoculation of bacteria (T4, T5 and T6), the contents of HOAc-ex-
tractable Cd and Ni kept declining. At 60 days, compared to control
(without bacteria inoculation), the contents of Cd and Ni in T1 - T6
decreased 6.26–15.33% and 13.31–19.53%, respectively. This result
was coincident with the primary isolation experiment in LB liquid
culture medium, which illustrated that the isolated strains performed
better on Ni immobilization than Cd. Besides, it was remarkable that
the contents of HOAc-extractable Cd and Ni were also fluctuated in the
control treatment without bacterial inoculation, which might cause by
the activity of indigenous microorganisms.
Moreover, the re-inoculation of bacteria resulted to 61.27–128.50%
and 23.69–39.66% higher immobilization rates on Cd and Ni, which
demonstrated the re-inoculation was propitious to the remediation
process. In addition, compared to T1 and T2 treatments, the combine
application of strains L5 and L6 individually resulted to 54.30% and
65.90% higher immobilizing rates on Cd, which was consistent with the
research that demonstrated the combined application of microbes could
Y. Wang, et al. Ecotoxicology and Environmental Safety 192 (2020) 110294

Fig. 7. Bacterial community structures under different treatments on the genus level and the abundance is presented in terms of a percentage of the total effective
bacterial sequences in the sample (A). Bar plots of Wilcoxon rank-sum test at the genus level (B: control group and L5 inoculated group; C: control group and L6
inoculated group).

7
Y. Wang, et al.
promote microbes on contaminant tolerating and remediation (Gullotto
et al., 2014). In specific, the combined application of two fungi was
reported obtaining better remediation effects on Cd and endosulfan
contaminated soil through the mutual promoted effects both on fungi
growth and toxicity tolerance (Wang et al., 2017b).
3.5. Alteration of enzyme activities in soil
As shown in Fig. 6, substantial increase of soil enzymes was ob-
served with the inoculation of bacteria in 30 days (Table S4). With the
inoculation of strains for 60 days, acid phosphatase activity increased
by 10.18–26.97%, and the activities of FDA hydrolysis, urease, in-
vertase as well as dehydrogenase obviously (p < 0.05) increased
9.42–29.18%, 59.68–134.55%, 79.19–210.92% and 6.57–174.39%,
respectively, which was mainly correlated to the reduce of heavy metal
availability in soil (Fig. 5). Wang et al. reported that the immobilization
of heavy metals could increase the activities of acid phosphatase and
urease in soil. In the present study, the increase of soil enzyme activities
could be explained as following aspects: (1) the massive microorgan-
isms in the added materials that had the capacity to stimulate the ac-
tivity of soil enzymes; (2) the functional groups on the inoculated
bacteria, such as carboxyl, phenolic, alcohol and carbonyl could react
with heavy metal ions in soil by forming metal complexes to reduce the
toxicity of heavy metal and improve the microbial activity (Nannipieri,
1994; Wang et al., 2017a).
Soil enzymes activities were influenced by soil microbial activity,
plant root system secretion, plant and soil fauna residues decomposition
activity (Suneetha and Khan, 2010). In this experiment, exogenous
nutrient was not added into soil, the amount of nutrients in soil was
limited, which would affect the normal activities of heterotrophic mi-
croorganisms in soil. Therefore, as shown in Fig. 6, the activities of
enzymes decreased at 60 days. Li et al. (2016) reported that bacterial
inoculation played significant roles in promoting the activities of FDA
hydrolysis, acid phosphatase and dehydrogenase in Cd and Ni co-con-
taminated soil. But, similar to our study, these enzyme activities also
presented decrease tendency in final. Wang et al. (2016) demonstrated
that the activity of ligninolytic enzymes in soil also decreased with
time, which was caused by the poor colonization of the inoculated
bacteria, nutrition exhaustion and competition with the indigenous
microorganisms. In addition, various proteins that secreted by bacteria
could be applied as substrates for soil enzymes (Rajkumar et al., 2012).
Therefore, it was also remarkable that the activities of soil enzymes
showed a significant recovery after 15 days of the re-inoculation of
bacteria.
3.6. The determination of soil microorganism diversity
Soil bacterial diversity was determined after 60 days of bacterial
inoculation. 272,920 efficient sequences consisted of 118488940 bases
were obtained from 7 treatments (Table S5). From the results, the
average sequence length was 434.15 bases and the OTU amount of each
sample was 511–599. Chao and Ace were commonly utilized as species
richness indicators used on the number of OTUs (Chao, 1984). Shannon
and Simpson indexes were also widely used to analyze microbial
community diversity (Gotelli, 1995). Compared to control, the diversity
indexes presented better tendency in general. Specifically, Shannon
index increased 4.73–19.90% and Simpson index decreased
35.73–66.07% with the isolated strain inoculations. The experimental
results also demonstrated that the bacterial diversity was positively
related to the content of bio-available heavy metal in soil. Owing to the
immobilizing bacteria inoculation, the availability of heavy metals in
soil were inhibited, which decreased the environmental stress to mi-
crobes in soil. The HOAc-extractable Cd and Ni contents in T4 (in-
oculated with L5 for twice) decreased by 15.33% and 19.54%, respec-
tively. Accordingly, the indexes of Ace, Chao, Shannon in T4 increased
5.89%, 6.87% and 16.17%, respectively. Meanwhile, the Simpson index
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Ecotoxicology and Environmental Safety 192 (2020) 110294
decreased 65.17%, which indicated that the diversity and species
richness were improved with the inoculation of strain L5. While, with
the comparison of Ace, Chao, Shannon index and Simpson indexes
among treatments and control, the repeated bacteria inoculation played
no significant role in whether microbial species richness or diversity.
The structures of bacterial community with different treatments on
the genus level were presented in Fig. 7A. The results illustrated that
the consistence of soil bacteria species in control and other treatments
was similar, but the abundance of bacteria in each genu was different.
In the present experiment, at genus level, the abundance of Nocardioides
bacteria in control group was obviously higher than other treatments,
which occupied about 24% of the control OTU and was reported to own
abilities to adapt to heavy metal polluted environmental through re-
acting with heavy metal copper, zinc and cadmium (Abdulla, 2009;
Shrestha et al., 2013). But, with the inoculation of L5 and L6 the pro-
portion of Nocardioides decreased significantly (Fig. 7B and C). Besides,
it was noteworthy that the proportion of Bacillus sp. obviously increased
234.71% with the inoculation of strain L6 in T2 treatment. Researches
illustrated that the community structure of soil bacteria was sensitive to
the change of heavy metal contents in soil (Wu et al., 2018; Yin et al.,
2016), resulting to the soil bacterial structure change. Wu et al. (2019a)
also indicated that the inoculation of exogenous bacteria significantly
influenced the microbial community of soil and the proportion of
exogenous bacteria significantly increased due to its colonization. In
addition, with the excellent heavy metal resistant capacity, Bacillus sp.
Was the most frequently isolated bacteria from heavy metal con-
taminated soil and might be the dominant genus in heavy metals pol-
luted areas. The increase proportion of Bacillus in soil might indicate:
(1) the heavy metals toxicity in soil was inhibited that resulted to the
increase of abundance of Bacillus in soil (Zampieri et al., 2016); (2)
Bacillus sp. had been successfully colonized in soil (Xiao et al., 2017).
4. Conclusion
In conclusion, the strains with the capacities of both tolerating and
immobilizing high concentrations of Cd and Ni have been isolated from
the combined contaminated soil. Pot experiment with the initial Cd and
Ni contaminated soil indicated that the contents of bio-available heavy
metals in soil were decreased and soil ecology including soil enzyme
activities and bacterial diversity was ameliorated with the inoculation
of the isolated strains, individually and associatively. Additionally, the
combined inoculation of strains L5 and L6 was propitious to reduce
heavy metal bioavailability in soil and alleviate the stress of metals to
soil ecology. Our study suggested that inoculation of heavy metal re-
sistant and immobilizing bacteria was a promising method to solve co-
heavy metal polluted soil problems.
Acknowledgement
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 Technology Project of Sichuan
Province (2019JDRC0092, 2018RZ0110). The authors also wish to
thank Professor Guanglei Cheng and Hui Wang from Sichuan University
for the technical assistance.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ecoenv.2020.110294.
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