Chemosphere 197 (2018) 33e41

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Bioremediation of cadmium- and zinc-contaminated soil using

Rhodobacter sphaeroides
Weihua Peng, Xiaomin Li, Jingxiang Song, Wei Jiang, Yingying Liu, Wenhong Fan*
School of Space and Environment, Beihang University, Beijing, 100191, PR China

h i g h l i g h t s

Rhodobacter sphaeroides had a remediation effect on soil contaminated with metals.

Available metal fractions were transformed to less accessible and inert fractions.

Cd phytoavailability was significantly reduced in treated soils.

Rhodobacter sphaeroides bioremediation enhanced Zn/Cd ratio in wheat.

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

Article history: Bioremediation using microorganisms is a promising technique to remediate soil contaminated with
Received 9 June 2017 heavy metals. In this study, Rhodobacter sphaeroides was used to bioremediate soils contaminated with
Received in revised form cadmium (Cd) and zinc (Zn). The study found that the treatment reduced the overall bioavailable frac-
3 January 2018
tions (e.g., exchangeable and carbonate bound phases) of Cd and Zn. More stable fractions (e.g., Fe-Mn
Accepted 5 January 2018
Available online 6 January 2018
oxide, organic bound, and residual phases (only for Zn)) increased after bioremediation. A wheat
seedling experiment revealed that the phytoavailability of Cd was reduced after bioremediation using
Handling Editor: Martine Leermakers R. sphaeroides. After bioremediation, the exchangeable phases of Cd and Zn in soil were reduced by as
much as 30.7% and 100.0%, respectively; the Cd levels in wheat leaf and root were reduced by as much as
Keywords: 62.3% and 47.2%, respectively. However, when the soils were contaminated with very high levels of Cd
Soil and Zn (Cd 54.97e65.33 mg kg1; Zn 813.4e964.8 mg kg1), bioremediation effects were not clear. The
Heavy metal study also found that R. sphaeroides bioremediation in soil can enhance the Zn/Cd ratio in the harvested
Bioremediation wheat leaf and root overall. This indicates potentially favorable application in agronomic practice and
Rhodobacter sphaeroides
biofortification. Although remediation efficiency in highly contaminated soil was not significant,
Cadmium
R. sphaeroides may be potentially and practically applied to the bioremediation of soils co-contaminated
Zinc
by Cd and Zn.
© 2018 Elsevier Ltd. All rights reserved.

1. Introduction Cadmium (Cd) is a non-essential heavy metal that is toxic, can-

Heavy metals contamination in soils is becoming an increasingly
urgent problem throughout the world (Li and Zhou, 2012; Komarek
et al., 2013; Bolan et al., 2014; Liang et al., 2014). Pedogenic pro-
cesses and anthropogenic activities associated with industrial
processes, manufacturing, domestic and industrial waste material
disposal, and phosphate fertilizer application are the major sources
of metal enrichment in soils (Wu et al., 2010; Boussen et al., 2013;
Saifullah et al., 2013; Bolan et al., 2014; Sarwar et al., 2015).
* Corresponding author.
E-mail address: fanwh@buaa.edu.cn (W. Fan).
cerogenic, and teratogenic. It does not have a biological role in
living tissue (Meda et al., 2007; Li and Zhou, 2012; Saifullah et al.,
2013; Sarwar et al., 2015, 2017; Rebekic and Lon cari
c, 2016). Cd in
soil is of great concern, because it threatens biodiversity, agricul-
tural productivity, food safety, and human health as it is trans-
mitted through the food chain (Saifullah et al., 2013; Sarwar et al.,
2015; Tavarez et al., 2015). Zn is also a common heavy metal in
contaminated soil. Unlike Cd, Zn is an important essential element,
and has important regulatory roles in a number of biological pro-
cesses, such as facilitating electron transfers for proteins and acting
as co-factors of numerous enzymes (Sarwar et al., 2017). However,
all heavy metals may cause negative ecological effects when they
exceed toxic limits. For example, overdoses of Zn can cause harmful

https://doi.org/10.1016/j.chemosphere.2018.01.017
0045-6535/© 2018 Elsevier Ltd. All rights reserved.
34 W. Peng et al. / Chemosphere 197 (2018) 33e41
effects such as dizziness and fatigue (Sarwar et al., 2017).
Several studies have demonstrated that Zn can competitively
inhibit Cd movement and accumulation in crops. As such, Zn
fertilization has been regarded as a viable agronomic practice or
biofortification approach to reduce Cd levels (Hart et al., 2002,
2005; Meda et al., 2007; Khoshgoftarmanesh et al., 2013;
Saifullah et al., 2013; Fahad et al., 2015a; Sarwar et al., 2015;
Tavarez et al., 2015). Thus, Zn fertilization is very likely to pro-
duce the co-contamination of Cd and Zn in soil, particularly when
Zn fertilization is used to reduce Cd levels and increase Zn levels in
Zn-deficient soil (Khoshgoftarmanesh et al., 2013; Fahad et al.,
2015a, 2015b). It is common to find soil that is contaminated
with both Cd and Zn. For example, one study found that Cd and Zn
concentrations were as high as 42 and 4813 mg kg1, respectively,
in soil in the Jalta mining area (Boussen et al., 2013). Thus, studying
the co-contamination of Cd and Zn in soil has realistic and signif-
icant applications.
Remediating metal-contaminated soils is important because
these soils generally cover large areas that are rendered ineligible
for sustainable agriculture (Komarek et al., 2013; Ahemad, 2014;
Chirakkara et al., 2016). Different physical, chemical, and biolog-
ical techniques are used to remove heavy metals and metalloids
from soils (Wu et al., 2010; Komarek et al., 2013; Bolan et al., 2014;
Sarwar et al., 2017). In general, remediation strategies can be
classified into two categories: mobilization and immobilization
(stabilization), which can be used both in situ and ex situ
(Vangronsveld et al., 2009; Bolan et al., 2014). Stabilization tech-
niques have shown to be cost-efficient, safe, and the least
destructive alternatives. These techniques include chemical stabi-
lization (using different stabilizing amendments to reduce
contaminant mobility, bioavailability and bioaccessibility), phy-
tostabilization (using higher plants and associated microorganisms
to immobilize contaminants in the root zone) and their combina-
tion (so-called aided phytostabilization) (Wu et al., 2010; Komarek
et al., 2013; Bolan et al., 2014). Using plants and microbes is
preferred with stabilization techniques, because of their cost-
effectiveness, environmental friendliness, and fewer side effects
(Vangronsveld et al., 2009; Wu et al., 2010; Dixit et al., 2015).
Moreover, many studies have found that, microbes, especially rhi-
zospheric microorganisms (e.g., mycorrhizal fungi, symbiotic bac-
teria and free living rhizobacteria), play an important roles in
phytoremediation, and microbial-assisted phytoremediation has
become a hot spot (Ahemad, 2014; Dixit et al., 2015; Sarwar et al.,
2017). Microremediation itself is also an important strategy of soil
bioremediation (Wu et al., 2010; Mani and Kumar, 2013). Further-
more, microorganism immobilization has significant advantages
with respect to remediation cost and environmental compatibility.
For example, it was reported that biostimulation costs were only
about half of the costs of conventional stabilization (Mani and
Kumar, 2013). Therefore, more scholars have focused on immobi-
lizing heavy metals in soils using microorganisms.
Rhodobacter sphaeroides is a gram-negative, phototropic purple
non-sulphur bacterium with several metabolic pathways depend-
ing on the growth conditions (Calvano et al., 2014). This bacterium
has drawn great attention because of its ability to grow in both
microaerobic and anaerobic light conditions. It uses different sub-
strates as carbon and energy sources, and uses ammonium and/or
nitrate as a nitrogen source. It may also use sulphide or thio-
sulphate as an electron donor under photosynthetic conditions
(Madukasi et al., 2010; Calvano et al., 2014). R. sphaeroides has a
high tolerance of abiotic stress conditions, including the presence
of both organic and inorganic pollution (Li et al., 2016, 2017). Most
importantly, this bacterium can decrease the mobility of heavy
metals by generating sulfides (Bai et al., 2008; Jiang and Fan, 2008;
Fan et al., 2012; Li et al., 2016, 2017). Sulfides have a very low
solubility product constant and aid in heavy metal precipitation,
recycling, and reuse (Muyzer and Stams, 2008; Kumar et al., 2014;
Fonti et al., 2015). Thus, R. sphaeroides is widely used for environ-
mental remediation, especially in wastewater treatment (Li et al.,
2017). R. sphaeroides had been used to degrade different contami-
nants (e.g., phosphorus, atrazine, salts, radionuclide and heavy
metal) sediment mud; studies have also evaluated its removal ef-
ficiency, impacting factors, mechanisms, and the phytotoxicity after
bioremediation (Takeno et al., 1999; Du et al., 2011; Panwichian
et al., 2012; Sasaki et al., 2012a, 2012b). Moreover, previous
research has applied R. sphaeroides to restore soils contaminated
with Cd and Pb (Fan et al., 2012; Li et al., 2016). However, there have
been few studies concentrating on bioremediation of co-
contamination of Cd and Zn or other heavy metals in soil using
R. sphaeroides.
To address this knowledge gap, this study had the following
objectives: 1) compare the bioremediation effects of R. sphaeroides
on soils contaminated with different levels of Cd and Zn; 2) analyze
the remediation efficiency and mechanisms associated with the
chemical fractions of heavy metals and wheat phytoavailability;
and 3) discuss the possible interactions between Zn and Cd.
2. Materials and methods
2.1. Strain
The bacterial strain used for soil bioremediation was isolated
from the oil field injection water in DaQing, Heilongjiang Province,
China. The strain was identified as R. sphaeroides based on its
morphological and physical bio-chemical characteristics and its
gene sequence of 16S rRNA (Fan et al., 2012; Li et al., 2016). Postgate
C culture medium was used for strain enrichment and cultivation
(Postgate, 1979).
2.2. Background soil
The background soil used for this study was collected from the
Lianxiangqiao experimental field at the Chinese Academy of Agri-
cultural Sciences in Beijing, China. After the soil was naturally air-
dried, gravels and large organic scraps were removed from the
sample. The sample was then sieved through a 0.83 mm nylon
mesh. In general, the main heavy metals in this background soil
were lower than background values found in Beijing soil (Table S1).
2.3. Preparation of spiked soils with different pollution level
After sieving pretreatment, each 1.5 kg background soil sample
was sub-divided and placed in a 2.0 L glass beaker. In each beaker,
different amounts of Cd(NO3)2 (10.0 mg mL1) and Zn(NO3)2
(100.0 mg mL1) spiked solutions were added to simulate different
pollution levels in the soils. There were a total of eight test groups,
named S1 (no Zn and Cd added), S2, S3, S4, S5, S6, S7 and S8,
respectively. The beakers were sealed with preservative film, stir-
red once daily, and incubated at room temperature for 20 days, to
reach adsorption equilibrium. Each test group had three duplicates.
Table 1 lists the Cd and Zn concentrations in each test group.
2.4. Bioremediation experiment
The remediation tests were performed in the 2.0 L glass beakers.
After 20 d spiking, 300 mL of the strain liquid (approximately
2  108 of R. sphaeroides, CFU ¼ 6.7  105 mL1) (Fan et al., 2012)
was directly added into each test group. Deionized water was then
added to maintain 1800 mL of overlying water. After the soil was
fully mixed with the strain, the beakers were sealed with
 W. Peng et al. / Chemosphere 197 (2018) 33e41
Table 1
Cd and Zn concentrations in each test group (mean ± standard deviation, n ¼ 3, mg kg1).
Test groups S1 S2 S3
Cd 0.12 ± 0.05 6.95 ± 0.03 11.95 ± 0.19
Zn 69.89 ± 1.64 190.99 ± 4.40 310.40 ± 1.41
preservative film and incubated in a biochemical incubator at 30  C
for a 50 d bioremediation period.
2.5. Sample collecting and testing
During the 50 d bioremediation, soil samples were collected
from each test group at 0 d (before bioremediation), and at 14 d and
50 d. Both the total heavy metals levels and their speciations in the
soil samples were analyzed. Each soil sample was acid digested
(HNO3:HClO4 ¼ 3:2) and total heavy metals were analyzed using
inductively coupled plasma-atomic emission spectrometry (ICP-
AES, IRIS Intrepid-II). The speciations of heavy metals in soil were
determined using the improved Tessier sequential extraction pro-
cedure (Fan et al., 2002). This procedure which divided the heavy
metals combined with soil into five chemical fractions: exchange-
able (F1), carbonate bound (F2), Fe-Mn oxide (F3), organic bound
(F4), and residual phases (F5). The pH, oxidation reduction potential
(ORP) and sulfate in supernatant were also measured, using pre-
viously described methods (Fan et al., 2012). A reference material of
stream sediment (GBW-07309) was used for quality control.
2.6. Wheat seedling experiments
Wheat seeds (Lunxuan 987) obtained from the Chinese Acad-
emy of Agricultural Sciences were used to evaluate the remediation
efficiency of R. sphaeroides in each test group. The soil in each test
group before bioremediation and after 50 d bioremediation was
used for this part of the experiment. Wheat seedling experiments
were prepared and conducted based on previous research (Fan
et al., 2012; Li et al., 2016). After harvest, the wheat leaves and
roots in each group were washed, separated, dried, weighed, acid
digested (HNO3:HClO4 ¼ 3:2), and then analyzed with ICP-AES (IRIS
Intrepid-II).
2.7. Data analysis
One-way ANOVA (LSD method) was performed to identify the
differences of Zn and Cd in soils during bioremediation (0 d, 14 d
and 50 d), including total content and geochemical fractions. P-
values of less than 0.05 were considered statistically significant.
Two-tailed t-test was used to compare the difference of Cd, Zn, and
Zn/Cd ratio in wheat (e.g., leaf and root) harvested from the soil
before and after 50 d bioremediation within each test group. The
results of variance homogeneity for ANOVA, and the P-values for t-
test analysis were listed in Table S2 and Table S3, respectively. Data
analyses were conducted using IBM SPSS Statistics 19; OriginPro 8
was used to plot graphs.
3. Results
3.1. Total content of Cd and Zn
During bioremediation, the total Cd (Fig. S1a) and Zn (Fig. S1b)
concentrations in the soil of each test group did not significantly
change. The total Cd (Fig. S1c) and Zn (Fig. S1d) concentrations
were significantly positively correlated with their sum of
geochemical fractions (P < .001). This indicates that the sample
treatment process and test method were reliable.
35
S4 S5 S6 S7 S8
25.29 ± 0.04 37.66 ± 1.70 46.52 ± 0.82 54.97 ± 0.38 65.33 ± 1.63
431.8 ± 6.79 498.4 ± 13.86 587.7 ± 2.12 813.4 ± 23.19 964.8 ± 12.45
3.2. Change of Cd and Zn speciations
Fig. 1 and Fig. 2 show the absolute content of Cd and Zn
geochemical fractions in soils, respectively, during bioremediation.
Before bioremediation (black bar), as the total Cd and Zn increased
in spiked soils, the geochemical fractions of Cd and Zn gradually
increased from test group S1 to S8. In general, F1 and F3 were the
dominant fractions for Cd; Zn was predominantly associated with
F3 and F5.
For Cd (Fig. 1), F5 was not detected in any test group. After 50 d
bioremediation, F1 (Fig. 1a) and F2 (Fig. 1b) decreased in the test
groups overall, and F3 (Fig. 1c) and F4 (Fig. 1d) increased overall.
These results were particularly clear in the test groups with high
heavy metal levels (e.g., test groups S5-S8). In test groups S5-S8, F1
and F2 decreased by 13.3e30.7% (mean value 20.3%) and
20.7e42.4% (mean value 33.8%), respectively; while F3 and F4
increased by 6.8e33.5% (mean value 24.0%) and 24.3e48.9% (mean
value 33.5%), respectively.
For Zn (Fig. 2), after 50 d bioremediation, F1 (Fig. 2a), and F2
(Fig. 2b) in each test group were significantly decreased overall,
while F3 (Fig. 2c) and F5 (Fig. 2e) increased; F4 (Fig. 2d) decreased
in the test groups having low Cd and Zn (e.g., test groups S1-S5). In
test groups S1-S8, F1 and F2 were significantly reduced by
48.0e100.0% (mean value 69.3%) and 88.2e100.0% (mean value
97.8%), respectively. F3 (except for test groups S1 and S5) and F5
(except for test group S1) increased by 5.3e15.0% (mean value
10.0%) and 12.4e28.4% (mean value 19.1%), respectively. In test
group S1-S5, F4 was reduced by 15.4e56.8% (mean value 25.5%).
3.3. Change of Cd and Zn in wheat leaf and root
Fig. 3 shows Cd and Zn concentrations in wheat leaf and root
before and after bioremediation. As the Cd and Zn in spiked soils
increased, the Cd and Zn concentrations in wheat leaf and root also
increased overall. A correlation analysis shows that (Fig. 4), the
heavy metals (e.g., Cd and Zn) in wheat (e.g., leaf and root) were
significant positively correlated with the levels in the spiked soils
(P < .001). In general, the wheat root had higher Cd and Zn levels
than in the leaf. Before and after bioremediation, the translocation
factors (ratio of metal concentration in shoots to roots) of Cd were
0.12e0.29 and 0.13e0.54, respectively, while the translocation
factors of Zn were 0.12e0.58 and 0.15e0.59, respectively (Table S4).
Fig. 3 also shows that after 50 d bioremediation, Cd concentra-
tions in the wheat leaf and root were significantly reduced in most
spiked groups, with the exception of test groups S7-S8. Those two
test groups were highly contaminated and the results had a large
error (Fig. 3a and b). In test groups S2-S6, the Cd concentrations in
the wheat leaf and root were reduced by 17.4e62.3% and
18.6e47.2%, respectively. However, this was not observed for Zn. In
contrast, in most test groups (e.g., S5-S8), the Zn concentrations in
the wheat leaf after bioremediation were even higher than before
bioremediation (Fig. 3c). The linear fitting of heavy metal levels
between wheat and soil also indicated that Cd in the wheat leaf
(Fig. 4a) and wheat root (Fig. 4b), as well as Zn in the wheat root
(Fig. 4e), were reduced overall after bioremediation. Nevertheless,
Zn in the wheat leaf after bioremediation was generally higher than
before bioremediation (Fig. 4d, black line). Cd translocation factor
(Fig. 4c) was not obviously changed after bioremediation, while Zn
36 W. Peng et al. / Chemosphere 197 (2018) 33e41

Fig. 1. Cd geochemical fractions in soils during bioremediation (F1, F2, F3, and F4 represent Cd exchangeable phase, carbonate bound phase, Fe-Mn oxide phase, and organic bound
phase, respectively; different lowercase letters represent the significant difference during bioremediation within each test group using one-way ANOVA, while different uppercase
letters represent the significant difference between test groups at a sampling date).

translocation factor (Fig. 4f) was increased overall. mechanism (Chen et al., 2006; Lee et al., 2009; Liang et al., 2014).
In this study, the bioavailable fractions (e.g., F1 and F2) of Cd in
test soils were reduced overall (Fig. 1). In contrast, more stable
4. Discussions
fractions (e.g., F3 and F4) increased. This was particularly marked
for test groups S5-S8 (P < .05), which had high Cd and Zn concen-
4.1. Bioremediation results
trations. This indicated that Cd chemical fractions in the soil had
been changed by the R. sphaeroides bioremediation, and bioavail-
The toxicity and bioavailability of heavy metals depend on their
ability and toxicity were reduced. Previous research showed that
speciation, not the total concentration (Qian et al., 2009; Fonti et al.,
R. sphaeroides transformed more available fractions of heavy metals
2015). Sequential extraction is a useful tool to examine the
(e.g., Cd and Pb) into less accessible and inert fractions, reducing
bioavailability of heavy metals in soil, which critically influences
metal mobility and phytoavailability (Jiang and Fan, 2008; Fan et al.,
the plant's metal uptake (Lee et al., 2009; Vangronsveld et al., 2009;
2012; Li et al., 2016). Moreover, it was concluded that metal sulfide
Komarek et al., 2013; Bolan et al., 2014). In general, the stability of
formation was the main bioremediation mechanism of
the metal speciation, determined using the Tessier method, was as
R. sphaeroides (Bai et al., 2008; Jiang and Fan, 2008; Fan et al., 2012;
follows: F1 was the lowest, followed by F2, F3, F4, and F5 (most
Li et al., 2016a, 2017). This was the main reason why F1 and F2 of Cd
stable) (Peng et al., 2009; Bolan et al., 2014). F1 and F2 can be easily
decreased overall during bioremediation, while F3 and F4 of Cd
mobilized/solubilized, with detrimental effects on ecosystem
increased. However, the F5 of Cd was not detected after bioreme-
health (Shin and Kim, 2015). F3 and F4 are the most important
diation. This may be because, under these experimental conditions,
components for metal binding. Due to their strong complexation
the remediation time (50 d) was not long enough to form the most
effect with heavy metals, these two fractions can decrease heavy
stable mineral form (e.g., F5) (Li et al., 2016).
metal bioavailability (Burton, 2010). However, under changing
From the perspective of geochemical fractions, the bioremedi-
conditions, the heavy metal associated with F3 and F4 can also be
ation effect for Zn was highly significant (Fig. 2), because F1 and F2
mobilized/solubilized (Wang et al., 2014; Fonti et al., 2015), and
(P < .05) of Zn were significantly reduced; F5 of Zn significantly
may represent the potentially mobile component (Peng et al., 2009;
increased (with the exclusion of uncontaminated test group S1,
Shin and Kim, 2015). F5 entrapped within crystal minerals is usu-
P < .05); the potentially mobile component (e.g., F3 and F4) was
ally considered stable, with low mobility and no toxicity (Chen
also increased in some test groups, especially for F3. This indicated
et al., 2006; Wang et al., 2014; Fonti et al., 2015). Thus, the
that Zn bioavailability and toxicity decreased after bioremediation.
changing metal speciation is usually used to assess the remediation
The wheat seedling experiment provided the direct evidence for
efficiency of heavy metal in soil, as well as to reveal the remediation
 W. Peng et al. / Chemosphere 197 (2018) 33e41 37

Fig. 2. Zn geochemical fractions in soils during bioremediation (F1, F2, F3, F4, and F5 represent Zn exchangeable phase, carbonate bound phase, Fe-Mn oxide phase, organic bound
phase, and residual phase, respectively; different lowercase letters represent the significant difference during bioremediation within each test group using one-way ANOVA, while
different uppercase letters represent the significant difference between test groups at a sampling date).

the Cd bioremediation effect, because the Cd in the wheat leaf and
root of most test groups were both significantly reduced. The
exception was with test groups S7-S8, which were highly
contaminated with significant error rates (Fig. 3a and b). However,
unlike Cd, the Zn concentrations in the wheat leaf and root were
not clearly reduced. In contrast, after bioremediation, Zn concen-
trations in the wheat leaf and root of some test groups were even
higher than before bioremediation, particular for Zn in the wheat
leaf (Figs. 3c and 4d).
Although high Cd and Zn levels in the wheat root were still
observed after bioremediation, Fig. 4 indicated that both Cd (Fig. 4b,
red line) and Zn (Fig. 4e, red line) in wheat roots were less
accumulated overall after bioremediation. From this perspective,
R. sphaeroides bioremediation of soil reduced Cd and Zn accumu-
lation in wheat root.
It is difficult to compare the remediation efficiency of different
soils contaminated by different heavy metals, due to factors such as
different soil physical-chemical properties, initial heavy metal
concentrations, remediation method, remediation time, and
extraction procedures. Despite this challenge, this study attempted
to compare R. sphaeroides biostabilization efficiency with previous
studies, mainly from the perspective of Cd and Zn chemical speci-
ation changes.
Chen et al. (2006) used rock phosphate to remediate soils
38 W. Peng et al. / Chemosphere 197 (2018) 33e41
Fig. 3. Cd and Zn in wheat leaf and root harvested before and after bioremediation (* and ** represent the significant difference before and after bioremediation within each test
group at P < .05 and P < .01 level using t-test, respectively).
contaminated with Cd (4.5e6.66 mg kg1), Pb
(275.6e338.8 mg kg1) and Zn (328.78e637.9 mg kg1). After 110 d
remediation, rock phosphates (grain size <35 mm) transformed the
non-residual fractions of heavy metals to a residual fraction, with
increments of 30.8e31.7% Cd, 30.3e34.6% Pb, and 25.8e26.3% Zn,
respectively. They found that rock phosphate also reduced plant
uptake (Brassica oleracea L.) shoots for Cd (19.6e50.0%), Pb
(21.9e51.4%), and Zn (22.4e34.6%). Lee et al. (2009) investigated
different amendments (e.g., limestone, red-mud, and furnace slag)
to remediate soil contaminated with Cd, Pb, and Zn, with initial
concentrations of 10.81, 1295, and 545 mg kg1, respectively. The
study found that these amendments also shifted metal distribu-
tions from the exchangeable to the carbonate and FeeMn oxide
fractions. Moreover, red-mud was the most effective treatment in
decreasing heavy-metal concentrations in lettuce, reducing lettuce
uptake of Cd, Pb, and Zn by 86%, 58%, and 73%, respectively. Liang
et al. (2014) used sepiolite and palygorskite as amendments to
remediate Cd-polluted paddy soil at an in situ field-scale. Applying
sepiolite and palygorskite modified the fraction of Cd in soil from
phytoavailable to less phytoavailable phases, for example, these
two amendments reduced the F1 of Cd by 8.9e20.3% and
6.5e14.6%, respectively. The study also found that, after chemical
immobilization, Cd concentrations in brown rice decreased, indi-
cating the decrease in phytoavailable Cd concentrations in paddy
soils.
In this study, after R. sphaeroides bioremediation, the F1 of Cd
and Zn were reduced by 13.3e30.7% (in test groups S5-S8) and
48.0e100.0% (in all test groups), respectively. Cd concentrations in
the wheat leaf and root were reduced by 17.4e62.3% and
18.6e47.2% (in test groups S2-S6), respectively. Thus, when
compared with previous research, this study's R. sphaeroides
bioremediation achieved effects that are comparable with chemical
immobilization approaches, and may be both faster and more
efficient.
In a previous study under similar experimental conditions,
when Cd levels in the spiked soil were set at <100 mg kg1, the F1
of Cd was reduced by 27e46% using R. sphaeroides (Fan et al.,
2012). However, in this study, even though the Cd in the spiked
soil was 65.33 mg kg1, the F1 of Cd was only reduced by
13.3e30.7% (in test groups S5-S8). This was significantly lower
than previous research results, and suggests that remediation
efficiency varies due to different contamination conditions. This
also suggests that soil contaminated with multiple metals (e.g., Cd
and Zn) is likely more complex and more difficult to restore than
soil contaminated with a single metal (e.g., Cd). Chirakkara et al.
(2016) also summarized the complexities of mixed contamina-
tion. Combining of phytoremediation and microorganism has now
been considered as a promising strategy to enhance the remedi-
ation efficiency of contaminated soil, particularly mixed
contaminated soil. It was found that, R. sphaeroides can promote
plant growth (e.g., cucumber) via producing of indole acetic acid
and/or organic acids (Kang et al., 2015). Moreover, Jiao et al. (2015)
concluded that R. sphaeroides can also assist plants (e.g., cabbage
and spinach) to degrade petroleum hydrocarbon. Thus,
R. sphaeroides may have potential implications for microbial-
assisted phytoremediation. Better remediation effect of mixed
contaminated soil may be achieved by combining R. sphaeroides
with phyoremediation.
 W. Peng et al. / Chemosphere 197 (2018) 33e41 39

Fig. 4. The correlations between heavy metals (Cd, Zn and their translocation factor) in wheat (leaf and root) and their content in soil before and after bioremediation (a-c for Cd, e-f
for Zn).

4.2. Interactions between Zn and Cd
Trace element interactions can influence the extent to which
metals and micronutrients are taken up and transported to plant
tissues (Sarwar et al., 2015; Tavarez et al., 2015). Cd and Zn in-
teractions remain controversial; however, most studies have
concluded that Zn can act as a competitive ion for Cd absorption
from soil by roots. Zn may also interact with Cd within the plant's
transport system, due to its chemical similarity with Cd (Meda
et al., 2007; Li and Zhou, 2012; Sarwar et al., 2015; Tavarez et al.,
2015; Rebeki c and Loncaric, 2016). As such, Zn competitively in-
hibits Cd movement and accumulation in crops (Hart et al., 2002,
2005; Meda et al., 2007; Khoshgoftarmanesh et al., 2013;
Saifullah et al., 2013; Fahad et al., 2015a; Sarwar et al., 2015;
Tavarez et al., 2015). For example, Hart et al. (2002, 2005, 2006)
demonstrated that Zn2þ and Cd2þ compete for transport by a
common carrier at the plasma membrane of wheat root cells. This
inhibits Cd uptake at the plasma membrane of root epidermal and
cortical cells, and reduces grain Cd concentrations in durum wheat.
Increasing Zn/Cd ratio in crop grain using Zn fertilization is
considered a viable agronomic practice or biofortification approach
to reduce Cd and enhance Zn (Hart et al., 2005; Khoshgoftarmanesh
et al., 2013; Saifullah et al., 2013; Sarwar et al., 2015; Tavarez et al.,
2015), particularly in Zn-deficient soils (Khoshgoftarmanesh et al.,
2013; Fahad et al., 2015a, 2015b). Studies have also found that the
effect of Zn on Cd inhabitation and accumulation is influenced by
40 W. Peng et al. / Chemosphere 197 (2018) 33e41
Fig. 5. Zn/Cd ratio in wheat leaf (a) and root (b) harvested from the soil before and after bioremediation (* and ** represent the significant difference before and after bioremediation
within each test group at P < .05 and P < .01 level using t-test, respectively).
crop variety and soil properties, as well as the pattern, amount, and
time of Zn fertilization (Khoshgoftarmanesh et al., 2013; Saifullah
et al., 2013; Fahad et al., 2015b; Sarwar et al., 2015; Tavarez et al.,
2015).
In this study, after the soils were treated with R. sphaeroides, the
Zn/Cd ratio in the harvested wheat leaf and root of each test group
was enhanced overall (Fig. 5). The increasing of Zn/Cd ratio in
wheat leaf and root has great significance for increasing Zn/Cd ratio
in wheat grain. Thus, from this perspective, R. sphaeroides biore-
mediation improved Cd and Zn uptake, translocation, and accu-
mulation. This suggests a potentially positive application for
restoring soil contaminated with Cd and Zn. However, as the Cd and
Zn in the test group soils increased, the Zn/Cd ratios in both the
wheat leaf and root gradually decreased, particularly for test groups
S7 and S8. After bioremediation, the Zn/Cd ratios in the wheat root
of these two test groups were even lower than before bioremedi-
ation (Fig. 5b). This indicated that when the soil was highly
contaminated by Cd and Zn (Cd 54.97e65.33 mg kg1; Zn
813.4e964.8 mg kg1), the R. sphaeroides bioremediation effect
were not as clear. Thus, Cd and Zn levels are critical for bioreme-
diation efficiency; this is directly reflected in their levels in the
wheat leaf and root (Fig. 3).
From the perspective of geochemical fractions, the bioavailable
fractions (e.g., F1 and F2) of Cd (Fig. 1) and Zn (Fig. 2) in soil were
both reduced after bioremediation. Whereas, after wheat leaf and
root were harvested from soils treated by R. sphaeroides, Zn levels
were not significantly reduced. This differs from Cd. For most of the
test groups, the Zn concentrations in the wheat leaf and root were
even higher after bioremediation than before it (Fig. 3c and d).
Moreover, it was observed that Zn translocation factor was also
increased overall after bioremediation (Fig. 4f). This may be
because Cd and Zn are two different functional elements. Cd is one
of the most toxic non-essential elements, whereas Zn is an
important essential element (Meda et al., 2007; Li and Zhou, 2012;
Saifullah et al., 2013; Sarwar et al., 2015, 2017; Rebeki c and
Loncaric, 2016). The relationships between heavy metals (Cd and
Zn) in wheat (leaf and root) and their exchangeable phase content
in soil also indicated that wheat showed different metabolism
patterns for these two heavy metals (Fig. S2). Although F1 and F2
levels in soils were significantly reduced after bioremediation
(Fig. 2), other Zn chemical forms (e.g., F3 and F4 acting as the
potentially mobile component) could be taken up by wheat root
through the rhizosphere's biogeochemical activities. Microorgan-
isms (e.g., plant growth promoting bacteria and arbuscular
mycorrhizal fungi) in the rhizosphere can modify the chemical
composition of root exudates and soil pH. As such, they either
increase the mobilization of essential mineral elements in the
rhizosphere or inhibit uptake of toxic mineral elements from the
soil solution (Meda et al., 2007; Philippot et al., 2013; Ahemad,
2014; Sarwar et al., 2017).
Thus, under combined pollution stress, the ability of wheat
cellular homeostasis was enhanced after R. sphaeroides bioreme-
diation. This resulted in a higher Zn/Cd ratio in the wheat leaf and
root, which was expected in soil remediation and biofortification.
However, the adjustment mechanism associated with the wheat's
uptake of Cd and Zn was still not clear, nor was their translocation
and accumulation during the wheat growth process.
5. Conclusions
In summary, using R. sphaeroides as a bioremediation agent
stabilized Cd and Zn contaminants in soil. The bioremediation ef-
fects were indirectly reflected through changes in Cd and Zn
chemical fractions, and directly confirmed using a wheat seedling
experiment. The study found that R. sphaeroides bioremediation
reduced the bioavailable fractions (e.g., F1 and F2) of Cd and Zn in
soil, decreased Cd and Zn accumulation in wheat, and enhanced the
Zn/Cd ratio in wheat. R. sphaeroides bioremediation is comparable
to chemical immobilization approaches in terms of remediation
efficiency, and may result in faster and more efficient remediation.
However, Cd and Zn contaminant concentrations are critical to
bioremediation efficiency. When soil had very high Cd and Zn levels
(Cd 54.97e65.33 mg kg1; Zn 813.4e964.8 mg kg1), the bioreme-
diation effects were not clear. At these contaminant levels, the Cd
and Zn concentrations in the wheat leaf and root after bioremedi-
ation were not significantly reduced, and were even higher than
before bioremediation. Moreover, as Cd and Zn levels in soils
increased, the Zn/Cd ratios in the wheat leaf and root gradually
decreased.
Further research is needed to enhance the bioremediation effi-
ciency of R. sphaeroides in highly contaminated soil; to reveal the
bioremediation mechanisms from the perspective of the microor-
ganism community; and to explore the adjustment mechanism
that wheat uses to uptake, translocate, and accumulate Cd and Zn.
Moreover, a feasibility study is needed to assess the bioremediation
possible with R. sphaeroides on a pilot scale. That work would then
inform future practical applications in soil restoration.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (51778031, 21707006 and 51708012), Beijing
 W. Peng et al. / Chemosphere 197 (2018) 33e41 41

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