Water Air Soil Pollut (2017) 228:295
DOI 10.1007/s11270-017-3484-4
The Contribution of Pyrene Degrading Bacteria and Chemical
Reagents to Scirpus triqueter Phytoremediation of Pyrene
and Ni Co-contaminated Soil
Xiaoxin Hu & Xinying Zhang & Xiaoyan Liu
Liya Cao & Jing Chen & Zhuhao Huo
Received: 31 March 2017 / Accepted: 12 July 2017
# Springer International Publishing AG 2017
Abstract Phytoremediation of organic pollutant and
heavy metal (HM) co-contaminated soils shows many
advantages and can be improved by adding chemical
reagents or inoculating with degrading bacteria. In this
study, pot culture experiments were performed to ex-
plore the effects of chemical reagents (nitrilotriacetic
acid and alkyl polyglucoside), pyrene degrading bacte-
ria HD-1, and their combination on phytoremediation
efficiency for pyrene and nickel (Ni) co-contaminated
soil by Scirpus triqueter. After a 60-day culture, plant
biomass, pyrene dissipation from soil, Ni accumulation
in plant, and Ni accessibility in co-contaminated soil
were determined. Results showed that although the ap-
plication of chemical reagents alone had no apparent
effect on plant growth, their combination with the
Xinying Zhang and Xiaoyan Liu Equal contributors.
Highlights
Bacteria and chemical reagents could alleviate the growth
inhibition of plants
Ni in S. triqueter increased with the application of bacteria and
chemical reagents
Bacteria and chemical reagents could improve bioaccessible
fraction of Ni in soil
S. triqueter, chemical reagents, bacteria significantly enhanced
pyrene dissipation
X. Hu : X. Zhang (*) : X. Liu (*) : L. Cao : J. Chen :
Z. Huo
Laboratory of Environmental Remediation, School of
Environmental and Chemical Engineering, Shanghai University,
No.99, Shangda Road, Baoshan District, Shanghai 200444, China
e-mail: Zxyshu@shu.edu.cn
e-mail: lxy999@shu.edu.cn
&
introduced HD-1 alleviated the inhibition effects on
plant growth in co-contaminated soil. The dissipation
of pyrene in the soil with plant (P), soil with bacteria
(NPB), soil with chemical reagents (NPC) and soil with
both of them (PBC) were 35.49, 51.36, 42.89, and
59.78%, respectively, and were higher than NP
(19.52%) with neither of them. The Ni concentration
in Scirpus triqueter of group with bacteria (PB), group
with chemical reagents (PC) and group PBC increased
to 100.40, 80.97 and 87.77 mg kg1 respectively when
compared with that of group P (46.04 mg kg1) without
bacteria or chemical reagents. Besides, inoculation with
HD-1 or/and adding chemical reagents caused Ni to
shift from less bioavailable forms to more bioavailable
forms. This study suggested the contribution of pyrene
degrading bacteria and chemical reagents to Scirpus
triqueter phytoremediation of pyrene and Ni co-
contaminated soil.
Keywords Pyrene . Ni . Scirpus triqueter . Pyrene
degrading bacteria . Nitrilotriacetic acid . Alkyl
polyglucoside
1 Introduction
In nature, co-contaminations of heavy metal (HM) and
organic pollutants are ubiquitous, and the harmfulness is
obviously stronger than the single pollution (Lu et al.
2014; Zhao et al. 2016). Among heavy metals, nickel
(Ni) is frequently found in soil, which have caused
widespread concern due to its coexistence with
295 Page 2 of 9
polycyclic aromatic hydrocarbon (PAH), especially
pyrene, at many sites and threats to human health
(Mielke et al. 2001; Reddy 2010). As a promising
solution, phytoremediation is regarded as a suitable
technology because of its effectiveness and lower cost
(Hou et al. 2016; Romeh et al. 2016). In previous
investigations, the ecological risk of HM and PAH was
found in sediments from Huangpu River. Scirpus
triqueter (S. triqueter), with an extensive fibrous root
system, was a dominant wetland species along this river.
The large specific surface area of S. triqueter root helped
to promote the efficiency of phytoremediation (Zhang
et al. 2013). Considering limited capabilities of plants to
remove organic pollutants, inoculation of specific
strains with degradation capacity (such as pyrene
degrading bacteria) was suggested as a strategy for
improving remediation efficiency of co-contaminated
soil (Chaudhry et al. 2005). However, the remediation
efficiency is also closely related to the availability of
contaminants. PAH and HM due to their poor availabil-
ity in soil may result in lower removal in bacteria
assisted phytoremediation, which needed to be assisted
by chemical reagents including chelating agents and
surfactants.
The application of chelating agents can desorb toxic
metals from soil and enhance phytoremediation process
by increasing the plant available fraction, uptake, and
transport amounts of metals (Lee and Sung 2014). For
example, Lan et al. (2013) reported that addition of three
chelants (biodegradable ethylene diamine tetraacetate,
nitrilotriacetic, and anionic polyacrylamide) could in-
crease significantly the Cd concentrations in the shoots
of Siegesbeckia orientalis L. Zaier et al. (2010) also
found that ethylene diaminetetraacetic acid had a signif-
icant positive effects on metals (Zn, Mn, and Pb) uptake
and translocation in Brassica napus. Surfactants which
are amphiphilic chemicals can enhance the water solu-
bility of hydrophobic compounds and are used widely in
promoting microbial remediation for PAH (Gonzlez
et al. 2011; Mao et al. 2015). For example, Boonchan
et al. (1998) tested various synthetic surfactants for their
ability to enhance the PAH microbial degradation.
Bautista et al. (2009) indicated that using surfactants is
very effective for assisting a consortium composed by
three degrading bacteria in the PAH-biodegradation pro-
cess. Evidences from chelating agents enhanced
phytoremediation and surfactant assisted microbial bio-
remediation experiments have shown different degrees
of effectiveness for the removal of HM or PAH,
Water Air Soil Pollut (2017) 228:295
exhibiting the importance of studying the combined
system for the remediation of co-contaminated soil.
As a biodegradable chelating agent, nitrilotriacetic
acid (NTA) was reported to greatly increase plant-
uptake of heavy metals from soil (Robinson et al.
2000; Xie et al. 2015). Alkyl polyglucoside (APG), a
safe and environmentally friendly non-ionic surfactant,
has been proven to be effective in enhancing the bio-
availability of PAH and beneficial to the degradation of
pyrene (Liu et al. 2013; Madsen and Kristensen 1997).
In our previous study, the synergistic effect of NTA and
APG for pyrene or lead in phytoremediation has been
investigated, which demonstrated that they could be
potentially applied to enhance phytoremediation of co-
contaminated soil (Chen et al. 2016b). However, few
research focus on the remediation efficiency of co-
contaminated soil under the combined application of
chemical reagents (NTA and APG), pyrene degrading
bacteria and plant. Moreover, little is known about the
contribution of the three factors on remediation process.
Therefore, the objectives of this study were to (1)
investigate the effects of chemical reagents, pyrene-
degrading bacteria, and their combination on plant
growth, the dissipation of pyrene, as well as on the
accumulation of Ni in plant and the accessibility of Ni
in co-contaminated soil; and (2) explore preliminarily
the contribution of chemical reagents, pyrene-degrading
bacteria, and S. triqueter on the dissipation of pyrene
from co-contaminated soil.
2 Materials and Methods
2.1 Chemicals
APG1214 used in the test was bought from the China
Research Institute of Daily Chemical Industry. All other
reagents, analytical grade at least, which were purchased
from Sinopharm.
2.2 Soil Preparation
The soil sample (air-dried, 2 mm sieved) was gained
from the topsoil (020 cm) without exposing previously
to Ni and pyrene contamination in Shanghai University,
China. The uncontaminated soil consists of 7.4% clay,
60.4% silt, and 32.2% sand, and the basic properties are
as follows: pH 8.3, total nitrogen 0.5 mg kg1, total
phosphorus 1.2 mg kg 1 , and organic matter
Water Air Soil Pollut (2017) 228:295
19.6 g kg1. Contaminated soil was made by adding the
stock solution of pyrene-acetone firstly. After few days,
the Ni (NiCl2) solution was added. The contaminated
soil was aged for about 2 months after all the solvents
volatilized. The final concentrations of Ni and pyrene in
the soil were measured as 296.1 and 356.9 mg kg1,
respectively. The concentrations were chosen according
to the screening value of Ni and pyrene for soil risk
assessment (Beijing Bureau of Quality and Technical
Supervision DB11/T 8112011).
2.3 Preparation of Bacterial Suspension
Bacillus subtilis named HD-1 was once isolated from
sludge collected in a wastewater treatment plant, which
was chosen as pyrene degrading bacterium in terms of
the high removal ratio of pyrene. The 16S rRNA gene
sequence was deposited in the GenBank database under
the accession number KY581582.
HD-1 was incubated in Luria-Bertani (LB) liquid
medium (30 C, 150 rpm) until the exponential phase
was reached (Tao et al. 2017). Then cells were collected
by centrifugation, washed three times with sterile water.
Subsequently, they were suspended in sterile water
(OD600 adjusted to 1.0).
2.4 Experimental Design
The experimental design is shown in Table 1. The pots
containing 500 g soil were set up in greenhouse and
Table 1 Experimental design of different treatments
Treatments Description
CK S. triqueter, in uncontaminated soil
P S. triqueter, in contaminated soil
NP Without S. triqueter, in contaminated soil
PB S. triqueter, inoculated with HD-1, in contaminated
soil
NPB Without S. triqueter, inoculated with HD-1, in
contaminated soil
PC S. triqueter, with NTA and APG, in contaminated soil
NPC Without S. triqueter, with NTA and APG, in
contaminated soil
PBC S. triqueter, inoculated with HD-1, with NTA and
APG, in contaminated soil
NPBC Without S. triqueter, inoculated with HD-1, with
NTA and APG, in contaminated soil
Page 3 of 9 295
every group had three replicates. Bacterial suspensions
(50 mL pot1) were applied to groups PB, NPB, PBC,
and NPBC. Then S. triqueter (12 similar seedlings
pot1) were moved into groups P, PB, PC, and PBC.
Forty-five days later, the mixture of NTA and APG was
watered into the pots every 2 days according to Table 1.
In order to avoid secondary pollution caused by high
dose of chemical reagents, both NTA and APG concen-
tration were 40 mg L1 (Liu et al. 2013). The solution
pH was adjusted to 8.30 in order to keep the soil prop-
erty unchanged. Sixty days later, plants were harvested
and measured the length. All the plant and soil samples
were stored in 80 C refrigerator after freeze-drying.
2.5 Analysis of Pyrene and Ni
2.5.1 Analysis of Ni
Different fractions of extraction of Ni in soil were per-
formed according to the Tessier procedure (Chen et al.
2017; Tessier et al. 1979). Exchangeable fraction: First,
add 8 mL MgCl2 solution (1 M, pH = 7) to 0.25 g soil.
Second, shake the mixture at 25 C for 1 h. Third,
centrifugate the mixture at 4000 rpm for 15 min to get
supernatants. Associated to carbonates fraction: First,
add 8 mL NaOAc solution (1 M, pH = 5) to the residual
soil. Second, shake the mixture at 25 C for 5 h. Third,
centrifugate the mixture at 4000 rpm for 15 min to get
supernatants. Associated to Fe-Mn oxides fraction:
First, add 20 mL NH2OHHCl (0.04 M) to the residual
soil in HOAc solution (m/v, 25%). Second, shake the
mixture at 96 C for 6 h. Third, centrifugate the mixture
at 4000 rpm for 15 min to get supernatants. Associated
to organic matter fraction: First, add 3 mL HNO3 solu-
tion (0.02 M) and 5 mL H2O2 solution (m/v, 30%) to the
residual soil and shake at 85 C for 2 h. Then add 3 mL
H2O2 solution (m/v, 30%) and shake at 85 C for 3 h.
Second, add 5 mL NH4OAc solution (3.2 M) and shake
at 25 C for 30 min. Third, centrifugate the mixture at
4000 rpm for 15 min to get supernatants. Residual
fraction: HNO3-HClO4 (4:1, v/v) was added into the
residual soil for digestion for about 2 h at 220 C. Then
HF-HClO4 (v/v, 5:1) was added for further digestion at
220 C for about 2 h. Total Ni in plant samples were
extracted based on the method by Chen et al. (2016b)
and digested by using the above method. Ni in soil or
plant was quantified with inductively coupled plasma
optical emission spectrometry (ICPOES) after filtered
295 Page 4 of 9 Water Air Soil Pollut (2017) 228:295

through 0.45 m of microfiltration membrane. The in- 2.6 Statistical Methods

strumental parameters were shown in Table 2.
Each of the data in the work was the mean value (SD)
of three independent replicates. SPSS software program
2.5.2 Analysis of Pyrene
(version 19.0 for Windows) was used for statistical
analysis. The differences among treatments were ana-
The soil (2 g) with 1 g Na2SO4 was extracted in an
lyzed and considered statistically significant when
ultrasonic bath (45 kHz, 300 W) for 30 min in 10-mL
P < 0.05 based on Duncans test.
mixed solvents (dichloromethane and acetone (1:1, v/v))
followed by centrifugation for 20 min at 4000 rmp.
After centrifugation, the supernatant was removed and
the process above was repeated for three times. The
3 Results and Discussion
extracts were then rotary evaporated (40 C), exchanged
to 2-mL cyclohexane, and filtered through 4 g of silica
3.1 Growth of S. triqueter with Different Treatments
gel with 10 mL of mixed solvents (hexane and dichlo-
romethane (1:1, v/v)). Then, the samples were evaporat-
In this experiment, the existence of pyrene and nickel
ed, reconstituted in 1.0 mL of methanol, and passed
restrained the growth of plant, which resulted in a 20.92
through a 0.22-m Teflon filter. The concentration of
and 39.27% reduction in root and shoot length, respec-
pyrene was quantified by HPLC (LC-20AT). Quantifi-
tively, when compared to the group planting with
cation was performed by the external standard method.
S. triqueter in uncontaminated soil (Fig. 1). Although
The HPLC system was fitted with a 4.6 250 mm
the plant length was reduced, S. triqueter was able to
reverse phase C18 column and an ultraviolet detector.
survive in co-contaminated soil with high concentra-
The mobile phase was HPLC-grade methanol and the
tions of pyrene and Ni during the whole pot trial, which
flow rate was kept at 1 mL min1 (40 C). The wave-
might be due to its high tolerance to pyrene and Ni.
length was 245 nm. The sample solution (10 L) was
Compared with P group, it was observed that inocula-
injected into the HPLC system by an autosampler and
tion with HD-1 alleviated the inhibition effects on root
the limit of detection (LOD) for pyrene was
and shoot length to some extent in the soil spiked with
0.024 g g1. The dissipation of pyrene from soil was
calculated as following equation: 35
0
! 30
a shoot root
C pyrene
The length of plants (cm)

Dpyrene 1  100% 25 c bc b
C pyrene 20
c

0 15
where Dpyrene is the dissipation of pyrene, C pyrene is the 10
concentration of pyrene in contaminated soil after the 5
test, and Cpyrene is the initial concentration of pyrene in 0.0
contaminated soil. 1.2
2.4
b ab b b
3.6
Table 2 ICPOES instrumental parameters employed to Ni a
determination CK P PB PC PBC
Fig. 1 The roots and shoots length of S. triqueter with different
Parameters Values
treatments. (Uncontaminated soil with S. triqueter
cultivation(CK); Co-contaminated soil with S. triqueter
Frequency of RF generator 40.68 MHz
cultivation(P); Co-contaminated soil inoculated of HD-1 with
RF power 1.2 kW S. triqueter cultivation(PB); Co-contaminated soil added chemical
Plasma gas flow rate 19 L min1 reagents with S. triqueter cultivation(PC); Co-contaminated soil
Solution uptake rate 1.2 ml min1 inoculated of HD-1 and added chemical reagents with S. triqueter
cultivation (PBC). Error bars denote the standard deviations.
Integration time 10 s Mean and standard deviation of three replicates (n = 3). Different
Sample integration number 3 letters above the columns indicate statistically significant differ-
Analysis wavelength 231.604 nm ences in the roots and shoots length of S. triqueter among treat-
ments (P < 0.05))
Water Air Soil Pollut (2017) 228:295 Page 5 of 9 295

70
Pyrene dissipation from soil (%)
a
60 bc
c Dissipation of pyrene in soil with different treatments
c
50
d chemical reagents, and degrading bacteria on the remov-
40 e al of pyrene. The dissipation of 53.24, 49.48, and
30
f tion, and their combination in the presence of
20
10
P NP PB NPB PC NPC PBC NPBC
Fig. 2 Dissipation of pyrene from soil with different treatments.
(Co-contaminated soil with(P) or without (NP) S. triqueter culti-
vation; co-contaminated soil inoculated of HD-1 with (PB) or
without (NPB) S. triqueter cultivation; co-contaminated soil added
chemical reagents (NTA and APG) with (PC) or without (NPC)
S. triqueter cultivation; co-contaminated soil inoculated of HD-1
and added chemical reagents (NTA and APG) with (PBC) or
without (NPBC) S. triqueter cultivation. Error bars denote the
standard deviations. Mean and standard deviation of three repli-
cates (n = 3). Different letters above the columns indicate statisti-
cally significant differences in the dissipation of pyrene from soil
among treatments (P < 0.05))
pyrene and Ni. This phenomenon may be due to the
beneficial microbes-plant interaction in the rhizosphere.
Similarly, Xiao et al. (2013) found that inoculation with
carbendazim degrading strains could increase the bio-
mass of S. alfredii in Cd and carbendazim co-
contaminated soil. Besides, although no notable effect
on plant growth was found in adding chemical reagents
alone group (PC group), there was a significant increase
in shoot length when adding the mixture of NTA and
APG to the soil inoculated of HD-1 in comparison with
the group planting with S. triqueter alone (P group) in
co-contaminated soil.
3.2 Dissipation of Pyrene in Co-contaminated Soil
b
(Fig. 2) was detected to investigate the effects of plant,
59.78% pyrene was measured at the end of 60 days in
groups of HD-1 inoculation, chemical reagents applica-
S. triqueter, respectively, while 35.49% of pyrene was
removed from contaminated soil with S. triqueter culti-
vation alone. It was observed that the dissipation in
groups without S. triqueter cultivation was lower than
that in groups with S. triqueter cultivation. Additionally,
S. triqueter, chemical reagents, and degrading bacteria
HD-1 as well as their combination significantly en-
hanced the dissipation of pyrene from co-contaminated
soil (Table 3). And the relative contributions of three
factors influencing pyrene dissipation were as follows:
degrading bacteria > chemical reagents > plant.
Results of the present study showed that the contri-
butions of S. triqueter to the dissipation of pyrene in soil
varied with pyrene degrading bacteria inoculation and
chemical reagents application. When pyrene-degrading
bacteria were added to the soil, pyrene could be metab-
olized efficiently, especially in the presence of plant
(Brookes and McGrath 1984). To date, the combination
of plants and degrading bacteria has been applied to
remove PAH from soils (Daane et al. 2001; Sheng and
Gong 2006). With the discovery of solubilization caused
by surfactants, the notion that surfactants might provide
an effective means of enhancing biodegradation of PAH
from soil gained credence. The dissipation of pyrene
increased significantly in groups PC and NPC, which
might be due to the solubilization caused by surfactant
APG or its contribution as a carbon source for soil

Table 3 Results of ANOVA for

plant (S. triqueter), chemical Treatments Sum of squares d.f. F value P value
reagents (NTA and APG), and
microbe (pyrene degrading Plant 307.973 1 66.768 0.000*
bacteria HD-1) interactions Microbe 1974.704 1 428.114 0.000*
Chemical reagents 866.887 1 187.940 0.000*
Plant microbe 101.745 1 22.058 0.000*
Plant chemical reagents 18.660 1 4.045 0.061
Microbe chemical reagents 265.624 1 57.587 0.000*
Plant microbe chemical reagents 51.409 1 11.145 0.004*
Error 73.801 16
*P < 0.01
295 Page 6 of 9 Water Air Soil Pollut (2017) 228:295

120
the mixture of NTA and APG to pyrene and Pb co-
a
Concentration of Ni in plants

contaminated soil (Chen et al. 2016b). Greater dissipa-

100 tion of pyrene was evident when chemical reagents,
b
b HD-1, and S. triqueter were applied jointly compared
(mg kg-1)

80 with other groups, which indicated the beneficial plant

60
c
40
P PB
Fig. 3 Concentration of Ni in S. triqueter with different treat-
ments. (Co-contaminated soil with S. triqueter cultivation (P); co-
contaminated soil inoculated of HD-1 with S. triqueter cultivation
(PB); co-contaminated soil added chemical reagents with
S. triqueter cultivation (PC); co-contaminated soil inoculated of
HD-1 and added chemical reagents with S. triqueter cultivation
(PBC). Error bars denote the standard deviations. Mean and
standard deviation of three replicates (n = 3). Different letters
above the columns indicate statistically significant differences in
concentration of Ni in S. triqueter among treatments (P < 0.05))
microbes to improve the dissipation of pyrene in soil. In
addition, some surfactants was reported to increase the
microbe numbers in soil (Liao et al. 2015) and dehydro-
genase activities (Shi et al. 2009), which may promote
the biodegradation of PAH. This result was in agreement
with the findings of our previous studies which adding
bacteria-chemical reagents interaction (P < 0.01) in
removing pyrene from contaminated soil. Taken togeth-
er, the results show that planting S. triqueter together
with adding chemical reagents and inoculating with
pyrene degrading bacteria could be an effective strategy
for the removal of pyrene from pyrene-Ni co-contami-
PC PBC
nated soil.
3.3 Effects on the Accumulation of Ni in Plant
and the Accessibility of Ni in Soil
Unlike pyrene, decontamination of Ni from contaminat-
ed soils mainly relies on the removal by plants, because
metal cannot be degraded. In order to investigate the
effects of adding chemical reagents or/and inoculation
of pyrene degrading bacteria in co-contaminated soil on
the accumulation of Ni and the accessibility of Ni for
assimilation by S. triqueter, Ni concentrations in plants
and different fractions of Ni in soil with different treat-
ments were measured (Figs. 3 and 4). The Ni concen-
tration in S. triqueter of groups PB, PC, and PBC
increased to 100.40, 80.97, and 87.77 mg kg 1 ,

300 c b bc bc a bc a a

270
bc c
Ni in soil (mg kg-1)

240 d c cd
a c ab
ab a
ab ab
210 b b bc
c
30
a b
c c
a
20 cd
d
c
10 a a
b b b
d c
d
0
P NP PB NPB PC NPC PBC NPBC
Residual Associated to organic matter Associated to Fe-Mn oxides
Associated to carbonates Exchangeable
Fig. 4 Different fractions of Ni with different treatments in soil. and added chemical reagents (NTA and APG) with (PBC) or
(Co-contaminated soil with (P) or without (NP) S. triqueter culti- without (NPBC) S. triqueter cultivation. Error bars denote the
vation; co-contaminated soil inoculated of HD-1 with (PB) or standard deviations. Mean and standard deviation of three repli-
without (NPB) S. triqueter cultivation; co-contaminated soil added cates (n = 3). Different letters in the columns indicate significant
chemical reagents (NTA and APG) with (PC) or without (NPC) differences in fractions of Ni subgroups among treatments
S. triqueter cultivation; co-contaminated soil inoculated of HD-1 (P < 0.05))
Water Air Soil Pollut (2017) 228:295 Page 7 of 9 295

respectively, when compared with the Ni concentration chemical speciations and enhance phytoextraction by
of group P (46.04 mg kg1). promoting the exchangeable fraction of Ni in soil.
As we know, the availability of heavy metal in soil is
crucial to plants during the phytoremediation process.
Thus, improving the accessibility of Ni is of importance
4 Conclusions
for phytoextraction. Among five fractions, exchangeable
fraction of metal was considered to be most suitable for
The present results indicated that the introduced pyrene
plant uptake (Chen et al. 2016a). The Ni concentration in
degrading bacteria and its combination with chemical
S. triqueter in group PB was higher than that in group P.
reagents could alleviate the inhibition effects on the
And it was observed that the exchangeable fraction of Ni
growth of S. triqueter while application of chemical
in groups PB and NPB was higher than that in groups P
reagents alone had no apparent effect on plant growth
and NP, which indicated that the inoculation of HD-1
in pyrene and Ni co-contaminated soil. S. triqueter,
lead to the increase of exchangeable fraction of Ni in soil.
chemical reagents, degrading bacteria HD-1 as well as
The phenomenon might be explained that biosurfactants
their combination significantly enhanced the dissipation
might be produced by Bacillus subtilis (Nitschke and
of pyrene from co-contaminated soil. And the relative
Pastore 2006), which could dissolve the bound forms
contributions of three factors influencing pyrene dissi-
and then promote mobility of metals (Juwarkar et al.
pation were as follows: degrading bacteria > chemical
2007), consequently, Ni uptake. Similarly, the Ni con-
reagents > plant. In addition, inoculation with HD-1 or/
centration in S. triqueter in groups PC and PBC was
and adding chemical reagents resulted in increases of Ni
higher than that in group P, which may be due to the
in S. triqueter by improving its bioaccessible fractions
higher exchangeable fraction of Ni in soil caused by
and the shift of chemical speciations of metals in soil. In
NTA and APG. NTA was reported to facilitate the solu-
conclusion, planting S. triqueter together with added
bilization of metals (de Arajo and do Nascimento 2010)
chemical reagents and inoculated pyrene degrading bac-
and form complex with metals (Bucheli-Witschel and
teria could be a potential effective strategy for the reme-
Egli 2001). Moreover, some surfactants could increase
diation of pyrene and Ni co-contaminated soil.
metal concentration in solution or improve the perme-
ability of root barriers to the complexes (Almeida et al.
Acknowledgements The work was funded by the National Nat-
2009; Di Gregorio et al. 2006), which might increase the
ural Science Foundation of China (Nos. 41373097, 21677093).
exchangeable fraction Ni and favor metal absorption into
plants. Besides, no significant difference was observed Compliance with Ethical Standards
between the S. triqueter Ni concentrations of groups PC
and PBC, which indicated that the inoculation of HD-1 Conflicts of Interest The authors declare that they have no
did not lead to the enhanced phytoextraction of Ni in the competing financial interest.
presence of NTA and APG. As for groups PB and PBC,
the Ni concentration in S. triqueter decreased when
adding NTA and APG to the co-contaminated soil inoc-
ulated with HD-1 in comparison with the group inocu-
lated with HD-1 alone which may be due to the interac- References
tion among HD-1, chemical reagents (NTA and APG),
and S. triqueter. Besides the Fe-Mn oxide fractions, Ni Almeida, C. M. R., Dias, A. C., Mucha, A. P., Bordalo, A. A., &
was mainly found in the residual fraction, suggesting that Vasconcelos, M. T. S. D. (2009). Influence of surfactants on
the Cu phytoremediation potential of a salt marsh plant.
Ni was a relatively immobile element. It was also indi-
Chemosphere, 75, 135140.
cated in a study by Li and Shuman (1996). The concen- Bautista, L. F., Sanz, R., Molina, M. C., Gonzlez, N., &
trations of Ni in five fractions were different among Snchez, D. (2009). Effect of different non-ionic surfac-
groups NP, NPB, NPC, and NPBC, suggesting that tants on the biodegradation of PAHs by diverse aerobic
inoculation with HD-1 or/and adding chemical reagents bacteria. International Biodeterioration & Biodegradation,
63, 913922.
redistributed Ni from some to the other fractions. These Beijing Bureau of Quality and Supervision (2011). Screening
results illustrated that inoculation with HD-1 or/and Levels for Soil Environmental Risk Assessment of Sites
adding chemical reagents may lead to a shift of metal DB11/T 8112011. [in Chinese].
295 Page 8 of 9
Boonchan, S., Britz, M. L., & Stanley, G. A. (1998). Surfactant-
enhanced biodegradation of high molecular weight polycyclic
aromatic hydrocarbons by Stenotrophomonas maltophilia.
Biotechnology and Bioengineering, 59, 482494.
Brookes, P. C., & McGrath, S. P. (1984). Effect of metal toxicity
on the size of the soil microbial biomass. European Journal
of Soil Science, 35, 341346.
Bucheli-Witschel, M., & Egli, T. (2001). Environmental fate and
microbial degradation of aminopolycarboxylic acids. FEMS
Microbiology Reviews, 25, 69106.
Chaudhry, Q., Blom-Zandstra, M., Gupta, S. K., & Joner, E.
(2005). Utilising the synergy between plants and rhizosphere
microorganisms to enhance breakdown of organic pollutants
in the environment (15 pp). Environmental Science and
Pollution Research, 12, 3448.
Chen, T., Liu, X., Zhang, X., Chen, X., Tao, K., & Hu, X. (2016a).
Effect of alkyl polyglucoside and nitrilotriacetic acid com-
bined application on lead/pyrene bioavailability and dehy-
drogenase activity in co-contaminated soils. Chemosphere,
154, 515520.
Chen, T., Liu, X., Zhang, X., Hou, Y., Chen, X., & Tao, K.
(2016b). Enhanced Scirpus triqueter phytoremediation of
pyrene and lead co-contaminated soil with alkyl
polyglucoside and nitrilotriacetic acid combined application.
Journal of Soils and Sediments, 16, 20902096.
Chen, X., Liu, X., Zhang, X., Cao, L., & Hu, X. (2017).
Phytoremediation effect of Scirpus triqueter inoculated
plant-growth-promoting bacteria (PGPB) on different frac-
tions of pyrene and Ni in co-contaminated soils. Journal of
Hazardous Materials, 325, 319326.
Daane, L. L., Harjono, I., Zylstra, G. J., & Hggblom, M. M.
(2001). Isolation and characterization of polycyclic aromatic
hydrocarbon-degrading bacteria associated with the rhizo-
sphere of salt marsh plants. Applied and Environmental
Microbiology, 67, 26832691.
de Arajo, J. D. C. T., & do Nascimento, C. W. A. (2010).
Phytoextraction of lead from soil from a battery recycling
site: the use of citric acid and NTA. Water, Air, and Soil
Pollution, 211, 113120.
Di Gregorio, S., Barbafieri, M., Lampis, S., Sanangelantoni, A.
M., Tassi, E., & Vallini, G. (2006). Combined application of
Triton X-100 and Sinorhizobium sp. Pb002 inoculum for the
improvement of lead phytoextraction by Brassica juncea in
EDTA amended soil. Chemosphere, 63, 293299.
Gonzlez, N., Simarro, R., Molina, M., Bautista, L., Delgado, L.,
& Villa, J. (2011). Effect of surfactants on PAH biodegrada-
tion by a bacterial consortium and on the dynamics of the
bacterial community during the process. Bioresource
Technology, 102, 94389446.
Hou, Y., Liu, X., Zhang, X., Hu, X., & Cao, L. (2016).
Rhizosphere phytoremediation with Cyperus rotundus for
diesel-contaminated wetlands. Water, Air, and Soil
Pollution, 227, 26.
Juwarkar, A. A., Nair, A., Dubey, K. V., Singh, S. K., & Devotta, S.
(2007). Biosurfactant technology for remediation of cadmium
and lead contaminated soils. Chemosphere, 68, 19962002.
Lan, J., Zhang, S., Lin, H., Li, T., Xu, X., Li, Y., Jia, Y., & Gong,
G. (2013). Efficiency of biodegradable EDDS, NTA and
APAM on enhancing the phytoextraction of cadmium by
Siegesbeckia orientalis L. grown in Cd-contaminated soils.
Chemosphere, 91, 13621367.
Water Air Soil Pollut (2017) 228:295
Lee, J., & Sung, K. (2014). Effects of chelates on soil microbial
properties, plant growth and heavy metal accumulation in
plants. Ecological Engineering, 73, 386394.
Li, Z., & Shuman, L. M. (1996). Redistribution of forms of zinc,
cadmium and nickel in soils treated with EDTA. Science of
the Total Environment, 191, 95107.
Liao, C., Liang, X., Lu, G., Thai, T., Xu, W., & Dang, Z. (2015).
Effect of surfactant amendment to PAHs-contaminated soil
for phytoremediation by maize (Zea mays L.) Ecotoxicology
and Environmental Safety, 112, 16.
Liu, F., Zhang, X., Liu, X., Chen, X., Liang, X., He, C., Wei, J., &
Xu, G. (2013). Alkyl Polyglucoside (APG) amendment for
improving the phytoremediation of Pb-PAH contaminated
soil by the aquatic plant Scirpus triqueter. Soil and
Sediment Contamination, 22, 10131027.
Lu, M., Zhang, Z. Z., Wang, J. X., Zhang, M., Xu, Y. X., & Wu, X.
J. (2014). Interaction of heavy metals and pyrene on their
fates in soil and tall fescue (Festuca arundinacea).
Environmental Science & Technology, 48, 11581165.
Madsen, T., & Kristensen, P. (1997). Effects of bacterial inocula-
tion and nonionic surfactants on degradation of polycyclic
aromatic hydrocarbons in soil. Environmental Toxicology
and Chemistry, 16, 631637.
Mao, X., Jiang, R., Xiao, W., & Yu, J. (2015). Use of surfactants
for the remediation of contaminated soils: a review. Journal
of Hazardous Materials, 285, 419435.
Mielke, H. W., Wang, G., Gonzales, C., Le, B., Quach, V., & Mielke,
P. (2001). PAH and metal mixtures in New Orleans soils and
sediments. Science of the Total Environment, 281, 217227.
Nitschke, M., & Pastore, G. M. (2006). Production and properties
of a surfactant obtained from Bacillus subtilis grown on
cassava wastewater. Bioresource Technology, 97, 336341.
Reddy, K. R. (2010). Technical challenges to in-situ remediation
of polluted sites. Geotechnical and Geological Engineering,
28, 211221.
Robinson, B. H., Mills, T. M., Petit, D., Fung, L. E., Green, S. R.,
& Clothier, B. E. (2000). Natural and induced cadmium-
accumulation in poplar and willow: implications for
phytoremediation. Plant and Soil, 227, 301306.
Romeh, A. A., Khamis, M. A., & Metwally, S. M. (2016).
Potential of Plantago major L. for phytoremediation of
lead-contaminated soil and water. Water, Air, and Soil
Pollution, 227, 19.
Sheng, X. F., & Gong, J. X. (2006). Increased degradation
of phenanthrene in soil by Pseudomonas sp. GF3 in
the presence of wheat. Soil Biology and Biochemistry,
38, 25872592.
Shi, F. G., Hao, X. Z., Zhou, D. M., & Qian, Y. (2009).
Remediation of the combined polluted soil by growing rye-
grass enhanced by EDDS/rhamnolipid. Journal of Agro-
Environment Science, 28, 18181823.
Tao, K., Liu, X., Chen, X., Hu, X., Cao, L., & Yuan, X. (2017).
Biodegradation of crude oil by a defined co-culture of indig-
enous bacterial consortium and exogenous Bacillus subtilis.
Bioresource Technology, 224, 327332.
Tessier, A., Campbell, P. G., & Bisson, M. (1979). Sequential
extraction procedure for the speciation of particulate trace
metals. Analytical Chemistry, 51, 844851.
Xiao, W., Wang, H., Li, T., Zhu, Z., Zhang, J., He, Z., &
Yang, X. (2013). Bioremediation of Cd and carbendazim
co-contaminated soil by Cd-hyperaccumulator Sedum
Water Air Soil Pollut (2017) 228:295
alfredii associated with carbendazim-degrading bacterial
strains. Environmental Science and Pollution Research,
20, 380389.
Xie, J., Liu, Y., Zeng, G., Liu, H., Zheng, B., Tang, H., Xu, W.,
Sun, Z., Tan, X., Nie, J., Jiang, Z., Gan, C., & Wang, S.
(2015). The effects of P. aeruginosa ATCC 9027 and NTA on
phytoextraction of Cd by ramie (Boehmeria nivea (L.) Gaud).
RSC Advances, 5, 6750967517.
Zaier, H., Ghnaya, T., Rejeb, K. B., Lakhdar, A., Rejeb, S., &
Jemal, F. (2010). Effects of EDTA on phytoextraction of
Page 9 of 9 295
heavy metals (Zn, Mn and Pb) from sludge-amended soil with
Brassica napus. Bioresource Technology, 101, 39783983.
Zhang, X., Liu, X., Chai, W., Wei, J., Wang, Q., Li, B., & Li, H.
(2013). The use of proteomic analysis for exploring the
phytoremediation mechanism of Scirpus triqueter to pyrene.
Journal of Hazardous Materials, 260, 10011007.
Zhao, B., Che, H., Wang, H., & Xu, J. (2016). Column flushing of
phenanthrene and copper (II) co-contaminants from sandy
soil using Tween 80 and citric acid. Soil and Sediment
Contamination, 25, 5063.