Science of the Total Environment 697 (2019) 134148

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Science of the Total Environment

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

Application of Simplicillium chinense for Cd and Pb biosorption and

enhancing heavy metal phytoremediation of soils
Zhongmin Jin a,b, Songqiang Deng c, Yuchen Wen a, Yifeng Jin a, Lin Pan a, Yanfu Zhang a, Tom Black b,
Kevin C. Jones b, Hao Zhang b, Dayi Zhang d,⁎
a
College of Agriculture, Forestry and Life Science, Qiqihar University, Qiqihar 161006, PR China
b
Lancaster Environment Centre, Lancaster University, Lancaster LA1 2YW, United Kingdom
c
Research Institute for Environmental Innovation (Suzhou), Tsinghua, Suzhou 215163, PR China
d
School of Environment, Tsinghua University, Beijing 100084, PR China

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

• First study on metal biosorption capabil-

ity of fungus Simplicillium chinense
• Difference in Cd and Pb biosorption
mechanisms by Simplicillium chinense
• 28.6–48.0% more metal removal from
soils via fungi-mediated
phytoremediation
• Rhizosphere-fungi interaction explains
the enhanced phytoremediation
performance.

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

Article history: Phytoremediation is an effective approach to control soil heavy metal pollution. This study isolated a fungus
Received 18 May 2019 strain from soils contaminated by cadmium (Cd) and lead (Pb) in Zhalong Wetland (China), which was
Received in revised form 25 August 2019 identified as Simplicillium chinense QD10 via both genotypic and phenotypic analysis. The performance
Accepted 26 August 2019
and mechanism of S. chinense QD10 in Cd and Pb adsorption was unraveled by morphological analysis
Available online 27 August 2019
and biosorption test, and its roles in ameliorating phytoremediation by Phragmites communis were tested
Editor: F.M. Tack in pot-experiments. Cd biosorption was attributed to the formation of Cd-chelate, whereas Pb was predom-
inantly adsorbed by extracellular polymeric substances. Metal biosorption followed Langmuir isotherm,
Keywords: and the maximum biosorption capacity was 88.5 and 57.8 g/kg for Cd and Pb, respectively. Colonized in
Cadmium soils, such biosorption behavior of S. chinense QD10 can generate gradients of available Cr or Pb and drive
Lead their enrichment. Accordingly, S. chinense QD10 amendment significantly enhanced the phytoextraction
Biosorption of Cd and Pb by P. communis, possibly attributing to rhizospheric enrichment of Cd or Pb and defending ef-
Phytoremediation fects on plants, explained by the significant removal of acid-extractable and reducible metals in soils and the
Phytoextraction
increase of Cd and Pb content in P. communis tissues. The present study explored the mechanisms of

⁎ Corresponding author.
E-mail address: zhangdayi@tsinghua.edu.cn (D. Zhang).

https://doi.org/10.1016/j.scitotenv.2019.134148
0048-9697/© 2019 Elsevier B.V. All rights reserved.
2 Z. Jin et al. / Science of the Total Environment 697 (2019) 134148
S. chinense QD10 in Cd and Pb biosorption and proved its potential in ameliorating the phytoremediation
performance at metal contaminated sites.
1. Introduction
Metal contamination is one of the key environmental concerns for its
persistence, accumulation and toxicity in the environment (Dong et al.,
2010). Due to the global industrialization and urbanization, metals are
found in either air, soil or water, threatening human health and ecosys-
tems (Jiang et al., 2019; Liu et al., 2013). Some metals can cause
genotoxicity, immunotoxicity and genomic instability, with further
risks to induce cancer (Leonard et al., 2004). They can even accumulate
in vital organs and glands in human, such as heart, brain, kidney, bone
and liver (Valko et al., 2005). In China, some of the urban soils are seri-
ously contaminated by metals, and the mean concentrations of Cr, Cu,
Pb, Zn, Ni, and Cd are 78.4, 115, 1.35 × 103, 266, 99.5 and 1.58 mg/kg
soil, respectively (Wei and Yang, 2010). It is reported that 64.8% of the
soils from 1.4 million ha lands irrigated with wastewater are contami-
nated by metals, accounting for 16% of total cultivated lands in China
(Tan et al., 2011). Using plant to remove pollutants from the environ-
ment, phytoremediation is solar driven and sustainable. Many plants
have been reported with the strong capabilities for phytoremediation,
such as Brassica juncea for Zn (Glick, 2003), Arundo donax L. and
Miscanthus sp for Zn and Cr (Barbosa et al., 2015), Sedum alfredii
Hance for Cd (He et al., 2019; Yang et al., 2004), Elsholtzia haichowensis
Sun for Cu (Deng et al., 2018), and Zea mays L. and Scrophularia scoparia
for Pb (Hadi and Bano, 2010; Nouri et al., 2011).
In the soil ecosystem, the major soil biomass and biodiversity are
formed by microorganisms. Their presence in the rhizosphere plays im-
portant roles in trace metal phytoremediation (Khan, 2005). The
amendment of microbes, e.g., fungi, is an emerging technology to im-
prove the phytoremediation efficiency in soils (Desai et al., 2016;
Gomez-Sagasti et al., 2012; Ullah et al., 2015), explained by several
mechanisms. Firstly, plant growth-promoting fungi (PGPF) in the rhizo-
sphere promote the plant biomass, indirectly increasing the uptake effi-
ciency of trace metals (Rajkumar et al., 2012). For instance, various PGPF
encourage the nutrient and metal (Mn, Fe, Cu and Zn) uptake by sor-
ghum (Dhawi et al., 2016). The plants can be promoted by inoculation
with phosphate-solubilizing fungi (Whitelaw, 2000), and Trichoderma
harzianum increases phosphates and micronutrients solubility to im-
prove plant growth (Altomare et al., 1999). Endophytic fungi can partic-
ularly improve phytoremediation by secreting phytohormones,
enhancing nutrient availability and protecting against biotic and abiotic
stresses (El-Deeb et al., 2012; Hamayun et al., 2012). Some fungi con-
taining extracellular or intracellular detoxification systems can alleviate
the toxicity of trace metals for both plants and soil microbes, and thus
improve plant biomass (Zhao et al., 2015). Secondly, PGPF also improve
the metal accumulation in plant tissues by secreting phytohormones
(Zhuang et al., 2007). For example, arbuscular mycorrhizal fungi con-
tribute to the accumulation of trace metals in metallophytes in non-
toxic form (Gaur and Adholeya, 2004). Thirdly, microbes can improve
phytoremediation by direct biosorption (Chen et al., 2005). Aspergillus
niger and Aspergillus flavus are metal-resistant fungi and show remark-
able metal adsorption capacity of Cr, Cu and Pb over a wide range of
temperature and pH (Iram et al., 2015; Kapoor et al., 1999).
Only limited fungal strains have been investigated for their con-
tribution to phytoremediation, and there are still many unknown
fungi to be studied on their roles in heavy metal removal in soils. In
the present study, we isolated a fungal strain (Simplicillium chinense
QD10) with resistance to Cd and Pb. Our work aimed to evaluate the
biosorption capability, isotherms and kinetics of Cd and Pb by
S. chinense QD10, and assess the improvement of Cd and Pb removal
© 2019 Elsevier B.V. All rights reserved.
efficiency by Phragmites communis in pot experiments supplemented
with S. chinense QD10. P. communis is selected because it has great
potential in metal phytoremediation in soils, waters and sediments
contaminated by Cd, Mn, Pb and Cu, attributed to its satisfactory
metal tolerance, relatively high accumulation capacity and economic
benefits as a raw material for paper production (Liu et al., 2006; Lu
et al., 2017; Ning et al., 2019). These findings provided deeper in-
sights into the mechanisms and application of S. chinense QD10 as
bio-adsorbents for effective phytoremediation of heavy metal con-
taminated soils.
2. Materials and methods
2.1. Cultivation medium and strain isolation
In the present study, the soil samples were collected from Zhalong
Wetland (Latitude 47°32′30″, Longitude 124°37′50″, Qiqihar City,
China) in October 2015, transferred to the lab immediately and stored
at 4 °C. The 10 g soil samples were added into 90 mL sterile water,
and shaken at 160 rpm and 28 °C for 2 h. After 30 min settlement, the
soil suspension was serially diluted and spread on potato dextrose
agar (PDA) plate. The PDA medium was consisted of 200 g potato,
20 g glucose and 20 g agarose in 1000 mL deionized water and
autoclaved. After cultivation at 28 °C for 5 days, the colonies were se-
lected and re-inoculated in PDA medium with 100 mg/L Cd (CdCl2) or
200 mg/L Pb (PbCl2). The positive colonies were further selected and in-
oculated in PDA medium with increasing Cd and Pb concentrations until
1000 mg/L and 1500 mg/L, respectively. The colonies were further inoc-
ulated in PDA medium with Cd\\Pb mixture (800 mg/L and 1500 mg/L,
respectively) until the Cd- and Pb-tolerant fungal colonies appeared.
Without specific statement, all the chemicals were analytical grade
and purchased from Tianjin Kaitong Chemical Reagents (China).
2.2. Genotypic and phenotypic analysis
After cultivated on PDA medium for 7 days, the isolated fungal colo-
nies were collected and resuspended in deionized water. The fungal hy-
phae were separated and fixed on the slide, and the images were taken
by a microscope (OLYMPUS CX31, Japan). The details of testing fungal
resistance to metals and DNA extraction were listed in supplementary
data. The sequence of 18S rRNA gene indicated high similarity (97%)
of the isolated fungal strain to Simplicillium chinense, named as
Simplicillium chinense QD10.
Biolog PM01 plate (Biolog, USA) was used to examine the carbon
metabolic profiles of S. chinense QD10, following previous reported
method (Zhang et al., 2015). The 1.5 mL of washed S. chinense QD10
cells were resuspended in 13.5 mL minimal medium and added into
PM01 (95 carbon sources) plate. In each well, 1.5 μL Biolog Redox Dye
Mix A (100×) was added. The plate was incubated at 30 °C for 7 days
and the color development was read every 4 h by measuring the absor-
bance at 590 nm wavelength (respiratory unit, RU) by a multimode mi-
croplate reader (FLUOstar Omega, BMG Labtech, UK), following our
previous instructions (Zhang et al., 2015). The RU data were collected
and analyzed by MARS software (BMG Labtech, UK). The relative respi-
ration index (RRI) represented the growth of S. chinense QD10 in a spe-
cific carbon source comparing to the control (no carbon source
addition), calculated as the ratio of RU (Wang et al., 2016b).
 Z. Jin et al. / Science of the Total Environment 697 (2019) 134148
2.3. Lead and cadmium biosorption
Without specifically stated, all the biosorption isotherm experi-
ments were carried out at 30 °C and pH 7.0. Briefly, the 0.4 g fungal
cells (dry cell weight, DCW) were added into 100 mL heavy metal solu-
tion with different concentrations (Cd: 10, 30, 50, 100, 150 and
200 mg/L; Pb: 10, 30, 50, 100, 150 and 200 mg/L; Cd/Pb mixture: 10/
10, 30/30, 50/50, 100/100, 150/150 and 200/200 mg/L). The experimen-
tal details for the impacts of biomass, temperature and pH on metal
biosorption by S. chinense QD10 were provided in supplementary data.
2.4. Phytoremediation experiment
P. communis seeds were collected from Zhalong Wetland in October
2015. After cultivation for 6–8 months, the tillers of P. communis were
further separated into individual ones, and those healthy tillers with
the same weight and height were selected for phytoremediation
test to ensure identical biomass at the beginning of experiments.
The soil samples were collected from the plantation in Qiqihar Univer-
sity and air dried. These soils contained clay (30.6%), sand (31.2%) and
silt (39.2%), representing typical dark brown soils in northern China
and having similar physiochemical properties as those metal-
contaminated sites in Qiqihar city. The measurement of the soil proper-
ties mainly followed the Chinese Technical Specification for Soil Envi-
ronmental Monitoring (HJ/T 166-2004) according to previous study
(Lin et al., 2019). Soil pH was 7.3 and the contents of organic matters,
total nitrogen, available nitrogen, total phosphate and available potas-
sium were 42.1, 2.12, 0.29, 1.64 and 170 g/kg, respectively.
After removing large objects, the soils were blended and sieved
through a 2-mm pore-size screen to remove stones and plant debris.
Subsequently, 1000 g soils were mixed well with 50 mL Cd
(400 mg/L) and Pb (2000 mg/L) stock solution, and aged for 6 months
at room temperature. The total Cd and Pb concentration in soil was
22.4 mg/kg (dry soil weight, DSW) and 138 mg/kg DSW, respectively.
Cd concentration fitted well with Soil Environmental Quality Risk Con-
trol Standard for Soil Contamination of Development Land (Chinese na-
tional standard, 20 mg/kg), whereas Pb concentration followed the
contamination level at most Pb-contaminated sites in Qiqihar city, rang-
ing from 50 to 200 mg/kg. In pot experiment, the 4 tillers of P. communis
were cultivated in 1000 g soils, amended with or without S. chinense
QD10 and mixed well as the following six treatments: negative control
with neither P. communis nor S. chinense QD10 (CK), P. communis only
(PC), low S. chinense QD10 amendment (5 mg DCW S. chinense, SCL),
high S. chinense QD10 amendment (10 mg DCW S. chinense, SCH),
P. communis with low S. chinense QD10 amendment (PC + SCL) and
P. communis with high S. chinense QD10 amendment (PC + SCH). The
pot experiments were carried out at 30 °C for 30 days in biological trip-
licates, and the soils and plant tissues were harvested for further
analysis.
2.5. Morphological and chemical analysis
The morphology of S. chinense QD10 was analyzed by scanning elec-
tron microscope (SEM, Quanta200, USA), together with the element
analysis by energy-dispersive X-ray spectroscopy (EDS). X-ray photo-
electron spectroscopy (XPS) was used to analyze the fungal surface be-
fore and after heavy metal biosorption, performed with an ESCALAB
250Xi X-ray photoelectron spectrometer (Thermo Scientific, USA),
using a monochromatized focused Al-Kα X-ray source (1486.6 eV).
The extraction of total amount, acid-extractable, reducible, oxidizable
and residual fractions of Cd and Pb in soils and P. communis tissues
followed sequential extraction procedures (Tessier et al., 1979; Wang
et al., 2016a), and the details were in supplementary data. The concen-
trations of Cd and Pb were analyzed by inductively coupled plasma
mass spectrometry (ICP-MS, X-series 2, Thermo Scientific, USA). The
standard calibration solutions contained the mixture of Cd and Pb in
3
0.1 M HNO3 and the concentration ranged from 0 to 100 μg/L. Internal
standards were spiked and the recovery rates of Cd and Pb were
90.2%–103.5%. All the HMs concentrations were corrected according to
reference recovery.
2.6. Data analysis
All the treatments were carried out in biological triplicates. The
biosorption isotherm of Cd and Pb on S. chinense QD10 cells followed
the relationship between the adsorbed metal ions and their equilibrium
concentration in aqueous phase. The Langmuir isotherm model repre-
sents the monolayer adsorption mechanism, as described in Eq. (1).
Freundlich isotherm model demonstrates both monolayer and multi-
layer adsorptions by considering the heterogeneous surfaces possessing
different sorption energy sites, as shown in Eq. (2).
K L Ce
Q e ¼ Q max ð1Þ
1 þ KLCe
Q e ¼ K F C e1=n ð2Þ
Here, Qe (g/kg DCW) refers to the absorbed metals on S. chinense
QD10, and Ce (g/L) represents the equilibrium metal concentration in
the supernatant. Qmax (g/kg DCW) is the maximum biosorption capacity
for monolayer adsorption in Langmuir isotherm model, and KL (L/mg) is
the Langmuir constant associated with adsorption energy. KF (g/kg
DCW) represents the adsorption capacity in both monolayer and multi-
layer mechanism in Freundlich isotherm model, and 1/n is the heteroge-
neous sorption sites. Either Langmuir or Freundlich isotherm model is
expressed in the linear form as shown in Eqs. (3) and (4), respectively.
Ce 1 Ce
¼ þ ð3Þ
Q e Q max ∙K L Q max
1
lgQ e ¼ lgK F þ lgC e ð4Þ
n
The translocation factor (TF) was calculated as the ratio of metal
concentration in shoots to that in roots. All the biosorption efficiencies
and residual Cd/Pb fractions in soils were analyzed by two-way analysis
of variance (ANOVA) by SPSS 13.0 and the significant level was p =
0.05.
3. Results and discussion
3.1. Characteristics of Simplicillium chinense QD10
Of all the 13 isolates from the soils in Zhalong Wetland, S. chinense
QD10 was identified with strong tolerance to Cd and Pb. S. chinense
QD10 isolate had aerial mycelia with white fluffy appearance (Fig. 1A),
and the diameter of the colonies achieved 2–3 cm after 48 h. Sexual
structures were observed under the microscope (Fig. 1B), and there
were ascomata with spore droplets. The hyphae of S. chinense QD10
was smooth and nearly translucent, forming significantly elongated
neck at the tapered tip. The carbon metabolic profiles of S. chinense
QD10 were listed in Fig. S1. S. chinense QD10 could effectively utilize
D-glucose-6-phosphate, D-galactonic acid-g-Lactone, L-glutamine,
Tween 80, glycolic acid and methylpyruvate (RRI N 1.0). The moderately
utilized carbon sources (0.5 b RRI b 1.0) included D-saccharic acid, glyc-
erol, Tween 40, α-ketobutyric acid. α-D-Lactose, α-Hydroxyglutaric
acid-g-lactone, α-hydroxybutyric acid, adenosine, glycyl-L-aspartic
acid, fumaric acid, bromosuccinic acid, glyoxylic acid, D-cellobiose, ino-
sine, Gly-Glu, tricarballylic acid, mono-methylsuccinate, D-malic acid, L-
malic acid, p-hydroxyphenyl acetic acid, m-hydroxyphenyl acetic acid
and 2-aminoethanol. To the best of our knowledge, this is the first
study unravelling the carbon metabolic profiles of genus Simplicillium
4 Z. Jin et al. / Science of the Total Environment 697 (2019) 134148

(A) (B) (C)

(D ) (E ) (F)

Uniform Pb aggregation Anomalous Cd aggregation Pb/Cd co-aggregation

Fig. 1. Morphology of S. chinense QD10 on PDA plate (A) or by microscopy (B). Results of scanning electron microscope (SEM) and energy-dispersive X-ray spectroscope (EDS) illustrate
the biosorption of Cd and Pb on S. chinense QD10 cells. (C) No heavy metal amendment; (D) Pb treatment; (E) Cd treatment; (F) Pb-Cd binary treatment.

via phenotypic assay, and the results suggest that S. chinense QD10 can (50 mg/L), the growth curves of S. chinense QD10 remained the same
utilize a wide range of carbon sources. comparing to the control, and there was no significant difference in
Before metal biosorption, the surface of S. chinense QD10 was growth rate and biomass production (p N 0.05). When exposed to
smooth (Fig. 1C), and neither Cd nor Pb was observed from the EDS re-
sults (Fig. S2A and Table S1). Post-exposure to Pb solution, Fig. 1D illus-
0.60
trated the uniform Pb aggregation on S. chinense QD10 cells, consistent
with EDS data (Fig. S2B). Oppositely, some anomalous Cd aggregations
appeared after biosorption (Fig. 1E), and the atomic Cd signals were 0.50
only detectable on this crystal-shaped structure (0.03%). The different
aggregation patterns between Cd and Pb strongly hinted their different
biosorption mechanisms, which has not been reported previously. Var- 0.40
ious principles of metal binding to the microbes have been reported, in-
Biomass (g/L)

cluding ion exchange, chelation and adsorption (Gupta, 2015). Our

results suggested the heterogeneous Cd biosorption might be explained 0.30
by the specific deposition of Cd ions in the fibrillary and spaces of the
structural polysaccharide or peptidoglycan on the membrane of
S. chinense QD10, forming the Cd-chelates, whereas the biosorption of 0.20
Pb was attributed to direct adsorption by extracellular polymeric sub-
stances (EPS). When exposed to Cd\\Pb mixture (Fig. 1F), the aggrega-
tion became shorter and rod-shaped, and EDS results confirmed the co- 0.10
existence of Cd (0.02%) and Pb (0.01%) at these aggregative sites
(Fig. S2D). XPS spectra (Fig. S3) further proved the presence of Cd and
0.00
Pb on cell membrane. 0 20 40 60 80 100
Time (day)
3.2. Tolerance to Pb and Cd
CK Cd-25 Cd-50 Cd-100
S. chinense QD10 could tolerate Cd and Pb, as illustrated in Fig. 2. In Pb-50 Pb-100 Pb-200 Cd-25/Pb-50
medium without Cd or Pb amendment, S. chinense QD10 had the highest
Cd-50/Pb-100 Cd-100/Pb-200
growth rate. After a lag phase in the first 18 h, the growth rate of
S. chinense QD10 increased to 5.98 × 10−3 g DCW/(h·L). After 96 h, Fig. 2. Growth curve of S. chinense QD10 exposure to different concentrations of Cd and Pb.
the biomass maintained around 0.465 g DCW/L and stepped into the Cd exposure was 25, 50 and 100 mg/L, and Pb exposure was 50, 100 and 200 mg/L. In Cd-
stationary phase. Postexposure to low-level Cd (25 mg/L) and Pb Pb binary treatment, the concentrations of Cd/Pb were 25/50, 50/100 and 100/200 mg/L.
 Z. Jin et al. / Science of the Total Environment 697 (2019) 134148 5

higher Cd (N50 mg/L) and Pb (N100 mg/L), the growth rate of S. chinense Table 1
QD10 slightly declined (p b 0.05) and the biomass was b0.400 g DCW/L. Parameters of Qmax and KL in Langmuir isotherms model and KF and n in Freundlich iso-
therms model for Pb and Cd biosorption.
The binary exposure of Cd and Pb aggravated the inhibition of growth
rate, suggesting the synergistic effects of metallic ion on fungal growth. Isotherm Model equation Parameter Cd Pb
Previous studies reported that S. chinense had great adaptive tolerance Langmuir KLC Qmax (g/kg DCW) 88.5 57.8
Q ¼ Q max
potential to Al, Cr or Pb via induced-tolerance training (Chen et al., 1 þ KLC KL (L/g) 21.1 11.9
2017). Our results furtherly proved that S. chinense can tolerate Cd R2 0.981 0.973
Freundlich Q = KFC1/n KF (g/kg DCW) 28.9 6.57
and Cd\\Pb combined pollution, suggesting that the tolerance of
1/n 0.560 0.544
S. chinense on heavy metal might be general and non-specific, which R2 0.918 0.920
can broaden their application range in complex environment.

A positive relationship was observed between biomass of S. chinense

3.3. Heavy metal biosorption isotherm
Both Langmuir and Freundlich models fitted well with the
biosorption isotherms of Cd and Pb on S. chinense QD10, as illustrated
in Fig. 3. From Table 1, Qmax for Cd and Pb was 88.5 and 57.8 g/kg
DCW in Langmuir isotherm, respectively. The Langmuir constant (KL)
was 21.1 L/mg for Cd, significantly higher than that for Pb (11.9 L/mg).
Similarly, Freundlich isotherm also showed higher biosorption capacity
of Cd (KF, 28.9 g/kg DCW) than that of Pb (6.57 g/kg DCW). The
biosorption capacities of Cd and Pb in our present study were similar
with previous studies, e.g., the Pb biosorption of fungal Penicillium sp.
MRF-1 followed both Langmuir and Freundlich isotherms, and Qmax
and KL was 56.8–72.5 and 0.13–9.39 g/kg DCW, respectively
(Velmurugan et al., 2010). The stronger biosorption capability towards
Cd than Pb might be attributed to the active adsorption and detoxifica-
tion of Cd, which was aggregated on specific active sites (Fig. 1D and F).
QD10 and biosorption efficiency (Fig. 3B). The highest Cd and Pb re-
moval efficiency achieved 59.6% and 75.2% when the fungal biomass
content was 1.20 g/L. The biosorption followed the same parameters
in the isotherms described above (Table 1). Higher fungal biomass con-
tent facilitated the maximum biosorption, whereas lower fungal bio-
mass declined heavy metal biosorption capability, consistent with
other heavy metal biosorption by previous studies (Gupta and
Balomajumder, 2015; Mukhopadhyay et al., 2007; Pakshirajan and
Swaminathan, 2009; Park and Chon, 2016; Velmurugan et al., 2010).
3.4. Heavy metal biosorption kinetics
The heavy metal biosorption dynamics showed a fast biosorption in
the first 60 min (Fig. 4A), and the removal efficiency of Cd and Pb
achieved 49.1% and 62.4%, respectively. The adsorption capacity was
24.6 and 31.2 g/kg DCW for Cd and Pb, achieving 27.8%–54.0% of the

40.0
(A) 400 (B)
30.0
300
Ce (mg/L)
Ce (mg/L)

20.0
200

10.0
100

0.0
0
0 0.2 0.4 0.6 0.8 1 1.2
0 50 100 150 200 250
Biomass (g/L)
C0 (mg/L)
Cd Pb
Cd Pb

10.0 5.0
(C) y = 0.0173x + 1.4557
(D) y = 0.5442x + 0.8178
8.0 R² = 0.9726 R² = 0.9195
4.0
Ln(Qe) (mg/L)
Ce/Qe (L/g)

6.0
3.0
4.0
2.0 y = 0.5596x + 1.4605
2.0 y = 0.0113x + 0.5369 R² = 0.9182
R² = 0.9805
0.0 1.0
0 100 200 300 400 1 2 3 4 5 6 7
Ce (mg/L) Ln(Ce) (mg/L)

Cd Pb Cd Pb

Fig. 3. Cd and Pb biosorption isotherms of S. chinense QD10 for different Cd or Pb concentrations (A) and fungal biomass (B). Experimental data fitted well with Langmuir (C) and
Freundlich (D) isotherms, respectively. C0 and Ce (g/L) represent the initial and equilibrium metal concentration in the supernatant, respectively. Qe (g/kg DCW) represents the
absorbed metals on S. chinense QD10.
6 Z. Jin et al. / Science of the Total Environment 697 (2019) 134148
(A) 100%
80%
60%
C/C0
40%
20%
0%
0 50 100
Time (min)
Cd Pb
Fig. 4. (A) Cd and Pb biosorption dynamics of S. chinense QD10. (B) First order adsorption kinetics of S. chinense QD10 for both Cd and Pb. C0 and C (g/L) represent the metal concentration in
the supernatant at time 0 and t, respectively.
maximum biosorption capacity in Langmuir isotherms. The residual Cd
and Pb concentrations remained stable afterwards. Such two-stage
biosorption behavior was observed previously that the rapid initial
biosorption was attributed to the direct interaction between the heavy
metal ions and the binding sites on fungal membrane, and the subse-
quent slow adsorption was explained by the slower transportation of
metals into cytoplasm or slow intracellular diffusion (Saglam et al.,
2002). In the present study, the heavy metal biosorption in the rapid
biosorption stage followed the pseudo-first order kinetics (Fig. 4B),
and the biosorption constant was 1.05 × 10−2 and 1.77 × 10−2 g/
(mg·min) for Cd and Pb, respectively. Our results fitted well with the
most applied models and data describing the heavy metal biosorption
process (Zabochnicka-Swiatek and Krzywonos, 2014). About 83.1%
and 81.8% biosorption of Cu and Pb by Aspergillus niger and Aspergillus
flavus was achieved within the first 10 min (Iram et al., 2015). The
pseudo-first order adsorption constant of Aspergillus niger B77 was
(1.78–2.71) × 10−2 and (0.86–1.44) × 10−2 g/(mg·min) for Cu and
Cd, respectively (Tsekova et al., 2010). The Pb biosorption by Penicillium
sp. MRF-1 had the pseudo-first order adsorption constant of 2.41–4.67
g/(mg·min) (Velmurugan et al., 2010).
3.5. Factors affecting biosorption performance
Temperature was a key environmental variable significantly altering
the biosorption efficiency of S. chinense QD10, as shown in Fig. S4A. The
biosorption capability (60.4% for Cd and 38.3% for Pb) was highest at 30
°C and significantly declined when the temperature increased to 45 °C.
The interaction between metal ions and binding sites on fungal mem-
brane is dependent on temperature (Kapoor and Viraraghavan, 1995).
Temperature causes the chemical moieties ionization and affects the
stability and configuration of fungal membrane (Oka et al., 2005).
Since 30 °C is the optimal growth temperature for S. chinense QD10 in
the present study, the highest activities of the binding sites are expected
to achieve the best biosorption performance. Similarly, most of fungal
strains were reported to have the highest heavy metal biosorption ca-
pacities under their optimal growth conditions (Iram et al., 2015).
Though higher temperature might promote the biosorption interaction
between metals and fungal cells, it might damage the binding sites and
reduce the heavy metal transportation (Zabochnicka-Swiatek and
Krzywonos, 2014).
The pH value is another factor affecting the biosorption process, at-
tributing to its impacts on both heavy metal solubility/mobility and
the dissociation of functional groups on fungal membrane. There was
a positive relationship between the removal efficiency of Cd/Pb and
(B) 100%
y = 0.0177x
R² = 0.9594
80%
60%
Ln(C0/C)
40%
y = 0.0105x
R² = 0.9751
20%
0%
150 200 0 20 40 60 80
Time (min)
Cd Pb
pH (3.0 to 9.0, p b 0.05, Fig. S4B). Accordingly, the biosorption efficiency
increased from 5.63% (pH = 3) to 52.3% (pH = 9) for Cd and 9.81% (pH
= 3) to 76.6% (pH = 9) for Pb, respectively. Metal biosorption was re-
ported to depend on pH, related to the affinity of the binding sites on
bacterial or fungal cells and the logarithmic values of the metal's first
hydrolysis constant (Pagnanelli et al., 2003). The active binding sites,
which are high-molecular weight compounds, are affected by pH. The
ionization of EPS was pH-dependent and reported to affect the
biosorption capacities of metals (Wang et al., 2014). The biosorption
functional groups behave differently among pH conditions, contributing
to the change of heavy metal biosorption efficiency (Farooq et al., 2010).
For instance, H3O+ ions can compete with heavy metal ions for the
binding sites of carboxylic group under acidic condition (pH b 7.0), con-
sequently reducing the availability of the bindings sites and biosorption
capacities (Comte et al., 2008). It explained the lower biosorption per-
formance under low pH conditions (Fig. S4B), consistent with previous
studies on the metal biosorption behavior to species-dependent carbox-
ylic functional groups (Comte et al., 2008; Guibaud et al., 2005). Cd
biosorption by Exiguobacterium sp. increased with pH up to 7.0 and
remained constant when pH was above 7.0 (Park and Chon, 2016).
When pH value was above 7.0, the increasing binding sites were avail-
able to absorb metals (Zabochnicka-Swiatek and Krzywonos, 2014),
and the metal ions tended to bind to low affinity ligands such as hy-
droxyl and carboxyl groups (Saeed and Iqbal, 2003). Meanwhile, Cd2+
and Pb2+ cations can react with OH– and form the hydroxide precipita-
tion on cell membrane (Mavrov et al., 2006). Hence, the stronger
biosorption efficiency of Cd and Pb under alkaline condition in this
study was attributed to the precipitation and fungal biosorption
together.
3.6. Contribution of S. chinense QD10 to phytoremediation
Different forms of Cd and Pb in soils were measured via stepwise ex-
traction after S. chinense QD10 amendments. Fig. 5A illustrated that
acid-extractable, reducible and residual Cd in soils slightly declined,
whereas oxidizable Cd increased. The content and speciation change
of Pb in soils behaved similar as Cd (Fig. 5B). From the results of Two-
way ANOVOA (Table S2), Cd and Pb concentration of all forms signifi-
cantly declined in the treatments with P. communis, except for oxidiz-
able Pb (p = 0.145). The removal efficiency was 20.0% (acid-
extractable), 32.2% (reducible), 4.21% (oxidizable) and 13.8% (residual)
for Cd, and 27.6% (acid-extractable), 22.7% (reducible), 4.96% (oxidiz-
able) and 25.1% (residual) for Pb, respectively. Acid-extractable and
 Z. Jin et al. / Science of the Total Environment 697 (2019) 134148 7

(A) 15.0 (B) 120.0

100.0
12.0

Cd concentration (mg/kg)

Pb concentration (mg/kg)
80.0
9.0

60.0

6.0
40.0

3.0
20.0

0.0 0.0
CK SCL SCH PC PC+SCL PC+SCH CK SCH PC+SCL
Treatment Treatment

HCl extractable Cd Oxidizable Cd Reducible Cd Residual Cd HCl extractable Pb Oxidizable Pb Reducible Pb Residual Pb

Fig. 5. Soil Cd (A) and Pb (B) concentrations of different forms after P. communis phytoremediation. CK represents the negative control with neither P. communis nor S. chinense QD10; SCL
represents low S. chinense QD10 (5 mg DCW/kg) treatment; SCH represents high S. chinense QD10 (10 mg DCW/kg) treatment; PC represents P. communis treatment; PC + SCL represents
P. communis with low S. chinense QD10 (5 mg DCW/kg) treatment; PC + SCH represents P. communis with high S. chinense QD10 (10 mg DCW/kg) treatment.

reducible metals were reported to have higher solubility and mobility, against parasitic nematodes and bacteria (Zhao et al., 2013). Comparing
easier to be taken by P. communis than other fractions. to Cd, the removal efficiency of acid-extractable Pb was slightly higher
Previous studies have defined metal-accumulating plants as those (46.8%–48.0%), but lower for reducible Pb (28.6%–30.3%) and residual
have metal concentrations over 100 times higher than other species Pb (31.4%–34.0%), as illustrated in Fig. 5B. The oxidizable Cd and Pb
(van der Ent et al., 2013), whereas metal-tolerating plants can survive were both significantly higher in fungi-mediated phytoremediation
and maintain growth conditions in metal-rich soils but cannot accumu- treatments (PC + SCL and PC + SCH) than those with S. chinense
late metals (Mertens et al., 2005; Phillips, 1977). Normally, they are dis- QD10 only (SCL and SCH). There was no significant difference in Cd or
tinguished by the metal contents in tissues that metal-accumulating Pb removal efficiencies between PC + SCL and PC + SCH treatments,
have significantly higher tissue metal contents than those in soils showing minimal impacts of initial dosage of S. chinense QD10 on
(McGrath and Zhao, 2003). For phytoremediation, both metal- phytoremediation. Our study indicated that P. communis could effec-
accumulating and metal-tolerating capabilities are important to extract tively remove Cd and Pb from soils as phytoextractor, and fungal coloni-
and remove maximum metals from contaminated soils (Clemens, zation was more crucial for phytoremediation than fungal dosage,
2006), and it is therefore necessary to evaluate the removal of Cd and owing to the fast growth of S. chinense QD10 in the rhizosphere.
Pb from soils and their accumulation level in tissues of P. communis. Fig. 6 illustrated high accumulation of metals in roots and shoots of
In the present study, S. chinense QD10 amendment significantly im- P. communis. In PC treatment, the dry weight of P. communis did not
proved the removal of Cd and Pb from soils. From Fig. 5A, the fungi- show significant different comparing to CK treatment (data not
mediated treatments (PC + SCL and PC + SCH) achieved 37.3%–42.3%, shown), demonstrating P. communis is a Cd- and Pb-tolerating plant.
42.3%–48.0% and 37.5%–42.9% removal efficiency of acid-extractable, re- The accumulated Cd and Pb concentration was 2.71 and 20.7 g/kg in
ducible and residual Cd, respectively. It might be attributed to the roots and 0.59 and 5.56 g/kg in shoots, respectively. The metal accumu-
biosorption and accumulation of Cd on S. chinense QD10 within the rhi- lation level in this study was similar to previous works on heavy metal
zosphere and the beneficial effects of S. chinense QD10 on helping plant accumulation by different plants, e.g., the Cd concentration in

(A) 8.0 (B) 30.0

6.0
20.0
4.0
Pb concentration (g/kg)
Cd concentration g/kg)

10.0
2.0

0.0 0.0

2.0
10.0
4.0
20.0
6.0

8.0 30.0
PC PC+SCL PC+SCH PC PC+SCL PC+SCH
Treatment Treatment

Root Shoot Root Shoot

Fig. 6. Cd (A) and Pb (B) in the roots and shoots of P. communis with or without S. chinense QD10 amendment. PC represents P. communis treatment; PC + SCL and PC + SCH refer to the
P. communis treatments with low (5 mg DCW/kg) and high (10 mg DCW/kg) S. chinense QD10 amendment, respectively.
8 Z. Jin et al. / Science of the Total Environment 697 (2019) 134148
Phragmites australis (3.69 g/kg in roots and 0.08 g/kg in shoots) (Iannelli
et al., 2002). S. chinense QD10 amendment significantly enhanced the
accumulation of Cd and Pb in P. communis tissues, and its initial dosage
had a significant contribution to the increasing metal accumulation (p
= 0.01, Fig. 6). The Cd concentration in roots increased 48.0%
(4.01 g/kg) and 100.4% (5.42 g/kg) in PC + SCL and PC + SCH treat-
ments, respectively. For shoots, the Cd concentration increased to
1.87 g/kg (217% higher in PC + SCL treatment) and 2.02 g/kg (243%
higher in PC + SCH treatment). Similar results were observed for Pb ac-
cumulation. In addition, the content of Cd and Pb was 22.4 and
138 mg/kg in soils and 0.59 and 5.56 g/kg in shoots of P. communis.
The bioconcentration factor of Cd and Pb was 26.4 and 40.3 respectively,
further proving P. communis as a Cd/Pb accumulator and candidate for
phytoextraction. Our results were consistent with Wang's study that
the uptake of metals (Cu, Zn, Pb and Cd) by Elsholtzia splendens was sig-
nificantly enhanced by the addition of` arbuscular mycorrhizal fungi
(Wang et al., 2005).
Results in the present study suggested the significant removal of Cd
and Pb, indicating that fungi-mediated phytoremediation showed bet-
ter performance than sole plants. It might be explained by the strong
biosorption capacity of S. chinense QD10 towards Cd and Pb, which gen-
erates gradients of available Cd or Pb in soils, drives their enrichment in
rhizosphere, and enhances metal uptake by P. communis. Additionally,
S. chinense has been proved to be a mutually beneficial symbiosis and
benefit host plants, e.g., S. chinense can be used as a control reagent
against plant parasitic nematodes and promotes plant growth (Zhao
et al., 2013). Some previous studies also showed that arbuscular mycor-
rhizal fungi and Penicillium fungi increased the dry weight of Elsholtzia
splendens shoots, further improved the contents of Cu, Zn and Pb in
shoots (Wang et al., 2007). It was also reported that ectendomycorrhiza
fungi had significant impacts on Cd uptake and development of Pinus
pinaster seedlings (Sousa et al., 2012). All these results suggested
fungi-mediated phytoremediation is a feasible bioaugmentation ap-
proach for heavy metal removal from contaminated soils.
The shoot-to-root ratio, evaluated by the translocation factor (TF),
reflected the metal translocation within the plant organs. Among the
three treatments, S. chinense QD10 amendment significantly increased
the TF of Cd from 0.218 (PC treatment) to 0.466 (PC + SCL treatment)
and 0.372 (PC + SCH treatment), whereas it remained unchanged for
Pb (0.269 in PC treatment, 0.273 in PC + SCL treatment and 0.279 in
PC + SCH treatment). The different biosorption mechanisms of Cd and
Pb on S. chinense QD10 might explain the difference that the heteroge-
neous adsorption of Cd consequently formed the Cd-chelates (Fig. 1E),
which might improve the availability and mobility of Cd and enhance
its translocation within plant tissues. From the study on heavy metal up-
take by Elsholtzia splendens, chitosan amendment significantly in-
creased the metal translocation owing to the formation of chitosan-
metal chelates (Wang et al., 2007; Wang et al., 2005). Since Pb was
absorbed mainly by EPS and no Pb-chelate was formed, its TF did not
alter between the treatments with and without S. chinense QD10
amendment.
4. Conclusion
This work isolated and identified a new soil fungal strain, S. chinense
QD10, which showed a strong capability to adsorb Cd and Pb following
different mechanisms. Cd biosorption was attributed to the formation of
Cd-chelates, whereas Pb was predominantly adsorbed by EPS.
S. chinense QD10 showed satisfactory tolerance performance on Cd
and Pb at 50 and 100 mg/L, respectively, and the biosorption fitted
well with both Langmuir and Freundlich isotherms. S. chinense QD10
amendment can significantly enhance the phytoremediation of Cd and
Pb, potentially attributing to the rhizospheric enrichment of Cd or Pb
and defending effects on plants mediated by S. chinense QD10. The pres-
ent work introduced a new fungal strain with great potential for
enhancing phytoremediation, which might contribute to better practi-
cal phytoremediation in metal contaminated wetland or soils.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
This study was supported by the Natural Science Foundation of Hei-
longjiang Province (No. C201240) and Science and Technology Research
Project of the Department of Education, Heilongjiang Province (No.
12531754). DZ also acknowledges the support of Chinese Government's
Thousand Talents Plan for Young Professionals.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2019.134148.
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