J Soils Sediments (2015) 15:10291038
DOI 10.1007/s11368-015-1076-8
SEDIMENTS, SEC 4 SEDIMENT-ECOLOGY INTERACTIONS RESEARCH ARTICLE
Bioleaching of heavy metals from contaminated sediments
by the Aspergillus niger strain SY1
Xiangfeng Zeng & Shuhe Wei & Lina Sun &
David A. Jacques & Jiaxi Tang & Meihua Lian &
Zhanhua Ji & Jun Wang & Jianyu Zhu & Zixiang Xu
Received: 24 February 2014 / Accepted: 23 January 2015 / Published online: 11 February 2015
# Springer-Verlag Berlin Heidelberg 2015
Abstract
Purpose The objective of this study was to investigate the
bioleaching of heavy metals from contaminated sediments
by Aspergillus niger strain SY1. To achieve this, three targets
were identified: (1) identify organic acids produced by the
isolated A. niger strain SY1 from contaminated sediments,
(2) compare the leaching ability and transformation of chem-
ical speciation of heavy metals during the bioleaching pro-
Responsible editor: Gijs D. Breedveld
X. Zeng : S. Wei (*) : Z. Ji
Key Laboratory of Pollution Ecology and Environment Engineering,
Institute of Applied Ecology, Chinese Academy of Sciences,
Shenyang 110016, Peoples Republic of China
e-mail: shuhewei@iae.ac.cn
L. Sun : J. Tang : M. Lian
Key Laboratory of Regional Environment and Eco-Remediation
(Ministry of Education), Shenyang University, Shenyang 110073,
Peoples Republic of China
X. Zeng : Z. Ji
University of Chinese Academy of Sciences, Beijing 100039,
Peoples Republic of China
D. A. Jacques
Energy Research Institute, School of Process, Environmental and
Materials Engineering, University of Leeds, Leeds LS2 9JT, UK
J. Tang
Department of Land and Environment, Shenyang Agricultural
University, Shenyang 110866, Peoples Republic of China
X. Zeng : J. Wang : J. Zhu
Center for Environmental Biotechnology, The University of
Tennessee, Knoxville 37996, TN, USA
Z. Xu
Key Laboratory of Systems Microbial Biotechnology, Tianjin
Institute of Industrial Biotechnology, Chinese Academy of Sciences,
Tianjin 300308, Peoples Republic of China
cesses, and (3) determine the toxic characteristic of sediment
before and after bioleaching.
Materials and methods The contaminated sediment was col-
lected from the dredging of the Xihe River, China. The
A. niger strain SY1 was isolated from this sediment.
Bioleaching experiments were carried out in 250 ml
autoclaved conical flasks with 10 g autoclaved sediment,
1 ml of spore suspension, and 99 ml culture medium; the
flasks were placed in a shaking incubator (220 rpm) at 30 C
for 7 days. Toxicity characteristic leaching procedure (TCLP)
tests were carried out according to USEPA-SW846 Method
1311, and the wheat and earthworm toxicity tests were carried
out according to OECD BGuidelines for the Testing of
Chemicals.^ Fractionation of heavy metals was undertaken
by the three-step sequential extraction procedure. The metab-
olites were determined with a HPLC system.
Results and discussion There was 11.5 % leaching efficiency
of Pb from the polluted sediment in the one-step bioleaching
process; while in the two-step bioleaching process, the highest
extraction efficiency of Pb was 65.4 %. In one-step
bioleaching, 93.5 % Cd, 62.3 % Cu, and 68.2 % Zn were
leached out; whereas, the highest metal extraction efficiencies
of Cd, Cu, and Zn were 99.5, 56, 71.9, and 76.4 %, respec-
tively, in two-step bioleaching. After the bioleaching, the
metals remaining in the sediment were mainly found in the
stable fractions. Cd, Pb, Cu, and Zn concentrations in extract-
ed liquor of TCLP tests were reduced to far below the levels in
two Chinese standards, and the sediment after bioleaching had
a lower toxicity on wheat and earthworm.
Conclusions A. niger strain SY1 can effectively remove
heavy metals in contaminated sediment. The bioleaching
efficiencies of heavy metals in the two-step bioleaching
were better than that in one-step bioleaching. After the
bioleaching, metals remaining in the sediment were main-
ly found in the stable fractions, and the toxicity of it was
reduced to a level for it to be used safely in landfill or
1030
used in land application. A. niger strain SY1 is a cost-
effective, environmentally friendly, and efficient
bioleacher of heavy metals.
Keywords Aspergillus niger . Bioleaching . Fractionation .
Heavy metals . Toxicity
1 Introduction
Due to rapid industrialization and urbanization, heavy metal
(e.g., Pb, Cd, Cu, and Zn) contamination of sediment has
become one of the major environmental problems in China
and all over the world (Chen et al. 2007; Peng and Song
2009). The increasing amounts of contaminated sediments
and high concentrations of toxic pollutants raise a serious
concern (Mulligan et al. 2001; Chen and Lin 2009). Recently,
great efforts have been focused on the treatment of contami-
nated sediment due to its potential danger to ecosystems and
human health (Yang et al. 2009; Beolchini et al. 2013). For a
successful remediation, alongside controlling the pollutants at
sources and building sewer systems, dredging of contaminat-
ed sediment is one of the main approaches available (Mulligan
et al. 2001; Chen et al. 2007). Most of the dredged sediment
contains a high concentration of toxic pollutants such as heavy
metals and it is possible that metals may diffuse into the
groundwater or enter the food chain through landfill or land
application (Chen and Lin 2004; Seidel et al. 2004). It is
therefore necessary to remove heavy metals from contaminat-
ed sediments before said landfill, land application, or other
utilization. Physical and chemical technologies to remove tox-
ic heavy metals from contaminated sediments have become
less and less attractive because of high-energy requirements,
high cost, low efficiencies, and operational difficulties (Akinci
and Guven 2011; Fang et al. 2011).
Currently, a bioleaching approach offers a promising cost-
effective alternative for the removal of heavy metals from
low-grade mine tailings (Escobar et al. 2010), residues (Chan
and Dudeney 2008), and contaminated soils and sediments
(Kumar and Nagendran 2007; Fang et al. 2011), with the
advantages of lower costs, lower energy requirements, envi-
ronmental safety, and operational flexibility (Pathak et al.
2009). Filamentous fungi have the potential to leach metals
from different substances through the production of weak or-
ganic acids that form water-soluble complexes with metals
(More et al. 2010). Because Aspergillus niger (A. niger) is
generally more tolerant than bacteria to the toxicity of heavy
metals, and the organic acids they produce form complexes
with heavy metals and render these less toxic, A. niger is one
of the most widely used fungi in bioleaching (Ren et al. 2009).
As well as this, A. niger can produce organic acids (such as
citric acid, oxalic acid, and gluconic acid) using carbohydrates
J Soils Sediments (2015) 15:10291038
found in wastes such as date fruit syrup, cheese whey, corn-
cobs, and cane molasses, which dramatically reduces the cost
of bioleaching and makes it more applicable (Del Mundo et al.
2009; Sankaran et al. 2010).
Until now, A. niger has been mostly used for leaching
heavy metals from low grade ores (Mulligan et al. 2001), mine
tailings (Seh-Bardan et al. 2012), contaminated soils (Ren
et al. 2009), and industrial wastes, such as fly ash (Karlfeldt
Fedje et al. 2010), nickeliferous laterites (Simate et al. 2010),
and spent fluid catalytic cracking catalyst (Mafi Gholami et al.
2012). Recently, two strains of A. niger (DSM 2182 and DSM
2466) have been used to bioleach heavy metals from dredged
sediments (Sabra et al. 2012). However, it was found that the
bioleaching yields were relatively limited (<50 %), with the
exception of Mn. The bioleaching efficiencies of A. niger on
heavy metals were not only based on the total content of heavy
metals, but were also based on the fractions and toxic effects
of heavy metals and the tolerance of A. niger to said metals.
The selection of heavy metal-resistant fungi and their adapta-
tion training are necessary before bioleaching treatment of
sediments. To forecast the mobility, bioavailability, and toxic-
ity of heavy metals and the potential hazard to the ecosystem
and human health of the sediments after bioleaching using
A. niger, it is essential to find out the transformation of chem-
ical forms of heavy metals during bioleaching, However, the
information available on the bioleaching process of contami-
nated sediments by A. niger is limited. The objectives of this
study were therefore to: (1) identify organic acids produced by
the isolated A. niger strain SY1 from contaminated sediments,
(2) compare the leaching ability and transformation of chem-
ical speciation of heavy metals during the bioleaching pro-
cesses, and (3) determine the toxic characteristic of sediment
before and after bioleaching.
2 Materials and methods
2.1 Sediment
The contaminated sediment used in this study was collected
from the dredging process of the Xihe River, near Qianmiaozi
park, Shenyang, China (413910 N, 123624 E). There were
no specific permissions required for these locations/activities,
and the field studies did not involve endangered or protected
species. The Xihe River has a total length of 78.2 km and has
received 500,000 kg day1 of untreated treated municipal and
industrial waste as well as 550 kg day1 of contaminants since
1962. This equates to 40 % of the total wastewaters and 60
70 % of the total contamination discharged by the city of
Shenyang, respectively. Samples of the upper 020 cm depth
of sediment were taken with a van Veen grab in April 2012.
They were then placed into sealed plastic bags and returned to
the laboratory. In the laboratory, all gravels and large organic
J Soils Sediments (2015) 15:10291038 1031

materials were removed from the sediment and the sediment 2.3 Spore collection and inoculum preparation
was stored in sealed plastic bags and kept at 4 C for further
study. Sediment samples were air dried, passed through a 2- A fungi strain, originally isolated from the heavy metal-
mm sieve, and then mechanically mixed to ensure homogene- contaminated sediment from Xihe River in Shenyang, Liao-
ity and stored prior to experiment. ning Province, China, was identified as A. niger. Identification
was achieved by sequencing 26S rDNA (D1/D2) and ITS.
The 26S rDNA sequence size was 557, and the ITS sequences
2.2 Sequential extraction study size was 533. The similarity of gene sequence of 26S rDNA
and ITS5 showed 100 % agreement with A. niger. Adaptation
Fractionation of heavy metals present in sediment was of the fungus was carried out through a series of sub-cultures
carried out by the three-step sequential extraction proce- after exposure to the sediment used in the study. The strain
dure developed by the Measurement and Testing Pro- was cultured in a Chashi liquid medium composed of 90 g l1
gram of the European Commission (BCR; Perez- glucose. For the inoculum preparation, adapted A. niger strain
Santana et al. 2007). The extractions were conducted in SY1was incubated three times on potato dextrose agar (PDA)
50 ml polypropylene centrifuge tubes to minimize loss of slants using a sterile platinum loop at 30 C for 5 days. The
sediment. Between successive extractions, separation was resulting spores were harvested in 0.1 % Tween 80 solution
achieved through centrifugation at 3,500g for 20 min. and used to inoculate the raw liquid at 2109 spores per liter.
Mild extractant aliquots were acidified and stored in Then 1 ml of spore suspension was added to 99 ml medium in
polypropylene bottles at 4 C to prevent the growth of a 250-ml flask. Flasks containing Chashi liquid medium and
bacteria before later analysis. The supernatant was fil- sediment were autoclaved at 115 C for 30 min to achieve
tered and analyzed for heavy metals by an atomic ab- sterilization.
sorption spectrophotometer (AA240, Varian, USA). Be-
fore carrying out the next extraction, the residue was 2.4 Bioleaching experiments
washed with 8 ml of distilled water and centrifuged for
20 min; this supernatant was discarded. Minimal volume Bioleaching experiments were carried out in 250 ml
of rinse water was used to avoid excessive solubilization autoclaved conical flasks. In the one-step bioleaching pro-
of solid material. The details of extraction steps are sum- cess, 10 g autoclaved sediment and 1 ml of spore suspen-
marized in Table 1. sion were added into 99 ml culture medium. A control
experiment was carried out in parallel without inoculation.
All flasks were agitated in a rotary shaking incubator
Table 1 Steps of the heavy metal extract fractions (220 rpm) at 30 C for 7 days. In the two-step bioleaching
Step Extract fraction Extract reagent and method process, 1 ml of spore suspension was inoculated in 99 ml
of medium for 2 days (step 1). After 2 days of cultivation,
Step A Exchangeable/carbonate 20 ml 0.11 mol l1 acetic acid, 2 g autoclaved sediment was added and bioleached in a
fraction (B1) shaken at room temperature shaking incubator (220 rpm) at 30 C for 5 days (defined
for 16 h
as the second step). All the experiments were run in trip-
Step B Fe-Mn oxide fraction 20 ml 0.1 mol l1
(B2) hydroxylammonium chloride,
licate. Before filtration, the samples were weighed and the
shaken at room temperature water loss due to evaporation was replenished with dis-
for 16 h tilled water every day.
Step C Organic fraction (B3) (1) 5 ml 8.8 mol l1 hydrogen During the bioleaching experiment, the variation of pH was
peroxide (pH 2 adjusted with measured at selected time intervals. After the bioleaching pro-
17 mol l1 HNO3), covered
cess, the mixtures of sediment, mycelia, and liquid medium
and the digested at room
temperature for 1 h, with were filtered, and then, the mycelia adhering to the sediment
occasional manual shaking were sought out. The sediment was washed with deionized
and 1 h at 85 C; (2) 5 ml water three times. Thereafter, the sediment was air-dried for
hydrogen peroxide, covered
24 h. Five grams of the sediment from the assay were digested
and digested at 85 C for 1 h;
and (3) 25 ml as described in Section 2.2 and 2.6.
1 mol l1ammonium acetate
(pH 2), shaken at room 2.5 Toxic characteristic leaching procedure
temperature for 16 h
Step D Residues fraction (B4) Acid digestion describe as the
The toxicity characteristic leaching procedure (TCLP) tests of
determination of total content
of heavy metal in sediment the sediment before and after bioleaching were carried out
according to USEPA-SW846 Method 1311. The wheat and
1032 J Soils Sediments (2015) 15:10291038

Table 2 Physicochemical characteristics of the sediment

Parameter pH Organic matter Total N Total P Total K Cd Pb Cu Zn

(%) (mg kg1) (mg kg1) (mg kg1) (mg kg1) (mg kg1) (mg kg1) (mg kg1)

Value 6.6 10.7 1,089.4 1,165.0 472.7 33.3 915.1 384.9 887

earthworm toxicity tests were carried out according to ISO
11269-2, 2012 and ISO 11268-1, 1993, respectively.
2.6 Analytical methods
The physical and chemical properties of the sediment were
determined by using conventional agricultural analysis
methods. The total heavy metal content in the sediment was
determined by subjecting the sediment to an acid digestion
mixture (HCl, HNO3, HClO4 and HF) that was heated with
an electric heating plate. The digestion solution was diluted
with 1 % (v/v) nitric acid for heavy metals analysis, The full
description of this method is available in USEPA standard
3050B.
The metabolites of A. niger in the leaching liquid, such as
pyruvic acid, citric acid, oxalic acid, malic acid, and succinic
acid, were determined with a HPLC system (Agilent 1100
Series, USA) equipped with a Bio-Rad Aminer HPX-87-H
column (300 mm 7.8 mm) and a diode array detector
(DAD) at 210 nm. The mobile phase was 4 mmol l1 H2SO4
at a flow rate of 0.6 ml min1 and the operation was carried out
at 30 C. An external standard method was used to quantify
organic acids and glucose.
3 Results and discussion
3.1 Physicochemical characteristics of sediment
environment and human health. Reduction or elimination of
these heavy metals before land application or other utilization
is therefore necessary (Chen and Lin 2009).
The different forms of heavy metals represent different en-
ergy states, and this affects the efficiency of bioleaching.
Metals in exchangeable, carbonate and Fe/Mn oxide-bound
fractions are considered to be relatively mobile, dangerous,
and bioavailable. Compared to this, the organic matter and
residual fractions are considered to be more stable and non-
bioavailable (Naresh Kumar and Nagendran 2009; Kim et al.
2012). The results of the BCR studies are presented in Table 3.
As shown, there was a wide variation in the form of heavy
metals present in the sediment sample. Cadmium in sediment
was mainly present in the Fe/Mn oxide-bound fraction
(15.27 mg kg1), followed by the exchangeable/carbonate
fraction (14.05 mg kg1), the organic fraction
(3.54 mg kg1), and the residual fraction (0.43 mg kg1). Cad-
mium in the exchangeable, carbonate and Fe/Mn oxide-bound
fraction accounted for >88 % of the total Cd content. This
demonstrates that the Cd in the sediment was easily
bioleached out. Lead and Cu content differed greatly with
the majority being found in the organic fraction (196.13 and
190.08 mg kg1) and the residual fraction (537.68 and
152.42 mg kg 1 ). For Zn, the residual fraction was
422.15 mg kg1, followed by the Fe/Mn oxide-bound fraction
(280.01 mg kg1), the organic fraction (164.88 mg kg1), and
the exchangeable/carbonate fraction (19.96 mg kg1).

The basic properties of the sediment before bioleaching are
shown in Table 2. The initial sediment pH was found to be
near to neutral (6.6), the organic matter content was 10.7 %
and the total N, total P, and total K content were 1.0, 1.2, and
0.5 %, respectively. The sediment was highly contaminated
with heavy metals and the concentrations of heavy metals in
the sediment were 33.3 mg kg1 Cd, 915.1 mg kg1 Pb,
384.9 mg kg1 Cu, and 887 mg kg1 Zn. These concentrations
are extremely high, posing a significant hazard for the aquatic
3.2 Production of organic acids and pH variation
One-step process and two-step process experiments were per-
formed by growing A. niger in the presence of 10 % (w/v)
sediment. The pH of samples taken from the culture was de-
termined at regular time intervals and at the end of the
leaching experiment, the concentrations of organic acids were
determined. Figure 1 indicates that the pH decreased steadily
from 6.4 to 2.5 in the two-step process, and the final pH was

Table 3 Fractionation of the

heavy metals present in the Cd Pb Cu Zn
sediment (mg kg1)
B1 (exchangeable/carbonate fractions) 14.051.2 33.192.1 6.40.8 19.960.3
B2 (Fe/Mn oxide-bound fractions) 15.271.0 148.6110.3 362.3 280.018.1
B3 (organic fractions) 3.540.8 196.136.9 190.087.1 164.883.2
B4 (residual fractions) 0.430.4 537.689.2 152.426.4 422.159.6
Raw sediment 33.3 915.1 384.9 887
J Soils Sediments (2015) 15:10291038 1033

8
pyruvic acid had a higher concentration in the one-step
7 bioleaching process than that in the two-step bioleaching.
6
two-step bioleaching
pH value (U)

5 3.3 Extraction of heavy metals

one-step bioleaching
4 control
3 stepcan affect the bioleaching efficiencies of heavy metals.
2 However, due to the difference in heavy metals, the effects
36h
1 were different (Naresh Kumar and Nagendran 2009; Deng
0
0 1 2 3 4 5 6 7
Bioleaching time (day)
Fig. 1 Change of pH value during bioleaching
1.7 after a 7 day bioleaching period. The rate of pH decrease in
the one-step process was slower than that of the two-step
process, with a final pH of 2.1. It should be noted, however,
that there was a lag phase of 36 h during the one-step
bioleaching process before there was any notable drop in
pH. The decrease in pH was principally because of the bio-
production of organic acids although some other metabolites
may be produced by fungi (Ren et al. 2009; Qu et al. 2013).
In this study, oxalic acid, citric acid, glucose acid, pyruvic
acid, succinic acid, lactic acid, and acetic acid were deter-
mined after bioleaching and all of these acids were detectable.
The retention time and concentrations of the organic acids are
listed in the Table 4. The contents of the organic acids differ
because the production of organic acids depends on many
factors, such as the carbon source, the ratio of nitrogen and
phosphate in the medium, pH and heavy metal content. The
total organic acid concentrations were 8,045 mg l1 in one-
step bioleaching and 9,054.6 mg l1 in two-step bioleaching
after 7 days. A large amount of glucose acid, oxalic acid and
citric acid was generated both in the one-step and two-step
bioleaching process. Lactic acid and acetic acid
(<0.3 mg l1) were only detected in small quantities in both
process. Succinic acid in the two-step bioleaching process was
more than in one-step bioleaching process. However, the
Different bioleaching approachese.g., one-step and two-
et al. 2012). Figure 2 shows the bioleaching efficiencies of
heavy metals (Pb, Cd, Cu, and Zn) in the one-step and two-
step bioleaching processes. The results show that the Pb
bioleaching efficiency in the two-step bioleaching was signif-
icantly higher than that in the one-step bioleaching. For the
other heavy metals considered (Cd, Cu and Zn), a higher
bioleaching efficiency was observed in the two-step
bioleaching process than the one-step bioleaching process.
For example, 11.5 % Pb was leached from the polluted sedi-
ment in one-step bioleaching, whereas in two-step
bioleaching, the highest extraction efficiency of Pb was
65.4 %. In one-step bioleaching, 93.5 % Cd, 62.3 % Cu, and
68.2 % Zn were leached out, and the highest metal extraction
efficiencies of Cd, Cu and Zn in the two-step bioleaching
process were 99.5, 56, 71.9, and 76.4 % for Pb, Cd, Cu, and
Zn, respectively. Only a small of amount of heavy metals (Pb
4.2, Cd 6.0, Cu 1.7, and Zn 2.3 %) were extracted in the
control experiment. For one-step and two-step bioleaching,
the bioleaching efficiency of heavy metals from sediment de-
creased in the following order: Cd > Zn > Cu > Pb.
These results can be attributed to three reasons, namely: the
decline of the pH value; the production of organic acids; and
bioaccumulation (Sullivan et al. 2012). The pH value de-
creased with the production of organic acids during the growth
of A. niger in the one-step process, but it did not decrease as
much compared with the two-step process (as shown in
Fig. 1). According to the pH, some organic acids had been
produced by A. niger before being added into the sediment.
Once the sediment was added into the pre-cultured medium
containing the A. niger, the heavy metals were solubilized

Table 4 Production of organic

acids after a 7-day bioleaching Item Retention Equation of Correlation Content in one-step Content in two-step
period time (min) quantification coefficient bioleaching bioleaching (mg l1)
(mg l1)

Oxalate 6.927 y=1,569.6x 0.9999 6048.7 5751.3

Citric acid 7.65 y=1,375.7x 0.9998 367.5 260.2
Gluconic acid 8.194 y=775.42x 0.999 1616.4 3035.1
Pyruvic acid 9.811 y=1,124.6x- 0.9988 10.225 3.664
35.56
Succinic acid 11.329 y=785.19x 0.9999 2.085 3.914
Lactic acid 12.01 y=1,103.8x 1.0000 0.266 0.188
Acetic acid 14.556 y=842.91x 0.9996 0.234 0.184
1034 J Soils Sediments (2015) 15:10291038

two-step bioleaching
120 treatment process. As Fig. 3 shows, the total content of heavy
Percentage of bioleaching (%)

one-step bioleaching
a
100
a control metals and their relative fractions were changed due to the
a a bioleaching process.
80 a a
a Changes in concentration of Cd bonded to different frac-
60
tions during bioleaching are shown in Fig. 3a. Compared with
40

20 b
c b b b 50
B4

Cd fraction content(mg kg )
0

-1
Pb Cd Cu Zn 40 B3
B2
He avy metals 30 B1
Fig. 2 Different bioleaching methods of A. niger
20

10
rapidly and chelated tightly by the organic acids already pro-
duced in the two-step bioleaching (Arwidsson et al. 2010). 0
one-step two-step control original
Meanwhile, the addition of the contaminated sediment result- bioleaching bioleaching bioleaching
ed in some toxicity to the growth of A. niger, but after 2 days
the A. niger cultivated freely and had a higher tolerance to 1000 B4

Pb fraction content(mg kg-1)

heavy metals than the spore. So, a different metabolite was
B3
involved in the two-step leaching compared to the with one- 800
B2
step leaching. Bio-accumulation occurs during fungal
600 B1
bioleaching and enhances metal leaching by altering the equi-
librium metal concentration in the suspension (Ren et al. 400
2009; Deng et al. 2012) and thus higher bioleaching efficien-
200
cies of heavy metals were obtained in the two-step process.
Cadmium was comparatively more easily removed from 0
soil than Zn, Cu and Pb at low pH. This is related to the one-step two-step control original
chemical forms and the characteristics of heavy metals and bioleaching bioleaching bioleaching
the interaction between the heavy metal and the sediment
surface (Krishnamurti and Naidu 2002). Since the Cd concen- 500
tration was sizeable in the exchangeable/carbonate fraction B4
Cu fraction content (mgkg )
-1

400 B3
(42.2 %), the Fe-Mn oxide fraction (45.9 %) and the organic
B2
fraction (10.6 %), these fractions of cadmium were more eas-
300 B1
ily able to be bioleached than the residues fraction (1.3 %).
The bioleaching efficiencies of Zn was comparatively higher 200
and this is due to the low affinity interaction between Zn and
the soil surface (Burckhard et al. 1995). However, the higher 100
affinity interaction with Cu/Pb and the sediment surface and
0
their stable chemical forms in the sediment led to their lower one-step two-step control original
bioleaching efficiencies (Deng et al. 2012, 2013). In addition, bioleaching bioleaching bioleaching
the selective biosorption of Cu by mycelia of the A. niger was 1000
weaker than for the other metals, and insoluble salts are B4
Zn fraction content(mg kg )
-1

formed when oxalate and Pb are in the same liquid (Silver 800 B3
B2
and Phung 1996). They may also be other factors influencing
600 B1
the lower bioleaching efficiency of Cu and Pb.
400
3.4 Fractions of heavy metal before and after bioleaching
200
The mobility and bioavailability of the metals depend on the
0
chemical fractions and binding forms in sediment (Naresh one-step two-step control original
Kumar and Nagendran 2009). In order to determine the suit- bioleaching bioleaching bioleaching
ability of sediments after bioleaching, it is necessary to exam- Fig. 3 Fraction contents of heavy metals (Cd, Cu, Pb, and Zn) before and
ine the changes in the fractions likely to occur during the after bioleaching (one-step/two-step)
J Soils Sediments (2015) 15:10291038 1035

Table 5 Toxicity characteristic leaching procedure (TCLP) results of raw sediment and bioleached sediment (heavy metal concentration in
extracted fluid; mg l1)

Heavy metals Raw sediment One-step bioleached Two-step bioleached Identification Pollution control
sediment sediment standarda standardb

Cd 1.1950.125 0.0280.002 0.0090.001 1 0.5

Pd 1.8890.318 0.3870.031 0.2390.026 5 5
Cu 2.5220.241 0.9210.056 0.5620.034 50 50
Zn 7.1810.535 3.6520.126 2.4480.141 50 75
a
Identification standard for hazardous wastes-identification for extraction procedure toxicity, National Environmental Agency, China (GB 5085.3-1996)
b
Standard for pollution control on the security landfill site for hazardous wastes, National Environmental Agency, China (GB 18598-2001)

original bioleaching, Cd in the sediment after one-step
bioleaching was mainly present in the organic fraction
(1.74 mg kg 1 ), followed by the residual fraction
(0.38 mg kg 1 ), the Fe/Mn oxide-bound fraction
(0.05 mg kg1), and the exchangeable/carbonate fraction
(0.03 mg kg1), while Cd in the sediment after two-step
bioleaching was mainly present in the residual fraction
(0.40 mg kg 1 ), followed by the organic fraction
(0.07 mg kg 1 ), the Fe/Mn oxide-bound fraction
(0.07 mg kg1), and the exchangeable/carbonate fraction
(0.04 mg kg1). Over 99.6 % of the exchangeable/carbonate
fraction and the Fe/Mn oxide-bound fraction were leached out
in the one-step process. There was also a decrease of the or-
ganic fraction (50.8 %) and the residual fraction (13.2 %). In
the two-step bioleaching process, there were different changes
of heavy metal fractions. During this process, all of the frac-
tions except for the residual fraction (7.1 %) were dramatically
decreased (99.6 %). In the control experiment, only a
low decrease of all the fractions (<6 %) was detected.
As a whole, bioleaching efficiency of Cd in two-step
bioleaching was better than in one-step bioleaching, es-
pecially the organic fraction which was completely solu-
bilized. This is because the pH was lower in two-step
bioleaching than in one-step bioleaching, implying that
Cd was exposed to a more acidic environment. Cadmi-
um, remaining in the sediment, was mainly bonded to
120
100
the residual fraction, which is inert and the complexation
is stronger (Naresh Kumar and Nagendran 2009).
Fractional variation of Pb in the sediment is shown in
Fig. 3b. Before bioleaching, Pb was mainly bonded to the
residual fraction (58.7 %). The organic fraction, the Fe/Mn
oxide-bound fraction and exchangeable/carbonate fraction
were 21.4, 18.4, and 1.9 %, respectively. The exchangeable/
carbonate fraction, Fe/Mn oxide-bound fraction and organic
fraction decreased to 19.5 %, 16.2 % and 11.7 %, however, the
residual fraction increased to 60.6 % after the one-step
bioleaching. During two-step bioleaching, 81.6 % of the Fe/
Mn oxide-bound fraction and 72.7 % of the organic fraction
were solubilized, and both the residual fraction and the
exchangeable/carbonate fraction also decreased. The
bioleaching efficiency of Pb in the two-step process was better
than in the one-step bioleaching process. This result was
caused by the different species and concentrations of organic
acids produced by A. niger during the one-step and two-step
bioleaching processes.
The fraction variation of Cu in the sediment is shown in
Fig. 3c. Before bioleaching, Cu was mainly bonded to the
organic fraction (49.4 %) and the residual fraction (39.6 %).
Similar changes occurred in the one-step and two-step
bioleaching processes. For example, 75.3 % and 79.8 % of
the Fe/Mn oxide-bound fraction were decreased in the one-
step and two-step bioleaching processes, respectively. Al-
though the total content of the exchangeable/carbonate frac-
tion decreased, their ratio in the sediment after bioleaching
100 showed an slight increase, which may be helpful in the next
extraction step. The residual fraction increased to 68.9 % and
80 71.3 % after the one-step and two-step bioleaching processes,
Germination rate (%)

80
Inhibition rate (%)

Weight respectively, indicating that Cu prefers to be associated with a

60
60 Shoot elongation stable fraction.
Root elongation
40
Germination rate 40
Changes in concentration of Zn in sediment are shown in
20
Fig. 3d. Prior to bioleaching, Zn was mainly bonded to the
20 residual fraction (47.6 %), followed by the Fe/Mn oxide-
0
bound fraction (31.6 %), the organic fraction (18.6 %) and the
original bioleaching control one-step two-step
-20 bioleaching bioleaching 0 exchangeable/carbonate fraction (2.3 %). The Fe/Mn oxide-
Wheat bound fraction and the organic fraction all decreased in both
Fig. 4 Toxic effects of sediment on wheat, before and after bioleaching the one-step and two-step bioleaching processes. The Fe/Mn
1036
Fig. 5 Toxic effects of sediment
60
on earthworm, before and after
bioleaching
Inhibition rate (%)
50
40
30
20
10
0
oxide-bound fraction showed an especially large decrease
which can be attributed to the low pH levels at the end of
bioleaching. The ratio of the exchangeable/carbonate fraction
increased a small amount during the one-step bioleaching and
no change was observed during two-step bioleaching. The re-
sidual fraction increased to 77.1 % and 79.9 % after the one-
step and two-step bioleaching processes, respectively. The dis-
solution of the Fe/Mn oxide-bound fraction and the increase of
the residual fraction imply that the Zn in the sediment after
bioleaching was stable and had a low bioavailability.
3.5 Toxic characteristic of sediment before and
after bioleaching
The TCLP tests of the sediments before and after bioleaching
are presented in Table 5. The Cd concentration was
1.195 mg l1 in the TCLP extract of the raw sediment. This
exceeded both the pollution control standard (0.5 mg l1) and
the identification standard (1 mg l1) that is regulated by the
National Environmental Agency of China. It is therefore nec-
essary to treat the sediment in order to reduce the hazardous
metal contents before land disposal. After one-step and two-
step bioleaching, the Cd concentrations were reduced to as
low as 0.028 and 0.009 mg l1, respectively. Meanwhile, the
results also showed that during the TCLP tests, Pb, Cu, and Zn
concentrations in the extracted liquor were reduced to levels
far below those required in the two standards. It is evident that
A. niger was able to successfully decrease the mobility of
heavy metals and detoxify the contaminated sediment. The
sediments after bioleaching can be landfilled safely.
In order to evaluate the toxicity of sediment on land plants,
inhibition of weight, shoot elongation, root elongation, and
germination rates for wheat (Triticum aestivum) were deter-
mined (Fig. 4). It was noted that the weight, shoot elongation,
and root elongation of wheat planted on original sediment
were restrained. The inhibition rates were determined to be
J Soils Sediments (2015) 15:10291038
one-step bioleaching
two-step bioleaching
c control
c
original bioleaching
c
c
a a
b
b
Weight Deat h
Earthworm
44.7, 33.8, and 98.8 %, respectively. In addition, only
70.0 % of the seeds germinated. Inhibition rates of weight,
shoot elongation, and root elongation all decreased in both
the one-step and two-step bioleaching processes, especially
the inhibition rate of the root elongation which showed a large
decrease as low as 3.75 % in the one-step bioleaching sedi-
ment. Meanwhile, the root elongation of wheat increased
3.6 % in the sediment that underwent one-step bioleaching;
this means that there was not only no toxicity to wheat, but in
fact advanced growth. Germination rates also improved rising
to 85.0 and 90.0 % in the one-step and two-step bioleaching
processes, respectively, equating to a 15 and 20 % improve-
ment, respectively, when compared to original bioleaching.
The sensitivity order of the measured parameters of the wheat
was as follows: root elongation > shoot elongation > weight >
germination rate. The inhibition effects of the sediments to the
wheat were, in decreasing order: original bioleaching (raw
sediment) > control > one-step bioleaching > two-step
bioleaching. The influence of sediments on phytotoxicity var-
ied and depended on the type of sediment, and plant endpoints
evaluated. Similarly to earlier studies (e.g., Oleszczuk and
Hollert 2011), root elongation proved to be the least sensitive
parameter. Most published studies indicate that the evaluation
of seed germination is a more sensitive parameter that root
elongation, shoot elongation, and weight. The inhibition of
root growth was a very sensitive parameter for all tested sed-
iments. The toxic effects of the contained sediments on wheat
varied depending on kind, content, and fraction of heavy
metals. As discussed above, the total content of easily
bioleached heavy metals and the inhibition of wheat had a
clear relationship. This means that bioleaching using A. niger
can not only decrease the total content of heavy metals, but
also decrease the toxicity of sediment to land plants.
The importance of earthworms for heavy metal
biomagnification in terrestrial ecosystems is widely recog-
nized (De Silva et al. 2009). In this study, the inhibition rate
J Soils Sediments (2015) 15:10291038
and death rate of earthworms were detected to evaluate the
toxicity of sediment on land animals. The toxic effects of the
sediments on wheat varied depending on the kind, content,
and fraction of heavy metals (Fig. 5). Before bioleaching,
the weight of earthworms decreased by 46.4 %, compared
with the clean soil, and 33.3 % of the earthworms died
throughout the 28 day exposure period; whereas in the clean
soil control, no mortality was recorded. The inhibition rate of
weight decreased after both one-step and two-step
bioleaching. The earthworms (16.7 %) died in a one-step
bioleaching sediment, and all of the earthworms were alive
at the end of exposure in two-step bioleaching sediment. The
results from this study confirm that the bioleaching using
A. niger is an efficient method to decrease the toxicity of
sediment to land animals.
4 Conclusions
A. niger strain SY1 can effectively remove heavy metals in
contaminated sediment at 10 % (w/v) addition rate of sediment
to culture medium. The bioleaching efficiencies of heavy
metals in two-step bioleaching are better than that in one-
step bioleaching. After the bioleaching, metals remaining in
the sediment were mainly found in stable fractions. The toxic
tests showed that the bioavailability and toxicity of heavy
metals decreased after bioleaching and the sediments can be
safely landfilled or used in land applications.
Acknowledgments This work was supported by the National Natural
Science Foundation of China (21277150,31270540, 31070455,
31370829, 51174239, and 40971184), Ministry of Science and Technol-
ogy (2011DFA91810), Ministry of Environmental Protection
(2012ZX07202-004), the National Science & Technology Pillar Program
(2012BAC17B04), Hi-tech research and development program of China
(2012AA06A202), Natural Science Foundation of Liaoning Province,
China (201102224), Natural Science Foundation of Shenyang City, Chi-
na (F13-067-2-00), the Geping green action-environmental research and
education B123 project^ of Liaoning Province, China (CEPF2011-123-1-
1), and the State Scholarship Fund organized by China Scholarship Coun-
cil (CSC2013). The authors would also like to thank Paula McNamee and
Dr. Xiaoman Yu for their constant support.
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