J Soils Sediments (2012) 12:900908

DOI 10.1007/s11368-012-0520-2

SOILS, SEC 3 I REMEDIATION AND MANAGEMENT OF CONTAMINATED OR DEGRADED LANDS I RESEARCH ARTICLE

Bioleaching of heavy metals from sewage sludge

by Acidithiobacillus thiooxidansa comparative study
Ye-Ming Wen & Qing-Ping Wang & Caixian Tang &
Zu-Liang Chen

Received: 19 December 2011 / Accepted: 1 April 2012 / Published online: 27 April 2012
# Springer-Verlag 2012

Abstract
Purpose To understand the bioleaching of metals from
sludge by Acidithiobacillus thiooxidans, the aims of this
study were to evaluate the experimental conditions affecting
the efficiency of removal of the metals, including solids
concentration, initial pH, sulfur concentration and inoculum
level were examined, and following the bioleaching mechanism was proposed.
Materials and methods A. thiooxidans were isolated from
collected sludge samples containing bacteria from Fuzhou
Jingshan sewage treatment plant, and identification of bacteria
by sequencing the 16 s rDNA gene sequences. Conditions
affecting the bioleaching and application were conducted by
batch experiments. The analysis of Cr, Cu, Pb, and Zn was
carried out using an atomic absorption spectrophotometer, and
the pH and oxidationreduction potential (ORP) were measured using a pH meter and an ORP meter.
Results and discussion The results show that a high metal
leaching efficiency was achieved at low solid concentrations
due to decreases in buffering capacity. In addition, the best
conditions of the bioleaching included 2 % (w/v) solid concentration, 5.0 gL1 sulfur concentration, and 10 % (v/v)
inoculum concentration, where the removal efficiencies of
Responsible editor: Bernd Markert
Y.-M. Wen : Q.-P. Wang : Z.-L. Chen (*)
School of Environmental Science and Engineering,
Fujian Normal University,
Fuzhou 350007 Fujian Province, Peoples Republic of China
e-mail: zlchen@fjnu.edu.cn
C. Tang
Department of Agricultural Sciences, La Trobe University,
Bundoora,
Melbourne, VIC 3086, Australia

Cr, Cu, Pb, and Zn in sewage sludge was 43.6 %, 96.2 %,

41.6 %, and 96.5 %, respectively.
Conclusions We found that the bioleaching of Zn was governed by direct and indirect mechanisms, while the bioleaching of Cu, Pb, and Cr was mainly dominated by the
bioleaching indirect mechanism. After processing with the
proposed techniques, the heavy metals in the sewage sludge
did meet the requirement of the national standards.
Keywords Acidithiobacillus thiooxidans . Bioleaching .
Heavy metal . Sewage sludge

1 Introduction
In recent years, the quantity of the total sludge generated all
over the world has increased dramatically, the disposal of
which is one of the serious environmental concern (Babel
and Dacera 2006; Pathak et al. 2009; Lombardi et al. 2001).
Since the origin of the sewage and its treatment from the
sewage treatment plant varies, and sewage sludge contains
high concentrations of toxic metals (Lombardi and Garcia
2002), the disposal of untreated sludge to land shows a
potential hazard to human health and to the environment
(Tsai et al. 2003). For these reasons, it is necessary to remove
the heavy metals from the sludge before being used on land.
Various chemicals such as chelating agents such as ethylene
di-amine tetraacetic acid and nitrilotriacetic acid (Lo and Chen
1990) have been reported for extraction of metals from sludge.
However, the practical application of the chemical processes
is still limited due to the requirement of large amount of
chemicals, the high operating cost, the operational difficulties,
and the secondary pollution problems associated with them
(Pathak et al. 2009; Kumar and Nagendran 2008). As such,

J Soils Sediments (2012) 12:900908

the microbial method is one of the options that can be used to

remove heavy metals in the sludge due to its low cost and low
energy requirement (Hsu and Harrison 1995).
Bioleaching, which is based on the ability of microorganisms to transform metals from the solid waste into soluble
and extractable forms, has been reported to be an efficient
and economical method for removal of heavy metals from
the sludge (Babel and Dacera 2006; Pathak et al. 2009). In
bioleaching process, Acidithiobacillus thiooxidans can be
used in leaching of metals from sewage sludge, where A.
thiooxidans oxides sulfur to sulfuric acid to obtain energy
from the oxidation process, leading to cause sludge acidification and to make heavy metal solubilization (Babel and
Dacera 2006; Pathak et al. 2009). The oxidation and acid
production by A. thiooxidans are the primary impetuses of
solubilization of heavy metals (Zhang et al. 2009). A number of studies on bioleaching of heavy metals using A.
thiooxidans have recently been reported, where the reports
concerned only one condition impacting on the bioleaching
such as the pH, solids concentration, energy source, and
leaching microorganisms (Babel and Dacera 2006; Pathak
et al. 2009). However, this is limited to understanding the
mechanisms of bioleaching because of its lacking evidence.
For example, the effect of sulfur on bioleaching of heavy
metals from mine tailings bacteria has been recently
reported, the results have indicated that 2 % sulfur substrate
concentration is optimal for bacterial activity and metal
solubilization (Liu et al. 2008). The influence of initial pH
of the system on bioleaching of heavy metals from metalcontaminated soils has also been reported, which highlights
that pH plays an important role in metal solubilization and
that microorganisms can utilize elemental sulfur as an energy source (Kumar and Nagendran 2007). The effects of
sludge solids concentration on the bioleaching process
in a batch bioreactor have been reported, where a decrease in pH, an increase in oxidationreduction potential (ORP), and the reduction of metal were associated
with increases in the concentration and the buffering
capacity of sludge solids (Chen and Lin 2004a, b).
However, the different forms of heavy metal in the
sludge vary according to the type of sludge, the characteristics of the metal, and the method employed for
the treatment of the sewage. To understand the bioleaching process, it is therefore necessary to systemically
investigate various conditions affecting the bioleaching
of heavy metals from sludge.
For these reasons, this study systematically investigated the bioleaching of Cr, Cu, Pb, and Zn from
sewage sludge using A. thiooxidans. The experimental
conditions affecting the efficiency of removal of the
metals, including solids concentration, initial pH, sulfur
concentration, and inoculum level were examined, and
following, the bioleaching mechanism was proposed.

901

The feasibility of the removal of metal ions such as

Cu, Cr, Pb, and Zn in sewage sludge using A. thiooxidans was demonstrated under the optimal conditions.

2 Materials and methods

2.1 Sewage sludge source and its characterization
The sludge sample containing bacteria was collected from
Fuzhou Jingshan sewage treatment plant. The sludge sample
was transported back to the laboratory and stored at 4C
prior to use. The sample was divided into two parts. One
part was used for isolating A. thiooxidans, another part was
used for bioleaching experiments. The subsample was sterilized by autoclaving (30 min at 120C) and was characterized. The measured characteristics of the sludge sample
were listed in Table 1.
2.2 Isolation and identification of indigenous
sulfur-oxidizing bacteria
For enrichment of sulfur-oxidizing bacteria, the Waksman
liquid medium of the following composition was used: 0.2 g
L1 (NH4)2SO4, 3.0 gL1 K2HPO4, 0.5 gL1 MgSO47H2O,
0.25 gL1 CaCl22H2O, 10 gL1 S0 as an energy source.
The initial pH of the Waksman liquid medium was 4.0. Test
sludge (10 mL with solids concentration of 4 % (w/v)) was
taken in a 250-mL flask containing 100 mL of the Waksman
liquid medium. The flasks were mounted on a shaking
incubator (Shanghai Sukun CO., Ltd. SKY-2102) and shaken
at 150 rpm at 30C; the pH of the medium was monitored.
When the pH reached 2.0, 10 mL of the acidified sludge was
transferred to the second flask containing 100 mL of the
Waksman liquid medium under the same conditions. After
three generations of enrichment, the isolate was inoculated on
the Waksman solid medium, and the pure culture for further
use was obtained after three successive subcultures (Kumar
and Nagendran 2007). Molecular identification of the isolated
culture was done by sequencing the 16 s rDNA gene
sequences.

Table 1 The selected characteristics of sewage sludge

Characteristics

Solids (%)
Organic matter (%)
Total P (g kg1 dry sludge)
Total N (g kg1 dry sludge)
pH

Heavy metals
(mg kg1 dry sludge)
17.6
40.6
95.9
10.9
7.66

Zn
Cu
Cr
Pb

1,732
2,430
285
204

902

2.3 Experimental conditions affecting the bioleaching

and application
Laboratory scale bioleaching experiments were carried out
in 250-mL Erlenmeyer flasks containing 150 mL of the
sludge samples, which was agitated at 30C and 150 rpm
on a gyratory shaker for 24 h to raise its temperature and
initial ORP, which provided a suitable condition for the
growth of A. thiooxidans. The factors affecting the metal
leachability using A. thiooxidans includes five solids concentrations (2 %, 4 %, 6 %, 8 %, and 10 % (w/v)), five initial
pH (2, 3, 4, 5, and 6) which were achieved by adjustment
using 2 N H2SO4, five levels of sulfur (2, 5, 10, 15, and 20 g
L1) and five inoculum concentrations of 108~109 cells/mL
(2 %, 5 %, 10 %, 15 %, and 20 %). All experiments were
performed in replicate and were incubated in a gyratory
incubator at 30C and 150 rpm. During the bioleaching
process, the water loss due to evaporation was replenished
with distilled water daily.
To test the removal efficiency of metals under the optimal
conditions, a sludge slurry (solids concentration, 2 % (w/v)) of
135 mL contained in a 250-mL flask was agitated at 30C,
150 rpm for 24 h. A 10 % volume of active bacterium
suspension was added to the sludge slurry with an initial cell
concentration of 108~109 cells per milliliter and followed by
the addition of 0.75 g elemental sulfur. A control run without
inoculum of bacteria was also carried out in order to compare
the results. The whole set-up was weighed and incubated as
described above.
2.4 Analytical methods
The pH and ORP were measured using a pH meter
(Shanghai Precision and Scientific Instrument Co., Ltd.,
PHS-3 C) and an ORP meter (Shanghai Kang-Yi
Instruments Co., Ltd., ORP-421), respectively. Samples
(15 mL) were collected at 2-day intervals from the flasks
for analyses of heavy metals. The samples were centrifuged
at 7,000 rpm for 10 min, and the supernatant was filtered
through a 0.45-m membrane filter. The analysis of Cr, Cu,
Pb, and Zn was carried out using an atomic absorption
spectrophotometer (AA240, Varian, USA).

3 Results and discussion

3.1 Identification of A. thiooxidans
A. thiooxidans isolated from the sewage sludge grew well on
a medium containing elemental sulfur as the energy source.
As shown in Fig. 1, microscopic examination of the culture
revealed the presence of gram negative, rod shaped, and
motile bacteria. The data showed that the isolated

J Soils Sediments (2012) 12:900908

microorganism was closely related to acidophilic sulfuroxidizing bacteria A. thiooxidans with 99 % similarity by
the classification, which was based on homology of its 16 S
rDNA gene sequences from the National Center for
Biotechnology Information databank using BLASTN (Lin
et al. 2010).

3.2 Changes in pH and ORP during bioleaching

The changes in pH and ORP during the bioleaching process
are shown in Fig. 2, when sludge concentration was 2 %,
sulfur concentration was 5 gL1, and inoculum concentration was 10 %. The activated A. Thiooxidans in the sludge
was able to alter oxided sulfur to sulfuric acid during their
growth. Consequently, the pH of the sludge medium decreased with increasing bioleaching time as shown in
Fig. 2a. A rapid drop in pH was observed in the first 6 days,
where the pH was reduced from 5.30 to 1.07 due to the
microbial oxidation of sulfur and the production of sulfuric
acid based on their bioleaching mechanisms (Pathak et al.
2009; Suzuki 2001). This also indicates that the sulfur can
be used as the energy source for the growth of A. thiooxidans. However, in the control medium, the pH decreased
gradually to 4.62 in the first 6 days, then increased to 5.42 in
the following 2 days, the final pH was stabilized at about
4.98. A slight increase in pH observed in the control indicates that chemical oxidation of sulfur did not occur, and its
alkaline range observed in the control can be attributed to
buffering action and release of carbonate substance from the
sewage sludge with basic nature (Kumar and Nagendran
2008). After 12 days of bioleaching, the pH of the control
was higher than those of the test treatments. Similar results
were reported on the study of the pH profile during bioleaching of metals resulted from anaerobical digestion by
sulfur-oxidizing bacteria (Villar and Garcia 2006).
In contrast with the trend of change in pH, as shown in
Fig. 2b, ORP increased rapidly from 300 to 445 mV in the
first 4 days of bioleaching, increased slightly to 505 mV in
the following 10 days and was then maintained until the end
of bioleaching. This is because the elemental sulfur was
oxidized to soluble sulfate, leading to decreased concentration of sulfur followed by increases in ORP (Kumar and
Nagendran 2008). However, the ORP of the control was
kept around 295 mV during the same bioleaching period.
These results show that the inoculation of A. thiooxidans
accelerated the sludge acidification resulted from oxidizing
sulfur to sulfuric acid and an increase in the ORP due to a
stimulated microbial activity. The high values of ORP together with the low pH are responsible for the solubilization
of metals (Lombardi and Garcia 2002). These results provide the evidence for the indirect bioleaching mechanism by
A. thiooxidans, where the sulfur in the sludge is oxidized

J Soils Sediments (2012) 12:900908

903

Fig. 1 The colony morphology

and individuals of A.
thiooxidans

depends on physical, chemical, and biological factors in the

system. To demonstrate the feasibility of using A. thiooxidans
with a high bioleaching efficiency of metals from the sludge,
unlike other reports (Chen and Lin 2004a, b; Kumar and
Nagendran 2007), it is necessary to systematically understand
the experimental conditions that affect the bioleaching process. Major factors are investigated in the following sections.

into sulfuric acid and leads to acidification of the sludge

medium (Jain and Tyagi 1993; Du et al. 1995).

3.3 Conditions affecting the bioleaching

In bioleaching of metals from sludge using A. thiooxidans,
both direct and indirect mechanisms have been proposed
(Pathak et al. 2009). In direct bioleaching, metal sulfides are
directly oxidized by the A. thiooxidans into soluble metal
sulfates according to the following reaction:
A. thiooxidans
MS 2O2 ! MSO4

3.3.1 Effort of sludge solids concentration on bioleaching

Figure 3 presents how sludge solids concentration affects
the solubilization of Zn, Cu, Pb, and Cr from the sludge
during the bioleaching process, with 10 gL1 sulfur concentration and 10 % inoculum concentration. Generally, the
bioleaching efficiency of Zn, Cu, Pb, and Cr decreased with
increasing solid sludge concentration. Longer bioleaching
times were required to produce a sufficient acidification of
the leaching solution at high sludge solid concentrations.
However, the removal efficiency of Pb was substantially
lower at high solids concentrations even with longer leaching times compared to other treatments. This is due to an
increased precipitation of PbSO4 at a high solids concentration (Ryu et al. 2003). For each metal, the highest removal
efficiency achieved was at 2 % solids. For example, 81.9 %
Zn, 75.3 % Cu, 47.5 % Pb, and 64.7 % Cr in the sludge were
removed in the first 6 days due to a faster reduction of pH at

In indirect bacterial leaching, the elemental sulfur in the

sludge is oxidized by A. thiooxidans into sulfuric acid,
which reduces the pH of the sludge medium, thereby enhancing the solubilization of the metal:
A. thiooxidans
S0 H2 O 1:5O2 ! H2 SO4

H2 SO4 materials  M ! materials  2H MSO4

where M is a bivalent metal. On the viewpoint of direct and

indirect mechanisms, the bioleaching efficiency clearly
6

500

5
450

ORP (mv)

pH

Fig. 2 Dynamics of pH and

oxidationreduction potential
(sludge concentration, 2 %;
sulfur concentration, 5 gL1;
inoculum concentration, 10 %;
temperature, 30C; agitation
speed, 150 rpm)

pH A.t
pH control

400

ORP A.t
ORP control

350

2
300

1
0

Time (d)

10

12

250

Time (d)

10

12

904
100

100

80

80

Removal of Cu (%)

Removal of Zn (%)

Fig. 3 Solubilization of a Zn, b

Cu, c Pb, and d Cr during the
bioleaching process as affected
by sludge solids concentrations
(sulfur concentration, 10 gL1;
initial pH, 7.66; inoculum
concentration, 10 %;
temperature, 30C; agitation
speed, 150 rpm)

J Soils Sediments (2012) 12:900908

60

40

2%
4%
6%
8%
10%

20

10

60

40
2%
4%
6%
8%
10%

20

12

Time (d)

10

12

Time (d)

60

c
2%
4%
6%
8%
10%

30

Removal of Cr (%)

Removal of Pb (%)

60

40

20
10
0

70

50

2%
4%
6%
8%
10%

50
40
30
20
10

Time (d)

this solid concentration (Ryu et al. 2003). The removal

efficiency for Zn, Cu, Cr, and Pb was increased to 93.1 %,
90.1 %, 64.3 %, and 49.2 %, respectively, by day 12 using
2 % solids concentration.
In comparison, 91.2 % Zn, 53.2 % Cu, 2.2 % Pb, and
18.5 % Cr were removed at solids concentration of 10 %.
This is because high solids concentrations result in high
buffering capacity, leading to an increased time to attain a
low pH (Xiang et al. 2000; Liu et al. 2007). In addition, the
increase in concentration of the leached metals also results
in toxicity that may inhibit the growth of the microorganisms (Cho et al. 2002). However, the removal of metals
increased significantly with time and then plateaued at the
values lower than 2 % solids concentration. Another reason
is that microorganisms are able to survive at higher solid
contents of the sludge (Henry et al. 1991), leading to complete cessation of metal bioleaching at solids concentration
of 10 % (Cho et al. 2002), and resulted in reduce of bioleaching solubilization. Nevertheless, this current study
shows that A. thiooxidans can be used effectively for bioleaching of heavy metals with the efficiency in the order of
Zn>Cu>Cr>Pb.
3.3.2 Effort of initial pH on bioleaching
Figure 4 shows the removal efficiency of the heavy metals at
various initial pH values at 2 % sludge concentration, 10 g

10

12

10

12

Time (d)

L1 a sulfur concentration and 10 % inoculum concentration. As shown in Fig. 4a, b, the higher removal efficiency
of Zn and Cu was observed at different initial pH, where
variations in the initial pH of the system did not influence
the bioleaching of Zn and Cu. In the Zn approach, the
removal efficiency varied only from 86.2 % to 88.1 % after
bioleaching for 12 days, although the highest removal efficiency was recorded at an initial pH of 6, while the lowest
value found at pH 2, but there was slightly difference.
However, the high range of solubilization of Zn as shown
in Fig. 4a is in agreement with the study on bioleaching of
heavy metals from contaminated soil using A. thiooxidans
(Kumar and Nagendran 2007). Similarly, the removal efficiency of Cu ranged from 83.4 % to 86.7 % after bioleaching for 12 days, indicating the initial pH values did not
significantly impact on the bioleaching of metals. Low
solubilization of Cu at initial pH 4 and 5 was reported while
carrying out bioleaching of heavy metals from aquatic sediments employing Acidithiobacillus ferroxidans (Chartier
and Couillard 1997).
In contrast, the solubilization pattern of Pb and Cr was
highly dependent on initial pH values (Fig. 4c, d). The
removal efficiency of Pb varied from 36.1 % at an initial
pH of 2 to 40.3 % at pH of 5 after bioleaching for 12 days.
The overall low Pb removal with solution pH was mainly
due to the precipitation of PbSO4 with a very low solubility
(Ksp 01.62108) (Lo and Chen 1990). It is this reason that

J Soils Sediments (2012) 12:900908

100

100

a
60

Removal of Cu (%)

80

Removal of Zn (%)

Fig. 4 Effect of initial pH on

solubilization of a Zn, b Cu, c
Pb, and d Cr during the
bioleaching process (sludge
concentration, 2 %; sulfur
concentration, 10 gL1;
inoculum concentration, 10 %;
temperature, 30C; agitation
speed, 150 rpm)

905

2
3
4
5
6

40

60
2
3
4
5
6

40
20

20
0

80

10

12

Time (d)

10

12

Time (d)
70

40
30
2
3
4
5
6

20
10
0

60

Removal of Cr (%)

Removal of Pb (%)

50

50
40
2
3
4
5
6

30
20
10

Time (d)

the efficiency of Pb solubilization was generally lower than

other metals in the bioleaching process. The similarly low
solubilization of Pb during bioleaching of heavy metals
from sludge employing sulfur-oxidizing bacteria was also
observed (Couillard and Mercier 1993). However, the removal efficiency of Cr ranged from 59.4 % at pH 2 to
64.5 % at pH 4 after 12 days of bioleaching. Since Cr
solubilization is pH dependent, extremely acidic conditions
tend to accelerate the solubilization of Cr (Fang and Zhou
2007). This finding is also supported by the study on the
microbial leaching processes using A. thiooxidans at neutral
pH without any initial pH adjustment (Ryu et al. 2003). In
addition, the results suggest that bioleaching of metals is
easily affected by pH. A pH of 11.5 was recommended for
a sufficient removal of all the types of heavy metals at
various sludge solids concentrations.
3.3.3 Effort of sulfur concentration on bioleaching
As shown in Eq. 3, the elemental sulfur is oxidized to form
sulfuric acid, resulting in acidification of the sludge medium. Consequently, the metals in sludge become soluble
during bioleaching. However, the batch study shows a major
portion of the sulfur used in bioleaching was not completely
oxidized (Chen and Lin 2004a, b). Therefore, the determination of sulfur concentration is of great significance in
developing an efficient bioleaching process. Figure 5 shows
the effect of sulfur concentration on the removal efficiency

10

12

10

12

Time (d)

of heavy metals when the sludge concentration of 2 %, and

the inoculum concentration of 10 % were used. Increasing
sulfur concentration from 0.5 and 15 gL1 significantly
increased the removal efficiency of metals after bioleaching
for 2 days. This may be due to the high pH buffer capacity
or the release of alkaline from sewage sludge during the
initial bioleaching (Liu et al. 2008). The increase in the
bioleaching efficiency of metal after 2 days of bioleaching
is due to a fast acidification rate of sewage sludge, leading to
enhanced solubilization of metals (Chen and Lin 2004a, b).
It can be seen from Fig. 5 that the removal efficiency of
metals from sewage sludge was in the decreasing order of
Zn>Cu>Cr>Pb. The higher removal efficiency of Zn and
Cu was achieved in the range of 90.199.8 % and 84.5
99.5 % after bioleaching for 12 days, respectively, while Cr
in the range of 18.466.8 % and Pb in the range of 9.8
43.6 %. Cr and Pb data are identical where obtained at sulfur
concentration of 0.5 to 15 gL1. The removal efficiency of
Cu and Zn is often high from sludge (Chen and Lin 2004a, b).
This is because Cu is mainly associated with sulfides/organic
matter which can be directly oxidized by A. thiobacilli, while
the bioleaching of Zn is found in more available and mobile
fractions (i.e., exchangeable, carbonate, and Fe/Mn oxide
fractions) (Chen and Lin 2000). The bioleaching efficiency
of Pb is generally lower than other metals because of the
formation of insoluble PbSO4 as discussed above (Tyagi
1992; Chen and Lin 2001). In addition, the low efficiency of
Cr can be attributed to its low solubilization at extremely

906
100

100

80

Removal of Cu (%)

Removal of Zn (%)

Fig. 5 Solubilization of a Zn, b

Cu, c Pb, and d Cr during the
bioleaching process as affected
by sulphur concentrations
(sludge concentration, 2 %;
initial pH, 7.66; inoculum
concentration, 10 %;
temperature, 30C; agitation
speed, 150 rpm)

J Soils Sediments (2012) 12:900908

60
0.5 g/L
2 g/L
5 g/L
10 g/L
15 g/L

40
20
0

10

80
60
0.5 g/L
2 g/L
5 g/L
10 g/L
15 g/L

40
20
0

12

Time (d)
50

70

10

12

10

12

60

40

0.5 g/L
2 g/L
5 g/L
10 g/L
15 g/L

30

Removal of Cr (%)

Removal of Pb (%)

Time (d)

20

0.5 g/L
2 g/L
5 g/L
10 g/L
15 g/L

50
40
30
20

10
10
0

Time (d)

acidic condition (Chen and Lin 2000). A higher concentration

of sulfur enables higher solubilization of metals via short-term
bioleaching but is not recommended because un-oxidized
sulfur remains in the sludge, and the disposal of sulfurcontaminated sludge on land may promote the acidification
of soil. At the sulfur concentration of 5 gL1, 99.8 % Zn,
99.5 % Cu, 37.8 % Pb, and 66.8 % Cr were removed within
12 days of bioleaching. Hence, 5 gL1 sulfur was recommended, and it was concluded that that sulfur was completely
oxidized to sulfuric acid.
3.3.4 Effort of initial inoculum concentration
on bioleaching
As described in Eqs. 2 and 3, A. thiooxidans can directly
oxidize metal sulfides into soluble metal sulfate, and also
oxidize the elemental sulfur to form sulfuric acid. Figure 6
shows the removal efficiency of heavy metals at various
initial inoculum concentrations with the sludge concentration of 2 % and the sulfur concentration of 5 gL1. It can be
seen that bioleaching of Zn and Cu from sewage sludge was
not strongly influenced by inoculum concentration with an
exception of 2 % inoculum concentration. The removal
efficiency of Zn and Cu ranged from 92.1 % to 96.5 % after
12 days of bioleaching when the inoculum concentration
was in the range of 220 % (v/v). Similar results were also
observed on the bioleaching of Zn and Cu from municipal
waste incineration fly ash by using sulfur-oxidizing bacteria,

10

12

Time (d)

where the inoculum addition had been greater than 2.5 %

(Ishigaki et al. 2005). Furthermore, Zn and Cu are easily
soluble in sludge during the bioleaching even at a low initial
inoculum concentration, where a high rate of acidification,
and a low final pH of the sewage sludge indicate effective
microbial sulfur oxidation and hence ability to leach heavy
metals (Ryu et al. 2003). In contrast, the removal efficiency
of Pb and Cr increased with increasing the initial inoculum
concentration. The removal efficiency of Pb and Cr were
about 6.626.7 % and 4.244.5 % after 8 days of bioleaching, indicating that the reduction of pH was fast with an
increased concentration of inoculum. This was because a
higher concentration of inoculum accelerated sludge acidification, leading to increases in bioleaching of Pb and Cr
(Pathak et al. 2009).
3.4 Removal of heavy metals from the sewage sludge
Bioleaching of Zn, Cu, Cr, and Pb from sludge is depicted in
Fig. 7 under the optimized conditions, including 2 % solid
concentration, 5.0 g/L sulfur concentration, and 10 % inoculum concentration. The removal efficiency of Zn, Cu, Pb,
and Cr was 95.5 %, 95.0 %, 41.0 %, and 43.4 % after 12 days
of bioleaching, respectively. Although the removal efficiency
of Zn was greater in the bioleaching sample (40.1 %) than in
the control (7.7 %), the removal efficiency in the control
sample was reasonably high (83.8) after 12 days (see
Fig. 7a). This is because Zn binding to sludge (i.e.,

J Soils Sediments (2012) 12:900908

100

100

a
Removal of Cu (%)

80

Removal of Zn (%)

Fig. 6 Effect of inoculum level

on solubilization of a Zn, b Cu,
c Pb, and d Cr during the
bioleaching process (sludge
concentration, 2 %; initial pH,
7.66; sulfur concentration, 5 g
L1; temperature, 30C;
agitation speed, 150 rpm)

907

60
2%
5%
10%
15%
20%

40
20
0

10

12

80
60
2%
5%
10%
15%
20%

40
20
0

14

Time (d)
35

d
Removal of Cr (%)

Removal of Pb (%)

25
2%
5%
10%
15%
20%

20
15
10

40
30
2%
5%
10%
15%
20%

20
10

10

12

Time (d)

exchangeable, carbonate, and Fe/Mn oxides fractions) was not

strong and hence easily exchanged (Chen and Lin 2004a, b). It
is concluded that Zn bioleaching by A. thiooxidans can be
governed by both direct and indirect mechanisms.

12

100

80

80

Removal of Cu (%)

Removal of Zn (%)

10

In the case of Cu, Pb, and Cr, it can be seen that removal
efficiency was remarkably different between the bioleached
sample and the control sample after 2 days of bioleaching,
indicating that the inoculation was an essential requirement

a
A.t
control

60
40
20
0

Time (d)

100

A.t
control

60
40
20

10

12

Time (d)

10

10

12

Time (d)

50

50

c
40

40

A.t
control

Removal of Cr (%)

Removal of Pb (%)

12

50

Fig. 7 The removal efficiency

of a Zn, b Cu, c Pb, and d Cr in
sewage sludge by bioleaching
(sludge concentration, 2 %;
initial pH, 7.66; sulfur
concentration, 5 gL1;
inoculum concentration, 10 %;
temperature, 30C; agitation
speed, 150 rpm)

10

60

30

Time (d)

30
20

A.t
control

30
20
10

10

0
0

Time (d)

10

12

Time (d)

12

908

for better acclimatization and growth of A. thiooxidans

(Kumar and Nagendran 2007). For example, 95.0 % Cu,
43.3 % Pb, and 41.0 % Cr in bioleached samples were removed after bioleaching for 12 days, while only 17.6 % Cu,
9.7 % Pb, and 10.4 % Cr were removed in control sludge
medium. This suggests that A. thiooxidans plays an important
role in the bioleaching of Cu, Pb, and Cr because these metals
strongly bind with the organic matter, carbonate, and Fe/Mn
oxide in sludge (Kumar and Nagendran 2007). These results
provide the evidence that the bioleaching of Cu, Pb, and Zn is
mainly dominated by bioleaching indirect mechanism.

4 Conclusions
The present study demonstrated that the isolated indigenous
sulfur-oxidizing bacteria A. thiooxidans can be used in the
bioleaching of heavy metals from sewage sludge. Our systematic studies show that solid concentration, pH, sulfur concentration, and inoculum concentration effectively influenced the
bioleaching of metals. A. thiooxidans can utilize elemental
sulfur as an energy source, resulting in low pH and high
ORP to facilitate the heavy metals leaching from sludge.
Our results indicate that the bioleaching of Zn was governed
by both direct and indirect mechanisms, while the bioleaching
of Cu, Pb, and Cr was mainly dominated by the bioleaching
mechanism. The bioleaching efficiency of Cr, Cu, Pb, and Zn
from the sewage sludge was 43.6 %, 96.2 %, 41.6 %, and
96.5 %, respectively, under the optimized conditions. This
study also demonstrates that using A. thiooxidans is a potential
technique to bioleach heavy metals from sewage sludge.
Acknowledgments The authors sincerely thank Fujian Normal University for financial support through the Min-Jiang Fellowship.

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