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Bioresource Technology 99 (2008) 41244129

Bioleaching of heavy metals from mine tailings by indigenous

sulfur-oxidizing bacteria: Eects of substrate concentration
Yun-Guo Liu *, Ming Zhou, Guang-Ming Zeng, Xin Wang,
Xin Li, Ting Fan, Wei-Hua Xu
College of Environmental Science and Engineering, Hunan University, Changsha 410082, PR China

Received 9 July 2007; received in revised form 29 August 2007; accepted 29 August 2007
Available online 24 October 2007

Abstract

The aim of this study was to determine the eect of substrate concentration (elemental sulfur) on remobilization of heavy metals from
mine tailings by indigenous sulfur-oxidizing bacteria. Also, the variation in the binding forms of heavy metals before and after bioleach-
ing was explored. This work showed the laboratory results of bioleaching experiments on PbZnCu mine tailings. The results showed
that 97.54% Zn, 97.12% Cu, and 44.34% Pb could be removed from mine tailings by the bioleaching experiment after 13 days at 2% w/v
substrate concentration. The results also indicated that substrate concentration 2% was found to be best to bacterial activity and metal
solubilization of the ve substrate concentration tested (0.5%, 1%, 2%, 3%, and 5%) under the chosen experimental conditions. The biole-
aching had a signicant impact on changes in partitioning of heavy metals.
 2007 Elsevier Ltd. All rights reserved.

Keywords: Bioleaching; Mine tailings; Heavy metal; Substrate concentration; Partitioning

1. Introduction Bioremediation of heavy metals has gained increased

attention since it is innovative, environmentally friendly
The mining and metallurgical activities that were partic- and economical (Tichy et al., 1998). Bioleaching processes
ularly intensive during the last century resulted in the gen- are based on the ability of microorganisms to transform
eration of huge amounts of mine tailings, including acid- solid compounds and result in soluble and extractable ele-
generating suldic tailings. Most of the tailings have been ments which can be recovered. Metal solubilization from
left without any management at mines of China. Their solid wastes or other solids is achieved through a variety
improper management in the past resulted in the mobiliza- of acidophilic and chemoautolithotrophic bacteria such
tion of heavy metals to the surrounding environment, con- as Acidithiobacillus thiooxidans and Acidithiobacillus ferro-
tributing to soil substrates contamination, soil texture oxidans. Other microorganism such as Leptospirillum ferro-
destruction, short of nutrient, ecological landscape destruc- oxidans was reported to bioleach zinc from the marmatite
tion, groundwater pollution, biological diversity decrease, otation concentrate eectively (Shi and Fang, 2005) and
etc. The presence of toxic heavy metals in mine tailings the fungal treatment was also found to promote extensive
caused lots of serious environmental problems. In order leaching of chromium from preservative-treated wood
to resolve the above problems, it is important to develop (Reyes, 2007). Because of the advantages of low cost and
a suitable and economical technology for removal of heavy environment friendliness and better eciency, bioleaching
metals from mine tailings. technology has been a great success for the mining industry
(Shi et al., 2006). The use of microorganism or its biomass
*
Corresponding author. Tel.: +86 731 8649208; fax: +86 731 8822829. for the recovery of metals from waste as well as the
E-mail address: axore@163.com (Y.-G. Liu). employment of plants for landll applications have

0960-8524/$ - see front matter  2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2007.08.064
 Y.-G. Liu et al. / Bioresource Technology 99 (2008) 41244129 4125

achieved growing attention (Umrania, 2006). In recent from mine tailings and variation in partitioning of chemical
years, bioleaching has proved to be a possible way to forms for heavy metals of mine tailings after the bioleach-
remove heavy metals from metal contaminated materials ing process.
such as anaerobically digested sewage sludge, contami-
nated river sediment, spent nickelcadmium batteries, tan-
nery sludge, and incinerator y ash (Wong et al., 2004; 2. Methods
Seidel et al., 1998; Chen and Lin, 2004; Zhu et al., 2003;
Fang and Zhou, 2007; Xu and Ting, 2004; Paul et al., 2.1. Sample and source and its characterization
2004). Our previous study had also proved that it was fea-
sible to remove heavy metals from mine tailings with the Mine tailings and bacteria-containing sludge used in this
use of the bioleaching remediation method (Liu et al., study were collected from the Shuikoushan tailing dam
2007). located 40 km south of Hengyang City, central Chinas
In this work we want to carry out bioleaching experi- Hunan province. The Shuikoushan mine mainly produced
ment of mine tailings by indigenous sulfur-oxidizing bacte- lead, zinc, and copper and the large-scale Kangjiawan mine
ria. The bacterium A. thiooxidans is active at low pH and of the Shuikoushan mine was the fourth largest lead and
can endure harsh conditions that exist in concentrated zinc mine in China with an annual ore-producing capacity
solutions of heavy metals. It can catalyze the oxidation of more than 400,000 tons and reserves 16,000,000 tons of
of elemental sulfur or reduced sulfur compounds to sulfuric lead and zinc.
acid and obtain energy from the oxidation process, and The mine tailings samples and bacteria-containing
cause bioacidication and solubilization of heavy metals. sludge were transported to lab and stored at 4 C prior
The oxidation and acid producing activity of sulfur-oxidiz- to their use. Mine tailings samples were dried in a glove
ing bacteria are the primary mode of solubilization of box which was continuously purged with oxygen-free nitro-
heavy metals in the bioleaching process. The solubilization gen. Then mine tailings were characterized before its use in
mechanism of bioleaching by A. thiooxidans can be direct the bioleaching experiment. The measured characteristics
and indirect. The direct and indirect mechanism can be of mine tailings were described below: The pH value of
described by the following equations (Rulkens et al., 1995): mine tailing was 6.30. Water content (%) and organic mat-
The direct mechanism ter (%) of mine tailings were 1.32, 1.37, respectively. Cu,
Zn, Pb, Fe, Ca, sulfate (mg/kg dry weight), and carbonates
A: thiooxidans
MS 2O2 ! MSO4 1 (as CaCO3 mg/kg) of mine tailings were 1332.4, 1169.1,
153.8, 4756.8, 38 749, 3208, 97 665, respectively.
The indirect mechanism In addition to the total metal content, the distribution of
metals among various fractions in the mine tailings is a use-
A: thiooxidans ful measure as partitioning information determines the
S0 H2 O 1:5O2 ! H2 SO4 ; 2 behavior of the metal in the environment and it allows an
H2 SO4 materials-M ! materials-2H MSO4 3 investigation of which metals, bound to which fractions,
are solubilized during bioleaching. So it is important to
where M was a bivalent metal. describe the partitioning of heavy metals into dierent frac-
The eectiveness of bioleaching is highly dependent on tions before and after their biological removal from mine
the physical, chemical and biological factors in the system tailings. Four binding fractions (F1: exchangeable, F2:
(Chen and Lin, 2001b). Among these aecting factors such reducible fraction or fraction associated with Fe and Mn
as nature of contaminated material, substrate concentra- oxides, F3: oxidizable-fraction associated with suldes
tion, solids concentration, temperature, oxygen, pH, oxida- and organic matter, F4: residual fraction) of heavy metals
tionreduction potential (ORP), bacterial strain and cell (Cu, Zn, and Pb) were analyzed using the four-step proce-
concentration (Bosecker, 1997), substrate concentration dure recommended by Community Bureau of Reference
plays an important role in optimization of the bioleaching (BCR) (Wang et al., 2005).
process. In this bioleaching process, elemental sulfur was In this study, the amounts of metals were determined by
generally added as the substrate for A. thiooxidans. The Atomic Adsorption Spectrophotometer (AAS). Binding
acid production and pH variation in the bioleaching pro- fractions of heavy metals in mine tailings were listed in
cess are inuenced by the concentration of added sulfur. Table 1.
However, the added elemental sulfur is not completely oxi-
dized to sulfuric acid and the remaining sulfur will lead to
the reacidication of treated mine tailings during the nal Table 1
Binding fractions of heavy metals in mine tailings
disposal (Chen and Lin, 2001a). Therefore, a complete
understanding of the factor sulfur concentration is impor- Heavy metals F1 (%) F2 (%) F3 (%) F4 (%)
tant to optimize the bioleaching processes. Cu 19.94 11.77 46.13 22.16
The objective of this work was to investigate the eect of Zn 15.52 9.90 38.77 35.81
Pb 15.02 18.60 10.40 55.98
substrate concentration on remobilization of heavy metals
4126 Y.-G. Liu et al. / Bioresource Technology 99 (2008) 41244129

2.2. Enrichment and acclimation of indigenous sulfur- organic matter of the tailings. The mine tailings slurry sam-
oxidizing bacteria ple taken from the reactor was centrifuged at a speed of
10,000 rpm for 20 min. The ltrate was analyzed for its sul-
Indigenous sulfur-oxidizing bacteria were enriched by fate concentrations according to the Standard Methods
adding bacteria-containing sludge from the Shuikoushan (APHA, 1995). Heavy metal (Cu, Zn, Pb, Ca, Fe) concen-
mine in 250 ml Erlenmeyer ask with 100 ml culture med- trations by atomic absorption spectrophotometer (Model
ium (1 g/L NH4Cl, 1 g/L KH2PO4, and 0.5 g/L ZEEnit700, Analytik Jena AG). The total heavy metals
MgCl2 H2O), at 2% inoculum (v/v). In this acclimation in the mine tailings were determined after HFHNO3
process, 2.15 g/L tyndallized elemental sulfur was trans- HCl digestion method (USEPA, 1995). All treatments
ferred to sludge suspension. This Erlenmeyer ask was and controls were done in duplicate.
placed on gyratory shaker and agitated at 180 rpm and at
28 C. The pH of the sludge was monitored. When the
pH value of sludge dropped under 2.0, 2 ml acidied sludge 3. Results and discussion
was then transferred to 100 ml fresh culture (with the
2.15 g/L tyndallized elemental sulfur) medium under the 3.1. Eect of dierent substrate concentration on pH, ORP,
same conditions. The inoculum for the bioleaching experi- and sulfates in bioleaching process
ment was obtained by repeating the above procedures until
the indigenous sulfur-oxidizing bacteria in the sludge had During the bioleaching process, elemental sulfur was
the highest rate of acidication. oxidized to sulfuric acid by the indigenous sulfur-oxidizing
bacteria, resulting in a decrease in pH and solubilization of
2.3. Bioleaching experiment metals. The variations of pH during the bioleaching pro-
cess with dierent sulfur concentration were shown in
The bioleaching was conducted in 250 ml Erlenmeyer Fig. 1.
asks with 100 ml same culture medium at ve dierent sul- From the gure, there was an initial rise in pH between
fur concentration (w/v) (0.5%, 1%, 2%, 3%, and 5%). The day 0 and day 2, perhaps caused by the buer or by the
asks were agitated at 28 C, 180 rpm on a gyratory sha- release of alkaline from mine tailings during the initial
ker. A blank Run (A1) without either inoculum of bacteria stage of bioleaching. In the control Run A1 without inocu-
was also carried out in order to compare the results. Biole- lation of indigenous sulfur-oxidizing bacteria, the pH
aching conditions were tabulated in Table 2. During the increased from 6.57 to 6.74 in the rst two days, then the
bioleaching process the water loss due to evaporation was nal pH was stabilized at about 6.58. In the other runs,
replenished with distilled water daily. the initial pH were under 7.0, because the 2% of inoculum
with the pH value under 2.0 was added, and a faster
decrease in pH was observed after two days and the nal
2.4. Chemical analysis pH was much lower than that of Run A1 due to the indig-
enous sulfur-oxidizing bacteria oxidation of sulfur.
During the experiment, the pH and ORP value were As indigenous sulfur-oxidizing bacteria have grown on
measured at half-day intervals, whereas sulfate and soluble sulfur, sulfuric acid accumulated in Erlenmeyer asks and
heavy metals (Cu, Zn, Pb) were measured at one-day inter- the mine tailings pH decreased to the pH value of about
val. Binding fractions of heavy metals were measured after 2.0 in 3, 5, 7.5, 8.5, and 13 days for sulfur concentrations
the bioleaching using the four-step procedure recom- of 2%, 1%, 3%, 5%, and 0.5%, respectively. It was apparent
mended by BCR. The pH and ORP values were measured that the mine tailings sulfur concentration signicantly
by pH and ORP analyzer (Model K100, Dr-Kornder). The aected the rate of pH decrease. It also can be found that
approximate gravimetric method (Raad, 1978) was used
for carbonates of the tailings and the WalkleyBlack wet
combustion method (Nelson and Sommer, 1982) for 7
6
Table 2 5
Experimental conditions for bioleaching 4
pH

Run no. Sulfur Temperature Rpm Inoculum Solid 3

concentration (C) dosage concentration 2
(%) (v/v)
1
A1 0.5 28 180 No 1% 0
(blank)
A 0.5 28 180 2% 1% 0 1 2 3 4 5 6 7 8 9 10 11 12 13
B 1 28 180 2% 1% Time (days)
C 2 28 180 2% 1%
A1 A B C D E
D 3 28 180 2% 1%
E 5 28 180 2% 1% Fig. 1. Eect of sulfur concentration on pH during bioleaching.
 Y.-G. Liu et al. / Bioresource Technology 99 (2008) 41244129 4127

pH decreased fastest at the sulfur concentrations of 2% the concentration of sulfate increased fastest at 2% sulfur
than at the other ones. An increase in the sulfur concentra- concentration of the ve substrate concentration tested.
tion leads to an increase in the adsorption of bacteria onto It was found that the metabolic nal product, sulfate was
sulfur particles which enhances the acidication rate. produced with time and the rate of sulfate production also
Therefore the rate of pH reduction increased with an increased with the increase of sulfur concentration in sulfur
increase in feed sulfur concentration in sulfur concentra- concentrations of 0.52%. However, by comparison
tions of 0.52%. However, the oxidation of elemental sul- between test C and tests E, D, it was deduced that a lot
fur was inhibited by a higher sulfur concentration of the sulfur powder added in the bioreactor was not oxi-
(greater than 2%). dized (utilized) during the bioleaching process at a higher
Changes in oxidationreduction potential (ORP) also sulfur concentration (greater than 2%). It can be also
indicate the level of activity of sulfur-oxidizing bacteria. proved that higher sulfur concentration of mine tailings
Fig. 2 showed the changes in ORP with bioleaching time can not always induce the higher sulfate concentration
at dierent sulfur concentration. From Fig. 2, the ORP (Chen and Lin, 2001a).
of the control Run A1 was stabilized at about 50 during From Figs. 1, 2, 3 the results indicated that the rates of
the bioleaching period. In contrast, the ORP of the other pH reduction, ORP rise and sulfate production increased
runs increased by the oxidation of sulfur rapidly and then with the increase of sulfur concentration in sulfur concen-
gradually approached a limiting value within 13 days. trations of 0.52% because an increase in the sulfur concen-
Compared to the variation of pH in bioleaching, the vari- tration conduces to an increase in the adsorption of
ations of ORP were much related to those of pH. The bacteria onto sulfur particles. However, the rates of pH
increase of ORP had a similar trend with decrease of pH. reduction, ORP rise and sulfate production was inhibited
It can be also observed that higher or lower sulfur concen- by a higher sulfur concentration (greater than 2%).
tration of mine tailings induced a drop in the rate of ORP
increase.
The rate of sulfate production is an important indicator 3.2. Metal solubilization in bioleaching
of bioleaching eciency. Fig. 3 showed the trend of sulfate
production in the bioleaching process at dierent elemental Figs. 4, 5, 6 showed the eects of sulfur concentration on
sulfur concentration. It was apparent from the gure that the solubilization of heavy metals (Cu, Zn, Pb) from mine

100%
500
Solubilization (Cu)

80%
400
ORP (mv)

60%
300
200 40%

100 20%

0 0%
0 1 2 3 4 5 6 7 8 9 10 11 12 13 0 1 2 3 4 5 6 7 8 9 10 11 12 13
Time (days) Time (days)
A1 A B C D E A1 A B C D E

Fig. 2. Eect of sulfur concentration on ORP during bioleaching. Fig. 4. Bioleaching of Cu at dierent sulfur concentration.

10000 100%
Sulfate (mg/l)

8000 80%
Solubilization (Zn)

6000
60%
4000
40%
2000
20%
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13
0%
Time (days)
0 1 2 3 4 5 6 7 8 9 10 11 12 13
A1 A B C D E Time (days)
A1 A B C D E
Fig. 3. Eect of sulfur concentration on sulfate production during
bioleaching. Fig. 5. Bioleaching of Zn at dierent sulfur concentration.
4128 Y.-G. Liu et al. / Bioresource Technology 99 (2008) 41244129

50% optimal sulfur concentration for solubilization of total

extractable heavy metal was 2% under the chosen experi-
40%
Solubilization (Pb)

mental conditions. At 2% sulfur concentration the solubili-

zation eciency of bioleaching of Zn, Cu, Pb arrived at
30%
97.54%, 97.12%, 44.34% respectively, within 13 days.
20%

10% 3.3. Variation in the binding forms of heavy metals

0% The dierent forms of heavy metals stand dierent

0 1 2 3 4 5 6 7 8 9 10 11 12 13 energy states, and aect not only the eciency of bioleach-
Time (days) ing but also the bioavailability of heavy metals after biole-
A1 A B C D E aching. Metals in exchangeable, and Fe/Mn oxide-bound
fractions are considered to be more mobile, dangerous,
Fig. 6. Bioleaching of Pb at dierent sulfur concentration.
and bioavailable. The organic matter/sulde-bound and
residual metals are considered to be more stable and non-
tailings during the bioleaching process. By comparison bioavailable than metals in exchangeable, and Fe/Mn
between tests A, B, C, D and test E, it was evident that oxide-bound fractions. So it was necessary to describe the
heavy metals removal can be strongly inuenced by sulfur partitioning of heavy metals into dierent fractions before
concentration; lag phase was observed in the solubilization and after the bioleaching.
of Cu, Zn, Pb at higher and lower sulfur contents (Runs A The partitioning of heavy metals in control A1 and Run
and E). An increase in the sulfur concentration means an A was shown in Figs. 7 and 8. With control A1, for all of
increase in the availability of total surface area of sulfur the metals of control A1, F2: the Fe/Mn oxide-bound frac-
particles, which enhances the acidication rate. So the rate tions, and F4: residual forms had little change during the
of metal solubilization increased linearly with the increase running, while F3: the sulde-bound fractions of Cu, Pb,
of sulfur concentration in sulfur concentration range from and Zn showed a decrease because of the oxidizing envi-
0.5% to 2%. However, adding 3% or 5% of elemental sulfur ronment caused by aeration with agitation. As for F1 the
into the reactor did not improve the acidication rate exchangeable bound form, all of the metals showed an
appreciably compared to that of 2% of elemental sulfur. increasing trend during the experimental period.
Except for Pb, solubilization eciency of Cu, Zn was The bioleaching had a signicant impact on changes in
satisfactory. The solubilization eciency of bioleaching partitioning of heavy metals. Figs. 8 showed variation in
of Zn was highest at 97.5472.35%, and that of Cu was the binding forms of heavy metals in Run A before and after
higher at 97.1263.66% and that of Pb was lowest at the bioleaching process. Cu, mainly bound to F3: the sulde-
44.3414.32% of the three metals in the mine tailings. bound fractions and F1 the exchangeable bound fractions in
The solubilization of Zn, and Cu from mine tailings was raw mine tailings, was mainly bound to F4: residual fraction
signicantly higher than that of Pb at the same sulfur con- and F2 the Fe/Mn oxide-bound fractions after the bioleach-
centration. The eciency of Pb solubilization was not high ing process. Solubilization eciency of Pb was somewhat
in the bioleaching process because the solubilized Pb can unsatisfactory, but Pb was mainly bound to F4: residual
form into low solubility of PbSO4 (Ksp = 1.62 108) with fraction after the bioleaching process. So Pb remaining in
sulfate. the mine tailings was stable and nonbioavailable after the
In conclusion, sulfur concentration strongly aected the bioleaching process. A transformation of binding fractions
solubilization of heavy metals during the bioleaching pro- occurred during the bioleaching of Zn; mainly bound to
cess. From the experimental results, it was clear that the F3: the sulde-bound fractions and F1: the exchangeable

a 100% b 100% c 100%

80% 80% 80%
Percentage
Percentage

Percentage

60% 60% 60%

40% 40% 40%
20% 20% 20%
0% 0% 0%
0 13 0 13 0 13
Time (days) Time (days) Time (days)
F1 F2 F3 F4

Fig. 7. Variation in partitioning of chemical forms for Cu (a), Pb (b), and Zn (c) of control A1 after the experiment.
 Y.-G. Liu et al. / Bioresource Technology 99 (2008) 41244129 4129

a 100% b 100% c 100%

80% 80% 80%

Percentage
Percentage

Percentage
60% 60% 60%

40% 40% 40%

20% 20% 20%

0% 0% 0%
0 13 0 13 0 13
Time (days) Time (days) Time (days)

F1 F2 F3 F4

Fig. 8. Variation in partitioning of chemical forms for Cu (a), Pb (b), and Zn (c) of Run A after the bioleaching.

bound fractions before the bioleaching process, it was and Brettanomyces B65 in an air-lift reactor. Chemosphere 69, 303
mainly bound to F4: residual fraction and F2 the Fe/Mn 310.
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