Journal of Industrial and Engineering Chemistry 21 (2015) 451–458

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Journal of Industrial and Engineering Chemistry

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

Bioleaching of arsenic and heavy metals from mine tailings by pure

and mixed cultures of Acidithiobacillus spp.
Van Khanh Nguyen, Mu Hyun Lee, Hyung Jun Park, Jong-Un Lee *
Department of Energy and Resources Engineering, Chonnam National University, Gwangju 500-757, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Article history: Microbial removal of heavy metals in mine tailings from a Cu–Ag mine in the Philippines was
Received 22 June 2013 investigated. Effect of bacterial strains on bioleaching and fractionation of heavy metals were also
Received in revised form 28 January 2014 studied. Single and mixed cultures of Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans were
Accepted 5 March 2014
used separately to compare efficiency of metal solubilization. The results showed that mixed cultures
Available online 13 March 2014
were more efficient than each bacterium for Cu and As, while A. ferrooxidans demonstrated faster
extraction efficiency for Mn and Zn. The results of sequential extraction for residues indicated that pure
Keywords:
bacteria and mixed cultures mediated occurrence mode of heavy metals in different ways.
Heavy metals
Arsenic
ß 2014 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights
Bioleaching reserved.
Mine tailings
Acidithiobacillus ferrooxidans
Acidithiobacillus thiooxidans

1. Introduction A. thiooxidans. The direct and indirect mechanism can be described

by the following reactions (Eqs. (1)–(3)) [5].
The bioleaching technique has been increasingly applied to the Direct mechanism:
extraction and recovery of heavy metals from metallic ores and
A: thiooxidans
sediment, soil and sludge contaminated with heavy metals. It is an MeS þ 2O2 ! MeSO4 (1)
environmentally cleaner process when compared with physical
Indirect mechanism:
and chemical processes [1] and rarely emits atmospheric
contaminants. Bioleaching operations are relatively inexpensive S0 þ H2 O þ 1:5O2
A: thiooxidans
! H2 SO4 (2)
and simple to operate [2].
The bacteria commonly employed in the bioleaching processes H2 SO4 þ materials-Me ! materials-2H þ MeSO4 (3)
are found to be of genus Acidithiobacillus and Thiobacillus, such as
Acidithiobacillus ferrooxidans, A. thiooxidans and Thiobacillus thio- where Me is a bivalent metal.
parus [3]. Among them, A. thiooxidans uses reduced forms of On the other hand, A. ferrooxidans differs from A. thiooxidans by
inorganic S but not Fe2+ as an energy source. In addition, they are the fact that they derive energy from the oxidation of Fe2+ as an
highly acidophilic (pH 0.5 to 5.5, optimum pH 2 to 3.5), and can electron donor in addition to reduced S compounds. In the absence
decrease the pH of the leaching medium to 1.5 to 1.0 and even of oxygen, A. ferrooxidans is still able to grow on reduced inorganic
lower [4]. Metal solubilization mechanisms by A. thiooxidans can be S compounds using Fe3+ as an alternative electron acceptor [6]. The
direct or indirect. In the direct mechanism, metal sulfides can be mechanisms of bioleaching by A. ferrooxidans are usually discussed
oxidized into SO42 directly by these acidophilic bacteria. In the in terms of direct bacterial attack on sulfide minerals and indirect
indirect mechanism, H2SO4, a strong leaching agent, is generated oxidative dissolution of minerals by Fe3+ [7]. The direct dissolution
from the oxidation of elemental S or reduced S compounds by of minerals is caused by the attack on sulfide minerals by the
enzymatic system of the bacteria situated at the mineral surface
(Eq. (4)) [8–10].
A: ferrooxidans
MeS2 þ H2 O þ 3:5O2 ! Me2þ þ 2SO2
4 þ 2H
þ
(4)
* Corresponding author. Tel.: +82 62 530 1728; fax: +82 62 530 1729.
E-mail address: jongun@chonnam.ac.kr (J.-U. Lee). where MeS2 is an insoluble metal sulfide and Me2+ is a metal ion.

http://dx.doi.org/10.1016/j.jiec.2014.03.004
1226-086X/ß 2014 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
452 V.K. Nguyen et al. / Journal of Industrial and Engineering Chemistry 21 (2015) 451–458
In the indirect mechanism, the bacteria regenerate Fe3+ through
the oxidation of Fe2+ in solution and Fe3+ serves as a strong
oxidizing agent of sulfide minerals (Eqs. (5) and (6)) [11,12]
A: ferrooxidans
14Fe2þ þ 3:5O2 þ 14Hþ ! 14Fe3þ þ 7H2 O
MeS2 þ 8H2 O þ 14Fe3þ ! Me2þ þ 14Fe2þ þ 2SO2 þ respectively. The flasks were shaken at 200 rpm in a shaking
4 þ 16H
To date, many studies on the bioleaching of heavy metals from
mine tailings using A. thiooxidans are reported [13,14]. Some
investigations on bioleaching employed a pure or mixed cultures;
for example, bioleaching of chalcopyrite by pure and mixed
cultures of Acidithiobacillus spp. and Leptospirillum spp. [15,16],
bioleaching of As from medicinal realgar by pure and mixed
cultures of A. thiooxidans and A. ferrooxidans [17], and bioleaching
of sediment contaminated with heavy metals by pure and mixed
cultures of Acidithiobacillus spp. [4].
The objective of this research was to elucidate the microbial
removal efficiencies for Cu, Mn, Zn, and As from mine tailings taken
from a Cu–Ag mine in the Philippines. A set of batch experiments
was conducted to compare the efficiency of metal solubilization in
the bioleaching processes which used both pure and mixed
cultures of two Acidithiobacilli species, A. ferrooxidans and A.
thiooxidans as microbial agents for the same mine tailings and
under the same condition. The partition of heavy metals which
represents the mode of occurrence in the mine tailings was also
investigated through sequential extraction experiments.
2. Materials and methods
2.1. Sample characterization
The mine tailings used in this study were collected from a
floatation plant of Dizon Mine located in San Marcelino, Luzon,
Philippine. The target product of the mine is Cu and accompanied
precious metals are Ag and Au. The particle size of the sample was
lower than 0.180 mm in diameter. Prior to experiments, an amount
of mine tailings was air-dried for chemical analysis and bioleach-
ing experiments. The mine tailing samples were digested by aqua
regia and then analyzed for their total Fe, Mn, As, and heavy metals
(Cu, Zn, Co, Cr, Ni, Mo, and Pb) by an inductively coupled plasma
optical emission spectrometer (ICP–OES). For the digestion, 0.25 g
of an air-dried sample was digested with a concentrated HNO3–
HCl (1:3 v/v) mixture according to the Standard Methods of Korean
Ministry of Environment [18]. Then, the suspension was filtered
through a 0.45 mm nylon syringe filter, serially diluted and
introduced to the ICP–OES. The mine tailing sample was also
subjected to X-ray fluorescence (XRF) analysis to determine both
major and trace elements (Si, Al, Fe, Mg, Na, K, Ca, S, Ti, Cu, P, Cl, Mn,
Zn, Sr, Cr, and As).
2.2. Microorganisms and acclimation tests
The cultures of A. ferrooxidans and A. thiooxidans were obtained
from Microbial Geochemistry Laboratory, Chonnam National
University in Korea and used throughout this study. Bacterial
cells were cultivated in 250 mL of Medium 271 [19] in 500 mL
flasks located in a shaking incubator at 30 8C and 200 rpm. The
Medium 271 contains 2 g (NH4)2SO4, 0.5 g K2HPO4, 0.5 g
MgSO47H2O, 0.1 g KCl, and 0.02 g Ca(NO3)24H2O in 1000 mL
distilled water. In addition, 45 g FeSO47H2O and 10 g elemental S
were used as energy sources for A. ferrooxidans and A. thiooxidans,
respectively. The Medium 271, FeSO47H2O, and elemental S were
sterilized separately, cooled and mixed subsequently.
The acclimation tests were performed prior to bioleaching
experiments. In the acclimation tests, 25 mL subcultures of A.
ferrooxidans and A. thiooxidans were separately added in 250 mL of
the mine tailing suspension in the Medium 271 (1% solid contents,
w/v) in 500 mL Erlenmeyer flasks. The mine tailing samples were
autoclaved at 121 8C for 30 min before being suspended in the
(5) Medium 271. Then 1% of sterilized elemental S and Fe2+ were
added as energy sources for A. thiooxidans and A. ferrooxidans,
(6)
incubator at 30 8C. The pH and redox potential of acclimation tests
were monitored periodically and continued until pH of the media
dropped below 2.0 and then the inoculums for bioleaching
experiments were obtained.
The number of bacterial cells in inoculums was defined by
enumerating the colony forming unit (CFU) using the plate count
method on a sterile agar in a Petri dish. For A. thiooxidans
enumeration, Acidithiobacillus agar medium [20] was prepared
separately and poured into a Petri dish. The Acidithibacillus agar
medium contains 0.1 g NH4Cl, 3 g KH2PO4, 0.1 g MgCl26H2O, 0.1 g
CaCl2, 5 g Na2S2O35H2O, and 20 g technical agar (Becton,
Dickinson and Company Sparks, Maryland, USA) in 1000 mL of
distilled water, and pH was adjusted to 4.2 before it was autoclaved
at 121 8C for 15 min. For A. ferrooxidans enumeration, a modified
thiobacillus solid medium (TSM1) was prepared [21]. The TSM1
medium is composed of three solutions; Solution A, basalt salts: 3 g
(NH4)2SO4, 0.1 g KCl, 0.05 g K2HPO4, 0.5 g MgSO47H2O, 0.015 g
Ca(NO3)24H2O, and 0.25 g tryptone soya broth dissolved in this
order in 600 mL of distilled water, acidified with 1 M H2SO4 to pH
2.5 and autoclaved at 121 8C for 15 min and cooled to 60 8C.
Solution B: 22 g FeSO47H2O dissolved in 150 mL of distilled water
acidified to pH 2.5, filter-sterilized and warmed to 60 8C. Solution
C: 5 g agarose Biorad High mr 162–0001 in 250 mL distilled water,
autoclaved at 121 8C for 15 min and cooled down to 60 8C. Three
solutions were mixed and poured on a Petri dish. The inoculums
were diluted serially and spread on an agar plate, then the cultures
were incubated at 25 8C and the CFUs were counted at the end of
the incubation time.
2.3. Bioleaching experiments
The bioleaching experiments were conducted in duplicate in
500 mL flasks incubated in a shaking incubator at 30 8C and
200 rpm for 600 h. Eight flasks, each with a working volume of
200 mL containing the Medium 271 with filter-sterilized 45 g/L of
FeSO47H2O (9 g/L of Fe2+) for A. ferrooxidans, 1% (w/v) sterilized
elemental S for A. thiooxidans and mixed cultures, and the 5% (w/v)
sterile mine tailing sample was inoculated with 10% (v/v) bacterial
inoculums. The solid content was chosen in the range which was
suggested by Chen and Lin [22]. The inoculums of two bacterial
cultures (A. thiooxidans and A. ferrooxidans) were obtained from an
acclimation process with cell concentrations of 6  106 cells/mL
and 8  105 cells/mL, respectively. An abiotic control experimental
set was also carried out without bacterial inoculation. The
experimental sets were designed as shown in Table 1. During
the experiments, aliquot samples were periodically taken from
each flask and pH, redox potential, Fe2+, total Fe, SO42, soluble As
and heavy metals were determined.
2.4. Sequential extraction of heavy metals
The sequential extraction procedures proposed by Tessier et al.
[23] were performed to determine the binding forms of heavy
metals in the mine tailings. The details of extraction steps and
target phases are described in Table 2. In order to examine the
chemical forms of As solid phases in the mine tailings, a modified
procedure suggested by Ahn et al. [24] was employed. The modes
of As occurrence in the mine tailings were separated into an
ionically bound fraction, strongly adsorbed fraction, amorphous
 V.K. Nguyen et al. / Journal of Industrial and Engineering Chemistry 21 (2015) 451–458 453

Table 1
The experimental sets designed for this study.

Experimental run Mine tailings Medium Substrate Inoculum

A. thiooxidans 10 g 200 mL Medium 271 2 g elemental S 20 mL A. thiooxidans

A. ferrooxidans 10 g 200 mL Medium 271 9 g/L of Fe2+ 20 mL A. ferrooxidans
Mixed 10 g 200 mL Medium 271 2 g elemental S 10 mL A. thiooxidans + 10 mL A. ferrooxidans
Control 10 g 200 mL Medium 271 2 g elemental S None (20 mL Medium 271)

Table 2
Sequential extraction procedure for fractionation of heavy metals.

Step No. Target phases Extractants and condition

1 Exchangeable fraction 0.5 M magnesium chloride (MgCl2), pH 7.0, 1 h, room temperature,

2 Carbonate and specifically adsorbed fraction
3 Fe–Mn oxides bound fraction
4 Organic matter and sulfides bound fraction
5 Residual fraction
continuous agitation
1 M sodium acetate (CH3COONa), pH 5.0 (adjusted by acetic acid), 5 h, room
temperature, continuous agitation
0.04 M hydrochloride–ammonium hydroxide (NH2OH–HCl) in acetic acid 25% (v/v), 6 h, 95 8C,
occasional agitation
0.02 M nitric acid (HNO3) and 30% hydrogen peroxide (H2O2) (adjusted to pH 2.0 with nitric acid),
5 h, 85 8C, occasional agitation
3.2 M ammonium acetate (CH3COONH4) in 20% nitric acid, 1 h, room temperature, occasional agitation
Aqua-regia (35% HCl + 70% HNO3, 3:1 (v/v), 1 h, 78 8C, occasional agitation.

and poorly crystalline hydrous oxides of Fe–Mn fraction, crystal- equations:

line Fe oxyhydroxides fraction, and residual fraction (Table 3).
ML
Both of the procedures started with 1 g of a dry sample and Metal removalð%Þ ¼  100 (7)
MS
between each successive extraction, separation were done by
centrifugation at 15,000 rpm for 15 min. The supernatant was X
n
filtered by 0.45 mm Whatmann nylon syringe filter and introduced ML ðmgÞ ¼ C i  V i þ C i1  vi1 (8)
to ICP–OES to determine metals. Before each next extraction, the i¼2
residue was washed twice with distilled water and centrifuged for
15 min, and the supernatant was discarded. A minimal volume of MS ðmgÞ ¼ C t  m (9)
rinse water was used to avoid excessive solubilization of solid where ML represents the mass of heavy metals solubilized in
materials. leachate (mg), MS represents the mass of heavy metals in mine
tailings (mg) before bioleaching, Ci represents the metal concen-
2.5. Analytical techniques tration in the leachate at the sampling time i, Vi represents the
volume of leachate at the sampling time i, vi1 represents the
Redox potential and pH were measured by using a combination volume of the sample amount taken from experimental flasks at
electrode connected to Orion Model 230A+. The concentration of the sampling time i  1, Ct represents the initial metal content in
Fe2+ was measured using a modified ferrozine method [25] in the mine tailings, and m represents the mass of mine tailings.
which an amount of 0.1 mL sample was rapidly mixed with 4.9 mL
0.1% (w/w) ferrozine (3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine- 3. Results and discussion
40 ,400 -disulfonic acid sodium salt (C20H13N4NaO6S2)) solution in a
0.5% ammonium acetate buffer of pH 7.0 (adjusted by using 1 M 3.1. Characteristics of mine tailings and mode of heavy metal
NaOH). Then the samples were poured into cuvettes and optical occurrences
absorption was measured at 562 nm by using UV–vis spectropho-
tometer (UV Mini 1240, Shimadzu). Total Fe, As and heavy metals The metal contents of the mine tailings determined using ICP–
were determined by an inductively coupled plasma mass OES after aqua regia digestion and XRF analysis are given in Tables
spectrometer (ICP–MS; Agilent Technologies 7700 Series, USA). 4 and 5, respectively.
The concentration of SO42 was determined by ion chromatogra- The sequential extraction analysis revealed that 59.6% and
phy (IC; Metrohm 830 Series, Switzerland). 38.9% of Cu occurred as sulfide bound and residual fractions,
The percent removals of the metals (solubilization of metals respectively, in the mine tailings. Manganese appeared to be one of
from the mine tailings sample) were calculated by following the most labile metals in the sample because of its association with

Table 3
Sequential extraction procedure for fractionation of As.

Step No. Target phases Extractants and condition

1 Ionically bound fraction
2 Strongly adsorbed fraction
3 Amorphous and poorly crystalline
hydrous oxides of Fe–Mn fraction
4 Crystalline Fe oxyhydroxides fraction
5 Residual fraction
1 M magnesium chloride (MgCl2), pH 8.0, 2 h, room temperature, continuous
agitation
1 M sodium dihydrogen phosphate (NaH2PO4), pH 5.0, 12 h, room temperature,
continuous agitation
0.2 M ammonium oxalate (C2H8N2O4), pH 3.0, 2 h, room temperature,
continuous agitation in darkness
0.5 M sodium citrate (C6H5Na3O7) and 1 M sodium bicarbonate (NaHCO3),
1 h, 75 8C, occasional agitation
Sodium dithionite (Na2S2O4), 30 min, room temperature, continuous agitation
Aqua-regia (35% HCl + 70% HNO3, 3:1 (v/v), 1 h, 78 8C, occasional agitation.
454 [(Fig._1)TD$IG]
V.K. Nguyen et al. / Journal of Industrial and Engineering Chemistry 21 (2015) 451–458

Table 4 8
The selected metal contents of the mine tailings
determined by ICP–OES analysis.

Elements Concentrations
6
(mean  S.D, n = 3) (mg/kg)

Fe 36,074.7  84.6
Cu 1,343.3  0.4 A. thiooxidans

pH
Mn 1,335.5  11.5 4 A. ferrooxidans
Zn 142.6  0.9
Mixed
As 53.8  1.6
Co 12.0  0.3 Control
Cr 26.6  0.1 2
Ni 18.7  0.0
Mo 19.6  4.3
Pb 20.7  1.0

S.D: standard deviation. 0

0 100 200 300 400 500 600
Time (hours)
Table 5
Fig. 1. Variation of pH over time during bioleaching process of the mine tailings by
Chemical composition of the mine tailings determined
pure and mixed cultures.
by XRF analysis.

Elements Concentrations (%)

O 48.610 Along with the pH reduction, the oxidation of Fe2+ and S caused
Si 30.896 an increase in redox potential of the media over time during the
Al 9.172 bioleaching process (Fig. 2). The flasks with A. ferrooxidans showed
Fe 3.674
the highest redox potential in comparison with A. thiooxidans and
Na 2.228
Mg 1.863 mixed cultures. It reached a steady state, from 282 mV to 570 mV,
K 1.625 after only 90 h, and finally reached to 593 mV after 600 h. The high
Ca 0.747 redox potential of the experimental set cultured with A.
S 0.413
ferrooxidans was mainly due to the bacterial oxidation of Fe2+ to
Ti 0.334
Cu 0.156
Fe3+ and maintenance of the dissolved state of Fe3+ and thus high
Mn 0.109 ratio of Fe3+/Fe2+ in the solution [26]. The maximum redox
P 0.083 potential value in the flasks inoculated with mixed cultures
Cl 0.023 reached 516 mV after 257 h incubation and then dropped to
Zn 0.018
405 mV at the end of the bioleaching process. This could be due to
Zr 0.013
Sr 0.013 bacterial types in mixed cultures and their cell concentrations
Cr 0.007 according to competition between two bacteria [4]. It was likely
As 0.007 that a slight dominance of A. thiooxidans over A. ferrooxidans
appeared after 300 h. The redox potential in the flasks with A.
thiooxidans increased from 287 mV to 404 mV and kept stable
readily extractable fractions; that is, exchangeable fraction (4.2%), during the experiment while the redox potential of the control
carbonate and specifically adsorbed fraction (21.5%), and Fe–Mn flasks varied between 132 mV and 254 mV.
oxides bound fraction (56.3%). Only 9.9% of Mn occurred as residual
fraction in the mine tailings. The studied elements were ranked 3.3. Production of SO42 by A. thiooxidans and mixed cultures
according to decreasing mobility order as follows; Mn > Zn >
Cu > As. Even though most of As (90.3% of total) existed as residual The variation of SO42 production is another indicator of
fraction in the mine tailings which is the most stable fraction against [(Fig._2)TD$IG]
bioleaching efficiency and it is usually in correlation with pH
environmental change, it can still pose a risk to the surrounding
environment when the mine tailings are discharged and dispersed. 700
This is because 5.1% of the total As which occurred as ionically bound
and strongly adsorbed fractions are available to be removed from the 600
Redox potential (mV)

mine tailings by natural weather factors.

500
3.2. Variation of pH and redox potential
400
Variations of pH over time during the bioleaching process with
300
different bacterial cultures are shown in Fig. 1. The pH decreasing
ability of different bacterial cultures can be ranked as decreasing
200
order as follows, A. thiooxidans  Mixed cultures > A. ferrooxidans.
The pH reduction induced by bacteria can be explained by Eqs. (2), A. thiooxidans A. ferrooxidans
100
(4), and (6). The pH of the flasks inoculated with A. thiooxidans
Mixed Control
decreased significantly within the first 257 h of the experiment from
0
pH 2.5 to 1.1, then reached a steady state and finally obtained pH 0.9
0 100 200 300 400 500 600
after 600 h of incubation. The pH propagation of A. thiooxidans and
Time (hours)
mixed cultures was quite similar. In the flasks with A. ferrooxidans,
pH went down to 1.5. The pH did not drop significantly in the control Fig. 2. Variation of redox potential over time during bioleaching process of the mine
flasks which were conducted without bacterial inoculation. tailings by pure and mixed cultures.
[(Fig._3)TD$IG]
140
A. thiooxidans
120 Mixed
Control
100
Normalized SO 42- (g/L)
80
60
40
20
0
0 100 200
Time (hours)
Fig. 3. Variation of SO42 production over time during bioleaching process with A.
thiooxidans and mixed cultures.
reduction (Eqs. (1) and (2)). The variation of SO42 concentration
during the bioleaching process in the cases of inoculation with A.
thiooxidans and mixed cultures can be seen in Fig. 3. Increasing
production of SO42 over time during the bioleaching process was
due to the oxidation of sulfide minerals and elemental S, which
proceeded with the similar trend of pH reduction and redox
potential increase. As shown in Fig. 3, SO42 concentration in the
flasks with A. thiooxidans achieved a slightly higher value than
mixed cultures and reached the maximum value (120 g/L) after
600 h of incubation. The SO42 concentration in the flasks without
bacterial inoculation was quite stable over experimental time.
3.4. Oxidation of Fe2+ by A. ferrooxidans
As shown in Eqs. (5) and (6), Fe3+, a strong oxidant, can
remarkably mediate heavy metals removal from mine tailings,
especially from metal sulfides. The soluble Fe3+ is extremely
unstable in nearly all environmental conditions except for low pH
and high redox potential, since it promptly forms iron oxides or
hydroxides and is removed from solution by precipitation.
Therefore, production and maintenance of dissolved Fe3+ in the
leaching media are the vital conditions for effective bioleaching
results.
In Fig. 4, the disparity between the input amount of Fe2+ (9 g/L)
and the total dissolved Fe which was observed at the given time
stands for the amount of precipitated Fe. Likewise, the difference
between total dissolved Fe in solution and soluble Fe2+ represents
the amount of aqueous Fe3+. Ferrous ion initially added was rapidly
oxidized to Fe3+ after only 90 h. A small amount of Fe2+
(approximately 7 mg/L) was maintained in the media till the
end of the experiment. The aqueous Fe3+ in the media varies from 2
to 3 g/L during the bioleaching process.
3.5. Heavy metal solubilization
The concentrations of heavy metals leached out of the mine
tailings over time during the bioleaching process are shown in
Fig. 5. The solubilization of heavy metals from the mine tailings
was expressed, at each sampling time, as an increase of
concentration of metals in the solution from the beginning of
the experiment to the last sampling time.
The higher solubilization efficiency of Cu was obtained from the
flasks inoculated with mixed cultures when compared with pure
cultures of A. thiooxidans and A. ferrooxidans (Fig. 5(a)). After only
[(Fig._4)TD$IG]
V.K. Nguyen et al. / Journal of Industrial and Engineering Chemistry 21 (2015) 451–458 455
10
Total dissolved Fe
8 Fe (II)
Dissolved Fe (g/L)
6
4
2
300 400 500 600 0
0 100 200 300 400 500 600
Time (hours)
Fig. 4. Variation of total dissolved Fe and Fe2+ concentrations in the flasks with A.
ferrooxidans during bioleaching process.
137 h, Cu solubilization efficiency was 34.4%, 31.5%, and 40.8%, for
A. thiooxidans, A. ferrooxidans and mixed cultures, respectively,
then increased slightly and reached 47.1% at the end of incubation
time for mixed cultures. Majority of Cu occurred in mine tailings as
sulfide bound fraction, the combination of the individual
bioleaching mechanisms of A. ferrooxidans and A. thiooxidans
therefore demonstrated a potential for application of mixed
culture for recovery of Cu from mine tailings.
The solubilization mechanisms of Mn and Zn from the mine
tailings are quite similar during the bioleaching process (Fig. 5(b)
and (c)). A. ferrooxidans was likely to be the best option among the
employed bacterial cultures since it extracted more amounts of Mn
and Zn in a kinetically fast way when compared with A. thiooxidans
and mixed cultures. Nearly all of Mn was removed out of the mine
tailings after only approximately 137 h of incubation. At the end of
the experiment, 78.4% of total Zn was removed from the mine
tailings by A. ferrooxidans. A small amount of Mn and As which
existed as a mobile fraction was removed from the mine tailings in
control flasks (Fig. 5(b) and (d)).
It was noted that Mn showed the highest solubilization
efficiency, i.e. 99.5%, 94.8%, 91.1% for A. ferrooxidans, mixed
cultures and A. thiooxidans, respectively (Fig. 6). The solubiliza-
tion efficiency of metals from the mine tailings was
Mn > Zn > As > Cu in decreasing order (Fig. 6). Generally, the
metal solubilization efficiency in the bioleaching process depends
on the mode of occurrence regarding heavy metals in the mine
tailings. Heavy metals in exchangeable, carbonate adsorbed, and
Fe–Mn oxides bound fractions are considered to be easier to
solubilize and are bioavailable. On the contrary, the organic
matter and sulfides bound and residual fractions are stable and
non-bioavailable [27]. Since about 82% of total Mn existed as a
mobile fraction in the mine tailings, the solubilization efficiency of
Mn showed the highest. In the experiments, the bioleaching
process could solubilize all heavy metals occurred as mobile
fractions and a part of stable fractions.
Though most of As in the mine tailings occurred as stable
fractions (90% of As existed as residual fraction), the As removal
efficiency was not very low (Fig. 6). More than 50% of total As was
removed from the mine tailings after 90 h by A. ferrooxidans
(Fig. 5(d)). However, As removal efficiency achieved the highest
value (72.2%) with mixed cultures at the end of the experiment.
According to removal efficiency, the effect of the bacterial cultures
on As removal can be ranked in decreasing order as follows, Mixed
cultures > A. ferrooxidans > A. thiooxidans (Fig. 6). At the first stage
[(Fig._5)TD$IG]
456 V.K. Nguyen et al. / Journal of Industrial and Engineering Chemistry 21 (2015) 451–458

35 70

30 60

Soluble Mn (mg/L)
25 50
Soluble Cu (mg/L)

20 40

15 30
A. thiooxidans
10 A. ferrooxidans 20
Mixed
5 Control 10

0 0
0 100 200 300 400 500 600 0 100 200 300 400 500 600
Time (hours) Time (hours)

(a) Cu (b) Mn

6 2.5

5
2.0

Soluble As (mg/L)
Soluble Zn (mg/L)

4
1.5
3
1.0
2

0.5
1

0 0.0
0 100 200 300 400 500 600 0 100 200 300 400 500 600
Time (hours) Time (hours)

(c) Zn (d) As
Fig. 5. Concentrations of (a) Cu, (b) Mn, (c) Zn, and (d) As leached out of the mine tailings over time during bioleaching process.

of incubation, the solubilization of As by A. thiooxidans and mixed during the bioleaching process and served as an energy source for
cultures is lower than that of A. ferrooxidans. However, the A. ferrooxidans in mixed culture leaching solution.
solubilization of As by mixed cultures increased rapidly after 300 h
and obtained the highest efficiency at the end of incubation. This 3.6. Variation in mode of occurrences of As and heavy metals after
could be due to the enhancement in activity of A. ferrooxidans by bioleaching
the increase of Fe2+ which was readily extracted from mine tailings
[(Fig._6)TD$IG] The distribution of metals after bioleaching represents not only
the efficiency of bioleaching but also the stability and bioavail-
ability of metals remained in the mine tailings after bioleaching.
A. thiooxidans Metals in exchangeable, carbonates bound and Fe–Mn oxides
100
A. ferrooxidans bound fractions are considered to be more mobile, dangerous, and
Solubilization efficiency (%)

Mixed bioavailable than other forms; whereas, the metals in sulfides and
80
organic matter bound and residual fractions are considered more
stable and non-bioavailable. The results of sequential extraction
60 after bioleaching indicated that the bioleaching had a significant
impact on the changes in mode of occurrences of metals in the
residues (Fig. 7). The bioleaching process removed a significant
40
part of sulfides bound and residual fractions of Cu which was
considered to be most stable in the mine tailings. After the
20 process, only a small amount of Mn existed in the residues as the
exchangeable form. A. ferrooxidans extracted all of Zn from Fe–Mn
0 oxides bound and sulfides bound fractions. There was an
Cu Mn Zn As important change in the distribution of As in the mine tailings
after bioleaching with A. ferrooxidans. Arsenic in the residues
Fig. 6. Removal efficiency of heavy metals after 600-hour incubation. existed as ionically bound, amorphous Fe–Mn bound and residual
[(Fig._7)TD$IG] V.K. Nguyen et al. / Journal of Industrial and Engineering Chemistry 21 (2015) 451–458 457

Fig. 7. Distribution of (a) Cu, (b) Mn, (c) Zn, and (d) As in the mine tailings before and after bioleaching process. Raw: mine tailings before bioleaching, A.t: residues after
bioleaching using A. thiooxidans, A.f: residues after bioleaching using A. ferrooxidans, Mixed: residues after bioleaching using mixed cultures of bacteria, Control: residues after
leaching using the medium without bacterial inoculum. Refer to the text for detailed meanings of the legends.

fractions and the proportion of these three fractions was almost
equal.
4. Conclusions
In this research, the pure and mixed cultures of Acidithioba-
cillus spp. were used to investigate the effect of bacterial strains on
the bioleaching efficiency of mine tailings. After 600 h of
incubation, A. ferrooxidans was found to be the most effective
bacteria compared to A. thiooxidans and mixed cultures for
bioleaching of Mn and Zn from the mine tailings; meanwhile,
mixed cultures showed the higher efficiency on bioleaching of Cu
and As from the mine tailings when compared with pure cultures.
According to the results of sequential extraction, the mobility of
the heavy metals in the mine tailings could be ranked in a
decreasing order as follows, Mn > Zn > Cu > As. At the end of the
experiment, the highest solubilization efficiency was noted with
Mn and the lowest removal efficiency was found with Cu which
has high proportion of stable fractions in the mine tailings among
the studied heavy metals. The efficiency of heavy metals
solubilization from the mine tailings was Mn > Zn > As > Cu in
decreasing order. The high solubilization efficiency of Mn was
likely due to the fact that the occurrence of Mn is a mobile form in
the mine tailing and thus Mn is the most mobile metal in the mine
tailings. Besides the mobile forms of the heavy metals, a
significant amount of their stable forms could even be solubilized
by the bioleaching process.
From the results of the study, it was noted that the selection
of bacterial strains to be used for the bioleaching process should
depend on the kind of heavy metals that need to be recovered
from mine tailings for practical applications. To achieve
satisfactory solubilization and recovery efficiencies of metals
from the mine tailings, it is important to choose the proper
bacterial strains and to provide the optimum conditions for
bacterial growth.
Acknowledgment
The study was financially supported by a grant from Korea Mine
Reclamation Corporation, 2013.
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