Bioprocess Biosyst Eng (2012) 35:433440

DOI 10.1007/s00449-011-0582-3

ORIGINAL PAPER

Column bioleaching of low-grade mining ore containing high level

of smithsonite, talc, sphaerocobaltite and azurite
Sadia Ilyas Ruan Chi H. N. Bhatti
I. A. Bhatti M. A. Ghauri

Received: 4 July 2011 / Accepted: 8 August 2011 / Published online: 27 September 2011
 Springer-Verlag 2011

Abstract Present work describes the bioleaching potential of metals from low-grade mining ore containing
smithsonite, sphaerocobaltite, azurite and talc as main
gangue minerals with adapted consortium of Sulfobacillus
thermosulfidooxidans strain-RDB and Thermoplasma acidophilum. Bioleaching potential improved markedly by
added energy source, acid preleaching and adaptation of
microbial consortium with mixed metal ions. During whole
leaching period including acid preleaching stage of 960 h
and bioleaching stage of 212 days about 76% Co, 70% Zn,
84% Cu, 72% Ni and 63% Fe leached out.
Keywords Column bioleaching
Low-grade sulfide ore

Consortium of moderate thermophiles

Introduction
The continued depletion of high-grade ores, high cost of
fuel, strict environmental laws and energy intensive nature
Electronic supplementary material The online version of this
article (doi:10.1007/s00449-011-0582-3) contains supplementary
material, which is available to authorized users.
S. Ilyas
R. Chi (&)
Hubei Key Laboratory of Novel Reactor and Green Chemical
Technology, School of Chemical Engineering and Pharmacy,
Wuhan Institute of Technology, Wuhan 430073, China
e-mail: awns1@yahoo.com
H. N. Bhatti
I. A. Bhatti
Department of Chemistry and Biochemistry,
University of Agriculture, Faisalabad, Pakistan
M. A. Ghauri
National Institute for Biotechnology and Genetic Engineering
(NIBGE), Faisalabad, Pakistan

of pyrometallurgical processing has led to enhanced efforts

in recovering metals from low-grade ores [15]. To extract
metals from low-grade ores and refractory gold ores is a
multibillion dollar business worldwide. Millions of tones of
low-grade ores of varied nature wait to develop efficient
and economically attractive extractive process [68].
In China, Daye, Huangshi and Yangxin areas are rich in
copper sulfide minerals, with explored copper reserves totaling 3.6 million tons. Daye Nonferrous Metals group (DNMC)
owns six copper mines that are Tonglushan, Fengshandong,
Tongshankou, Chimashan, Dayetongchang and Xinye.
DNMC produced more than 20,000 tons of copper concentrate, 61,600 tons of crude copper, and 45,600 tons of electrolytic copper. It is a large mining, dressing, smelting and
processing complex [9]. However, a mass of copper containing discarded ore left during exhausting rich ore which
recognized to be anthropogenic and lethal to many kinds of
living beings including human beings. Because of highenergy demand, technical implementation and enforcement of
strict environmental laws, the existing strategies for highgrade ores are not probable for low-grade ores and there is a
need to choose an economically attractive process.
Biohydrometallurgy, an interdisciplinary field involving
geomicrobiology, microbial ecology, microbial biochemistry and hydrometallurgy, is a novel promising technology
for recovering valuable metals from discarded ores [10].
Metal-winning route based on the activity of microorganisms, offer a possibility to get metal ions from mineral
reserves not accessible by conventional techniques.
Microbes such as bacteria and fungi convert metal compounds into their water soluble forms and are biocatalysts
of microbial leaching or bioleaching [11, 12].
Because of rather high levels of smithsonite, sphaerocobaltite, azurite and talc present in the main gangue
minerals, there were some difficulties during bioleaching of

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ore. So shake flask bioleaching experiments carried out in

our previous studies and some leaching factors were optimized by Tagushi orthogonal array designing [13]. So far,
no report is available in literature for column bioleaching
of copper bearing sulfide ore containing rather high levels
of smithsonite, sphaerocobaltite, azurite and talc. Considering that column bioleaching treated as simulation of heap
bioleaching, the purpose of this work is to bioprocess Daye
low-grade copper-bearing sulfide ore using column reactor
and then test the technical possibility for heap bioleaching.

Bioprocess Biosyst Eng (2012) 35:433440

thermosulfidooxidans and Thermoplasm acidophilum.

S. thermosulfidooxidans was collected from Reko Diq
copper ore deposits [14], Pakistan. After isolation, purification, 16S rDNA gene amplification, sequencing and
homology checking (98%) by NCBI blast search, it was
submitted to GenBank for accession number (GQ228448).
While T. acidophilum was obtained from culture collection
of Wuhan Institute of Technology, Wuhan, China and its
origin was Xinjing coal deposits in China.
Culture conditions

Materials and methods

Mineral characteristics
The low-grade sulfide ore used in the column bioleaching
test was obtained from Daye, Hubei province of China.
Mineralogical analysis of the ore sample showed the
presence of main sulfide minerals as 8.2% bornite, 0.3%
Pentlandite, 0.8% chalcocite, 0.8% linnaeite, 0.3% wurtzite
and 0.3% sphalerite. The 20% smithsonite, 3% talc, 25%
sphaerocobaltite, 10% azurite were the main gangue
minerals.
Chemical leaching of ore
Finely powdered sample (1.0 g each) of low-grade copper
containing sulfide ore was refluxed with 100 mL of aqua
regia in round bottom flask for 1 h. The solution allowed to
cool at room temperature and filtered through filter paper
(Whatman No.42). The dissolved metals from ore sample
were determined by atomic absorption spectrophotometery
(varienAA10/20) and the percentage of these metals in the
sample was calculated.
Preparation of ore samples for bioleaching studies
Statistically representative samples of the ore were taken
and large pieces were crushed in a jaw-crusher. Then all
pieces were ground to smaller particles using disc-grinding
machine (FRITSCH Pulverisette, Germany). The final
grinding of the ore was carried out using ring grinder
(FRITSCH Pulverisette, Germany). ASTM Sieves were
used to separate the particles according to their sizes. Ore
sample used as column charge was screened to different
particle sizes and the fine ore particle size was controlled
less than 0.1 mm (mass fraction).
Microorganisms
Acidophilic moderately thermophilic bacterial cultures
were used in these studies. These included Sulfobacilllus

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Iron-tryptone soya broth (FeTSB) medium, developed by

Johnson et al. [15] was used with some modifications
to obtain and maintain the growth of S. thermosulfidooxidans. The FeTSB medium comprised of (g/L):
MgSO.47H2O, 0.50; (NH4)2SO4, 0.15; KCl, 0.05;
KH2PO4, 0.05; Ca(NO3)2, 0.01 and TSB, 0.25. The
solution pH was adjusted to 2.0 using sulfuric acid and
autoclaved at 121 C and 15 psi for 15 min. Filter sterilized ferrous sulfate solution was added to the solution to
a final concentration of 50 mM, before inoculation. After
gaining rich growth, the cell mass of cultures was harvested by centrifugation at 10,000 rpm for 20 min. The
cell pellet was washed twice with autoclaved distilled
water having pH adjusted at 2.0 with 2.0 M sulfuric acid
and finally suspended in sterilized distilled water and
preserved at 4 C for inoculation in the further experiments. The acidophilic heterotrophs was grown in the
same medium but supplemented with glucose (1% w/v)
instead of ferrous sulfate as energy source at pH 2.0. The
above mentioned liquid media were supplemented with
0.5% (w/v) agarose to prepare solid media.
Prestrategies for bioleaching test
Because of high-level of carbonaceous materials as main
gangue and oxidation of ore from surface, target ore was
pretreated with acid and mixed with 10% pyrite before
loading to column. For improved bioleaching potential,
adaptation of the consortium of moderate thermophiles to
mixed metal ions (Co, Zn, Cu, Ni, Fe) was performed
through serial subculturing and gradually increasing to
concentrate metal ions in the medium.
Bioleaching studies
First bioleaching parameters were determined in small
columns then several large columns of 85 cm height with
an internal diameter of about 15 cm were used to perform
bioleaching test. These columns were made of 30 lm thick
stainless steel. A HDP support plate with multiple 80 lm
holes was used for allowing air to be introduced below the

Bioprocess Biosyst Eng (2012) 35:433440

plate and dispersed uniformly over the ore in the columns.

A coating of support rock, low in carbonate and other acidconsuming minerals, sized 100120 lm, was placed in the
bottom of the columns before loading 80 kg charge. Column bioleaching was carried out under purely ambient
temperature conditions. Changes in temperature were noted
throughout the leaching test. Thermocouples were installed
to measure temperature changes of column charge, feed
solution and ambient air.
In preleaching test, pH of the ore was stabilized for
estimating acid consumption and preparation of the ore
surface for bacterial attack in the next bioleaching stage.
The charge was preleached with acidic solution at the rate
of 92 mL/min at different feed solution pH values and
particle sizes. Acidic solution was applied to the surface of
the column charge by using a garden sprinkler head and
allowed to pass through the washed charge by gravity and
recirculated through a side pipe with a peristaltic pump.
Containers with a capacity of 18 L collected the effluent
and injected feed solution in the columns. The pH of the
effluent was maintained after recirculation of required
amount of concentrated sulfuric acid, periodic removal of
effluent and addition of autoclaved water. After stabilization of pH of effluents at 22.2, irrigation of preleaching
was stopped. During this whole test permeability of columns, slumping, solution collection and preferential flows
were checked and parameters optimized.
During bioleaching studies each column fed with two
liters of autoclaved FeTSB medium having pH 2.0 was
adjusted with sulfuric acid. Then experimental column was
inoculated with 10% (v/v) inoculum of adapted consortium
with a cell density of about 3 9 107cells/mL. The feed
solution level was kept at an appropriate height by forcing
the air upwards through the column charge and clean air
provided through a rotameter.
Samples of effluents both from preleaching and bioleaching were collected periodically and analyzed for metal
ions concentration and the acid balance. When analysis of
leaching solution was marked to limit bioleaching, irrigation stopped to allow the column effluents to drain off and
the column contents were washed to remove remaining
metal ions. Column charge was washed with dilute sulfuric
acid solution followed by distilled water. Column then was
unloaded and the residues dried and prepared for final
analysis.
Sampling procedure
Effluents of preleaching and bioleaching were first filtered
through Whatman No. 1 filter paper to remove solid particles and then centrifuged at 10,000 rpm for 10 min to
remove bacterial cells and supernatant was analyzed for
Co, Zn, Cu, Ni and Fe ions concentration.

435

Analytical techniques
Free bacteria in solution were counted by direct counting
with phase contrast microscope using a counting chamber
(Neubauer). Soluble metal ions (Co, Zn, Cu, Fe and Ni) in
leached solution were analyzed using atomic absorption
spectrophotometer (Varian AA-400). The solid residues
were air dried and samples taken for chemical analysis and
X-ray diffraction (XRD). Ferric, ferrous and total iron
concentration in the solution were determined by spectrophotometeric method using 5-sulfosalicylic acid and 110
ortho phenanthroline [16]. The pH of the leaching solutions
was monitored at room temperature with a pH meter calibrated with a low pH buffer. The redox potential (Eh) of
the leaching solution was measured with a Pt electrode in
reference to a saturated Ag/AgCl electrode.
Results and discussions
Chemical analysis of Daye copper ore
Chemical analysis of the Daye ore used in these studies
was carried out to find out the concentrations of various
metal ions present in the material (Table 1).
Adaptation of microorganisms to metal ions
Effects of metal ions on the growth of moderately thermophilic chemolithotrophic bacteria before and after
adaptation were noted as shown in Fig. 1a and b. Before
adaptation bacterial culture grew well with 20 g/L mixed
metal ions, reproduced after a lag phase of 11 days with
20 g/L mixed metal ions and died during 37 days incubation period with 25 g/L mixed metal ions. While after
adaptation of nearly 1 year, bacterial cultures reproduced
well with 20 g/L mixed metal ions and gave rich growth
after a lag phase of 7 days with 25 g/L metal ions. With
28 g/L metal ions, bacterial cultures grew slowly with a lag
phase of 12 days and died with 30 g/L mixed metal ions
during an incubation period of 37 days as depicted in
Fig. 1b. It might be because of the reason that when the
microorganisms acclimatized to tolerate increasing concentration of metal ions by repeated subculturing on metal
Table 1 Chemical analysis of Daye ore
Metals

Concentration (% w/w)

Fe

7.2 0.04 0.05

Cu

8.0 0.04 0.03

Ni
Co

1.2 0.06 0.02

0.3 0.03 0.02

Zn

0.8 0.04 0.02

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Bioprocess Biosyst Eng (2012) 35:433440

bioleaching in the next stage. Some tests took more than

130 days to stabilize the pH at 22.2. These tests were
postponed also because of financial reasons. Other tests
performed for bioleaching and the results are listed in
Table 3.
The main gangue minerals were easily pulverized by
sulfuric acid. It can be seen from the results that smaller
particle size needed smaller time to extract metals however
lead to rapid pulverization of the ore but larger particle size
lead to better permeability however needed longer time so
both of these tests were excluded. The optimized leaching
conditions depend on the balance between these leaching
parameters. Irrigation and aeration rates were also optimized. The optimized irrigation rates were 6570 and
4548 L/m2/h for preleaching and bioleaching process
respectively. The optimized aeration rate was 180250 L/h.
Bioleaching studies

Fig. 1 a Effect of mixed metal ions on the growth of the consortium

of moderately thermophiles before adaptation. b Effect of mixed
metal ions on the growth of the consortium of moderately thermophiles after adaptation

containing media, there were physiological changes that

eased the bacteria to cope with the high metal concentration in the media during bioleaching. Many genetic systems
are known in bacteria for maintaining intracellular
homeostasis of essential metal ions and for getting resistance against toxic metals [17]. Among these, the wellstudied genetic mechanisms of metal resistance in bacteria
include heavy metal efflux systems [18] and the presence
of metal binding proteins [19, 20].
Bioleaching parameters optimization
The preleaching of the Daye ore at various particle sizes
and feed solution pH values were examined in small columns and the results are listed in Table 2.
Some tests ended because of solution accumulation in
columns (Table 2). These columns failed to perform

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When the leaching parameters were optimized in small

column reactors, experimental tests were performed using
large column reactors.
The preleaching pH and irrigation rate of feed solution
were adjusted at pH 0.8 and 6570 L/m2/h, respectively.
Effluents redox potential, pH of experimental test and
abiotic control are shown in Figs. 2 and 3.
It could be seen from Fig. 2 that effluents redox
potential remained in the range of 340450 mV. After
672 h of recirculation, pH started to decrease steadily and
after 960 h of recirculation, pH remained at almost 2.0 in
each column as shown in Fig. 3. This showed that preleaching process was a typical acid leaching process [21].
During this whole process permeability of columns
remained well and no slumping, solution accumulation and
preferential flows were found in columns and 46% Fe, 12%
Zn, 5.5% Cu, 15% Ni and 3.5% Co leached out.
Column bioleaching studies
The changes in pH values during 212 days of bioleaching
experiment are shown in Fig. 4. At first, slight increase in
pH was noted during first 32 days of bioleaching that
remained consistent with the delayed growth of bacteria
and depicted that the stage was dominated by chemical
acid leaching instead of bioleaching. Then from day 33, pH
started decreasing because of the hydrolysis of the metal
ions released in the medium because of microbial action
[22] and continued up to day 160 with pH 2.0. Trend of pH
marked that this stage was typically bioleaching stage
instead of chemical acid leaching. Then pH started to
decrease slowly and decreased to a value of 1.87 from day
161 to 185. Then no significant change in pH was noted till
the end of bioleaching test. So pH remained in the range of

Bioprocess Biosyst Eng (2012) 35:433440

437

Table 2 Optimization studies in small columns

pH 0.2

pH 0.4

pH 0.6

pH 0.8

pH 1.0

B20 lm

-(3 d)*

-(5 d)*

-(8 d)*

-(12 d)*

-(14 d)*

B50 lm

-(6d)*

-(8 d)*

-(13 d)*

-(18 d)*

-(20 d)*

B70 lm

-(10 d)
*

-(15 d)*

-(23 d)*

-(29 d)*

-(33 d)*

B100 lm

-(15 d)
*

-(27 d)*

-(33 d)*

-(35 d)*

?(40 d; Fe 30%, Cu 2.0%,

Ni 2.3, Zn 1.0%, Co
3.1%)**

B120 lm

-(23
d)*

-(32 d)*

-(39 d)*

?(44 d; Fe 45%, Cu 3.8%,

Ni 6.3, Zn 3.5%, Co
2.8%)**

?(65d; Fe 35%, Cu 2.9%,

Ni 3.3, Zn 2.0%, Co 3.5)**

B150 lm

-(27
d)*

-(40 d)*

?(55 d; Fe 37%, Cu 2.0, Ni

5.1%, Zn 2.2%, Co 3.5%)

(73 d; Fe 40%, Cu 2.5, Ni

5.3, Zn 2.2%, Co 3.5%)

?(77 d; Fe 36%, Cu 2.5%,

Ni 5.3, Zn 2.3%, Co
3.5%)**

B200 lm

-(39
d)*

?(67d; Fe 41%, Cu 1.9%,

Ni 2.0, Zn 2.0%, Co)

?(80 d; Fe 35%, Cu 3.5%,

Ni 4.3, Zn 3.0%, Co)

-(130 d)***

-(130 d)***

B300 lm

-(45
d)*

?(85 d; Fe 39%, Cu 2.8%,

Ni 4.0%, Zn 3.2%, Co
2.8%)

-(130 d)***

-(130 d)***

-(130 d)***

d days
* Pulverization of the ore by sulfuric acid led to an accumulation of solution in column and test was aborted after pre-leaching operation
?** Permeability of the ore kept well, no solution accumulation was found in the column during pre-leaching operation, considered for
verification test
*** pH of ore does not stabilize at 22.2 after 130 days of pre-leaching operation yet the permeability of ore kept well, test was aborted due to
economic reasons

Table 3 Selective bio-leaching studies in small columns

Column number

Metal extraction (%)

Particle
size B 100 lm

?(120 d; Fe 37, Cu 38, Ni 20, Zn 15, Co 12)

Pre-leaching pH 1.0
Particle size B 120
Pre-leaching pH 0.8

?(120 d; Fe 33, Cu 88, Zn 74, Co 79, Ni

75)**

Particle size B 120

?(210 d; Fe 32, Cu 85, Co 75, Ni 73, Zn 72)

Pre-leaching pH 1.0
Particle size B 150
Pre-leaching pH 0.6

?(250 d; Fe 35, Cu 79, Ni 75, Zn 75, Co 70)

Particle size B 150

?(310 d; Fe 35, Cu 68, Ni 70, Zn 70, Co 65)

Pre-leaching pH 0.8
Particle size B 150,

?(340 d; Fe 34, Cu 59, Ni 69, Zn 64, Co 56)

Pre-leaching pH 1.0
Particle size B 200

?(370 d; Fe 33, Cu 50, Ni 61, Zn 65, Co 48)

Pre-leaching pH 0.4
Particle size B 200

?(380 d; Fe 32, Cu 49, Ni 60, Zn 62, Co 43)

Pre-leaching pH 0.6
Particle size B 300

?(395 d; Fe 30, Cu 47, Ni 54, Zn 50, Co 45)

Pre-leaching pH 0.4
d days; ?** permeability of the ore kept well, no solution accumulation was found in the column

Fig. 2 Changes in redox potential during pre-leaching

1.852.6 throughout the bioleaching process. While in

abiotic control pH remained almost in the range of 3.33.9.
Iron species present in the effluents of two columns were
monitored and results have been presented in Fig. 5. It can
be seen from Fig. 5 that ferric concentration increased
slowly during first 32 days but total iron and ferrous concentration at the start increased in a linear pattern and

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Bioprocess Biosyst Eng (2012) 35:433440

Fig. 3 Changes in pH during pre-leaching

Fig. 5 Changes in total iron, ferrous and ferric during column
bioleaching

Fig. 4 Changes in pH during column bioleaching

Fig. 6 Changes in redox potential during column bioleaching

redox potential of effluents also increased slowly as shown

in Fig. 6, coinciding with low growth of bacterial cultures
and domination of acid leaching stage. Then from day 33 to
day 56 the ferric ions concentration started to increase
while ferrous ions concentration started to decrease linearly
and from day 66 to day 185 concentrating ferric ions
increased up to 1,690 mg/L (Fig. 5) and high value of
redox potential 652 mV was observed (Fig. 6). This trend
credited to bioleaching stage instead of acid leaching and
indicative of good bacterial activity as seen from Fig. 6.
Then from day 186 to 212 concentration of ferric ions
started to decrease a little and value of redox potential
decreased from 682 to 485 mV respectively, indicative of
regressive bioleaching and deceleration phase of bacteria.
Production of Fe3? ions during oxidation of iron containing

sulfide minerals resulted in more positive values of redox

potential of leach suspension [23].
The metal ions solubilization calculated from the analysis of the effluents during the whole leaching period is
shown in Fig. 7.
These dissolution trends of metals were different in
preleaching and bioleaching. Only a small quantity of zinc,
copper, cobalt and nickel dissolution was observed during
the pre-acid consuming test. These dissolution trends of
nickel and zinc were linear while copper and cobalt dissolved slowly but iron dissolved more than other metals
during this preleaching period.
During whole bioleaching process about 76, 70, 84,
72, 63% of Co, Zn, Cu, Ni and Fe leached out, respectively, with consortium of metal-adapted cultures of

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439

Pyrite addition acts as an added energy source and

contributes toward rich bacterial growth and effective
bioleaching process.
Acid preleaching of ore lessened the time required to
stabilize the pH and provide optimum conditions for
bacterial attack on ore surface and economical probability therefore depends upon the accessibility of cheap
acid and prestrategies before bioleaching operation.

Acknowledgments This work was supported by Changjiang

scholars and innovation research team in universities (IRT0974),
Higher Education Commission of Pakistan and National Institute for
Biotechnology and Genetic Engineering (Industrial biotechnology
division) of Pakistan.

Fig. 7 Percent metals solubilization during whole leaching process

S. thermosulfidooxidans and T. acidophilum with sulfuric

acid consumption of 342.5 kg/t of ore. These results suggested that dissolution of iron mainly contributed to acid
leaching but dissolution of copper and cobalt mainly contributed to bioleaching. Dissolution of nickel and zinc was
contributed both by acid leaching and bioleaching. High
leaching rates in present study were because of the synergistic effects of acidophilic heterotrophs on the growth of
S. thermosulfidooxidans. Furthermore, Ni, Cu, Co, Zn and
Fe leaching rates no longer increased from day 186 to 212,
pointing to wind up bioleaching test (Fig. 7).
In case of uninoculated control the percent leach abilities were significantly low about 5.2, 13.8, 6.3, 15.9 and
49.3% for Co, Zn, Cu, Ni and Fe, respectively. This pattern
showed that the leaching of metal ions from the low-grade
sulfide ore of copper because of chemical oxidation was
insignificant and major contribution was from bioleaching
for dissolution of metals.

Conclusions

During whole leaching period including acid preleaching stage of 960 h and bioleaching stage of
212 days about 76% Co, 70% Zn, 84% Cu, 72% Ni and
63% Fe leached out with mixed consortium of metaladapted cultures of S. thermosulfidooxidans and T. acidophilum with sulfuric acid consumption of 342.5 kg/t
of ore at column reactor level.
Preadaptation of the microorganisms to high metal ions
concentration improves the metal solubilization rates as
they can survive under high concentration of metal ions.
Using consortia of moderate thermophiles also improve
bioleaching process because of the synergistic affects
of acidophilic heterotrophs on the growth of
S. thermosulfidooxidans.

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