Hydrometallurgy 127-128 (2012) 99103

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Hydrometallurgy
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Technical note

Effect of agitation intensity on the biooxidation process of refractory gold ores by

Acidithiobacillus ferrooxidans
Li-Xin Sun, Xu Zhang , Wen-Song Tan, Ming-Long Zhu
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Various views persist about the effect of the agitation intensity on the mineral biooxidation process, but the
Received 21 January 2012 mechanism remains unclear. In this study, the effects of the agitation intensity on the biooxidation process of
Received in revised form 12 July 2012 refractory gold ores by Acidithiobacillus ferrooxidans were investigated in experimental stirred tank bioreac-
Accepted 16 July 2012
tors (STRs). The results demonstrated that both the biooxidation rates and the concentration of cells de-
Available online 1 August 2012
creased with the agitation intensity. The higher agitation intensity not only damaged the suspended cells
Keywords:
but also prevented the adsorption of cells onto the mineral surface. The oxygen uptake rate (OUR), the
Refractory sulde gold ores Fe 2+ oxidation activity and the rus gene expression of A. ferrooxidans decreased with the agitation intensity
Agitation intensity during the biooxidation process, which might be responsible for the decrease of the biooxidation rates with
Biooxidation process the agitation intensity.
Acidithiobacillus ferrooxidans 2012 Elsevier B.V. All rights reserved.

1. Introduction
Biooxidation of suldic-refractory gold minerals is primarily applied
for the pretreatment of refractory sulde gold ores. Currently, the com-
mercial biooxidation process has been successfully used in the gold pro-
cessing (Dew et al., 1997). The stirred tank reactors (STRs), which
equipped with a suitable impeller, are widely used for the biooxidation
of sulde minerals at the industrial or laboratory scale due to their tech-
nical and economical advantage (Rossi, 2001).
Agitation in biooxidation reactors plays important roles, for exam-
ple: to obtain a high degree of homogeneity for the gasliquidsolid
system; to enhance the mass transfer rates of the oxygen and carbon
dioxide from the gas to the liquid phase; to enhance the heat transfer
rate to a certain level, which is necessary to keep operation tempera-
ture within the recommended range. Therefore, increased agitation
intensity could improve the mass transfer rates and accelerate the
dissolution of mineral in the bioreactor (Wang et al., 2010).
However, the microbial growth and the biooxidation rates would be
inhibited when the agitation intensity beyond a certain level (Oolman,
1993). It was suggested (Doran, 1995; Thomas, 1990) that the exposure
of microorganisms to a strong shear environments could result in the
lysis of cells, the inhibition of biomass growth or product synthesis, the
denaturation of extracellular proteins, the change in morphology, the
thickening of the cell wall, the impediment of the attachment of bacteria
to solid substrate, etc. d'Hugues et al. (1997) found that the excessive tur-
bulence, which associated with high agitation intensity during the pyrite
biooxidation process, would affect the efciency of the biooxidation by
Corresponding author. Tel.: +86 21 64252536; fax: +86 21 64252250.
E-mail address: zhangxu@ecust.edu.cn (X. Zhang).
limiting the bacterial productivity and reducing the contact chance
between the bacterial and solid substrate. Deveci (2002) showed that
during the biooxidation process in STRs, the loss of viability of the bacte-
rial cells was mainly resulted from the mutual friction between the solid
particles. Still, the mechanism of the effects of the agitation intensity on
the biooxidation process remains unclear.
In this work, the effects of the agitation intensity on the refractory gold
ores biooxidation rates, the growth and the activity of Acidithiobacillus
ferrooxidans, the Fe2+ oxidation activity, and the expression of rus gene
were investigated in the experimental STRs, respectively.
2. Material and methods
2.1. Sample
The low-grade refractory gold ores used in the experiments were
kindly supplied by Tiancheng Co. Ltd., Shandong, P.R. China, contained
24.9 g/t Au, 96.1 g/t Ag, 21.30% Fe, 20.09% S, 7.74% C, 4.59% As, 0.02%
Cu, 0.31% Pb and 0.13% Zn. X-ray diffraction (XRD) analysis showed
that the sample contained pyrite and quartz as the major mineral
components.
2.2. Microorganism and culture medium
The strain of A. ferrooxidans was also provided by Tiancheng Co. Ltd.
The culture medium (iron-free 9 K medium) was developed from the
9 K medium (Silverman and Lundgren, 1959). The composition was
as follows (g/L): (NH4)2SO4: 3.0; KCl: 0.1; K2HPO4: 0.5; MgSO4 7H2O:
0.5; Ca(NO3)2: 0.01; FeSO47H2O: 44.2. All chemical reagents used
were of analytical grade.

0304-386X/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.hydromet.2012.07.007
100 L.-X. Sun et al. / Hydrometallurgy 127-128 (2012) 99103
2.3. Batch biooxidation experiments
The batch biooxidation experiments were carried out in three 1.5 L
STRs with a working volume of 1 L, and the medium composition was
as follows: 900 ml iron-free 9 K medium, 100 ml inoculum and 100 g
ores (105130 m) as energy source. The agitation was provided by a
six-bladed 45o downward pitched blade turbine (PBT) impeller. A
sparger was situated below the impeller. The aeration rates of dry air
were measured by a gas rotameter and controlled by a valve attached
to the rotameter. The biooxidation conditions were as follows: 41 C,
pH 1.5, the impeller speeds in STRs were 300, 500 and 700 rpm, respec-
tively. The aeration rates were adjusted from 0.02 to 2 L/min, to keep
the DO concentration at 3.1 ppm during the biooxidation process for
all the experiments.
2.4. Bacterial adsorption experiment
Cells in the exponential growth phase were collected and suspended
in 1 L iron-free 9 K medium (41 C, pH 1.5) in three 1.5 L STRs working
at impeller speeds of 300, 500 and 700 rpm respectively. Then the min-
eral sample (100 g) was pulp in the cell suspension and start timing.
The samples were withdrawn at regular intervals and centrifuged at
2500 rpm for 1 min to settle the mineral particles. The cell number in
the supernatant was recorded. The difference between the cell number
before and after adsorption was assumed to be the number of adsorbed
cells on the mineral surface.
2.5. Analytical methods
The pH and the DO concentration of the suspension were measured
by a pH meter (Mettler model FE20) and a DO meter (OXYFERM 225,
Hamilton). Mineral dissolution was followed by measuring the iron
concentration in solution. The concentration of Fe 2 + was deter-
mined by titration with potassium dichromate in the presence of
N-phenylanthranilic acid as an indicator (Vogel, 1961). The Fe 3+ was
determined by titration EDTA at pH 2 in the presence of sulphosalicylic
acid as an indicator (Davis and Jacobsen, 1960). Total iron concentration
in the liquid phase was the summation of both above. The OUR was
measured using the dynamic gas-out/gas-in method (Bandyopadhyay
et al., 1967). The amount of suspended cells in solution was measured
by direct microscopic counting using a Pettrof-Hauser-type cell counter.
The ores oxidation ratio () and oxidation rate () were calculated
as follows (Gleisner et al., 2006):
 
Feti Fet o =Fep  100% 1
    higher at 700 rpm than that at 300 rpm. These results indicate that the
Fet j Fet i = t j t i 2 biooxidation process was inhibited at the agitation intensity of 700 rpm.
where [Fe]ti and [Fe]tj were the iron concentrations in the liquid at two
consecutive time points, [Fe]t0 and [Fe]p were the iron concentrations in
the liquid and in the ores before biooxidation respectively.
2.6. Fe 2+ oxidation activity
The Fe 2+ oxidation activity of A. ferrooxidans, at different agitation
intensity was determined by measuring the Fe 2+ oxidized in the
reaction mixture under aerobic conditions (Sugio et al., 2007). The re-
action mixture was composed of 2 ml 0.1 M alanine-SO42 buffer (pH
3.0), washed intact cells, and 1 ml ferrous sulfate (CFe 2+
= 0.2 g/L).
The total volume was 3.0 ml. The reaction was carried out by shaking
the reaction mixture at 41 C. A sample of the reaction mixture
(0.2 ml) was withdrawn every 15 s and the concentration of Fe 2+
was determined spectrophotometrically by the o-phenanthroline
method (Saywell and Cunningham, 1937). Protein concentration
was measured by the method of Bradford (1976) using crystalline
bovine serum albumin as the reference protein. The Fe 2+ oxidation
activity was dened as the decrease of ferrous ion concentration per
second per gram of dry cells.
2.7. Quantitative real-time PCR
The relative expression levels of rus gene of A. ferrooxidans grown at
different agitation intensity were determined by real-time PCR, using
the Mx3000 Real-Time PCR System (Stratagene, America). Cells were
collected during the exponential phase by ltration through a 0.22 m
pore size membrane and the total cellular RNA was extracted using
the TRIzol reagent (TianGen Biotech, Beijing) according to the
manufacturer's instructions.
The synthesis of cDNA from RNA samples was performed using
the 1st Strand cDNA synthesis kit (MBI, America), according to the
manufacturer's instructions. Then reaction mixtures were incubated
at 50 C for 30 min, and the reaction were ended by heating at 85 C
for 5 min.
Quantitative RT-PCR experiments were performed with the Mx3000
Real-Time PCR System and the Maxima SYBR Green q-PCR Master Mix
Kit (MBI, America) were used. The reaction mixture contained 12.5 l of
SYBR Green PCR Master Mix (Biotools), 1 l of cDNA, 0.5 l of each
primer, nuclease-free water added to a total of 25 l. The amplication
program consisted of 1 cycle of 95 C for 5 min, and then 40 cycles of
95 C for 30s, 58 C for 60s and 72 C for 60s. The real-time PCR primers
were 5-CGCGATGGCCGGTACTCTGGA-3 and 5-CCGCCGCGACCACATG
TACAGT-3 for the rus gene, 5-AATCCAAGAAGAAGCACCG-3 and 5-CCA
CTGATGTTCCTCCAG-3 for the 16S rRNA (house-keeping gene).
The threshold cycle (Ct) values were determined using the curve
analytical software of the Mx3000 (Stratagene, American). The fold
change in the target gene was determined by the formula 2 Ct
(Schmittgen and Livak, 2008), where Ct = (Ct from the experi-
mental condition) (Ct from control). The Ct was calculated by
the difference between the Ct from the target gene and that from
the reference gene (16S rRNA).
3. Results and discussion
3.1. Effects of agitation intensity on the mineral oxidation
The concentration of Fe 2+ and Fe 3+ in solution and the ores oxi-
dation ratio during the mineral biooxidation process at various agi-
tation intensity are shown in Figs. 13. It is found that the concentration
of Fe3+ and the oxidation ratio at the agitation speed of 700 rpm were
lower than those at 300 rpm. However, the concentration of Fe2+ was
The effects of the agitation intensity on the biooxidation rates ()
are shown in Fig. 4. It is illustrated that the oxidation rates increased
during the rst 150 h and then decreased. On the other hand, the
biooxidation rates decreased with the agitation intensity. The highest
oxidation rate was about 0.1 g/L/h at 300 rpm, but decreased to
0.066 g/L/h at 700 rpm. Grifn and Luinstra (1989) also reported
that the increase in the agitation intensity would lead to the decrease
of the biooxidation rates using mesophilic cultures of acidophilic
bacteria.
3.2. Effects of agitation intensity on the concentration of suspended and
adsorbed cells
The effect of the agitation intensity on the growth of suspended cells
during the biooxidation process is shown in Fig. 5. It can be seen that the
concentration of suspended cells increased initially and after 185 h
reached a plateau. Besides, the concentration of suspended cells de-
creased as the agitation intensity increased from 300 to 700 rpm. It
 L.-X. Sun et al. / Hydrometallurgy 127-128 (2012) 99103
Fig. 1. Evolution of the concentration of Fe2+ in the biooxidation process at various
agitation intensity.
seems that the higher agitation intensity would damage the suspended
cells.
The effect of the agitation intensity on the adsorption process be-
havior of A. ferrooxidans on the mineral at an initial cell concentration
of about 5.0 10 8cell/ml is in Fig. 6. It can be seen that the concentra-
tion of adsorbed cells increased rstly, then kept at constant at all of
the agitation intensity. The adsorption equilibrium was all obtained
after about 45 min. However, the cell density on the mineral surface
at 700 rpm was less than that at 300 rpm. The equilibrium cell con-
centration was about 4.8 10 8cell/g at 300 rpm and decreased to
4.28 10 8cell/g at 700 rpm. It is suggested that the adsorption of
A. ferrooxidans onto the surface of mineral was restrained by the
higher agitation intensity. While it was known that the adsorbed
cells could play an important role in biooxidation process (Harneit
et al., 2006). Thus, this may be one of the reasons for the decreased
of biooxidation rate at the higher agitation intensity.
3.3. Effect of agitation intensity on the metabolic activity of A. ferrooxidans
The oxygen uptake rate (OUR), which can reect the metabolic
activity of the cells in the culture, has been used for optimizing the fer-
mentation process (Garcia-Ochoa et al., 2010). The OUR of bacteria was
based on the presumption that only the active cells would oxidize fer-
rous iron and hence consume oxygen (Deveci, 2002). Fig. 7 shows
that the effect of agitation intensity on the OUR of A. ferrooxidans during
Fig. 2. Evolution of the concentration of Fe3+ in the biooxidation process at various
agitation intensity.
101
Fig. 3. Evolution of the ores oxidation ratio in the biooxidation process at various
agitation intensity.
the biooxidation process. It can be seen that OUR increased at the bacte-
rial exponential growth phase, reached the maximum at the late period
of cell growth and then decreased. Besides, the OUR decreased with the
agitation intensity. The maximum OUR was about 0.036 mg/L/s at
300 rpm, and decreased sharply to 0.026 mg/L/s at 700 rpm.
It is well known that the increasing of the agitation intensity could
improve the mass transfer rates in STRs. However, the high agitation
intensity will damage the cell. From the study of Deveci (2002, 2004)
and Liu et al. (2007), it was conrmed that the activity of cells would
decrease due to the collision and friction produced by solid particles.
The 9 K culture experiments of A. ferrooxidans with and without glass
beads at different agitation intensity had been carried out in experi-
mental STRs (the data were not shown). And the results were consis-
tent with the reports of Deveci and Liu. While the increase in the
agitation intensity would give rise to the probability and frequency
of the collisions between the particles (Cherry and Papoutsakis,
1986), which will ultimately cause the extent of the cell damage
and the loss of bacteria metabolic activity. Therefore, the OUR of
A. ferrooxidans during the biooxidation process decreased with the
agitation intensity.
3.4. Effect of agitation intensity on the Fe 2+ oxidation activity and the
expression of rus genes of A. ferrooxidans
In aerobic conditions, A. ferrooxidans obtains energy from the oxi-
dation process of ferrous ion to ferric ion using molecular oxygen as
the terminal electron acceptor in respiratory chain (Ingledew, 1982).
Fig. 4. Evolution of the biooxidation rates at various agitation intensity.
102 L.-X. Sun et al. / Hydrometallurgy 127-128 (2012) 99103
Fig. 5. Evolutions of the suspended cell concentration at various agitation intensity.
Meanwhile, cells also get reducing power derived from the respiratory
chain. Both of them are necessary for many subsequent metabolic pro-
cesses, including CO2 xation (Quatrini et al., 2006).
The Fe 2+ oxidation activity of A. ferrooxidans could reect the
electron transport rate of respiratory chain. The Fe 2+ oxidation activ-
ity of A. ferrooxidans during the exponential growth phase was deter-
mined and shows in Fig. 8. The results indicate that the Fe 2+
oxidation activity decreased with the agitation intensity. And it was
almost 45% higher at a speed of 300 rpm than that at 700 rpm. The
higher Fe 2+ oxidation activity, the higher electron transport rate of
respiratory chain. Namely, bacteria could get the more energy and re-
ducing power at lower agitation intensity, resulting in an increase in
the metabolic activity of cells.
Various redox proteins involved in electron transfer process had
been isolated and characterized in A. ferrooxidans. Among these,
rusticyanin (encoded by rus gene), which is a 16.5-kDa periplasmic
copper protein, has received a special attention, since it might consti-
tute as much as 5% of the total soluble proteins in A. ferrooxidans dur-
ing autotrophic growth on iron (Cox and Boxer, 1978). It has been
suggested that rusticyanin probably functions as an electron reservoir
in such a way that it readily took up electrons available at the outer
membrane and channeled them down the respiratory pathway.
The study of Yarzbal et al. (2003) shown that the iron oxidation
took place only when the rusticyanin content was high enough inside
the cell. Liu et al. (2010) also found that overexpressing the rus gene
by generating the engineered strain of A. ferrooxidans (pTRUS) could
Fig. 6. Concentration of adsorbed cells on mineral at various agitation intensity.
Fig. 7. Evolution of the OUR during the biooxidation at various agitation intensity.
improve the Fe 2+ oxidation activity. In order to study the mechanism
of the agitation intensity on the Fe 2+ oxidation activity, the effect of
the agitation intensity on the rus gene expression of A. ferrooxidans
was investigated. The relative transcription levels of the rus gene
are shown in Fig. 9. The expression of the rus gene at 300 rpm was
about a 17-fold higher than that at 700 rpm. Therefore, the rus gene
expression decreased with the agitation intensity, which might be re-
sponsible for the decrease of the Fe 2+ oxidation activity.
4. Conclusions
In this study, the effects of the agitation intensity on the refractory
gold ores bioxidation process by A. ferrooxidans were investigated in
experimental STRs. The results demonstrated that the concentration
of Fe 3 +, the oxidation ratio and the biooxidation rates decreased
with agitation intensity. However, the concentration of Fe 2+ was
higher at 700 rpm than those at 300 rpm. The biooxidation process
by A. ferrooxidans was restricted at the high agitation intensity. The
higher agitation intensity would damage the suspended cells and pre-
vent the adsorption of cells onto the mineral surface, which would
lead to the reduction in the concentration of suspended and adsorbed
cells. The OUR, the Fe 2+ oxidation activity and the rus gene expres-
sion of A. ferrooxidans during the biooxidation process decreased
with the increase of the agitation intensity. The maximum OUR was
about 0.036 mg/L/s at 300 rpm, and decreased sharply to 0.026 mg/L/s
at 700 rpm. The Fe2+ oxidation activity and the rus gene expression
were 1.45-fold and 18-fold at 300 rpm as high as those at 700 rpm,
respectively. They all might be responsible for the decrease of the
biooxidation rate with the agitation intensity.
Fig. 8. The Fe2+ oxidation activity of A. ferrooxidans during the exponential growth
phase at various agitation intensity.
 L.-X. Sun et al. / Hydrometallurgy 127-128 (2012) 99103
Fig. 9. Effect of agitation intensity on the expression of the rus gene during the
biooxidation of A. ferrooxidans (rus gene expression at 700 rpm condition was desig-
nated as control).
Acknowledgments
This study was nancially supported by the Open Project Funding of
State Key Laboratory of Bioreactor Engineering of China and the National
High Technology Research and Development Program of China (No.
2007AA060904 and No. 2012AA061503).
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