Journal of Bioscience and Bioengineering

VOL. 121 No. 5, 536e542, 2016

www.elsevier.com/locate/jbiosc

Adaptation of a mixed culture of acidophiles for a tank biooxidation of refractory

gold concentrates containing a high concentration of arsenic

Jeongsik Hong,1, z Rene A. Silva,1, z Jeonghyun Park,1 Eunseong Lee,1 Jayhyun Park,2 and Hyunjung Kim1, *

Department of Mineral Resources and Energy Engineering, Chonbuk National University, 567 Baekje-daero, Deokjin-gu, Jeonju, Jeonbuk 561-756, Republic of Korea1 and R&D Team,
Institute of Mine Reclamation Corporation, Coal Center, 30 Chungjin-dong, Jongno-gu, Seoul 110-727, Republic of Korea2

Received 29 January 2015; accepted 11 September 2015

Available online 23 October 2015
We adapted a mixed culture of acidophiles to high arsenic concentrations to confirm the possibility of achieving more
than 70% biooxidation of refractory gold concentrates containing high arsenic (As) concentration. The biooxidation
process was applied to refractory gold concentrates containing approximately 139.67 g/kg of total As in a stirred tank
reactor using an adapted mixed culture of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. The per-
centage of the biooxidation process was analyzed based on the total As removal efficiency. The As removal was moni-
tored by inductively coupled plasma (ICP) analysis, conducted every 24 h. The results obtained with the adapted culture
were compared with the percentage of biooxidation obtained with a non-adapted mixed culture of A. ferrooxidans and
A. thiooxidans, and with their respective pure cultures. The percentages of biooxidation obtained during 358 h of re-
action were 72.20%, 38.20%, 27.70%, and 11.45% for adapted culture, non-adapted culture, and pure cultures of
A. thiooxidans and A. ferrooxidans, respectively. The adapted culture showed a peak maximum percentage of bio-
oxidation of 77% at 120 h of reaction, confirming that it is possible to obtain biooxidation percentages over 70% in gold
concentrates containing high As concentrations.
Ó 2015, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Tank biooxidation; High arsenic concentration; Gold concentrates; Acidithiobacillus ferrooxidans; Acidithiobacillus thiooxidans]

Biooxidation is based on the ability of microorganisms to
separate the valuable metal from the refractory concentrate by
solubilizing the undesirable minerals. The solubilization of the
undesirable minerals, such as sulfide minerals (such as FeAsS and
FeS2), promotes the increase of the concentration of the valuable
metal (e.g., gold) and facilitates the interaction between the gold
and its extracting medium (1e3). It has been demonstrated that
depending on the process temperature, the degree of oxidation of
the mineral matrix (i.e., total arsenic released) generated by the
bacteria is directly proportional to the gold recovery in the subse-
quent processes (e.g., cyanidation) (4), and that more than 70% of
oxidation of the mineral matrix, can increase the gold recovery
yield up to more than 90% (5,6).
The most common bacteria used in the biooxidation process
include iron (II) and sulfur-oxidizing bacteria (Acidithiobacillus fer-
rooxidans), sulfur-oxidizing bacteria (Acidithiobacillus thiooxidans),
iron(II)-oxidizing bacteria (Leptospirillum ferrooxidans), and several
others such as Acidithiobacillus caldus, Sulphobacillus sp. and Fer-
roplasma sp. (7e9). Bacteria have the ability to generate oxidizing
agents such as iron (III) ions and/or protons in the system. This
generation of oxidizing agents serves as a preliminary step for the
biooxidation mechanism (10,11). Biooxidation occurs under the
same extraction mechanism as bioleaching (i.e., contact, non-
* Corresponding author. Tel.: þ82 63 270 2370; fax: þ82 63 270 2366.
E-mail address: kshjkim@jbnu.ac.kr (H. Kim).
z
The first two authors contributed equally to the work.
contact, and cooperative mechanism); however, unlike bio-
leaching, during biooxidation processes, the valuable metal is not
solubilized. Previous studies have shown that the non-contact
mechanism is likely to be the most influential mechanism during
bioleaching processes and thus the biooxidation process (12e16).
Due to the high influence of the non-contact mechanism, the pro-
duction of the oxidizing agent (i.e., Fe3þ) is important before the
addition of gold concentrates. The highest generation of oxidizing
agent in the solution can be observed by a stabilization in the
oxidation-reduction potential (ORP) in pure cultures of iron-
oxidizing bacteria and stabilization in the pH in pure cultures of
sulfur-oxidizing bacteria (17e19).
Previous studies reported that mixing different kinds of bacteria
produced synergistic effects that were capable to improving the
bioleaching efficiency for metals such as Cu, As, Fe, and Zn (20e23).
Among the previous reports, Nguyen et al. (22) and Zhang et al. (23)
reported that As leaching efficiency in mine tailings and As-bearing
sulfide minerals with mixed cultures was enhanced, as compared
with the pure cultures, respectively (22,23).
A. ferrooxidans has been shown to be tolerant to several heavy
metals (24,25). However, it has been proposed that As is a toxic
element that can affect bacterial growth and leaching efficiency
(17,26). Previous studies have conducted succesfully the adaptation
of acidophiles to high concentrations of As (up to 35,000 ppm) to
improve As extraction efficiency (27e30). Nevertheless, the range
of the concentration of As in gold concentrates is variable and not
limited to the concentrations used in previous studies (5); thus,

1389-1723/$ e see front matter Ó 2015, The Society for Biotechnology, Japan. All rights reserved.
http://dx.doi.org/10.1016/j.jbiosc.2015.09.009
VOL. 121, 2016
TABLE 1. Chemical composition of the gold concentrates from the Terek-Sai mine in
Kyrgyzstan (g/kg).
As Cu Pb Fe S Zn
139.67 4.43 0.18 27.50 102.11 1.98
more systemic studies on the adaptation of acidophiles to higher
concentrations of As in gold concentrates are required.
In the present study, the adaptation of a mixed culture of
A. ferrooxidans and A. thiooxidans was conducted to obtain a mixed
culture bacteria that is capable of oxidizing >70% of the refractory
gold concentrate containing a high concentration of As (approxi-
mately 139.67 g/kg). Previous studies have stated a linear rela-
tionship between the percent of biooxidation and As removal, and a
direct relationship with an improvement in gold recovery (5,6). It
has been stated that biooxidation of >70% of the gold concentrates
can increase the gold recovery up to 90% in subsequent gold re-
covery processes (5). The results obtained with the adapted culture
were compared with the biooxidation yield obtained using non-
adapted pure and mixed cultures of A. ferrooxidans and
A. thiooxidans. The biooxidation experiments were conducted in a
stirred tank reactor with continuous air injection. The addition of
gold concentrates to the pure culture of A. ferrooxidans and mixed
culture was conducted after stabilization of ORP in the solution. In
the pure culture of A. thiooxidans the addition of gold concentrates
was conducted after stabilization of the pH and ORP. During the
biooxidation experiments, changes in pH, ORP, concentration of Fe
in bulk solution [i.e., total iron (Fe), ferric ion (Fe3þ), and ferrous ion
(Fe2þ)], were evaluated to understand and confirm their correlation
with biooxidation processes.
MATERIALS AND METHODS
Gold concentrates The gold concentrates were obtained from the Terek-Sai
gold mine in Kyrgyzstan, with a particle size determined to be 74 mm (AccuSizer 780/
SIS, PSS-NICOMP). Once the gold concentrates were dried out to constant weight and
room temperature, approximately 2 kg of sample were placed in a closed 1 L glass
bottle for a three series of autoclaving at 121 C and 0.12 MPa during 30 min (AC-
12, Jeio Tech) to eliminate the effect of any indigenous bacteria (28). XRD analyses
were conducted before and after the sterilization to discard any major
modification in the chemical structure of the concentrates (data not shown). The
XRD measurements confirmed that the gold concentrates used for the
biooxidation process were composed mainly of quartz (SiO2), FeS2, and FeAsS. The
XRD spectra were collected within the 2q range from 10 to 50 , with a 0.01 step
and 1 s/step1 counting time, on Bruker D8 HRXRD (Germany), with Cu Ka
radiation (l ¼ 0.154606 nm, 40 kV, 40 mV), and a SolX detector. To measure the
heavy metal concentration, inductively coupled plasma (ICP, Optima7300DV,
PerkinElmer) analysis was conducted. According to the ICP analysis, the As
concentration was determined to be 139.67 g/kg, which was higher than that of
other heavy metals (Cu, Pb, Zn; Table 1).
Bacterial cultures In the present study, A. ferrooxidans was used as an iron-
oxidizing bacteria (KCTC 4516), and A. thiooxidans (KCTC 4515) as the sulfur-
oxidizing bacteria. The two bacterial species were provided by the Korea Research
Institute of Bioscience and Biotechnology (KRIBB). DSMZ medium 882 was used to
culture A. ferrooxidans, and A. thiooxidans was cultured in JCM medium 93. DSMZ
medium 882 was sterilized at 112 C, while JCM medium 93 at 121 C during
30 min as proposed by the standardized methodology of each culture medium.
The bacteria were cultured up to the stationary growth phase (in the case of
A. ferrooxidans, at 150 rpm and 25 C for 48 h, and in the case of A. thiooxidans, at
150 rpm and 30 C for 72 h), in an aerobic environment. Bacteria were
concentrated by centrifugation (8240 g, 10 min, 4 C) after stationary phase was
reached. The bacterial concentrations were determined using a counting chamber
(Buerker-Tuerk Chamber, Marienfeld Laboratory Glassware, Germany) under a
phase-contrast microscopy (ODEO-2003 Triple, Iponacology).
Stirred tank reactor experiments The As removal efficiency was used to
obtain an indirect percentage of the biooxidation of gold concentrates containing
high amounts of As (139.67 g/kg) (5,6). The As removal efficiency was analyzed using
pure cultures of A. ferrooxidans, A. thiooxidans, a mixed culture, and an adapted
mixed culture of them. The biooxidation experiments were conducted in
triplicated at 5% (w/v) of pulp density using a tank reactor (Pyrex 1 L reactor)
with 800 mL of working volume (Fig. 1). The stirring speed and temperature were
TANK BIOOXIDATION OF GOLD CONCENTRATES 537
FIG. 1. Schematic representation of the tank bioreactor used in the present study (Pyrex 1 L
reactor with 800 mL of working volume, air injection 5 L/min, and 400 rpm at 30 C).
kept constant at 400 rpm and 30 C using an agitator and a constant-temperature
water bath, respectively. The initial pH was adjusted to 1.8, and the air injection
rate to 5 L/min. The bacterial concentrations of A. ferrooxidans and A. thiooxidans
were adjusted to 5  108 cells/mL for pure culture experiments, and a
combination of 2.5  108 cells/mL of each culture (i.e., 5  108 cells/mL total) for
mixed culture. For all conditions, the biooxidation experiments were conducted in
triplicate. The addition of the concentrates to the tank reactor was conducted
after the stabilization of the growth of the microorganisms. The time for the
stabilization of the pure culture of A. ferrooxidans and the non-adapted mixed
culture (i.e., A. ferrooxidans and A. thiooxidans) was previously determined in
separated experimental runs (data not shown) where the microbial stabilization
was identified after the stabilization of the solution ORP at potentials higher than
600 mV (4). Meanwhile the time identified for the microbial stabilization in pure
culture of A. thiooxidans was identified after the stabilization of the solution pH
and ORP (31). The stabilization period for the pure cultures and the non-adapted
mixed culture was intended to increase the concentration of oxidizing agent in
the biooxidation solution, and hence, obtain higher extraction efficiencies in
shorter periods of time (32,33).
The pH and ORP of the reaction solution were measured at intervals of 24 h with
a Hanna HI 2211 pH/ORP meter. To evaluate the changes in the concentration of As,
Fe, and S, aliquots of 2 mL of the solution were collected every 24 h and filtered
through a syringe filter (0.45 mm) for ICP analysis. The concentration of Fe2þ was
measured by UV spectrophotometry (HS-3300, Humas) using the o-phenanthroline
method (34). The Fe3þ concentration level of the solution was determined by
calculating the difference between total Fe and Fe2þ. For the pure culture of
A. thiooxidans and the mixed culture of A. ferrooxidans and A. thiooxidans, 3.8 g of
sulfur were added to the culture medium as an initial energy source of the bacteria.
To evaluate the development of the biooxidation reaction the concentration of SO24
was measured instead of the Fe2þ concentration (35). The SO2 4 was obtained with
the analysis proposed by the American Public Health Association (APHA) (36).
Bacterial adaptation The adaptation of each bacteria was carried out sepa-
rately in DSMZ medium 882 in the presence of FeSO4,7H2O and elemental sulfur as
an energy source, and gold concentrates as the source of As. The bacterial adaptation
followed the procedures of Wang et al. (27), consisting in four different subculture
sets in which the addition of the energy source decreased (20, 10, 5, and 0 g of
FeSO47H2O; and 3, 1.5, 0.5, and 0 g of elemental sulfur, respectively) while the
addition of gold concentrate increased (0, 5, 10, and 10 g of gold concentrate) (27).
Each subculture was cultivated three times until the bacteria reached a stationary
phase and a stable solution pH and ORP. After stabilization of each subculture, it
was used as inoculum for the following subculture, until the final subculture
containing the highest solid concentration (i.e., 10 g) and lowest energy source
(i.e., 0 g of FeSO47H2O and elemental sulfur). The bacterial adaptation was
conducted under constant aeration and agitation, fixed temperature of 30 C, and
a constant agitation speed of 200 rpm. After the bacterial culture was confirmed
538 HONG ET AL.
FIG. 2. As removal behavior from the refractory gold concentrates over time using
adapted (diamonds) and non-adapted (triangles) mixed cultures of A. ferrooxidans and
A. thiooxidans, and their respective pure cultures (squares and circles, respectively).
The biooxidation experiments were conducted in triplicate at 30 C, pulp density of 5%
(w/v), and initial pH of 1.8.
to be adapted, biooxidation experiments were conducted in triplicated using a
bacterial concentration of 1.0  108 cells/mL, with the addition of gold
concentrates at same time of inoculation. The sampling of pH, ORP, and metals
concentration was conducted every 24 h as described in the above section.
RESULTS
Biooxidation of gold concentrates In Fig. 2, the total As
removal efficiency is shown for pure cultures of A. ferrooxidans,
A. thiooxidans, a non-adapted mixed culture of A. ferrooxidans and
A. thiooxidans (non-adapted culture), and an adapted mixed
culture of A. ferrooxidans and A. thiooxidans (adapted culture). As
shown, the lowest As removal efficiency was obtained by the
pure culture of A. ferrooxidans (11.45% As removal), followed by
the pure culture of A. thiooxidans (27.7% As removal). The non-
adapted culture showed an As removal efficiency of 38.2%,
meanwhile the highest As removal efficiency was observed with
the adapted culture at 72.2%. These As removal efficiencies were
obtained during 358 h of biooxidation reaction.
The addition of the gold concentrate varied in time for each
bacterial culture. For the pure culture of A. ferrooxidans and the
non-adapted culture, the addition of gold concentrates was carried
out after 24 h of the inoculation of the culture medium, a time at
which the stabilization of the ORP was observed higher than
600 mV (4). For the pure culture of A. thiooxidans, the addition time
was carried out at 70 h, when the stabilization of the pH and ORP of
the culture medium was observed. Finally, for the adapted culture,
the addition of gold concentrates was carried out at the same time
of the inoculation of the culture medium.
As shown in Fig. 2, for the pure culture of A. ferrooxidans,
removal of As increased up to 5.50% shortly after the gold
concentrate were added. The As removal efficiency continued to
increase until it reached 10.4% As removal at 48 h of reaction. It
remained almost stable until the end of experiment, reaching the
final 11.45% As removal. For the pure culture of A. thiooxidans, the
arsenic removal increased rapidly up to 9.4% after the addition of
the gold concentrates. The As removal remained almost stable, at
around 9.7%, during the following 70 h of reaction. Finally, an in-
crease in the As removal efficiency was observed, and the As
removal efficiency reached a final 27.7% of removal. In the case of
the non-adapted culture, there was an increase in the As removal of
J. BIOSCI. BIOENG.,
FIG. 3. ORP (A) and pH (B) changes of reacting solution according to reaction time using
an adapted (diamonds) and non-adapted (triangles) mixed culture of A. ferrooxidans
and A. thiooxidans, and their respective pure cultures (squares and circles, respec-
tively). The biooxidation experiments were conducted in triplicate at 30 C, pulp
density of 5% (w/v), and initial pH of 1.8.
11.6% soon after the addition of the concentrates. Following the
initial increase, an additional 48 h was necessary to increase the As
removal up to 12.2%. After this small increase, the removal effi-
ciency increased significantly until the end of the experiment,
reaching a total of 38.2% of As removal efficiency. For the adapted
culture, the As removal showed a moderate increase during the first
48 h of reaction, reaching a total of 7.6% of removal. A rapid increase
occurred in the following 48 h of reaction, reaching a total of 69.0%
by 96 h of reaction. The As removal efficiency peaked, with the
maximum at 120 h of reaction, reaching 77.0% of removal efficiency.
After this point, the As removal decreased down to 72.2% by the end
of the experiment.
Oxidation-reduction potential and pH In Fig. 3 the trend
obtained for the ORP (Fig. 3A) and pH (Fig. 3B) are presented. In
Fig. 3A, a rapid increase in the ORP can be observed for the pure
culture of A. ferrooxidans and the non-adapted culture before the
addition of gold concentrates. Both cultures reached stability and
their maximum ORP value at 24 h of reaction at 600 mV and
612 mV, respectively. After the addition of the gold concentrates,
the ORP values in both cultures decreased, down to 320 mV for
the pure culture of A. ferrooxidans and 329 mV for the non-
adapted culture. This was the lowest ORP value for the non-
adapted culture and it was reached at 90 h of reaction. After this
VOL. 121, 2016
FIG. 4. Total Fe (A), Fe2þ (B), and Fe3þ (C) changes in reacting solution according to
reaction time using an adapted (diamonds) and non-adapted (triangles) mixed culture
of A. ferrooxidans and A. thiooxidans, and their respective pure cultures (squares and
circles, respectively). The biooxidation experiments were conducted in triplicate at
30 C, pulp density of 5% (w/v), and initial pH of 1.8.
point, the non-adapted culture started to increase its ORP until the
end of the experiment. On the other hand, the pure culture of
A. ferrooxidans did not increase its ORP value throughout the rest
of the experiment. Although A. thiooxidans does not uses Fe2þ/
Fe3þ as the most important redox couple, the oxidation of S0 to
SO2
4 can be indirectly described by the ORP trend observed along
the biooxidation reaction (31,37). The analysis of the sulfate
concentration trend (data not shown) in the biooxidation
TANK BIOOXIDATION OF GOLD CONCENTRATES 539
medium for pure culture of A. thiooxidans was corroborated to be
in accordance with the ORP trend obtained (Fig. 3A). The trend
obtained for the pure culture of A. thiooxidans showed an initial
ORP increase, up to 428 mV, before the addition of the gold
concentrates at 70 h of reaction. After the addition of
concentrates, a small ORP decrease occurred and it took another
70 h to observe another increase in the ORP. The ORP for the pure
culture of A. thiooxidans did not decrease after this point. In the
case of the adapted culture, the ORP trend increased up to
546 mV within the first 96 h of reaction and then remained
almost constant.
As shown in Fig. 3B, the initial pH of the biooxidation process
was corrected to 1.8 in all the cultures analyzed. It can be seen that
the pH of the solution for the pure culture of A. ferrooxidans
increased to almost 3.00 from the initial 1.80. The highest value of
pH was observed at 125 h of reaction and the pH remained almost
constant throughout the experiment. The pH of the pure culture of
A. thiooxidans decreased, down to 1.22 in the first 70 h of reaction.
The addition of gold concentrates increased the pH trend and it
took the pure culture of A. thiooxidans another 70 h to start
decreasing the pH again. After 140 h of reaction, no increase in the
pH was observed. In the case of the non-adapted culture bacteria,
an increase in the pH was observed before the addition of gold
concentrates at 24 h. The pH continued increasing after the addi-
tion of gold concentrates reaching a highest point at 2.65 at 70 h of
reaction. After this point, the pH started to decrease and no incre-
ment was observed along the rest of the experiment. In relation to
the adapted culture, there was a stable pH of 1.80 in the first 32 h of
reaction, followed by a brief increase up to 1.90. After this point, as
well as the non-adapted culture, the pH started to decrease and no
increase was observed through the rest of the experiment.
Total iron, ferrous and ferric ion concentrations In Fig. 4,
the trends obtained for the iron behavior regarding the total Fe
(Fig. 4A), Fe2þ (Fig. 4B), and Fe3þ (Fig. 4C) are presented. As seen,
the total Fe increased for all the cultures along the biooxidation
reaction. The highest increase was observed in the adapted
culture, reaching a total Fe concentration of 14.2 g/L. The second
highest was obtained with the pure culture of A. thiooxidans,
showing an increment that was only observed after the addition
of gold concentrates at 70 h of reaction. The final concentration of
total Fe was 2.93 g/L. This increment was not enough to reach the
concentration obtained by the pure culture of A. ferrooxidans with
a final concentration of 4.00 g/L. With the pure culture of
A. ferrooxidans, the increase of the total Fe concentration was
significantly low, and the highest increment was observed at the
time of the addition of the gold concentrates. The concentration
remained almost constant after 24 h of reaction. The non-adapted
culture showed a slight increase along the reaction time, reaching
the highest concentration (5.65 g/L) at 285 h of reaction. After
this point, the total Fe concentration decreased until the end of
the experiment, lowering the concentration down to 5.00 g/L.
Fig. 4B shows the Fe2þ behavior along the biooxidation reaction.
The pure culture of A. ferrooxidans, the non-adapted culture, and
the adapted culture followed the same trend before the 24 h of
reaction, reaching a concentration below 0.1 g/L. After the addition
of gold concentrates, the pure culture of A. ferrooxidans and non-
adapted culture showed increased Fe2þ concentrations, up to an
almost constant 3.5 g/L throughout the experiment. On the other
hand, the adapted culture remained almost constant.
The behavior of Fe3þ is shown in Fig. 4C. The pure culture of
A. ferrooxidans and non-adapted culture showed similar trends.
After the addition of gold concentrates in both cultures, the con-
centration of Fe3þ decreased to a concentration below 1.0 g/L. The
concentration of Fe3þ of the non-adapted culture showed a slight
increase after 120 h, reaching the highest concentration (2.5 g/L) at
540 HONG ET AL.
285 h of reaction. After this point, the Fe3þ concentration decreased
until the end of the experiment, lowering the concentration down
to 2.0 g/L. On the other hand, the concentration of the pure culture
of A. ferrooxidans did not increase throughout the experiment. The
concentration of Fe3þ for the adapted culture increased rapidly up
to 14.0 g/L and remained stable at this concentration.
DISCUSSION
In the present study, the biooxidation of gold refractory con-
centrates was analyzed based on the total As removal efficiency (5)
obtained with an adapted mixed culture of A. ferrooxidans and
A. thiooxidans. The mixed culture was adapted to high concentra-
tions of As to increase the percentage of biooxidation up to 70%.
This biooxidation percentage has been shown to be responsible for
an increase in gold recovery, up to 90%, in subsequent procedures
(e.g., cyanidation) (5,6). The As removal efficiency obtained by the
adapted culture was compared with the As removal efficiency,
obtained by a non-adapted mixed culture of A. ferrooxidans and
A. thiooxidans and with their respective pure cultures. The per-
centage of the biooxidation reached by each culture is shown in the
Results section.
The As oxidation occurs by the action of the synergetic effect
generated by the mixed culture bacteria. As a first step the bacteria
produce strong oxidizing agents (i.e., Fe3þ and H2SO4) (9,38,39), as
expressed by Eqs. 1 and 2 (5,11,19):
A:f
4Fe2þ þO2 þ4Hþ /4Fe3þ þ2H2 O (1)
A:t
S0 þH2 O þ 1:5O2 /H2 SO4 (2)
The strong oxidizing agents are then responsible for oxidizing
the As, resulting in the liberation of the refractory concentrates.
This oxidation is well known to occur by a polysulfide pathway
previously described by Shippers and Sand (38) and clearly exem-
plified by Rohwerder et al. (15). The total oxidation of As can be
summarized as stated in Eq. 3 (5,11):
FeAsS þ 7Fe3þ þ4H2 O/8Fe2þ þH3 AsO4 þS0 þ5Hþ (3)
As expressed in Eq. 3, a higher concentration of Fe3þ ions will
increase the As removal efficiency. This can be observed in As
removal efficiency trend (Fig. 2) at the moment of the addition of
the gold concentrates. The addition of the gold concentrates in the
system with pure culture of A. ferrooxidans and the non-adapted
culture was conducted at 24 h of reaction, time in which the bac-
teria had produced a high concentration of Fe3þ ions (Fig. 4C). On
the other hand, in the system for the adapted bacteria, the addition
of gold concentrates was conducted at the moment of the inocu-
lation, in which the Fe3þ ions concentration was almost non-
existent. After 24 h of the addition of the concentrates, the sys-
tems containing a rich concentration of Fe3þ ions were capable of
generating an As removal efficiency that almost doubled the per-
centage of As removal obtained with the system without Fe3þ in the
adapted culture. These results confirmed the importance of the
production of Fe3þ ions during the biooxidation process regarding
the iron-oxidizing bacteria (32,33). The results of the analysis of the
sulfate production in the sulfur-oxidizing bacteria system (data not
shown) resembled the production of a strong oxidizing agent.
Production of a high concentration of sulfate ions in the system of
the pure culture of A. thiooxidans produced a rapid increase in the
As removal efficiency (Fig. 2). After the addition of concentrates in
the pure culture of A. thiooxidans, there was a rapid increase in the
As removal efficiency, which followed the same pattern observed in
the system with high Fe3þ ion concentration.
J. BIOSCI. BIOENG.,
FIG. 5. Bacterial concentration change in reacting solution over time using an adapted
(diamonds) and non-adapted (triangles) mixed culture of A. ferrooxidans and
A. thiooxidans, and their respective pure cultures (squares and circles, respectively).
The biooxidation experiments were conducted in triplicate at 30 C, pulp density of 5%
(w/v), and initial pH of 1.8.
After the addition of the concentrates, a decrease in Fe3þ was
observed (Fig. 4C). Consumption of Fe3þ in the process represents
its reduction to Fe2þ and the oxidation of the metal sulfide in the
gold concentrates; consequently, the ORP of the reaction solution
decreased (Fig. 3A). Variations in the ORP are due to the production
of Fe3þ by A. ferrooxidans (see Eq. 1) and the production of Fe2þ in
the oxidation of As (see Eq. 3); a higher production of Fe3þ repre-
sents a higher ORP (17e19). One noteworthy observation is that in
Fig. 4C, even in the presence of the iron-oxidizing bacteria, the
concentration of Fe3þ, and its respective ORP (Fig. 3A), did not in-
crease at the same rate as it did before the addition of the con-
centrates. This reduction in the production rate of Fe3þ could
indicate that the bacterial metabolism had slowed down or was
even cancelled by the addition of the gold concentrates, which
represents a low oxidation of Fe2þ (Fig. 4B) and decrease of ORP
(Fig. 3A). Previous studies have reported that the cancellation of
activity of A. ferrooxidans could be due to the presence of the re-
sidual flotation reagents, such as collector, frother, and/or activator,
that remained on the surface of the gold concentrates (40,41), or by
the high concentration of As (17,26). However, none of these
negative effects was observed in the results obtained for the As
removal behavior obtained with the adapted culture. These find-
ings indicate that the adapted culture was capable of overcoming
the high concentration of As, and that the adaptation could have
also worked to reduce the negative effects of the residual flotation
reagents.
The pure culture of A. thiooxidans was the only pure culture
capable of overcoming the negative effects of the addition of the
concentrates. This represents a recuperation in the bacterial activity
in the system and can be observed in the increase of the As removal
efficiency (Fig. 2). In a pure culture of A. thiooxidans, the normal
bacterial activity is expected to decrease the pH trend, as observed
in Fig. 3B. As stated in Eq. 2, A. thiooxidans oxidizes elemental sulfur
to sulfuric acid, resulting in the decrease in pH. However,
the decrease in the pH was interrupted after the addition of the
concentrates, presumably by the buffering effect of the alkaline
carbonate substances found in the concentrates (42e44).
A. thiooxidans required a period of approximately 70 h to counteract
the buffering effect of the concentrates and continued to decrease
the pH. A similar pattern was also observed for the non-adapted
culture. After the addition of the concentrates, a similar period of
VOL. 121, 2016
FIG. 6. XRD patterns obtained for the refractory gold ores before and after the bio-
oxidation process.
time was observed before the decrease in the pH trend (Fig. 3B).
Additionally, Eq. 2 proposes that the oxidation of sulfur to sulfate by
a healthy bacteria leads to a decrease in the concentration of free
electrons in the solution, resulting in an increase in ORP (31,37). The
time needed by the pure culture of A. thiooxidans and the non-
adapted culture to start showing an increase in the ORP trend af-
ter the addition of concentrates (Fig. 3A) was similar to the 70 h
period observed for the pH and As removal efficiency. Furthermore,
a similar lag period is observed in the As removal efficiency and
bacterial growth for non-adapted culture and the pure culture of
A. thiooxidans (Figs. 2 and 5, respectively). This correlation between
the time the pure culture of A. thiooxidans needed to recuperate the
activity in the system and the time needed by the non-adapted
culture shows the importance of A. thiooxidans in the bio-
oxidation of As from gold concentrates in a mixed culture. However,
as stated in Results, the As removal efficiency obtained by the pure
culture of A. ferrooxidans was observed to be higher than the one
observed by A. thiooxidans before the negative effect of the con-
centrates cancelled the bacterial metabolism of A. ferrooxidans. The
cancelation of the bacterial metabolism can be observed by
analyzing the bacterial growth presented in Fig. 5. As it is shown,
the adapted mixed culture of A. ferrooxidans and A. thiooxidans was
capable to overcome the negative impact of the addition of the
concentrates by conserving the bacterial growth almost unaffected.
However, the increase in the bacterial concentration for the pure
cultures and the non-adapted culture was interrupted close to the
time in which the addition of the concentrates was conducted. It is
also possible to observe that the pure cultures required a longer
period to start increasing its bacterial concentration. It is worthy to
mention that the decrease in the bacterial concentration for the
adapted culture at the end of the experiment could be due to the
saturation of As in the system.
Therefore, by comparing the results obtained for the As removal
efficiency between the pure cultures and the non-adapted culture,
it can be concluded that the synergistic effect of the mixed culture
of A. ferrooxidans and A. thiooxidans is needed to obtain a higher As
removal efficiency. At the same time, the improvement in the As
removal efficiency seen with the adapted culture, shows that, with
adaptation, it is possible to obtain the 70% of biooxidation needed
to improve the gold extraction efficiency up to 90% in subsequent
procedures. In accordance to the results obtained, the differences in
the XRD patterns for the concentrates after each biooxidation
condition proved that the adapted culture was the one that greatly
TANK BIOOXIDATION OF GOLD CONCENTRATES 541
modified the patterns obtained for arsenopyrite supporting the
high arsenic extraction (Fig. 6).
In conclusion, the biooxidation of refractory gold concentrates
containing a high concentration of As (approximately 139.67 g/kg)
was analyzed based on the total As removal efficiency obtained
with an adapted mixed culture of A. ferrooxidans and A. thiooxidans.
The As removal efficiency obtained by the adapted culture was
compared with the As removal efficiency obtained by a non-
adapted mixed culture of A. ferrooxidans and A. thiooxidans and
with their respective pure cultures. A. ferrooxidans was used as an
iron-oxidizing bacteria, while A. thiooxidans was used as a sulfur-
oxidizing bacteria. The highest As removal efficiency was ob-
tained by the adapted mixed culture at 72.20% of biooxidation,
followed by the non-adapted mixed culture with a biooxidation
efficiency of 38.20%. The pure culture of A. thiooxidans showed
27.70% biooxidation, while the lowest biooxidation was observed
with the pure culture of A. ferrooxidans with only 11.45%. With
these results, it was shown that it is possible to achieve a bio-
oxidation rate above 70% for refractory gold concentrates contain-
ing approximately 14% (w/w) of As.
The comparison between the pure culture and the mixed culture
showed the capabilities of the mixed culture to overcome threats
due to the physicochemical characteristics of the concentrates. In
addition, it was possible to observe the importance of the adapta-
tion of the mixed culture of bacteria because the non-adapted
bacteria were not capable of reaching even half of the 70% of
biooxidation.
ACKNOWLEDGMENTS
This work was supported by the Mine Reclamation Corporation
Research Fund, South Korea, and the Ministry of Education (MOE)
and National Research Foundation of Korea (NRF) through the
Human Resource Training Project for Regional Innovation
(2015H1C1A1035930).
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