Adaptation of a mixed culture of acidophiles for a tank biooxidation of refractory gold concentrates containing a high concentration of arsenic

Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e7, 2015 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 xxx

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 percentage of the biooxidation process was analyzed based on the total As removal efficiency. The As removal was monitored 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 reaction 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 biooxidation 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 subsequent 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 ferrooxidans), sulfur-oxidizing bacteria (Acidithiobacillus thiooxidans), iron(II)-oxidizing bacteria (Leptospirillum ferrooxidans), and several others such as Acidithiobacillus caldus, Sulphobacillus sp. and Ferroplasma 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: [email protected] (H. Kim). z The first two authors contributed equally to the work.

contact, and cooperative mechanism); however, unlike bioleaching, 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 production 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 ironoxidizing 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

Please cite this article in press as: Hong, J., et al., Adaptation of a mixed culture of acidophiles for a tank biooxidation of refractory gold concentrates containing a high concentration of arsenic, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.09.009

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TABLE 1. Chemical composition of the gold concentrates from the Terek-Sai mine in Kyrgyzstan (g/kg). As 139.67

Cu

Pb

Fe

S

Zn

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 (approximately 139.67 g/kg). Previous studies have stated a linear relationship 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 recovery processes (5). The results obtained with the adapted culture were compared with the biooxidation yield obtained using nonadapted 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 (AC12, 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 ironoxidizing bacteria (KCTC 4516), and A. thiooxidans (KCTC 4515) as the sulfuroxidizing 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

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 SO2 4 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 separately 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

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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 nonadapted 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 increase 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

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, respectively). 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 efficiency 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 nonadapted culture. This was the lowest ORP value for the nonadapted culture and it was reached at 90 h of reaction. After this

Please cite this article in press as: Hong, J., et al., Adaptation of a mixed culture of acidophiles for a tank biooxidation of refractory gold concentrates containing a high concentration of arsenic, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.09.009

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J. BIOSCI. BIOENG., 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 addition 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 increment 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.

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

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 nonadapted 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 concentration 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

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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 concentrates 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 concentrations 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 percentage 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 A:t

S0 þH2 O þ 1:5O2 /H2 SO4

(1) (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 exemplified 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 bacteria 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 inoculation, in which the Fe3þ ions concentration was almost nonexistent. After 24 h of the addition of the concentrates, the systems containing a rich concentration of Fe3þ ions were capable of generating an As removal efficiency that almost doubled the percentage 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.

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 represents 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 increase at the same rate as it did before the addition of the concentrates. 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 residual 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 findings 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

Please cite this article in press as: Hong, J., et al., Adaptation of a mixed culture of acidophiles for a tank biooxidation of refractory gold concentrates containing a high concentration of arsenic, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.09.009

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FIG. 6. XRD patterns obtained for the refractory gold ores before and after the biooxidation 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 nonadapted culture to start showing an increase in the ORP trend after 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 biooxidation 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 concentrates 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

J. BIOSCI. BIOENG., 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 nonadapted 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 sulfuroxidizing bacteria. The highest As removal efficiency was obtained 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 biooxidation rate above 70% for refractory gold concentrates containing 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 adaptation 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). References 1. Langhans, D., Lord, A., Lampshire, D., Burbank, A., and Baglin, E.: Biooxidation of an arsenic-bearing refractory gold ore, Miner. Eng., 8, 147e158 (1995). 2. Makita, M., Esperon, M., Pereyra, B., Lopez, A., and Orrantia, E.: Reduction of arsenic content in a complex galena concentrate by Acidithiobacillus ferrooxidans, BMC Biotechnol., 4, 22 (2004). 3. Gahan, C. S., Sundkvist, J. E., and Sandstrom, A.: Use of mesalime and electric arc furnace (EAF) dust as neutralising agents in biooxidation and their effects on gold recovery in subsequent cyanidation, Miner. Eng., 23, 731e738 (2010). 4. Gahan, C. S., Sundkvist, J. E., Engstrom, F., and Sandstrom, A.: Utilisation of steel slags as neutralising agents in biooxidation of a refractory gold concentrate and their influence on the subsequent cyanidation, Resour. Conserv. Recycl., 55, 541e547 (2011). 5. Lindström, E. B., Gunneriusson, E., and Tuovinen, O. H.: Bacterial oxidation of refractory sulfide ores for gold recovery, Crit. Rev. Biotechnol., 12, 133e155 (1992). 6. Lynn, N. S.: The bioleaching and processing of refractory gold ore, JOM, 49(4), 24e26 (1997). 7. Gahan, C. S., Sundkvist, J. E., and Sandstrom, A.: A study on the toxic effects of chloride on the biooxidation efficiency of pyrite, J. Hazard. Mater., 172, 1273e1281 (2009). 8. Gahan, C. S., Cunha, M. L., and Sandstrom, A.: Comparative study on different steel slags as neutralising agent in bioleaching, Hydrometallurgy, 95, 190e197 (2009). 9. Gonzalez, R., Gentina, J. C., and Acevedo, F.: Biooxidation of a gold concentrate in a continuous stirred tank reactor: mathematical model and optimal configuration, Biochem. Eng. J., 19, 33e42 (2004). 10. Sand, W., Gehrke, T., Jozsa, P. G., and Schippers, A.: (Bio) chemistry of bacterial leaching e direct vs. indirect bioleaching, Hydrometallurgy, 59, 159e175 (2001). 11. Sand, W., Gerke, T., Hallmann, R., and Schippers, A.: Sulfur chemistry, biofilm, and the (in)direct attack mechanism e a critical-evaluation of bacterial leaching, Appl. Microbiol. Biotechnol., 43, 961e966 (1995).

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Please cite this article in press as: Hong, J., et al., Adaptation of a mixed culture of acidophiles for a tank biooxidation of refractory gold concentrates containing a high concentration of arsenic, J. Biosci. Bioeng., (2015), http://dx.doi.org/10.1016/j.jbiosc.2015.09.009