Arsenopyrite Dissolution and Bioscorodite Precipitation by Acidithiobacillus ferrivorans ACH under Mesophilic Condition

: Arsenopyrite is the most abundant arsenic-bearing sulﬁde mineral in the lithosphere, usually associated with sulﬁde gold ores. The recovery of this highly valuable metal is associated with the release of large quantities of soluble arsenic. One way to mitigate the effects of high concentrations of arsenic in solution is to immobilize it as scorodite precipitate, a more stable form. Hence, we addressed the scorodite formation capacity (under mesophilic conditions) of psychrotolerant Acidithiobacillus ferrivorans ACH isolated from the Chilean Altiplano. Bio-oxidation assays were performed with 1% arsenopyrite concentrate as unique energy source and produced solids were evaluated by X-ray diffraction (XRD) and QEMSCAN analysis. Interestingly, the results evidenced scorodite generation as the main sub-product after incubation for 15 days, due to the presence of the microorganism. Moreover, the QEMSCAN analysis support the XRD, detecting a 3.5% increase in scorodite generation by ACH strain and a 18.7% decrease in arsenopyrite matrix, implying an active oxidation. Finally, we presented the ﬁrst record of arsenopyrite oxidation capacity and the stable scorodite production ability by a member of A. ferrivorans species under mesophilic conditions.


Introduction
Arsenopyrite (FeAsS) is the most abundant arsenic-bearing sulfide mineral in the lithosphere, whereby it has become important for the mineral processing industries that have been focusing their interest on finding new technologies to recover commercially valuable metals such as copper, nickel, and gold from these ores with lower economic and energy costs [1,2]. Low grade refractory sulfide gold ores are usually associated with high amounts of pyrite and arsenopyrite. In such ores, gold is dispersed as submicroscopic particles and the efficient recovery of this finely dispersed metal is very difficult by conventional methods (e.g., cyanidation) without pretreatment [3]. In this context, the bio-oxidation of refractory sulfide gold ores prior to cyanidation could be one alternative sulfur and pyrite as energy source and exhibited a broad range of growth temperatures ranging between 4-28 • C [24], and it is also able to tolerate high Cu(II) concentrations [25]. Following this, A ferrivorans ACH was cultured at 28 • C with 120 rpm of constant agitation in modified 1X Mackintosh (MAC) medium (50X, per liter): 50 mM (NH 4 ) 2 SO 4 ; 26.65 mL H 2 SO 4 ; 7.8 mM K 2 HPO 4 ; 6.2 mM MgCl 2 ·6H 2 O; 50 mM CaCl 2 ·2H 2 O and 50 mL of trace elements [26].

Bio-Oxidation Experiments
The bio-oxidation experiments were performed in 500 mL Erlenmeyer shake flasks containing 200 mL of MAC medium with 1% arsenopyrite pulp (concentrate) as energy source and 10% bacterial inoculum; incubated for 20 days at 28 • C with 120 rpm of constant agitation. The arsenopyrite concentrate was obtained from the "Comercializadora de Minerales Viacha" mining company (La Paz-Bolivia, Ciudad de la Paz, Bolivia). Next, bacterial growth was monitored by planktonic unstained cell counting by phase-contrast microscope (CX21, Olympus, Tokyo, Japan) using a Neubauer chamber. Additionally, the redox potential and pH values were measured by an Ag/AgCl electrode (Model 420A, ORION, Shanghai, China). As and Fe total in solution during the bio-oxidation assays were obtained using Perkin Elmer Optima 7000 DV model inductively coupled plasma optical emission spectrometry (ICP-OES, Waltham, MA, USA). The assays were set to recollect the produced solids after 5, 10, 15 and 20 days of incubation. Additionally, control assays were prepared exactly the same without the bacteria. Three independent experiments with at less three technical replicates each were performed.

Mineralogical Characterization
The control of arsenopyrite pulp and the bio-oxidation solid products were characterized by mineralogical identification through different approaches: (i) X-ray diffraction was carried out with a diffractometer (D5000, Siemens, Munich, Germany). The powder patterns were analyzed using DiffracPlus and TOPAS (Total Pattern Analysis Software). Crystalline phases were identified using the database of the International Center for Diffraction Data (ICDD); (ii) X-ray fluorescence using a spectrometer (SRS 3000, Siemens); (iii) Quantitative Evaluation of Minerals by Scanning Electron Microscopy analysis-QEMSCAN ® (E430, CSIRO, Canberra, Australia) and (iv) Dual optical microscopy (BX51, Olympus, Japan).

Search for Arsenic Resistance Genetic Determinants in the ACH Strain Genome
Some known and described arsenic response genes for the Acidithiobacillus genus were used as reference and queried to identify their presence in the ACH strain genome (GeneBank Accesion: GCA_018854855.1) using BLAST.

Characterization of Arsenopyrite Concentrate
The preliminary analysis of the arsenopyrite showed a chemical composition of 27.7% iron, 32.1% arsenic and 18.8% sulfur. In parallel, through optical microscopy, we visualized the mineralogical species present at the sample (Figure 1), which was composed mainly by arsenopyrite and pyrite ( Figure 1A,B,D), while chalcopyrite and gold particles were detected in a minor proportion ( Figure 1B  Additionally, to corroborate the aforementioned results, we performed an X-ray diffraction (XRD) to the same arsenopyrite pulp to estimate mineralogical composition. The diffractogram pattern evidenced the presence of arsenopyrite, pyrite and quartz as the main mineralogical species in the sample. The major proportion detected was for arsenopyrite with 38.9% followed by pyrite (24.4%) and quartz with a minor proportion of 5.5% ( Figure 2). In addition, through X-ray fluorescence spectrophotometry we estimated that the sample contained trace amounts of nickel (Ni), cobalt (Co), antimony (Sb), copper (Cu), zinc (Zn), lead (Pb) and bismuth (Bi) ( Table 1). Additionally, to corroborate the aforementioned results, we performed an X-ray diffraction (XRD) to the same arsenopyrite pulp to estimate mineralogical composition. The diffractogram pattern evidenced the presence of arsenopyrite, pyrite and quartz as the main mineralogical species in the sample. The major proportion detected was for arsenopyrite with 38.9% followed by pyrite (24.4%) and quartz with a minor proportion of 5.5% ( Figure 2). Additionally, to corroborate the aforementioned results, we performed an X-ray diffraction (XRD) to the same arsenopyrite pulp to estimate mineralogical composition. The diffractogram pattern evidenced the presence of arsenopyrite, pyrite and quartz as the main mineralogical species in the sample. The major proportion detected was for arsenopyrite with 38.9% followed by pyrite (24.4%) and quartz with a minor proportion of 5.5% ( Figure 2). In addition, through X-ray fluorescence spectrophotometry we estimated that the sample contained trace amounts of nickel (Ni), cobalt (Co), antimony (Sb), copper (Cu), zinc (Zn), lead (Pb) and bismuth (Bi) ( Table 1). In addition, through X-ray fluorescence spectrophotometry we estimated that the sample contained trace amounts of nickel (Ni), cobalt (Co), antimony (Sb), copper (Cu), zinc (Zn), lead (Pb) and bismuth (Bi) ( Table 1). First, the ACH strain cells were initially adapted to grow in the presence of different pulp concentrations of arsenopyrite (1, 5, 10 and 12%, respectively; Figure 3) and the growth rate was negatively affected by concentrations higher than 5%. Additionally, the iron oxidation capacity decreased, evidenced by the low redox potential values in 5, 10 and 12% pulp concentrations (361 mV, 318 mV and 300 mV, respectively; Figure 3). On the other hand, the pH values remained close to 1.8 in presence of 1% pulp, while at higher concentrations of pulp, pH values remained close to 1.8 ( Figure 3). Hence, the best growth rate was obtained with the 1% pulp condition ( Figure 4). Furthermore, the cell growth (exponential phase) started after 5 days of lag phase, reaching between 5.0 × 10 6 -3.2 × 10 7 cell mL −1 , increasing one order of magnitude after 20 days incubation of growth. Besides, the redox potential value also increased up to 560 mV by this time (Figure 4). Thus, according to these results, a 1% pulp was selected as experimental condition for the bio-oxidation assays.

Growth of A. ferrivorans ACH Cells Using Arsenopyrite as Energy Source
First, the ACH strain cells were initially adapted to grow in the presence of different pulp concentrations of arsenopyrite (1, 5, 10 and 12%, respectively; Figure 3) and the growth rate was negatively affected by concentrations higher than 5%. Additionally, the iron oxidation capacity decreased, evidenced by the low redox potential values in 5, 10 and 12% pulp concentrations (361 mV, 318 mV and 300 mV, respectively; Figure 3). On the other hand, the pH values remained close to 1.8 in presence of 1% pulp, while at higher concentrations of pulp, pH values remained close to 1.8 ( Figure 3). Hence, the best growth rate was obtained with the 1% pulp condition ( Figure 4). Furthermore, the cell growth (exponential phase) started after 5 days of lag phase, reaching between 5.0 × 10 6 -3.2 × 10 7 cell mL −1 , increasing one order of magnitude after 20 days incubation of growth. Besides, the redox potential value also increased up to 560 mV by this time (Figure 4). Thus, according to these results, a 1% pulp was selected as experimental condition for the bio-oxidation assays.

Growth of A. ferrivorans ACH Cells Using Arsenopyrite as Energy Source
First, the ACH strain cells were initially adapted to grow in the presence of different pulp concentrations of arsenopyrite (1, 5, 10 and 12%, respectively; Figure 3) and the growth rate was negatively affected by concentrations higher than 5%. Additionally, the iron oxidation capacity decreased, evidenced by the low redox potential values in 5, 10 and 12% pulp concentrations (361 mV, 318 mV and 300 mV, respectively; Figure 3). On the other hand, the pH values remained close to 1.8 in presence of 1% pulp, while at higher concentrations of pulp, pH values remained close to 1.8 ( Figure 3). Hence, the best growth rate was obtained with the 1% pulp condition ( Figure 4). Furthermore, the cell growth (exponential phase) started after 5 days of lag phase, reaching between 5.0 × 10 6 -3.2 × 10 7 cell mL −1 , increasing one order of magnitude after 20 days incubation of growth. Besides, the redox potential value also increased up to 560 mV by this time (Figure 4). Thus, according to these results, a 1% pulp was selected as experimental condition for the bio-oxidation assays.

Arsenopyrite Dissolution and Bioscorodite Precipitation by A. ferrivorans ACH
Solid residues recovered from A. ferrivorans ACH bio-oxidation experiments were characterized by optical microscopy, XRD and QEMSCAN. The dissolution of arsenopyrite concentrates by ACH action was evidenced by optical microscopy from day 15 onwards, as we saw the unperturbed mineral structure for the first 10 days ( Figure 5A,B). However, after 15 days, the arsenopyrite structure drastically changed, displaying an irregular surface, due to microbial activity ( Figure 5C). Finally, after 20 incubation days, arsenopyrite looked almost completely dissolved ( Figure 5D). This could potentially enhance the release of more valuable components contained in this refractory matrix.

Arsenopyrite Dissolution and Bioscorodite Precipitation by A. ferrivorans ACH.
Solid residues recovered from A. ferrivorans ACH bio-oxidation experiments were characterized by optical microscopy, XRD and QEMSCAN. The dissolution of arsenopyrite concentrates by ACH action was evidenced by optical microscopy from day 15 onwards, as we saw the unperturbed mineral structure for the first 10 days ( Figure 5A,B). However, after 15 days, the arsenopyrite structure drastically changed, displaying an irregular surface, due to microbial activity ( Figure 5C). Finally, after 20 incubation days, arsenopyrite looked almost completely dissolved ( Figure 5D). This could potentially enhance the release of more valuable components contained in this refractory matrix. Similarly, the XRD pattern suggests that after 5 days of incubation arsenopyrite was the predominant solid, without any significant change in the mineral matrix ( Figure 6A), despite the fact that an increase in the redox potential values was detected at this period of time ( Figure 4). Subsequently, after 10 days of incubation, the XRD pattern showed changes in the pulp mineral species, detecting arsenopyrite, elemental sulfur and pyrite ( Figure 6B). Interestingly, after 15 days of incubation, the XRD patterns revealed the presence of bioscorodite (FeAsO4 2H2O), in addition to arsenopyrite, pyrite, elemental sulfur and quartz ( Figure 6C). Therefore, the bioscorodite precipitated is produced due to the bacterium ability to oxidize both iron and arsenic contained in the arsenopyrite, generating a stable precipitate which is rich in both elements. Finally, after 20 incubation days, the XRD result show that scorodite is one predominant mineral species in the culture, produced under mesophilic conditions ( Figure 6D). Similarly, the XRD pattern suggests that after 5 days of incubation arsenopyrite was the predominant solid, without any significant change in the mineral matrix ( Figure 6A), despite the fact that an increase in the redox potential values was detected at this period of time ( Figure 4). Subsequently, after 10 days of incubation, the XRD pattern showed changes in the pulp mineral species, detecting arsenopyrite, elemental sulfur and pyrite ( Figure 6B). Interestingly, after 15 days of incubation, the XRD patterns revealed the presence of bioscorodite (FeAsO 4 2H 2 O), in addition to arsenopyrite, pyrite, elemental sulfur and quartz ( Figure 6C). Therefore, the bioscorodite precipitated is produced due to the bacterium ability to oxidize both iron and arsenic contained in the arsenopyrite, generating a stable precipitate which is rich in both elements. Finally, after 20 incubation days, the XRD result show that scorodite is one predominant mineral species in the culture, produced under mesophilic conditions ( Figure 6D). Additionally, we performed a QEMSCAN analysis on both the arsenopyrite pulp and the solids obtained during bio-oxidation assays to estimate the percent of arsenopyrite dissolution and bioscorodite generation by the ACH strain activity. Firstly, the results for the pulp composition supports those of X-ray fluorescence indicating arsenopyrite as the main sulfide mineral in the sample with 35.5% followed by other As-Fe-Ni sulfides (15.1%) and nickel/cobalt associated arsenopyrite sulfides (13.5%) ( Table 2). Secondly, the ACH strain was capable of dissolving the arsenopyrite matrix from 35.5% to 16.8% after 20 days of incubation. In addition, the microorganism was capable of dissolving other types of arsenopyrite such as those associated with nickel and cobalt (13.5% to 1.1%), and other arsenic sulfides (Table 2).  Additionally, we performed a QEMSCAN analysis on both the arsenopyrite pulp and the solids obtained during bio-oxidation assays to estimate the percent of arsenopyrite dissolution and bioscorodite generation by the ACH strain activity. Firstly, the results for the pulp composition supports those of X-ray fluorescence indicating arsenopyrite as the main sulfide mineral in the sample with 35.5% followed by other As-Fe-Ni sulfides (15.1%) and nickel/cobalt associated arsenopyrite sulfides (13.5%) ( Table 2). Secondly, the ACH strain was capable of dissolving the arsenopyrite matrix from 35.5% to 16.8% after 20 days of incubation. In addition, the microorganism was capable of dissolving other types of arsenopyrite such as those associated with nickel and cobalt (13.5% to 1.1%), and other arsenic sulfides (Table 2). It is important to highlight the mineral mass appearance of As-bearing sulfates, reaching 32% after 15 days of incubation. Interestingly, the scorodite synthesis by the presence of ACH strain was also determined, increasing the mineral mass from 1.9% to 5.4% (Table 2), which corroborates the patterns obtained previously by XRD.
Finally, we determined the As and Fe concentrations in solution after the arsenopyrite bio-oxidation assays (Figure 7). We observed an increase in total Fe and As concentration over time, both concomitant with the grow of A. ferrivorans ACH showed previously (Figure 4). This potential means that the ACH strain is capable of tolerating higher concentrations of arsenic; however, the respective tolerance tests, as determined by Minimal Inhibitory Concentrations (MIC), are pending a future study.
It is important to highlight the mineral mass appearance of As-bearing sulfates, reaching 32% after 15 days of incubation. Interestingly, the scorodite synthesis by the presence of ACH strain was also determined, increasing the mineral mass from 1.9% to 5.4% (Table 2), which corroborates the patterns obtained previously by XRD.
Finally, we determined the As and Fe concentrations in solution after the arsenopyrite bio-oxidation assays (Figure 7). We observed an increase in total Fe and As concentration over time, both concomitant with the grow of A. ferrivorans ACH showed previously (Figure 4). This potential means that the ACH strain is capable of tolerating higher concentrations of arsenic; however, the respective tolerance tests, as determined by Minimal Inhibitory Concentrations (MIC), are pending a future study.

Discussion
Some authors have described that the rates of oxidation and dissolution for sulfide minerals (such as arsenopyrite) are enhanced by microorganism metabolic processes, because they could use those to generate energy [27][28][29]. The ability of those organisms has led to their use for mineral ores pretreatment, which include sulfide ores containing valuable metals such as gold [29]. Thus, the arsenopyrite dissolution by microorganisms has been previously reported by many authors [27,28,30]. Additionally, four mechanisms of action for arsenopyrite dissolution have been suggested according to the following equations:

Discussion
Some authors have described that the rates of oxidation and dissolution for sulfide minerals (such as arsenopyrite) are enhanced by microorganism metabolic processes, because they could use those to generate energy [27][28][29]. The ability of those organisms has led to their use for mineral ores pretreatment, which include sulfide ores containing valuable metals such as gold [29]. Thus, the arsenopyrite dissolution by microorganisms has been previously reported by many authors [27,28,30] The microorganism grows initially on the surface of arsenopyrite where ferrous iron (as FeSO 4 ), arsenite (as arsenous acid (H 3 AsO 3 )) and elemental sulfur are generated (Equation (1)). Later, due to increased concentrations of ferrous iron (Fe +2 ), the microorganisms are capable of oxidizing this to ferric iron (Fe +3 ) (Equation (2)). Then, the ferric iron (as Fe 2 (SO 4 ) 3 ) generated by the microorganism is used for attaching (chemically) to the mineral surface producing ferrous iron, arsenite, sulfuric acid and elemental sulfur (Equation (3)). Finally, the elemental sulfur (S 0 ) turns into sulfuric acid to maintain the low pH values required by the microorganisms (Equation (4)) [31].
The principal microorganisms involved in arsenopyrite dissolution are acidophilic, iron and/or sulfur oxidizing and capable of resisting high arsenic concentration in solution [29]. In this context, here we reported for the first time the capacity of Acidithiobacillus ferrivorans to use arsenopyrite concentrate as energy source under mesophilic conditions. However, previous studies have described this species as a psychrotolerant, able to oxidize iron, sulfur, and reduced inorganic sulfur compounds, and to solubilize pyrite as energy source [18,19,24]. Moreover, the capacity to grow using arsenopyrite as unique energy source by ACH strain is evidenced in Figure 4, where the increase in bacterial cellular density is one order of magnitude after 20 days of incubation. In addition, the increased redox potential was concomitant with the proliferation of cell number detected in our assays ( Figure 4). Therefore, it can be suggested that bacteria promote an active oxidation of the arsenopyrite concentrate reported previously in some group of archaea such as Sulfobacillus and Acidianus. In the particular case of the Acidithiobacillus genus, previous reports determined the capacities of arsenic resistance, specifically in some A. ferrooxidans strain grown in the presence of arsenopyrite [32,33], suggesting that this genus is tolerant to arsenic. In addition, mixed mesophilic adapted culture (enriched in A. caldus and L. ferriphilum) have shown high tolerance to arsenic in packed-bed columns and continuous bioleaching reactors of arsenopyrite from mine tailings [34,35].
Bacterial attachment on minerals increases leaching activities due to a higher interaction space between the cells and mineral surface [36], enhancing the oxidation of refractory minerals such as arsenopyrite. In this context, our results show the real capacity of ACH strain to oxidize arsenopyrite, dissolving almost entirely the matrix by action of the ferric iron generated by the microorganisms after 15 days (Equation (2)) ( Figure 5). In addition, microorganism's presence generates more drastic changes on the arsenopyrite surface compared to the use of a Fe 2 (SO 4 ) 3 chemical solution [37]. On the other hand, depending on the oxidizing conditions, a great number of sub-products could be generated from the arsenopyrite oxidation, which could inhibit the correct mineral oxidation due to the formation of surface passivation layers [30]. Additionally, these passivation layers might be composed by elemental sulfur, arsenolite, jarosite, iron hydroxides, amorphous ferric arsenate/scorodite and ferric phosphate [38]. Therefore, to describe these sub-products and understand their possible negative effects, a combination of different approaches (X-ray diffraction (XRD), scanning electron microscopy (SEM) and QEMSCAN) was the most convenient strategy to analyze the surface chemistry during arsenopyrite bio-oxidation.
Interestingly, the XRD patterns revealed that the principal sub-products generated by ACH strain were sulfur, pyrite and scorodite ( Figure 6). Additionally, a similar result reported the presence of ferric arsenates and sulfur on bio-oxidized arsenopyrite by Acidthiobacillus ferrooxidans [27]. Additionally, the transformation of arsenopyrite to jarosite, sulfur and ferric arsenate when metabolized by Acidithiobacillus caldus at 45 • C was described [38]. Nevertheless, in none of these cases was scorodite identified; only an unidentified, amorphous ferric arsenate was reported. Likewise, the formation of jarosite, sulfur and silica were reported due the presence of A. thiooxidans, due to a mine tailing (mainly composed by arsenopyrite) oxidation [39]. Additionally, similar results using two different moderate thermophile bacterial consortia (1: Sulfobacillus thermosulfidooxidans with A. caldus, and 2: Ferroplasma thermophilum with A. caldus) were described, for which jarosite and sulfur were the main sub-products generated in both cases [31].
Additionally, the comparative oxidation of arsenopyrite employing three different systems evidenced the sulfur formation in all the cases. Nevertheless, only in the acidic systems (sterile acid) was scorodite formation detected after 18 days of incubation [40]. In our work, jarosite formation was not detected; one possible reason is the assays parameters, such as pH values (below 3 but above 1.5) (Figure 3), as the formation of this compound requires lower pH values (pH 1.5) [41]. In addition, the QEMSCAN analysis performed to the solids generated by A. ferrivorans ACH arsenopyrite oxidation corroborate the previously XRD obtained results, detecting a 5.4% scorodite formation after 20 days and an 18.7% arsenopyrite matrix decrease (Table 2). Additionally, we identified an increase in As-bearing sulfate generated by A. ferrivorans ACH, detecting 32.1% after 20 days. These results could be attributed to the formation of some precipitate of sulfates such as tooelite (Fe 6 (AsO 3 ) 4 (SO 4 )(OH) 4 ·4H 2 O), a ferric arsenic sulfate generated by some microorganisms capable of oxidizing ferrous iron and not arsenite [42], like our strain. The biogenic of tooelite by several A. ferrooxidans strains (CC1, B5, B20 and the type strain ATCC 23270) which depend on iron rate oxidation was reported; however, the biogenic tooelite mechanisms have not been elucidated to date [43].
The microorganism ability to oxidize arsenopyrite should imply their capacity to tolerate high concentrations of soluble arsenic. In our case, we propose that the ACH strain can tolerate concentrations over 2 g/L of As, because we determined the As and Fe concentrations in solution after the arsenopyrite bio-oxidation assays (Figure 7). We observed that the increase in total Fe over time is concomitant with bacterial growth (Figure 4), and as the oxidation process takes place, the bacteria could tolerate higher arsenic concentrations (Figure 7). It is important to mention that this is the first report describing the ability of an A. ferrivorans species member to grow in arsenopyrite concentrate, in turn generating scorodite under mesophilic conditions. Moreover, a preliminary analysis of the A. ferrivorans ACH genome revealed the presence of the ars operon involved in arsenic resistance, in the five genes conformation: arsA, arsenical pump-driving ATPase; arsD, arsenical resistant operon trans-actin repressor; arsC, arsenate reductase; arsR, arsenical resistant operon repressor; arsB, arsenic efflux pump (Supplementary Table S1) [44]. Hence, the presence of this classical arsenic resistance operon would provide to the ACH strain the necessary functional potential to cope with toxic environmental arsenic concentrations. Moreover, the main mechanism relies on the arsenate (As(V)) reduction to arsenite (As(III)) by ArsC, for its later expulsion by ArsB. Nevertheless, the presence of an arsenite oxidase was not detected, like in others Acidithiobacillus genus strain [32], which would be responsible for the As(III) to As(V) oxidation, which is required for the ferric arsenate (scorodite) precipitate formation at low pH values [12,15]. However, we have to consider thermodynamic aspects: at low pH values, the arsenite (As(III)) is found as arsenous acid (H 3 AsO 3 ), which should be easily oxidized to arsenate (As(V)) (as arsenic acid, H 3 AsO 4 ) by the ferric ions action, according to the following equation: Nevertheless, some authors have reported that the kinetics of the reaction is very slow and almost no oxidation of As(III) occurs [45]. Conversely, other authors described that in presence of pyrite, the reaction speed increase and the oxidation of As(III) to As(V) can be observed [46]. Thus, this observation aligns with our results due to presence of pyrite ( Figure 6 and Table 2). In other words, the capacity of the ACH strain to oxidize pyrite, could favor the As(III) to As(V) oxidation (Equation (5)) as it has been reported before [23]. Additionally, the higher concentration of biogenic ferric iron would allow scorodite precipitation (Equation (6)) [45].
The role of microorganisms is mainly to keep high the redox potential (Eh > 450 mV) and a high concentration of ferric ion in the solution, catalyzing the reoxidation of ferrous ion [45]. The chemical mechanisms described previously could be answer as to why the ACH strain is capable of generating scorodite (biogenic) despite not having an arsenite oxidase in his genome, potentially enhancing its use in bio-oxidation processes of refractory ore.

Conclusions
In this study, we provided the first evidence of arsenopyrite concentrate oxidation capacity conducted by the psychrotolerant iron-sulfur oxidizing A. ferrivorans ACH under mesophilic condition reducing from 35.5% to 16.8% arsenopyrite matrix. This strain was effectively adapted to high concentrations of As, which was reflected in As concentrations quantified after the bio-oxidations assays, in turn generating scorodite as sub-products of the reaction, this being the most stable ways to discard arsenic in mining industries.
Our ACH strain was capable of generating 5.4% bioscorodite after 20 days of incubation. On the other hand, we demonstrated that higher pulp concentration effects their biooxidation capabilities, with 1% being the best pulp concentrations assayed. In general, the bio-oxidation of the gold-containing sulfide ores is used as a pretreatment which can decrease the consumption of lixiviants used for gold solubilization in subsequent parts of the operation, ultimately increasing the gold yields. The results of this study suggest the feasibility of using A. ferrivorans ACH to remove As from As-rich minerals under mesophilic condition, as an alternative to conventional processes actually employed at higher temperature. Future studies in this area are required to understand the resistance mechanisms used by microorganisms to thrive under adverse conditions, which could be coupled with other advantageous capacities (as bioscorodite precipitation), generating biotechnological applications to solve industrial problems with a focus on the efficient use of natural resources, and minimizing environmental impacts.