Role of Ag+ in the Bioleaching of Arsenopyrite by Acidithiobacillus ferrooxidans

Arsenopyrite (FeAsS) is often associated with gold, but pre-treatment is necessary prior to gold leaching, mainly due to the gold encapsulation in the matrix of FeAsS. Bio-oxidation is attractive and promising, largely due to its simplicity, low cost and environmental friendliness. A critical problem that still impedes the large-scale applications of this green technology is its slow leaching kinetics. Some metal ions such as Ag+ have previously been found to expedite the bioleaching process. In this paper, the role of Ag+ in the arsenopyrite bioleaching by Acidithiobacillus ferrooxidans was investigated in detail by bioleaching experiments and a series of analyses including thermodynamics, X-ray diffraction (XRD), scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Experimental results suggested that addition of 5 mg/L Ag+ to the leaching system could significantly improve the final As leaching efficiency from 30.4% to 47.8% and shorten the bioleaching period from 19 days to 15 days. Thermodynamic analysis indicates that Ag+ destabilises As2S2, As2S3 and S0 via forming Ag2S, which is confirmed by the XRD analysis on the phase transformation during bioleaching. SEM and XPS analyses further showed that Ag+ removed the passivating film consisting mainly of As2S2, As2S3 and S0 because Ag2S formed on the arsenopyrite surface from the start bioleaching of 36 h. In the presence of Fe3+, Ag2S could easily be dissolved to Ag+ again, likely leading to the establishment of the Ag+/Ag2S cycle. The bacteria utilised the two synergistic cycles of Fe3+/Fe2+ and Ag+/Ag2S to catalyse the bioleaching of arsenopyrite.


Introduction
In nature, significant amounts of gold are often found in arsenopyrite (FeAsS) [1][2][3]. However, most gold in the arsenopyrite is difficult to leach, mainly because the gold tends to be encapsulated in the mineral matrix [4,5]. It is, thus, necessary to pre-treat arsenopyrite gold ores before leaching to improve gold extraction. Currently, four pre-treatment methods are commonly used, i.e., oxidative roasting, chemical oxidation, pressure oxidation and biological oxidation [6,7]. Amongst them, bio-oxidation is a simple, low-cost and eco-friendly technology that is appropriate to extract valuable metals from a series of ores/concentrates and waste materials with substantially reduced environmental pollution. In the past decade, this promising green technology has drawn significant attention, and its applications in bio-hydrometallurgy have also been on the increase [8][9][10].
Although, many advantages can be provided from bio-oxidation, the problem of slow leaching kinetics still impedes its large-scale application [8,11]. A range of metal ions, including Cu 2+ , Bi 3+ , Co 2+ , Hg 2+ and Ag + have, therefore, been used as catalysts to enhance the dissolution kinetics and shorten the bioleaching period [12,13]. Much attention has been paid to Ag + among these metal ions. Significant Metals 2020, 10 researches had suggested that Ag + is an efficient catalyst in improving the leaching yields and kinetics of valuable metals from a wide range of copper sulphide minerals, such as chalcopyrite [14][15][16][17]. Ag + was also shown to effectively enhance the bioleaching of arsenic sulphide minerals, such as realgar and orpiment [18,19]. However, few research effects have been made to investigate the effects of Ag + on the bioleaching of arsenopyrite. Research has reported that as 2 mg·L −1 Ag + is added to leaching solution, the bioleaching efficiency of As, from arsenopyrite, could be improved by~23% for the same leaching duration [20]. Maybe the catalytic effect of Ag + is attributed to an autocatalytic role of Ag + or formation of an autocatalytic surface. The reason why Ag + enhanced the bioleaching of arsenopyrite, and how Ag + played its role in the process, remain unclear.
In our recent research [13,21,22], a compact passivating film was found to form on the surface of arsenopyrite from the beginning stage of bioleaching (<36 h), and this film would severely hinder the subsequent leaching process. The formation of this passivating film that was shown to consist mainly of As 2 S 2 , As 2 S 3 and S 0 was considered as the main reason for the slow leaching kinetics and long leaching periods. Based on these research findings, this paper reports results from a detailed investigation into the role of Ag + in the bioleaching of pure arsenopyrite in 9K culture medium containing Acidithiobacillus ferrooxidans (A. ferrooxidans). This investigation starts with a series of bioleaching experiments on natural samples of pure arsenopyrite. The leachate and leached solid samples were respectively analysed with inductively coupled plasma-atomic emission spectrometry (ICP-AES) for the As and Fe concentrations and with X-ray diffraction (XRD), scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) for mineralogical phase, morphology and surface composition. In addition, thermodynamic analyses were conducted to elucidate the reasons for the improved bioleaching kinetics by Ag + . The possible mechanisms by which the Ag + enhanced the bioleaching of arsenopyrite were also proposed in this paper.

Thermodynamic Calculations
The software, HSC Chemistry 6.0 [23], was used to construct the Eh−pH diagrams for the FeAsS−H 2 O and FeAsS−Ag + −H 2 O systems. The temperature and pressure were set at 25 • C and 1 atm, respectively. Under the leaching conditions of [Fe 2+ ] 0.16 M, [FeAsS] 0.06 M (10 g/L) and [Ag + ] 0.046 mM (5 mg/L; when Ag + was present in the leaching system), the predominance areas for Fe, As and S as well as Ag species on relevant Eh (versus the standard hydrogen electrode (SHE)) and pH scales were presented in Eh−pH diagrams. The available thermodynamic data used in the calculations are listed in Appendix A Table A1.

Minerals, Strain and Media
The high-purity arsenopyrite sample investigated had a composition of Fe 1.00 As 0.99 S 0.97 and only contained impurities of Si 0.132%, Ni 0.062% and Co 0.034% assayed by X-ray fluorescence analysis. Either particles or cuboids of the arsenopyrite samples were used in this study. The arsenopyrite particles (over 90%, −74 µm) were obtained by wet-milling in a ball mill whilst the cuboids (~15 mm × 15 mm ×~5 mm) were prepared using a cutter. Air-tight plastic bags were used to store the samples to minimise oxidation.
The bacteria of A. ferrooxidans and 9K culture medium containing FeSO 4 ·7H 2 O 44.8 g/L, (NH 4 ) 2 SO 4 3.0 g/L, K 2 HPO 4 ·3H 2 O 0.50 g/L, KNO 3 0.14 g/L, Ca(NO 3 ) 2 ·4H 2 O 0.01 g/L and MgSO 4 ·7H 2 O 0.5 g/L were utilised for the bioleaching of arsenopyrite. A total of 100 mL 9K culture medium and 15 mL inoculum were first added to 250 mL Erlenmneyer flasks and then shaken in an orbital thermostat shaker under 160 rpm and 30 • C to culture and sub-culture A. ferrooxidans. The medium was adjusted by careful addition of~3 M H 2 SO 4 to have an initial pH value of 1.8. In the presence of 10 g/L arsenopyrite particles, the bacteria had been sub-cultured for three months before the bioleaching experiments.
Silver nitrate (AgNO 3 ) was utilised as the silver cation (Ag + ) source. The reagents used in the bioleaching experiments of arsenopyrite and the cultivation of bacteria were all analytically pure. De-ionised water was utilised throughout all experiments.

Bioleaching Experiment
Using the same experimental method and conditions for the cultivation of bacteria, the growth of A. ferrooxidans and the bioleaching of arsenopyrite by A. ferrooxidans proceeded simultaneously. The addition of Ag + to the leaching solution ranged from 1 to 20 mg/L. For the bioleaching of arsenopyrite particles, the pulp density was 10 g/L of FeAsS. Samples from the supernatant were withdrawn at regular intervals during the bioleaching process for chemical analysis. During the bioleaching process, the solution potential and pH values were also measured and recorded. All solution potentials were reported relative to the SHE. The pulp was filtered, washed with copious de-ionised water at least ten times, and dried in a vacuum oven at 35 • C overnight, to attain the residue for the subsequent mineralogical phase study. The morphology and surface composition analyses were conducted on the arsenopyrite cuboids. Prior to each bioleaching experiment, silicon carbide papers of 800, 1500 and 3000 grit were used sequentially to polish the cuboids, and then the contaminants on the surface of cuboids were cleaned ultrasonically in alternate baths of 5 M HCl, methanol and water for 5 min [24,25].

Analytical Methods
The concentrations of As and Fe in leachate were determined by ICP-AES (PS-6, Baird). Potassium dichromate titration method was used to determine the concentration of Fe 2+ . The difference between the concentration of the total Fe and Fe 2+ obtained the Fe 3+ concentration. A pH meter (PHSJ-4A, Shanghai Leici, Shanghai, China) equipped with a Pt electrode and Ag/AgCl (saturated KCl, Orion) reference electrode was used to measure the values of solution potential and pH. An X-ray diffractometer (D/Max 2500, Rigaku, Hiroshima, Tokyo, Japan) was used to determine the mineralogical phases of the bioleached residue. An SEM (Helios NanoLab G3 UC, FEI, Hillsboro, OR, USA) was used to examine the morphology of leached cuboids. The Ag species on the surface of the leached cuboid was analysed with XPS (ESCALAB250Xi, Thermo Fisher, Waltham, MA, USA).

Effect of Ag + Addition on Arsenopyrite Bioleaching
The kinetic results from the arsenopyrite bioleaching are shown in Figure 1. As the leaching behaviour of As from arsenopyrite acts as a critical indicator for the bioleaching of arsenopyrite [13,21,22], the effect of Ag + on arsenopyrite bioleaching can be evaluated by its influence on the change of As leaching efficiency during bioleaching, as shown in Figure 1a. In contrast with the slow bioleaching kinetics of As without Ag + , the addition of Ag + to the solution was shown to substantially enhance the bioleaching of As, and thus, arsenopyrite as manifested by the marked rise in the leaching efficiency of As during bioleaching. The optimal concentration of Ag + was shown to be 5 mg/L. Under this Ag + level, the final leaching efficiency of As was noticeably increased from 30.4% without Ag + to 47.8%. The bioleaching period was also shortened from 19 days to 15 days. The presence of Ag + made the bioleaching of As much faster than the situation without Ag + from the initial leaching stage. After leaching 1 day, the As leaching efficiency was markedly increased from 2.4% without Ag + to 12.3% with 5 mg/L Ag + . Obviously, when Ag + (5 mg/L) was contained in the solution, the passivation of the arsenopyrite surface that took place initially during bioleaching, as reported in our recent researches [13,21], was greatly weakened and even disappeared. When the concentration of Ag + exceeded 5 mg/L, the bioleaching deteriorated as the As leaching efficiency started to decrease. This phenomenon can be explained by the fact that an excess of Ag + have detrimentally affected the reproduction and activity of bacteria, and thus, the bioleaching of As.
of Ag + (5 mg/L) was found to cause a much lower Fe 3+ concentration and a higher Fe 2+ concentration ( Figure 1c) and thus a lower level of solution potential ( Figure 1b). However, a much higher leaching efficiency of As (Figure 1a) was still achieved, further confirming the catalytic effect of Ag + (5 mg/L). As will be demonstrated in the following sections, a series of analyses of thermodynamics, mineralogical phase, morphology and surface composition were conducted to elucidate why, and how, Ag + played this catalytic effect.

Thermodynamics of Arsenopyrite Dissolution in the Presence of Ag +
The EhpH diagrams of FeAsSH2O and FeAsSAg + H2O systems are presented in Figure 2a-c and 2a'-d', respectively. Under the optimal addition of Ag + (5 mg/L), Figure 1b,c show the variation of pH and solution potential, as well as concentrations of iron ions including Fe 3+ , Fe 2+ and total Fe during bioleaching. As shown in Figure 1b, a similar variation trend of pH was found regardless if Ag + was present in the solution. The pH rose noticeably with the growth of bacteria in the initial leaching of 3 days. At the later stage (>3 days), the pH decreased, mainly due to the oxidative dissolution of arsenopyrite [13,21]. The bioleaching process is closely related to the solution potential, which is largely determined by the Fe 3+ /Fe 2+ couple in the leaching system. A similar change trend between the solution potential and the Fe 3+ concentration was clearly seen from Figure 1b,c. A greater solution potential can offer a stronger driving force for the bioleaching of arsenopyrite [13,21]. The presence of Ag + (5 mg/L) was found to cause a much lower Fe 3+ concentration and a higher Fe 2+ concentration (Figure 1c) and thus a lower level of solution potential (Figure 1b). However, a much higher leaching efficiency of As (Figure 1a) was still achieved, further confirming the catalytic effect of Ag + (5 mg/L). As will be demonstrated in the following sections, a series of analyses of thermodynamics, mineralogical phase, morphology and surface composition were conducted to elucidate why, and how, Ag + played this catalytic effect. In the pH range of 0-3 that is a typical acidic bioleaching condition, Figure 2a-c show that, in the absence of Ag + , an Eh higher than −0.4 V can drive the oxidative dissolution of arsenopyrite. In In the pH range of 0-3 that is a typical acidic bioleaching condition, Figure 2a-c show that, in the absence of Ag + , an Eh higher than −0.4 V can drive the oxidative dissolution of arsenopyrite. In the Eh−pH region of −0.4-0.4 V and 0-3, the predominant species of Fe, As and S are Fe 2+ (Figure 2a), As 2 S 2 /As 2 S 3 (Figure 2b), and As 2 S 2 /As 2 S 3 /S 0 (Figure 2c), respectively. As demonstrated previously [13,21], the solid species of As 2 S 2 , As 2 S 3 and S 0 are the main intermediate products that would form a passivating film on the arsenopyrite surface, causing a severe hindrance to the bioleaching process. Under an Eh higher than 0.2-0.4 V, H 3 OFe 3 (SO 4 ) 2 (OH) 6 in Figure 2a,c is shown to be the main phase for the Fe and S species while HAsO 3 and H 2 AsO 4 − in Figure 2b are the main phase for the As species. Note that, in the 9K culture medium, H 3 OFe 3 (SO 4 ) 2 (OH) 6  show that, within pH of 0-3, a much lower Eh (> −0.44 -−0.62 V) is required to leach the Fe from FeAsS as Fe 2+ to the leaching solution, whilst a much higher Eh (>0.3-0.5 V) is needed for the transformation of Fe 2+ to H 3 OFe 3 (SO 4 ) 2 (OH) 6 . The predominance areas of As and S species, i.e., As 2 S 2 , As 2 S 3 and S 0 in Figure 2b,c change to those of HAsO 2 (a) and Ag 2 S as shown in Figure 2b',c'. In common with the formation of Fe 2+ , HAsO 2 (a) and Ag 2 S can also be formed at a lower Eh (> −0.44-−0.62 V). Therefore, thermodynamically, the presence of Ag + makes As 2 S 2 , As 2 S 3 and S 0 unstable by forming Ag 2 S. In addition, Figure 2d' shows that the predominant Ag species is Ag 2 S, which is readily converted to Ag + at an Eh higher than 0.3-0.5 V. This means that the oxidative dissolution of Ag 2 S into the solution can easily take place in the presence of the Fe 3+ /Fe 2+ couple (E • = 0.771 V versus SHE).

Mineralogical Phase Transformation
To determine the effect of Ag + (5 mg/L) on the transformation of mineralogical phase, XRD surveys were performed on bioleached arsenopyrite particles for different days. As the passivation took place from the onset of bioleaching [13,21], the phase change was investigated for the initial leaching stage (<3 days). The XRD diffractograms are presented in Figure 3. the EhpH region of −0.4-0.4 V and 0-3, the predominant species of Fe, As and S are Fe 2+ (Figure 2a), As2S2/As2S3 (Figure 2b), and As2S2/As2S3/S 0 (Figure 2c), respectively. As demonstrated previously [13,21], the solid species of As2S2, As2S3 and S 0 are the main intermediate products that would form a passivating film on the arsenopyrite surface, causing a severe hindrance to the bioleaching process. Under an Eh higher than 0.2-0.4 V, H3OFe3(SO4)2(OH)6 in Figure 2a,c is shown to be the main phase for the Fe and S species while HAsO3 -and H2AsO4 − in Figure 2b are the main phase for the As species. Note that, in the 9K culture medium, H3OFe3(SO4)2(OH)6 and K + can readily form jarosite [KFe3(SO4)2(OH)6] [26,27].
Comparing Figure 2a-c with 2a'-c', the addition of 0.046 mM (5 mg/L) Ag + to the FeAsSH2O system leads to significant changes in the predominance regions for Fe, As and S species. Figure 2a and a' show that, within pH of 0-3, a much lower Eh (> −0.44 -−0.62 V) is required to leach the Fe from FeAsS as Fe 2+ to the leaching solution, whilst a much higher Eh (>0.3-0.5 V) is needed for the transformation of Fe 2+ to H3OFe3(SO4)2(OH)6. The predominance areas of As and S species, i.e., As2S2, As2S3 and S 0 in Figure 2b,c change to those of HAsO2 (a) and Ag2S as shown in Figure 2b',c'. In common with the formation of Fe 2+ , HAsO2 (a) and Ag2S can also be formed at a lower Eh (> −0.44-−0.62 V). Therefore, thermodynamically, the presence of Ag + makes As2S2, As2S3 and S 0 unstable by forming Ag2S. In addition, Figure 2d' shows that the predominant Ag species is Ag2S, which is readily converted to Ag + at an Eh higher than 0.3-0.5 V. This means that the oxidative dissolution of Ag2S into the solution can easily take place in the presence of the Fe 3+ /Fe 2+ couple (E° = 0.771 V versus SHE).

Mineralogical Phase Transformation
To determine the effect of Ag + (5 mg/L) on the transformation of mineralogical phase, XRD surveys were performed on bioleached arsenopyrite particles for different days. As the passivation took place from the onset of bioleaching [13,21], the phase change was investigated for the initial leaching stage (<3 days). The XRD diffractograms are presented in Figure 3.
Without the addition of Ag + , the phases of realgar (As2S2) and orpiment (As2S3) occurred after bioleaching 1 day (Figure 3a). Elemental sulphur (S 0 ) and jarosite [KFe3(SO4)2(OH)6] were also found after 3 days (Figure 3b). With the addition of Ag + at 5 mg/L, however, Figure 3a,b shows that the passivating phases of As2S2, As2S3 and S 0 disappeared with the formation of acanthite (Ag2S). This agrees well with the thermodynamic predictions in Section 4.2.

Morphology and Surface Composition Changes
SEM and XPS analyses were further conducted to reveal the effect of Ag + (5 mg/L) on the morphology and surface composition of the bioleached arsenopyrite cuboids. During the bioleaching process, considerable amounts of jarosite were observed to form after around 5 days whether Ag + was added to the solution or not. However, our recent research suggested that the precipitates of Without the addition of Ag + , the phases of realgar (As 2 S 2 ) and orpiment (As 2 S 3 ) occurred after bioleaching 1 day (Figure 3a). Elemental sulphur (S 0 ) and jarosite [KFe 3 (SO 4 ) 2 (OH) 6 ] were also found after 3 days (Figure 3b). With the addition of Ag + at 5 mg/L, however, Figure 3a,b shows that the passivating phases of As 2 S 2 , As 2 S 3 and S 0 disappeared with the formation of acanthite (Ag 2 S). This agrees well with the thermodynamic predictions in Section 4.2.

Morphology and Surface Composition Changes
SEM and XPS analyses were further conducted to reveal the effect of Ag + (5 mg/L) on the morphology and surface composition of the bioleached arsenopyrite cuboids. During the bioleaching process, considerable amounts of jarosite were observed to form after around 5 days whether Ag + was added to the solution or not. However, our recent research suggested that the precipitates of jarosite only formed a loose and porous overlayer, instead of a passivating film, on the surface of arsenopyrite [13,21]. The surface was investigated just for the initial leaching stage (i.e., 36 h) to avoid the interference from jarosite. The SEM images and XPS spectrum are presented in Figures 4  and 5 jarosite only formed a loose and porous overlayer, instead of a passivating film, on the surface of arsenopyrite [13,21]. The surface was investigated just for the initial leaching stage (i.e., 36 h) to avoid the interference from jarosite. The SEM images and XPS spectrum are presented in Figures 4 and 5, respectively. As clearly seen from Figure 4a, a compact passivating film was formed on the arsenopyrite surface. Note that the film was intact in the solution, and the appearance of the cracks occurred only after drying. The film was shown to consist mainly of As2S2, As2S3 and S 0 in our recent researches [13,21]. After bioleaching in the presence of Ag + (5 mg/L), no passivating film was observed on the surface, as presented in Figure 4b. As shown in the above XRD analysis, the disappearance of the phases of As2S2, As2S3 and S 0 due to the addition of Ag + is responsible for the removal of this passivating film.
In addition, Figure 5 shows the XPS spectrum of Ag 3d on the outmost surface of the leached arsenopyrite cuboid at the Ag + level of 5 mg/L. Two obvious peaks of Ag 3d(3/2) and Ag 3d(5/2) were exhibited. The Ag 3d(5/2) peak centring at the binding energy of 368.1 eV reflects the typical peak of Ag2S [28,29].  jarosite only formed a loose and porous overlayer, instead of a passivating film, on the surface of arsenopyrite [13,21]. The surface was investigated just for the initial leaching stage (i.e., 36 h) to avoid the interference from jarosite. The SEM images and XPS spectrum are presented in Figures 4 and 5, respectively. As clearly seen from Figure 4a, a compact passivating film was formed on the arsenopyrite surface. Note that the film was intact in the solution, and the appearance of the cracks occurred only after drying. The film was shown to consist mainly of As2S2, As2S3 and S 0 in our recent researches [13,21]. After bioleaching in the presence of Ag + (5 mg/L), no passivating film was observed on the surface, as presented in Figure 4b. As shown in the above XRD analysis, the disappearance of the phases of As2S2, As2S3 and S 0 due to the addition of Ag + is responsible for the removal of this passivating film.
In addition, Figure 5 shows the XPS spectrum of Ag 3d on the outmost surface of the leached arsenopyrite cuboid at the Ag + level of 5 mg/L. Two obvious peaks of Ag 3d(3/2) and Ag 3d(5/2) were exhibited. The Ag 3d(5/2) peak centring at the binding energy of 368.1 eV reflects the typical peak of Ag2S [28,29]. In summary, the presence of 5 mg/L Ag + substantially enhanced the bioleaching efficiency and kinetics (Section 4.1.). The XRD, SEM, XPS and thermodynamic results, presented in Sections 4.2.-4.4. demonstrated that the inclusion of 5 mg/L Ag + in the leaching system could lead to the disappearance of the visible passivating film on the arsenopyrite surface in bioleaching without Ag + . The removal of the passivating film that can form at the initial leaching stage (< 36 h) explains why Ag + catalyses the whole bioleaching process. As clearly seen from Figure 4a, a compact passivating film was formed on the arsenopyrite surface. Note that the film was intact in the solution, and the appearance of the cracks occurred only after drying. The film was shown to consist mainly of As 2 S 2 , As 2 S 3 and S 0 in our recent researches [13,21]. After bioleaching in the presence of Ag + (5 mg/L), no passivating film was observed on the surface, as presented in Figure 4b. As shown in the above XRD analysis, the disappearance of the phases of As 2 S 2 , As 2 S 3 and S 0 due to the addition of Ag + is responsible for the removal of this passivating film.
In addition, Figure 5 shows the XPS spectrum of Ag 3d on the outmost surface of the leached arsenopyrite cuboid at the Ag + level of 5 mg/L. Two obvious peaks of Ag 3d (3/2) and Ag 3d (5/2) were exhibited. The Ag 3d (5/2) peak centring at the binding energy of 368.1 eV reflects the typical peak of Ag 2 S [28,29].
In summary, the presence of 5 mg/L Ag + substantially enhanced the bioleaching efficiency and kinetics (Section 4.1). The XRD, SEM, XPS and thermodynamic results, presented in Sections 4. 2-4.4 demonstrated that the inclusion of 5 mg/L Ag + in the leaching system could lead to the disappearance of the visible passivating film on the arsenopyrite surface in bioleaching without Ag + . The removal of the passivating film that can form at the initial leaching stage (< 36 h) explains why Ag + catalyses the whole bioleaching process.

Possible Mechanisms for the Ag + Catalysed Bioleaching of Arsenopyrite
Based on the above experimental and analytical results, the possible mechanism for the silver-catalysed bioleaching of arsenopyrite can be proposed as presented in Figure 6. In addition, the possible reactions involved in the bioleaching process are listed in Table 1. Based on the above experimental and analytical results, the possible mechanism for the silvercatalysed bioleaching of arsenopyrite can be proposed as presented in Figure 6. In addition, the possible reactions involved in the bioleaching process are listed in Table 1.   Figure 6a shows that, in the leaching solution without Ag + , the bacteria oxidise the Fe 2+ to Fe 3+ , which will in turn oxidise the arsenopyrite to achieve its leaching, as shown by Equations (1)-(3) in Table 1. With the bioleaching of arsenopyrite, As2S2, As2S3 and S 0 are also produced in situ on the surface of arsenopyrite particles. Although these solid products could be further oxidised according to reactions (7)-(9) listed in Table 1, they still formed a passivating film at the initial bioleaching stage, causing a severe impedance to the subsequent leaching process.   Figure 6a shows that, in the leaching solution without Ag + , the bacteria oxidise the Fe 2+ to Fe 3+ , which will in turn oxidise the arsenopyrite to achieve its leaching, as shown by Equations (1)-(3) in Table 1. With the bioleaching of arsenopyrite, As 2 S 2 , As 2 S 3 and S 0 are also produced in situ on the surface of arsenopyrite particles. Although these solid products could be further oxidised according to reactions (7)-(9) listed in Table 1, they still formed a passivating film at the initial bioleaching stage, causing a severe impedance to the subsequent leaching process.
With the addition of Ag + to the solution (Figure 6b), the oxidative dissolution of arsenopyrite would proceed according to Equations (4)-(6) with much lower ∆G r 0 values than those of Equations (1)-(3) ( Table 1). In the process, Ag 2 S was formed on the surface of arsenopyrite, largely preventing the formation of the passivating products (i.e., As 2 S 2 , As 2 S 3 and S 0 ) on the basis of Equations (10)- (12), as evidenced by the results from XRD and XPS analyses. In addition, Ag 2 S itself is a conductor that is beneficial for the transfer of electrons in the leaching process. In the presence of Fe 3+ , Ag 2 S can also be oxidised by the Fe 3+ /Fe 2+ couple according to reaction (13), releasing Ag + back to the solution. Therefore, as presented in Figure 6b, a catalytic cycle of Ag + /Ag 2 S was also established from the catalytic cycle of Fe 3+ /Fe 2+ . The two catalytic cycles led to the elimination of the passivating film, as evidenced by the SEM images, i.e., the much improved bioleaching of arsenopyrite.

Conclusions
Experimental results from the arsenopyrite bioleaching by A. ferrooxidans in 9K culture medium suggested that addition of 5 mg/L Ag + to the solution had no significant influence on the pH, but resulted in a lower level of the solution potential. However, the bioleaching of arsenopyrite was still substantially improved from the onset of leaching. On the one hand, the final leaching efficiency of As was significantly increased from 30.4% to 47.8%; on the other hand, the bioleaching period was noticeably shortened from 19 days to 15 days. This is attributed to the fact that the presence of Ag + in the solution leads to the removal of the passivating film (being composed mainly of As 2 S 2 , As 2 S 3 and S 0 [13,21]) that could be formed initially on the surface of arsenopyrite. Thermodynamic and XRD analyses demonstrated that, during the bioleaching of arsenopyrite, Ag + could destabilise As 2 S 2 , As 2 S 3 and S 0 through formation of Ag 2 S, which would easily be dissolved to Ag + by the Fe 3+ /Fe 2+ couple. SEM and XPS analyses further suggested that the main difference the Ag + caused was that the formation of the passivating film was prevented by Ag + via forming Ag 2 S on the arsenopyrite surface. On the basis of the combined results from bioleaching experiments, thermodynamic and phase analyses, and surface characterisation, a possible mechanism for the silver catalysed bioleaching of arsenopyrite is put forward that the two synergistic cycles of Ag + /Ag 2 S and Fe 3+ /Fe 2+ were utilised by the bacteria to improve the leaching of arsenopyrite. The results in this paper can also offer useful information for the extraction of valuable metals from other As-and S-bearing materials.
Author Contributions: Y.Z. and Q.L. conceived and designed the study; Q.L. contributed reagents/materials/analysis tools; Y.Z. performed the experiments; X.L. and Y.Z. analysed the data; X.L. wrote the paper. All authors have read and agreed to the published version of the manuscript.