Synchrotron Radiation Based Study of the Catalytic Mechanism of Ag + to Chalcopyrite Bioleaching by Mesophilic and Thermophilic Cultures

Zhenyuan Nie 1, Weiwei Zhang 1, Hongchang Liu 1,2,*, Jinlan Xia 1, Wei Zhu 3, Duorui Zhang 1, Lei Zheng 4, Chenyan Ma 4, Yidong Zhao 4 and Wen Wen 5 1 Key Lab of Biometallurgy of Ministry of Education of China, School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China; zynie@csu.edu.cn (Z.N.); zhangweiwei@gig.ac.cn (W.Z.); jlxia@csu.edu.cn (J.X.); drzhang@csu.edu.cn (D.Z.) 2 CAS Key Laboratory of Mineralogy and Metallogeny, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, China 3 College of Science, Hunan University of Technology, Zhuzhou 41000, China; greatcsu@163.com 4 Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 10049, China; zhenglei@mail.ihep.ac.cn (L.Z.); macny@ihep.ac.cn (C.M.); zhaoyd@ihep.ac.cn (Y.Z.) 5 Shanghai Synchrotron Radiation Facility, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201204, China; wenwen@sinap.ac.cn * Correspondence: liuhongchang@gig.ac.cn; Tel.: +86-20-85290341


Introduction
Chalcopyrite is the most abundant copper mineral amongst the copper sulfides and accounts for more than 70% of the copper minerals in the world [1].In recent decades, due to the excessive consumption of copper concentrate for industrial applications, there is an urgent need to recover copper from low-grade sulfide ores [2,3].Bioleaching technology is a promising method to recover copper from those ores with advantages such as low-cost and environment-friendly [4].However, one common problem limiting the application of this technology to industrial applications is that the copper extraction during bioleaching of chalcopyrite is still low [5,6].

Bioleaching Experiment
For bioleaching experiments, all cultures were incubated in 500 mL Erlenmeyer flasks containing 2 g chalcopyrite and 200 mL sterilized basal medium, with initial pH 1.8 and inoculated cell density 4  10 7 cells/mL.The cultivation of the mixed mesophilic culture and mixed thermophilic culture was performed in rotary shakers (SHZ-GW) at 170 r/min for 28 days, with constant temperatures at 30 °C and 48 °C, respectively.Before experiment, different concentrations of Ag2SO4 were added to each culture condition.The concentrations of Ag + in solution were presented as 0.1%, 0.2%, 0.3% of the weight of silver to the weight of chalcopyrite, that is, 10, 20 and 30 mg/L, respectively.The bioleaching experiments in the absence of Ag + and the sterile experiment in the presence of 0.1%, 0.2% or 0.3% of Ag + served as controls.The experiments were conducted in triplicate at each condition.During bioleaching, evaporated water was compensated with sterilized ultra-pure water based on weight loss at 24-h and 12-h intervals for the mesophilic culture and thermophilic culture, respectively.

Leaching Parameters Determination
During bioleaching and sterile control experiments, solutions were taken out at 1 day or 2 days intervals to determine cell densities, pH and redox potential (ORP) values and the concentrations of Cu 2+ ([Cu 2+ ]), Fe 3+ ([Fe 3+ ]) and total iron ([TFe]) according to previous study [17].Briefly, cell numbers were counted with an Olympus microscope (BX43).The pH value was determined with a pH meter (PHS-3C).The ORP value was measured with a platinum electrode, using a calomel electrode

Bioleaching Experiment
For bioleaching experiments, all cultures were incubated in 500 mL Erlenmeyer flasks containing 2 g chalcopyrite and 200 mL sterilized basal medium, with initial pH 1.8 and inoculated cell density 4 × 10 7 cells/mL.The cultivation of the mixed mesophilic culture and mixed thermophilic culture was performed in rotary shakers (SHZ-GW) at 170 r/min for 28 days, with constant temperatures at 30 • C and 48 • C, respectively.Before experiment, different concentrations of Ag 2 SO 4 were added to each culture condition.The concentrations of Ag + in solution were presented as 0.1%, 0.2%, 0.3% of the weight of silver to the weight of chalcopyrite, that is, 10, 20 and 30 mg/L, respectively.The bioleaching experiments in the absence of Ag + and the sterile experiment in the presence of 0.1%, 0.2% or 0.3% of Ag + served as controls.The experiments were conducted in triplicate at each condition.During bioleaching, evaporated water was compensated with sterilized ultra-pure water based on weight loss at 24-h and 12-h intervals for the mesophilic culture and thermophilic culture, respectively.

Chalcopyrite Surface Compositions Analyses
The chalcopyrite surface compositions were analyzed by SR-XRD and XANES spectroscopy.Before these analyses, the solid samples were gently washed three times with diluted sulfuric acid and diluted hydrochloric acid, respectively, to prevent the possible contamination from solution species and dried in vacuum in a vacuum drying oven (DZF-6020, Shaanxi Aoxin Electronic Technology, Shaanxi, China) and stored in nitrogen atmosphere at −70 • C. SR-XRD was detected at BL14B1 beamline of Shanghai Synchrotron Radiation Facility, Shanghai, China, at a step size of 0.01 • and a dwell time of 0.5 s with X-ray energy 10 keV and spot size 0.5 × 0.5 mm.XANES spectroscopy of S and Fe speciation was performed at 4B7A and 4B7B beamlines of Beijing Synchrotron Radiation Facility, Beijing, China.The S K-edge XANES spectra were scanned between 2460 eV and 2520 eV at step width of 0.2 eV in fluorescence mode at ambient temperature.The Fe L-edge XANES spectra data were recorded from 706 to 728 eV with step size of 0.1 eV in total electron yield mode at ambient temperature.The S K-edge and Fe L-edge XANES spectra were then normalized to the maxima of the absorption spectra and fitted for their linear combinations (LC) using reference spectra with IFEFFIT program according to previous description [20][21][22].
[Fe 3+ ]/[TFe] curves for all bioleaching experiments showed similar trends, rapidly increasing to ~100% from day 0 to day 2 and then unchanging (Figure 2c).ORP values of all bioleaching experiments (Figure 2d) increased rapidly to reach 500-550 mV initially and then changed slightly, however, the ORP values of bioleaching experiments in the presence of 0.1-0.3% of Ag + were greater than that without Ag + on days 0-8 but lower on days 16-28.
The pH values for the bioleaching experiment without Ag + (Figure 2e) slowly increased initially, then remained unchanged and then slightly decreased.The pH values for the bioleaching experiment with 0.1% of Ag + (Figure 2e) slowly increased to 1.89 on days 0-8, increased more rapidly to 2.65 on days 8-20 and then changed slightly.While the pH for the bioleaching experiment with 0.2% and 0.3% of Ag + (Figure 2e) increased to 2.17 on day 16 and 2.26 on day 12, respectively and then slowly decreased to 2.10 on day 18 and 2.19 on day 20, respectively and then increased again.The cell densities (Figure 2f) for all bioleaching experiments showed similar trends, increasing to their greatest values Minerals 2018, 8, 382 5 of 16 and then changing slightly, while the values of the cell density for the bioleaching experiments with Ag + was slightly greater than that without Ag + at their stationary growth phases.
In contrast, [Cu 2+ ] in all sterile experiments at 30 • C (Figure 2a) slowly increased, up to 0.093~0.095g/L on day 28, corresponding to ~2.8% copper extraction rate.[TFe] in all sterile experiments (Figure 2b) slowly reached ~0.09 g/L on day 28.The percentage values of [Fe 3+ ] to [TFe] for all sterile experiments were zero (Figure 2c).The ORP values increased to 288 mV on day 2 and then remained unchanged (Figure 2d).The pH values increased to 1.87 on day 4 and then changed slightly (Figure 2e).for all sterile experiments were zero (Figure 2c).The ORP values increased to 288 mV on day 2 and then remained unchanged (Figure 2d).The pH values increased to 1.87 on day 4 and then changed slightly (Figure 2e).
The percentage of [Fe 3+ ] to [TFe], ORP and cell densities for all bioleaching experiments by thermophilic culture (Figure 3c-f) showed similar trends to that by mesophilic culture (Figure 2c-f), rapidly increasing to the greatest values and then keeping steady.The pH values in the thermophilic and mesophilic systems were different.For bioleaching by thermophilic culture, the pH values in the presence of 0.2-0.3% of Ag + (Figure 3e) increased slightly to pH 1.93-1.95on day 4, decreased to pH 1.79-1.83on day 6, increased to the highest pH values (2.35-2.47) on days 14-16 and then decreased.The pH values for the bioleaching experiments with 0.1% of Ag + (Figure 3e) showed similar trends to that with 0.2-0.3% of Ag + but the greatest value was only ~2.The pH values for the bioleaching experiments without Ag + (Figure 3e) changed slightly until day 14 and then gradually decreased.
In contrast, the leaching parameters in all sterile experiments at 48 • C (Figure 3a) showed similar trends to that at 30 • C (Figure 2a), except that the [Cu 2+ ] (0.17-0.20 g/L), [TFe] (0.18-0.19 g/L) and ORP (328-334 mV) of the former on day 28 were slightly greater than the latter (with 0.093~0.095g/L of [Cu 2+ ], 0.11 g/L of [TFe] and ORP 304-306 mV).The percentage of [Fe 3+ ] to [TFe], ORP and cell densities for all bioleaching experiments by thermophilic culture (Figure 3c-f) showed similar trends to that by mesophilic culture (Figure 2c-f), rapidly increasing to the greatest values and then keeping steady.The pH values in the thermophilic and mesophilic systems were different.For bioleaching by thermophilic culture, the pH values in the presence of 0.2-0.3% of Ag + (Figure 3e) increased slightly to pH 1.93-1.95on day 4, decreased to pH 1.79-1.83on day 6, increased to the highest pH values (2.35-2.47) on days 14-16 and then decreased.The pH values for the bioleaching experiments with 0.1% of Ag + (Figure 3e) showed similar trends to that with 0.2-0.3% of Ag + but the greatest value was only ~2.The pH values for the bioleaching experiments without Ag + (Figure 3e) changed slightly until day 14 and then gradually decreased.

Surface Morphologies of Leaching Residues
The surface of chalcopyrite in the sterile experiments at 30 • C and 48 • C remained unchanged by comparing with original chalcopyrite (Figure 4a).
only some granules precipitated on the mineral surface on day 12 (Figure 4b) and day 28 (Figure 4c), while the surfaces of chalcopyrite in the presence of 0.3% of Ag + suffered from serious corrosion, with many etch pits spreading on the surface on day 12 (Figure 4d) and structured granules and porous residues forming on day 28 (Figure 4e).
The surface morphologies of chalcopyrite during bioleached in the presence of thermophilic culture without Ag + (Figure 4f-g) and with Ag + (Figure 4h-i) showed more serious corrosion than that by mesophilic culture, especially for that by thermophilic culture with 0.3% of Ag + (Figure 4i), the size of the residues became smaller and the surface contained more serious etch pits on its surface.The residues were loose combined on the surface of chalcopyrite or fallen off from the mineral particles.

SR-XRD Analysis
The leach residues on day 28 were analyzed using SR-XRD, as shown in Figure 5.For all sterile experiments at 30 °C and 48 °C without Ag + and with 0.3% of Ag + , only little S 0 and jarosite formed.After bioleaching, the XRD patterns became more complex.For the residues after bioleaching by mesophilic culture and thermophilic culture in the absence of Ag + , the diffraction signal of jarosite and S 0 were detected, while for bioleaching by both cultures in the presence of 0.3% of Ag + , jarosite, S 0 and covellite were detected.It should be noted that, for the bioleaching experiments by both cultures with 0.3% of Ag + , only a little chalcopyrite phase was left while jarosite and S 0 were as the main phases, suggesting that the chalcopyrite dissolution was significantly promoted by Ag + added for both mesophilic culture and thermophilic culture and this result was consistent with the results of [Cu 2+ ] curves (Figure 2a and Figure 3a).The corrosion of chalcopyrite in the presence of mesophilic culture but absence of Ag + was slight, only some granules precipitated on the mineral surface on day 12 (Figure 4b) and day 28 (Figure 4c), while the surfaces of chalcopyrite in the presence of 0.3% of Ag + suffered from serious corrosion, with many etch pits spreading on the surface on day 12 (Figure 4d) and structured granules and porous residues forming on day 28 (Figure 4e).
The surface morphologies of chalcopyrite during bioleached in the presence of thermophilic culture without Ag + (Figure 4f-g) and with Ag + (Figure 4h-i) showed more serious corrosion than that by mesophilic culture, especially for that by thermophilic culture with 0.3% of Ag + (Figure 4i), the size of the residues became smaller and the surface contained more serious etch pits on its surface.The residues were loose combined on the surface of chalcopyrite or fallen off from the mineral particles.

SR-XRD Analysis
The leach residues on day 28 were analyzed using SR-XRD, as shown in Figure 5.For all sterile experiments at 30 • C and 48 • C without Ag + and with 0.3% of Ag + , only little S 0 and jarosite formed.After bioleaching, the XRD patterns became more complex.For the residues after bioleaching by mesophilic culture and thermophilic culture in the absence of Ag + , the diffraction signal of jarosite and S 0 were detected, while for bioleaching by both cultures in the presence of 0.3% of Ag + , jarosite, S 0 and covellite were detected.It should be noted that, for the bioleaching experiments by both cultures with 0.3% of Ag + , only a little chalcopyrite phase was left while jarosite and S 0 were as the main phases, suggesting that the chalcopyrite dissolution was significantly promoted by Ag + added for both mesophilic culture and thermophilic culture and this result was consistent with the results of [Cu 2+ ] curves (Figures 2a and 3a).

Fe L-Edge and S K-Edge XANES
The Fe and S speciation compositions due to bioleaching by both cultures in the presence of 0.3% Ag + and in the absence of Ag + were analyzed using Fe L-edge and S K-edge XANES spectroscopy, respectively.Results are shown in Figures 6-8.
For Fe L-edge XANES analyses, the spectra of reference samples chalcopyrite, bornite and jarosite showed clear differences in their relative intensities of peaks A and B and in their peak positions of peak B (Figure 6a).For the Fe L-edge XANES spectrum of chalcopyrite, the intensity of peak A was greater than that of peak B, while for bornite and jarosite, it was the opposite.For the Fe L-edge XANES spectra of bornite and jarosite, the position of peak B of the former was 0.3 eV less

Fe L-Edge and S K-Edge XANES
The Fe and S speciation compositions due to bioleaching by both cultures in the presence of 0.3% Ag + and in the absence of Ag + were analyzed using Fe L-edge and S K-edge XANES spectroscopy, respectively.Results are shown in Figures 6-8.

Discussion
The bioleaching experiments showed that Ag + significantly promotes the dissolution of chalcopyrite by both mesophilic and thermophilic cultures.For both cultures, within the tested concentrations of Ag + (0.1-0.3%), the greater the concentration of Ag + added, the greater the copper For Fe L-edge XANES analyses, the spectra of reference samples chalcopyrite, bornite and jarosite showed clear differences in their relative intensities of peaks A and B and in their peak positions of peak B (Figure 6a).For the Fe L-edge XANES spectrum of chalcopyrite, the intensity of peak A was greater than that of peak B, while for bornite and jarosite, it was the opposite.For the Fe L-edge XANES spectra of bornite and jarosite, the position of peak B of the former was 0.3 eV less than the latter.It can be seen from Figure 6b-e that the Fe L-edge XANES spectra of leach residues in all the sterile experiments changed slightly.However, for the Fe L-edge XANES spectra of all bioleaching residues, the intensity of peak B gradually increased and became larger than that of peak A after 12 days, 4 days, 4 days and 1 day of bioleaching by mesophilic culture without Ag + , mesophilic culture with 0.3% of Ag + , thermophilic culture without Ag + and thermophilic culture with 0.3% of Ag + , respectively.This indicates that the dissolution of chalcopyrite was promoted by Ag + added to both cultures and it also indicated that the dissolution of chalcopyrite by thermophilic culture was greater than that by mesophilic culture whether in the presence of Ag + or not.It was of particular interest that the peak at 714.0 eV was observed in the spectra of chalcopyrite bioleached by mesophilic culture with Ag + on days 4-12 and in the spectra of chalcopyrite bioleached by thermophilic culture with Ag + on days 1-4, indicating the iron deficient secondary mineral bornite-like species formed.
For S K-edge XANES analyses, the spectra of the reference samples chalcopyrite, bornite, chalcocite, covellite, S 0 and jarosite showed significant difference in their peaks positions and intensities at 2470, 2472 and 2482.7 eV (Figure 7a).For all sterile experiments, the S K-edge XANES spectra changed slightly on comparison to the original chalcopyrite (Figure 7b-e).For bioleaching by mesophilic culture in the absence of Ag + (Figure 7b), the intensity of the peak at 2470 eV remained unchanged and the intensity of the peak at 2482.7 eV increased a little.With 0.3% of Ag + (Figure 7c), the intensity of the peak at 2470.0 eV significantly decreased, the intensity of the peak at 2482.7 eV significantly increased and a new peak at 2472.8 eV appeared from day 4.For bioleaching experiments by thermophilic culture in the absence of Ag + (Figure 7d), the peak at 2470 eV gradually decreased and the peak at 2482.7 eV gradually increased.With Ag + (Figure 7e), similar trends were observed to the mesophilic culture with Ag + .
The LC fitting results (Figure 8) of S K-edge XANES spectra in Figure 7b-e with the reference spectra in Figure 7a showed that, after 28 days of bioleaching, the content of chalcopyrite species decreased to 78%, 27%, 69% and 7% for the bioleaching experiments by mesophilic culture without Ag + , mesophilic culture with 0.3% of Ag + , thermophilic culture without Ag + and thermophilic culture with 0.3% of Ag + , respectively.Results also showed that the new S 0 , jarosite, chalcocite, covellite and bornite species detected during all bioleaching experiments.The contents of bornite and covellite species, as well as S 0 species for the bioleaching in the presence of both cultures with 0.3% of Ag + (Figure 8b,d) were significantly greater than without Ag + (Figure 8a,c).

Discussion
The bioleaching experiments showed that Ag + significantly promotes the dissolution of chalcopyrite by both mesophilic and thermophilic cultures.For both cultures, within the tested concentrations of Ag + (0.1-0.3%), the greater the concentration of Ag + added, the greater the copper extraction obtained.When 0.3% of Ag + was added, the copper extraction after 28 d of bioleaching increased from 17% to 68% by mesophilic culture (Figure 2a) and from 36% to 94% by thermophilic culture (Figure 3a).It was also confirmed that dissolution of chalcopyrite was more significantly promoted by the thermophilic culture than by mesophilic culture, with greater copper leaching, which was consistent with previous publication [10,17].
According to Equations ( 1) and ( 2), the catalytic effect of Ag + on chalcopyrite dissolution would result in greater Fe 2+ and S 0 .The released Fe 2+ and S 0 could be further oxidized by bioleaching cultures, because of their iron and sulfur-oxidizing capabilities (Equations ( 3) and ( 4)) [23], resulting in [TFe], [Fe 3+ ]/[TFe], pH, ORP and cell density in the bioleaching solutions (Figure 2b-f, Figure 3b-f).It should be noted that the curves of each leaching parameter showed a similar trend during bioleaching if the degree of the parameter change is ignored, indicating the addition of Ag + did not change the bacterial sulfur/iron-oxidizing capability of the mesophilic and thermophilic cultures.The effect of Ag + on chalcopyrite bioleaching by the mesophilic and thermophilic cultures in the present study is significantly different with that by the extremely thermophilic archaeal strain A. manzaensis [17].According to Xia et al. [17], the presence of 0.02%-0.1% Ag + could significantly promote copper extraction from chalcopyrite bioleaching by A. manzaensis and the optimal concentration of catalyst [Ag + ] was 0.05%.No more promotion occurred when [Ag + ] was higher than 0.1%; when [Ag + ] was > 0.05%, the iron-oxidizing capability of the archaeal cells decreased significantly [17].These differences were probably caused by the cellular structures and resistant capacity to Ag + between bacterial and archaeal cells [24].
With increasing [TFe] (mainly [Fe 3+ ]), the pH value increased (Equation 3), resulting in the formation of jarosite (Equation ( 5)), which was also accelerated by Ag + added for both mesophilic and thermophilic cultures.Some researchers suggested that the formation of jarosite could hinder chalcopyrite dissolution [25,26] but that is still uncertain [27].The present study showed that jarosite rapidly formed on day 12 and day 4 for bioleaching experiments by the mesophilic and thermophilic cultures, respectively and gradually became a major phase.During the rapid formation of jarosite, chalcopyrite still dissolved rapidly, suggesting jarosite probably not hinder copper extraction for both cultures with and without Ag + .
The M + represent a monovalent cation or group, such as K + , Na + , H 3 O + and NH 4 + .
Other intermediates formed during bioleaching were S 0 and secondary minerals, bornite, chalcocite and covellite (Figures 5-8).Among these intermediates, S 0 species have also been proposed as one major intermediate hindering chalcopyrite dissolution [28], however it is still controversial [29].Though microbes with sulfur-oxidizing capability could utilize S 0 as an energy source, S 0 could be continually formed and accumulated on chalcopyrite surface as a thin film associating with other intermediates, suggesting that it is inefficiently utilized by sulfur-oxidizing microorganisms.Some methods have been proposed to eliminate the S 0 layers, for example, adding surfactant [30].However, in the present study, the greater the amount of S 0 formed during the bioleaching experiment by both mesophilic and thermophilic cultures with 0.3% of Ag + did not hinder chalcopyrite dissolution in comparison to leaching without Ag + .Miller and Portillo [11] suggested that the presence of Ag + could prevent the formation of an impervious S 0 layers on the chalcopyrite surface and instead a porous sulfur film.In the present study, it was also confirmed by surface morphologies of chalcopyrite residues after bioleaching by both mesophilic and thermophilic cultures with Ag + , which was looser than that of without Ag + (Figure 4), which were also in accordance with Liu et al. [17].
For bioleaching of chalcopyrite, the metal-deficient secondary minerals, such as bornite, chalcocite and covellite, were formed mainly due to the preferential dissolution of Fei.The present study showed that more bornite like species formed for bioleaching experiments by both mesophilic and thermophilic cultures in the presence of Ag + (Figure 6, Figure 8).From the results of LC fitting, on days 4-8 for the mesophilic culture and on days 8-12 for the thermophilic culture, bornite was 5~6 times greater than that without Ag + (Figure 8).Meanwhile, the copper extraction rate during this period was almost the highest for the bioleaching experiment with Ag + by comparison with the other bioleaching experiments.According to previous study [31], bornite is easier to dissolve than chalcopyrite.The formation of bornite like species has been speculated to be a key reason for the accelerated dissolution of chalcopyrite during bioleaching at greater pH (>2.00) and smaller solution ORP (<450 mV) [32] and the formation of bornite during bioleaching by the archaeal strain in the presence of Ag + has been proposed to contribute the acceleration of chalcopyrite dissolution at 65 • C [17].In the present study, it was also found that bornite species could be formed even at a greater ORP (~550 mV), suggesting that the addition of Ag + could promote the release of iron and the formation of bornite like species in the presence of the mesophilic and thermophilic cultures with Ag + .
It should be noted that, in a proposed model for chalcopyrite dissolution, chalcocite and bornite were formed initially and then gradually transformed to covellite [33][34][35].In the present study, we also found that the chalcocite and bornite species formed at the early stage (Figure 8) and then transformed to covellite for both mesophilic and thermophilic cultures in the absence of Ag + .After adding Ag + , the chalcocite species was found to be no different to that in the absence of Ag + , while the covellite species was found ~6 times greater than that in the absence of Ag + , suggesting that covellite was probably mainly transformed from bornite under current conditions.On the other hand, according to Yang et al. [36], the accumulation of covellite at the end of chalcopyrite bioleaching could slow down copper extraction.In the present study, we also found the copper extraction was increased slowly on days 20-28, meanwhile covellite was rapidly accumulated, suggesting that covellite like species probably contributes the passivation of chalcopyrite in the presence of the mesophilic and thermophilic cultures.Thus, it can be speculated that the rapid dissolution of chalcopyrite by both mesophilic and thermophilic cultures in the presence of catalyst Ag + is also probably due to the rapid transformation of chalcopyrite to bornite and the conversion of bornite to covellite, however, the accumulation of covellite could be a key reason that copper dissolution slowing down at the late stage of the bioleaching.In addition, it is also indicated that the rapid formation of bornite and the conversion of bornite to covellite for the bioleaching of chalcopyrite in the presence of Ag + is not at all affected by types of bioleaching cultures and their growth temperatures.

Conclusions
Bioleaching experiment has shown that Ag + can significantly promote the dissolution of chalcopyrite by both mesophilic and thermophilic cultures.In addition, the dissolution of chalcopyrite can be more significantly promoted by the thermophilic culture than that by the mesophilic culture, with more serious corrosion on the mineral surfaces.XRD and XANES analyses showed that the leaching intermediates S 0 , jarosite and secondary minerals bornite, chalcocite and covellite formed during all bioleaching experiments.The formation of S 0 or jarosite on chalcopyrite bioleaching probably had no negative effect on chalcopyrite bioleaching in the presence of Ag + .For the bioleaching experiments by both mesophilic and thermophilic cultures, the formation of bornite and covellite was distinctly promoted by addition of Ag + , providing insight that the catalytic mechanism of Ag + with regard to chalcopyrite bioleaching by mesophilic culture and thermophilic culture is probably by promoting the rapid formation of bornite and the conversion of bornite to covellite.

Figure 4 .
Figure 4. Scanning electron microscopy (SEM) micrographs of original chalcopyrite (a) and chalcopyrite bioleached by mesophilic culture without Ag + on day 12 (b) and day 28 (c) and in the presence of 0.3% Ag + on day 12 (d) and day 28 (e); SEM micrographs of chalcopyrite bioleached by thermophilic culture without Ag + on day 12 (f) and day 28 (g) and in the presence of 0.3% Ag + on day 12 (h) and day 28 (i).

Figure 4 .
Figure 4. Scanning electron microscopy (SEM) micrographs of original chalcopyrite (a) and chalcopyrite bioleached by mesophilic culture without Ag + on day 12 (b) and day 28 (c) and in the presence of 0.3% Ag + on day 12 (d) and day 28 (e); SEM micrographs of chalcopyrite bioleached by thermophilic culture without Ag + on day 12 (f) and day 28 (g) and in the presence of 0.3% Ag + on day 12 (h) and day 28 (i).

Figure 5 .
Figure 5. SR-XRD patterns of leaching residues by mesophilic culture and in sterile control at 30 °C without Ag + and with 0.3% of Ag + (a) and SR-XRD patterns of leaching residues by thermophilic culture and in sterile control at 48 °C without Ag + and with 0.3% of Ag + (b).

Figure 5 .
Figure 5. SR-XRD patterns of leaching residues by mesophilic culture and in sterile control at 30 • C without Ag + and with 0.3% of Ag + (a) and SR-XRD patterns of leaching residues by thermophilic culture and in sterile control at 48 • C without Ag + and with 0.3% of Ag + (b).

28 Figure 7 .Figure 7 .
Figure 7. Normalized S K-edge XANES spectra of reference sulfur-containing compounds (a), chalcopyrite bioleached by mesophilic culture from day 1 to day 28 and in the sterile control at 30 °C on day 28 without Ag + (b) and with 0.3% of Ag + (c) and the spectra of chalcopyrite bioleached by thermophilic culture from day 1 to day 28 and in the sterile control at 48 °C on day 28 without Ag + (d) and with 0.3% of Ag + (e).

Figure 7 .Figure 8 .
Figure7.Normalized S K-edge XANES spectra of reference sulfur-containing compounds (a), chalcopyrite bioleached by mesophilic culture from day 1 to day 28 and in the sterile control at 30 °C on day 28 without Ag + (b) and with 0.3% of Ag + (c) and the spectra of chalcopyrite bioleached by thermophilic culture from day 1 to day 28 and in the sterile control at 48 °C on day 28 without Ag + (d) and with 0.3% of Ag + (e).

Figure 8 .
Figure 8. Sulfur speciation compositions during chalcopyrite bioleaching by mesophilic culture and in the sterile control at 30 • C without Ag + (a) and with 0.3% of Ag + (b), and the compositions during chalcopyrite bioleaching by thermophilic culture and in the sterile control at 48 • C without Ag + (c) and with 0.3% of Ag + (d), by the LC fitting of the unknown spectra from Figure 7b-e with the reference spectra from Figure 7a.