Bio-Oxidation of a Double Refractory Gold Ore and Investigation of Preg-Robbing of Gold from Thiourea Solution

Article Bio-Oxidation of a Double Refractory Gold Ore and Investigation of Preg-Robbing of Gold from Thiourea Solution

Rui Xu, Qian Li, Feiyu Meng *, Yongbin Yang, Bin Xu, Huaqun Yin and Tao Jiang School of Minerals Processing and Bioengineering, Central South University, Changsha 410083, China; [email protected] (R.X.); [email protected] (Q.L.); [email protected] (Y.Y.); [email protected] (B.X.); [email protected] (H.Y.); [email protected] (T.J.) * Correspondence: [email protected]; Tel.: +86-135-4050-3028

Received: 13 August 2020; Accepted: 4 September 2020; Published: 10 September 2020

Abstract: Carbonaceous sulfidic gold ores are commonly double refractory and thus require pretreatment before gold extraction. In this paper, the capacity of pre-bio-oxidation can simultaneously decompose sulfides or deactivate carbonaceous matters (CM) from a double refractory gold ore (DRGO) using pure cultures of A. ferrooxidans or L. ferrooxidans, and a mixed culture containing A. ferrooxidans and L. ferrooxidans was investigated. The results showed that direct thiourea leaching of the as-received DRGO yielded only 28.7% gold extraction, which was due to the encapsulation of sulfides on gold and the gold adsorption of CM. After bio-oxidation, thiourea leaching of the DRGO resulted in gold extraction of over 75–80%. Moreover, bio-oxidation can effectively reduce the adsorption of carbon to gold. XRD, SEM-EDS and FTIR analysis showed that many oxygen-containing groups were introduced on the surface of DRGO during bio-oxidation, while the C=C bond was cleaved and the O–C–O and C–N bonds were degraded, causing a decrease in active sites for gold adsorption. Moreover, passivation materials such as jarosite were formed on the surface of DRGO, which might reduce the affinity of CM for gold in solutions. In addition, the cleavage of the S–S band indicated that sulfides were oxidized by bacteria. This work allows us to explain the applicability of pre-bio-oxidation for degrading both sulfides and CM and increasing gold recovery from DRGO in the thiourea system.

Keywords: double refractory gold ore; bio-oxidation; preg-robbing; thiourea leaching; green technology

1. Introduction Environmentally friendly gold extraction from refractory ores has gradually dominated gold production, with a decrease in the quality of gold ores [1]. Currently, refractory gold ores account for around one-third of the total gold production of natural ores, and their refractory behavior is mainly due to the fact that the fine gold particles are often encased in sulfide matrices such as pyrite and arsenopyrite, or the leached gold are preg-robbed by carbonaceous matters (CM) [2–4]. In some cases, the gold ore contains both sulfide minerals and CM, and it can be classified as a double refractory gold ore (DRGO), which is usually abandoned for economic reasons [5]. In such ores, the gold is encased in sulfides, which prevents direct contact between the leaching reagent and gold particles, thus interfering with the dissolution of the gold [6]. Moreover, CM has a good affinity for dissolved gold complex (preg-robbing), leading to gold losses [7,8]. Commercially, pretreatment of the DRGO is required in order to degrade sulfides and deactivate CM prior to leaching to improve gold extraction [9]. Bio-oxidation has been proven to be a viable pretreatment process for DRGO, which can reduce the consumption of chemical solvents for gold leaching in subsequent operations. It presents

Metals 2020, 10, 1216; doi:10.3390/met10091216 www.mdpi.com/journal/metals Metals 2020, 10, 1216 2 of 16 several attractive features such as environmental friendliness and low cost [10–12]. The commonly used large-scale leaching bacteria now mainly include Acidithiobacillus ferrooxidans (A. ferrooxidans), Leptospirillum ferrooxidans (L. ferrooxidans) and Acidithiobacillus thiooxidans [13]. These autotrophic bacteria are reliant on oxidizing ferrous (Fe2+) to ferric (Fe3+) ion or oxidizing sulfur compounds or elemental S to H2SO4 as their energy source [2,14]. This enables bacteria to oxidize and decompose the sulfide matrices, releasing the entrapped gold. In addition, there are two main types of views on the interaction between microorganisms and CM, reflecting the passivation and degradation of CM by microorganisms and their metabolites. It is reported that Phanerochaete chrysosporium (P. chrysosporium) can secrete manganese peroxidase, lignin peroxidase and oxidative enzymes during metabolism, which are capable of degrading aromatic CM [15–17]. Ofori-Sarpong et al. [18,19] used P. chrysosporium to deactivate different grades of CM and found that biotransformation of CM reduced the graphitization of carbon and increased the oxygen content and average pore diameter, which limited the adsorption of gold by CM. Amankwah et al. [20] also pointed out in a two-stage microbial process that Streptomyces setonii has the ability to degrade CM, and gold extraction increased by 13.6% after degradation of CM. This may be due to the production of alkaline metabolites which was responsible for CM degradation. Though there have been numerous studies on biological pretreatment of DRGO, the results are rather controversial, and there is no general agreement about whether A. ferrooxidans and L. ferrooxidans can simultaneously decompose sulfides and deactivate CM. Moreover, according to previous studies, the mixed bacteria could result in synergistic effects that can improve the bio-oxidation efficiency [21–23]. Up to now, there has not been a clear correlation between the mixture of these two dominant strains and the efficiency of the bio-oxidation process. Thus, it is interesting to compare the behavior of A. ferrooxidans, L. ferrooxidans and a mixed culture composed of A. ferrooxidans and L. ferrooxidans in a bio-oxidation of DRGO system. Cyanidation is generally used for leaching gold ores because of its high stability, low cost and simple process [24,25]. However, its poor selectivity, slow leaching kinetics and harmful environmental effects have called for an alternative lixiviant. Thiourea has been considered to be a promising alternative to cyanide due to its high leaching rates and good selectivity [26], and it can be used to leach refractory gold ores containing sulfides (such as pyrite and arsenopyrite) or carbonaceous sulfides [27]. The initial leaching rate of gold at high concentration thiourea is comparable to that of cyanide leaching [28]. Thiourea is a water-soluble organic compound, which is relatively stable in acidic solutions but decomposes rapidly in basic solutions. Therefore, thiourea leaching of gold is normally carried out in a pH range of 1–2 [29]. In acidic solutions, thiourea dissolves gold and forms a cationic complex; this reaction is rapid and gold extractions can reach 99% [30]. Previous research has revealed that gold particles only dissolve in thiourea solutions in the presence of oxidizing agents, and ferric sulfate has been shown to be the most effective [31]. The dissolution of gold in thiourea solutions can be expressed as the following reaction. + ( ) + 3+ = ( ) )+ + 2+ Au 2CS NH2 2 Fe Au CS NH2 2 2 Fe (1)

Unlike the cyanide system, no adequate explanation for gold adsorption by CM in the thiourea system has been made in previous studies. The available literature indicates that the ability of CM to + adsorb gold in cyanide, thiourea and thiosulfate systems is in the order of Au(CN)2− > Au(CS(NH2)2)2 3 > Au(S2O3)2 − [32]. This conclusion has been proven by Ofori-Sarpong and Osseo-Asare [19]. It has also been reported that CM can adsorb not only gold but also thiourea, and the adsorbed gold is diﬃcult to elute [33]. Considering the potential of thiourea to be used as an alternative to cyanide, it is necessary to evaluate the behavior of preg-robbing of CM in the thiourea system. The objective of this study was to investigate the ability of decomposing sulﬁdes and decreasing gold adsorption on CM by using pure cultures of A. ferrooxidans or L. ferrooxidans and a mixed culture composed of A. ferrooxidans and L. ferrooxidans in a thiourea system, so as to assess the potential of A. ferrooxidans and L. ferrooxidans for the pretreatment of DRGO. This work can help to expand the Metals 2020, 10, 1216 3 of 16 current understanding of the biological pretreatment of DRGO by A. ferrooxidans and L. ferrooxidans, especially the impact of the pretreatment on the preg-robbing of CM for gold in the thiourea system.

2. Experimental

2.1. Materials The DRGO was obtained from a gold mine in Yunnan Province, China. The ore was ﬁnely ground to a particle size of 90.0% < 0.074 mm. The chemical component analysis showed that the gold content in this ore was 18.05 g/t, and the ore had high contents of C (6.95%) and S (20.76%) (Table1). The elemental and organic carbon that has a preg-robbing eﬀect on gold accounted for 75.40% of the total carbon [8]. Mineralogical studies of Au showed that only 1.72% of gold was exposed and 87.31% was encapsulated in arsenopyrite and pyrite.

Table 1. Chemical composition and the phase constitution analysis of C and Au of the double refractory gold ore.

Constituent Au * C As Fe SiO2 CaO MgO S Content/wt% 18.05 6.95 1.62 19.6 27.68 4.26 2.00 20.76 Phase of C Elemental carbon Organic carbon Carbonate Total Content/wt% 4.74 0.50 1.71 6.95 Distribution/% 68.20 7.20 24.60 100.00 Encapsulated in Encapsulated Encapsulated Encapsulated Phase of Au Exposed gold Total arsenopyrite in pyrite in oxides in silicates Content * 0.31 12.07 3.69 1.87 0.11 18.05 Distribution/% 1.72 66.87 20.44 10.36 0.61 100.00 * Unit g/t.

2.2. Microorganisms A. ferrooxidans and L. ferrooxidans were obtained from the Key Lab of Biohydrometallurgy of Ministry of Education, Central South University, Changsha, China. Bacterial cells were cultivated in 100 mL of 9 K medium in 250 mL Erlenmeyer ﬂasks were located in a shaking incubator at 30 ◦C and 160 rpm. The initial solution pH was adjusted to 1.8 using 3 M sulfuric acid. The 9 K medium contains 3 g (NH ) SO , 0.1 g KCl, 0.5 g MgSO 7H O, 0.5 g K HPO , 0.01 g Ca(NO ) in 1000 mL 4 2 4 4· 2 2 4 3 2 deionized water. In addition, 44.7 g/L of FeSO 7H O was supplemented as the energy source for 4· 2 strains. For domestication, the microorganisms were serially cultured in 9 K medium in the presence of 10 g/L DRGO particles for three months prior to bio-oxidation experiments. All chemical reagents used in this study were analytically graded, and all solutions were prepared with ultrapure water.

2.3. Bio-Oxidation Experiments The bio-oxidation experiments were conducted in 250 mL flasks containing 100 mL 9 K medium, and the pulp density was 10 g/L of DRGO. Inoculations were separately achieved with 20 mL inoculum of A. ferrooxidans and L. ferrooxidans which had adapted to the DRGO (Section 2.2), and the cell concentration in the two inoculated solutions was around 108 cells/mL. The flasks were located in a shaking incubator at 160 rpm and 30◦ for 8 days, with an initial pH of 1.8. In this study, A. ferrooxidans and L. ferrooxidans were mixed in 1:1 (v/v) culture to obtain a mixed culture. The experimental conditions of bio-oxidation using the mixed culture were the same as those using pure cultures. Parallel experiments without bacterial inoculation, but with the same medium and DRGO, were performed as sterile control. During the bio-oxidation process, pH and redox potential (Eh) were measured daily, and aliquot samples were periodically taken from the supernatant of each flask for determining total concentrations Metals 2020, 10, 1216 4 of 16 of Fe and As, as well as total organic carbon (TOC). The pulp from the bio-oxidation experiment was filtered with a neutral filter paper at different phases, rinsed with copious ultrapure water and dried in a vacuum dryer at 35 ◦C overnight to obtain the residue for the subsequent preg-robbing studies, thiourea leaching process and other analyses.

2.4. Preg-Robbing Experiments The thiourea leaching solution with known gold concentration (2.612 mg/L) was used for the preg-robbing experiments. Triplicate samples of as-received and bio-oxidized DRGO (each weighing 0.5 g) were contacted with 100 mL of 2.612 mg/L gold solution and agitated with a magnetic stirrer at a rotating speed of 400 rpm at 30 ◦C for 3 h. The initial pH of the solution was adjusted to around 1.5. After the preg-robbing experiments, the residual solutions were subsequently separated by filtration for the analysis of gold concentration. The difference in gold concentration between as-received and bio-oxidized DRGO gave an indication of the effect of A. ferrooxidans and L. ferrooxidans on preg-robbing for gold by the DRGO.

2.5. Thiourea Leaching Experiments Leaching experiments were carried out in a 200 mL beaker using an agitator. Each beaker contained 10 g samples of as-received and bio-oxidized DRGO, respectively, and the liquid–solid ratio was set at 5:1. Then, each beaker was supplemented with 14 g/L of ferric sulfate, and the initial pH was adjusted to 1.5. Finally, thiourea was added to each beaker in a quantity of 6 g/L. The formed pulp was agitated at 400 rpm, 30 ◦C and the experiments lasted for 4 h. Triplicate leaching experiments were performed under identical conditions. After the experiment, the pulp was ﬁltered and the concentration of gold was measured.

2.6. Thermodynamic Calculation The bio-oxidation process was based on the redox reactions occurring in an aqueous electrochemical system. To illustrate the thermodynamic probability of the formation of intermediate products during the bio-oxidation process of arsenopyrite and pyrite, the Eh pH diagrams of Fe-As-S-H O and Fe-S-H O − 2 2 systems were implemented using the thermodynamic software HSC Chemistry 6.0. In addition, some 2 special species, such as H3OFe3(SO4)2(OH)6,H2S and SO4 −, are so thermodynamically stable that they would be preferentially shown in the E–pH diagram. Therefore, to reﬂect the dissolution of arsenopyrite and pyrite and the possible intermediate products in the process of thiourea leaching, the thermodynamic calculation was conducted by considering or neglecting some of the special species. The thermodynamic data were available from the software itself. The temperature and pressure were respectively set at 25 ◦C and 1 atm in the process of plotting the diagrams, within the ranges of Eh ( 1–1 V) and pH value (0–6). Moreover, the concentration of Fe2+ used to plot the diagrams was − 0.16 mol/L, and the concentration of FeAsS was 7.4 10 4 mmol/L and FeS was 3 10 4 mmol/L × − 2 × − (i.e., around 10 g/L DRGO).

2.7. Analysis Methods The pH and Eh were measured using a pH meter (PHSJ-4A, Shanghai Leici, Shanghai, China) equipped with a Pt electrode and a standard Ag/AgCl (saturated KCl) as a reference electrode. The total concentrations of Fe, As and Au in the solutions were detected by inductively coupled plasma–atomic emission spectrometer (ICP-AES) (PS-6, Baird, TX, USA). TOC concentration in the sample was analyzed using a total organic carbon analyzer (LCPH, Shanghai, China). The contents of C, Fe, As and S in the DRGO were determined by X-ray fluorescence (XRF, Panakot, Netherlands). Mineralogical compositions of as-received and bio-oxidized DRGO were analyzed by X-ray diffractometer (XRD) (D/Max 2500, Rigaku, Tokyo, Japan). The morphology of as-received and bio-oxidized DRGO was examined using a scanning electron microscope (SEM) (Helios NanoLab G3 UC, FEI, Hillsboro, OR, USA) equipped with X-ray energy dispersive analysis (EDS). The chemical bonds and functional groups Metals 2020, 10, 1216 5 of 16 of as-received and bio-oxidized DRGO were analyzed by Fourier transform infrared spectroscopy (FTIR) (IRAffinity-1, Shimadzu, Kyoto, Japan), and the KBr disk method was employed in the wavelength 1 1 range of 4000 to 400 cm− with a resolution of 4 cm− .

3. Results and Discussion

3.1. Bio-Oxidation Behavior of DRGO Variations in the parameters of the solution over time during the bio-oxidation process of DRGO usingMetals different 2020, 10 bacterial, x FOR PEER cultures REVIEW are shown in Figure1. As can be seen from Figure1a, the pH increased5 of 16 from 1.8 to 2.26–2.29 within 1 day for all samples except for the sterile control, in which it did not 3. Results and Discussion change significantly throughout the entire process. This was due to the fact that A. ferrooxidans and 2+ 3+ + L. ferrooxidans3.1. Bio-Oxidationwere able Behavior to oxidize of DRGO Fe to Fe to obtain energy, with a mass of H being consumed at this time, and it could be summarized by Equation (3). Furthermore, the pH increase might be caused Variations in the parameters of the solution over time during the bio-oxidation process of DRGO by the decomposition of a small amount of alkaline mineral presented in the DRGO [34]. After this using different bacterial cultures are shown in Figure 1. As can be seen from Figure 1a, the pH period, pH started to decrease due to the fact that the oxidation and decomposition of sulfide produced increased from 1.8 to 2.26–2.29 within 1 day for all samples except for the sterile control, in which it aciddid (Equations not change (4)–(7)). significantly The throughout Eh generally the indicatedentire process. bacterial This was activity due to the and fact bio-oxidation that A. ferrooxidans efficiency duringand the L. ferrooxidans bio-oxidation were process able to oxidize (Figure Fe1b).2+ to The Fe3+ Ehto obtain within energy, the range with a of mass 330–390 of H+ mVbeing was consumed detected in the sterileat this control, time, and while it could a remarkable be summarized initial by increase Equation in Eh(3). values Furthermore, was detected the pH inincrease the bacterial might be system, reachingcaused 595–627 by the decomposition mV after 8 days, of a small which amount was conduciveof alkaline mineral to decomposing presented in the the sulfideDRGO [34]. minerals After [35]. Thisthis was period, mainly pH because started the to decrease Eh in the due bio-oxidation to the fact that solution the oxidation was related and decomposition to the Fe3+/Fe of2+ sulfideratio using the Nernstproduced equation acid (Equations (Equation (4)–(7)). (2)), The and Eh it wasgenera documentedlly indicated thatbacterial the oxidationactivity and rate bio-oxidation of Fe2+ in the bio-oxidationefficiency processduring the was bio-oxidation 105 to 106 timesprocess faster (Figure than 1b). that The in Eh the within non-bio-oxidation the range of 330–390 process mV at was low pH detected in the sterile control, while a remarkable initial increase in Eh values was detected in the (<3) [36]. Figure1c clearly shows that the total Fe concentration decreased for all the samples as the bacterial system, reaching 595–627 mV after 8 days, which was conducive to decomposing the sulfide bio-oxidation reaction proceeded, except for the sterile control. This was because Fe3+ was extremely minerals [35]. This was mainly because the Eh in the bio-oxidation solution was related to the Fe3+/Fe2+ unstableratio inusing almost the Nernst all environmental equation (Equation conditions (2)), and except it was for documented high Eh and that low the pH,oxidation and itrate could of Fe rapidly2+ formin hydroxides the bio-oxidation or iron process oxides was such 105 as to jarosite 106 times (Equation faster than (8)). that in the non-bio-oxidation process at low pH (<3) [36]. Figure 1c clearly shows that the total Fe concentration decreased for all the samples 3+ RT Fe 3+ as the bio-oxidation reaction proceeded,E = exceptEθ + for theln sterile control. This was because Fe was (2) extremely unstable in almost all environmental conditionsZF Fe2 +except for high Eh and low pH, and it could rapidly form hydroxides or iron oxides such as jarosite (Equation (8)).

2.4 650 (a) (b) Mixed culture 600 2.2 A. ferrooxidans L. ferrooxidans 550 Sterile control Mixed culture 2.0 500 A. ferrooxidans

pH L. ferrooxidans

Eh/mV Sterile control 1.8 450

400 1.6

350 1.4 012345678 12345678 Time/d Time/d 9.5 110 (c) (d) 9.0 100

8.5 90 Mixed culture 8.0 Mixed culture A. ferrooxidans 80 A. ferrooxidans 7.5 L. ferrooxidans L. ferrooxidans Sterile control 70 7.0 Sterile control 60 6.5 6.0 rate/% leaching As 50

Total Fe concentration/(g/L) 5.5 40 5.0 30 12345678 12345678 Time/d Time/d Figure 1. Cont.

Metals 2020, 10, 1216 6 of 16 Metals 2020, 10, x FOR PEER REVIEW 6 of 16

50 (e) 45 40 35 30 25 20 Mixed culture 15 A. ferrooxidans

TOC TOC concentration/(mg/L) L. ferrooxidans 10 Sterile control 5 12345678 Time/d FigureFigure 1. Variations 1. Variations in in the the (a )(a pH;) pH; ( b(b)) Eh; Eh; ((cc)) totaltotal Fe Fe concentration; concentration; (d) ( dAs) Asleaching leaching rate;rate; (e) TOC (e) TOC concentrationconcentration over over time time during during the the oxidation oxidation processprocess of of DRGO. DRGO.

Figure1d generally shows that both A. ferrooxidans and3+ L. ferrooxidans were able to decompose θ RT Fe arsenopyrite (that encapsulated 66.87% ofEE=ln gold)+ in DRGO, and the As removal efficiency of(2) mixed ZF Fe2+ cultures achieved the highest value (98.8%). As will be shown in the subsequent thiourea leaching experiments,Figure this 1d result generally is consistent shows that with both the A. goldferrooxidans recovery and rate L. ferrooxidans (>80%). In were terms able of removalto decompose efficiency, the earsenopyriteffect of the (that different encapsulated cultures 66.87% on As of removalgold) in DRGO, could and beranked the As removal as follows: efficiency mixed of mixed cultures > L. ferrooxidanscultures achieved> A. ferrooxidans the highest> valusterilee (98.8%). control. As Thewill be oxidation shown in of the As subsequent could be explained thiourea leaching as stated in experiments, this result is consistent with the gold recovery rate (>80%). In terms of removal Equation (5). It is well known that this oxidation occurs through a polysulfide pathway previously efficiency, the effect of the different cultures on As removal could be ranked as follows: mixed describedcultures by > ShippersL. ferrooxidans and > Sand A. ferrooxidans [37] and > clearly sterile control. confirmed The oxidation by Rohwerder of As could et al. be [38 explained]. The changes as in TOCstated concentration in Equation in(5). the It is solutions well known are shownthat this in oxidation Figure1 e,occurs which through presented a polysulfide a trend ofpathway increasing first andpreviously then decreasingdescribed by during Shippers the and oxidation Sand [37] process and clearly of CM,confirmed indicating by Rohwerder that some et al. organic [38]. The carbon fractionschanges in the in TOC ore were concentration solubilized in the when solutions the sulfur are shown and arsenic in Figure were 1e, oxidized. which presented After 4 a days, trend the of TOC valuesincreasing tended tofirst decrease and then until decreasing the end during of the the experiment, oxidation process which of might CM, indicating be caused that by thesome lack organic of energy, restrainingcarbon thefractions bacterial in the activity, ore were and solubilized the TOC when started the sulfur to adhere and arsenic to the were surface oxidized. of the After ore. In4 days, addition, the changethe TOC range values of tended TOC concentration to decrease until in the the end sterile of the system experiment, was weaker which might than the be caused bacterial by systems,the whichlack was of possiblyenergy, restraining caused by the its bacterial much slower activity, chemical and the TOC oxidation. started to adhere to the surface of the ore. In addition, the change range of TOC concentration in the sterile system was weaker than the bacterial systems, which was possibly caused by itsbacteria much slower chemical oxidation. 4Fe2+ + O + 4H+ 4Fe3+ + 2H O (3) 2 bacteria 2 2+ +⎯⎯⎯⎯→→ 3+ (3) 4Fe + O22 + 4H 4Fe + 2H O 0 bacteria + 2 2S + 3O2 + 2H2O 4H + 2SO4 − (4) 0+2bacteria→ − 2S + 3O + 2H O ⎯⎯⎯⎯→ 4H + 2SO (4) 3+ 222+ 40 + FeAsS + 5Fe + 3H2O 6Fe + H3AsO3 + S + 3H (5) 3+→→ 2+ 0 + (5) FeAsS + 5Fe3+ + 3H233 O 6Fe + H AsO + S+ + 3H 2Fe + 2H3AsO4 2FeAsO4 + 6H (6) 3+→ + 2Fe3+ + 2H AsO → 2FeAsO2+ + 6H+ 2 (6) FeS + 14Fe + 8H34O 15Fe + 16H 4 + 2SO − (7) 2 2 → 4 − 3+ FeS+ + 14Fe3+ + 8H O 2→ 15Fe 2+ + 16H + + 2SO 2 + (7) 3Fe + K 22+ 6H O + 2SO − KFe (SO ) (OH) 4+ 6H (8) 2 4 → 3 4 2 6 − 3Fe3+ + K + + 6HFeS O + 2SO2Fe3+ 2→ KFe3Fe2+ (SO+ 2S )0 (OH) + 6H + (8) (9) 242 → 3426 3+→ 2+ 0 (9) 3.2. Characterization of DRGO FeS2 + 2 Fe 3Fe + 2S

3.2.1.3.2. Mineralogical Characterization Composition of DRGO Analysis by XRD The XRD patterns of as-received, bio-oxidized and sterile control DRGO are shown in Figure2. 3.2.1. Mineralogical Composition Analysis by XRD It can be seen that the DRGO consisted of muscovite, graphite, quartz, arsenopyrite and pyrite, etc. Evidently,The photocatalysts XRD patterns of with as-received, the bio-oxidized bio-oxidized DRGO and sterile exhibited control similar DRGO XRD are shown patterns, in Figure with 2. peaks It can be seen that the DRGO consisted of muscovite, graphite, quartz, arsenopyrite and pyrite, etc. at 17.37◦ (012), 28.78◦ (021), 45.55◦ (303) and 50.08◦ (220), which corresponded to the jarosite or ammoniojarosite phase. This was consistent with the results in Figure1d, meaning that the oxidative dissolution of Fe-bearing sulﬁdes was caused by A. ferrooxidans and L. ferrooxidans but not observed obviously in the sterile control. This ﬁnding was consistent with the work of Nazari et al. [39] in Metals 2020, 10, x FOR PEER REVIEW 7 of 16

Evidently, photocatalysts with the bio-oxidized DRGO exhibited similar XRD patterns, with peaks at 17.37° (012), 28.78° (021), 45.55° (303) and 50.08° (220), which corresponded to the jarosite or ammoniojarosite phase. This was consistent with the results in Figure 1d, meaning that the oxidative dissolutionMetals 2020, 10 ,of 1216 Fe-bearing sulfides was caused by A. ferrooxidans and L. ferrooxidans but not observed7 of 16 obviously in the sterile control. This finding was consistent with the work of Nazari et al. [39] in which A. ferrooxidans was used to investigate jarosite formation during the bioleaching of a pyrite. whichPreviousA. ferrooxidansstudies suggestedwas used that to investigatethe formation jarosite of jarosite formation in duringbioleaching the bioleaching systems reduces of a pyrite. the dissolutionPrevious studies kinetics suggested of metallic that the sulfide formation [40,41]. of jarosite However, in bioleaching it was systemsfound in reduces this study the dissolution that the precipitationkinetics of metallic of jarosite sulfide had [40 ,41little]. However, effect on itthe was bioleaching found in this process. study that As thepreviously precipitation reported of jarosite [42], jarositehad little presented effect on as the fluffy bioleaching or woolly process. spherical As previously particles, loosely reported packed [42], jarosite in a porous presented structure as flu onffy the or woollysurface, spherical and a similar particles, conclusion loosely packed was reached in a porous by previous structure works on the [43,44]. surface, It and should a similar be noted conclusion that, wasconsidering reached that by previous the sterile works control [43, 44had]. It no should significant be noted effect that, on considering the oxidation that of the DRGO, sterile no control analysis had wasno significant conducted e ffinect the on subsequent the oxidation process. of DRGO, no analysis was conducted in the subsequent process.

j n j q-Quartz, c-Carbon, m-Muscovite, t-Stibnite, p-Pyrite, a-Arsenopyrite, n-Ammoniojarosite, j-Jarosite j j j j j j j Mixed culture j n j

j j j j j j j A. ferrooxidans j n j

j j j j j j j L. ferrooxidans

Intensity (a.u.) m q c t m m p a t p mp a p a t t p q a qm a j mt Sterile q c m m m t m t p p a t p a t ap t p q a a qm q m As-received

10 20 30 40 50 60 70 2-Theta (°) Figure 2. XRDXRD diffraction diffraction patterns for the as-receive as-received,d, bio-oxidized and sterile control DRGO. 3.2.2. Morphology Analysis by SEM-EDS 3.2.2. Morphology analysis by SEM-EDS High-magnification SEM images and EDS data of DRGO using different cultures are shown in High-magnification SEM images and EDS data of DRGO using different cultures are shown in Figure3. In Figure3a, the SEM image showed that the DRGO surface was smooth before treatment, Figure 3. In Figure 3a, the SEM image showed that the DRGO surface was smooth before treatment, and the atomic percentage of C, As and S in the as-received DRGO was 16.87%, 0.91% and 22.07%, and the atomic percentage of C, As and S in the as-received DRGO was 16.87%, 0.91% and 22.07%, respectively (spot a-1). After 1 day of bio-oxidation, there were many small crystalline flocs visible on respectively (spot a-1). After 1 day of bio-oxidation, there were many small crystalline flocs visible the mineral surface, as seen in Figure3b,e,h. According to the EDS analysis, the atomic percentages of on the mineral surface, as seen in Figure 3b,e,h. According to the EDS analysis, the atomic percentages elemental C, O and S on the mineral surface were significantly increased, indicating that DRGO was of elemental C, O and S on the mineral surface were significantly increased, indicating that DRGO oxidized and CM was released. In addition, it was accompanied by the formation of a large amount was oxidized and CM was released. In addition, it was accompanied by the formation of a large of elemental sulfur and precipitates with high oxygen content (Equations (5), (8) and (9)). However, amount of elemental sulfur and precipitates with high oxygen content (Equations (5), (8) and (9)). the element content varied greatly in different ore sites—this was because the bacterial attachment to However, the element content varied greatly in different ore sites—this was because the bacterial the metal sulfide did not occur randomly. In other words, these bacteria, such as A. ferrooxidans, were attachment to the metal sulfide did not occur randomly. In other words, these bacteria, such as A. preferentially attached to sites with visible defects on the surface [38]. Within 3 days of bio-oxidation, ferrooxidans, were preferentially attached to sites with visible defects on the surface [38]. Within 3 holes and cracks were clearly shown in Figure3c,f,i. This suggested that the crystal lattice structure days of bio-oxidation, holes and cracks were clearly shown in Figure 3c,f,i. This suggested that the of the DRGO surface was partly disrupted following bio-oxidation. Meanwhile, the content of O on crystal lattice structure of the DRGO surface was partly disrupted following bio-oxidation. the DRGO surface was increased, but the C, As and S contents were decreased, suggesting that the Meanwhile, the content of O on the DRGO surface was increased, but the C, As and S contents were DRGO was further oxidized and elemental sulfur was oxidized by bacteria (Equation (4)). After 6 decreased, suggesting that the DRGO was further oxidized and elemental sulfur was oxidized by days of bio-oxidation, the mineral surface became granular and larger cracks or pits also appeared on bacteria (Equation (2)). After 6 days of bio-oxidation, the mineral surface became granular and larger the surface, and the SEM images (Figure3d,g,j) show that it was covered with many solid particles. cracks or pits also appeared on the surface, and the SEM images (Figure 3d,g,j) show that it was According to the XRD analysis (Figure2), it could be deduced that jarosite and ammoniojarosite were covered with many solid particles. According to the XRD analysis (Figure 2), it could be deduced that formed. In particular, the corrosion on mineral surfaces seemed more severe in the mixed culture jarosite and ammoniojarosite were formed. In particular, the corrosion on mineral surfaces seemed (Figure3j) than pure cultures. more severe in the mixed culture (Figure 3j) than pure cultures.

Metals 2020, 10, 1216 8 of 16 Metals 2020, 10, x FOR PEER REVIEW 8 of 16

35 a-1 a-2 a-3

15 Content/(at %)

0 C O As Si S K Sb Fe

FigureFigure 3. 3.SEM SEM images with with EDS EDS data data (at.%) (at.%) for forthe theas-received as-received sample sample (DRGO) (DRGO) and the and DRGO the after DRGO afteroxidizing oxidizing 1 d, 13 d,d and 3 d 6 and d in 6 9 d K in culture 9 K culture medium. medium. (a) As-received; (a) As-received; (b–d) A. ferrooxidans (b–d) A. ferrooxidans; (e–g) L. ; (e–ferrooxidansg) L. ferrooxidans; (h–j) ;(Mixedh–j) Mixedculture. culture.

Metals 2020, 10, 1216 9 of 16 Metals 2020, 10, x FOR PEER REVIEW 9 of 16

3.2.3. Chemical Chemical Bonds and Functional Groups Analysis by FTIR FTIR analysis was further carried out to verify the conversion of DRGO in the bio-oxidationbio-oxidation process. By using differential differential spectra, where the “ “zero-moment”zero-moment” spectra (the spectra collected before the treatment) treatment) were were subtracted subtracted from from a series a series of spectrograms of spectrograms collected collected at different at different times, the times, difference the differencespectra obtained spectra would obtained contribute would contribute to the understanding to the understanding of the evolution of the of evolution chemical of structural chemical structuralchanges [45 changes,46]. The [45,46 negative]. The peaks negative in difference peaks spectra in difference mean the spect lossra of chemical mean the structure, loss of whilechemical the positivestructure, peaks while are the a sign positive of the peaks formation are ofa sign new of chemical the formation structures of [46new]. Aschemical expected, structures the FTIR [46] spectral. As peaks’expected, shape the andFTIR trend spectral of samples peaks’ shape were basicallyand trend the of samesamples as shownwere basically in Figure the4a–c, same indicating as shown that in Figtheseure treatments 4a–c, indicating had a parallel that these influence treatments on the structure had a and parallel composition influence distribution on the structure of functional and compositiongroups in bio-oxidized distribution DRGO. of functional The differences groups in in the bio bio-oxidized-oxidized DRGO samples. The were differences mainly concentrated in the bio- oxidizedin the peaks samples and the were intensity mainly of concentrated some functional in the groups. peaks and Compared the intensity to the of FTIR some spectra functional of as-received groups. 1 ComparedDRGO, the main to the peaks FTIR of spectra bio-oxidized of as samples-received included DRGO, a thestrong main broad peaks peak of of bio 3419-oxidized cm− , which samples was −1 1 includedattributed a tostrong O-H broad stretching peak vibration of 3419 c ofm water, which or was Fe oxyhydroxides attributed to O [-47H]. stretching The new vibration peak at 1427 of water cm− −1 orcould Fe oxyhydroxides be assigned to [47] the. Thev3 stretching new peak vibrationat 1427 cm of thecould C-O be bondassigned [48 ],to suggestingthe v3 stretching that avibration mass of ofoxygen-containing the C-O bond [48] groups, suggesting were introducedthat a mass on of theoxygen surface-containing of DRGO groups after bio-oxidation.were introduced Peaks on the at 1 1 −1 −1 2 1238surface cm − of and DRGO 1079 after cm − biocould-oxidation. be assigned Peaks to at the 1238 stretching cm and vibration 1079 cm of SO could4 − [ 49 be], assigned which was to due the 2− 2 1 stretchingto the bio-oxidation vibration of of pyriteSO4 and[49], arsenopyrite, which was due producing to the bio SO-4oxidation− ions. The of pyritepeak at and around arsenopyrite, 971 cm− producingcorresponded SO4 to2− ions. O–H The bending peak a vibration,t around 971 which cm−1 was corresponded ascribed to to the O adsorption–H bending of vibration, water onto which the wasmineral ascribed surface to the [50]. adsorption of water onto the mineral surface [50].

40 40 -1 (a) -1 (b) 1238 cm 1427 cm-1 1238 cm 1427 cm-1 -1 20 3419 cm -1 20 3419 cm-1 971 cm 971 cm-1 -1 1079 cm-1 1079 cm 0 0

-1 -20 1376 cm 1141 cm-1 -20 1376 cm-1 1141 cm-1 -1 1051 cm-1

Absorbance 1051 cm Absorbance -1 1617 cm-1 1617 cm 603 cm-1 1 day bio-oxidation 603 cm-1 1 day bio-oxidation -40 3 days bio-oxidation -40 3 days bio-oxidation 6 days bio-oxidation 6 days bio-oxidation

-60 -60 725 cm-1 725 cm-1 4000 3500 3000 2500 2000 1500 1000 500 4000 3500 3000 2500 2000 1500 1000 500 Wavenumber/cm-1 Wavenumber/cm-1 40 (c) 1238 cm-1 1427 cm-1 20 3419 cm-1 971 cm-1 1079 cm-1 0

-1 -20 1376 cm 1617 cm-1 1141 cm-1 Absorbance -1 1 day bio-oxidation 1051 cm 603 cm-1 -40 3 days bio-oxidation 6 days bio-oxidation

-60 725 cm-1 4000 3500 3000 2500 2000 1500 1000 500 Wavenumber/cm-1 Figure 4. 4. FTIRFTIR spectra spectra (di (differenceﬀerence spectrum) spectrum) of DRGO of DRGO after after oxidizing oxidizing 1, 3 and 1, 36 in and 9 K 6 culture in 9 K medium. culture (medium.a) A. ferrooxidans (a) A. ferrooxidans;(b) L. ferrooxidans; (b) L. ferrooxidans;(c) Mixed; ( culture.c) Mixed culture. In addition, negative peaks also occurred at 1617 cm 1, 1376 cm 1, 1141 cm 1, 1051 cm 1 and In addition, negative peaks also occurred at 1617 cm−1−, 1376 cm−1,− 1141 cm−1, −1051 cm−1 and− 603 603 cm 1. The C=C band around 1617 cm 1 was considered to be characteristic of the graphite cm−1. The− C=C band around 1617 cm−1 was considered− to be characteristic of the graphite hexagonal hexagonal ring structure [51]. However, there was no signiﬁcant interaction between highly ordered ring structure [51]. However, there was no significant interaction between highly ordered structures structures (such as graphite) and infrared radiation, and this absorption band might be due to the (such as graphite) and infrared radiation, and this absorption band might be due to the presence of presence of polar functional groups or the irregularities of the aromatic structure, such as carboxylic polar functional groups or the irregularities of the aromatic structure, such as carboxylic acid groups acid groups or phenolic groups attached to aromatic rings [51]. Peaks at 1376 cm 1 and 1141 cm 1 or phenolic groups attached to aromatic rings [51]. Peaks at 1376 cm–1 and 1141 cm–1− were designated− to the stretching vibration of the O–C–O and C–N bands [52], respectively, suggesting that A. ferrooxidans and L. ferrooxidans could remove the aromatic compounds contained in the DRGO in the

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wereMetals designated 2020, 10, x FOR to the PEER stretching REVIEW vibration of the O–C–O and C–N bands [52], respectively, suggesting10 of 16 that A. ferrooxidans and L. ferrooxidans could remove the aromatic compounds contained in the DRGO −1 1 in theprocess process of bio-oxidation. of bio-oxidation. The peak The peakat 1051 at cm 1051 referred cm− referred to the bending to the bending vibration vibration of the O–H of the band O–H band[52], [52 which], which was was decreased, decreased, and this and might this might be due be to due the tofact the that fact some that substances some substances of DRGO of were DRGO werecoated coated with with the the sediment sediment or orconverted converted from from hydrophobic hydrophobic to tohydrophilic hydrophilic nature nature brought brought about about by by bio-oxidation. In addition, the peak at 603 cm−11 was referenced as the stretching vibration of the S–S bio-oxidation. In addition, the peak at 603 cm− was referenced as the stretching vibration of the S–S bandband [53 [53],], which which indicated indicated that that sulfides sulfides were were oxidized oxidized byby A. ferrooxidans andand L.L. ferrooxidans ferrooxidans. . TheseThese results results implied implied that that DRGO DRGO could could be eff ectivelybe effectively oxidized oxidized by A. ferrooxidans by A. ferrooxidansand L. ferrooxidans and L. withferrooxidans time. However, with time. the intensityHowever, of the some intensity functional of some groups functional tended groups to increase tended faster to increase in mixed faster culture in mixed culture than in pure cultures, which was consistent with the results obtained from the changes than in pure cultures, which was consistent with the results obtained from the changes in various indexes in various indexes in solutions and the analyses of XRD, SEM and EDS in previous sections. In in solutions and the analyses of XRD, SEM and EDS in previous sections. In addition, bio-oxidation by addition, bio-oxidation by A. ferrooxidans and L. ferrooxidans could significantly decrease the content A. ferrooxidans and L. ferrooxidans could significantly decrease the content of aromatic structures and of aromatic structures and introduce large quantities of oxygen-containing groups on the surface of introduce large quantities of oxygen-containing groups on the surface of DRGO. This phenomenon DRGO. This phenomenon might result in reduced preg-robbing for gold in the subsequent thiourea mightleaching result process. in reduced preg-robbing for gold in the subsequent thiourea leaching process. 3.3. The Process of Sulfides Bio-Oxidation 3.3. The Process of Sulfides Bio-Oxidation GoldGold occurred occurred mainly mainly in arsenopyritein arsenopyrite and and pyrite pyrite in the in DRGO the DRGO used inused this in study, this andstudy, the and diagrams the ofdiagrams arsenopyrite of arsenopyrite and pyrite and oxidation pyrite oxidation in Eh–pH in Eh–pH coordinates coordinates are presented are presented in Figure in Figure5. Since 5. Since the bio-oxidationthe bio-oxidation process process was carriedwas carried out under out under acidic acid conditions,ic conditions, the the relevant relevant range range of pHof pH values values was 0–3,was where 0–3, thewhere phase the changes phase changes were considered. were considered As indicated,. As indicated, arsenopyrite arsenopyrite was relatively was relatively unstable andunstable it could and be oxidizedit could be when oxidized Eh > when0.4 VEh under > −0.4 bio-oxidation V under bio-oxidation conditions conditions (Figure5 a–c).(Figure When 5a–c). the − solutionWhen exhibitedthe solution a exhibited low Eh ( a0.4 low V–0.4 Eh (− V),0.4 V–0.4 iron existedV), iron mostlyexisted asmostly Fe2+ ,as while Fe2+, while arsenic arsenic and sulfurand − 0 presentedsulfur presented mainly asmainly As2S 2as, AsAs22S32,,H As32SAsO3, H3AsOand3 Sand. InS0. theIn the solutions solutions of of high high Eh Eh (> (>0.2 0.2 V V (pH (pH 3)–0.4 3)– V (pH0.4 V 0)), (pH the 0)), elements the elements of iron, of iron, arsenic arsenic and and sulfur sulfur in in sulfide sulfide latticeslattices were were oxidized oxidized into into FeO·OH, FeO OH, − − − 2− 2 − · HAsOHAsO3−/3H/H3AsO3AsO44//HH22AsO44− andand HS HS2O23O/S3−2O/S32O/HS3 −2O/HS4 , 2respectively.O4−, respectively. Furthermore, Furthermore, jarosite was jarosite easily was + + easilyformed formed between between H3OFe H3OFe3(SO34)(SO2(OH)4)26(OH) and 6Kand in K9 Kin medium 9 K medium [54], and [54 ],the and precipitate the precipitate could couldcoat the coat surface of arsenopyrite in the presence of jarosite, FeO·OH, As2S2, As2S3 and S0, which would0 impede the surface of arsenopyrite in the presence of jarosite, FeO OH, As2S2, As2S3 and S , which would the subsequent oxidation and leaching process. However, As· 2S2, As2S3 and S0 could be oxidized0 by impede the subsequent oxidation and leaching process. However, As2S2, As2S3 and S could be oxidizedincreasing by increasing Eh (>0.4 V). Eh (>0.4 V).

Figure 5. Eh–pH diagrams of Fe-As-S-H2O systems: (a) Fe species; (b) As species; (c) S species and Figure 5. Eh–pH diagrams of Fe-As-S-H2O systems: (a) Fe species;2+ (b) As species; (c) S species and4 Fe- Fe-S-H2O systems: (d) Fe species; (e) S species. Conditions: [Fe ]total 0.16 M, [FeAsS] 7.4 10− mM, S-H2O systems: (d) Fe species; (e) S species. Conditions: [Fe2+]total 0.16 M, [FeAsS] 7.4 × 10−4 mM,× [FeS2] [FeS ] 3 10 4 mM; 25 C and 1 atm. 3 2× 10−×4 mM;− 25 °C and◦ 1 atm.

Metals 2020, 10, 1216 11 of 16 Metals 2020, 10, x FOR PEER REVIEW 11 of 16

The data presented in the phase diagram for pyrite in the course of bio-oxidation (Figure 5d,e) The data presented in the phase diagram for pyrite in the course of bio-oxidation (Figure5d,e) show show that the oxidation potential of pyrite was −0.26 V to 0.33 V within the pH range of 0–3. The that the oxidation potential of pyrite was 0.26 V to 0.33 V within the pH range of 0–3. The oxidation oxidation potential of pyrite was significantly− higher than arsenopyrite, indicating that the pyrite was potential of pyrite was significantly higher than arsenopyrite, indicating that the pyrite was more more difficult to oxidize than arsenopyrite [55]. In the subsequent deeper oxidation process, the iron difficult to oxidize than arsenopyrite [55]. In the subsequent deeper oxidation process, the iron and 3+ 0 2- 2+ and sulfur species became3+ Fe0 , S and S2 2Ox at a higher Eh (>0.33 V), while2+ Fe , elemental iron and sulfur species became Fe ,S and S2Ox − at a higher Eh (>0.33 V), while Fe , elemental iron and HS− HS- occurred at a lower Eh (<−0.26 V). It can be seen from the data that the S0 was gradually oxidized occurred at a lower Eh (< 0.26 V). It can be seen from the data that the S0 was gradually oxidized and and the thiosulphate stabilizing− effect could dominate with the increase in oxidation potential, which the thiosulphate stabilizing effect could dominate with the increase in oxidation potential, which could could accelerate the overall gold dissolution [56]. accelerate the overall gold dissolution [56]. The above thermodynamic analysis demonstrated that the gold encapsulated in both The above thermodynamic analysis demonstrated that the gold encapsulated in both arsenopyrite arsenopyrite and pyrite could be liberated by bio-oxidation. In addition, the possible equilibrium and pyrite could be liberated by bio-oxidation. In addition, the possible equilibrium equations involved equations involved in the bio-oxidation of arsenopyrite and pyrite are listed in Appendix A. in the bio-oxidation of arsenopyrite and pyrite are listed in AppendixA.

3.4. Preg-Robbing Preg-Robbing Studies Studies of DRGO in Thiourea System The effect effect of didifferentfferent bio-oxidation treatments on gold adsorption in the thiourea system is presented in Figure 66.. ItIt cancan bebe observedobserved thatthat thethe adsorptionadsorption capacitycapacity ofof as-receivedas-received DRGODRGO toto goldgold was 0.157 mgmg/g/g withinwithin 33 h.h. AfterAfter bio-oxidation, bio-oxidation, there there was was a a decrease decrease in in the the preg-robbing preg-robbing capacity capacity of allof allsamples, samples, and and the the overall overall decrease decrease in preg-robbing in preg-robbing capacity capacity was was around around 14%. 14%. It was It was thus thus very very clear clear that thatA. ferrooxidans A. ferrooxidansand L.and ferrooxidans L. ferrooxidanspossessed possessed the ability the ability to inactivate to inactivate CM inCM DRGO in DRGO and reduceand reduce its gold its goldadsorption adsorption activity. activity. It has It been has been reported reported that thethat graphitic the graphitic structure structure of CM of wasCM importantwas important for gold for goldadsorption adsorption from from the thiourea the thiourea system system [19]. However,[19]. However, there wasthere an was introduction an introduction of oxygen of oxygen groups groups on the onCM, the which CM, hadwhich the had potential the potential to reduce to the reduce degree the of degree graphitization of graphitization and thus decrease and thus gold decrease adsorption gold adsorptionactivity. In addition,activity. In the addition, cleavage ofthe the cleavage aromatic of Cthe=C aromatic band and C=C the degradationband and the of aromaticdegradation O-C-O of aromaticand C-N bondsO-C-O reducedand C-N the bonds affinity reduced of CM the for affinity gold in of solution CM for [57 gold]. Another in solution possibility [57]. Another was the possibilityreduction in was the the specific reduction surface in the area specific via blocking surface the area pores via ofblocking CM by the biochemicals pores of CM produced by biochemicals from the producedmicrobial metabolism,from the microbial hence reducing metabolism, gold hence adsorption reducing [58]. gold In addition, adsorption the coating[58]. In ofaddition, passivation the coatingmaterials of such passivation as jarosite materials reduced such the easffective jarosite contact reduced between the effective CM and contact gold. This between conclusion CM and was gold. also Thisreported conclusion in previous was also research reported from in ourprevious team [rese59],arch where from graphite our team was [59], used where as a graphite substitute was for used CM. asThis a substitute observation for might CM. This contribute observation to the might increase contri in goldbute extraction to the increase from thein gold bio-oxidized extraction samples from the in bio-oxidizedthe process of samples thiourea in leaching. the process of thiourea leaching.

0.21 35

0.18 30 0.157 0.15 25

0.12 20

0.085 0.09 0.08 0.079 15

0.06 10 Adsorption rate (%)

Adsorption capacity /(mg/g) capacity Adsorption 0.03 5

0 0 As-received A. ferrooxidans L. ferrooxidans Mixed Figure 6. Eﬀect of diﬀerent treatments on gold adsorption by DRGO. Figure 6. Effect of different treatments on gold adsorption by DRGO. 3.5. Thiourea Leaching 3.5. Thiourea Leaching Leaching of gold was carried out in acidic thiourea solutions (Figure7). It can be seen that the as-receivedLeaching DRGO of gold yielded was onlycarried around out in 28.7% acidic of goldthiourea extraction solutions by direct(Figure thiourea 7). It can leaching, be seen indicating that the as-receivedthat the present DRGO sample yielded was very only refractory. around The28.7% recovery of gold loss extraction was mainly by caused direct bythiourea the inaccessibility leaching, indicatingof thiourea that reagent the present to gold particlessample was and very the preg-robbing refractory. The of CMrecovery for gold loss in was solutions. mainly After caused biological by the inaccessibility of thiourea reagent to gold particles and the preg-robbing of CM for gold in solutions. After biological pre-treatment, the gold recovery rate reached 78.69% (A. ferrooxidans) and 75.31% (L.

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Metals 2020, 10, x FOR PEER REVIEW 12 of 16 pre-treatment, the gold recovery rate reached 78.69% (A. ferrooxidans) and 75.31% (L. ferrooxidans), ferrooxidans), and the DRGO oxidized by mixed culture resulted in higher gold extraction of 80.07%. and the DRGO oxidized by mixed culture resulted in higher gold extraction of 80.07%. It can be It can be seen that the mixed culture did not show any obvious advantage over the pure A. ferrooxidans seen that the mixed culture did not show any obvious advantage over the pure A. ferrooxidans and and L. ferrooxidans in this study. This is inconsistent with previous reports [22,60]. However, this is L. ferrooxidans in this study. This is inconsistent with previous reports [22,60]. However, this is possibly possibly because the capacity of the strain varies depending on its source, pre-adaptation to minerals, because the capacity of the strain varies depending on its source, pre-adaptation to minerals, the pH the pH value of the solution and bacterial growth conditions [61]. In addition, compared with A. value of the solution and bacterial growth conditions [61]. In addition, compared with A. ferrooxidans, ferrooxidans, L. ferrooxidans has a stronger affinity for ferrous iron and is less sensitive to the inhibition L. ferrooxidans has a stronger affinity for ferrous iron and is less sensitive to the inhibition of ferric of ferric iron [60]. Therefore, the use of mixed culture did not show obviously better bio-oxidation iron [60]. Therefore, the use of mixed culture did not show obviously better bio-oxidation results than results than pure cultures, which may be related to the inferior iron oxidation ability of A. ferrooxidans pure cultures, which may be related to the inferior iron oxidation ability of A. ferrooxidans at high at high potential. This finding is similar to the results reported earlier by Yang et al. [61] and Falco et potential. This finding is similar to the results reported earlier by Yang et al. [61] and Falco et al. [62]; al. [62]; both found that the synergistic effect of microorganisms with different metabolic pathways both found that the synergistic effect of microorganisms with different metabolic pathways did not did not play an important role in their study. play an important role in their study.

2 90

1.6 70 1.42 1.45 1.36 60

1.2 rate/% 50

40 0.8 30 0.52 20 Gold recovery

Gold concentration/(mg/L) Gold 0.4 10

0 0 As-received A. ferrooxidans L. ferrooxidans Mixed Figure 7. Effect of different treatments on gold recovery rates of DRGO. Figure 7. Effect of different treatments on gold recovery rates of DRGO. 4. Conclusions 4. Conclusions This paper demonstrated that the bio-oxidation of a typical DRGO using A. ferrooxidans and L. ferrooxidansThis papercould demonstrated decompose that sulfides the bio-oxidation and simultaneously of a typical reduce DRGO the gold using adsorption A. ferrooxidans capability and ofL. CMferrooxidans in a thiourea could system.decompose Investigations sulfides and showed simultaneously that A. ferrooxidans reduce theand goldL. ferrooxidansadsorption capabilitywere able toof oxidizeCM in a Fe thiourea2+ to Fe 3system.+ and thus Investigations increase Eh showed in the solutions, that A. ferrooxidans which was and conducive L. ferrooxidans to decomposing were able the to sulfides.oxidize Fe This2+ to result Fe3+ and was thus also increase proven by Eh thermodynamic in the solutions, analysis. which was The conducive preg-robbing to decomposing capacity for gold the wassulfides. reduced This byresult more was than also 14% proven after by bio-oxidation. thermodynamic XRD, analysis. SEM-EDS The and preg-robbing FTIR analyses capacity showed for gold that A.was ferrooxidans reduced byand moreL. ferrooxidans than 14% afterwere bio-oxidation. able to destroy XRD, the graphitization SEM-EDS and structure FTIR analyses by the showed introduction that ofA. oxygen-containingferrooxidans and L. ferrooxidans groups, thus were decreasing able to thedestroy active the sites graphitization necessary for structure gold adsorption. by the introduction Moreover, biochemicalsof oxygen-containing were produced groups, by thus microbial decreasing metabolism the active and passivationsites necessary materials for gold such adsorption. as jarosite wereMoreover, formed biochemicals on the surface were of produced the DRGO, by which microbia preventedl metabolism effective and contact passivation between materials CM and such gold. as Injarosite addition, were the formed S-S band on the was surface cleaved, of the indicating DRGO, which that sulfides prevented were effective oxidized contact by bacteria. between Thiourea CM and leachinggold. In resultsaddition, have the shown S-S band that goldwas extractionscleaved, indicating of over 75% that were sulfides achieved were for oxidized both pure by and bacteria. mixed cultures.Thiourea However,leaching results the use have of mixed shown culture that gold did extractions not show obviously of over 75% better were bio-oxidation achieved for results both pure than pureand mixed cultures. cultures. Preliminary However, results the use were of promising,mixed culture though did not there show was obviously still partial better gold bio-oxidation adsorption. Therefore,results than with pure optimized cultures. parameters Preliminary in results the process were of promising, bio-oxidation, though higher there levels was of still gold partial extraction gold andadsorption. lower levels Therefore, of preg-robbing with optimi arezed feasible. parameters in the process of bio-oxidation, higher levels of gold extraction and lower levels of preg-robbing are feasible. Author Contributions: R.X. and Q.L.: Data curation, Writing, Original draft preparation, Conceptualization, Methodology,Author Contributions: Fundingacquisition. R.X. and Q.L.: F.M.: Data Visualization, curation, Writing, Investigation, Original Writing—Reviewing draft preparation, and Conceptualization, Editing, Funding acquisition. Y.Y.: Software. B.X. and H.Y.: Writing—Reviewing and Editing. T.J.: Supervision, Validation. AllMethodology, authors have Funding read and acquisition. agreed to the F.M.: published Visualizatio versionn, of Investigation, the manuscript. Writing—Reviewing and Editing, Funding acquisition. Y.Y.: Software. B.X. and H.Y.: Writing—Reviewing and Editing. T.J.: Supervision, Validation. All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by the National Key Research and Development Program of China (grant

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Funding: This research was funded by the National Key Research and Development Program of China (grant No. 2018YFE0110200), the National Natural Science Foundation of China (grant No. 51574284) and Science and Technology Planning Project of Yunnan Province (grant No. 2018ZE001) Conﬂicts of Interest: The authors declare no conﬂict of interest.

Appendix A

Table A1. Possible equilibrium equations involved in the bio-oxidation of arsenopyrite and pyrite.

0 Bio-Oxidation of Arsenopyrite and Pyrite ∆G 298/(kcal/mol) No. 3+ 2+ 0 + FeAsS + 5Fe + 3H2O = 6Fe + H3AsO3 + S + 3H 81.66 1 3+ 2+ + − 2FeAsS + 16Fe + 9H2O = 18Fe + 2HAsO3− + HS2O3− + 15H 211.199 2 3+ 2+ − 6FeAsS + 12Fe = 3As2S2 + 18Fe 322.269 3 3+ 2+ 0 + − FeAsS + 3H2O + 6Fe = 7Fe + S + HAsO3− + 5H 91.489 4 3+ 2 2+ + − 4FeAsS + 4H2O + 12Fe = As2S3 + S − + 16Fe + 2AsO2− + 8H 194.989 5 3+ 0 2 2+ + − 2FeAsS + 6H2O + 10Fe = S + 2HAsO3− + S − + 12Fe + 10H 126.894 6 3+ 2 2+ + − 2As2S2 + 15H2O + 16Fe = 4HAsO3− + 2S − + HS2O3− + 16Fe + 25H 67.163 7 3+ 2 2+ + − As2S3 + 9H2O + 10Fe = 2HAsO3− + S − + HS2O3− + 10Fe + 15H 42.212 8 3+ 2+ + 2 − FeS2 + 14Fe + 8H2O = 15Fe + 16H + 2SO4 − 134.77 9 3+ 2+ 0 − FeS2 + 2Fe = 3Fe + 2S 19.164 10 3+ 2+ + − FeS2 + 3H2O + 6Fe = 7Fe + HS2O3− + 5H 47.385 11 0 3+ 2 + 2+ − S + 4H2O + 6Fe = SO4 − + 8H + 6Fe 57.803 12 3+ + − Fe + 3H2O = Fe(OH)3 + 3H 5.503 13 2Fe3+ + 4H O = 2FeO OH + 6H+ 1.07 14 2 ·

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