Contribution of Sessile Acidophiles on Chalcopyrite Bioleaching Under Controlled Redox Potentials

Abstract

Although the bioleaching of secondary copper sulfides has been industrialized for decades, the application of chalcopyrite bioleaching remains under development because of its low leaching rate. The effect of contact microbes on chalcopyrite leaching is still unclear due to the technical challenges in separating the contact (sessile micro-organisms) and the non-contact (planktonic micro-organisms) processes. Chalcopyrite bioleaching experiments were conducted using a novel device that stabilizes the redox potential and distinguishs between the microbial contact and non-contact effects. The contribution of the microbial “contact mechanism” in chalcopyrite leaching was quantified considering different redox potentials, compared to the “non-contact mechanism”. Based on the copper leaching kinetics and morphology of the leaching residue, it was demonstrated that the leaching rate of chalcopyrite was significantly influenced by the redox potential (850 mV > 650 mV > 750 mV), from 6.30% to 14.02% in 8 days leaching time. At each redox potential, the chalcopyrite leaching rate was 9.3%–30.6% higher with the presence of sessile microbes than without sessile microbes. Analysis of the leached chalcopyrite surface using time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectrometry (XPS) revealed the formation of polysulfide and elemental sulfur at the surface. While the contacted sulfur oxidized the microbes, here, the Acidithiobacillus caldus preferred sessile at the chalcopyrite surface rather than Leptospirillum ferriphilum. Sulfur-oxidizing bacteria reduced the elemental sulfur content at the leach residue surface, thus playing an important role in degrading the sulfur passivation layer. In chalcopyrite bioleaching, the “contact mechanism” was primarily explained by sulfur-oxidizing bacteria promoting chalcopyrite oxidation through the removal of sulfur intermediates, while the “non-contact mechanism” was explained by ferrous-oxidizing microbes influencing the redox potential.

1. Introduction

Although the heap bioleaching of secondary copper ore has been industrialized for decades, the bioleaching of chalcopyrite (CuFeS₂) ore has not largely been applied due to the low leaching kinetics, usually lower than 50% recovery at a leaching period of one year [1,2,3,4]. Acidic solutions composed of sulfuric acid and ferric sulfate are particularly common in chalcopyrite leaching studies. In the sulfuric acid condition, Fe³⁺ acts as an oxidizing agent, and the variation in the redox potential, as well as microbial activity, is a key factor in determining the leaching rate of chalcopyrite [5].

Chalcopyrite can be attacked and degraded by Fe³⁺ and protons, with polysulfide and elemental sulfur as the main intermediates [1,6]. These bioleaching acidophilic microorganisms mainly have the abilities of oxidizing iron and oxidizing sulfur [7]. Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum, as iron-oxidizing microorganisms, promote the oxidation of ferrous iron, regenerate the oxidant ferric iron, and increase the solution potential, thus playing a significant role in enhancing chalcopyrite leaching. Acidithiobacillus caldus and Acidithiobacillus thiooxidans, as sulfur-oxidizing microorganisms, can remove intermediate sulfur layers, thereby increasing the leaching rate of chalcopyrite. The promoting effect of microorganisms on chalcopyrite leaching was reflected not only in their action on the mineral but also in their influence on the environment. They regenerate the oxidant ferric ions, which affects the redox potential, and both impact the chalcopyrite leaching process [8,9,10].

In 1964, Silverman and Ehrlich first proposed that mineral dissolution by acidophiles could be achieved either via the “direct mechanism” or the “indirect mechanism”, in which bacteria oxidize Fe²⁺ to regenerate Fe³⁺ [11]. These two mechanisms provided important theoretical support for the development of biohydrometallurgy over the decades [12]. Although some researchers suggest that small pits on the ore surface reflect the impact of “contact leaching”, chemical oxidation can also cause pit formation [13,14]. Scientists later discovered that microbial mineral leaching was accompanied by complex electrochemical and biochemical behaviors, while there was no such direct leaching by enzymes [15]. Sand and others further proposed that iron and sulfur oxidation were both indirect leaching regardless [5,16], and then the concepts of “contact” and “non-contact” leaching have been widely accepted [17]. Contact leaching considers that cells attach to the surface of sulfide minerals, meaning that the electrochemical processes leading to the dissolution of sulfide minerals occur at the interface between the microbial cell and the mineral sulfide surface. This space is filled with extracellular polymeric substances (EPSs), a mixture of polysaccharides, proteins, lipids, and nucleic acids [16,18]. Contact leaching can be understood as the adsorption of microorganisms on the surface of sulfide minerals, leading to faster oxidation of Fe²⁺, as well as the oxidation of the intermediate sulfur during chalcopyrite oxidation [5]. Non-contact leaching is basically exerted by planktonic microorganisms, which oxidize iron(II) ions in solution [5]. The regenerated iron(III) ions get into contact with a mineral surface, where they are reduced, and the sulfide moiety is oxidized.

However, the contribution of “contact leaching” has not been fully verified, as the influence of “non-contact leaching” was not excluded in studies. Redox potential is the critical factor for chalcopyrite oxidation [2]; thus, the microbial-influenced redox potential also significantly affects the chalcopyrite leaching rate. This challenge makes it difficult to precisely determine the contribution of contact and non-contact microbes to the oxidation process of chalcopyrite. Therefore, it is particularly important to conduct in-depth research on the microbial leaching of chalcopyrite under controlled solution redox potential to maintain constant “non-contact leaching” when studying the mechanism of “contact leaching” on chalcopyrite. During microbial leaching, most microorganisms are adsorbed onto the mineral surface [7]. This further emphasize the importance of the contact microbes on chalcopyrite leaching.

A novel device was established to stabilize the redox potential, as well as to separately investigate the contribution of contact and non-contact microbes. The controlled redox potential can make the constant microbial a non-contact effect, allowing for the investigation of the contribution of contact microorganisms to chalcopyrite leaching. In addition, the importance of redox potential on chalcopyrite, also explained as non-contact leaching, can be reflected. The morphology of the mineral surface was observed through scanning electron microscopy (SEM), and the valence states and distribution of different elements on the residue surface were analyzed using X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). By studying the surface products of residue samples as well as the microbial community composition, we can infer the role of attached microorganisms on chalcopyrite bioleaching.

2. Materials and Methods

2.1. Samples and Microbes

A highly pure crystal chalcopyrite sample was collected from a mine at Guilin City, Guangxi Province. Ore was crushed to about 2 mm, and gangue impurities were removed using a plier. Then, the ore was ground to a particle size of less than 200 μm, and wet sieving was used to select the ore particles in the range of 74 μm to 105 μm for leaching tests. X-ray diffraction testing (XRD, Smartlab, Tokyo, Japan) revealed no diffraction peaks from other crystals, indicating the high purity of the sample (Figure 1). To remove the oxide layer on the sample surface, the powder sample was washed in a 3 mol L⁻¹ hydrochloric acid solution at 50 °C for 30 min. Subsequently, anhydrous ethanol was added, and the sample was cleaned multiple times using an ultrasonic cleaner. After cleaning, the sample was filtered and washed with deionized water to ensure complete removal of impurities. Finally, the cleaned samples were placed in a vacuum drying oven for drying at −20 °C for 4 h, and then uniformly mixed for subsequent experiments.

To observe the changes in the surface morphology of chalcopyrite and for the ToF-SIMS testing after leaching, the chalcopyrite was cut into cubes approximately 10 mm × 10 mm × 3 mm. After polishing the surface, the cubes were cleaned with hydrochloric acid and ethanol to ensure surface cleanliness.

The microbial consortium used in this experiment was sourced from the industrial heap bioleaching plants of copper sulfide at Monywa mine, Myanmar [19,20]. To cultivate the microorganisms, an optimized 9K medium was used, with the following specific composition: (NH4)₂SO₄ 3 g/L, KCl 0.1 g/L, K₂HPO₄ 0.5 g/L, MgSO₄·7H₂O 0.5 g/L, Ca(NO₃)₂ 0.01 g/L, FeSO₄ 5 g/L, and S⁰ 2 g/L, pH 1.5, at 35 °C in the oscillator. Once the microorganisms had reached the stationary phase, they were separated from the medium by filtration using a 0.22 μm filter membrane. The microbes on the membrane were then washed down with the sulfuric acid solution at pH 1.5, aided by ultrasonic treatment at 40 kHz, to remove any residual iron and sulfur prior to being used in the subsequent batch experiments.

2.2. Apparatus and Batch Experiments

The experiment was conducted in a device that can control the redox potential and also allow or not allow microbes to contact the minerals (Figure 2). This system mainly consisted of four parts: the redox potential control unit (6, 9, 10-1), the leaching unit (1-1, 2-1, 3-1, 4), the microbial culture and separation unit (1-2, 2-2, 3-2, and 8), and the solution circulation system (10-2 and 7) [14]. The redox potential control unit uses H₂O₂ or ascorbic acid solution to control the redox potential of the solution. The redox potential of the solution in the leaching unit is measured using a platinum ring electrode and an Ag/AgCl reference electrode. If the redox potential is lower or higher, the pump will activate and pump the H₂O₂ or ascorbic acid solution to the flask. A 1000 mL three-neck flask was also used as the microbial culture container, with isolation between flasks achieved through a filtration plate containing a 0.22 μm filter membrane if needed. For “non-contact leaching”, the membrane can keep the microbes only in the solution but not in contact with the minerals, while for “contact leaching”, no membrane was installed in the filtration plate, allowing microorganisms to circulate with the solution and to contact the chalcopyrite. To maintain the solution balance between the two culture flasks, a peristaltic pump was used to pump the solution, ensuring the ionic concentration in both culture flasks remained consistent.

Batch experiments using the fine powders or chalcopyrite cubes described in Section 2.1 employed a 9K medium. The medium was prepared by mixing ferrous sulfate and ferric sulfate, with a total iron concentration of 2.5 g/L and a pH adjusted to 1.5 using H₂SO₄. A different ratio of ferrous iron and ferric iron was added to form redox potentials of about 650 mV, 750 mV, and 850 mV (vs. SHE), respectively. Eight grams of chalcopyrite powder were added to the leaching flask with 1 L solution. During the experiment, an ORP controller and peristaltic pump were used to precisely adjust the solution potential, maintaining it at 650 mV, 750 mV, and 850 mV (vs. SHE), respectively, at 35 °C. Microorganisms were added to the microbial culture flask, and their concentration was adjusted to 1 × 10⁷ cells mL⁻¹. During the leaching, solution samples were taken every 24 h, and Cu concentration was determined using an inductively coupled plasma optical emission spectrometer (ICP-OES, Termofisher, Waltham, MA, USA). After leaching, the residue was filtered and washed with dilute H₂SO₄ at a pH of 1.5, of which about the half residue was dried in a low-temperature vacuum drying oven for XPS and HPLC assay, and the remaining half of the material was used for extracting contact microorganisms. The leaching of the chalcopyrite cubes for surface morphology observation and ToF-SIMS (TOF.SIMS5-100, ION-TOF, Munster, Germany) testing were performed with a similar procedure to that above.

2.3. Surface Morphology by SEM

The chalcopyrite samples after biological oxidation underwent a series of pre-treatment steps before scanning electron microscope (SEM, Hitachi SU8010, Tokyo, Japan) analysis. The chalcopyrite residue samples were placed in a 3% glutaraldehyde solution and fixed at 4 °C for 24 h. After fixation, the chalcopyrite samples were sequentially immersed in 50%, 75%, and 99% ethanol solutions for 10 min each. Following dehydration, the samples were dried in a ventilated environment for 2 h. The chalcopyrite samples then underwent a gold sputtering treatment after drying. The accelerating voltage for the SEM was set to 15 kV.

2.4. Surface Components by XPS

The surface copper, iron, and sulfur species of the chalcopyrite residues under various treatments were analyzed using an X-ray photoelectron spectrometer (ESCALAB 250Xi, Thermo Fisher Scientific, Waltham, MA, USA). The incident radiation consisted of monochromatic Al Kα X-rays (1486.7 eV) at 150W (15 kV, 15 mA). The base pressure in the analysis chamber was 2.31 × 10⁻¹⁰ mbar, which increased to 2.31 × 10⁻⁸ mbar during sample analysis. Survey scans were conducted to identify the elements present, while high-resolution scans were performed to determine the oxidation states and composition of sulfur and iron. Data processing and fitting were carried out using the Avantage v5.938 software package and the relative sensitivity factor (RSF) library. The spectra were charge-referenced to the adventitious C 1s peak at 248.8 eV, and the smart background subtraction method was applied.

2.5. Copper, Iron, and Sulfur Distributions by Tof-SIMS

The copper, iron, and sulfur distributions at different depths were determined using ToF-SIMS (TOF.SIMS5-100, ION-TOF, Munster, Germany). ToF-SIMS can analyze the compositional resolution of substances ranging from 10⁻⁶ to 10⁻⁹. By adjusting the primary ion current density, information about the surface monolayer of atoms and molecules can be determined. A Bi⁺ primary ion beam was used with an incident power of 30 keV at a 45° angle, and a scanning area of 100 μm × 100 μm. The secondary ion polarity and mass range were 0 to 3000. The sputtering positive ion beam was O²⁺, while the sputtering negative ion beam was Cs⁺, with an incident power of 2 keV at a 45° angle. The sputtering rate was 0.54 nm s⁻¹.

2.6. Quantification of Elemental Sulfur by HPLC Assay

One gram of chalcopyrite residue was cleaned three times in ethanol using an ultrasonic treatment at a frequency of 40 thousand Hz. The treated residue was subjected to centrifugal separation at high speed for 5 min afterward, and the supernatant was collected. The sulfur content in the supernatant was tested using high-performance liquid chromatography (HPLC). Hypersil-ODS-5μ chromatographic column was used as the separation medium, with pure methanol as the elution solvent, and a constant flow rate of 1 mL/min was set [21].

2.7. Microbial Community Analysis

An optical binocular microscope and a hemocytometer counting method were used to determine the number of microorganisms in the solution. The Fast DNA Spin kit was used for the efficient extraction of microbial DNA, followed by the detection of microbial communities both in the solution and adhered to the mineral surface. Using primers F515 (5′-GTGCCAGCMGCCGCGGTAA-3′) and R806 (5′-GGACTACVSGGGTATCTAAT-3′), the microbial 16S rRNA was amplified. The high-throughput sequencing of the amplification products was performed using Illumina MiSeq sequencing technology, and through data filtering and merging, more accurate and comprehensive microbial community information was obtained and classified into different operational taxonomic units (OTUs). To further identify the microbial species represented by these OTUs, representative sequences of each OTU were selected and compared using BLAST (Blastn, NCBI, Bethesda, MD, USA) against the NCBI database.

3. Results and Discussion

3.1. Morphological Characteristics of Oxidized Chalcopyrite Cube Surface

The morphology of chalcopyrite residues was imaged by SEM, as shown in Figure 3. It was reported that two suitable redox potential ranges exist during the chalcopyrite leaching process: the low-potential leaching interval (550 mV to 680 mV, vs. SHE) and the high-potential leaching interval (greater than 820 mV, vs. SHE) [22]. Between 680 mV and 820 mV, the oxidative dissolution resulted in more metal-deficient polysulfide on the chalcopyrite surface, thus its more passive effect will decrease the chalcopyrite leaching. Morphological analysis showed that the corrosion degree of chalcopyrite in the suitable redox potentials was higher than that outside the passive redox potential. At each redox potential, regardless of whether the microorganisms were sessile, the corrosion on the chalcopyrite surface was similar. Small cracks and pores were observed at 650 mV, with an average channel width of about 0.2 μm, while at 850 mV, more severe corrosion occurred, with an average channel width of about 0.8 μm. In contrast, at 750 mV, the chalcopyrite surface was smoother than at the other two redox potentials. The cracks formed when microorganisms were attached were slightly wider than when no microorganisms were present, suggesting that microorganisms can accelerate chalcopyrite leaching.

3.2. Copper Leaching Kinetics

Copper leaching kinetics were calculated using the copper concentration in the flasks during the leaching experiment (Figure 4). Whether the microorganisms were attached onto chalcopyrite or not, the redox potential had a significant impact on the leaching of chalcopyrite. When the solution potential was at 850 mV and 650 mV, the chalcopyrite leaching rate was higher (9.98% and 12.85% without the contact microbe, and 11.85% and 14.02% with the contact microbes, respectively), while at 750 mV, the leaching rate was slower (6.30% and 8.23% without and with the contact microbes, respectively). This is in accordance with the morphology image. This result is in accordance with previous studies that between about 680 and 720 mV, a passive layer more likely formed, thus decreasing the chalcopyrite leaching rate [2,9]. This means that the ferrous-oxidizing microbes do not always promote the leaching; sometimes, they may inhibit the leaching kinetics if the redox potential falls to a redox potential of 680–720 mV (the redox potential is mainly determined by the Fe²⁺/Fe³⁺ ratio). In the absence of sessile acidophiles, where only iron ions are acting on the surface, the leaching rate can represent the contribution of the “non-contact leaching”. The kinetics obtained by subtracting the leaching rate with attached acidophiles from the rate without attached microorganisms at the same redox potential represent the contribution of the “contact leaching” of the attached microorganisms. So, the extent of chalcopyrite kinetics increases in the treatment with contacted leaching compared to the treatments without contact leaching, reflecting the promoting effect of the contact microbes. Copper leaching kinetics under all three redox potentials suggest that both the contact and non-contact mechanisms contribute to the chalcopyrite oxidation.

3.3. Chalcopyrite Residue Surface Component Assay

The sulfur S 2p spectra of the leach residue are shown in Figure 5, including sulfate (SO₄²⁻), polysulfide (S_n²⁻), elemental sulfur (S⁰), and sulfide (S²⁻). The fitting peak with a binding energy of 161.1–161.8 eV in the S 2p3/2 region corresponds to S²⁻ in the surface phase of chalcopyrite, the binding energy of 163.0–164.7 eV corresponds to polysulfides and elemental sulfur in the surface phase of chalcopyrite, and the binding energy of 168.0–169.0 eV corresponds to SO₄²⁻ from the post-experiment solution [23]. By separating sulfur species and calculating their amounts, a pathway of chalcopyrite oxidation can be speculated, and differences between treatments can be determined.

The percentages of various sulfur species under different redox potential conditions after the experiments are shown in

Table 1. Species and contents of sulfur on the surface of chalcopyrite residue after leaching with or without contact microbes (+M, −M) under different redox potentials (650, 750, and 850 mV (vs. SHE)).

	650 mV − M	650 mV + M	750 mV − M	750 mV + M	850 mV − M	850 mV + M
Species	%	%	%	%	%	%
S22−	53.88	52.04	56.85	52.40	17.27	21.01
Sn2−/S0	39.12	28.7	34.66	25.65	45.76	34.86
SO42−	6.98	19.26	8.49	21.95	36.97	44.13

. When the redox potential of the solution was at 750 mV, compared to the condition with sessile microbes, the amount of elemental sulfur on the chalcopyrite residue surface was significantly higher than when there are no sessile microbes. This observation indicates that sessile microbes can metabolize elemental sulfur, thereby accelerating the leaching efficiency of chalcopyrite. The percentage of S_n²⁻ and S⁰ was about 10 percentage points lower in the treatment with contact microbes than that without contact microbes.

Although XPS provides valuable information about the sulfur species on the chalcopyrite residue surface, it has limitations in precisely measuring the elemental sulfur content. Therefore, high-performance liquid chromatography (HPLC) was used to more accurately quantify the amount of elemental sulfur (Figure 6). In the oxidation process of chalcopyrite, the first sulfur-containing ion released is the thiosulfate ion (S₂O₃²⁻). S₂O₃²⁻ then generates unstable intermediate substances during the oxidation process, but ultimately transforms into elemental sulfur (S⁰) and sulfate (SO₄²⁻) [24,25]. Under the redox potential at 650 mV, 750 mV, and 850 mV, without sessile microbes, each gram of chalcopyrite residue contains about 600, 400, and 800 μg of elemental sulfur, respectively. These results suggest that in the absence of adsorbed microbes, the oxidation rates at different redox potentials were positively correlated with the amount of elemental sulfur generated. In taking the 750 mV potential as an example, with the contact microbes, the elemental sulfur content on each gram of chalcopyrite residue was only about 25% that of the treatment with no contact microbes. Similar situations also occur at potentials of 650 mV and 850 mV. This indicates that the elemental sulfur generated during the chalcopyrite leaching process was oxidized by the sessile microbes.

The Fe 2p3/2 spectra of the leached fine residue and ore cubes are shown in Figure 7, and the species compositions of iron in the leach residue are presented in

Table 2. Composition and proportion of Fe 2p valence states on the chalcopyrite surface after leaching at 750 mV (vs. SHE) with or without contact microbes (+M, −M).

	750 mV − M	750 mV + M
Species	%	%
FeS2	100	28.6
FeSO4	0	71.4

. The splitting peaks at 707 eV and 708 eV correspond to the FeS₂ phase in the chalcopyrite matrix, while the splitting peak at 712 eV corresponds to FeSO₄ in chalcopyrite. Additionally, the fitting peak at a binding energy of 711 eV indicates the presence of Fe³⁺ [26]. Under constant potential conditions, the valence state of iron on the chalcopyrite surface did not undergo significant changes, regardless of the presence of contact microbes. The iron in the leached residue existed in the form of Fe (II), while the iron in the ore cubes existed in the form of Fe (III). This is because the ore cubes have a smaller contact area compared to the leach residue, producing less secondary products and secondary products. Previous researchers had pointed out that contact microbes promoted the leaching of chalcopyrite by inhibiting the formation of jarosite on the chalcopyrite surface [27], while in this study, the contact microbes did not significantly influence the formation of secondary minerals, maybe because the pH during the leaching is about 1.3, while low pH is not suitable for jarosite formation [28,29].

The Cu 2p spectra of the chalcopyrite cube surface are shown in Figure 8. The proportion of Cu (II) and Cu (I) in the ore pieces is stable under constant redox potential, and the presence of microbes had little effect on the copper species and contents (

Table 3. Composition and proportion of Cu 2p valence states on the surface of chalcopyrite at 850 mV (vs. SHE) with or without contact microbes (+M, −M).

	850 mV + M	850 mV − M
Species	%	%
Cu+	76.05	77.46
Cu2+	23.95	22.54

). It was reported that the Cu ion in chalcopyrite crystal was mainly Cu⁺ [24,30]; the Cu²⁺ here may also include some oxidized Cu on the chalcopyrite surface.

It was suggested that the contact microbes did not fundamentally alter the leaching mechanism of chalcopyrite. When analyzing the leaching rate in combination with these surface tests, under constant “non-contact” conditions, microbial “contact” action promoted chalcopyrite leaching, and the microbial “contact” action mainly eliminates the intermediate sulfur at the reaction interface. It was reported that the oxidation of Fe²⁺ could occur on the membranes, while the oxidation of sulfur need the transportation of sulfur onto the microbe’s membranes more [7]. Also, the element sulfur was not dissolved in the solution, so it was more likely absorbed on the mineral surface; thus, the contact leaching was more important for sulfur. The EPS helps the microbes attach to the minerals and contact the sulfur compounds [31].

3.4. Chalcopyrite Elemetal Distribution Assay by ToF-SIMS

ToF-SIMS provided the elemental distribution of copper, iron, and sulfur at different depths on the surface of the leached chalcopyrite cubes. The Cu and Fe distribution was analyzed in the positive-ion mode, while the sulfur species were analyzed in the negative-ion mode at an area range of 100 μm × 100 μm (Figure 9). The Cu/Fe ration for the leached chalcopyrite was normalized against that of a freshly polished sample, where the Cu/Fe ratio was assumed to be 1 across the entire range of depth (Figure 10). At the very surface of a few nm in rage, maybe it was influenced by the Cu and Fe ions absorbed on the cubes, while below a few nm, it showed the reacted layer of the chalcopyrite cubes. The Cu/Fe ratio in the treatment without contact microbes was lower than that with contact microbes at a depth < 60 nm. According to the S/Cu and S/Fe ratio, the treatment without sessile microbes had more sulfur than the treatment with sessile microbes. The S/Cu and S/Fe ratios finally reached a value of about 2, indicating the unreacted crystal bulk at a depth of >100 nm.

Leached mineral samples with major peaks belonged to sulfur species at m/z = 32 and 64. Previous work demonstrated that the intensity ratio of S2/S1 can be used to illustrate the extent of polymerization of sulfide species [1,32], although low amounts of S3, S4, and S5 were also detected. With the sputter depth increasing, S2/S1 came to a constant ratio. Unlike in the positive mode, where the Cu/Fe ratio can be assumed to be 1 for a fresh sample, there is no standard ratio for S2/S1 for pure chalcopyrite; thus, the numerical value of the normalized S2/S1 intensity ratio does not have physical significance. A higher S2/S1 ratio was measured on the non-contact leaching chalcopyrite surface than the contact leaching chalcopyrite surface, suggesting that the surface species on the non-contact leached surface have a higher degree of sulfur polymerization. This result, together with previous XPS and HPLC results, proved the contact microbes cause the elimination of intermediate sulfur compounds.

3.5. Microbial Community in Chalcopyrite Residue

The composition study of sessile microbes helped us understand their role in copper sulfide ore leaching. It was found that the population mainly consisted of two types of microorganisms. The dominant microbial species in the inoculated population was the iron-oxidizing bacterium L. ferriphilum, accounting for approximately 70%. This microorganism can only use Fe²⁺ as an energy source. The sulfur-oxidizing bacterium was A. caldus, accounting for about 30%. This microorganism can only use reduced sulfur as an energy source and cannot utilize sulfide minerals directly as energy. Other microorganisms account for less than 4%.

As shown in Figure 11, significant changes were observed in the adsorbed microbial population compared to the initial inoculated microbial population. Under these three different redox potential conditions, the abundance of sulfur-oxidizing bacteria A. caldus on the copper sulfide ore residue surface was significantly higher than their abundance in the solution. At the solution redox potential of 650 mV, 750 mV, and 850 mV, the proportion of sulfur-oxidizing bacteria on the residue surface was increased to 65%, 53%, and 88%, respectively, much higher than that in the solution as inoculated. This finding suggests that elemental sulfur generated during copper sulfide ore leaching can promote the abundance of sulfur-oxidizing bacterium on the residue surface.

The effects of pure and mixed microbial strains on the copper sulfide ore leaching process have been widely studied [7]. Research has shown that copper sulfide ore can be oxidized by microorganisms with iron-oxidizing ability [33,34], as these microorganisms can oxidize Fe²⁺ to Fe³⁺, and Fe³⁺ acts as an important oxidizing agent in acidic solutions. The sulfur-oxidizing microorganism, such as A. caldus, which mainly possesses sulfur-oxidizing abilities, has been confirmed not to directly participate in the oxidation of copper sulfide ore [35,36]. Nevertheless, the addition of sulfur-oxidizing microorganisms can significantly accelerate the oxidation rate of copper sulfide ore using mixed microorganisms, further proving that sulfur-oxidizing microorganisms play an indirect, promoting role in the oxidation process of copper sulfide ore [37,38]. The mechanism behind this phenomenon is that A. caldus can eliminate the passivation effect caused by the sulfur layer on the surface of copper sulfide ore [39]. In addition, as the proportion of sulfur-oxidizing microorganisms in the microbial community increases, their overall sulfur-oxidizing ability is also enhanced [40,41].

All microbes, whether sessile microbes or planktonic microbes, participate in the indirect effect on chalcopyrite leaching. The redox potential was manually controlled in this study, which cannot reflect the actual contribution of microbial ferrous oxidation. It was assumed that the microbes on the mineral surface were more abundant than those in the solution during bioleaching, because minerals offer a solid attachment surface for a continuous substrate supply, create a favorable microenvironment and protection, and enable efficient electron transfer for their metabolic processes [7]. So, the sessile iron-oxidizing microbes may also contribute a lot to the solution redox potential. These results suggest that the “non-contact mechanism”, i.e., mainly the regeneration of oxidizing agents by microorganisms and their influence on the potential, also played a significant role in the leaching of chalcopyrite; the “contact mechanism” of attached microorganisms, i.e., mainly sulfur oxidation, promotes chalcopyrite oxidation. A similar mechanism has been proved in pyrite bioleaching [13,14]. The difference is that pyrite and chalcopyrite favor different redox potentials. For pyrite, the rest potential is about 650 mV (vs. Ag/AgCl). Below this redox potential, pyrite is barely oxidized, while above this redox potential, higher redox potential resulted in a higher pyrite leaching rate [42]. So, higher microbial ferrous-oxidizing activity is favorable for pyrite oxidation. But for chalcopyrite, the redox potential at which the chalcopyrite leaching is passivized is around 680–750 mV [24], which means lower or higher ferrous oxidation activity is favorable but not medium activity.

4. Conclusions

This study, using a novel device, investigated the contribution and mechanism of sessile microbes on the bioleaching of chalcopyrite under the different controlled redox potentials (to simulate the different constant non-contact effects). This device can also be used in research on the bioleaching of other minerals. It clearly separated and quantified the contact and non-contact leaching, as well as the mechanism related to the function of ferrous- and sulfur-oxidizing microbes. The bioleaching rate of chalcopyrite was significantly related to the solution redox potential, with the order of 850 mV > 650 mV > 750 mV, regardless of the presence of sessile microbes. In the bioleaching system, the redox potential is determined mainly by the ferrous oxidizing activity, mainly representing the non-contact microbes. When compared with bioleaching with only non-contact microbes, the chalcopyrite bioleaching kinetics were higher in the treatments with the contact and non-contact microbes together, suggesting the contribution of contact leaching. It was proved using different analytic tools that the contact microbes were mainly sulfur-oxidizing microbes rather than ferrous-oxidizing microbes, which help in the elimination of the intermediate sulfur compounds. This also has implications for the industrial chalcopyrite bioleaching in relation to bioleaching microbial cultivation and regulation.