Difference in Biological Oxidation Between High-Sulfur Coal and Pure Pyrite at Different pH Levels

Abstract

In this study, Acidthiobacillus ferrooxidans LX5 was used as an experimental microbial strain, and differences in biological oxidation between high-sulfur coal and pure pyrite were thoroughly investigated over 18 days in acidic environments with initial pH values of 1.70, 2.00, 2.30, and 2.60. The results showed that the pyrite bio-oxidation efficiency in the coal biological desulfurization system exceeded that in the pure pyrite bio-oxidation system at the same initial pH. The net increase in SO₄²⁻ concentration in the coal biological desulfurization system increased with increasing initial pH values, consistent with the net increasing trend in SO₄²⁻ in the pure pyrite biological oxidation system. The net increase in SO₄²⁻ concentration in the high-sulfur coal biological oxidation system with an initial pH of 2.60 reached 4589.06 mg/L after 18 days. The density of A. ferrooxidans LX5 in both systems increased with increasing initial pH values. With increasing initial pH levels, the inorganic sulfur (pyritic sulfur and sulfate sulfur) removal efficiencies increased in both the coal biological desulfurization and pyrite biological oxidation systems, reaching 88.28% and 9.25%, respectively, at an initial pH of 2.60. The results are of great significance for better understanding the biological desulfurization process of coal.

1. Introduction

Coal remains a very important natural resource, accounting for over 65% of global fossil energy production [1]. In China, coal consumption accounts for nearly 60% of the total energy production [2]. However, the sulfur content in coal presents a significant environmental challenge, particularly due to the emission of sulfur dioxide when burning high-sulfur coal. The release of sulfur dioxide contributes to the formation of acid rain [3,4]. Acid rain is a pervasive issue, with Chinese acid rain typically having a pH in the range of 3.00–4.50 [5]. Acidic precipitation inflicts damage on ecosystems, resulting in the mortality of both fauna and flora, while also hastening the degradation of limestone structures and historical artifacts [6]. Furthermore, acid rain erodes building structures, bridges, dams, and industrial machinery. It poses risks to individuals who consume water from acidic sources or eat fish from contaminated rivers. Additionally, acid rain modifies the physicochemical properties of soil, leading to the depletion of essential plant nutrients [7,8,9,10]. China has abundant high-sulfur coal resources, accounting for one-quarter of the total coal reserves [11]. Consequently, the advancement of high-sulfur coal desulfurization technologies is central to alleviating these environmental impacts, both in China and around the world.

The predominant forms of sulfur in coal are inorganic and organic. Inorganic sulfur can be primarily categorized into coal-derived pyrite and sulfate sulfur. Typically, when the total sulfur content in coal exceeds 2%, it is predominantly present in inorganic form [12]. Conversely, when the total sulfur content in coal is less than 0.5%, it is primarily present in organic form [13,14]. Coal that contains more than 3% total sulfur is categorized as high-sulfur coal. Consequently, the desulfurization process for high-sulfur coal should focus on eliminating inorganic sulfur, with coal-derived pyrite being the most significant inorganic form present [15,16], accounting for 60–70% of the total sulfur content [17]. Therefore, the desulfurization of high-sulfur coal must place particular emphasis on the removal of coal-derived pyrite.

Conventional methods for removing coal-derived pyrite involve the physical flotation method, which exploits the differences in surface properties between pyrite and organic matter in coal [18,19], as well as the chemical method of oxidizing and dissolving pyrite through the use of oxidizing agents and acidic or alkaline solutions [20]. These processes may result in issues such as increased operational costs, reduced calorific value of the coal, and elevated carbon dioxide emissions. Therefore, it is necessary to develop economical and low-pollution coal desulfurization technologies. In recent years, researchers have shown widespread interest in microbial desulfurization due to its low operating costs, mild reaction conditions, and the advantage of not affecting the calorific value of coal [21,22]. A. ferrooxidans is a microorganism commonly used for the microbial desulfurization of coal [23]. However, low desulfurization efficiency has become a critical issue limiting the application of biological desulfurization. He et al. [14] studied coal desulfurization using MTWL-7, and achieved a removal efficiency for coal-derived pyritic sulfur of 47% after 40 days. Investigating the constraining factors that influence microbial desulfurization is crucial for improving desulfurization efficiency. Moreover, investigating the differences in biological oxidation efficiency between high-sulfur coal and pure pyrite is expected to provide insight into the mechanism of the biological desulfurization of coal. Unfortunately, there is currently very limited information available on this aspect.

The initial pH serves as a critical limiting factor that influences the microbial leaching desulfurization process, affecting microbial proliferation and the secretion of extracellular polymeric substances (EPSs), thereby directly or indirectly affecting the biological desulfurization efficiency of high-sulfur coal. However, information concerning the impact of initial pH on coal desulfurization is very limited. Most researchers have merely compared the dissolution efficiency of pyrite with the oxidation efficiency of Fe²⁺ iron, or have simply contrasted the desulfurization efficiencies of coal following bioleaching at varying initial pH levels [24,25]. For example, Liu et al. [12] found that the dissolution efficiency of iron and sulfate ions was highest at an initial pH of 2.50 when studying pyrite. Subsequently, they conducted further coal desulfurization research by controlling the initial pH at this value. Atkins et al. [26] achieved the highest efficiency of Fe²⁺ oxidation at an initial pH of 2.00. Likewise, they conducted further coal desulfurization research by controlling the initial pH at this value. It is evident that employing various assessment methods may lead to divergent experimental outcomes. Therefore, a systematic evaluation is necessary to assess the differences in biological oxidation between high-sulfur coal and pure pyrite at different initial pH values.

In view of the above, this research focuses on high-sulfur coal and pure pyrite, assessing the impact of initial pH on the bio-oxidation of these substrates facilitated by A. ferrooxidans. The findings are crucial for a more comprehensive understanding of the biological desulfurization process of coal.

2. Materials and Methods

2.1. Preparation of High-Sulfur Coal and Pyrite Samples

The coal samples used in this study were collected from a coal washing plant (111.61° E, 36.57° N) in Linfen City, Shanxi Province, China. Coal samples were dried in a drying oven and then ground and sieved to a size range of 0.075–0.150 mm. The total sulfur, pyritic sulfur, organic sulfur, and sulfate sulfur contents in a typical sample were 3.18%, 1.86%, 1.12%, and 0.06%, respectively. According to the Chinese national standard “Classification of quality of coal” (GB/T 15224.2-2021)[27], the studied material can be classified as high-sulfur coal. All coal data are reported on a moisture-free basis.

Pyrite samples were soaked in 1 mol/L hydrochloric acid for 1 h, and then washed with deionized water until the pH of the filtrate was consistent with that of deionized water. The samples were dried in a drying oven and then ground and sieved to a size range of 0.075–0.150 mm. The purity of the pyrite was 88.54%.

2.2. Preparation of Bacterial Suspension

A. ferrooxidans LX5 was obtained from the Solid Waste Research Institute, Nanjing Agricultural University, Jiangsu Province, China. A. ferrooxidans LX5 cells were grown in a series of 250 mL glass conical flasks, each containing 135 mL of modified 9K liquid medium and 15 mL of A. ferrooxidans LX5 inoculum [28,29]. The conical flasks were placed in a rotary shaker and incubated at 28 °C and 180 r/min for 2–3 days, allowing sufficient time for the Fe(II) to be completely bio-oxidized within the system. The above steps were repeated twice more with fresh 15 mL aliquots of the cultured bacterial suspension. The bacteria were centrifuged at 4 °C and 12,000 r/min for 15 min, the supernatant was discarded, and the remaining cells were washed twice with sulfuric acid (pH 2.50) before being suspended in sulfuric acid at a volume ratio of 30:1 (bacterial cell solution/sulfuric acid). The bacterial abundance in the suspension, as measured by 16S rRNA gene copies, was 1.5 × 10⁹ copies/mL.

2.3. Coal Biological Desulfurization and Pyrite Bio-Oxidation by A. ferrooxidans LX5

2.3.1. Coal Biological Desulfurization by A. ferrooxidans LX5

The total duration of the experiment was 21 days, which consisted of 3 days of adjusting the initial pH and 18 days of the biological desulfurization process. The biological desulfurization process was carried out in a series of 250 mL shaker flasks. Culture medium (1 mL; iron-free 9K medium; the concentration of inorganic salts in this medium is 10 times that in conventional 9K culture medium) and deionized water (148 mL) were mixed with portions (10 g) of high-sulfur coal in flasks, and the mixtures were adjusted to pH 1.70, 2.00, 2.30, and 2.60, respectively, with 3 mol/L H₂SO₄ solution. After the pH of each system had stabilized, A. ferrooxidans LX5 bacterial suspension (1 mL) was added to each flask. The flasks were then sealed with sterilized eight-layer gauze, placed in a thermostatted shaker at 28 °C, and agitated at 180 r/min to perform coal biological desulfurization. Volatilization of water from the system was compensated for by periodically adding deionized water. The four treatments were designated as “coal pH 1.70”, “coal pH 2.00”, “coal pH 2.30”, and “coal pH 2.60”. All treatments were performed in triplicate. The pH and oxidation–reduction potential (ORP) were monitored at intervals of 24 h, and aliquots (1 mL) were withdrawn from the flasks at intervals of 72 h, filtered through 0.22 μm nitrocellulose membranes, and the total Fe, Fe²⁺, Fe³⁺, and SO₄²⁻ concentrations therein were determined. The chemical composition of the extracellular polymeric substances (EPSs) secreted by the A. ferrooxidans LX5 in the systems was analyzed at the end of the experiment by mixing the triplicate systems for each treatment. Aliquots (5 mL) were withdrawn from the mixed solution obtained from the triplicate systems at intervals of 144 h and bacterial density was measured. Desulfurized coal samples were obtained from each system by filtering the mixtures through Whatman No. 4 filter paper. Subsequently, the desulfurized coal samples were oven-dried at 50 °C to constant weight, and the contents of pyritic sulfur, inorganic sulfur (pyritic sulfur and sulfate sulfur), and total Fe therein were determined.

2.3.2. Pyrite Bio-Oxidation by A. ferrooxidans LX5

The reaction conditions for the pyrite bio-oxidation experiments were similar to those for the coal biological desulfurization system, but with pyrite (0.4 g) as the substrate, this being equivalent to the pyrite content in the coal biological desulfurization system. The initial pH values of each reaction system were again adjusted to 1.70, 2.00, 2.30, and 2.60, respectively, and then A. ferrooxidans solution (1 mL) was added to each flask, as described for the coal biological desulfurization system. Volatilization of water from the system was compensated for by adding distilled water. The four treatments were designated as “pyrite pH 1.70”, “pyrite pH 2.00”, “pyrite pH 2.30”, and “pyrite pH 2.60”. All treatments were performed in triplicate. Determinations of pH, ORP, and total Fe, Fe³⁺, Fe²⁺, and SO₄²⁻ concentrations and EPS chemical components, bacterial density measurement times, and measurement methods for the system were the same as those for the coal biological desulfurization system. At the end of the reaction, bio-oxidized pyrite samples were obtained from each system by filtering the mixtures through Whatman No. 4 filter paper. Subsequently, the bio-oxidized pyrite samples were oven-dried at 50 °C to constant weight, and then their mineral morphologies, XPS patterns, and inorganic sulfur contents were determined.

2.4. Analytical Procedures

A Coulomb sulfur analyzer was used to determine total sulfur content (KZDL-3, Hebei Haibi Instrument Co., Ltd., Hebi City, Hebei Province, China). The method described by Atkin et al. [30] was employed to measure the contents of pyritic sulfur and sulfate sulfur in the coal. Using a pH and ORP meter (Model pHS-3C, Shanghai Yueping Scientific Instruments Co., Ltd., Shanghai, China), pH and ORPs were measured with accuracies of ±0.01 and ±1, respectively. The concentrations of total Fe and Fe²⁺ in a solution were determined by the 1,10-phenanthroline method. The concentration of Fe³⁺ was calculated by subtracting the concentration of Fe²⁺ from the total Fe concentration [31]. The SO₄²⁻ concentration in a solution was measured by ion chromatography (IC6200, Anhui Wanyi Science and Technology Co., Ltd., Hefei, Anhui Province, China). EPSs in the system were extracted by the EDTA method. Specifically, aliquots (15 mL) of the mixtures were treated with an equal volume of 2% EDTA-2Na solution for 3 h. The reaction mixture was centrifuged at 12,000 r/min at 4 °C, and the EPSs in the obtained supernatant were determined. For this, the crude EPS solution was placed in a dialysis bag with a molecular weight cut-off of 3500 and dialyzed at 4 °C in distilled water for 2 days to remove impurities. Protein and polysaccharide concentrations in the EPS were measured by the Coomassie Brilliant Blue G-250 method and the anthrone method, respectively [32,33]. Bacteria contents were determined by withdrawing aliquots (5 mL) from the mixtures and submitting them to quantitative fluorescence PCR [34]. Pyrite morphologies were inspected by field-emission scanning electron microscopy (SEM, JSM-7001F, Tokyo, Japan), and surface compositions were determined by X-ray photoelectron energy spectroscopy.

2.5. Statistical Analysis

All data obtained in this study were analyzed using Microsoft Excel 2019 software. The experimental data shown in the figures are presented as the mean ± standard deviation to indicate their reproducibility and reliability. OriginPro 21 software was used for drawing figures.

3. Results and Discussion

3.1. Effect of Initial pH on pH and ORP in the Coal Desulfurization and Pyrite Bio-Oxidation Systems

Pyrite bio-oxidation by A. ferrooxidans would lead to the increase in the iron ion and H ⁺ concentration in solution. Therefore, changes in pH and ORP are commonly used to evaluate the processes of coal desulfurization and pyrite bio-oxidation [35,36,37]. Figure 1 shows the changes in pH and ORP during the studied reactions. With increasing reaction time, the ORPs of each system gradually increased and the pH gradually decreased. The net decreases in pH in the coal desulfurization systems were greater than those in the pyrite bio-oxidation systems at the same initial pH. At an initial pH of 1.70, 2.00, 2.30, and 2.60, these differences amounted to 0.04, 0.10, 0.22, and 0.26, respectively. The changes in the ORPs showed a similar phenomenon. At initial pH values of 1.70, 2.00, 2.30, and 2.60, the ORPs at the end of the reaction were greater for the coal desulfurization systems than for the pyrite bio-oxidation systems by 42 mV, 38 mV, 11 mV, and 20 mV, respectively.

This indicates that coal desulfurization systems react more rapidly than pyrite bio-oxidation systems in the same acidic environment. It is noteworthy that within the first day, the ORPs of the coal desulfurization systems increased more rapidly, with an increase of 89 mV or more. This may be attributed to the presence of iron oxides (such as Fe₂O₃), which can be dissolved by sulfuric acid during the pH adjustment process. The released Fe³⁺ attacks the pyrite to produce Fe²⁺ [38]. The ORP is positively correlated with the Fe³⁺/Fe²⁺ ratio; with the addition of A. ferrooxidans, Fe²⁺ is rapidly oxidized, leading to a rapid increase in the ORP of the system [37].

3.2. Effect of Initial pH on Fe and SO₄²⁻ Concentrations in the Coal Desulfurization and Pyrite Bio-Oxidation Systems

Pyrite is bio-oxidized in the presence of O₂ and A. ferrooxidans as follows [19,37]:

$$4\mathrm{Fe}{\mathrm{S}}_2+15{\mathrm{O}}_2+2{\mathrm{H}}_2\mathrm{O}\stackrel{A.\mathit{ferrooxidans}}{\to }2\mathrm{F}{\mathrm{e}}_2{\left(\mathrm{S}{\mathrm{O}}_4\right)}_3+2{\mathrm{H}}_2\mathrm{S}{\mathrm{O}}_4$$

(1)

$$\mathrm{F}{\mathrm{e}}_2(\mathrm{S}{\mathrm{O}}_4{)}_3+\mathrm{Fe}{\mathrm{S}}_2+3{\mathrm{O}}_2+2{\mathrm{H}}_2\mathrm{O}\to 3\mathrm{FeS}{\mathrm{O}}_4+2{\mathrm{H}}_2\mathrm{S}{\mathrm{O}}_4$$

(2)

$$4\mathrm{FeS}{\mathrm{O}}_4+{\mathrm{O}}_2+2{\mathrm{H}}_2\mathrm{S}{\mathrm{O}}_4\stackrel{A.\mathit{ferrooxidans}}{\to }2\mathrm{F}{\mathrm{e}}_2(\mathrm{S}{\mathrm{O}}_4{)}_3+2{\mathrm{H}}_2\mathrm{O}$$

(3)

Reactions according to Equations (1)–(3) describe the relationship between FeS₂ and liquid-phase Fe²⁺, Fe³⁺, and SO₄²⁻. In the presence of A. ferrooxidans, pyrite is dissolved to produce Fe³⁺ and H₂SO₄ [Equation (1)]. Fe³⁺ can then attack pyrite to produce Fe²⁺ and H₂SO₄ [Equation (2)]. The Fe²⁺ produced is oxidized by A. ferrooxidans in the system [Equation (3)]. It is clear that the concentrations of Fe²⁺, Fe³⁺, and SO₄²⁻ ions present in the liquid phase of the reaction system can serve as effective proxies for gauging the efficiency of the pyrite bio-oxidation process.

3.2.1. Effect of Initial pH on Total Fe, Fe³⁺, and Fe²⁺ Concentrations in the Coal Desulfurization and Pyrite Bio-Oxidation Systems

Figure 2 illustrates the variations in total Fe, Fe³⁺, and Fe²⁺ concentrations in each system. Initially, the coal desulfurization systems contain Fe³⁺, which originates primarily from the dissolution of iron oxides (such as Fe₂O₃) by sulfuric acid during the pH adjustment process.

The initial total Fe and Fe³⁺ concentrations showed an increasing trend with decreasing initial pH values. The initial total Fe concentrations in the coal desulfurization systems with an initial pH of 1.70, 2.00, 2.30, and 2.60 were 181.41, 158.15, 167.96, and 119.48 mg/L, respectively, of which Fe³⁺ accounted for 173.17, 148.51, 137.30, and 109.84 mg/L, respectively. In contrast, there was evidently no Fe³⁺ in the pyrite bio-oxidation systems at the initial time. This observation implies that the generation of Fe³⁺ within the pyrite bio-oxidation systems is primarily reliant on the biological reaction facilitated by A. ferrooxidans. With increasing reaction time, the total Fe and Fe³⁺ concentrations in the coal desulfurization and pyrite bio-oxidation systems gradually increased. At the end of the reaction, the net increases in total Fe concentration for “coal pH 1.70”, “coal pH 2.00”, “coal pH 2.30”, and “coal pH 2.60” were 432.86, 488.68, 405.45, and 369.87 mg/L, respectively, and the net increases in Fe³⁺ concentration were very close to the increases in total iron content in each system. It is evident that the concentrations of total Fe and Fe³⁺ tended to increase as the initial pH was lowered, contrary to the behavior observed in the pyrite bio-oxidation systems. The concentrations of total Fe and Fe³⁺ in the pyrite bio-oxidation systems increased as the initial pH was increased. Moreover, the concentrations of total Fe and Fe³⁺ in the pyrite bio-oxidation systems were lower than those in the coal desulfurization systems in the respective acidic environments. At the end of the reaction, the concentrations of total Fe in the treatments “pyrite pH 1.70”, “pyrite pH 2.00”, “pyrite pH 2.30”, and “pyrite pH 2.60” were 55.74, 71.19, 86.97, and 106.11 mg/L, respectively. The Fe³⁺ concentrations were 41.64, 70.52, 85.85, and 104.99 mg/L, respectively. Figure 2c depicts the variations in Fe²⁺ concentration in the pyrite bio-oxidation and coal desulfurization systems. Evidently, the Fe²⁺ concentrations in the coal desulfurization systems were higher than those in the pyrite bio-oxidation systems after the same reaction time, reflecting the higher Fe³⁺ concentration in the former, which can facilitate the production of Fe²⁺ [Equation (2)].

3.2.2. Effect of Initial pH on SO₄²⁻ Concentration in the Coal Desulfurization and Pyrite Bio-Oxidation Systems

Figure 3 shows the variations in SO₄²⁻ concentrations in the coal desulfurization and pyrite bio-oxidation systems. The initial SO₄²⁻ concentrations in the coal desulfurization and pyrite bio-oxidation systems increased with the lower initial pH. The initial SO₄²⁻ concentrations in the coal desulfurization systems with initial pH values of 1.70, 2.00, 2.30, and 2.60 were 5026.67, 3145.78, 2123.51, and 2035.41 mg/L, respectively. In corresponding acidic environments, the initial SO₄²⁻ concentrations in the pyrite bio-oxidation systems were lower than those in the coal desulfurization systems due to the absence of buffer substances. Nevertheless, the change trend matches that of the coal desulfurization systems. The initial SO₄²⁻ concentrations in the pyrite bio-oxidation systems with an initial pH of 1.70, 2.00, 2.30, and 2.60 were 2328.80, 1746.94, 847.25, and 259.21 mg/L, respectively.

With increasing reaction time, the concentration of SO₄²⁻ in each system exhibited an upward trend. During the initial stage of the reaction, the concentrations of SO₄²⁻ in the coal desulfurization systems exhibited a significant upward trend, comparable with those in the pyrite bio-oxidation systems. After 3 days, the SO₄²⁻ concentrations in the “coal pH 1.70”, “coal pH 2.00”, “coal pH 2.30”, and “coal pH 2.60” treatments increased by 2042.86, 2976.93, 2306.87, and 1383.69 mg/L, respectively, and then the rates of increase leveled off. Three reasons for the rapid increase in SO₄²⁻ concentration in the coal desulfurization system may be envisaged. (1) Although the coal and pure pyrite particle sizes were consistent, coal-derived pyrite has a smaller particle size compared to pure pyrite, offering a larger surface area for A. ferrooxidans attachment, which may improve the reaction efficiency [39]. (2) A. ferrooxidans often adsorbs onto the surface of pyrite through its secreted EPS. The introduction of acid dissolves Fe³⁺ ions from iron oxide, which may be complexed by EPS to enhance A. ferrooxidans adsorption and electron transfer, promoting pyrite dissolution [40,41]. (3) The dissolution of Fe³⁺ facilitates the oxidation of pyrite. At the end of the reaction, both the pyrite bio-oxidation and coal desulfurization systems exhibited similar trends, with the net increases in SO₄²⁻ concentration increasing with higher initial pH. The net increases in SO₄²⁻ concentration in the coal desulfurization systems were higher than those in the pyrite bio-oxidation systems in the respective acidic environments. Among the coal desulfurization systems, “coal pH 2.60” showed the highest net increase in SO₄²⁻ concentration, amounting to 4589.26 mg/L, around 1.43, 1.32, and 1.08 times those in the “coal pH 1.70”, “coal pH 2.00”, and “coal pH 2.30” treatments, respectively. Among the pyrite bio-oxidation systems, “pyrite pH 2.60” showed the highest net increase in SO₄²⁻ concentration, amounting to 596.64 mg/L, around 2.02, 1.17, and 1.05 times those of “pyrite pH 1.70”, “pyrite pH 2.00”, and “pyrite pH 2.30”, respectively.

3.3. Effect of Initial pH on Bacterial Density and EPS in the Coal Desulfurization and Pyrite Bio-Oxidation Systems

The proliferation of bacteria and the secretion of EPS have important impacts on the bio-oxidation of pyrite [42,43]. To thoroughly investigate the impact of initial pH on the coal desulfurization and pyrite bio-oxidation systems, we determined the primary constituents of the EPS (mainly existing in the forms of proteins and polysaccharides) [44] in each system at the end of the reaction, as well as the bacterial densities of each system on days 6, 12, and 18, as shown in Figure 4.

Figure 4a illustrates the changes in protein concentration in the coal desulfurization and pyrite bio-oxidation systems with different acidic environments. In the coal desulfurization system, the protein concentration in each treatment showed no significant difference, except for a significant reduction at an initial pH of 1.70. In the pyrite bio-oxidation systems, the protein concentration broadly increased with increasing initial pH. It can be seen that initial pH had a more significant effect on protein secretion in the pyrite bio-oxidation system. Polysaccharide levels in both the coal desulfurization and pyrite bio-oxidation systems increased with increasing initial pH (Figure 4b). In the coal desulfurization systems, polysaccharide concentrations at an initial pH 1.70, 2.00, 2.30, and 2.60 were 0, 12.41, 20.10, and 23.18 mg/L, respectively. For the pyrite bio-oxidation systems, polysaccharide concentrations at the corresponding pH values were 0, 2.60, 3.55, and 4.88 mg/L, respectively. Evidently, polysaccharide secretion in the coal desulfurization system exceeded that in the pyrite bio-oxidation system at the corresponding initial pH, with the exception of pH 1.70. Figure 4c,d show the changes in bacterial density in the coal desulfurization and pyrite bio-oxidation systems with different acid environments. In all of the treatments except “coal pH 1.70” and “pyrite pH 1.70”, bacterial density increased as the reaction proceeded. Thus, it can be inferred that A. ferrooxidans growth was significantly hindered at an initial pH of 1.70. In the coal desulfurization system, as the initial pH was increased, the bacterial density broadly increased. The final bacterial densities were 0.03 × 10⁶, 0.17 × 10⁶, 0.90 × 10⁶, and 1.48 × 10⁶ copies/mL for initial pH values of 1.70, 2.00, 2.30, and 2.60, respectively. The variation pattern of bacterial density in the pyrite bio-oxidation system was similar to that in the coal desulfurization system. The final bacterial densities were 0.01 × 10⁵, 0.26 × 10⁶, 2.64 × 10⁶, and 6.62 × 10⁶ copies/mL at initial pH values of 1.70, 2.00, 2.30, and 2.60, respectively. As shown in Figure 4d, the increasing trend in A. ferrooxidans density and polysaccharide secretion was consistent with the increasing trend in SO₄²⁻ concentration, which also increased when raising the initial pH. This indicates that, without interference from iron oxides, the initial pH mainly affects the dissolution process of pyrite by influencing the growth of bacteria in the system.

3.4. Analysis of Inorganic Sulfur and Pyritic Sulfur Removal in the Coal Desulfurization and Pyrite Bio-Oxidation Systems

Figure 5a,b illustrate that the inorganic sulfur (inorganic sulfur here represents the total of pyritic sulfur and sulfate sulfur in coal) removal efficiencies in the coal desulfurization and pyrite bio-oxidation systems increased with increasing initial pH. The highest inorganic sulfur removal efficiencies were observed at an initial pH of 2.60 for both the coal desulfurization and pyrite bio-oxidation systems, amounting to 88.28% and 9.25%, respectively. The reasons for the similar change laws of inorganic sulfur removal efficiency in the coal desulfurization and pyrite bio-oxidation systems are different. Inorganic sulfur removal efficiency in the coal desulfurization system is determined by a combination of the bio-oxidation of coal-derived pyrite and the replacement of CO₃²⁻ by SO₄²⁻ in coal [12]. As the initial pH is increased, SO₄²⁻ replacement in the system is suppressed, and the total sulfur removal approaches the pyritic sulfur removal efficiency (Figure 5c).

The main reason why the inorganic sulfur removal efficiency of the pyrite bio-oxidation system increases with increasing initial pH is that this promotes the growth of A. ferrooxidans, which in turn promotes the dissolution of pyrite. An increase in pH promotes the secretion of EPS from A. ferrooxidans (Figure 4), thereby improving the bio-oxidation efficiency of pyrite. However, this change also leads to a decrease in the solubility of iron compounds (Figure 5d), which in turn reduces the efficiency of indirect reactions [Equation (2)], resulting in no significant difference in the removal efficiencies of pyrite sulfur at varying initial pH values (Figure 5c).

Meanwhile, although a relatively high coal desulfurization efficiency was achieved in this study, the time required for coal desulfurization through bio-oxidation is still relatively long. Exploring the combined technology of biological flotation and bio-oxidation may be a direction for coal biological desulfurization in the future. This combination has the potential to substantially reduce desulfurization time while maintaining optimal coal desulfurization efficiency.

3.5. Effect of Initial pH on the Morphology and Chemical Composition of Bio-Oxidized Pyrite

As shown in Figure 6, the surface of original pyrite was relatively smooth, whereas after bio-oxidation it showed some erosion pits. The largest erosion pit was formed after bio-oxidation at an initial pH of 2.60. Figure 7 illustrates that with an increase in the initial pH, the ratios S⁰/FeS₂ and SO₄²⁻/FeS₂ both increase progressively. It can be seen that the biological oxidation of pyrite produces S⁰, and S⁰ is mainly generated from the chemical oxidation of pyrite by Fe³⁺ [45].

4. Conclusions

It is confirmed that pH has a significant impact on the biological desulfurization of coal and pyrite bio-oxidation. In the coal desulfurization system, higher pH levels boost bacterial growth and EPS secretion (particularly of polysaccharides), which are beneficial for the removal of sulfur from coal-derived pyrite. However, iron oxide dissolution decreases at higher pH levels, reducing the removal of sulfur from coal-derived pyrite. In the pyrite bio-oxidation systems, the release of Fe and SO₄²⁻ ions is less extensive than in the coal desulfurization systems with different acidic environments. As the initial pH is increased, the growth of systemic bacteria and the secretion of EPS are promoted, enhancing the dissolution of Fe and SO₄²⁻ ions in the system. Moreover, as the initial pH is increased, the chemical composition of the bio-oxidized pyrite shows gradual increases in the S⁰/FeS₂ and SO₄²⁻/FeS₂ ratios.