The Stress Effect and Biomineralization of High Phosphorus Concentration on Acid Mine Drainage Treatment Mediated by Acidithiobacillus ferrooxidans

Abstract

Acid mine drainage (AMD), containing large quantities of heavy metals and acidic components, poses a severe threat to the environment and human health. Acidithiobacillus ferrooxidans (A. ferrooxidans) plays a crucial role in the treatment of AMD, but its activity is significantly influenced by environmental conditions. This study systematically analyzes the stress effect of high phosphorus concentration on A. ferrooxidans during AMD treatment and its biomineralization effect. The results indicate that with phosphorus concentrations ranging from 0 g/L to 2 g/L, the system’s pH and Fe²⁺ oxidation rate initially decrease and then increase, with higher phosphorus concentrations delaying the time of increase. When the phosphorus concentration exceeds 2 g/L, both pH and Fe²⁺ oxidation rates generally show a downward trend. The morphology and elemental composition of the precipitates obtained under different phosphorus concentrations exhibit significant differences, indicating that phosphorus concentration notably affects the oxidation activity of A. ferrooxidans and its mediated biomineralization process. Under high phosphorus concentrations, the activity of A. ferrooxidans is inhibited, hindering the Fe²⁺ oxidation process and resulting in the formation of a large quantity of amorphous ferric phosphate precipitates. The findings provide a scientific basis for optimizing AMD treatment technologies, suggesting that reasonable control of phosphorus concentration in practical applications can improve AMD treatment efficiency and pretreatment effects.

1. Introduction

Acid mine drainage (AMD) is one of the most destructive and challenging environmental issues associated with mining activities. AMD contains large amounts of SO₄²⁻, Fe, and other heavy (and semi-metal) elements [1,2], which not only severely threaten the ecological environment but also damage land and surface water resources, posing potential hazards to human health [3,4]. Therefore, seeking efficient, economical, and environmentally friendly AMD treatment methods is a significant research direction in the field of environmental science.

Currently, AMD treatment methods mainly include chemical neutralization, sulfide precipitation, constructed wetlands, adsorption, and microbial methods. Chemical neutralization involves adding alkaline substances to neutralize acidic wastewater, but this is costly and may lead to secondary pollution [1,2,5]. Sulfide precipitation effectively removes heavy metals but is complex to operate and requires strict control of reaction conditions [6]. Constructed wetlands use the synergistic effects of plants and microorganisms to naturally remove pollutants; they are low-cost and easy to maintain but have treatment efficiency affected by seasonal and environmental changes [7]. Adsorption uses porous materials to adsorb heavy metals; it is simple to operate but has limited adsorption capacity, requiring periodic replacement of adsorbents [8]. The fixed sulfate-reducing bacteria method can achieve sulfate conversion and biological recovery of metals from acidic metal-containing water, but the initial biomass of sulfate-reducing bacteria is very low, with long start-up times and high environmental requirements [9]. In summary, each method has its advantages and disadvantages, often necessitating the combination of multiple technologies to enhance treatment efficiency and reduce costs. Thus, exploring environmentally friendly and efficient AMD treatment methods is of great significance.

Acidithiobacillus ferrooxidans (A. ferrooxidans) is a Gram-negative, chemolithoautotrophic, acidophilic aerobic bacterium capable of oxidizing Fe²⁺ and reducing sulfides, producing iron oxides and sulfuric acid, playing a key role in AMD bioremediation. The optimum growth temperature and pH are 28–30 °C and 2.5, respectively [10]. A. ferrooxidans can tolerate organic compounds and metal ions within a certain concentration, which is one of the main reasons for its survival in extreme environments such as metal mines, coal mines, and sludge in sewage treatment plant. In recent years, A. ferrooxidans has become a research hotspot for AMD pretreatment due to its high efficiency in oxidizing iron sulfides. Under the mediation of A. ferrooxidans, it utilizes CO₂ as the only carbon source; Fe²⁺ in AMD is oxidized to Fe³⁺, which is hydrolyzed to form secondary iron minerals such as jarosite and schwertmannite [11,12]. Schwertmannite has strong adsorption and co-precipitation effects on Mn, Cr, Cu, and As [3], effectively removing soluble iron and SO₄²⁻ from AMD [4]. As a microorganism, the biomineralization process of A. ferrooxidans is significantly affected by environmental factors, including pH, temperature, CO₂ concentration, nutrient elements, induction of added minerals, and the types and concentrations of anions and cations [13,14,15,16].

Phosphorus is an essential nutrient element and a necessary component of nucleic acids, phospholipids, and ATP in cells, participating in the formation of ATP and ADP. It plays an important role in energy accumulation and conversion, as well as physiological metabolism. During the metabolism of A. ferrooxidans, an appropriate amount of phosphorus can significantly enhance its Fe²⁺ oxidation ability and biomineralization efficiency [17]. However, when phosphorus concentration is too high, it stresses the iron redox reactions, altering the metabolic activities and treatment effects of A. ferrooxidans, thereby reducing the efficiency of AMD treatment [18]. Furthermore, excess phosphorus may stimulate the formation of secondary minerals, affecting subsequent treatment outcomes [19]. However, systematic research on the specific roles and stress effects of phosphorus in A. ferrooxidans-mediated AMD treatment is still lacking.

This study aims to systematically analyze the effect of phosphorus concentration on A. ferrooxidans during AMD treatment. Using a modified phosphorus-free 9K liquid medium under batch conditions, the study investigates the influence of phosphorus concentration on the formation of biogenic secondary iron minerals mediated by A. ferrooxidans. The study examines the pH, Fe²⁺ oxidation rate, total iron (TFe) precipitation rate, composition, and mineral phase of the precipitates in the reaction system to elucidate the following: (1) the effect of phosphorus concentration on A. ferrooxidans in AMD treatment; (2) the impact of phosphorus concentration on the metal removal efficiency and precipitate products mediated by A. ferrooxidans, revealing the role of phosphorus in A. ferrooxidans-mediated AMD treatment. This research provides a scientific basis for optimizing AMD biotreatment technologies.

2. Materials and Methods

2.1. Test Materials

Preparation of Modified Phosphorus-Free 9K Liquid Medium: The medium was prepared by dissolving (NH₄)₂SO₄ 3.0 g, K₂SO₄ 0.50 g (substituted for K₂HPO₄), MgSO₄·7H₂O 0.50 g, KCl 0.10 g, and Ca(NO₃)₂·4H₂O 0.01 g in 1 L of deionized water, and adjusting the pH to 2.5 with 1:1 H₂SO₄. The phosphorus stock solution was prepared using NaH₂PO₄·2H₂O, with a phosphorus concentration of 50 g/L. Strain: The strain used in the experiments was A. ferrooxidans (ATCC 23270), collected from an acid mine pit. Resting Cells of A. ferrooxidans: A. ferrooxidans was inoculated into the 9K medium at 10% (v/v) and incubated on a shaker at 28 °C and 180 r/min for expansion. The cultivation was stopped in the late exponential growth phase (approximately 2–3 days), and the resulting secondary minerals were removed by filtration using qualitative filter paper. The filtrate was centrifuged at 10,000× g (4 °C, 10 min) to collect the cells, which were then washed three times with pH 1.5 acid (prepared with H₂SO₄) to remove ions. The cells from the 250 mL culture system were resuspended in 5 mL of pH 2.5 acid (prepared with H₂SO₄), concentrated 50 times to obtain A. ferrooxidans concentrated cell suspension, with a cell density of approximately 10⁹ cells/mL [20].

2.2. Experimental Methods

Seven treatments were set according to phosphorus concentration, with nutrients provided by the modified phosphorus-free 9K liquid medium. The phosphorus concentrations were set to 0, 0.5, 1, 2, 3, 4, and 5 g/L, labeled as “P-0 g/L,” “P-0.5 g/L,” “P-1 g/L,” “P-2 g/L,” “P-3 g/L,” “P-4 g/L,” and “P-5 g/L,” respectively. The substrate was FeSO₄·7H₂O, with a Fe²⁺ concentration of 160 mmol/L. Each treatment was inoculated with 1 mL of concentrated cell suspension, and the total reaction volume was 250 mL. The system pH was adjusted to 2.50 ± 0.02 with 1:1 H₂SO₄. Each treatment was set up with three replicates, and the cultures were shaken at 180 r/min and 28 °C. During the cultivation, the solution pH was periodically monitored, and 1 mL liquid samples were filtered (0.22 μm filter membrane) to analyze changes in Fe²⁺ and TFe. Sampling times were 12 h, 24 h, 36 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h. At the end of cultivation, the precipitates were filtered using neutral qualitative filter paper, washed three times with pH 1.5 deionized water (prepared with 1:1 H₂SO₄), and then washed three times with deionized water. The collected minerals were dried at 60 °C to constant weight, weighed, and stored in a desiccator containing anhydrous silica gel for future use.

2.3. Measurement Methods and Data Analysis

pH: The pH of the solution was measured using a pHS-3C pH meter (Shanghai LeiCi Factory, Shanghai, China). Fe²⁺ Concentration: Measured using the o-phenanthroline colorimetric method. TFe: Fe³⁺ was reduced to Fe²⁺ using hydroxylamine hydrochloride, followed by measurement using the o-phenanthroline colorimetric method. Calculation Methods: Fe²⁺ oxidation rate and TFe precipitation rate were calculated using standard methods.

The TFe precipitation rate was determined as follows:

$$\mathrm{T}\mathrm{F}\mathrm{e}\left(\%\right)=\left({\mathrm{T}\mathrm{F}\mathrm{e}}_{\mathrm{i}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}}-{\mathrm{T}\mathrm{F}\mathrm{e}}_{\mathrm{t}}\right)/{\mathrm{T}\mathrm{F}\mathrm{e}}_{\mathrm{i}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}}\times 100,$$

where ${\mathrm{T}\mathrm{F}\mathrm{e}}_{\mathrm{i}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}}$ represents the initial iron concentration and ${\mathrm{T}\mathrm{F}\mathrm{e}}_{\mathrm{t}}$ is the iron concentration at t (hours) of reaction time.

The oxidation rate of Fe²⁺ was calculated as follows:

$${\mathrm{F}\mathrm{e}}^{2+}\left(\mathrm{\%}\right)=\left({\mathrm{F}\mathrm{e}}_{\mathrm{i}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}}^{2+}-{\mathrm{F}\mathrm{e}}_{\mathrm{t}}^{2+}\right)/{\mathrm{F}\mathrm{e}}_{\mathrm{i}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}}^{2+}\times 100,$$

where ${\mathrm{F}\mathrm{e}}_{\mathrm{i}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}}^{2+}$ denotes the initial Fe²⁺ concentration and ${\mathrm{F}\mathrm{e}}_{\mathrm{t}}^{2+}$ is the Fe²⁺ concentration at t (hours) of reaction time.

In the same experiment, three sets of parallel samples were utilized to obtain error bars.

XRD: X-ray diffraction analysis was performed using an X’Pert 3 Powder diffractometer (PANalytical, The Netherlands) under the following conditions: tube voltage 50 kV, tube current 150 mA, scanning range 10–80° (2θ), step size 0.02°, scanning speed 5°/min, with a Cu target (curved crystal monochromator). FTIR: Fourier transform infrared spectroscopy was conducted using a Frontier FT-IR spectrometer (PerkinElmer, Shanghai, China). SEM: Scanning electron microscopy was carried out using an S-4800 microscope (Hitachi, Japan) with an accelerating voltage of 5.0 kV.

The mean and standard deviation of each data point were calculated using Microsoft Excel^® 2019. All figures were generated using Origin^® 9.0 software.

3. Results

3.1. Effect of Phosphorus on pH Changes during Acid Mine Drainage Treatment

The presence of phosphorus affects pH changes during AMD treatment, as indicated in Figure 1.

As shown in Figure 1, based on the pH changes under different phosphorus concentrations, the reaction system can be divided into three groups: 0 g/L, 0.5–2 g/L, and above 2 g/L. At a phosphorus concentration of 0 g/L, the system pH initially rises and then falls. The initial rise is due to the oxidation of Fe²⁺ by A. ferrooxidans, which consumes H⁺, leading to an increase in pH. Subsequently, Fe³⁺ is hydrolyzed into minerals, generating H⁺ and causing the pH to decrease [21].

At phosphorus concentrations of 0.5 g/L to 2 g/L, the pH value initially decreases and then rapidly increases. The higher the phosphorus concentration, the later the increase in pH. At phosphorus concentrations of 0 g/L, 0.5 g/L, 1.0 g/L, and 2.0 g/L, the pH starts to increase at 0 h, 30 h, 92 h, and 119 h, respectively. This is because the added phosphorus source, NaH₂PO₄·2H₂O, is acidic, causing an initial significant drop in pH. As the reaction progresses, A. ferrooxidans begins to consume H⁺, but NaH₂PO₄·2H₂O has a buffering capacity, which increases with concentration. Therefore, with phosphorus concentrations of 0.5 g/L to 2 g/L, the pH increase is significantly delayed.

At phosphorus concentrations above 2 g/L, the system pH generally shows a downward trend, and the rate of decline is slightly higher than that in systems with phosphorus concentrations below 2 g/L. This may be due to the increased acidity of the reaction system with higher phosphorus concentrations, affecting the reaction between Fe²⁺, OH⁻, and PO₄³⁻, forming precipitates and reducing the Fe²⁺ available for oxidation, thus decreasing the H⁺ consumption capacity. Figure 3 also supports this point. Additionally, the increased Na⁺ concentration (from NaH₂PO₄·2H₂O) might change the osmotic pressure between the solution system and bacterial cells, affecting the Fe²⁺ oxidation process [22], inhibiting A. ferrooxidans activity, and preventing effective bio-oxidation, leading to a continuous pH decrease.

3.2. Effect of Phosphorus on Fe²⁺ Oxidation Rate during Acid Mine Drainage Treatment

The presence of phosphorus affects Fe²⁺ oxidation rate changes during AMD treatment, as indicated in Figure 2.

As shown in Figure 2, based on the changes in Fe²⁺ oxidation rate under different phosphorus concentrations, the system can be divided into three groups: 0 g/L, 0.5–2 g/L, and above 2 g/L.

At a phosphorus concentration of 0 g/L, the Fe²⁺ oxidation rate rises rapidly, reaching nearly 100% within 24 h. At phosphorus concentrations of 0.5 g/L to 2 g/L, the Fe²⁺ oxidation rate initially rises and then levels off. The higher the phosphorus concentration, the slower the oxidation rate increase. At phosphorus concentrations of 0.5 g/L, 1 g/L, and 2 g/L, the Fe²⁺ oxidation rate reaches nearly 100% at 72 h, 113 h, and 161 h, respectively. This is because the added phosphorus source, NaH₂PO₄·2H₂O, is acidic, inhibiting A. ferrooxidans activity. As the reaction progresses, A. ferrooxidans gradually adapts to the environment and begins to oxidize Fe²⁺. The oxidation rate curve changes align with the pH curve changes in Figure 1, confirming the impact of the acidic environment on A. ferrooxidans.

At phosphorus concentrations above 2 g/L, the Fe²⁺ oxidation rate is generally lower and increases slowly. At phosphorus concentrations of 3 g/L, 4 g/L, and 5 g/L, the Fe²⁺ oxidation rate reaches approximately 51%, 42%, and 48% after 168 h, respectively. This is due to the inhibition or inactivation of A. ferrooxidans at high phosphorus concentrations, affecting the Fe²⁺ oxidation rate [22]. Compared to existing studies [23,24], the inhibition observed in this study is more pronounced, possibly due to differences in phosphorus sources. When the phosphorus source is potassium phosphate, A. ferrooxidans is completely inhibited at PO₄³⁻ concentrations ≥500 mmol/L. However, in this study, A. ferrooxidans becomes gradually inactivated at phosphorus concentrations above 0.5 mg/L, possibly due to the influence of cations. Generally, K⁺ significantly enhances the Fe²⁺ oxidation process by A. ferrooxidans, while the Na⁺ used in this study does not have this function, affecting A. ferrooxidans’s tolerance to phosphorus.

3.3. Effect of Phosphorus on TFe Precipitation Rate and Total Precipitation in Acid Mine Drainage Treatment

The presence of phosphorus affects TFe precipitation rate and total precipitate mass changes during AMD treatment, as indicated in Figure 3.

As shown in Figure 3, based on the changes in TFe precipitation rate and total precipitate mass under different phosphorus concentrations, the system can be divided into three groups: 0 g/L, 0.5–2 g/L, and above 2 g/L.

At a phosphorus concentration of 0 g/L, the TFe precipitation rate rises rapidly within 48 h, reaching approximately 37%, and then gradually stabilizes (Figure 3a). The total precipitate mass is 1.87 g (Figure 3b). This indicates that under phosphorus-free conditions, when Fe²⁺ is completely oxidized by A. ferrooxidans, iron can efficiently precipitate and form stable mineral phases.

At phosphorus concentrations of 0.5 g/L to 2 g/L, the TFe precipitation rate shows a significant downward trend, and the decline rate slows with increasing phosphorus concentration. At phosphorus concentrations of 0.5 g/L to 2 g/L, the TFe precipitation rate reaches approximately 24.9%, 17.7%, and 22.4% at 168 h, respectively, when Fe²⁺ is completely oxidized by A. ferrooxidans (Figure 3a). The total precipitate mass is 1.29 g, 1.35 g, and 1.43 g, respectively (Figure 3b). The total precipitate mass does not correspond to the precipitation rate, which is due to the increased addition of NaH₂PO₄·2H₂O, causing Fe³⁺ to react with excess PO₄³⁻ and OH⁻, forming precipitates and leading to an increase in total precipitate mass with increasing phosphorus concentration [21].

At phosphorus concentrations above 2 g/L, the TFe precipitation rate is generally higher and rises rapidly. At phosphorus concentrations of 3 g/L, 4 g/L, and 5 g/L, the TFe precipitation rate reaches approximately 47.1%, 44.1%, and 47.4% after 168 h, respectively (Figure 3a). The total precipitate mass significantly increases to 3.31 g, 3.05 g, and 3.37 g, respectively (Figure 3b). High phosphorus concentrations significantly enhance iron precipitation efficiency but also lead to changes in precipitate structure and composition (

Table 1. Elemental analysis of the precipitate obtained under different phosphorus concentration conditions.

Group	Percentage of Each Element
	K (wt%)	N (wt%)	Na (wt%)	P (wt%)	Fe (wt%)	SO42− (wt%)
0 g/L phosphorus concentration	0.66	3.83	0.13	0.00	15.33	10.19
0.5 g/L phosphorus concentration	0.61	1.30	0.38	3.47	17.65	7.92
1 g/L phosphorus concentration	0.50	1.73	0.70	4.99	14.35	7.18
2 g/L phosphorus concentration	0.05	1.23	0.46	14.57	13.41	0.48
3 g/L phosphorus concentration	0.11	1.13	0.30	12.94	13.69	1.27
4 g/L phosphorus concentration	0.07	1.00	0.27	14.12	12.49	0.34
5 g/L phosphorus concentration	0.06	1.01	0.28	14.26	12.53	0.39
KFe3(SO4)2(OH)6	7.80				33.50	38.30
NH4Fe3(SO4)2(OH)6		2.92			35.00	40.00
H3OFe3(SO4)2(OH)6					34.80	40.00
NaFe3(SO4)2(OH)6			4.38		28.28	12.87

, Figure 5). This is because under high phosphorus concentrations, the increased acidity from NaH₂PO₄·2H₂O further strengthens the chemical reactions between unoxidized Fe²⁺, Fe³⁺, and excess PO₄³⁻ and OH⁻, resulting in a continuous increase in TFe precipitation rate and total precipitate mass with increasing phosphorus concentration [18].

3.4. XRD Analysis of Precipitates Obtained under Different Phosphorus Concentrations

The presence of phosphorus affects the XRD spectra of the precipitate changes during AMD treatment, as indicated in Figure 4.

As shown in Figure 4, based on the XRD spectra of the precipitates obtained under different phosphorus concentrations, the system can be divided into two groups: 0 g/L to 1 g/L and above 1 g/L.

At phosphorus concentrations of 0 g/L to 1 g/L, the XRD spectra of the precipitates show distinct crystalline peaks, indicating that A. ferrooxidans can effectively catalyze the oxidation of Fe²⁺ at low phosphorus concentrations, resulting in the formation of highly crystalline mineral phases. These mineral phase characteristic peaks are very similar to the XRD spectra of jarosite (KFe₃(SO₄)₂(OH)₆), ammoniojarosite (NH₄Fe₃(SO₄)₂(OH)₆), natrojarosite (NaFe₃(SO₄)₂(OH)₆), and carphosiderite (H₃OFe₃(SO₄)₂(OH)₆).

At phosphorus concentrations above 1 g/L, the XRD spectra of the precipitates show very weak or no obvious crystalline peaks. This indicates that as the phosphorus concentration increases, the Fe²⁺ oxidation process is significantly affected, leading to insufficient Fe³⁺ supply and hindered Fe³⁺ hydrolysis, resulting in the formation of poorly crystalline mineral phases. The precipitates at this stage are likely composed of amorphous minerals such as Fe₃(PO₄)₂, Fe(OH)₃, amorphous iron phosphate (AIP), amorphous ferric hydroxide (AFH), and amorphous iron sulfate (AIS) [25,26,27].

3.5. FTIR Analysis of Precipitates Obtained under Different Phosphorus Concentrations

The presence of phosphorus affects the FTIR spectra of the precipitate changes during AMD treatment, as indicated by Figure 5.

As shown in Figure 5, based on the FTIR spectra of the precipitates obtained under different phosphorus concentrations, the system can be divided into two groups: 0 g/L to 1 g/L and above 1 g/L.

At phosphorus concentrations of 0 g/L to 1 g/L, the FTIR spectra of the precipitates show -OH stretching vibration absorption peaks at 3430–3410 cm⁻¹, H-O-H deformation vibration absorption peaks at 1400–1640 cm⁻¹, and SO₄²⁻ group vibration peaks at 1005–1125 cm⁻¹. This indicates that the product FeOOH has a certain adsorption capacity for SO₄²⁻ [28]. Peaks at 1082 cm⁻¹ and 627 cm⁻¹ correspond to SO₄²⁻ stretching vibrations, 1002 cm⁻¹ corresponds to -OH deformation vibrations, and at around 508 cm⁻¹ appears the FeO₆ octahedral vibration peak, indicating the presence of jarosite in the precipitates [29].

At phosphorus concentrations above 1 g/L, the FTIR spectra of the precipitates show P-O vibration peaks around 1050 cm⁻¹ and Fe-O-P stretching vibration peaks around 550 cm⁻¹, suggesting that the precipitates obtained under these conditions are mainly composed of iron phosphate.

Therefore, under phosphorus concentrations of 0 g/L to 1 g/L, the precipitates are composed of jarosite and other jarosites, while at phosphorus concentrations above 1 g/L, the precipitates are mainly composed of amorphous iron phosphate.

3.6. SEM-EDS Analysis of Precipitates Obtained under Different Phosphorus Concentrations

The presence of phosphorus affects the morphology and elemental composition of the precipitate changes during AMD treatment, as indicated in Figure 6 and Table 1.

As shown in Figure 6 and Table 1, the morphology and elemental composition of the precipitates obtained under different phosphorus concentrations show significant differences. With increasing phosphorus concentration, the morphology of the precipitates changes noticeably. At a phosphorus concentration of 0 g/L (Figure 6A), the precipitates exhibit more regular crystalline morphology. At phosphorus concentrations of 0.5 g/L (Figure 6B) and 1 g/L (Figure 6C), the precipitates gradually become loose and granular. At phosphorus concentrations above 2 g/L (Figure 6D–G), the introduction of phosphate significantly alters the microstructure of the precipitates, resulting in agglomeration and condensation phenomena, forming irregular particle shapes and presenting as amorphous granular structures. These changes indicate that with increasing phosphorus concentration, the oxidative capacity of A. ferrooxidans is inhibited, causing the formed mineral phases to gradually transition from highly crystalline to amorphous structures.

The EDS elemental analysis results (Table 1) further confirm this viewpoint. With increasing phosphorus concentration, the phosphorus (P) content in the precipitates increases significantly, while the iron (Fe) content gradually decreases. At a phosphorus concentration of 0 g/L, the precipitates are primarily iron oxides with almost no phosphorus content. At phosphorus concentrations of 0.5 g/L to 2 g/L, the phosphorus content gradually increases, and the iron content gradually decreases, indicating the increasing formation of iron phosphate precipitates. At phosphorus concentrations of 3 g/L to 5 g/L, the phosphorus content increases significantly, and the iron content decreases significantly, indicating the primary formation of iron phosphate precipitates.

Additionally, at phosphorus concentrations of 0 g/L to 1 g/L, the precipitates contain lower amounts of K, Na, and P, and higher amounts of Fe, indicating that the composition mainly includes jarosite and other jarosites (Figure 4 and Figure 5). At phosphorus concentrations above 1 g/L, the phosphorus content gradually increases, and the iron content gradually decreases, indicating the increasing formation of iron phosphate precipitates, consistent with the patterns observed in Figure 4, Figure 5 and Figure 6.

4. Discussion

The present study systematically investigated the impact of varying phosphorus concentrations on the activity of Acidithiobacillus ferrooxidans (A. ferrooxidans) and the subsequent biomineralization processes during acid mine drainage (AMD) treatment. The findings reveal crucial insights into optimizing phosphorus management to enhance AMD treatment efficiency and address both immediate and long-term environmental impacts.

The experimental results indicate that phosphorus concentration exerts a significant influence on the pH and Fe²⁺ oxidation rate during AMD treatment. At phosphorus concentrations between 0 g/L and 2 g/L, the pH and Fe²⁺ oxidation rates initially decrease and then increase. Higher phosphorus concentrations delay the recovery time, suggesting that A. ferrooxidans initially struggles to adapt to increased phosphorus levels before eventually resuming activity. However, when phosphorus concentrations exceed 2 g/L, both pH and Fe²⁺ oxidation rates generally decline, indicating substantial inhibition of A. ferrooxidans activity. This inhibition is likely due to the increased acidity and osmotic stress imposed by high phosphorus levels, which hinder the bio-oxidation processes essential for AMD remediation. Phosphorus concentration significantly affects the formation and composition of secondary iron minerals. At low phosphorus concentrations (0 g/L to 1 g/L), highly crystalline mineral phases such as jarosite and schwertmannite are formed. Schwertmannite, in particular, has strong adsorption and co-precipitation effects on heavy metals such as Mn, Cr, Cu, and As [3]. These properties make schwertmannite an effective mineral for reducing the mobility and environmental impact of heavy metals in AMD. The presence of these crystalline minerals indicates efficient Fe²⁺ oxidation and Fe³⁺ hydrolysis, contributing to the overall effectiveness of AMD treatment. Conversely, at high phosphorus concentrations (above 1 g/L), amorphous iron phosphate precipitates dominate. These amorphous structures are less effective in immobilizing heavy metals compared to their crystalline counterparts. The shift from crystalline to amorphous phases under high phosphorus conditions suggests reduced efficiency in both Fe²⁺ oxidation and the subsequent hydrolysis and precipitation processes, leading to less effective AMD treatment and potential challenges for subsequent remediation efforts.

The formation of amorphous iron phosphate precipitates under high phosphorus conditions could impair the stability and long-term immobilization of heavy metals. This could result in increased mobility of contaminants over time, posing a risk to downstream ecosystems and water quality. Furthermore, the reduced formation of schwertmannite under high phosphorus conditions diminishes the potential for co-precipitation of heavy metals, further compromising the effectiveness of AMD treatment.

The findings underscore the importance of optimizing phosphorus levels in AMD treatment. Managing phosphorus concentration to stay below 0.5 g/L can enhance A. ferrooxidans activity, promote the formation of beneficial crystalline minerals such as jarosite and schwertmannite, and improve overall treatment efficiency. Beyond this range, the inhibitory effects on A. ferrooxidans and the formation of less effective amorphous precipitates could compromise the long-term sustainability of AMD remediation efforts. Future research should focus on the detailed mechanisms underlying the inhibitory effects of high phosphorus concentrations on A. ferrooxidans. In conclusion, by maintaining appropriate phosphorus levels, it is possible to improve the efficiency of AMD treatment, reduce environmental risks, and ensure the sustainability of remediation efforts. The formation and effectiveness of secondary minerals, particularly schwertmannite, play a pivotal role in the successful treatment of AMD and the long-term immobilization of heavy metals.

5. Conclusions

This experiment focused on studying the effects of different phosphorus concentrations on the activity of Acidithiobacillus ferrooxidans and the formation of secondary iron minerals. Through shake flask experiments, the process of A. ferrooxidans-mediated secondary iron mineral formation in an iron-rich sulfate environment was simulated, and the impact of high phosphorus concentration on the A. ferrooxidans-mediated acid mine drainage treatment process was analyzed. The conclusions are as follows:

At phosphorus concentrations ranging from 0 g/L to 2 g/L, the pH and Fe²⁺ oxidation rates initially decreased and then increased, with higher phosphorus concentrations causing a delayed increase.

Above 2 g/L, both the pH and Fe²⁺ oxidation rates generally showed a downward trend, indicating significant inhibition of A. ferrooxidans activity by high phosphorus concentrations.

At 0 g/L phosphorus concentration, the TFe precipitation rate and total precipitate mass were the highest. Between 0.5 g/L and 2 g/L phosphorus concentrations, the TFe precipitation rate decreased while the total precipitate mass increased with increasing phosphorus concentration. Above 2 g/L phosphorus concentration, both the TFe precipitation rate and total precipitate mass significantly increased due to the formation of a large quantity of amorphous iron phosphate precipitates.

Low phosphorus concentrations (0–1 g/L) resulted in highly crystalline mineral phases such as jarosite and other jarosites being formed, whereas high phosphorous concentrations (above 1 g/L) led to the formation of amorphous iron phosphate precipitates, which significantly affected mineral phase crystallinity.

The removal of excess phosphorus is necessary prior to pretreating AMD rich in Fe²⁺ with A. ferrooxidans.

The results of this study may help provide insight to the activity of Acidithiobacillus ferrooxidans and the formation of secondary iron minerals, and provide optimized conditions and theoretical guidance for the treatment of AMD.