Assessment of the induced effect of selected iron hydroxysulfates biosynthesized using Acidithiobacillus ferrooxidans for biomineralization of acid mine drainage

Many sulfide mineral tailings are produced during the development and utilization of mineral resources (Huang et al. 2020; Hu et al. 2021). The accumulated tailings are exposed and react with oxygen and water in the air under the action of microorganisms, producing a large amount of acid mine drainage (AMD) rich in Fe²⁺, Fe³⁺, ⁠, and other heavy metals (such as Cu, Cr, and Cd; Akcil & Koldas 2006; Song et al. 2022). Moreover, a significant amount of H⁺ is produced in the formation of AMD, and the pH of the drainage is generally approximately 2–4 (Tabelin et al. 2018, 2020). Acid and heavy metals are the main pollutants in AMD, which cause great harm to the environment and human health (Kefeni et al. 2017). Neutralization is currently the most common method of treating AMD (Wang et al. 2019), and engineering utilization accounts for more than 90% of its application (Herrera et al. 2007). By adding an alkaline neutralizer to AMD, heavy metals could react with OH⁻ to form hydroxide precipitation under alkaline conditions, which could be removed by filtering.

Additionally, the pH of the drainage is increased at the same time to achieve the purpose of AMD treatment. Currently, lime or limestones are the most commonly used neutralizers. Neutralization can treat AMD of any concentration and nature, and it has the advantages of mature technology, simple operation, excellent adaptability, and good treatment effect (Markovic et al. 2020). However, a large amount of toxic waste residue, consisting of CaSO₄, Fe(OH)₃, and toxic metal oxides, is produced in this method, which will easily cause secondary pollution to the environment if not treated properly. Therefore, finding a new environmentally friendly AMD treatment method is of great significance.

Acidithiobacillus ferrooxidans (A. ferrooxidans) is a chemoautotrophic bacterium that widely exists in AMD. It mainly obtains energy by oxidizing Fe²⁺, thereby fixing the carbon source in the air to promote its own growth without other energy sources (Raquel et al. 2007). In recent years, many studies have shown that the content of heavy metals in AMD is significantly lower than the theoretical value during the discharge process, and it is also accompanied by the natural passivation of arsenic (Gagliano et al. 2003; Regenspurg & Peiffer 2005). That is because Fe³⁺ biooxidized by Fe²⁺ under the action of A. ferrooxidans is further hydrolyzed to form secondary iron hydroxysulfate minerals (Egal et al. 2009). As a result, Fe²⁺, Fe³⁺, H⁺, and were consumed in the process, and the content of heavy metals in AMD was greatly reduced by the adsorption and coprecipitation of secondary iron minerals. In neutralization, as Fe²⁺ needs to be precipitated at a high pH, it usually needs to be converted to Fe³⁺ by preparation before adding lime. Using the bio-oxidation of A. ferrooxidans, Fe²⁺ can be rapidly and completely oxidized, which reduces the processing time. Moreover, most metal and sulfate ions in the solution were removed via hydrolysis precipitation, which greatly reduced the amount of lime added in the subsequent neutralization treatment; therefore, only a small amount of calcium sulfate needs secondary treatment. Furthermore, the secondary iron minerals produced in the process can be used as environmental adsorption materials after collection (Xiong et al. 2008; Song et al. 2014). Due to the advantages of prebiomineralization by A. ferrooxidans, many scholars have investigated how to consciously regulate and strengthen the biomineralization process and apply it to water treatment in recent years (Zhou 2017; Naidu et al. 2019; Wang et al. 2019, 2021; Song et al. 2022). For instance, Zhou (2017) develops a novel passive biomineralization–limestone ditch treatment system through several oxidation–reduction cycles via A. ferrooxidans and Acidiphilic iron-reducing bacteria. Song et al. (2022) purposed an approach for treating AMD using a cyclic bio-oxidation and electroreduction process.

Schwertmannite and jarosite have excellent adsorption and coprecipitation effects on heavy metals and metalloids, and developing them as a new type of environmental mineralogy materials for AMD treatment is of great environmental significance and application value. Promoting the mineralization ability of various ions is a new direction for the treatment of AMD (Bao et al. 2018). However, there remain many defects in the biosynthesis of secondary iron minerals. For example, the yield of schwertmannite synthesis is low; hence, the removal effect of ions and in solution is not ideal. This research focuses on obtaining a large amount of secondary iron minerals in a short time to effectively remove metal ions and in the solution. In recent years, some scholars have proposed adding crystal seeds in a simulated AMD environment to stimulate mineralization through seed induction and have conducted a series of experiments. For example, Wang & Zhou (2011) reported the removal of soluble ferrous iron in AMD through the formation of biogenic iron oxysulfate precipitates facilitated by diatomite and quartz sand. However, the promoting effect of those two kinds of exogenous seeds is relatively slow, and the obtained products are often mixed with impurities, which are difficult to separate. Therefore, schwertmannite and jarosite were selected as exogenous seeds to study the effect of their addition on the formation of sulfate iron minerals in AMD environments. Wang et al. used jarosite as a seed crystal for the first time and studied the effect of its addition on jarosite formation in an AMD environment (Wang et al. 2013). The results show that jarosite can effectively promote the formation of sulfate iron minerals through seed crystal stimulation. However, there is currently no report on the effect of schwertmannite as a seed crystal on mineral formation. The difference in the effect of the two minerals as seed crystals to induce mineralization is unclear, and their mechanism of action is unknown.

Therefore, this study intends to add endogenous crystal seeds, schwertmannite and jarosite, to a simulated AMD environment under optimized conditions according to the previous research results and to examine the effects of their addition amount and addition method on Fe²⁺ oxidation and Fe³⁺ mineralization in the biosynthesis of secondary iron hydroxysulfate minerals. Moreover, this study aims to analyze the influence mechanism and provide optimized conditions and theoretical guidance for iron and removal in AMD.

A. ferrooxidans bacteria were obtained from China General Microbiological Culture Collection Center (CGMCC) and the accession no. was CGMCC No. 0727, which were cultured in a 9K medium developed by Silverman & Lundgren (1959) and refrigerated at 4 °C. The components (analytically pure) of the medium were as follows: 1-L deionized water, 3.00 g of (NH₄)₂SO₄, 0.10 g of KCl, 0.50 g of K₂HPO₄, 0.50 g of MgSO₄·7H₂O, and 0.01 g of Ca(NO₃)₂. Before the experiment, to prevent the A. ferrooxidans cells from being inactive due to prolonged refrigeration, they were resuscitated first, and the culture was expanded to the amount required for the experiment (Liao et al. 2009). Briefly, in a series of 250 mL Erlenmeyer flasks, 15 mL of the refrigerated bacterial suspensions was inoculated into a 9K liquid medium, keeping the total reaction volume at 150 mL. Furthermore, 6.63 g FeSO₄·7H₂O was added as an energy substance. After shaking it to dissolve, we adjusted the system's pH to 2.50 with 1:1 H₂SO₄. The Erlenmeyer flasks were then incubated in a shaker at 28 °C and 180 rpm until the late logarithmic growth phase was reached (approximately 72 h). Finally, the cultured suspensions were filtered through a Whatman No. 4 filter paper to remove the iron hydroxysulfate mineral precipitates formed during the cultivation process. The filtrates were centrifuged at 10,000 × g for 10 min to precipitate bacterial cells, which were then washed three times with acidic deionized water (pH 2.00) and pure water to remove various heavy metal ions. Next, these cells were suspended with H₂SO₄ (pH 2.50), and finally, A. ferrooxidans resting cell suspensions were obtained.

Biosynthesis of jarosite was performed by oxidizing FeSO₄ with A. ferrooxidans cells in the presence of K⁺, as reported by Bai et al. (2012). The most suitable conditions for the formation of jarosite by biomineralization were adopted for the synthesis, including the initial pH, temperature, inoculation amount of bacteria, initial Fe²⁺ concentration, and Fe/K molar ratio (Bai & Zhou 2011a, 2011b; Song et al. 2018). Briefly, in a series of 500 mL Erlenmeyer flasks, 30 mL of A. ferrooxidans cell suspensions was introduced to a solution containing 13.26 g FeSO₄·7H₂O and 1.38 g of K₂SO₄ with a Fe/K molar ratio of 3:1. Then, deionized water was added to keep the total reaction volume at 300 mL. The system's pH was adjusted to 2.50 with 1:1 H₂SO₄. The above flasks containing 10% (v/v) inoculum and FeSO₄ were subsequently incubated in a shaker at 28 °C and 180 rpm. After reaction for 96 h, the yellow minerals formed in the systems were harvested by filtering through a Whatman No. 4 filter paper. We washed them three times with acidic deionized water (pH 2.00) and pure water to remove soluble impurities. Finally, we dried them at 50 °C to constant weight and stored them in a desiccator for later use. A certain amount of minerals was identified using a scanning electron microscope (SEM) and X-ray diffraction (XRD).

By oxidizing FeSO₄ with A. ferrooxidans cells, biogenic schwertmannite can be synthesized according to the method described by Liao et al. (2009). Furthermore, the most suitable conditions for the formation of schwertmannite had been adopted (Song et al. 2018). Briefly, in a series of 500 mL Erlenmeyer flasks, 30 mL of A. ferrooxidans cell suspensions was introduced to a solution containing 13.26 g of FeSO₄·7H₂O. Then, deionized water was added to keep the total reaction volume at 300 mL. Next, we adjusted the system's pH to 2.50 with 1:1 H₂SO₄. The above flasks containing 10% (v/v) inoculum and FeSO₄ were subsequently incubated in a shaker at 28 °C and 180 rpm. The red minerals formed in the solution were collected via filtration in the same manner and identified via SEM and XRD.

For the experiment of induced mineral synthesis by jarosite, briefly, in a series of 250 mL Erlenmeyer flasks, 15 mL of A. ferrooxidans cell suspensions were added to a solution containing 6.63 g of FeSO₄·7H₂O and 0.69 g of K₂SO₄ with a Fe/K molar ratio of 3:1. Then, deionized water was added to keep the total reaction volume at 150 mL. Next, the system's pH was adjusted to 2.50 with 1:1 H₂SO₄ to obtain a synthetic solution of jarosite. According to the addition amount and addition method of the presynthesized jarosite listed in Table 1, five different treatments were arranged as follows: Treatment 1 was the abovementioned jarosite synthesis solution; Treatment 2 was the jarosite synthesis solution with 5 g/L jarosite seed crystals added before the initiation of Fe²⁺ oxidation; Treatment 3 was the jarosite synthesis solution with 10 g/L jarosite seed crystals added before the initiation of Fe²⁺ oxidation; Treatment 4 was the jarosite synthesis solution with 5 g/L jarosite seed crystals added before the initiation of Fe²⁺ oxidation and after the oxidation was complete. Treatment 5 was the jarosite synthesis solution with 10 g/L jarosite seed crystals added after the Fe²⁺ oxidation was complete. Each treatment was performed in triplicate, and all Erlenmeyer flasks were subsequently incubated in a shaker at 28 °C and 180 rpm.

For the experiment of induced mineral synthesis by schwertmannite, briefly, in a series of 250 mL Erlenmeyer flasks, 15 mL of A. ferrooxidans cell suspensions was added to a solution containing 6.63 g of FeSO₄·7H₂O. Then, deionized water was added to keep the total reaction volume at 150 mL. Next, the system's pH was adjusted to 2.50 with 1:1 H₂SO₄ to obtain a synthetic solution of schwertmannite. According to the addition amount and addition method of the presynthesized schwertmannite, five different treatments were arranged as listed in Table 1. Each treatment was performed in triplicate, and all Erlenmeyer flasks were subsequently incubated in a shaker at 28 °C and 180 rpm.

The pH, Fe²⁺ concentration, TFe concentration, and concentration of each Erlenmeyer flask were dynamically monitored by taking a 2 mL sample filtered through a 0.22 μm membrane and subsequently converting Fe²⁺ oxidation efficiency, TFe precipitation efficiency, and precipitation efficiency. After reaction for 96 h, the jarosite or schwertmannite produced in each treatment was harvested by filtering through a Whatman No. 4 filter paper. We washed them three times with acidic deionized water (pH 2.00) and pure water to remove soluble impurities and finally dried them at 50 °C. Then, we calculated and compared the net dry weight of jarosite or schwertmannite in each treatment solution.

The pH of the solution was measured using a digital pH meter (pHS-3C, China). The Fe²⁺ and TFe concentrations were determined using the 1,10-phenanthroline method (APHA 2005). When it was necessary to distinguish between Fe³⁺ and Fe²⁺, Fe²⁺ concentration was measured before and after reduction using excess ascorbic acid, and Fe³⁺ concentration was calculated based on the difference between the two measurements. Other metal elements were determined using a Shimadzu ICPS-7500 inductively coupled plasma atomic emission spectrometer (ICP-AES; Shimadzu, Japan; Todolí & Mermet 2006). The concentration was determined using barium chromate spectrophotometry (ASTM 2005). A SU8010 SEM (Hitachi, Japan) was used to observe the mineral morphologies (Weaver et al. 2010). The crystal structures of the collected minerals were determined using an X-ray diffractometer (Bruker D8, Germany; Fewster 2004).

After Fe²⁺ oxidation was complete (after 48 h), 5 and 10 g/L jarosite were added to ‘Treatment 4’ and ‘Treatment 5,’ respectively. At this time, although the addition of seed crystals could still stimulate mineralization and make the solution pH drop faster, the promoting effect was lower than that of adding before the initiation of Fe²⁺ oxidation. After 96 h of reaction, the pH of ‘Treatment 1’ without jarosite dropped to 1.82, and the pH of ‘Treatment 2’ added with 5 g/L jarosite only before the initiation of Fe²⁺ oxidation dropped to 1.75. ‘Treatment 3,’ ‘Treatment 4,’ and ‘Treatment 5’ were all added with a total amount of 10 g/L jarosite, but the pH of each treatment solution differed due to the influence of the addition method. The pH of ‘Treatment 3’ added with 10 g/L jarosite before the initiation of Fe²⁺ oxidation dropped to 1.73. The pH of ‘Treatment 4,’ in which 5 g/L jarosite was added twice before the initiation of Fe²⁺ oxidation and after the oxidation of Fe²⁺, was complete and dropped to 1.75. The pH of ‘Treatment 5’ added with 10 g/L jarosite only after Fe²⁺ oxidation was complete, dropped to 1.79.

For FeSO₄–H₂O systems added with schwertmannite, after 12 h, the pH of ‘Treatment 1’ and ‘Treatment 5’ without adding schwertmannite remained around 2.50. Conversely, the pH of the experimental group added with schwertmannite showed a significant downward trend. The more schwertmannite was added, the faster the pH dropped. Thereafter, with the prolongation of the reaction time, the hydrolysis of Fe³⁺ in the solution dominated, and the pH of each treatment solution decreased significantly. Studies have shown that the pH of the reaction system affects the synthesis of schwertmannite. When pH < 2.10, schwertmannite will no longer be synthesized in the solution, the hydrolysis of Fe³⁺ will be stagnant, and the pH of the solution will remain unchanged (Bigham et al. 1990). Therefore, the pH of all treatment solutions dropped to approximately 2.10 after a period of time and no longer changed; only the reaction time was different. The pH of ‘Treatment 3’ dropped to 2.10 at 36 h. Moreover, the pH of ‘Treatment 2’ and ‘Treatment 4’ reached 2.10 at 48 h. Conversely, the pH of ‘Treatment 1’ and ‘Treatment 5’ dropped to 2.10 at 60 h, which was the longest time.

For K₂SO₄–FeSO₄–H₂O systems added with jarosite, during the Fe²⁺ oxidation period (0–48 h), the only variable between the different solutions was the additional amount of jarosite. In this stage, the precipitation efficiency of TFe was significantly positively correlated with the amount of jarosite added, which was the most obvious in 0–24 h. After 48 h, the precipitation efficiency of TFe in ‘Treatment 1’ and ‘Treatment 5’ without adding jarosite was 41.8%, whereas that in ‘Treatment 2’ and ‘Treatment 4’ with 5 g/L jarosite added was approximately 50.0%. Furthermore, ‘Treatment 3’ added with 10 g/L jarosite, which was the largest amount of jarosite added in the early stage of the reaction, had the highest precipitation efficiency of 54.5% in 48 h. This had the same conclusion as the changing trend of pH in each treatment solution. That is, the addition of jarosite effectively promoted the hydrolysis and mineralization of Fe³⁺ at the initial stage of the reaction, so that the pH of the solution was lower than that of the treatment solution without seed crystals added.

After 48 h, Fe²⁺ in the solution was completely oxidized, 5 and 10 g/L jarosite were added to ‘Treatment 4’ and ‘Treatment 5,’ respectively. Observing the trend of TFe precipitation efficiency after 48 h in the figure, we discovered that the addition of jarosite at this time could still promote the hydrolysis and mineralization of Fe³⁺, increasing the precipitation efficiency of TFe. However, this promoting effect was not as significant as adding before the initiation of Fe²⁺ oxidation. After 96 h of reaction, the precipitation efficiency of ‘Treatment 1’ without jarosite added was 61.8%, and the precipitation efficiency of ‘Treatment 2’ with 5 g/L jarosite added only before the initiation of Fe²⁺ oxidation was 67.7%. ‘Treatment 3,’ ‘Treatment 4,’ and ‘Treatment 5’ were all added at a total amount of 10 g/L seed crystals, but the TFe precipitation efficiencies were not the same due to the influence of the addition method. The precipitation efficiency of ‘Treatment 3’ with 10 g/L jarosite added before the initiation of Fe²⁺ oxidation was 70.0%. Furthermore, the precipitation efficiency of ‘Treatment 4,’ in which 5 g/L jarosite was added twice before the initiation of Fe²⁺ oxidation and after Fe²⁺ oxidation, was 68.2%. The precipitation efficiency of ‘Treatment 5’ with 10 g/L jarosite added only after the oxidation of Fe²⁺ was 64.6%.

The synthesis of jarosite required the consumption of Fe³⁺ and ⁠. The higher the TFe precipitation efficiency, the more jarosite synthesized and the more consumed. Therefore, and TFe precipitation efficiencies of each treatment solution had a similar changing trend, which together reflected the influence of jarosite addition on the hydrolysis and mineralization of Fe³⁺ in the system. After 96 h, the precipitation efficiencies from ‘Treatment 1’ to ‘Treatment 5’ were 41.2, 47.0, 50.1, 48.2, and 44.0%, respectively.

For FeSO₄–H₂O systems added with schwertmannite, each treatment solution could reach the end of the reaction within 96 h and had the same TFe and precipitation efficiencies, only the time required was different. ‘Treatment 3’ reached the end of the reaction at 36 h, ‘Treatment 2’ and ‘Treatment 4’ reached the end of the reaction at 48 h, whereas ‘Treatment 1’ and ‘Treatment 5’ ended the reaction at 60 h. Finally, the precipitation efficiency of TFe in each treatment solution was approximately 33.0%, and the precipitation efficiency of was approximately 7.0%. The addition of schwertmannite in the solution before the start of the reaction did not improve the degree of Fe³⁺ hydrolysis but effectively shortened the mineral synthesis time.

Figure 5 shows that the addition of jarosite before the initiation of Fe²⁺ oxidation effectively improved the mineral yield within 96 h, and it was significantly positively correlated with the amount of seed crystals added. After 96 h, compared with ‘Treatment 1’ without adding jarosite, the yields of ‘Treatment 2’ and ‘Treatment 3’ with 5 or 10 g/L jarosite added only before Fe²⁺ oxidation increased by 17.6 and 26.0%, respectively. The addition of jarosite after Fe²⁺ oxidation was complete had a certain promoting effect on mineral production, but this effect was lower than that of adding jarosite before the oxidation started. The yield of ‘Treatment 4,’ in which 5 g/L jarosite was added twice before the initiation of Fe²⁺ oxidation and after Fe²⁺ oxidation, increased by 16.2%, which was lower than that of the ‘Treatment 3’ solution. Furthermore, the yield of ‘Treatment 5’ with 10 g/L jarosite added only after Fe²⁺ oxidation increased by 4.0%, which was significantly lower than that of the ‘Treatment 3’ solution.

Since each treatment solution added with schwertmannite had the same TFe and precipitation efficiencies at the end of the reaction, the dry weight of the resulting schwertmannite was basically the same. As shown in Figure 7, the dry weight of the schwertmannite produced in each treatment solution was approximately 0.75 g.

As the above results showed, the addition amount and method of jarosite or schwertmannite did not affect the Fe²⁺ oxidation efficiency by A. ferrooxidans but would change the hydrolysis and mineralization efficiency of the oxidation product Fe³⁺. However, there were some differences in the seed induction effect of the two minerals. With the same amount of seed crystals added, jarosite exhibited a much higher capacity to promote Fe³⁺ hydrolysis and mineralization than schwertmannite. Adding seed crystals before the initiation of Fe²⁺ oxidation (0 h) could significantly promote Fe³⁺ mineralization efficiency. With the increase of the additional amount of seed crystals, jarosite could not only shorten the time required for mineral synthesis but also improve the final mineral yield, whereas schwertmannite could only shorten the time required for mineral synthesis. When Fe²⁺ was completely oxidized to Fe³⁺ (48 h), the supplementary addition of jarosite could still effectively improve the hydrolysis and mineralization efficiency of Fe³⁺, but the addition of schwertmannite no longer affected the final mineralization degree.

Gibbs free energy (G) is a crucial thermodynamic state function in physical chemistry, and the direction and limit in which various thermodynamic processes proceed spontaneously can be judged by the Gibbs free energy change (ΔG). The formation of biogenic secondary iron minerals mediated by A. ferrooxidans can be carried out at normal temperature and pressure (20–30 °C, ∼101 kPa); it is scientific to use the standard molar Gibbs free energy (⁠⁠) (25 °C, 100 kPa) as a criterion for the formation of biogenic secondary iron minerals. Therefore, this study is the first attempt to use G as the criterion to discuss the thermodynamic mechanism of the difference in the seed induction effect between jarosite and schwertmannite.

Table 2 shows the for different reactions (Robie & Hemingway 1995; Drouet & Navrotsky 2003; Majzlan et al. 2004). In a single Fe³⁺ system, = 6.63 kJ·mol⁻¹ > 0 (reaction (2)), the mineralization reaction cannot proceed spontaneously; that is, Fe³⁺ cannot directly combine with to form schwertmannite. However, when K⁺ with a strong ability to induce vitriol is present in the system, in the jarosite mineralization process, = –22.20 kJ·mol⁻¹ < 0 (reaction (3)); thus, the reaction could proceed spontaneously. However, due to the small ⁠, the mineral synthesis process is relatively slow, and the effect of TFe and precipitation is affected, which is consistent with the results in Figure 4. After coupling with Fe²⁺ oxidation by A. ferrooxidans (reaction (1)), the of ideal schwertmannite and ideal jarosite reached −34.12 and −67.45 kJ·mol⁻¹, respectively, satisfying the condition that the reaction proceeds spontaneously with < 0. Therefore, the process of hydrolyzing Fe³⁺ to form schwertmannite can be spontaneously carried out, which cannot be achieved by itself. Additionally, the bio-oxidation of Fe²⁺ to synthesize jarosite is more favorable than schwertmannite.

Table 2

of different reactions

Reaction number .	Equation .	(kJ·mol−1) .
(1)	Fe2+(aq) + 1/4O2(g) + H+(aq) = Fe3+(aq) + 1/2H2O(l)	−45.25
(2)	Fe3+(aq) + 7/4H2O(l) + 1/8(aq) = 1/8Fe8O8(OH)6SO4(s) + 11/4H+(aq)	6.63
(3)	1/3K+(aq) + Fe3+(aq) + 2/3(aq) + 2H2O(l) = 1/3KFe3(SO4)2(OH)6(s) + 2H+(aq)	−22.20
Schwertmannite	(1) + (2)	−34.12
Jarosite	(1) + (3)	−67.45

Reaction number .	Equation .	(kJ·mol−1) .
(1)	Fe2+(aq) + 1/4O2(g) + H+(aq) = Fe3+(aq) + 1/2H2O(l)	−45.25
(2)	Fe3+(aq) + 7/4H2O(l) + 1/8(aq) = 1/8Fe8O8(OH)6SO4(s) + 11/4H+(aq)	6.63
(3)	1/3K+(aq) + Fe3+(aq) + 2/3(aq) + 2H2O(l) = 1/3KFe3(SO4)2(OH)6(s) + 2H+(aq)	−22.20
Schwertmannite	(1) + (2)	−34.12
Jarosite	(1) + (3)	−67.45

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In this study, we proposed the possibility of using jarosite and schwertmannite as crystal seeds to induce the rapid formation of minerals in a simulated AMD environment and obtained good results. However, this study was conducted as a laboratory simulation, not a pilot test. Furthermore, AMD has a complex composition and various metal ions; thus it is unclear whether it can produce similar results in wastewater. Moreover, this paper only discussed the first half of the treatment, i.e., biological mineralization, and did not study the following lime neutralization. The wastewater treatment was incomplete, and the advantages of lime neutralization over the traditional neutralization method were not realized. The reduced lime consumption cannot be calculated, including the analysis of economic benefits. Therefore, we can only say that it is possible to use minerals to induce mineralization, and their application to engineering needs further research.

The addition of jarosite and schwertmannite had no significant effect on the bio-oxidation of Fe²⁺ by A. ferrooxidans, but both could promote the hydrolysis of Fe³⁺ to form minerals. However, due to thermodynamic differences in the biosynthesis of jarosite and schwertmannite, the effect of seed stimulation was significantly different. The addition of jarosite can not only shorten the time required for mineral synthesis but can also effectively improve the mineral yield. However, the addition of schwertmannite can only shorten the mineral synthesis time. For the additional amount, the more the seed crystals added, the better the promoting effect. For the addition method, adding jarosite before and after Fe²⁺ oxidation could promote the hydrolysis and mineralization of Fe³⁺. However, the addition of schwertmannite after Fe²⁺ oxidation was complete no longer had a promoting effect. This can be explained using the of jarosite and schwertmannite biosynthesis. At normal temperature and pressure, the reaction of Fe³⁺ hydrolysis to form jarosite has a negative ⁠, whereas the reaction to form schwertmannite has a positive ⁠. When coupled with the biological oxidation of Fe²⁺ by A. ferrooxidans, the resulting Fe³⁺ hydrolysis to form schwertmannite could occur spontaneously, exhibiting a negative ⁠.

Furthermore, the formation of jarosite could show a larger negative ⁠. Therefore, the effect of jarosite addition on the hydrolysis and mineralization of Fe³⁺ was much greater than that of schwertmannite addition, and the induction effect of schwertmannite was terminated together with the synthesis reaction after Fe²⁺ oxidation was complete. The finding of this study has important implications for the efficient removal of soluble iron and sulfate in AMD.

This work was supported by the National Natural Science Foundation of China (No. 21906183) and the Fundamental Research Funds for the Central Universities (No. 2722023BY020, 202351415).

H.W. and Q.G. conceptualized methodology, designed this study, and wrote the original draft. Z.G., H.L., and H.L. carried out data curation and formal analysis. Y.S. and J.Y. wrote, reviewed and edited the original draft. All authors have read and agreed to the published version of the manuscript.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.