Composite strain and application thereof in treatment of coal gangue and acid mine drainage and leaching of rare earths

Abstract

The present application relates to a kind of composite strains and its application in coal gangue and acid mine drainage treatment and rare earth leaching, belong to environmental microbial repair, mine solid waste resource and rare earth biological leaching technical field.The composite strain of the present application includes Acidithiobacillus ferrooxidans WZ33 and acid deposit acidophilic thiobacillus WY33;The preservation number of the Acidithiobacillus ferrooxidans is: CGMCC No.37726;The preservation number of the acid deposit acidophilic thiobacillus is CGMCC No.38196.Acidithiobacillus ferrooxidans (A. ferrooxidans WZ33) in the present application is mainly responsible for ferrous oxidation, rapid acid production and Fe 3+ Oxidizing agent is generated;Acid deposit acidophilic thiobacillus (A. acidisediminis WY33) is mainly responsible for sulfur material oxidation, eliminate sulfur passivation film, continuously maintain low pH environment.The two form complementary synergic system, significantly improve leaching and processing efficiency, better than single strain.

Description

Technical Field

[0001] This invention relates to the fields of environmental microbial remediation, resource utilization of mine solid waste, and rare earth bioleaching, particularly to a composite bacterial strain and its application in the treatment of coal gangue and acid mine wastewater, and rare earth leaching. Specifically, it relates to a preserved composite microbial agent, particularly the integrated application of a composite agent of *Thiobacillus ferrooxidans* and *Thiobacillus acidophilus* in the desulfurization and stabilization of coal gangue, the synergistic treatment of acid mine wastewater (AMD), and the efficient bioleaching of rare earth elements. Background Technology

[0002] Existing technologies for treating acid mine wastewater (AMD) mostly employ *Thiobacillus ferrooxidans* as a single bacterium, or in combination with *Thiobacillus thiooxidans*, *Leptospira ferrooxidans*, *Chlorella vulgaris*, or biochar, to achieve effects such as ferrous oxidation, ferric salt precipitation, arsenic and heavy metal removal / passivation, and metal leaching. In existing technologies for rare earth bioleaching, *Thiobacillus ferrooxidans* is the primary bacterium used for the bioleaching of rare earth-containing materials such as phosphate rock; however, its combination with acid-precipitated *Thiobacillus acidophilus* for rare earth leaching in coal gangue is not observed. Existing technologies for coal gangue treatment often use combinations of *Thiobacillus ferrooxidans* + *Thiobacillus thiooxidans* / *Leptospira* to achieve in-situ leaching, ferrous leaching, or desulfurization of coal gangue, without utilizing acid-precipitated *Thiobacillus acidophilus*. However, the following technical problems still exist: (1) Single function: It can only realize one or two of the functions of AMD processing, rare earth leaching and coal gangue desulfurization, and cannot coordinate the three functions.

[0003] (2) Limitations of strain combinations: all are conventional combinations such as ferrooxidizobacterium + thiooxidizobacterium thiocyanate / leptospirobacter / algae, without ferrooxidizobacterium + acid deposit thiocyanate complex system.

[0004] (3) Insufficient adaptability: Single bacteria or conventional combinations in the coal gangue-AMD coupling system have insufficient acid resistance, heavy metal resistance, acid production capacity, and low rare earth leaching efficiency.

[0005] (4) Fragmented process: Coal gangue disposal, AMD treatment and rare earth recycling are carried out in stages, resulting in long process, high cost and no integrated solution.

[0006] (5) It is difficult to obtain a composite strain that has been preserved, has complementary functions, and is resistant to acid and heavy metals, so as to achieve the integration of three functions: desulfurization and stabilization of coal gangue, deep repair of AMD, and simultaneous and efficient leaching of rare earth.

[0007] Therefore, there is an urgent need to find a composite bacterial strain system that is functionally complementary, acid-resistant, and heavy metal-resistant, in order to achieve integrated treatment of coal gangue desulfurization and stabilization, deep AMD remediation, and efficient rare earth leaching. Summary of the Invention

[0008] To address the above technical problems, this invention provides a composite bacterial strain and its application in the treatment of coal gangue and acidic mine wastewater and rare earth leaching.

[0009] The first objective of this invention is to provide a composite bacterial strain comprising *Acidithiobacillus ferrooxidans* WZ33 and *Acidithiobacillus acidisediminis* WY33; the *Acidithiobacillus ferrooxidans* has the accession number CGMCC No. 37726, deposited at the China General Microbiological Culture Collection Center on February 5, 2026; the *Acidithiobacillus acidisediminis* has the accession number CGMCC No. 38196, deposited at the China General Microbiological Culture Collection Center on March 30, 2026.

[0010] A second objective of this invention is to provide a compound microbial agent comprising the aforementioned compound microbial strains.

[0011] In some embodiments of the present invention, the compound bacterial agent includes Acidithiobacillus ferrooxidans WZ33 bacterial solution and Acidithiobacillus acidizediminis WY33 bacterial solution. The volume ratio of Acidithiobacillus ferrooxidans WZ33 bacterial suspension to Acidithiobacillus acidisediminis WY33 bacterial suspension was 9:1 to 1:9.

[0012] In some embodiments of the present invention, the concentrations of both the *Acidithiobacillus ferrooxidans* WZ33 bacterial suspension and the acid-deposited *Acidithiobacillus acidisediminis* WY33 bacterial suspension are 10. 8 -10 9 CFU / mL.

[0013] A third objective of this invention is to provide a method for preparing the compound microbial agent as described above, comprising the following steps: Acidithiobacillus ferrooxidans WZ33 bacterial culture and acid-deposited acidithiobacillus acidisediminis WY33 bacterial culture were cultured independently, and then the two were mixed and compounded to obtain the compound bacterial agent.

[0014] The fourth objective of this invention is to provide an application of the composite strain or composite agent as described above in the treatment of acid mine wastewater (AMD).

[0015] In some embodiments of the present invention, the wastewater AMD treatment includes at least one of the following functions: ferrous oxidation, ferric salt precipitation, heavy metal removal, heavy metal passivation, and leaching and recovery of valuable metals.

[0016] In some embodiments of the present invention, the amount of the compound microbial agent is 5%-15% (v / v).

[0017] The fifth objective of this invention is to provide the application of the aforementioned composite strain or composite bacterial agent in desulfurization of coal gangue and leaching of iron, and rare earth leaching in rare earth-containing solid waste.

[0018] In some embodiments of the present invention, when treating coal gangue or rare earth-containing solid waste, the mass-to-volume ratio of the solid phase to the composite microbial agent is (1:10)-(1:20) g / mL.

[0019] In this invention, *A. ferrooxidans* WZ33 is mainly responsible for ferrous oxidation, rapid acid production, and the generation of Fe. 3+ Oxidizing agent; Acid deposits: Acidophilus acidisediminis WY33 is mainly responsible for the oxidation of sulfur-based substances, the removal of sulfur passivation films, and the maintenance of a low pH environment. The two form a synergistic system with complementary functions, significantly improving leaching and treatment efficiency, which is superior to that of a single strain.

[0020] The technical solution of the present invention has the following advantages compared with the prior art: 1. This invention achieves efficient desulfurization of coal gangue, reducing the risk of leaching pollution; at the same time, it achieves resource utilization by leaching ferrous iron and rare earth elements.

[0021] 2. This invention achieves synergistic treatment of AMD: it can treat AMD generated by coal gangue leaching in situ, and can also be directly applied to AMD generated in other mining areas, rapidly oxidizing ferrous iron, precipitating ferric compounds, removing heavy metals, increasing pH, and reducing sulfate.

[0022] 3. This invention enables efficient bioleaching of rare earth elements from rare earth-containing materials: it is not limited to coal gangue, but can be extended to other rare earth-containing ores such as phosphate rock and tailings, thereby improving resource recovery rate.

[0023] 4. This invention constructs a green, low-cost, and integrated mining solid waste-wastewater-resource recycling process, filling a gap in existing patents. Attached Figure Description

[0024] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Figure 1 This invention describes the cell morphology and physiological and biochemical characteristics of Acidithiobacillus ferrooxidans WZ33.

[0025] Figure 2 This is the phylogenetic tree of Acidithiobacillus ferrooxidans WZ33 of this invention.

[0026] Figure 3 This invention relates to the morphological and physiological-biochemical characteristics of Acidithiobacillus acidisediminis WY33.

[0027] Figure 4 This is the phylogenetic tree of Acidithiobacillus acidisediminis WY33 of the present invention.

[0028] Figure 5 This is a graph showing the changes of key indicators over time during the in-situ leaching process of coal gangue according to the present invention.

[0029] Figure 6 This is a graph showing the changes in leaching indicators of coal gangue in shake flasks under different proportions of microbial agents according to the present invention.

[0030] Figure 7 This is a graph showing the changes in key indicators of AMD treatment under different proportions of bacterial agents according to the present invention.

[0031] Figure 8 This refers to the rare earth leaching rate of ion-adsorption type rare earth ore under different bacterial agent ratios on day 25 of this invention. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0033] I. Core Microbial Components Disclosed in this Invention 1. Strain A: Acidithiobacillus ferrooxidans WZ33; Accession number: CGMCC No.37726; Depository institution: China General Microbiological Culture Collection Center; Deposit date: February 5, 2026.

[0034] 2. Strain B: Acidithiobacillus acidisediminis WY33; Accession number: CGMCC No.38196; Depository institution: China General Microbiological Culture Collection Center; Deposit date: March 30, 2026.

[0035] II. Functional Complementarity Mechanism of Strains 1. Acidithiobacillus ferrooxidans WZ33: Simultaneously oxidizes ferrous ions (Fe). 2+ ) and reduced inorganic sulfur compounds (such as S) 0 S2O3 2- This bacterium (such as thiosulfate) obtains energy through iron-sulfur coupled oxidation, accompanied by the generation of sulfuric acid and continuous acidification of the environment; it can drive the iron-sulfur biogeochemical cycle in extreme environments such as AMD and sulfide minerals, mediating heavy metal migration and mineral dissolution; this bacterium is also widely used in biometallurgy (leaching of metals such as copper, uranium, and yttrium), AMD treatment and bioremediation of sulfur-containing heavy metal polluted environments, and is one of the most mature ferrooxidizing thiobacilli currently studied and applied.

[0036] 2. *Acidithiobacillus acidisediminis* WY33: This bacterium uses reduced inorganic sulfur, such as elemental sulfur and tetrathionate, as its sole energy source. It generates sulfuric acid and protons through oxidation reactions to obtain energy and maintain an acidophilic environment. It does not possess the ability to oxidize ferrous metals. It can drive the sulfur cycle in acidic environments and, due to its high resistance to heavy metals, becomes an important member of the microbial community in AMD sediments. This bacterium has potential value in the biometallurgical leaching of metals from low-grade sulfide ores, the regulation of heavy metal precipitation in acidic mine wastewater, and the remediation of sulfur-contaminated soil and water.

[0037] 3. Synergistic effect: Acidithiobacillus acidisediminis WY33 and Acidithiobacillus ferrooxidans WZ33 can form a highly efficient iron-sulfur coupled oxidation system. The former specifically oxidizes sulfur to produce acid, accelerating the removal of sulfur from coal gangue and dissolving iron and rare earth elements; the latter rapidly oxidizes Fe. 2+ For Fe 3+ This not only promotes mineral decomposition and improves rare earth leaching efficiency, but also utilizes Fe... 3+ Hydrolysis and precipitation achieve iron fixation and pH recovery in AMD (acid-dependent oxidizing agents). These two processes complement and enhance each other, simultaneously achieving desulfurization of coal gangue, efficient leaching of iron and rare earth elements, and synergistic treatment of AMD.

[0038] III. Specific Technical Solutions 1. Preparation method of compound microbial agent The compound bacterial agent of this invention is obtained by separately isolating, purifying, and culturing Acidithiobacillus ferrooxidans WZ33 and Acidithiobacillus acidisediminis WY33, and then mixing them in a certain proportion. The specific preparation steps are as follows: 1) Culture medium 9K medium or Starkey medium was used for the proliferation culture of a single strain.

[0039] 2) Culture conditions: Each strain was cultured to the logarithmic growth phase under the following conditions: pH range: 1.5-3.5; Temperature: 25-35℃; Shaking speed: 150-200 r / min; Cultivate until the bacterial concentration reaches 10. 8 -10 9 CFU / mL.

[0040] 3) Compound preparation method of compound microbial agents Two single-strain bacterial solutions were combined in a specific volume ratio to obtain a compound bacterial agent. The preferred ratio range was: *A. ferrooxidans* WZ33 : *A. acidisediminis* WY33 = 1:1, 2:1; 4:1; 6:1; 9:1; 1:2; 1:4, 1:6; 1:9, with a further preferred ratio of 1:4. After thorough mixing, the compound bacterial agent was obtained and can be directly applied to coal gangue treatment, AMD remediation, and rare earth leaching. The culture medium for the compound bacterial agent was: ammonium sulfate 3 g / L, potassium chloride 0.1 g / L, dipotassium hydrogen phosphate 0.5 g / L, calcium nitrate 0.01 g / L, magnesium sulfate 0.5 g / L, ferrous sulfate 44.2 g / L; pH 2.0-2.5.

[0041] 2. Coal gangue treatment 1) Operating steps: A. Experiment 1: In-situ heap leaching of coal gangue at a stockpile.

[0042] Operating steps and parameters: Crush the coal gangue to a particle size of 10mm-50mm and pile it up; then inoculate the coal gangue pile with compound microbial agent at a dosage of 5%-15% (v / v); spray the coal gangue pile with liquid (initially water, then AMD), setting an appropriate number of nozzles according to the size of the pile, maintaining the moisture content of the pile at 30%-40%, spraying twice a day for 1-1.5 hours each time, with an interval of at least 4 hours between sprays, and ensuring the sprayed water is evenly atomized. Additives can be added. One or more nutrients: urea 0.5%-2.0% (w / v), ammonium sulfate 0.3%-1.5% (w / v), potassium dihydrogen phosphate 0.05%-0.3% (w / v), elemental sulfur 0.2%-1.0% (w / v), sodium thiosulfate 0.1%-0.5% (w / v), ferrous sulfate 0.2%-1.0% (w / v), magnesium sulfate 0.02%-0.1% (w / v), and a micronutrient mixture 0.005%-0.05% (v / v). Treatment time: 45-60 days.

[0043] B. Experiment 2: Laboratory Shaking Bottle Experiment Operating steps and parameters: First, prepare each purified strain to a concentration of approximately 10. 8 -10 9 A bacterial suspension of cells / mL was used as the "seed liquid" for the compound experiment; the liquid composition of the shake flask was: ammonium sulfate 3g / L, potassium chloride 0.1g / L, dipotassium hydrogen phosphate 0.5g / L, calcium nitrate 0.01g / L, magnesium sulfate 0.5g / L, and pyrite 1g / L; 5-15% (w / v) of coal gangue (100-200 mesh) was added to the shake flask; the "seed liquid" bacterial agent was added at a dosage of 5%-15% (v / v); the temperature was 25-35℃; the shaking speed was 150-200 r / min; and the treatment time was 25-40 days.

[0044] 3. AMD governance 1) Function: Oxidizes Fe 2+ →Fe 3+ It hydrolyzes and precipitates to form Fe(OH)3; removes heavy metals such as As, Pb, Cd, and Cu; and raises the pH from around 2.0 to 3.0-4.0.

[0045] 2) Operation method and parameters: First, inoculate the compound bacterial agent into AMD; then add nutrients (any one or more of the following: urea 0.5%-2.0% (w / v), ammonium sulfate 0.3%-1.5% (w / v), potassium dihydrogen phosphate 0.05%-0.3% (w / v), magnesium sulfate 0.02%-0.1% (w / v), and trace element mixture 0.005%-0.05% (v / v)); AMD will undergo iron biomineralization in the natural environment, forming iron minerals with rough surfaces and increased specific surface areas, which have the ability to adsorb and remove heavy metals; A. ferrooxidans WZ33 will... 2+ Oxidized to Fe 3+ Fe 3+ Hydrolysis precipitates to form Fe(OH)3; in addition, Fe 3+ The sulfurous acid and low-valent sulfides in AMD are then oxidized into sulfuric acid, causing the metals in the ore to be converted into sulfates and released.

[0046] 4. Rare earth bioleaching 1) H produced by the metabolism of complex bacteria + Fe 3+ Organic acids are used as the leaching system. This system selectively leaches rare earth elements such as La, Ce, Nd, and Y from coal gangue, achieving high leaching rates and low impurities.

[0047] 2) Operating methods and parameters: First, determine the rare earth content in the coal gangue (coal gangue in Guizhou contains rare earth elements in some areas and not in others); then prepare the purified strains to a concentration of approximately 10. 8 -10 9 A bacterial suspension of cells / mL; add 1:10-1:20 (w / v) coal gangue (100-200 mesh) to the bacterial suspension (Af bacteria: Aa bacteria = 1:1-1:9); temperature: 25-35℃; shaking speed: 150-200 r / min; treatment time: 25-40 days.

[0048] 5. The culture media used in the embodiments of the present invention are all conventional culture media, which can be obtained commercially, as detailed below: 9K liquid culture medium consists of solution A and solution B. Solution A consists of 3.0 g / L (NH4)2SO4, 0.1 g / L KCl, 0.5 g / L K2HPO4, 0.01 g / L Ca(NO3)2, and 0.5 g / L MgSO4·7H2O. The pH of the prepared solution A is adjusted to 2.0-2.5 using a 1:1 sulfuric acid solution, and then autoclaved at 121°C for 15 min. Solution B consists of 44.7 g / L FeSO4·7H2O, which is filtered through a 0.22 μm microporous membrane or sterilized under UV light for 30 min after preparation. After sterilization, solution A is cooled to room temperature and then thoroughly mixed with the sterilized solution B for subsequent use.

[0049] 9K solid culture medium consists of solutions A, B, and C. Solutions A and B are prepared using the same methods as 9K liquid culture medium. Solution C is a 7.5 g / L agarose solution dissolved by heating. After preparation, solution C is autoclaved at 121°C for 15 min. The sterilized solution A, cooled to 60°C, is then mixed 1:1 with the sterilized solution C, and the pH is adjusted to approximately 2.5 using a 1:1 sulfuric acid solution. Solution B is then quickly added and mixed. Finally, the mixed culture medium is dispensed into petri dishes and allowed to cool and solidify before use in subsequent experiments.

[0050] Starkey-S 0 The liquid culture medium consisted of 2.0 g / L (NH4)2SO4, 3.0 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.25 g / L CaCl2·2H2O, and 0.01 g / L FeSO4·7H2O. After preparation, the pH was adjusted to 2.0-2.5 using a 1:1 sulfuric acid solution, followed by autoclaving at 121°C for 20 min. An appropriate amount of bulk sulfur powder was added to the reagent bottle and dry-heat sterilized in an oven at 120°C for 2 h. Before inoculation, the sterilized sulfur powder was added to Starkey-S at a rate of 1-2% (w / v). 0 In liquid culture medium.

[0051] Starkey-Na₂S₂O₃ solid culture medium consists of solutions A, B, and C. Solution A: 6.0 g / L (NH₄)₂SO₄, 6.0 g / L KH₂PO₄, 1.0 g / L MgSO₄·7H₂O, 0.5 g / L CaCl₂·2H₂O, pH adjusted to 4.5-5.0, autoclaved at 121°C for 20 min; Solution B: 2% (w / v) agar powder, autoclaved at 121°C for 20 min; Solution C: 2.0 g Na₂S₂O₃·5H₂O and 0.006 g FeSO₄·7H₂O, dissolved in 10 ml deionized water, filtered through a 0.22 μm microporous membrane for sterilization. After solutions A and B have cooled to approximately 65°C, they are mixed in equal volumes, then 5% (v / v) solution C is added and mixed thoroughly to prepare solid plates.

[0052] Compound culture medium: ammonium sulfate 3g / L, potassium chloride 0.1g / L, dipotassium hydrogen phosphate 0.5g / L, calcium nitrate 0.01g / L, magnesium sulfate 0.5g / L, pyrite 1g / L.

[0053] Example 1: Isolation and Purification of Functional Bacteria 1. Isolation and purification of *Thiobacillus ferrooxidans* Take 10g of the collected microbial stock solution (taken from the bottom mud and leachate outlet of the coal gangue dump as the stock solution for screening *Thiobacillus ferrooxidans* / *Thiobacillus acidophilus* from acid sediment) and place it in a 250ml Erlenmeyer flask. Add 100ml of sterile water and shake at 180 rpm and 30℃ for 3 days. Then, add this solution to 9K liquid medium at a 10% inoculum and continuously enrich it at 180 rpm and 30℃ until the medium turns iron-reddish-brown. Next, inoculate with fresh 9K liquid medium at a 10% inoculum and continuously enrich for 5 generations (generally 4-5 days per generation), then dilute and plate. Use sterile 50wt% dilute sulfuric acid solution (pH 1.8) to treat the bacterial solution for 10 to 10 times the normal concentration. 6 The bacterial suspension was serially diluted several times, and then spread onto 9K solid medium and incubated at 30°C until yellowish-brown colonies were observed. After colony growth, suitable colonies were picked and inoculated into fresh 9K liquid medium for further cultivation. The alternating solid and liquid culture process was repeated until a pure single strain was isolated. The colonies exhibited irregular, flaky or star-shaped patterns ranging from orange-yellow to brownish-red, with a rough texture, feathery or petal-like edges, and a distinct rust-colored iron precipitation halo. Figure 1 (a) After Gram staining and observation, the bacterium was determined to be a Gram-negative bacterium. Figure 1 (b). Under SEM and TEM, this bacterium appears as a rod-shaped organism with blunt ends, approximately 15-18 μm in length and 3-5 μm in width. Figure 1 middle cd); 2. Identification was performed using 16S rRNA sequencing primers: 27F / 1492R (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'); the results are shown below: Submitted to NCBIGen Bank for comparison, homology ≥ 99%: 3. Phylogenetic tree (evolutionary tree) Construction method: Eight strains most closely related to the isolated strain at the genus level were selected from the GenBank database. A phylogenetic tree of these strains was constructed using the neighbor-joining method with DNAMAN software. Figure 2 As shown.

[0054] 2. Isolation and purification of *Thiobacillus acidophilus* from acid sediments The isolation and purification process is almost identical to that of *Thiobacillus ferrooxidans*. The difference lies in the culture medium; the acidophilic sulfur oxidizer uses Starkey-S... 0 Liquid culture medium and Starkey-Na2S2O3 solid culture medium. Furthermore, acidophilic sulfur oxidizing bacteria typically have a generation time of 7-8 days, and the culture medium does not turn reddish-brown during the cultivation process.

[0055] 3. Identification of *Thiobacillus ferrooxidans* / *Thiobacillus acidophilus* from acid sediments ( Figure 1-4 ) A small amount of bacterial culture was Gram-stained to determine whether it was Gram-positive or Gram-negative. Cells cultured to the logarithmic growth phase were collected by low-temperature high-speed centrifugation (4℃, 8000g). Cell morphology was observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). After multiple solid-liquid alternating cultures, the culture was diluted and plated for 12-14 days. Obvious colonies appeared on Starkey-Na2S2O3 solid medium, presenting as round, white colonies with neat edges. Figure 3 (a) After Gram staining and observation, the bacterium was determined to be a Gram-negative bacterium. Figure 3 (b). Under SEM and TEM, it appears as a short rod with blunt ends, approximately 7-15 μm in length and 3-6 μm in width. Figure 3 Simultaneously, genomic DNA was extracted from the strain, its 16S rRNA gene was amplified and Sanger sequencing was performed. The target fragment was amplified using universal primers 27F / 1492R. After sequence alignment, a phylogenetic tree was constructed based on the neighbor-joining (NJ) method, thereby achieving accurate identification and taxonomic analysis of the strain. Primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', primer 1492R: 5'-GGTTACCTTGTTACGACTT-3'.

[0056] Sequencing results: Acidithiobacillus acidisediminis 16SrRNA gene sequence of strain Acidithiobacillus acidisediminis WY33 3. Phylogenetic tree (evolutionary tree) Construction method: Eight strains most closely related to the isolated strain at the genus level were selected from the GenBank database. A phylogenetic tree of these strains was constructed using the neighbor-joining method with DNAMAN software. Figure 4 As shown.

[0057] Example 2: In-situ leaching test of coal gangue 1. Test sample Coal gangue was collected from a high-sulfur coal gangue stockpile in Xingren City, Guizhou Province. After being crushed and screened, samples of different particle sizes (10 mesh, 50 mesh, 150 mesh, and 200 mesh) were obtained. These samples were then dried and sealed for preservation to prevent oxidation. The coal gangue is a medium-high sulfur, low-ash silica-alumina type (grade 2 silica-alumina ratio). Its main chemical components are SiO2 38.86%, Al2O3 18.21%, Fe2O3 21.17%, and SO3 11.70%, with a loss on ignition of 30.26%. The main phases are quartz, pyrite, and albite. Fe mainly exists in the form of sulfides, while Si and Ti exist in the form of oxides.

[0058] 2. Test bacterial agent The combined bacterial agent of *Thiobacillus ferrooxidans* and *Thiobacillus acidophilus* obtained in Example 1 was used, with an initial bacterial concentration of 1.0 × 10⁻⁶. 8 CFU / mL, with a compounding ratio of 1:1 (the specific preparation method is as described above).

[0059] The compound microbial agent is prepared by the following method: 1) Culture medium 9K medium or Starkey medium was used for the proliferation culture of a single strain.

[0060] 2) Culture conditions: Each strain was cultured to the logarithmic growth phase under the following conditions: pH range: 2.25; Temperature: 30℃; Shaking speed: 180 r / min; Incubate until the bacterial concentration reaches 2.5 × 10⁻⁶. 9 CFU / mL.

[0061] 3) Compound preparation method of compound microbial agents The volume ratio of *A. ferrooxidans* WZ33 to *A. acidisediminis* WY33 was 1:1. After thorough mixing, a compound microbial agent was obtained that could be directly applied to coal gangue treatment, AMD remediation, and rare earth leaching. The culture medium for the compound microbial agent was: ammonium sulfate 3 g / L, potassium chloride 0.1 g / L, dipotassium hydrogen phosphate 0.5 g / L, calcium nitrate 0.01 g / L, magnesium sulfate 0.5 g / L, and ferrous sulfate 44.2 g / L; pH adjusted to 2.16.

[0062] 3. Leaching apparatus and scheme 1) Construct two sets of percolation columns (one blank and one composite bacteria column), each column is filled with 10kg of coal gangue (particle size 10mm-50mm), the bottom of the column is covered with a 1-2cm thick layer of crushed stone, and a leachate collection port is set up.

[0063] 2) Addition of microbial agent: Spray the compound microbial agent evenly onto the coal gangue pillar at a solid-liquid ratio of 10:1. After the first addition, add microbial solution once every 7 days (the amount added is 20% of the initial amount).

[0064] 3) Maintain a natural ambient temperature (15-28℃), keep the column moisture content at 30%-35%, spray twice a day for 1 hour each time, with a 6-hour interval between sprays, and ensure uniform atomization of the spray water. Nutrients can be added: 0.5% urea (w / v).

[0065] 4) Sampling plan: leachate samples were collected on days 0, 3, 7, 14, 21, 28, 35, 42 and 48 of leaching. Three parallel samples were collected at each time point and then mixed for testing.

[0066] 4. Detection Indicators and Methods 1) Total iron content: determined by ICP-MS.

[0067] 2) Sulfate content: Barium sulfate turbidimetric method was used.

[0068] 3) pH / ORP: measured on-site using a portable multi-parameter water quality analyzer.

[0069] 5. Test Results Depend on Figure 5 It can be seen that on day 48, the total iron and sulfate leaching amounts in the control group (CK) were 1.536 g / L and 3.23 g / L, respectively; while the total iron and sulfate leaching amounts in the 1:1 compound bacterial group were 12.252 g / L and 30.23 g / L, respectively.

[0070] Example 3: Laboratory shake-flask leaching test of coal gangue 1. Test sample The coal gangue was crushed and ground to 100t-200 mesh, and its basic physical and chemical properties were the same as in Example 2.

[0071] 2. Test bacterial agent Af bacteria (A. ferrooxidans WZ33) and Aa bacteria (A. acidisediminis WY33) were cultured separately until the logarithmic growth phase (concentration 1.0 × 10⁻⁶). 8 (CFU / mL), set 6 groups of bacterial suspension ratios: Control group 1: Blank group (no inoculation, only compound culture medium); Control group 2: Single Af bacteria group (100% Af); Control group 3: Single Aa bacteria group (100% Aa); Experimental group 1: Af:Aa=1:1 (v / v); Experimental group 2: Af:Aa=1:4 (v / v); Experimental group 3: Af:Aa=1:9 (v / v).

[0072] 3. Shake-flask test protocol 1) Use a 250mL conical flask, add coal gangue (10g / 100mL system) at 10% (w / v), and prepare a 100mL reaction system with 90mL compound culture medium + 10mL bacterial solution (inoculation amount 10%v / v).

[0073] 2) Cultivation conditions: constant temperature shaker, rotation speed 180 r / min, temperature 30℃, cultivation period 28 days.

[0074] 3) Sampling: Samples were taken on days 0, 1, 3, 7, 14, 21, and 28 of culture.

[0075] 4. Detection Indicators and Methods 1) Ferrous content: determined by potassium dichromate titration. Ferrous conversion rate: In the formula: η represents the Fe content in the solution. 2+ Conversion rate (%), C0 is the initial Fe in the system 2+ Concentration (g / L), C1 is the Fe concentration at a certain instant in the system. 2+ Concentration (g / L).

[0076] 2) Sulfate content: Barium sulfate turbidimetric method was used. Coal gangue sulfur leaching rate (desulfurization rate): In the formula: ξ is the sulfur leaching rate (%) in coal gangue, and C t V is the concentration of leachate (g / L), V is the volume (L), m0 is the initial mass of coal gangue added (g), and ω0 is the initial mass fraction of total sulfur (pyrite sulfur) in coal gangue (%).

[0077] 3) pH / ORP: Measured using a portable multi-parameter water quality analyzer.

[0078] 5. Test Results Depend on Figure 6 It can be seen that on day 28, the ferrous iron conversion rate and desulfurization rate of the blank group (CK) were 6.8% and 5.1%, respectively; the ferrous iron conversion rate and desulfurization rate of the single Af group were 74.8% and 65.2%, respectively; the ferrous iron conversion rate and desulfurization rate of the single Aa group were 46.5% and 79.8%, respectively; the ferrous iron conversion rate and desulfurization rate of the Af:Aa=1:1 group were 80.7% and 75.7%, respectively; the ferrous iron conversion rate and desulfurization rate of the Af:Aa=1:4 group were 85.2% and 83.2%, respectively; and the ferrous iron conversion rate and desulfurization rate of the Af:Aa=1:9 group were 82.9% and 87.2%, respectively.

[0079] Example 4: AMD Processing Experiment 1. AMD water sample AMD raw water was collected from a coal mining area in Guizhou Province. Basic water quality: pH=2.35, Fe... 2+ The concentration was 11.169 g / L, the sulfate concentration was 4.72 g / L, and the concentrations of heavy metals Pb and Cd were 128.6 mg / L and 113.5 mg / L, respectively.

[0080] 2. Test bacterial agent Based on the results of Example 3, six groups of bacterial agent ratios were set: Control group 1: Blank group (no bacterial inoculation) Control group 2: Single Af bacteria group (100% Af) Control group 3: Single Aa bacteria group (100% Aa) Experimental group 1: Af:Aa = 1:1 (v / v) Experimental group 2: Af:Aa = 1:4 (v / v) Experimental group 3: Af:Aa = 1:9 (v / v) The initial concentration of the bacterial culture was 1.0 × 10⁻⁶. 8 CFU / mL, inoculation amount 10% (v / v).

[0081] 3. Test Plan Use 250mL Erlenmeyer flasks, add 100mL of AMD raw water to each flask, inoculate with bacterial culture according to the ratio, and incubate on a constant temperature shaker at 180r / min and 30℃ for 7 days.

[0082] Sampling plan: Samples were taken on days 0, 1, 3, 5, and 7 to detect Fe. 2+ Sulfate and heavy metal concentrations.

[0083] 4. Detection Indicators and Methods 1) Ferrous content: determined by potassium dichromate titration. Ferrous conversion rate: In the formula: η represents the Fe content in the solution. 2+ Conversion rate (%), C0 is the initial Fe in the system 2+ Concentration (g / L), C1 is the Fe concentration at a certain instant in the system. 2+ Concentration (g / L).

[0084] 2) Sulfate content: The sulfate concentration in AMD was determined using the barium sulfate turbidimetric method (GB / T5750.5-2006). Sulfate removal rate: In the formula: ξ is the sulfate removal rate (%), C0 ， The initial sulfate concentration (g / L) for AMD, C t Let t be the sulfate concentration in the wastewater at time t (g / L).

[0085] 3) Heavy metals Pb and Cd: Inductively coupled plasma mass spectrometry (ICP-MS).

[0086] 5. Test Results Depend on Figure 7 It can be seen that on day 7, the ferrous conversion rate, sulfate removal rate, Pb removal rate, and Cd removal rate of the control group (CK) were 4.1%, 2.6%, 4.9%, and 3.5%, respectively; the ferrous conversion rate, sulfate removal rate, Pb removal rate, and Cd removal rate of the single Af group were 54.2%, 42.7%, 81.6%, and 75.3%, respectively; and the ferrous conversion rate, sulfate removal rate, Pb removal rate, and Cd removal rate of the single Aa group were 27.6%, 55.5%, 62.1%, and 58.4%, respectively. The Af:A ratio was... The ferrous conversion rate, sulfate removal rate, Pb removal rate, and Cd removal rate were 58.5%, 51.3%, 85.5%, and 73.2% for the Af:Aa=1:1 group, respectively; 68.9%, 65.6%, 93.4%, and 95.2% for the Af:Aa=1:4 group, respectively; and 63.1%, 73.8%, 88.7%, and 84.6% for the Af:Aa=1:9 group, respectively.

[0087] Example 5

[0088] 1. Rare earth ore samples A rare earth ore of ion adsorption type was selected from Guizhou Province. After crushing and grinding to 200 mesh, the basic composition was as follows: the total content of rare earth oxides (REO) was 1.23wt%, including 0.31wt% La2O3, 0.48wt% CeO2, 0.29wt% Nd2O3, and 0.15wt% Y2O3. The gangue minerals were kaolinite, quartz, and mica.

[0089] 2. Test bacterial agent Similar to Example 3, six groups of bacterial agent ratios were set: blank group, single Af group, single Aa group, Af:Aa = 1:1, 1:4, and 1:9, with an initial bacterial concentration of 1.0 × 10⁻⁶. 8 CFU / mL, inoculation amount 10% (v / v).

[0090] 3. Leaching test protocol 1) Using a 250mL Erlenmeyer flask, add coal gangue at 5% (w / v) (5g / 100mL system), and prepare a 100mL reaction system with 90mL of compound culture medium + 10mL of bacterial suspension (inoculum amount 10% v / v). Incubate on a constant temperature shaker at 180r / min and 30℃ for 25 days.

[0091] 2) Sampling plan: Samples were taken at 0, 5, 10, 15, 20 and 25 days respectively, filtered through a 0.22μm filter membrane, and the concentrations of La, Ce, Nd and Y in the leachate were measured.

[0092] 4. Detection Indicators and Methods Rare earth element concentration: Inductively coupled plasma mass spectrometry (ICP-MS) 5. Test Results Depend on Figure 8 It can be seen that on day 25, the leaching rates of La, Ce, Nd, and Y in the control group (CK) were 3.1%, 3.2%, 3%, and 3.2%, respectively; in the Af group, the leaching rates of La, Ce, Nd, and Y were 37.2%, 38.9%, 35.8%, and 33.5%, respectively; in the Aa group, the leaching rates of La, Ce, Nd, and Y were 43.1%, 44.8%, 41.5%, and 39.2%, respectively; and in the Af:Aa=1:1 group... The leaching rates of La, Ce, Nd, and Y were 47.8%, 49.6%, 46.3%, and 43.9%, respectively. In the Af:Aa=1:9 group, the leaching rates of La, Ce, Nd, and Y were 53.4%, 55.7%, 51.8%, and 49.5%, respectively. The leaching rate of Af:Aa=1:4 group was the highest, with leaching rates of La, Ce, Nd, and Y of 57.2%, 59.1%, 55.3%, and 52.8%, respectively.

[0093] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A compound bacterial strain, characterized in that, The samples include *Acidithiobacillus ferrooxidans* WZ33 and *Acidithiobacillus acidisediminis* WY33; the *Acidithiobacillus ferrooxidans* has the accession number CGMCC No. 37726, deposited at the China General Microbiological Culture Collection Center on February 5, 2026; the *Acidithiobacillus acidisediminis* has the accession number CGMCC No. 38196, deposited at the China General Microbiological Culture Collection Center on March 30, 2026.

2. A compound microbial agent, characterized in that, Includes the compound strain described in claim 1.

3. The compound microbial agent according to claim 2, characterized in that, The compound microbial agent includes Acidithiobacillus ferrooxidans WZ33 bacterial suspension and Acidithiobacillus acidizediminis WY33 bacterial suspension; The volume ratio of Acidithiobacillus ferrooxidans WZ33 bacterial suspension to Acidithiobacillus acidisediminis WY33 bacterial suspension was 9:1 to 1:

9.

4. The compound microbial agent according to claim 3, characterized in that, The concentrations of the *Acidithiobacillus ferrooxidans* WZ33 bacterial suspension and the acid-deposited *Acidithiobacillus acidisediminis* WY33 bacterial suspension were both 10. 8 -10 9 CFU / mL.

5. A method for preparing the compound microbial agent as described in claim 2 or 3, characterized in that, Includes the following steps: Acidithiobacillus ferrooxidans WZ33 and acid-deposited acidithiobacillus acidisediminis WY33 were cultured independently, and then the two were mixed to obtain the compound bacterial agent.

6. The application of the composite strain as described in claim 1 or the composite bacterial agent as described in claim 2 in the treatment of acidic mine wastewater (AMD).

7. The application according to claim 6, characterized in that, Wastewater AMD treatment includes at least one of the following functions: ferrous oxidation, ferric salt precipitation, heavy metal removal, heavy metal passivation, and leaching and recovery of valuable metals.

8. The application according to claim 6, characterized in that, The dosage of the compound microbial agent is 5%-15% (v / v).

9. The application of a composite bacterial strain as described in claim 1 or a composite bacterial agent as described in claim 2 or 3 in desulfurization of coal gangue and leaching of iron, and rare earth leaching in rare earth-containing solid waste.

10. The application according to claim 9, characterized in that, When treating coal gangue or rare earth-containing solid waste, the mass-to-volume ratio of the solid phase to the compound microbial agent is (1:10)-(1:20) g / mL.

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