Air bacillus bacterial agent for ecological restoration of coal gangue

Abstract

The present application relates to the technical field of microbial remediation, and discloses an air bacillus agent for ecological remediation of coal gangue, which comprises the following steps: impregnating and pretreating crushed straw with wood vinegar diluent, ventilating and air-drying to obtain activated straw organic material; inoculating white rot fungi, air bacillus and dark septate endophytic fungi with apatite powder into the activated straw, and then applying the mixture into the coal gangue. In the present application, the air bacillus continuously releases phosphate ions by biological dissolution of phosphorus, forms humus-iron (aluminum)-phosphate ternary mineral anchoring complex with iron / aluminum oxides and humus in the coal gangue, and secretes lipopeptide substances to maintain the stability of the bacterial community, so that the functional components synergistically act to significantly improve the humification efficiency and net accumulation of humus in the coal gangue.

Description

Technical Field

[0001] This invention relates to the field of microbial remediation technology, and more specifically, to an airborne Bacillus agent for the ecological remediation of coal gangue. Background Technology

[0002] Coal gangue is a large amount of solid waste generated during coal mining and washing. When accumulated and piled up, it creates extremely harsh habitat conditions. Pyrite ( Continuous oxidation and acid production cause the pH of coal gangue to be generally in the strong acid range of 2-4, and a large amount of free iron oxides accumulate on the surface of the stockpile. , The soil contains high levels of aluminum oxides, and excessive levels of heavy metals such as Cd, Pb, As, Cu, and Zn. It also suffers from extremely low organic matter content, poor soil structure, and weak water retention capacity. These multiple extreme conditions mean that the natural formation of humus typically takes decades or even over a century, severely hindering vegetation restoration and ecosystem reconstruction. To accelerate the ecological restoration of coal gangue, microbial remediation technology has been widely researched and applied. Wood vinegar pretreatment can dissolve the waxy layer of straw and promote the swelling of lignin-cellulose structure, thereby improving the invasion efficiency of cellulose-degrading bacteria on straw substrate and accelerating the initiation of humification. Dark-colored septate endophytic fungi can colonize for a long time in coal gangue environment with strong acid and high heavy metal pollution. Their hyphal walls are rich in melanin, which can copolymerize with humic precursors to form melanin-humic complexes with higher chemical stability. Phosphate released by bio-solubilization of apatite can form ternary mineral anchoring complexes with iron / aluminum oxides and humic substances in coal gangue, reducing humic substance leaching by rainfall. The study of the organic integration and synergistic effects of the above functional components is of great significance.

[0003] However, existing technologies present inherent technical contradictions when combining wood vinegar pretreatment with dark-colored septate endophytic fungi: wood vinegar contains methanol, which, after volatilization and concentration within the organic material microdomain, exerts cumulative metabolic toxicity on dark-colored septate endophytic fungi, inhibiting the normal function of their melanin synthesis-related enzyme systems, resulting in an inherent antagonism between the straw activation benefits of wood vinegar and the humic stabilization function of dark-colored septate endophytic fungi; in the initial stage of remediation of bare coal gangue land without host plants, the melanin synthesis metabolic pathway of dark-colored septate endophytic fungi remains in a low-activity state due to the lack of host plant signaling substances, and the humic stabilization function cannot be fully utilized in the remediation initiation stage; in addition, the primary humic substances produced by microbial degradation of organic materials lack effective chemical bonding with the surface of coal gangue minerals, and are largely lost with rainwater infiltration, resulting in a low net accumulation of humic substances. Summary of the Invention

[0004] This invention provides an airborne Bacillus agent for the ecological restoration of coal gangue, which solves the technical problems in related technologies, such as the inherent antagonistic contradiction between the activation of straw by wood vinegar pretreatment and the stabilization function of dark-colored endophytic fungi in humus, the low melanin synthesis activity of dark-colored endophytic fungi in the early stage of restoration, and the serious leaching of humus by rainfall.

[0005] This invention provides a method for preparing an airborne Bacillus agent for the ecological restoration of coal gangue, comprising the following steps: crushing straw, soaking it in a diluted wood vinegar solution for no less than 12 hours, and then air-drying it for no less than 48 hours. h, activated straw organic material is obtained; white-rot fungi, airborne Bacillus, methyl-trophic bacteria and dark-colored septate endophytic fungi are separately cultured to prepare various functional microbial agents; apatite powder is mixed with the activated straw organic material at a mass ratio of 1:5 to 1:10 and then inoculated with the various functional microbial agents to obtain airborne Bacillus agent; wherein, methyl-trophic bacteria are used to metabolize and degrade residual methanol in activated straw and secrete cytokinins to eliminate the metabolic toxicity of methanol to dark-colored septate endophytic fungi and activate the melanin synthesis of dark-colored septate endophytic fungi; white-rot fungi are used to degrade straw lignin into humic precursors; dark-colored septate endophytic fungi are used to synthesize melanin and copolymerize with the humic precursors to form a melanin-humic complex; airborne Bacillus is used to secrete organic acids to dissolve apatite and release dihydrogen phosphate ions, form a humic-iron (aluminum)-phosphate ternary mineral anchoring complex with coal gangue iron / aluminum oxides and humic substances, and secrete lipopeptide antibacterial active substances to antagonize miscellaneous bacteria.

[0006] Preferably, the straw is wheat straw, and the length of the crushed straw is 3 cm; the wood vinegar dilution is prepared by mixing wood vinegar stock solution and water at a volume ratio of 1:8.

[0007] Preferably, the white-rot fungi are cultured using a liquid fermentation process to a viable count of not less than [amount missing]. Solid bacterial agents were prepared by adsorption using vermiculite with a particle size of 1–3 mm as a solid carrier at a concentration of CFU / mL.

[0008] Preferably, the *Bacillus aeruginosa* is cultured using a liquid fermentation process to a viable count of not less than [amount missing]. CFU / mL.

[0009] Preferably, the methyltrophic bacteria are cultured using a liquid fermentation process to achieve an effective viable cell count of not less than [amount missing]. CFU / mL.

[0010] Preferably, the dark-colored septate endophytic fungus is propagated using a liquid fermentation process, and the total effective inoculum amount is not less than [amount missing]. A bacterial suspension of cells / mL.

[0011] Preferably, the particle size of the apatite powder is not greater than 74 μm, and the mass ratio of the apatite powder to the activated straw organic material is 1:7.

[0012] Preferably, the white-rot fungus oxidizes and cleaves straw lignin into phenolic compounds by secreting lignin peroxidase and manganese peroxidase, while simultaneously secreting laccase to... The phenolic compounds are catalyzed by electron acceptors to undergo single-electron oxidative polymerization, forming humic precursors that participate in the copolymerization reaction of melanin in dark-colored septate endophytic fungi.

[0013] Preferably, the cytokinin secreted by the methyltrophic bacteria is trans-zeatin type, and the trans-zeatin type cytokinin promotes the synthesis of dihydroxynaphthalene polymer melanin in the hyphal wall of dark-colored septate endophytic fungi through the polyketide synthesis pathway.

[0014] The beneficial effects of this invention are as follows: the combined application of wood vinegar pretreatment and methyl-trophic bacteria eliminates the inherent antagonistic contradiction between the straw activation benefits of wood vinegar and the humic stabilization function of dark-colored septate endophytic fungi, achieving positive synergy among the functional components; the cytokinins produced by methyl-trophic bacteria activate the melanin synthesis of dark-colored septate endophytic fungi under host plant-free conditions in the early stage of restoration, enabling the humic stabilization function to operate in the restoration initiation stage; Bacillus aerobicans continuously releases dihydrogen phosphate ions through bio-solubilization, forming a humic-iron (aluminum)-phosphate ternary mineral anchoring complex with iron / aluminum oxides and humic substances in coal gangue, firmly fixing the humic substances to the mineral surface and significantly reducing leaching losses from rainfall; Bacillus aerobicans secretes lipopeptide antibacterial active substances throughout the process to antagonize miscellaneous bacteria, ensuring the continuous and stable survival of each functional bacterial group in the extreme coal gangue environment; the synergistic effect of the above functional components significantly improves the humification efficiency of coal gangue and the net accumulation of humic substances, effectively promoting the ecological restoration process of coal gangue. Attached Figure Description

[0015] Figure 1 This is a grouped bar chart showing the change in net humic acid accumulation in each treatment group of the present invention with cultivation time; Figure 2 This is a graph showing the dynamic changes in methanol concentration in a micro-domain of organic materials. Figure 3 A graph showing the dynamic changes in melanin content in the hyphal walls of dark-colored, septate endophytic fungi in the microdomains of organic materials. Figure 4 A comparison chart of carbon mineralization rates for S1 and S2; Figure 5 A comparison diagram of the characteristics of humic components in S1 and S2; Figure 6 Comparison of the microscopic morphology of S1 (conventional humus) and S2 (melanin-humus complex) under field emission scanning electron microscopy (10,000x). Figure 7 Line graph comparing the concentration of dissolved organic carbon in leachate from different rainfall events; Figure 8 Line graph comparing the cumulative humic carbon loss from each rainfall event; Figure 9 This is an elemental surface distribution map obtained by field emission scanning electron microscopy combined with energy dispersive X-ray spectroscopy. Detailed Implementation

[0016] The subject matter described herein will now be discussed with reference to exemplary embodiments. It should be understood that these embodiments are discussed only to enable those skilled in the art to better understand and implement the subject matter described herein, and changes may be made to the function and arrangement of the elements discussed without departing from the scope of this specification. Various processes or components may be omitted, substituted, or added as needed in the examples. Furthermore, some features described in the examples may be combined in other examples.

[0017] Example 1

[0018] This embodiment discloses a method for preparing Bacillus airborne bacteria agent for the ecological restoration of coal gangue, the method comprising the following steps: Step 1: Crush agricultural waste wheat straw to a length of 3 cm; mix wood vinegar stock solution with water at a volume ratio of 1:8 to prepare wood vinegar dilution; immerse the crushed straw completely in the dilution at a liquid-to-solid ratio of 1:10 (straw dry weight (kg) to dilution volume (L)) for 18 hours to allow the effective components of wood vinegar to fully penetrate the straw; remove the straw and air dry it at 25°C under ventilation for 60 hours until low-boiling-point volatile components such as methanol are fully released, thus obtaining activated straw organic material.

[0019] Step 2: After the activated straw organic material is dried, prepare the following functional microbial agents respectively: (1) Expand the white rot fungus to an effective viable count according to the conventional liquid fermentation process. CFU / mL, the obtained bacterial suspension was uniformly adsorbed onto vermiculite solid carrier with a particle size of 1-3 mm to prepare a solid inoculum for white rot fungi; (2) Bacillus aerogenes was expanded to an effective viable count of CFU / mL according to the conventional liquid fermentation process of Bacillus. (3) Prepare solid spore powder by spray drying with CFU / mL; (4) Expand methyl nutrient bacteria to an effective viable count of CFU / mL using conventional liquid fermentation process. CFU / mL, the obtained bacterial suspension was uniformly adsorbed onto vermiculite solid carrier with a particle size of 1-3 mm to prepare methyl nutrient bacteria solid inoculum; (4) the dark-colored septate endophytic fungi were expanded according to conventional fungal liquid fermentation process to prepare effective inoculum (active mycelial fragments and spores) with a total amount of CFU / mL. A bacterial suspension of 1 / mL was uniformly adsorbed onto a vermiculite solid carrier with a particle size of 1–3 mm, and then dried to obtain a dark-colored, septate endophytic fungal solid inoculum.

[0020] Step 3: Select particles with a diameter no larger than 74 μm (200 mesh). Natural apatite powder with a content of 32% and heavy metal content such as Cd meeting the heavy metal limit requirements of GB / T 23349 mineral-derived fertilizers. Apatite powder and activated straw organic material obtained in step one are mixed evenly at a mass ratio of 1:7 (apatite: straw dry weight) to obtain activated straw mixture containing apatite.

[0021] Step 4: Evenly inoculate the solid inoculants of each functional microorganism prepared in Step 2 into the activated straw mixture containing apatite obtained in Step 3. After inoculation, the effective viable count of each functional component in the mixture reaches: white rot fungi CFU / g dry weight of straw, Bacillus aerogenes CFU / g straw dry weight, methyl nutrient bacteria Effective inoculum for CFU / g dry straw, dark-colored septate endophytic fungi (active hyphal fragments and spores) Bacillus aeruginosa inoculum was prepared by mixing each bacterium with the dry weight of straw in a saturated form (e.g., 1 g of straw).

[0022] Instructions for use of microbial agents: The bacterial agent was applied at a concentration of 20 kg / m³. 2 (Based on the dry weight of straw) Apply evenly to the surface layer of the coal gangue stockpile to be repaired (pH 2.8, Cd content 42 mg / kg, As content 35 mg / kg), and mix it with the surface coal gangue to a depth of 12 cm.

[0023] After application, methyl-trophic bacteria use the trace amounts of methanol remaining in the activated straw mixture as a carbon source for efficient aerobic metabolism, degrading the methanol into... and Simultaneously, during methanol metabolism, trans-zeatin-type cytokinin is synthesized and secreted. This cytokinin, under conditions without a pioneer plant host, activates dark-colored septate endophytic fungi to synthesize dihydroxynaphthalene-type melanin in the hyphal wall through the polyketide synthesis pathway. White-rot fungi colonize in the microdomains of organic matter (pH approximately 4–6), through processes such as glyoxal oxidase. The enzyme is produced extracellularly. Lignin peroxidase and manganese peroxidase thus As a co-substrate, straw lignin is oxidatively cleaved into humic precursors (phenolic compounds, lignin-modified fragments, etc.), and laccase is used as... The electron acceptor catalyzes the single-electron oxidation of phenolic compounds in humic precursors, generating phenoxy radicals. These radicals then copolymerize with dihydroxynaphthalene-based melanin from the hyphal wall of dark-colored septate endophytic fungi to form a melanin-humic complex with enhanced chemical stability. Airborne Bacillus colonizes in the micro-domains of organic materials, secreting organic acids such as citric acid and oxalic acid, which diffuse outwards into the acidic environment of coal gangue (pH 2-5), continuously dissolving apatite and releasing... , With coal gangue iron / aluminum oxides ( , The surface active sites and humic substances together form a ternary mineral anchoring complex of humic substances, iron (aluminum) and phosphate, which fixes the humic substances to the mineral surface through coordination bonds; Bacillus aerogenes secretes lipopeptide antibacterial substances such as irisin and surfactant throughout the process to maintain the dominant position of each functional bacterial group.

[0024] Example 2

[0025] This embodiment discloses a method for preparing Bacillus airborne bacteria agent for the ecological restoration of coal gangue, the method comprising the following steps: Step 1: Crush agricultural waste wheat straw to a length of 2 cm; mix wood vinegar stock solution with water at a volume ratio of 1:5 to prepare wood vinegar dilution; immerse the crushed straw completely in the dilution at a liquid-to-solid ratio of 1:8 (straw dry weight (kg) to dilution volume (L)) for 12 h; remove the straw and air dry it at 20°C under ventilation for 48 h to obtain activated straw organic material.

[0026] Step 2: (1) Expand the liquid fermentation culture of white rot fungi to the effective viable count. (2) Solid bacterial agent was prepared by adsorbing CFU / mL onto vermiculite with a particle size of 1-3 mm; (3) Bacillus aerogenes was fermented and cultured to the effective viable count. (3) Prepare a liquid bacterial suspension with CFU / mL and set aside; (4) Propagate the methyl nutrient bacteria by liquid fermentation to the effective viable count. CFU / mL, adsorbed onto vermiculite with a particle size of 1-3 mm to prepare a solid bacterial agent; (4) liquid fermentation and expansion of dark-colored septate endophytic fungi to prepare an effective inoculum (active mycelial fragments and spores) with a total amount of CFU / mL. A bacterial suspension of 100 cells / mL was adsorbed onto vermiculite with a particle size of 1–3 mm and dried to obtain a solid bacterial agent.

[0027] Step 3: Select particles with a diameter not exceeding 74 μm. Natural apatite powder with a content of 30% and a heavy metal content that meets the requirements of GB / T 23349 is mixed evenly with activated straw organic material at a mass ratio of 1:5 (apatite: straw dry weight) to obtain activated straw mixture containing apatite.

[0028] Step 4: The functional microbial agents prepared in Step 2 (Bacillus aeruginosa inoculated as a liquid suspension, and the other components inoculated as solid agents) are evenly inoculated into the activated straw mixture containing apatite prepared in Step 3. After inoculation, the effective viable count of each functional component reaches: white rot fungi CFU / g dry weight of straw, Bacillus aerogenes CFU / g straw dry weight, methyl nutrient bacteria CFU / g dry straw weight, dark-colored septate endophytic fungal effective inoculum Bacillus aeruginosa inoculum was prepared by mixing each bacterium with the dry weight of straw in a saturated form (e.g., 1 g of straw).

[0029] Instructions for use of microbial agents: Based on 10 kg / m 2 Apply evenly (based on the dry weight of straw) to the surface of the coal gangue stockpile to be repaired (pH 3.2, Zn content 280mg / kg), and mix it with the surface coal gangue to a depth of 10 cm.

[0030] Example 3

[0031] This embodiment discloses a method for preparing Bacillus airborne bacteria agent for the ecological restoration of coal gangue, the method comprising the following steps: Step 1: Crush agricultural waste corn stalks to a length of 4 cm; mix wood vinegar stock solution with water at a volume ratio of 1:10 to prepare wood vinegar diluted solution; according to the ratio of dry weight of straw (kg) to volume of diluted solution (L) of 1:12, completely immerse the crushed straw in the diluted solution and soak for 24 h; remove the straw and air dry it under ventilated conditions at 30°C for 72 h to obtain activated straw organic material.

[0032] Step 2: (1) Expand the liquid fermentation culture of white rot fungi to the effective viable count. (2) Solid bacterial agent was prepared by adsorbing CFU / mL onto vermiculite with a particle size of 1-3 mm; (3) Bacillus aerogenes was fermented and cultured to the effective viable count. (3) Prepare solid spore powder by spray drying with CFU / mL; (4) Expand the methyl nutrient bacteria by liquid fermentation to the effective viable count. CFU / mL, adsorbed onto vermiculite with a particle size of 1-3 mm to prepare a solid bacterial agent; (4) liquid fermentation and expansion of dark-colored septate endophytic fungi to prepare an effective inoculum (active mycelial fragments and spores) with a total amount of CFU / mL. A bacterial suspension of 100 cells / mL was adsorbed onto vermiculite with a particle size of 1–3 mm and dried to obtain a solid bacterial agent.

[0033] Step 3: Select particles with a diameter not exceeding 74 μm. Natural apatite powder with a content of 35% and a heavy metal content that meets the requirements of GB / T 23349 is mixed evenly with activated straw organic material at a mass ratio of 1:10 (apatite: straw dry weight) to obtain activated straw mixture containing apatite.

[0034] Step 4: Evenly inoculate the solid inoculants of each functional microorganism prepared in Step 2 into the activated straw mixture containing apatite obtained in Step 3. After inoculation, the effective viable count of each functional component reaches: white rot fungi CFU / g dry weight of straw, Bacillus aerogenes CFU / g straw dry weight, methyl nutrient bacteria CFU / g dry straw weight, dark-colored septate endophytic fungal effective inoculum Bacillus aeruginosa inoculum was prepared by mixing each bacterium with the dry weight of straw in a saturated form (e.g., 1 g of straw).

[0035] Instructions for use of microbial agents: Based on 30 kg / m 2 (Based on the dry weight of straw) Apply evenly to the surface of the coal gangue stockpile to be repaired (pH 2.3, Pb content 620 mg / kg, As content 52 mg / kg), and mix it with the surface coal gangue to a depth of 15 cm.

[0036] Example 4

[0037] This embodiment discloses a method for preparing Bacillus airborne bacteria agent for the ecological restoration of coal gangue, the method comprising the following steps: Step 1: Crush agricultural waste rice straw to a length of 5 cm; mix wood vinegar stock solution with water at a volume ratio of 1:7 to prepare wood vinegar dilution; immerse the crushed straw completely in the dilution at a liquid-to-solid ratio of 1:9 (straw dry weight (kg) to dilution volume (L)) for 20 h; remove the straw and air dry it at 28°C under ventilation for 65 h to obtain activated straw organic material.

[0038] Step 2: (1) Expand the liquid fermentation culture of white rot fungi to the effective viable count. (2) Solid bacterial agent was prepared by adsorbing CFU / mL onto vermiculite with a particle size of 1-3 mm; (3) Bacillus aerogenes was fermented and cultured to the effective viable count. (3) Prepare solid spore powder by spray drying with CFU / mL; (4) Expand the methyl nutrient bacteria by liquid fermentation to the effective viable count. CFU / mL, adsorbed onto vermiculite with a particle size of 1-3 mm to prepare a solid bacterial agent; (4) liquid fermentation and expansion of dark-colored septate endophytic fungi to prepare an effective inoculum (active mycelial fragments and spores) with a total amount of CFU / mL. A bacterial suspension of 100 cells / mL was adsorbed onto vermiculite with a particle size of 1–3 mm and dried to obtain a solid bacterial agent.

[0039] Step 3: Select particles with a diameter not exceeding 74 μm. Natural apatite powder with a content of 33% and heavy metal content meeting the requirements of GB / T 23349 is mixed evenly with activated straw organic material at a mass ratio of 1:8 (apatite: straw dry weight) to obtain activated straw mixture containing apatite.

[0040] Step 4: Evenly inoculate the solid inoculants of each functional microorganism prepared in Step 2 into the activated straw mixture containing apatite obtained in Step 3. After inoculation, the effective viable count of each functional component reaches: white rot fungi CFU / g dry weight of straw, Bacillus aerogenes CFU / g straw dry weight, methyl nutrient bacteria CFU / g dry straw weight, dark-colored septate endophytic fungal effective inoculum Bacillus aeruginosa inoculum (BSA) was prepared by mixing thoroughly and evenly with a gram of dry straw. Instructions for use of microbial agents: Based on 25 kg / m 2 (Based on the dry weight of straw) Apply evenly to the surface of the coal gangue stockpile to be repaired (pH 3.0, Cu content 185 mg / kg, Cd content 28 mg / kg), and mix it with the surface coal gangue to a depth of 13 cm.

[0041] Example 5

[0042] This embodiment discloses a method for preparing and using Bacillus airborne bacteria agent for the ecological restoration of coal gangue. The method includes the following steps: Step 1: Pretreatment of straw organic materials with wood vinegar Agricultural waste wheat straw was crushed to a length of 3 cm to increase the surface area accessible to microorganisms. Wood vinegar stock solution was mixed with water at a volume ratio of 1:8 to prepare a diluted wood vinegar solution. The crushed straw was completely immersed in the diluted solution at a liquid-to-solid ratio of 1:10 (straw dry weight (kg) to diluted solution volume (L)) for 18 hours to allow the effective components of the wood vinegar to fully penetrate the straw. After immersion, the straw was removed and allowed to stand and dry for 60 hours under good ventilation at 25°C until low-boiling-point volatile toxic components such as methanol were fully released, thus obtaining activated straw organic material.

[0043] The above pretreatment steps have the following effects: The organic acids, such as acetic acid, in the wood vinegar penetrate the straw and dissolve its surface wax layer, reducing the hydrophobic barrier of the wax layer to the invasion of functional microorganisms; the weakly acidic environment (pH 3-5) of the diluted solution causes moderate swelling of the straw lignin-cellulose complex structure, increasing the accessible surface area of ​​cellulose and hemicellulose, which is beneficial for the subsequent invasion and degradation by cellulose-degrading bacteria; the phenolic substances in the wood vinegar have an inhibitory effect on miscellaneous bacteria on the straw surface, reducing the competitive pressure of miscellaneous bacteria before inoculation with functional microorganisms; the aforementioned phenolic substances remain in the dried straw at low concentrations, which can serve as precursors for humification and polymerization reactions. The ventilation and drying step is a necessary prerequisite for the subsequent inoculation of various functional microorganisms, effectively avoiding the direct toxic inhibition of functional microbial communities by residual methanol in the wood vinegar.

[0044] Step 2: Preparation and compounding of functional microbial agents After the activated straw organic material has dried, the following functional microbial components are compounded in the form of liquid bacterial suspension or solid carrier inoculum, and applied to the coal gangue together with the activated straw organic material and apatite powder: (1) Preparation of lignin-degrading fungi Propagation to an effective viable cell count of not less than [number missing] using conventional liquid fermentation processes. CFU / mL, prepare bacterial suspensions or solid inoculum by adsorption onto vermiculite (particle size 1-3 mm) solid carrier. After being applied to coal gangue, the white-rot fungi colonize and degrade the activated straw in the organic micro-domain (pH approximately 4-6) formed by the activated straw organic material; lignin peroxidase and manganese peroxidase... As a co-substrate, straw lignin is continuously oxidized and cleaved to produce humic precursors (phenolic compounds, low molecular weight organic acids, lignin-modified fragments, etc.); laccase is used as... As an electron acceptor, it catalyzes the single-electron oxidation of the above-mentioned phenolic humic precursors to generate phenolic oxygen free radicals. These phenolic oxygen free radicals, as active intermediates for copolymerization with dihydroxynaphthalene-based melanin in the hyphal wall of dark-colored septate endophytic fungi, directly participate in the subsequent formation of melanin-humic complexes.

[0045] (2) Preparation of Bacillus aeruginosa Propagate using conventional Bacillus liquid fermentation process until the effective viable count is not less than [number missing]. CFU / mL, or prepared as a solid spore powder. After being applied to coal gangue, *Bacillus aeruginosa* performs the following two core functions: First, it secretes lipopeptide antibacterial active substances (such as ituronidin and surfactants) to antagonize other bacteria, maintaining the dominant position of the functional microbial community in the extreme coal gangue environment and ensuring the stability of the microbial community throughout the process; Second, it colonizes in the organic material microdomain (pH 4–6) and secretes organic acids such as citric acid and oxalic acid. These secreted organic acids diffuse outwards to the acidic environment of coal gangue (pH 2–5), continuously dissolving apatite through bio-phosphorus solubilization, driving phosphate ions to... The continuous release of phosphorus provides a source of energy for the anchoring of humic minerals and a continuous supply of phosphorus nutrition to various functional bacteria.

[0046] (3) Preparation of methyltrophic bacteria Propagation to an effective viable cell count of not less than [number missing] using conventional liquid fermentation processes. CFU / mL, bacterial suspensions were prepared or solid bacterial agents were prepared by adsorption onto vermiculite (particle size 1-3 mm) solid carriers. Methyltrophic bacteria, after being applied to coal gangue, used the trace amounts of methanol remaining in the dried straw organic material as a carbon source for efficient aerobic metabolism, degrading methanol into... and This approach eliminates the cumulative toxicity of methanol to dark-colored septate endophytic fungi from the perspective of material sources. At the same time, methyltrophic bacteria synthesize and secrete cytokinin-like active substances (mainly trans-zeatin type) during methanol metabolism. Under the initial conditions of bare coal gangue remediation without pioneer plant hosts, this maintains and promotes the melanin synthesis activity of dark-colored septate endophytic fungi, ensuring that the humic stabilization function of dark-colored septate endophytic fungi can be operational during the remediation initiation stage.

[0047] (4) Preparation of dark-colored septate endophytic fungi Propagation using conventional fungal liquid fermentation processes should yield an effective inoculum (active mycelial fragments and spores) with a total amount not less than [amount not specified]. A bacterial suspension of 1 / mL was prepared by adsorbing the above bacterial suspension onto a vermiculite (particle size 1-3 mm) solid carrier and drying it. Under the continuous promotion of cytokinins secreted by methyltrophic bacteria, dark-colored septate endophytic fungi maintained a high level of melanin synthesis (the main component of melanin in dark-colored septate endophytic fungi is dihydroxynaphthalene polymer, i.e., dihydroxynaphthalene melanin, which is biosynthesized through the polyketide synthesis pathway); the synthesized melanin undergoes a copolymerization reaction with humic precursors (phenolic compounds, etc.) produced by cellulose-degrading bacteria to form a melanin-humic complex; this complex has a highly cross-linked aromatic ring skeleton structure, and its chemical stability and resistance to mineralization degradation are significantly higher than those of conventional humic substances.

[0048] Step 3: Compounding of apatite powder Select apatite powder from natural mineral sources ( Particle size not greater than 74 μm The content is not less than 30%, and the content of heavy metals such as Cd, Pb, and As meets the requirements of GB / T 23349 for heavy metal limits in mineral-derived fertilizers. It is mixed with the activated straw organic material obtained in step one at a mass ratio of 1:7 (apatite: straw dry weight) to obtain an activated straw mixture containing apatite, which is then set aside.

[0049] Step 4: Preparation and application of Bacillus aeruginosa inoculant The functional microbial agents prepared in step two were uniformly inoculated into the activated straw mixture containing apatite prepared in step three. After inoculation, the effective viable count of each functional component in the mixture was ensured to reach: white-rot fungi not less than CFU / g straw dry weight, airborne Bacillus not less than CFU / g straw dry weight, methyltrophic bacteria not less than The effective inoculum (active hyphal fragments and spores) of dark-colored, septate endophytic fungi at a concentration of CFU / g dry straw weight is not less than [amount missing]. Bacillus aeruginosa inoculum was prepared by mixing thoroughly and evenly with 1 / g dry weight of straw; the inoculum was then applied at a concentration of 20 kg / m³. 2 (Based on the dry weight of straw) It is evenly applied to the surface layer of the coal gangue dump to be repaired and mixed with the surface coal gangue to a depth of 12 cm. The functional components work synergistically in the coal gangue environment in the following functional order to promote the humification process of coal gangue: (1) Methyltrophic bacteria use the trace amounts of methanol remaining in the activated straw mixture as a carbon source for efficient aerobic metabolism, degrading methanol into and It eliminates the cumulative toxicity of methanol to dark-colored septate endophytic fungi, while simultaneously synthesizing and secreting cytokinin-like active substances. (2) The cytokinins secreted by methyl-trophic bacteria promote the metabolic activity of dark-colored septate endophytic fungi hyphae, enabling dark-colored septate endophytic fungi to maintain a high level of melanin synthesis activity under the conditions of bare coal gangue land without pioneer plant hosts. (3) Lignin-degrading fungi (white rot fungi) colonize in the microdomain of organic materials (pH 4-6), and degrade and activate straw lignin through lignin peroxidase, manganese peroxidase and laccase to produce humic precursor substances (phenolic compounds, low molecular weight organic acids, lignin-modified fragments, etc.). (4) The melanin (dihydroxynaphthalene-based melanin) synthesized and accumulated in the hyphal wall by dark-colored septate endophytic fungi undergoes a copolymerization reaction with the humic precursor produced in step (3) to form a melanin-humic complex with a highly cross-linked aromatic ring skeleton structure. Its chemical stability and resistance to mineralization degradation are significantly higher than those of conventional humic substances. (5) Airborne Bacillus colonizes in the microdomain of organic materials (pH 4-6), secreting organic acids such as citric acid and oxalic acid, which diffuse outwards to the acidic environment of coal gangue (pH 2-5), continuously dissolving apatite and releasing The released With the large amount of iron oxides present in coal gangue ( , ) and aluminum oxide surface , Active sites undergo coordination binding, while the humic substances (including melanin-humic substance complexes) formed in step (4) are adsorbed onto the surface of the aforementioned metal oxide minerals, together forming a humic substance-iron (aluminum)-phosphate ternary mineral anchoring complex. This firmly binds the humic substances to the surface of the coal gangue iron / aluminum oxide minerals through coordination bonds, effectively preventing the loss of humic substances caused by rain leaching; the apatite dissolution process consumes It plays a partial neutralizing and buffering role in the extremely acidic environment of coal gangue, which helps to alleviate the stress of extreme acidity on functional microorganisms; the phosphorus continuously released by apatite provides phosphorus nutrition for various functional microorganisms. (6) Airborne Bacillus continuously secretes lipopeptide antibacterial active substances (such as isochorin and surfactant) throughout the process to antagonize other bacteria and maintain the dominant position of each functional group.

[0050] Experimental verification Experiment 1: The effect of wood vinegar pretreatment combined with compound microbial agent on the humification efficiency of coal gangue 1. Experimental Objective The study verified the synergistic effect of wood vinegar pretreatment on straw activation and the application of compound microbial agents on the accumulation of humic acid in coal gangue, demonstrating that the combined use of the two techniques (this invention) has a significantly higher humification efficiency than either treatment alone.

[0051] 2. Preparation of experimental samples Using the coal gangue (pH 2.8, Cd content 42 mg / kg, As content 35 mg / kg) used in Example 1 as the remediation matrix, four treatment groups were set up, with three replicates for each group: (1) CK (blank control): Untreated wheat straw (crushed to 3 cm) was subjected to a 20 kg / m³ feedstock treatment. 2 Coal gangue is directly applied without inoculating any microorganisms. Apatite powder is added at a ratio of 1:7 (apatite: dry weight of straw) and mixed with the surface coal gangue to a depth of 12 cm.

[0052] (2) T1 (Wood vinegar pretreatment only): The straw was pretreated according to the steps in Example 1 (wood vinegar stock solution: water = 1:8, soaked for 18 h, and dried at 25°C for 60 h) to obtain activated straw. Apatite powder (1:7) was added, and no microorganisms were inoculated. The straw was then treated at 20 kg / m³. 2 Add coal gangue and mix it to a depth of 12 cm.

[0053] (3) T2 (compound microbial agent only, untreated straw): Wheat straw (crushed to 3cm) that has not been pretreated with wood vinegar is mixed with apatite powder (1:7), and inoculated according to the inoculation density of each functional microorganism in step four of Example 1 (white rot fungi). CFU / g, Bacillus aerogenes CFU / g, Methyltrophic Bacteria CFU / g, effective inoculum of dark-colored septate endophytic fungi (pieces / g), at 20 kg / m 2 Add coal gangue and mix it to a depth of 12 cm.

[0054] (4) T3 (This invention is the same as in Example 1): Prepare the Bacillus aerogenes inoculum according to steps one to four of Example 1, at 20 kg / m 2 Add coal gangue and mix it to a depth of 12 cm.

[0055] 3. Experimental conditions Under natural light and with regular watering to maintain 60% field water holding capacity, the plants were cultivated for 56 days in an outdoor environment simulating a coal gangue dump, without the addition of any exogenous nutrients.

[0056] 4. Experimental Procedure (1) Before the experiment, coal gangue samples (0-12 cm depth) were taken to determine the initial humic acid content. The initial value was 0.6 g / kg dry weight.

[0057] (2) On days 14, 28, 42 and 56, 0-12 cm mixed soil samples were taken from each of the three replicates in each treatment group, air-dried and passed through a 2 mm sieve for humic acid extraction.

[0058] (3) Humic acid extraction: Weigh 5.0 g of air-dried sample, add 100 mL of 0.1 mol / L NaOH (containing 0.1 mol / L Na4P2O7), shake and extract for 24 h, centrifuge at 4000 r / min for 20 min, and collect the supernatant; add concentrated hydrochloric acid to the supernatant to adjust the pH to 1.0, let stand for 12 h, centrifuge and collect the precipitate, dry at 50°C and weigh, convert to g / kg dry weight, and calculate the net increment after deducting the initial background value.

[0059] 5. Experimental Results Table 1. Changes in net humic acid accumulation in each treatment group over cultivation time (unit: g / kg dry weight of coal gangue, after deducting the initial background value of 0.6 g / kg) Figure 1 A grouped bar chart showing the change in net humic acid accumulation in each treatment group over cultivation time.

[0060] 6. Analysis and Summary The net accumulation of humic acid in treatment T3 (in this invention) on day 56 (5.8 g / kg) was 2.9 times that of T1 (2.0 g / kg), 1.8 times that of T2 (3.2 g / kg), and 5.3 times that of CK (1.1 g / kg), representing a 427% increase relative to CK. This demonstrates a significant positive synergy between the activation of the waxy layer on the straw surface and the humification function of the compound microbial agent in the wood vinegar pretreatment.

[0061] The net accumulation of T3 on day 14 (1.2 g / kg) was on par with the level of T1 on day 28 (1.2 g / kg), and significantly exceeded the level of CK on day 28 (0.6 g / kg) by more than 2 times, indicating that the microbial agent of the present invention can significantly accelerate the initiation speed of the humification process.

[0062] Experiment 2: Verification of Methyltrophic Bacteria Eliminating Methanol Toxicity and Activating Melanin Synthesis in Dark-Colored Septate Endophytic Fungi 1. Experimental Objective The invention verifies the dual function of methyl-trophic bacteria in the micro-domain of straw organic materials pretreated with wood vinegar: degrading residual methanol to eliminate its metabolic toxicity to dark-colored septate endophytic fungi, and secreting trans-zeatin-type cytokinins during methanol metabolism to promote melanin synthesis in dark-colored septate endophytic fungi. This demonstrates that the invention eliminates the inherent antagonistic contradiction between the activation benefits of wood vinegar and the humic stabilization function of dark-colored septate endophytic fungi through methyl-trophic bacteria.

[0063] 2. Preparation of experimental samples Based on the wheat straw and coal gangue (pH 2.8) used in Example 1, three treatment groups (each with three replicates) were established according to differences in drying degree, and incubated in an indoor constant temperature (28°C) incubator for 28 days. Note: This experiment is a single-factor mechanism verification experiment. Each treatment group contained only methyltrophic bacteria and / or dark-colored septate endophytic fungi, deliberately excluding white-rot fungi and airborne Bacillus, in order to verify the causal chain of methyltrophic bacteria eliminating methanol toxicity and activating melanin synthesis in dark-colored septate endophytic fungi on a simplified substrate basis. (1) G1 (Methanol residue, only dark-colored septate endophytic fungi): After straw was pretreated with wood vinegar (1:8 dilution, soaking for 18h), it was dried at 25°C for only 12h (methanol was not fully dissipated, the initial methanol concentration in the organic material micro-domain was about 12.5 μL / g dry material), without inoculation with methyl-trophic bacteria, only with solid inoculum of dark-colored septate endophytic fungi (effective inoculum). (each grain / g dry weight of straw), add apatite powder (1:7), at 20 kg / m³ 2 Coal gangue was added.

[0064] (2) G2 (methanol residue, containing methyl-trophic bacteria and dark-colored septate endophytic fungi): The straw pretreatment and drying conditions were the same as G1 (initial methanol concentration was about 12.5 μL / g). Simultaneously, solid inoculum of methyl-trophic bacteria (inoculation density was the same as in Example 1) and solid inoculum of dark-colored septate endophytic fungi (same as in Example 1) were inoculated, and apatite powder (1:7) was added at 20 kg / m³. 2 Coal gangue was added.

[0065] (3) G3 (fully dried, only dark-colored septate endophytic fungi, methanol-free, cytokinin-free baseline group): After straw pretreatment, it was fully dried at 25°C for 60 h (methanol was fully dissipated, initial methanol concentration was about 0.1 μL / g), without inoculation with methyl-trophic bacteria, only inoculated with dark-colored septate endophytic fungi (same as in Example 1), and apatite powder (1:7) was added at 20 kg / m³. 2 Coal gangue was added.

[0066] 3. Experimental conditions The temperature was kept constant at 28°C, and the organic material sampling bottles were sealed during the incubation period for headspace methanol analysis. Apatite powder was added to each treatment group at a ratio of 1:7 to maintain consistency with Example 1, and organic material samples were taken every 7 days.

[0067] 4. Experimental Procedure (1) Methanol concentration determination (headspace gas chromatography): 1.0 g of organic material was placed in a 20 mL headspace vial, equilibrated at 60°C for 30 min, and 200 μL of headspace gas was injected into the gas chromatograph (capillary column HP-5, flame ionization detector, column temperature 40°C isothermal). The quantification was performed using the standard curve method. The unit was μL / g dry material.

[0068] (2) Extraction and determination of melanin from dark-colored septate endophytic fungi: Dark-colored septate endophytic fungal hyphae were picked from organic materials, 50 mg were weighed after freeze-drying, 1 mL of 1 mol / L NaOH was added, and the mixture was extracted in an 80°C water bath for 2 h. After centrifugation at 12000 r / min for 10 min, the absorbance of the supernatant was measured at 206 nm. The melanin content was converted to the standard curve of dihydroxynaphthalene melanin. The unit is mg / g of dry hyphae.

[0069] (3) Detection of trans zeatin (enzyme-linked immunosorbent assay): Organic material extract (0.01 mol / L phosphate buffer, pH 7.0) was collected on day 14 and day 28 and quantified using a trans zeatin enzyme-linked immunosorbent assay kit (detection limit 0.5 pmol / mL). Unit: pmol / mL.

[0070] 5. Experimental Results Table 2 Dynamic changes in methanol concentration in micro-fields of organic materials (unit: μL / g dry material) Table 3. Dynamic changes in melanin content in the hyphal walls of dark-colored septate endophytic fungi (unit: mg / g dry hyphae) Table 4. Concentration of trans-zeatin-type cytokinins (enzyme-linked immunosorbent assay, unit: pmol / mL) Figure 2 This is a graph showing the dynamic changes in methanol concentration in a micro-domain of organic materials.

[0071] Figure 3 This is a graph showing the dynamic changes in melanin content in the hyphal walls of dark-colored, septate endophytic fungi in the microdomains of organic materials.

[0072] 6. Analysis and Summary From Table 2 and Figure 2 It can be seen that the methanol concentration in G2 decreased from 12.5 μL / g to 3.1 μL / g on day 7 and to 0.5 μL / g on day 14 (close to the background level of G3), while G1 maintained 7.1 μL / g until day 28, confirming that methyltrophic bacteria can efficiently eliminate methanol residue in the microdomains of organic materials.

[0073] From Table 3, Table 4 and Figure 3 It can be seen that the melanin content of dark-colored septate endophytic fungi in G1 decreased from 0.42 mg / g to 0.36 mg / g, confirming the inhibitory effect of methanol on the melanin synthesis enzyme system; the melanin content of G3 (without methanol and cytokinin) increased to 0.95 mg / g; and the melanin content of G2 increased to 1.31 mg / g, which was significantly higher than that of G3. Moreover, the trans-zeatin concentration in the organic material extract of G2 reached 18.4 pmol / mL on day 14 (both G1 and G3 were below the detection limit), proving that methyltrophic bacteria secreted cytokinin while metabolizing methanol, which had an additional promoting effect on the melanin synthesis of dark-colored septate endophytic fungi.

[0074] Experiment 3: Verification of the chemical stability of the melanin-humic complex 1. Experimental Objective The melanin-humic complex formed by the copolymerization of dihydroxynaphthalene-based melanin synthesized by dark-colored septate endophytic fungi and humic precursors exhibits a significantly lower carbon mineralization rate and superior humic component characteristics compared to conventional humic substances, demonstrating the improvement effect of this invention on the quality and long-term stability of humic substances.

[0075] 2. Preparation of experimental samples Two groups of humus samples, S1 (conventional humus, control) and S2 (melanin-humus complex, this invention), were prepared independently. After 56 days of cultivation under simulated coal gangue dump conditions (temperature 20–28°C, natural light, field water holding capacity 60%), samples were extracted: S1 was prepared according to steps one through four of Example 1, but omitting the dark-colored septate endophytic fungus – 3 cm of wheat straw, diluted 1:8 with wood vinegar, soaked for 18 h, air-dried at 25°C for 60 h, and inoculated with white rot fungi using apatite:straw dry weight = 1:7. CFU / g, Bacillus aerogenes CFU / g, Methyltrophic Bacteria CFU / g, without inoculation with dark-colored septate endophytic fungi, at 20 kg / m 2 Apply coal gangue at pH 2.8, mix to a depth of 12 cm, and repeat 3 times for each step; S2 is performed according to steps one through four of Example 1, with all parameters being completely consistent with Example 1 (including effective inoculum of dark-colored septate endophytic fungi). The samples were collected from each treatment group (0–12 cm depth) and extracted with 0.1 mol / L NaOH (containing 0.1 mol / L Na4P2O7) by shaking for 24 h. The supernatant was collected by centrifugation (4000 r / min, 20 min), and desalted by dialysis (dialysis bag with a molecular weight cutoff of 3500 Da, dialysis with deionized water for 48 h, with water changed every 12 h). The supernatant was then freeze-dried to prepare humic powder. The only variable between S1 and S2 was whether or not dark-colored septate endophytic fungi were inoculated, which can accurately reflect the independent contribution of melanin copolymerization by dark-colored septate endophytic fungi to the stability of humic powder.

[0076] 3. Experimental conditions Carbon mineralization cultivation experiment: The substrate was kept at a constant temperature of 25°C in the dark, and the moisture content was adjusted to 70% of field capacity. The substrate was cultivated for 90 days, and samples were collected every 7 days. (Absorption by 0.5 mol / L NaOH alkaline solution). Scanning electron microscopy characterization: Field emission scanning electron microscopy, accelerating voltage 5 kV, observed after gold sputtering treatment of the sample surface.

[0077] 4. Experimental Procedure (1) Determination of carbon mineralization rate: Accurately weigh 0.200 g each of S1 and S2 (initial carbon content was pre-calibrated by an elemental analyzer), place them in a 100 mL wide-mouth bottle containing 2.0 g of sterilized quartz sand, adjust the water content, connect an alkali absorption tube containing 20 mL of 0.5 mol / L NaOH, and seal for incubation. Replace the alkali solution every 7 days, titrate with 0.5 mol / L HCl, and calculate the value at each time point. Release amount, cumulatively calculated 90-day carbon mineralization rate (%).

[0078] (2) Grading of humic components: Humic acid (precipitated by HCl after NaOH extraction) and fulvic acid (the part that does not precipitate by HCl) were separated according to the standard humic grading method of the International Humic Society, and quantified separately (g / kg) and the humic acid / fulvic acid ratio was calculated.

[0079] (3) Determination of humic aroma: Each sample was dissolved in 0.05 mol / L NaHCO3 buffer (pH 8.0), and the absorbance at 280 nm and 600 nm was measured using a UV-Vis spectrophotometer. The ratio represents the degree of aromatization.

[0080] (4) Field emission scanning electron microscopy characterization: freeze-dried humic powder was coated with conductive adhesive and then sputtered with gold. The surface micromorphology was observed and recorded under a field emission scanning electron microscope at 5000x and 10000x magnification, respectively.

[0081] 5. Experimental Results Table 5 Comparison of carbon mineralization rates between S1 and S2 (after 90 days of cultivation) Table 6 Comparison of Humic Component Characteristics between S1 and S2 Figure 4 A comparison chart of carbon mineralization rates for S1 and S2; Figure 5 A comparison diagram of the characteristics of humic components in S1 and S2.

[0082] Figure 6 Comparison of the microscopic morphology of S1 (conventional humus) and S2 (melanin-humus complex) under field emission scanning electron microscopy (10,000x).

[0083] Figure 6This image illustrates the differences in surface micromorphology between two humic samples at 10,000x magnification: the left image (conventional humic material) shows loose, amorphous organic aggregates with a smooth surface and particle sizes of 1–5 μm; the right image (melanin-humic material complex) shows dense, cross-linked aromatic polymer aggregates with a rough surface, containing fragments of melanized fungal hyphae with diameters of 2–3 μm, and exhibiting abundant nanoscale micropores, resulting in a more compact overall structure. Image scale bar: 2 μm; grayscale backscattered electron imaging mode.

[0084] 6. Analysis and Summary The 90-day carbon mineralization rate of S2 (melanin-humic complex) (8.9%) was only 23.3% of that of S1 (conventional humic matter, 38.2%), indicating an approximately 4.3-fold increase in resistance to mineralization degradation. The humic acid / fulvic acid ratio of S2 (3.20) was 5.7 times that of S1 (0.56), and the aroma index (0.31) was 3.9 times that of S1 (0.08), indicating that the introduction of dihydroxynaphthalene-based melanin significantly increased the proportion of high molecular weight stable components and the overall aromatization degree in humic matter.

[0085] Field emission scanning electron microscope images ( Figure 6 The study visually demonstrated the dense cross-linked microstructure of S2, which contrasted sharply with the loose amorphous morphology of S1, thus confirming from the perspective of material structure that melanin-humic matter copolymerization fundamentally improves the stability of humic matter.

[0086] Experiment 4: The inhibitory effect of apatite in the formation of ternary mineral anchoring complexes on humic substance leaching by rainfall. 1. Experimental Objective Verification of phosphorus release from apatite via bio-solubilization After forming a ternary mineral anchoring complex of humic-iron (aluminum)-phosphate with iron / aluminum oxides and humic substances from coal gangue, it can significantly reduce the carbon loss of humic substances caused by rainfall leaching, proving the key anchoring role of apatite components in the long-term accumulation of humic substances.

[0087] 2. Preparation of experimental samples Based on the parameters of Example 1, two treatment groups were set up (each with 3 replicates). A polyvinyl chloride experimental column (inner diameter 20 cm, height 30 cm, bottom filled with 18 cm thick coal gangue) was used; the treatment material was prepared at 20 kg / m³. 2 The application amount was evenly spread on top of the coal gangue, and then mixed to a depth of 12 cm to form a 12 cm mixed treatment layer; the total filling depth of the column was 30 cm, which constituted the experimental setup. (1) Group A (control without apatite): Bacillus aerogenes inoculum was prepared according to steps one to four of Example 1, but apatite powder was omitted. Other parameters (wheat straw 3 cm, wood vinegar diluted 1:8, soaking for 18 h, drying for 60 h, inoculation density and application rate of each functional microorganism 20 kg / m²) 2 All of these are completely consistent with Example 1, with coal gangue (pH 2.8) being added and mixed to a depth of 12 cm.

[0088] (2) Group B (containing apatite, same as in Example 1): Bacillus aeruginosa inoculum (containing apatite powder, apatite: straw dry weight = 1:7) was prepared exactly according to Example 1, at 20 kg / m 2 Add coal gangue (pH 2.8) and mix to a depth of 12 cm.

[0089] After two groups were pre-cultivated outdoors for 28 days (to initiate the humification process and form an initial humus reserve), the leaching experiment with rainfall began.

[0090] 3. Experimental conditions Simulated rainfall leaching apparatus: An outlet was installed at the bottom of the experimental column to collect the leachate. Each simulated rainfall event was 50 mm in intensity (approximately 1.57 L of water was uniformly sprayed onto the column surface), with a 14-day interval between rainfall events, for a total of 8 rainfall leaching events, lasting 16 weeks. After each rainfall event, all leachate (approximately 1.5 L / column) was collected, with the column cross-sectional area (0.031 m²) as the basis for the leaching. 2 This translates to approximately 48 L / m² of leachate. 2 .

[0091] 4. Experimental Procedure (1) After 28 days of outdoor pre-cultivation under experimental conditions, the leaching experiment was started.

[0092] (2) Collect all leachate immediately after each rainfall, filter it through a 0.45 μm filter membrane, and determine the dissolved organic carbon concentration (mg / L) using a total organic carbon analyzer (high temperature catalytic oxidation-non-dispersive infrared detection method).

[0093] (3) With a leaching liquid volume of 48 L / m 2 Calculate the amount of humic carbon lost (g / m³) during each rainfall event. 2 The total loss from the eight leaching processes was calculated cumulatively.

[0094] (4) After the 8th leaching, take the surface coal gangue (0-5 cm) of the experimental column of Group B and characterize the elemental distribution of the organic-inorganic complex on the mineral surface by field emission scanning electron microscopy combined with energy dispersive spectroscopy to observe the spatial co-distribution characteristics of C, P and Fe on the mineral surface.

[0095] 5. Experimental Results Table 7. Concentration of dissolved organic carbon and cumulative carbon loss of humic substances in leachate from each rainfall event. Figure 7 Line graph comparing the concentration of dissolved organic carbon in leachate from different rainfall events; Figure 8 Line graph comparing the cumulative carbon loss of humic substances from different rainfall events.

[0096] Figure 9 The image shows the elemental distribution of the surface by field emission scanning electron microscopy combined with energy dispersive X-ray spectroscopy; it also shows the elemental co-distribution characteristics of the humic-iron (aluminum)-phosphate ternary anchored complex on the surface of group B coal gangue minerals.

[0097] Figure 9 It consists of a main image on the left (backscattered electron imaging, 5000x, scale bar 5 μm) and four elemental surface distribution maps on the right (arranged in a 2×2 pattern): the left image shows goethite (FeOOH) and hematite ( The organic-mineral interface morphology is shown in the image. The Fe element map (red) shows the core enrichment of mineral particles, the C element map (green) shows the continuous distribution of humic substances along the mineral surface, the P element map (blue) shows that phosphate ions are precisely co-located at the organic-mineral interface, and the Fe-CP superimposed image (red / green / blue) visually demonstrates the interface co-distribution characteristics of the ternary mineral anchoring complex.

[0098] 6. Analysis and Summary After eight simulated rainfall leaching events, Group A (without apatite) experienced a cumulative humic carbon loss of 35.61 g / m³. 2 Group B (containing apatite, Example 1 of this invention) had a concentration of only 12.15 g / m³. 2 The carbon leaching loss of humic substances decreased by 65.9%. The dissolved organic carbon concentration in the leachate of Group A was as high as 142 mg / L in the first leaching and remained at 68 mg / L until the eighth leaching, indicating that humic substances continued to be lost in large quantities. The dissolved organic carbon concentration in Group B decreased rapidly and tended to stabilize from the fourth leaching (only 10 mg / L in the eighth leaching), indicating that humic substances were effectively fixed on the surface of the minerals after the formation of ternary mineral anchoring complexes.

[0099] Energy dispersive X-ray spectroscopy elemental distribution map ( Figure 9 This experiment visually demonstrates the spatial co-distribution of Fe, C, and P elements at the mineral surface interface, confirming the formation of a ternary mineral anchored complex of humic substances, iron (aluminum), and phosphate. This experiment also proves that apatite components are released through bio-solubilization. It forms a ternary mineral anchoring complex with iron / aluminum oxides and humic substances, which can reduce the carbon leaching loss of humic substances by about 2 / 3 and plays a key role in increasing the net accumulation of humic substances in coal gangue remediation areas.

[0100] The embodiments of the present invention have been described above. However, the embodiments are not limited to the specific implementation methods described above. The specific implementation methods described above are merely illustrative and not restrictive. Those skilled in the art can make more equivalent embodiments under the guidance of the present embodiments, and all of them are within the protection scope of the present embodiments.

Claims

1. A method for preparing an *Bacillus aeruginosa* inoculant for the ecological restoration of coal gangue, characterized in that, Includes the following steps: After crushing the straw, soak it in diluted wood vinegar solution for no less than 12 hours, and then air dry it for no less than 48 hours to obtain activated straw organic material. Each functional microbial agent was prepared by expanding and culturing white-rot fungi, airborne Bacillus, methyl nutrient bacteria and dark-colored septate endophytic fungi respectively. Apatite powder and the activated straw organic material are mixed at a mass ratio of 1:5 to 1:10 and then inoculated with the functional microbial agents to obtain Bacillus aerogenes agent; Among them, methyl-trophic bacteria are used to metabolize and degrade residual methanol in activated straw and secrete cytokinins to eliminate the metabolic toxicity of methanol to dark-colored septate endophytic fungi and activate the melanin synthesis of dark-colored septate endophytic fungi. White-rot fungi are used to degrade straw lignin into humic precursors; dark-colored septate endophytic fungi are used to synthesize melanin and copolymerize with the humic precursors to form a melanin-humic complex. Airborne Bacillus secretes organic acids to dissolve apatite and release dihydrogen phosphate ions, forming a humic-iron (aluminum)-phosphate ternary mineral anchoring complex with iron / aluminum oxides and humic substances in coal gangue, and secretes lipopeptide antibacterial active substances to antagonize miscellaneous bacteria.

2. The preparation method according to claim 1, characterized in that, The straw is wheat straw, and the length of the crushed straw is 3 cm; the wood vinegar dilution is prepared by mixing wood vinegar stock solution and water at a volume ratio of 1:

8.

3. The preparation method according to claim 1, characterized in that, The white-rot fungi are cultured using a liquid fermentation process to achieve an effective viable count of not less than [amount missing]. Solid bacterial agents were prepared by adsorption using vermiculite with a particle size of 1–3 mm as a solid carrier at a concentration of CFU / mL.

4. The preparation method according to claim 1, characterized in that, The *Bacillus aerobicans* was cultured using a liquid fermentation process to achieve an effective viable count of not less than [amount missing]. CFU / mL.

5. The preparation method according to claim 1, characterized in that, The methyltrophic bacteria are cultured using a liquid fermentation process to achieve an effective viable cell count of not less than [amount missing]. CFU / mL.

6. The preparation method according to claim 1, characterized in that, The dark-colored septate endophytic fungus is cultured using a liquid fermentation process, and the total effective inoculum is not less than [amount missing]. A bacterial suspension of cells / mL.

7. The preparation method according to claim 1, characterized in that, The particle size of the apatite powder is no greater than 74 μm, and the mass ratio of the apatite powder to the activated straw organic material is 1:

7.

8. The preparation method according to claim 1, characterized in that, The white-rot fungi oxidize and cleave straw lignin into phenolic compounds by secreting lignin peroxidase and manganese peroxidase, while simultaneously secreting laccase to... The phenolic compounds are catalyzed by electron acceptors to undergo single-electron oxidative polymerization, forming humic precursors that participate in the copolymerization reaction of melanin in dark-colored septate endophytic fungi.

9. The preparation method according to claim 1, characterized in that, The cytokinin secreted by the methyl-trophic bacteria is of the trans-zeatin type, which promotes the synthesis of dihydroxynaphthalene polymer melanin in the hyphal wall of dark-colored septate endophytic fungi via the polyketide synthesis pathway.

10. An airborne Bacillus agent for the ecological restoration of coal gangue, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 9.

Images