Coal gangue modifier based on microbial mineral mutual feeding, preparation method and application

Abstract

The present application relates to solid waste resource utilization technical field, especially to coal gangue modifier based on microbial mineral mutual feedback, preparation method and application, the modifier includes four layers gradient solid loading structure, including porous activated coal gangue carrier, deep layer functional microbial flora fixed in the internal pore of porous activated coal gangue carrier, surface layer functional microbial flora fixed on the outer surface of porous activated coal gangue carrier and calcium alginate gel slow-release embedding layer coated outside surface layer functional microbial flora, the modifier is used for coal gangue soil, or heavy metal contaminated soil remediation, or nutrient poor coal gangue substrate improvement, the preparation method is applied to the preparation of the modifier, and the present application realizes the accurate repair of different pollution scenes by the differentiation of microbial flora and gradient solid loading structure.

Description

Technical Field

[0001] This invention relates to the field of solid waste resource utilization technology, and in particular to a coal gangue modifier based on microbial-mineral interaction, its preparation method and application. Background Technology

[0002] Coal gangue is a solid waste generated during coal mining and washing. my country's cumulative stockpile exceeds 7 billion tons, with an annual increase of approximately 300-500 million tons. The large stockpiles of coal gangue not only occupy land resources but also cause serious environmental problems such as spontaneous combustion, release of harmful gases, and leaching of heavy metals. Utilizing coal gangue resources for soil improvement and ecological restoration is an important way to achieve its green disposal.

[0003] Existing technologies for soilification of coal gangue include physical crushing and screening, chemical activation treatment, and microbial fermentation treatment. Among these, microbial fermentation treatment technology has received widespread attention due to its environmental friendliness and low cost. Microorganisms can activate nutrients in coal gangue, promote the release of its fixed nutrients, and increase the available phosphorus content and enzyme activity in the soil. For example, using a double inoculation treatment of coal gangue matrix with phosphate-solubilizing Pseudomonas and dark-colored septate endophytic fungi can increase the available phosphorus content by 38.01% and the available potassium content by 80.00%.

[0004] However, the microbial fermentation treatment technology involves crushing and screening coal gangue, adding organic additives and microbial strains, fermenting it in a set environment, and then converting the carbonaceous material in the coal gangue into humic acid and forming granular structures to form soil. The humic acid also passivates pollutants. Microorganisms secrete EPS to form microcapsules that physically encapsulate heavy metals. Finally, the mixture is aged, decomposed, and granulated. However, the microbial fermentation treatment process still has the following shortcomings: (1) Microorganisms are mostly added directly in a free state, which is easily affected by environmental factors such as pH, temperature, and moisture, resulting in low survival rate and short action time; (2) The interaction between microorganisms and coal gangue is not ideal. The interaction mechanism has not been fully utilized, and there is a lack of engineering regulation of the microbial-mineral interaction relationship. Among them, the microbial-mineral interaction relationship refers to the activation of coal gangue to provide attachment sites and some nutrients for microorganisms. Microorganisms further promote the weathering of coal gangue and the release of nutrients through metabolic activities, such as secreting organic acids and extracellular polymers. The two form a positive cycle enhancement effect. (3) Existing passivation remediation technology lacks differentiated microbial community combination and layered immobilization design based on the type of soil pollution, such as acidic or alkaline or polymetallic soil environment, and corresponding matrix characteristics, resulting in inaccurate remediation effect and low efficiency.

[0005] To address the aforementioned issues, this invention proposes a coal gangue amendment based on microbial-mineral interaction and its preparation method. Functional microorganisms are immobilized within and on the surface of a porous carrier of activated and modified coal gangue through vacuum negative pressure adsorption, forming a four-layer gradient immobilization structure. Microcapsule particles are then encapsulated using calcium alginate gel, establishing a three-layer progressive slow-release system to achieve efficient remediation of various contaminated soils. Summary of the Invention

[0006] The purpose of this invention is to provide a coal gangue modifier based on microbial-mineral interaction and its preparation method, so as to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing a coal gangue amendment based on microbial-mineral interaction includes the following steps: S1: Obtain porous coal gangue, crush and ball mill it, screen out coal gangue powder of appropriate particle size, activate it with an activator, wash it until neutral and dry it to obtain porous activated coal gangue carrier. S2: Prepare deep functional microbial communities and surface functional microbial communities into deep bacterial suspensions and surface bacterial suspensions, respectively. S3: The deep bacterial suspension is fixed in the pores inside the activated porous coal gangue carrier by vacuum negative pressure adsorption, and a loading fixation is performed. S4: The surface bacterial suspension is immobilized on the surface of the porous activated coal gangue carrier that has been initially loaded, and a dense biofilm with a thickness of 5-20 μm is formed on the surface of the porous activated coal gangue carrier that has been initially loaded, and then subjected to secondary loading and immobilization. S5: Prepare the embedding agent solution, place the porous activated coal gangue carrier after secondary loading and fixation into the embedding agent solution and mix, and then drip it into the crosslinking agent solution to harden it, forming coal gangue spherical particles; S6: After removing the spherical coal gangue particles, rinse them with sterile water and place them in a vacuum freeze dryer. Freeze-dry them for 12 to 24 hours at -50 to -30°C and a vacuum degree ≤10Pa to obtain a coal gangue modifier based on microbial-mineral interaction.

[0008] Preferably, the activator in S1 is an alkaline activator or an acidic activator. The alkaline activator uses NaOH or KOH solution with a concentration of 0.5-3 mol / L, an activation temperature of 80-120℃, and an activation time of 3-5 hours. The acidic activator uses a concentration of 5-15%. The activation temperature is 80–100℃ and the activation time is 2–4 hours; or, HCl with a volume fraction of 10–20% is used, the activation temperature is 60–80℃ and the activation time is 1–3 hours.

[0009] Preferably, the deep functional microbial community in S2 includes one or more of phosphate-solubilizing bacteria, nitrogen-fixing bacteria, or silicate bacteria; the surface functional microbial community includes one or more of heavy metal passivating bacteria or dark-colored septate endophytic fungi; the phosphate-solubilizing bacteria can be one or more of Pseudomonas or alkali-resistant Bacillus megaterium; the nitrogen-fixing bacteria can be Azotobacter chrysotile; the heavy metal passivating bacteria are selected from one or more of acid-resistant desulfurization Vibrio, Bacillus subtilis, or exopolysaccharide-producing bacteria.

[0010] Preferably, the vacuum negative pressure adsorption fixation in S3 includes the following steps: placing the porous activated coal gangue carrier in a vacuum adsorption device, evacuating to -0.095 to -0.08 MPa, adding the prepared deep bacterial suspension, with a mass ratio of porous activated coal gangue carrier to deep bacterial suspension of 1:2 to 1:5, and maintaining this for 20 to 30 minutes; then removing the vacuum from the vacuum adsorption device and restoring it to normal pressure for 30 to 60 minutes; repeating the above operation 2 to 3 times, and then filtering and draining the deep bacterial suspension in the vacuum adsorption device to obtain a porous activated coal gangue carrier with a single load fixation.

[0011] Preferably, the specific steps of S4 are as follows: The porous activated coal gangue carrier with primary loading fixation is placed in the surface bacterial suspension, and the mass ratio of the porous activated coal gangue carrier with primary loading fixation to the surface bacterial suspension is 1:2 to 1:4. The carrier is shaken and adsorbed for 30 to 60 minutes at 20 to 35°C and 50 to 100 rpm. Heavy metal inactivating bacteria and dark-colored septate endophytic fungi form a 5 to 20 μm biofilm on the outer surface of the carrier. The surface bacterial suspension is then filtered to obtain the porous activated coal gangue carrier with secondary loading fixation.

[0012] Preferably, in step S5, the embedding agent solution is 200 mL of a 3% (w / w) sterile sodium alginate solution, with 0.3% (w / w) sodium carboxymethyl cellulose added as a dispersant, and the crosslinking agent solution is a 4% (w / w) sterile CaCl2 solution. The porous activated coal gangue carrier loaded with secondary microorganisms was slowly added to the encapsulation agent solution and stirred at 300 r / min for 15 minutes to form a uniform suspension. The suspension was then dripped into a 4% (w / w) sterile CaCl2 solution using a peristaltic pump at a dripping rate of 2-5 drops / second and a dropper height of 5-15 cm from the liquid surface. The solution was allowed to harden for 30-60 minutes to form spherical coal gangue particles.

[0013] A coal gangue modifier based on microbial-mineral interaction is applied to the aforementioned method for preparing a coal gangue modifier based on microbial-mineral interaction. The modifier is a composite particle with a four-layer gradient fixation structure, comprising: First layer: porous activated coal gangue carrier, wherein the porous activated coal gangue carrier has a three-dimensional interconnected pore structure; The second layer consists of deep functional microbial communities fixed in the pores inside the porous activated coal gangue carrier, wherein the pores constitute the pore colonization zone. The third layer consists of surface functional microbial communities fixed on the outer surface of the porous activated coal gangue carrier, forming a dense biofilm with a thickness of 5 to 20 μm on the surface of the porous activated coal gangue carrier. Fourth layer: A calcium alginate gel sustained-release embedding layer covering the outside of the third layer, with a thickness of 100-300 μm.

[0014] The amendment is used for the treatment of coal gangue soil, or for the remediation of metal-contaminated soil; or for the improvement of nutrient-poor coal gangue matrix.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a coal gangue modifier based on microbial-mineral interaction, which is designed with a four-layer gradient immobilization structure. Different microbial communities are colonized in the pores and surface of the carrier. Through a triple slow-release mechanism of gel barrier, pore adsorption and biofilm self-secretion, the effective survival period of functional microbial communities is extended to 60-120 days.

[0016] This invention provides a method for preparing a coal gangue modifier based on microbial-mineral interaction. Applied to the aforementioned microbial-mineral interaction-based coal gangue modifier, it achieves a microbial-mineral interaction mechanism through multi-layer gradient immobilization: the activated coal gangue carrier possesses abundant porous structure and surface active sites, providing physical protection and nutrients (such as Fe, Mg, Ca, etc.) for microorganisms; after colonization, microorganisms secrete organic acids, extracellular polysaccharides, iron carriers, and other substances through metabolic activities, further dissolving the coal gangue mineral lattice and releasing nutrients such as phosphorus, potassium, and sulfur. Simultaneously, it alters the pH and redox potential of the microenvironment, promoting the precipitation or adsorption of heavy metals. This bidirectional promoting effect constitutes a positive feedback loop, significantly improving the sustained action of the modifier; it achieves a synergistic function of "fast response - slow release," avoiding competitive inhibition by a single bacterial community; in particular, the use of vacuum negative pressure adsorption technology to forcibly press microorganisms into the pores inside the porous activated coal gangue carrier significantly improves the immobilization efficiency and uniformity, increasing the microbial loading by more than 50% compared to oscillatory adsorption, and resulting in a more uniform distribution; by adding dispersants and controlling the drop-addition spherical conditions, the problem of carrier dispersion in the encapsulating agent was solved, obtaining coal gangue spherical particles with uniform particle size and regular shape; coal gangue is a bulk solid waste, abundant in source and low in cost; the microbial culture and immobilization process is mature; the preparation process is carried out at room temperature and pressure, with low energy consumption, suitable for industrial-scale production.

[0017] This invention provides an application of a coal gangue amendment based on microbial-mineral interaction. Differentiated microbial community combinations and remediation mechanisms are established for acidic, alkaline, and multi-metal contaminated soils, as well as nutrient-poor coal gangue substrates. For acidic contaminated soils, acid-resistant desulfurizing Vibrio bacteria are selected, with sulfide precipitation as the primary remediation mechanism. For alkaline contaminated soils, alkali-resistant phosphate-solubilizing bacteria and extracellular polysaccharide-producing bacteria are selected, with phosphate precipitation and complexation adsorption as the primary mechanisms. For multi-metal contamination, a synergistic microbial community combination of acid-resistant desulfurizing Vibrio bacteria, extracellular polysaccharide-producing bacteria, and dark-colored septate endophytic fungi is employed. For nutrient-poor coal gangue substrates, nutrient-activating microbial communities such as nitrogen-fixing bacteria, phosphate-solubilizing bacteria, and silicate bacteria are strengthened. Through differentiated microbial community compatibility and gradient immobilization structures, precise remediation of different pollution scenarios is achieved, and adjustments can be made flexibly according to different pollution scenarios. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the preparation method of coal gangue amendment based on microbial-mineral mutual feedback as described in Embodiment 1 of the present invention; Figure 2 This is a curve comparing the survival periods of the amendment and free bacteria in the soil in Example 1 of the present invention. Figure 3 This is a comparison chart of the effective nutrient content of coal gangue substrates with and without additives after 60 days of cultivation in Example 1 of the present invention. Figure 4 This is a comparison chart of the TCLP leaching concentrations of heavy metals in metal-contaminated soils with and without amendments after 28 days of incubation, as shown in Example 1 of the present invention. Figure 5 This is a graph showing the change in the cumulative release rate of microorganisms with culture time when the modifier is applied in Example 1 of the present invention. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] In the following description of the invention, it should be noted that the terms "upper," "lower," "left," "right," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation or be constructed and operated in a specific orientation. The term "connection" simply indicates a connection between devices and has no special meaning.

[0021] Furthermore, the technical fields and installation methods involved in the embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0022] Specific Implementation Example 1: Please refer to... Figure 1 A method for preparing a coal gangue amendment based on microbial-mineral interaction includes the following steps: S1: Obtain porous coal gangue, crush and ball mill it, screen out coal gangue powder of appropriate particle size, activate it with an activator, wash it until neutral and dry it to obtain porous activated coal gangue carrier. Specifically, coal gangue from a certain mining area is crushed by a crusher, then ground to an appropriate particle size using a ball mill. The ball-milled coal gangue particles are then sieved to obtain coal gangue powder with uniform particle size. An activator is then added to activate the coal gangue powder.

[0023] In a preferred embodiment, the activator is an alkaline activator.

[0024] Specifically, 100g of coal gangue powder was added to 300mL of 2mol / L NaOH solution and stirred in a 90℃ water bath for 4 hours. After the reaction, the coal gangue powder was filtered, washed repeatedly with deionized water until the pH of the washing solution was neutral, and dried in an oven at 105℃ for 3 hours to obtain a porous activated coal gangue carrier.

[0025] The specific surface area of ​​the porous activated coal gangue carrier obtained after activation was 56.8 m² / g and the pore volume was 0.13 cm³ / g, which is significantly higher than the specific surface area of ​​8.6 m² / g and the pore volume of 0.03 cm³ / g of the coal gangue powder before activation.

[0026] The strong alkalinity of alkaline activators can disrupt the crystal structure of clay minerals such as kaolinite and illite in coal gangue, forming three-dimensional interconnected macropores, significantly increasing the specific surface area and pore volume; at the same time, it releases Ca... 2+ Mg 2+ Si 4+ The presence of nutrient ions enhances the activity of microbial enzymes and the stability of cell walls; it also increases the pH of the carrier, giving it buffering capacity, making it particularly suitable for acid-contaminated soils and providing local microenvironmental protection for acid-resistant microorganisms.

[0027] In a preferred embodiment, the activator is an acidic activator.

[0028] The acidic activator has a mass fraction of 5-15%. The activation temperature is 80–100℃, and the reaction time is 2–4 hours; or, the acidic activator is 10–20% HCl by volume, the activation temperature is 60–80℃, and the reaction time is 1–3 hours.

[0029] The strong acid properties of acidic activators dissolve carbonates and etch silicates, constructing interconnected microporous structures while releasing Fe. 2+ Al 3+ SO4 2- Equal components, of which Fe 2+ It can be used as a cofactor for acid-resistant desulfurization vibrio and sulfite reductase, and as a component of nitrogenase in nitrogen-fixing bacteria. SO4 2- It can serve as a direct electron acceptor for the metabolism of acid-resistant desulfurization Vibrio; HCl activation conditions can preserve the carrier framework while creating pores, thus preventing structural collapse.

[0030] S2: Prepare deep functional microbial communities and surface functional microbial communities into deep bacterial suspensions and surface bacterial suspensions, respectively. The deep functional microbial community includes one or more of phosphate-solubilizing bacteria, nitrogen-fixing bacteria, or silicate bacteria; the surface functional microbial community includes heavy metal passivating bacteria or dark-colored septate endophytic fungi. Specifically, phosphate-solubilizing bacteria can be one or more of Pseudomonas or alkali-resistant Bacillus megaterium; nitrogen-fixing bacteria can be Azotobacter chrysodontus; and heavy metal passivating bacteria can be one or more of acid-resistant desulfurizing Vibrio or exopolysaccharide-producing bacteria.

[0031] When the aforementioned phosphate-solubilizing bacteria, nitrogen-fixing bacteria, and heavy metal passivating bacteria are combined in various ways, the mutual influence, synergy, and complementary effects among the components of the amendment are enhanced, resulting in better improvement effects in coal gangue soil transformation, mining area ecological restoration, metal-contaminated soil remediation, and nutrient-poor coal gangue matrix improvement. Among these, metal-contaminated soil includes acidic heavy metal-contaminated soil, alkaline heavy metal-contaminated soil, and multi-metal composite-contaminated soil remediation.

[0032] In a preferred embodiment, *Pseudomonas* was inoculated in modified Pikovskaya liquid medium (modified phosphorus-free liquid medium for phosphate-solubilizing bacteria) and cultured with shaking at 28–30°C and 150–180 rpm for 24–36 hours; *Azotobacter chrysotile* was inoculated in Ashby nitrogen-free liquid medium and cultured with shaking at 30°C and 150–180 rpm for 24–36 hours.

[0033] The modified Pikovskaya liquid culture medium consisted of: 10.0 g glucose, 0.5 g (NH4)2SO4, 0.2 g KCl, 0.3 g MgSO4·7H2O, 0.03 g MnSO4·4H2O, 0.003 g FeSO4·7H2O, 5.0 g Ca3(PO4)2, 1000 mL distilled water, and a pH of 7.0-7.5.

[0034] Ashby's nitrogen-free liquid culture medium consists of: 10.0g mannitol, 0.2g KH2PO4, 0.2g MgSO4·7H2O, 0.2g NaCl, 0.1g K2SO4, 5.0g CaCO3, 1000mL distilled water, and pH 7.0-7.4.

[0035] Furthermore, after the culture was completed, the Pseudomonas aeruginosa and Azotobacter chrysophagus culture media were centrifuged at 5000 rpm for 10 minutes, resuspended in sterile physiological saline, and the concentration of each bacterial suspension was adjusted to 10. 9 CFU / mL.

[0036] The resuspended Pseudomonas bacterial suspension and Azotobacter chrysophagus bacterial suspension were mixed at a volume ratio of 1:1 to obtain a deep bacterial suspension.

[0037] In a preferred embodiment, the heavy metal passivating bacteria can be Bacillus subtilis. Bacillus subtilis is inoculated into LB liquid medium (Luria-Bertanny liquid medium) and cultured with shaking at 30-35°C and 150-200 r / min for 20-30 hours. Dark-colored septate endophytic fungi were inoculated onto Martin's medium for spore culture and then cultured with shaking at 25–28°C and 120–150 rpm for 5–7 days.

[0038] The LB liquid culture medium consists of: 10.0g peptone, 5.0g yeast extract, 10.0g NaCl, 1000mL distilled water, and pH 7.0-7.2.

[0039] Martin's medium consists of: 10.0g glucose, 5.0g peptone, 1.0g KH2PO4, 0.5g MgSO4·7H2O, 0.033g Bengal red, 0.03g streptomycin, 1000mL distilled water, pH 7.2-7.4.

[0040] Furthermore, the Bacillus subtilis culture medium was centrifuged at 5000 rpm for 10 minutes, resuspended in sterile physiological saline, and the concentration of each bacterial suspension was adjusted to 10. 9 CFU / mL; Centrifuge the dark-colored septate endophytic fungal culture medium at 8000 r / min for 15 minutes, resuspend in sterile physiological saline, and adjust the concentration of each bacterial suspension to 10. 7 spores / mL; The resuspended Bacillus subtilis suspension and the suspension of dark-colored septate endophytic fungi were mixed at a volume ratio of 2:1 to obtain the surface suspension.

[0041] The deep functional microbial community or the surface functional microbial community includes at least two microbial species with synergistic effects.

[0042] S3: The deep bacterial suspension is fixed in the pores inside the activated porous coal gangue carrier by vacuum negative pressure adsorption, and a loading fixation is performed. Specifically, take 50g of the porous activated coal gangue carrier prepared in step 1, place it in a vacuum barrel, seal the device, connect a vacuum pump, extract the air from the vacuum barrel, and input 200mL of deep bacterial suspension. The vacuum degree is -0.095 to -0.08MPa. The vacuum holding time for a single vacuuming is 20 to 30 minutes. After the vacuum adsorption device is de-vacuumed, it is restored to normal pressure and left to stand. Under atmospheric pressure, the bacterial suspension is further pressed into the carrier pores. Let it stand for 40 minutes. Repeat the above operation 2 to 3 times, keeping the adsorption temperature at 20 to 35°C. After the deep bacterial suspension adsorption is completed, filter with sterile gauze to remove excess deep bacterial suspension and obtain a porous activated coal gangue carrier that has been fixed by primary loading after draining. Due to the vacuum negative pressure, the microbial cells are saturated and pressed into the internal pores of the carrier, achieving deep immobilization.

[0043] S4: The surface bacterial suspension is immobilized on the surface of the porous activated coal gangue carrier that has been initially loaded, and a dense biofilm with a thickness of 5-20 μm is formed on the surface of the porous activated coal gangue carrier that has been initially loaded, and then subjected to secondary loading and immobilization. Specifically, the porous activated coal gangue carrier that adsorbs and fixes deep microbial communities is immersed in 200 mL of surface bacterial suspension. The mass ratio of the porous activated coal gangue carrier to the surface bacterial suspension for one-time loading and fixation is 1:2 to 1:4. The mass of the porous activated coal gangue carrier for one-time loading and fixation is the dry weight of the porous activated coal gangue carrier after the free deep bacterial suspension has been drained by S3 filtration and the one-time loading and fixation is completed.

[0044] The surface bacterial suspension and the porous activated coal gangue carrier fixed by the first loading were placed in a sterile container, such as an Erlenmeyer flask with a sterile breathable sealing film. After sealing, the mixture was shaken and adsorbed at 28°C and 80 r / min for 45 minutes, so that the surface microbial community in the surface bacterial suspension could attach to the outer surface of the porous activated coal gangue carrier and form a biofilm with a thickness of about 10 μm. The excess surface bacterial suspension was filtered off to obtain the porous activated coal gangue carrier after the second loading.

[0045] S5: Prepare an encapsulating agent solution including a dispersant, place the porous activated coal gangue carrier after secondary loading and fixation into the encapsulating agent solution and mix, and then drip it into the crosslinking agent solution to harden it, forming coal gangue spherical particles; The embedding agent solution uses a sodium alginate solution with a mass fraction of 2-3%, and adds sodium carboxymethyl cellulose with a mass fraction of 0.1-0.5% or attapulgite with a mass fraction of 0.5-2% as a dispersant. The crosslinking agent solution is a sterile CaCl2 solution with a mass fraction of 2-5%. When adding the solution, the dropping speed is controlled at 2-5 drops / second, the dropper is 5-15cm above the liquid surface, and the curing time is 30-60 minutes.

[0046] Preferably, the dropping rate of the crosslinking agent solution is 3 drops / second, the dropper is 10cm above the liquid surface, and the curing time is 40 minutes.

[0047] Specifically, a calcium alginate gel slow-release encapsulation layer with a thickness of 100–300 μm is formed on the surface of the spherical coal gangue particles.

[0048] S6: After removing the spherical coal gangue particles, rinse them with sterile water and place them in a vacuum freeze dryer. Freeze-dry them for 12 to 24 hours at -50 to -30°C and a vacuum degree ≤10Pa to obtain a coal gangue modifier based on microbial-mineral interaction.

[0049] A coal gangue modifier based on microbial-mineral interfeeding, using the aforementioned method for preparing a coal gangue modifier based on microbial-mineral interfeeding, wherein the modifier is a composite particle with a four-layer gradient immobilization structure, comprising: First layer: porous activated coal gangue carrier, wherein the porous activated coal gangue carrier has a three-dimensional interconnected pore structure; The second layer consists of deep functional microbial communities fixed in the pores inside the porous activated coal gangue carrier, wherein the pores constitute the pore colonization zone. Specifically, the pore colonization zone is where deep functional microbial communities attach from a free state to the pores inside the porous activated coal gangue carrier, survive, and establish stable micro-regions.

[0050] The third layer consists of surface functional microbial communities fixed on the outer surface of the porous activated coal gangue carrier, forming a dense biofilm with a thickness of 5 to 20 μm on the surface of the porous activated coal gangue carrier. Fourth layer: A calcium alginate gel sustained-release embedding layer covering the outside of the third layer, with a thickness of 100-300 μm.

[0051] Furthermore, the modifier employs a three-layer progressive sustained-release system: the first stage is the physical barrier and degradation control of the calcium alginate gel embedding layer, with a degradation half-life of 30–90 days; the second stage is the pore adsorption controlled release of the coal gangue carrier; and the third stage is the metabolic regulation and biofilm formation of the microorganisms themselves.

[0052] The initial release rate of the modifier (days 1-7) is ≤15%, and the effective survival period of the functional microorganisms is 60-120 days.

[0053] It should be noted that the functional microorganisms involved in this invention mainly include Pseudomonas, Azotobacter chrysophytes, Bacillus subtilis, and dark-colored septate endophytic fungi. In their free state, these microorganisms are subjected to environmental stresses (such as pH, moisture, and competition from indigenous microorganisms) after being directly applied to the soil, and their survival period is usually only 15 to 30 days. After being immobilized by this invention, the initial release rate is significantly reduced and the duration of metabolic activity of the microorganisms is extended through a four-layer gradient immobilization structure and a triple slow-release mechanism.

[0054] The improver described in Example 1 and the free bacteria control group were placed in a simulated soil environment (temperature 25℃, moisture content 20%), and samples were taken periodically to determine the number of viable bacteria; the results of the comparative experiment are shown in [reference needed]. Figure 2 The graph shows the comparison of the survival period of soil amendment and free bacteria in the soil. In the free bacteria control group, the same bacterial species were mixed once to prepare a uniform suspension and then directly added to the soil. The experimental results showed that after 30 days, the survival rate of microorganisms in the modifier was 45.6% of the initial value, while the survival rate of the free bacteria control group was only 8.3%. After 60 days, detectable viable bacteria were still present in the modifier, with a survival rate of approximately 12.8%, and the effective survival period reached more than 90 days. The survival rate was approximately 5.2% after 120 days. Free bacteria were essentially undetectable after 45 days. This verifies the significant effect of the immobilized structure of the present invention on extending the survival period of microorganisms by approximately 3 to 4 times.

[0055] The modifier described in Example 1 was mixed with coal gangue matrix at a mass ratio of 1:100 and cultured at 25°C. The available phosphorus and potassium contents were measured periodically. The control group, under the same conditions, did not add the modifier. The nutrient release effect of the coal gangue matrix was measured. See the comparative test results below. Figure 3 The graph shows a comparison of the effective nutrient content of coal gangue substrates with and without additives after 60 days of cultivation. Specifically, after 30 days, the effective phosphorus content in the treatment group increased by 42.3% and the effective potassium content increased by 76.8% compared to the control group. This indicates that microbial metabolism promoted the release of phosphorus and potassium from coal gangue, demonstrating the positive effect of the feedback mechanism.

[0056] In the described microbial-mineral feedback mechanism, the activated coal gangue carrier provides nutrients for the functional microorganisms. The activated coal gangue carrier possesses abundant porous structures and surface active sites, providing physical protection and essential nutrients (such as Fe, Mg, and Ca) for the microorganisms. After colonization, the microorganisms secrete organic acids, extracellular polysaccharides, and iron carriers through metabolic activities, further dissolving the coal gangue mineral lattice and releasing nutrients such as phosphorus, potassium, and sulfur. Simultaneously, they alter the pH and redox potential of the microenvironment, promoting the precipitation or adsorption of heavy metals. This bidirectional promoting effect constitutes a positive feedback loop, significantly enhancing the sustained effectiveness of the modifier.

[0057] The amendment described in Example 1 was mixed with metal-contaminated soil, while the control group received no amendment under the same conditions. The amendment effect was measured; the comparative test results are shown below. Figure 4 The image shows a comparison of the TCLP leaching concentrations of heavy metals in metal-contaminated soils with and without amendments after 28 days of incubation. Specifically, composite contaminated soil with lead content of 500 mg / kg and cadmium content of 10 mg / kg was treated with the amendment described in Example 1 at 5% of the soil mass, and cultured for 28 days at 25°C and 45% moisture content. TCLP leaching tests were conducted according to the HJ / T300-2007 method. The results showed that the Pb leaching concentration in the treated group decreased by 86.2% and the Cd leaching concentration decreased by 81.5% compared to the control group.

[0058] The soil amendment described in Example 1 was mixed with soil, and the microbial release curve of the soil amendment was determined; the test results are shown below. Figure 5 The graph shows the change in the cumulative release rate of the improver microorganisms over culture time. Specifically, testing showed that the release rate of the microorganisms was 12.3% in the initial stage (days 1-7), 42.6% cumulatively in the middle stage (days 8-30), and 68.5% cumulatively in the later stage (days 31-90), conforming to a first-order release kinetic model with a release rate constant k = 0.035 days. -1 It can be used once, has a long coverage time, and can provide continuous supply, making it suitable for scenarios that require a long-term, low-dose, stable supply.

[0059] Example 2 This embodiment provides a coal gangue modifier based on microbial-mineral interaction. The preparation method differs from that in Embodiment 1 in that the heavy metal passivating bacteria are acid-resistant desulfurizing Vibrio bacteria, the activator for the porous activated coal gangue carrier is 2 mol / L KOH, and the activation time is 4 hours at 100°C. Other operations are the same as in Embodiment 1.

[0060] The pH tolerance range of acid-tolerant desulfurization Vibrio is 4.0–6.5; the acid-tolerant desulfurization Vibrio was cultured in modified Postgate C liquid medium (modified sulfate-reducing bacteria medium C) at 30°C under anaerobic conditions for 72–96 hours.

[0061] Modified Postgate C liquid medium composition: 0.5g KH2PO4, 1.0g NH4Cl, 2.0g Na2SO4, 2.0g MgSO4·7H2O, 0.1g CaCl2·2H2O, 3.5mL sodium lactate, 0.5g FeSO4·7H2O, 1.0g yeast extract, 0.1g Vitamin C, 0.5g cysteine ​​hydrochloride, 1000mL distilled water, pH 7.0.

[0062] Composition of surface bacterial suspension: acid-resistant desulfurized Vibrio : dark-colored septate endophytic fungi = 2:1.

[0063] Example 3 This embodiment provides a coal gangue modifier based on microbial-mineral interaction. The preparation method differs from that in Embodiment 1 in that the phosphate-solubilizing bacteria are alkali-resistant Bacillus megaterium, and the heavy metal passivating bacteria are extracellular polysaccharide-producing strains. Other operations are the same as in Embodiment 1.

[0064] Bacillus megaterium was inoculated into modified Pikovskaya medium (modified phosphate-solubilizing liquid medium) and cultured with shaking at 30–35°C and pH 8.0–8.5 for 30–48 hours; the extracellular polysaccharide-producing strain was cultured in LB medium supplemented with 20 g / L sucrose at 30°C for 24–36 hours.

[0065] Composition of deep-layer bacterial suspension: Alkali-resistant Bacillus megaterium: Azotobacter chrysoprase = 1:1; Composition of the surface bacterial suspension: extracellular polysaccharide-producing bacteria: dark-colored septate endophytic fungi = 2:1.

[0066] Example 4 This embodiment provides a coal gangue modifier based on microbial-mineral interaction. The difference between its preparation method and that of Embodiment 1 is that the heavy metal passivating bacteria are selected from extracellular polysaccharide-producing strains and acid-resistant desulfurization Vibrio bacteria. Other operations are the same as in Embodiment 1.

[0067] The composition of the deep bacterial suspension was: Pseudomonas : Azotobacter = 1:1; Composition of the surface bacterial suspension: acid-resistant desulfurization Vibrio: extracellular polysaccharide-producing strains: dark-colored septate endophytic fungi = 3:1:1.

[0068] Example 5 This embodiment provides a coal gangue modifier based on microbial-mineral interaction. The difference between its preparation method and that of Embodiment 1 is that the deep functional microorganisms increase silicate bacteria and enhance the ratio of nitrogen-fixing bacteria and phosphate-solubilizing bacteria, while the surface functional microorganisms only use dark-colored septate endophytic fungi. Other operations are the same as in Embodiment 1.

[0069] Composition of deep bacterial suspension: Pseudomonas: Azotobacter: Silicate bacteria = 1:1:1; The surface bacterial suspension contains only dark-colored septate endophytic fungi, without the addition of heavy metal-insensitive bacteria, but retains the dark-colored septate endophytic fungi to promote plant growth and nutrient absorption.

[0070] The amendment described in Example 2 was applied to the improvement of acidic heavy metal contaminated soil (pH 4.5-5.5), the main pollutant of which was Cu. 2+ Cd 2+ .

[0071] Acid-resistant desulfurizing Vibrio bacteria utilize organic carbon sources such as sodium lactate as electron donors during metabolism, converting SO42- into hydrogen sulfide. 2- Restore to S 2- At the same time, it oxidizes organic matter into HCO3. - The S produced by reduction 2- With Cu 2+ Cd 2+ The reaction with heavy metal ions forms a very insoluble sulfide precipitate (CuS solubility product Ksp = 6 × 10⁻⁶). -36 CdS solubility product Ksp = 1 × 10 -28 In addition, the oxidation of organic carbon consumes H₂. + It can increase the pH value of the local microenvironment and further solidify heavy metal ions; at the same time, the activated coal gangue carrier has a certain degree of alkalinity, which can provide local microenvironment protection for acid-resistant desulfurization Vibrio bacteria.

[0072] Experiments showed that in Cu-Cd co-contaminated soil (Cu 300 mg / kg, Cd 5 mg / kg) with pH 5.0, adding the amendment described in this example at 5% of soil mass resulted in a 78.3% decrease in Cu TCLP leaching concentration and a 72.6% decrease in Cd concentration after 28 days of cultivation. The soil pH also increased from 5.0 to 6.2.

[0073] The amendment described in Example 3 was applied to the remediation of alkaline heavy metal contaminated soil (pH 8.0-8.5), the main pollutant of which was Pb. 2+ Cd 2+ .

[0074] Bacillus megaterium secretes organic acids, such as gluconic acid, citric acid, and oxalic acid, which, along with phosphatases, dissolve insoluble phosphates (such as calcium) in the soil. 10 (PO4)6(OH)2, FePO4·2H2O, etc.), releasing PO4 3- With Pb 2+ Cd 2+ The reaction forms a very insoluble phosphate precipitate: Pb 2+ +PO4 3- +5OH - →Pb5(PO4)3OH + 6H2O (basic lead phosphate Pb5(PO4)3OH solubility product Ksp = 2.5 × 10 -44 ), Cd 2+ +PO4 3- →Cd3(PO4)2 (Cd3(PO4)2 solubility product Ksp = 2.5 × 10 -33 The extracellular polymers secreted by the extracellular polysaccharide-producing strains contain a large number of functional groups such as carboxyl, hydroxyl, and phosphate groups, which adsorb Cd through complexation. 2+ and Pb 2+ It can also serve as a nucleation site for carbonate precipitation in an alkaline microenvironment, promoting the solidification of heavy metals in the form of carbonates.

[0075] Experiments showed that in Pb-Cd co-contaminated soil (Pb 600 mg / kg, Cd 8 mg / kg) at pH 8.2, adding the soil conditioner of this embodiment at 5% of soil mass and incubating for 28 days reduced the TCLP leaching concentration of Pb by 82.5% and Cd by 76.8%.

[0076] The amendment described in Example 4 was applied to the remediation of polymetallic contaminated soil (pH 6.5-7.5), the main pollutant of which was Pb. 2+ Cd 2+ Cu 2+ Zn 2+ wait.

[0077] Acid-resistant desulfurizing Vibrio bacteria simultaneously fix multiple heavy metal ions (CuS, CdS, PbS, ZnS, etc.) through sulfide precipitation mechanisms; phosphate-solubilizing bacteria supplement the fixation of some heavy metals (Pb) that readily form precipitates with phosphate ions through phosphate precipitation mechanisms. 2+ Cd 2 + (Extracellular polysaccharide-producing strains and auxiliary interception through biosorption; dark-colored septate endophytic fungi promote plant growth and form a synergistic protective barrier between microorganisms and minerals, with multiple mechanisms working together to achieve comprehensive remediation of multi-metal complex pollution.)

[0078] Experiments showed that in Pb-Cd-Cu-Zn composite contaminated soil (Pb 400 mg / kg, Cd 6 mg / kg, Cu 200 mg / kg, Zn 300 mg / kg) at pH 7.0, adding the soil conditioner of this embodiment at 5% of soil mass and incubating for 28 days resulted in a TCLP leaching concentration reduction of 81.2% for Pb, 75.5% for Cd, 74.8% for Cu, and 70.3% for Zn.

[0079] The improver described in Example 5 was applied to improve a nutrient-poor coal gangue matrix, which is a pure coal gangue matrix that is severely lacking in nutrients (N, P, K) and has a low organic matter content.

[0080] Nitrogen-fixing bacteria convert atmospheric nitrogen (N2) into NH3 using nitrogenase, increasing the available nitrogen content in the matrix. Phosphate-solubilizing bacteria secrete organic acids and phosphatases to convert insoluble phosphorus (such as phosphate rock and phosphate iron ore) in coal gangue into water-soluble phosphates, increasing the available phosphorus content. Silicate bacteria, through the secretion of organic acids and extracellular polysaccharides, disrupt the crystal structure of potassium feldspar and mica minerals in coal gangue, releasing potassium (K). + It is converted into available potassium that can be used by plants; the dark-colored septate endophytic fungi expand the root absorption range through the hyphal network, further promoting the absorption and utilization of nutrients; this combination provides precise supplementation for the nutrient-deficient characteristics of the substrate.

[0081] Experiments showed that when the amendment was mixed with coal gangue matrix at a mass ratio of 1:50 and cultured for 60 days, the available phosphorus content in the matrix increased by 56.7%, the available potassium content increased by 88.2%, and the total nitrogen content increased by 42.3% compared with the control. A pot experiment on ryegrass showed that the biomass of the treatment group with the amendment added was 2.8 times higher than that of the control group.

[0082] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that no immobilization and embedding treatment was performed. That is, the above-mentioned modifier was not used. The deep functional microorganisms, including Pseudomonas and Azotobacter chrysophagus, and the surface functional microorganisms, including Bacillus subtilis and dark septate endophytic fungi, from Example 1 were directly mixed in equal proportions to prepare a single free composite bacterial suspension. This suspension was applied directly to the coal gangue matrix without undergoing carrier negative pressure loading, surface biofilm fixation, and calcium alginate embedding processes.

[0083] After testing, the microbial survival rate dropped to 12.6% after 7 days, and almost no viable bacteria were detected after 30 days. The effective phosphorus and effective potassium contents increased by 18.5% and 32.1% respectively compared with the control group, significantly lower than those in Example 1. The passivation rates of heavy metals were 43.2% for Pb and 38.6% for Cd. The results show that the immobilization and embedding treatment of the present invention significantly improves the survival period and efficacy of microorganisms. Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the deep bacterial suspension was subjected to conventional oscillation adsorption, and the bacterial species in the deep bacterial suspension were mixed at one time.

[0084] Experimental results showed that the microbial load inside the carrier after oscillation adsorption was only 52.3% of that after vacuum negative pressure adsorption, and the total microbial load in the modifier was reduced by about 40%. After 30 days of cultivation in soil, the microbial survival rate of the oscillation adsorption group was 28.7%, which was significantly lower than the 45.6% of the vacuum negative pressure adsorption group. The results indicate that vacuum negative pressure adsorption significantly improves the immobilization efficiency and the survival period of microorganisms.

Claims

1. A method for preparing a coal gangue modifier based on microbial-mineral interaction, characterized in that: Includes the following steps: S1: Obtain porous coal gangue, crush and ball mill it, screen out coal gangue powder of appropriate particle size, activate it with an activator, wash it until neutral and dry it to obtain porous activated coal gangue carrier. S2: Prepare deep functional microbial communities and surface functional microbial communities into deep bacterial suspensions and surface bacterial suspensions, respectively. S3: The deep bacterial suspension is fixed in the pores inside the activated porous coal gangue carrier by vacuum negative pressure adsorption, and a loading fixation is performed. S4: The surface bacterial suspension is immobilized on the surface of the porous activated coal gangue carrier that has been initially loaded, and a dense biofilm with a thickness of 5-20 μm is formed on the surface of the porous activated coal gangue carrier that has been initially loaded, and then subjected to secondary loading and immobilization. S5: Prepare the embedding agent solution, place the porous activated coal gangue carrier after secondary loading and fixation into the embedding agent solution and mix, and then drip it into the crosslinking agent solution to harden it, forming coal gangue spherical particles; S6: After removing the spherical coal gangue particles, rinse them with sterile water and place them in a vacuum freeze dryer. Freeze-dry them for 12 to 24 hours at -50 to -30°C and a vacuum degree ≤10Pa to obtain a coal gangue modifier based on microbial-mineral interaction.

2. The method for preparing coal gangue amendment based on microbial-mineral interaction according to claim 1, characterized in that: The activator in S1 is either an alkaline or acidic activator. For alkaline activators, NaOH or KOH solution is used at a concentration of 0.5-3 mol / L, the activation temperature is 80-120℃, and the activation time is 3-5 hours. For acidic activators, a concentration of 5-15% is used. The activation temperature is 80–100℃ and the activation time is 2–4 hours; or, HCl with a volume fraction of 10–20% is used, the activation temperature is 60–80℃ and the activation time is 1–3 hours.

3. The method for preparing coal gangue amendment based on microbial-mineral interaction according to claim 1, characterized in that: The deep functional microbial community in S2 includes one or more of phosphate-solubilizing bacteria, nitrogen-fixing bacteria, or silicate bacteria; the surface functional microbial community includes one or more of heavy metal passivating bacteria or dark-colored septate endophytic fungi; phosphate-solubilizing bacteria may be one or more of Pseudomonas or alkali-resistant Bacillus megaterium; nitrogen-fixing bacteria may be Azotobacter chrysophagus; heavy metal passivating bacteria are selected from one or more of acid-resistant desulfurization Vibrio, Bacillus subtilis, or exopolysaccharide-producing bacteria.

4. The method for preparing coal gangue amendment based on microbial-mineral interaction according to claim 1, characterized in that: The vacuum negative pressure adsorption fixation in S3 includes the following steps: placing the porous activated coal gangue carrier in a vacuum adsorption device, evacuating to -0.095 to -0.08 MPa, adding the prepared deep bacterial suspension, with a mass ratio of porous activated coal gangue carrier to deep bacterial suspension of 1:2 to 1:5, and maintaining this for 20 to 30 minutes; then removing the vacuum from the vacuum adsorption device and restoring it to normal pressure for 30 to 60 minutes; repeating the above operation 2 to 3 times, and then filtering and draining the deep bacterial suspension in the vacuum adsorption device to obtain a porous activated coal gangue carrier with primary load fixation.

5. The method for preparing coal gangue amendment based on microbial-mineral interaction according to claim 1, characterized in that: The specific steps of S4 are as follows: The porous activated coal gangue carrier with primary loading fixation is placed in the surface bacterial suspension, and the mass ratio of the porous activated coal gangue carrier with primary loading fixation to the surface bacterial suspension is 1:2 to 1:

4. The carrier is shaken and adsorbed for 30 to 60 minutes at 20 to 35°C and 50 to 100 rpm. Heavy metal passivating bacteria and dark-colored septate endophytic fungi form a 5 to 20 μm biofilm on the outer surface of the carrier. The surface bacterial suspension is then filtered to obtain the porous activated coal gangue carrier with secondary loading fixation.

6. The method for preparing coal gangue amendment based on microbial-mineral interaction according to claim 1, characterized in that: In S5, the embedding agent solution is 200 mL of sterile sodium alginate solution with a mass fraction of 3%, and then 0.3% sodium carboxymethyl cellulose is added as a dispersant. The crosslinking agent solution is sterile CaCl2 solution with a mass fraction of 4%.

7. The method for preparing coal gangue amendment based on microbial-mineral interaction according to claim 6, characterized in that: The porous activated coal gangue carrier loaded with secondary microorganisms was slowly added to the encapsulation agent solution and stirred at 300 r / min for 15 minutes to form a uniform suspension. The suspension was then dripped into a 4% (w / w) sterile CaCl2 solution using a peristaltic pump at a dripping rate of 2-5 drops / second and a dropper height of 5-15 cm from the liquid surface. The solution was allowed to harden for 30-60 minutes to form spherical coal gangue particles.

8. A coal gangue modifier based on microbial-mineral interaction, applied to the preparation method of the coal gangue modifier based on microbial-mineral interaction as described in claims 1-7, characterized in that: The modifier is a composite particle with a four-layer gradient fixation structure, comprising: First layer: porous activated coal gangue carrier, wherein the porous activated coal gangue carrier has a three-dimensional interconnected pore structure; The second layer consists of deep functional microbial communities fixed in the pores inside the porous activated coal gangue carrier, wherein the pores constitute the pore colonization zone. The third layer consists of surface functional microbial communities fixed on the outer surface of the porous activated coal gangue carrier, forming a dense biofilm with a thickness of 5 to 20 μm on the surface of the porous activated coal gangue carrier. Fourth layer: A calcium alginate gel sustained-release embedding layer covering the outside of the third layer, with a thickness of 100-300 μm.

9. The application of the modifier according to claim 8, characterized in that, The amendment is used for the soilification of coal gangue; or for the remediation of metal-contaminated soil; or for the improvement of nutrient-poor coal gangue matrix.

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