Method for immobilizing growth-promoting rhizobacteria by taking decarburized gangue-based biochar as carrier, solid waste-based microorganism immobilization ball prepared by method and application of solid waste-based microorganism immobilization ball

Abstract

The invention discloses a method for immobilizing growth-promoting rhizobacteria by taking decarburized gangue-based biochar as a carrier, a solid waste-based microorganism immobilization ball prepared by the method and application of the solid waste-based microorganism immobilization ball, and relates to the technical field of solid waste utilization. The method comprises the following steps: preparing gangue-based zeolite by microwave activation and decarburization of coal gangue; preparing gangue-based biochar by utilizing phosphorus modification; and preparing the solid-waste-based microorganism solidification balls by utilizing an adsorption-embedding-crosslinking method. The decarburized gangue-based biochar is used as a carrier material for immobilizing the growth-promoting rhizosphere bacteria, and is used for restoring the sandy degraded soil in the northwest arid mining area. According to the technology, the survival time and functional activity of the growth-promoting rhizobacteria in the soil environment can be improved, and a new way is provided for consumption of bulk gangue solid waste. The method also has the technical advantages of improving the carbon source utilization rate of the gangue and increasing the phosphate fertilizer and the organic carbon library of the soil.

Description

Technical Field

[0001] The present invention relates to the technical field of solid waste utilization, and in particular to a method for fixing rhizosphere growth-promoting bacteria using decarbonized gangue-based biochar as a carrier, and solid waste-based microbial solidification balls prepared therefrom and applications thereof. Background Art

[0002] As the main solid waste generated in coal mining, coal gangue accounts for 1 / 3 of the coal mining volume. At present, the amount of open-air accumulation has exceeded 7 billion tons. It is urgent to develop ways to dispose of large quantities of coal gangue. 2 and Al 2 O 3 It is a common raw material for preparing ceramic membranes. 2 O 3 , CaO, TiO 2 It is a sintering aid for preparing ceramic membranes. Gangue-based porous ceramic membranes are mostly used for the adsorption of pollutants in wastewater. Gangue contains nutrients such as C, H, O, N, and S. After proper treatment, it can improve the porosity, connectivity and water content of the soil and can be used to prepare soil conditioners. In recent years, many scholars have modified gangue by thermal modification, acid-base modification and other means to prepare high-value-added environmental adsorption materials. Acid modification is to dissolve the acid-soluble metal ions in the gangue by acid leaching to increase the porosity of the material. Compared with acid modification, alkali modification, in addition to increasing the porosity of gangue, alkali fusion also converts part of the stable kaolinite in the gangue into metakaolinite with higher activity, which is used to prepare gangue-based zeolites with stronger adsorption performance. It is generally suitable for the remediation and treatment of contaminated soil, sewage and wastewater, and air pollution. However, the alkali fusion process needs to be maintained at a high temperature for a long time, and the required energy consumption is high. In the process, most of the carbon source in the gangue will be lost.

[0003] Rhizosphere growth-promoting bacteria, as beneficial bacteria that colonize around plant roots, can promote plant growth and improve plant stress resistance by fixing nitrogen, dissolving phosphorus, and producing antibiotics. However, the use of rhizosphere growth-promoting bacteria as soil conditioners also has problems such as short survival time of microorganisms, limited proliferation rate, and poor adaptability, which limits the effect of on-site application of rhizosphere growth-promoting bacteria. In order to solve these problems, microbial immobilization technology is particularly necessary.

[0004] Compared with free bacteria in the soil, microorganisms have higher cell density and activity after fixation, which can maximize the effectiveness of microorganisms in the soil environment. At present, the main microbial fixation technologies include adsorption, embedding, and cross-linking. The adsorption method refers to the adsorption of microorganisms on the surface of the immobilized carrier by electrostatic or surface tension to achieve the goal of microbial immobilization. It is an economical and practical method for microbial immobilization. However, after the microorganisms are combined with the immobilization material, they show poor stability and are easy to fall off the carrier, making it difficult to use them for a long time on site. The embedding method is to embed the microbial cells and microbial carriers in natural polymer gels so that the microorganisms will not be easily separated from the carrier. Currently, the commonly used embedding agents include sodium alginate, agar, polyvinyl alcohol, etc. The embedding method has the advantages of good fixation efficiency, difficult separation, good stability, and high mechanical strength, but the embedding method also has the disadvantages of complex preparation process and large mass transfer resistance. The cross-linking method refers to the application of a cross-linking agent to make the embedded microorganisms react further and connect with the embedding agent to form a porous network structure, thereby immobilizing the microorganisms. Commonly used cross-linking agents include chitosan, glutaraldehyde, etc. The cross-linking method has high microbial binding strength, good stability, and strong environmental adaptability. However, the single use of the cross-linking method has a violent reaction and leads to the loss of microbial activity.

[0005] References:

[0006] [1]Ruohan Z, Bing W, Xueyang Z, et al. Insights into Cr(VI)removalmechanismin water by facile one-step pyrolysis prepared coal gangue-biocharcomposite.[J]. Chemosphere, 2022, 299134334-134334.

[0007] [2] Junyuan G, Cheng C, Wenjing C, et al. Effective immobilization of Bacillus subtilis in chitosan-sodium alginate composite carrier for ammoniaremoval from anaerobically digested swine wastewater[J]. Chemosphere, 2021, 284131266-131266. Summary of the invention

[0008] Microwave technology provides a feasible alternative for the synthesis of gangue-based zeolite from coal gangue. It increases the reactivity of mineral components and carbon sources in coal gangue by consuming less energy, which not only improves the adsorption performance of gangue-based zeolite but also retains the carbon source.

[0009] Based on the above reasons, in response to the problems or defects existing in the prior art, the purpose of the present invention is to provide a method for fixing rhizosphere growth-promoting bacteria using decarbonized gangue-based biochar as a carrier, and the solid waste-based microbial solidification balls prepared therefrom and their applications, to solve or at least partially solve the above-mentioned technical defects existing in the prior art: the present invention uses decarbonized gangue-based biochar as a carrier material to fix rhizosphere growth-promoting bacteria, which is used to improve the degraded soil in coal mining wastelands, increase the survival time of rhizosphere growth-promoting bacteria in the soil environment, and enhance the soil nitrogen fixation capacity. At the same time, the gangue-based biochar preparation technology has the technical advantages of effectively utilizing gangue carbon sources and increasing soil phosphate fertilizers, ultimately achieving the purpose of ecologically high-value utilization of coal gangue and disposal of large quantities of gangue solid waste.

[0010] In order to achieve the above first object of the present invention, the technical solution adopted by the present invention is as follows:

[0011] A method for fixing rhizosphere growth-promoting bacteria using decarbonized gangue-based biochar as a carrier, comprising the steps of preparing gangue-based zeolite by microwave activation and decarbonization of coal gangue; preparing gangue-based biochar by phosphorus modification; and preparing solid waste-based microbial solidification balls by adsorption-embedding-crosslinking method.

[0012] Furthermore, in the above technical solution, the steps of preparing gangue-based zeolite by microwave activation and decarbonization of coal gangue are as follows:

[0013] (1) Decarbonization of coal gangue by microwave activation: coal gangue and calcium oxide (CaO) are mixed in a proportion, ball-milled to obtain coal gangue powder; then the coal gangue powder is mixed in a proportion with a NaOH solution, the obtained mixed reaction liquid is placed in a microwave reactor, and microwave activated for 35-45 minutes at 150-170° C. under stirring conditions to obtain an active aluminum silicate ore solution dissolved from the coal gangue; the ore solution is centrifuged, and the obtained precipitate is washed, dried, and then crushed to obtain carbon slag;

[0014] (2) Preparation of gangue-based zeolite: Silicon dioxide (SiO 2 ) is added to the active aluminum silicate ore solution in step (1), according to the chemical formula of analcime NaAlSi 2 O 6The atomic ratio of the elements in the mixture is adjusted to 1:1:2, and then the mixed solution is stirred and aged at room temperature; after the aging is completed, the aged solution is transferred to a reactor, and the reactor is sealed and heated to 180-220°C for constant temperature crystallization reaction for 8-12 hours; after the reaction is completed, the product is filtered, the filtered crystals are washed, and dried to precipitate the gangue-based zeolite.

[0015] Specifically, in step (1) of the above technical solution, adding calcium oxide for ball milling activation can better improve the surface activity of coal gangue; using microwave activation decarbonization process for activation digestion can not only more effectively dissolve the aluminum silicates in the coal gangue, but also retain the carbon in the gangue through microwave activation without destroying the carbon structure, so that the retained gangue carbon source can be further applied to the preparation of gangue-based biochar.

[0016] Furthermore, in step (1) of the above technical solution, the mass ratio of the coal gangue to calcium oxide is (5-15): 1. In a preferred embodiment of the present invention, the mass ratio of the coal gangue to calcium oxide is 10:1.

[0017] Furthermore, in step (1) of the above technical solution, the particle size of the coal gangue powder is less than 200 mesh.

[0018] Furthermore, in step (1) of the above technical solution, the solid-liquid ratio of the gangue powder to the NaOH solution is 1 part by mass: (20-50) parts by volume. In a preferred embodiment of the present invention, the solid-liquid ratio of the gangue powder to the NaOH solution is 1 part by mass: 30 parts by volume. Wherein: the mass parts and volume parts are based on g: ml.

[0019] Furthermore, in step (1) of the above technical solution, the concentration of the NaOH solution is 1-5 mol / L. In a preferred embodiment of the present invention, the concentration of the NaOH solution is 2 mol / L.

[0020] Furthermore, in step (2) of the above technical solution, the gangue and silicon dioxide (SiO 2 )’s mass ratio is 1:(0.1-0.2).

[0021] Furthermore, in step (2) of the above technical solution, the aging time is 3-5 hours. In a preferred embodiment of the present invention, the aging time is 4 hours.

[0022] Furthermore, in step (2) of the above technical solution, in a preferred embodiment of the present invention, the temperature of the crystallization reaction is 200° C., and the time of the crystallization reaction is 10 h.

[0023] Furthermore, in the above technical solution, the steps of preparing gangue-based biochar by phosphorus modification are specifically as follows:

[0024] (i) Phosphorus-modified raw materials: straw, the carbon slag, and the gangue-based zeolite are mixed according to a ratio, and then the mixed raw materials are mixed with a phosphate compound solution according to a ratio, and the mixed solution is fully stirred for 5-7 hours and then dried to obtain the phosphorus-modified raw materials;

[0025] (ii) Preparation of gangue-based biochar: The phosphorus-modified raw material is heated to 450-550° C. in an inert atmosphere and pyrolyzed at a constant temperature for 3-5 hours. After the pyrolysis is completed, the gangue-based biochar is obtained.

[0026] Furthermore, in step (i) of the above technical solution, in a preferred embodiment of the present invention, the mass ratio of the straw, carbon slag and gangue-based zeolite is 5:2:1.

[0027] Furthermore, in step (i) of the above technical solution, in a preferred embodiment of the present invention, the calcium phosphate salt compound is calcium dihydrogen phosphate (Ca(H 2 PO 4 ) 2 ).

[0028] Furthermore, in step (i) of the above technical solution, the concentration of the phosphate compound solution is 10 g / L.

[0029] Furthermore, in step (i) of the above technical solution, in a preferred embodiment of the present invention, the time for fully stirring the mixed liquid is 6 hours.

[0030] Specifically, in step (i) of the above technical solution, phosphorus and calcium are loaded onto the surface of gangue-based biochar through pyrolysis modification, which increases the active sites on the surface of gangue-based biochar and can also serve as nutrients in the soil to enhance the improvement effect on the topsoil.

[0031] Furthermore, in step (ii) of the above technical solution, in a preferred embodiment of the present invention, the pyrolysis temperature is 500° C., and the pyrolysis time is 4 hours.

[0032] Furthermore, in step (ii) of the above technical solution, the heating rate is 1-10°C·min -1 .

[0033] In a preferred embodiment of the present invention, the heating rate is 5°C·min -1 .

[0034] Furthermore, in the above technical solution, the steps of preparing solid waste-based microbial solidification balls by adsorption-embedding-crosslinking method are as follows:

[0035] (A) Preparation of bacterial solution: inoculate the rhizosphere growth-promoting bacteria agent in a nitrogen-fixing liquid culture medium, and then culture it in a shaker at 25-35°C for 90-100 hours; after the culture is completed, centrifuge, resuspend the obtained precipitate with a 0.85% NaCl solution with a suitable concentration for microbial growth, and adjust the concentration of the bacterial solution by absorbance to obtain a rhizosphere growth-promoting bacteria solution with an absorbance of 1.5-1.6;

[0036] (B) Immobilizing microorganisms by adsorption method: sterilizing the gangue-based biochar, then mixing the sterilized gangue-based biochar with the rhizosphere growth-promoting bacteria solution of step (A) according to a ratio, adjusting the pH value of the obtained mixed bacterial solution to 8, and placing it in a 30° C. shaker for adsorption for 30-40 hours to obtain microorganism-gangue-based biochar;

[0037] (C) Preparation of embedding agent and cross-linking agent

[0038] Dissolve sodium alginate in distilled water according to the ratio, mix well, and obtain the embedding agent;

[0039] Chitosan is dissolved in an acetic acid solution according to a ratio to obtain a chitosan-acetic acid solution; calcium chloride (CaCl 2 ), fully dissolved, and the pH value of the resulting liquid was adjusted to 7 to obtain a cross-linking agent;

[0040] (D) Preparation of solid waste-based microbial solidification balls

[0041] The microorganism-gangue-based biochar composite material of step (B) and the embedding agent of step (C) are mixed according to a ratio and stirred evenly; then the cross-linking agent of step (C) is added to the obtained mixed solution according to a ratio, and the mixture is reacted at room temperature for 11-13 hours; after the reaction is completed, the obtained product is washed and vacuum-freeze-dried to obtain the solid waste-based microorganism solidified ball.

[0042] Furthermore, in step (A) of the above technical solution, the rhizosphere growth-promoting bacteria are brown spherical nitrogen-fixing bacteria. The main function of the rhizosphere growth-promoting bacteria is to convert free nitrogen in the atmosphere into ammonium salts that can be directly used by the plant rhizosphere through nitrogenase, provide nutrients for plants, and react with insoluble phosphorus compounds to generate soluble phosphates, increase the content of phosphorus in the soil that can be absorbed by plants, and promote plant growth.

[0043] Furthermore, in step (A) of the above technical solution, in a preferred embodiment of the present invention, the rotation speed of the shaking table is 150r / min.

[0044] Furthermore, in step (A) of the above technical solution, in a preferred embodiment of the present invention, the rhizosphere growth-promoting bacteria are cultured on a shaker for 96 hours.

[0045] Furthermore, in step (A) of the above technical solution, in a preferred embodiment of the present invention, the absorbance of the rhizosphere growth-promoting bacteria solution is 1.5725.

[0046] Furthermore, in step (A) of the above technical solution, the specific composition of the nitrogen-fixing liquid culture medium is: 0.20 g / L KH 2 PO 4 , 0.80g / LK 2 HPO 4 , 0.20 g / L MgSO 4 , 0.10g / L CaSO 4 , 0.001g / L FeCl 3 , 0.001g / LNa 2 MoO 4 , 0.50g / L yeast powder, 20g / L mannitol.

[0047] Furthermore, in step (A) of the above technical scheme, in a preferred embodiment of the present invention, the method of inoculating the bacterial liquid is: dissolving the nitrogen-fixing liquid culture medium in distilled water according to a ratio and sterilizing; then, on a sterile operating table, using an inoculation loop to scrape the frozen rhizosphere growth-promoting bacteria, and transfer them to the obtained nitrogen-fixing liquid culture medium solution.

[0048] Preferably, in the above technical solution, the solid-to-liquid ratio of the nitrogen-fixing liquid culture medium to distilled water is 0.0218 g:1 mL.

[0049] Furthermore, in step (B) of the above technical solution, the solid-liquid ratio of the sterilized gangue-based biochar to the rhizosphere growth-promoting bacteria solution is 1g:(40-90)mL. In a preferred embodiment of the present invention, the solid-liquid ratio of the sterilized gangue-based biochar to the rhizosphere growth-promoting bacteria solution is 1g:50mL.

[0050] Furthermore, in step (B) of the above technical solution, the rotation speed of the shaking table is 150r / min.

[0051] Furthermore, in step (B) of the above technical solution, in a preferred embodiment of the present invention, the adsorption time is 36 hours.

[0052] Furthermore, in step (C) of the above technical solution, in a preferred embodiment of the present invention, the mass fraction of sodium alginate in the embedding agent is 4%.

[0053] Furthermore, in step (C) of the above technical solution, in a preferred embodiment of the present invention, the solid-liquid ratio of the chitosan to the acetic acid solution is 1 g:100 mL.

[0054] Furthermore, in step (C) of the above technical solution, the mass fraction of the acetic acid solution is 1-5%.

[0055] Furthermore, in step (C) of the above technical solution, in a preferred embodiment of the present invention, the mass ratio of chitosan to calcium chloride is 1:2.

[0056] Specifically, in step (C) of the above technical solution, sodium alginate is used as an embedding agent to embed microorganisms to form a stable solidified ball, which can protect the microorganisms from the influence of the external environment and maintain the activity and function of the bacteria, but the mechanical strength of the solidified ball is poor and the service life is short. The chitosan in the cross-linking agent is a natural polymer, and the amino group in the structure can combine with the carboxyl group in the sodium alginate molecular chain to form a polyelectrolyte membrane, thereby stabilizing the solidified ball and extending the service life.

[0057] Specifically, in step (C) of the above technical solution, the main function of the acetic acid solution is to dissolve chitosan which is difficult to dissolve in water under neutral or alkaline conditions; adding CaCl 2 The main role is: CaCl 2 Provides calcium ions (Ca 2 +) cross-links with alginate in sodium alginate to generate water-insoluble calcium alginate gel, which can further enhance the mechanical strength and stability of the microbial solidified ball.

[0058] Furthermore, in step (D) of the above technical solution, the usage ratio of the microorganism-gangue-based biochar to the embedding agent is 1 g:100 mL.

[0059] Furthermore, in step (D) of the above technical solution, the usage ratio of the microorganism-gangue-based biochar to the cross-linking agent is 1 g:100 mL.

[0060] Furthermore, in step (D) of the above technical solution, in a preferred embodiment of the present invention, the cross-linking time is 12 hours.

[0061] The second object of the present invention is to provide solid waste-based microbial solidification balls prepared by the method described above.

[0062] The third object of the present invention is to provide the use of the solid waste-based microbial solidification balls prepared by the above method in degrading the insoluble phosphorus in coal gangue.

[0063] Compared with the prior art, the present invention has the following beneficial effects:

[0064] (1) Compared with the traditional gangue porous material synthesis process, the present invention uses low-energy microwave activation decarbonization technology to activate and digest the gangue, while activating the silicate mineral lattice, separating and preserving the carbon source in the gangue. This method reduces the energy loss of the process and realizes the graded utilization of gangue carbon source and siliceous materials.

[0065] (2) The gangue-based biochar prepared by the phosphorus activation-pyrolysis process of the present invention not only adds the carbon retained in the microwave activation process to the gangue-based porous adsorption material, but also loads phosphorus and calcium to the surface of the material through activation modification, thereby increasing the active sites on the surface of the material and improving the phosphorus nutrient supply capacity of the barren soil. The present invention also converts mining solid waste into an environmentally friendly adsorption material, which can not only be used as a microbial loading material to improve the effectiveness of microorganisms in the environment, but also can achieve the purpose of effectively adsorbing pollutants in the environment, reducing environmental pollution, and improving the utilization rate of solid waste resources.

[0066] (3) Compared with the traditional process, the present invention adopts the gangue-based biochar embedding-crosslinking microorganism fixation technology. The use of solid waste such as gangue and straw not only reduces the cost of the loading material, but also can significantly improve the functional activity of rhizosphere growth-promoting bacteria in the soil environment, thereby enhancing the soil improvement effect of microbial agents.

[0067] (4) The present invention uses decarbonized gangue-based biochar as a carrier material to fix rhizosphere growth-promoting bacteria for the restoration of sandy degraded soil in the arid mining areas of Northwest China. This technology can not only improve the survival time and functional activity of rhizosphere growth-promoting bacteria in the soil environment, but also provide a new way to dispose of large quantities of gangue solid waste. This method also has the technical advantages of improving the utilization rate of gangue carbon sources and increasing soil phosphorus fertilizer and organic carbon pools. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0069] Figure 1 The XRD diffraction pattern of the gangue-based zeolite prepared in Example 1 of the present invention;

[0070] Figure 2 The XPS test result diagram of two kinds of gangue-based biochars obtained in Example 1 and Comparative Example 2 of the present invention; wherein: the gangue-based biochar (modified) is prepared in Example 1; the gangue-based biochar (unmodified) is prepared in Comparative Example 2;

[0071] Figure 3A comparison chart of the macroscopic adsorption amount of microorganisms by microorganism-gangue-based biochar at different solid-liquid ratios obtained in step 6 of Examples 1-6 of the present invention;

[0072] Figure 4 This is a comparison chart of PLFA biomarker amounts of the microorganism-gangue-based biochar obtained in Example 1, Comparative Example 1 and Step 6 of Example 1 of the present invention and the gangue-based biochar obtained in Step 4 of Example 1;

[0073] Figure 5 This is a comparison chart of the degradation effects of phosphorus in coal gangue obtained by Application Examples 1-3, Comparative Application Example 1 and Comparative Application Example 2 of the present invention;

[0074] Figure 6 This is a flow chart of a method for fixing rhizosphere growth-promoting bacteria using decarbonized gangue-based biochar as a carrier according to Example 1 of the present invention;

[0075] Figure 7 This is a schematic diagram of a method for fixing rhizosphere growth-promoting bacteria using decarbonized gangue-based biochar as a carrier according to Example 1 of the present invention. DETAILED DESCRIPTION

[0076] The gangue decarbonization described in the present invention refers to extracting carbon residues from the gangue by physical, chemical and other methods, so as to separate silicate mineral components from organic carbon sources and realize graded utilization of gangue resources.

[0077] The microbial immobilization technology described in the present invention refers to fixing a certain concentration of microbial liquid on a carrier material through adsorption, embedding, cross-linking and other methods. The carrier material has a protective effect on the microorganisms, allowing the microorganisms to maintain high-efficiency and long-lasting activity in an environment away from the culture medium. The microbial carrier material is also easy to recycle in water pollution control.

[0078] The rhizosphere growth-promoting bacteria described in the present invention are beneficial bacteria that colonize around the root system of plants. Through their own metabolic activities and interactions with soil, plants and microorganisms, they produce effects such as nitrogen fixation, phosphorus solubilization and antibiotic production, thereby promoting plant growth and improving plant resistance.

[0079] The main function of the immobilized carrier material is to provide a microenvironment suitable for the survival of microorganisms. The carrier material plays a key role in the immobilization of microorganisms. Among them, the most popular carriers are carbon-based materials, which are insoluble solids with high mechanical strength and adjustable surface properties. Compared with individual carbon materials or free cells, composite materials have more advantages in environmental remediation. The relatively high specific surface area ensures the loading of microorganisms, which is conducive to the contact between microorganisms and pollutants adsorbed on the surface and inside of the carrier. The hierarchical porosity provides a stable space for microorganisms and protects them from harm. Generally, carbon materials used as carriers include biochar, activated carbon and carbon nanotubes. Biochar is a common carbon material produced by anaerobic pyrolysis of carbon-rich biomass (such as crop straw, shells, rhizomes and sawdust). The adsorption effect of the porous structure of biochar is very obvious, and it has a good fixation effect on microorganisms. Activated carbon is composed of organic components produced by heating and activation. During the preparation process, the high temperature causes a sharp decrease in volatile carbon components and forms a microporous structure. Carbon nanotubes are flexible and dispersible, and can also capture microorganisms in a porous framework.

[0080] Table 1 Comparative analysis of the characteristics of the present invention and the prior art

[0081]

[0082] The present invention is further described in detail below through implementation cases. This implementation case is implemented based on the technology of the present invention, and a detailed implementation method and specific operation process are now given to illustrate that the present invention is creative, but the protection scope of the present invention is not limited to the following implementation cases.

[0083] The equipment and raw materials used in the present invention can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0084] For example, the rhizosphere growth-promoting bacteria used in the following embodiments of the present invention are brown spherical nitrogen-fixing bacteria, with the Latin name Azotobacter chroococcum, purchased from Shanghai Collection Biotechnology Center, CGMCC No. 1.824.

[0085] The absorbance measurement method used in the following embodiments of the present invention is as follows: the number of bacterial cells is determined by using an ultraviolet-visible spectrophotometer to measure the absorbance at a wavelength of 600 nm.

[0086] The calculation formula for the macroscopic adsorption capacity of microorganisms involved in the following embodiments of the present invention is:

[0087]

[0088] Where, OD 0 Represents the initial absorbance of the bacterial suspension, OD1 It is the absorbance of the supernatant after microbial adsorption.

[0089] Example 1

[0090] A method for fixing rhizosphere growth-promoting bacteria using decarbonized gangue-based biochar as a carrier in this embodiment specifically comprises the following steps:

[0091] Step 1: 10g of coal gangue and 1g of CaO are mixed evenly at a mass ratio of 10:1, and ball-milled in a ball mill for 30min to obtain coal gangue powder with a particle size of less than 200 mesh; 5g of the coal gangue powder after ball milling and activation is added to a 2mol / LNaOH solution at a solid-liquid ratio of 1g:30mL, added to a microwave reactor with magnetic stirring, and microwave activated at a temperature of 160°C for 40min to obtain an active aluminum silicate ore solution dissolved from coal gangue, and the ore solution is centrifuged in a centrifuge at 8000r / min for 5min, the supernatant is taken out, the precipitate is washed and dried, and crushed to obtain 2g of carbon slag.

[0092] Step 2: Mix coal gangue with SiO 2 Weigh 0.736 g of silicon dioxide according to the mass ratio of 1:0.1472. 2 Add the active aluminum silicate ore liquid suspension obtained from the centrifuged slag obtained in the above step 1, adjust the atomic ratio of sodium, aluminum and silicon in the ore liquid to 1:1:2, continue stirring and aging at room temperature for 4 hours; transfer the obtained aged product to a reactor, seal the reactor and heat it from room temperature to 200°C at a rate of 5°C / min for constant temperature crystallization for 10 hours, prepare gangue-based zeolite in the reactor, filter the reaction solution, wash the filtered crystalline material, and dry it in an oven at 50°C for 2 hours to obtain 1g of crystalline material, i.e., the gangue-based zeolite.

[0093] Step 3: Add the carbon slag and gangue-based zeolite obtained in steps 1 and 2 respectively to 5 g of straw, and mix the straw, carbon slag and gangue-based zeolite in a mass ratio of 5:2:1 to obtain a mixed raw material; mix the mixed raw material with Ca(H 2 PO 4 ) 2 Weigh 1.6 g Ca(H 2 PO 4 ) 2 , dissolved in 160 ml of distilled water to make Ca(H 2 PO 4 ) 2 The concentration of the solution was 10 g / L. Then the mixed raw materials were added and fully mixed and stirred for 6 hours, and dried in an oven at 60° C. until the moisture content was close to 0%, to obtain 8.23 ​​g of phosphorus-modified raw materials.

[0094] Step 4: Place the phosphorus-modified raw material obtained in step 3 into a tubular resistance furnace and heat it under N 2 At 5℃·min -1 The mixture was heated to 500 °C at a heating rate of 1.5 °C and maintained for 4 h to obtain 7.39 g of gangue-based biochar.

[0095] Step 5: Weigh 10.9 g of nitrogen-fixing liquid culture medium and dissolve it in 500 ml of distilled water. Sterilize it in a sterilizer at 120 ° C for 2 h. Use an inoculation loop to scrape the frozen rhizosphere growth-promoting bacteria and transfer them to the sterilized nitrogen-fixing liquid culture medium. Place it in a shaker at 30 ° C and 150 r / min for 96 h. Take it out and centrifuge it at 8000 r / min for 10 min. Discard the supernatant, collect the brown ball nitrogen-fixing bacteria, put it into 400 mL of NaCl solution with a mass fraction of 0.85% for resuspending, and measure the absorbance OD once. 0 , and a brown spherical nitrogen-fixing bacteria solution with an absorbance of 1.5725 was obtained.

[0096] Step 6: Sterilize 1g of the gangue-based biochar obtained in step 4 and place it in a shaker at 30°C and 150r / min for 36h at pH=8 with 50ml of the brown spherical nitrogen-fixing bacteria solution obtained in step 5. Measure the absorbance OD once after adsorption. 1 The experiment was set up with three groups of control repetitions, keeping other conditions unchanged, and setting the solid-liquid ratio to 1g:50ml to obtain microbial-gangue-based biochar.

[0097] Step 7: Heat 4g of sodium alginate and dissolve it in 100ml of distilled water to obtain an embedding agent. Dissolve 1g of chitosan in 100ml of distilled water containing 2wt% acetic acid to obtain a chitosan-acetic acid solution; add 2g of CaCl 2 and fully dissolved, the resulting solution was washed with 0.5 M NaHCO 3 , adjust the pH value to 7 to obtain a cross-linking agent, and sterilize the obtained embedding agent and cross-linking agent at high temperature for later use.

[0098] Step 8: Add 1 g of the microorganism-gangue-based biochar composite material prepared in step 6 to the embedding agent in the above step 7 and stir. After mixing, drip into the cross-linking agent in step 7 through a peristaltic pump. Cross-linking reaction is carried out at room temperature for 12 hours. The obtained material is rinsed with physiological saline and vacuum freeze-dried to obtain solid waste-based microbial solidified balls.

[0099] Example 2

[0100] The method of fixing rhizosphere growth-promoting bacteria using decarbonized gangue-based biochar as a carrier in this embodiment is basically the same as the method in Example 1, with the only difference being that the solid-liquid ratio in step 6 of this embodiment is 1 g:40 ml, and the other processes are the same as those in Example 1.

[0101] Example 3

[0102] The method of fixing rhizosphere growth-promoting bacteria using decarbonized gangue-based biochar as a carrier in this embodiment is basically the same as the method in Example 1, with the only difference being that the solid-liquid ratio in step 6 of this embodiment is 1 g:60 ml, and the other processes are the same as those in Example 1.

[0103] Example 4

[0104] The method of fixing rhizosphere growth-promoting bacteria using decarbonized gangue-based biochar as a carrier in this embodiment is basically the same as the method in Example 1, with the only difference being that the solid-liquid ratio in step 6 of this embodiment is 1 g:70 ml, and the other processes are the same as those in Example 1.

[0105] Example 5

[0106] The method of fixing rhizosphere growth-promoting bacteria using decarbonized gangue-based biochar as a carrier in this embodiment is basically the same as the method in Example 1, with the only difference being that the solid-liquid ratio in step 6 of this embodiment is 1g:80ml, and the other processes are the same as those in Example 1.

[0107] Example 6

[0108] The method of fixing rhizosphere growth-promoting bacteria using decarbonized gangue-based biochar as a carrier in this embodiment is basically the same as the method in Example 1, with the only difference being that the solid-liquid ratio in step 6 of this embodiment is 1g:90ml, and the other processes are the same as those in Example 1.

[0109] Comparative Example 1

[0110] The method for preparing a single solidified ball in this comparative example is prepared by adopting steps 5, 7 and 8 of Example 1, except that steps 1-4 and 6 are omitted in this comparative example.

[0111] Comparative Example 2

[0112] The present comparative example provides a method for preparing gangue-based biochar (unmodified), which adopts steps 1, 2, 3, and 4 of Example 1, with the only difference being that in step 3 of the present comparative example, the raw materials are not phosphorus-modified, that is, the product obtained in step 3 of the present comparative example is the unmodified raw material, and steps 5-8 are omitted, and the other processes are the same as those of Example 1.

[0113] Application Example 1

[0114] This application example uses the solid waste-based microbial solidification balls prepared in Example 1 to perform a degradation experiment on coal gangue, and calculates the effective phosphorus content that is easily absorbed by the soil after the insoluble phosphorus in the coal gangue is degraded. The specific application method is as follows:

[0115] 20 g of coal gangue powder with a particle size of 200 mesh and 20 g of the solid waste-based microbial solidification ball prepared in Example 1 were mixed evenly in a mass ratio of 1:1 and placed in a culture dish. Distilled water was added until the entire system was completely wetted. The mixture was placed in a constant temperature incubator at 30°C for 3d, 6d, 8d, and 10d. During the process, distilled water was added to the culture dish every day to keep the system moistened. After the mixture was finished, the effective phosphorus content in the coal gangue was measured using an ultraviolet spectrophotometer.

[0116] Application Example 2

[0117] The application method of this application example for degrading insoluble phosphorus in coal gangue is basically the same as that of Application Example 1, with the only difference being that the material used in this application example for degrading coal gangue is the microorganism-gangue-based biochar obtained in step 6 of Example 1.

[0118] Application Example 3

[0119] The application method of this application example for degrading insoluble phosphorus in coal gangue is basically the same as that of Application Example 1, with the only difference being that the material used in this application example for degrading coal gangue is the brown spherical nitrogen-fixing bacteria solution obtained in step 5 of Example 1.

[0120] Comparative application example 1

[0121] The application method of this comparative application example for degrading the insoluble phosphorus in coal gangue is basically the same as that of Application Example 1, with the only difference being that the material used in the comparative application example for degrading coal gangue is the single solidified ball prepared in Comparative Example 1.

[0122] Comparative Application Example 2

[0123] The application method of this comparative application example for degrading the insoluble phosphorus in coal gangue is basically the same as that of Application Example 1, with the only difference being that this comparative application example does not use any degradation material and only uses 20g of coal gangue (CK treatment).

[0124] Structural and performance testing:

[0125] The crystal material obtained in step 2 of the above embodiment 1 was subjected to XRD analysis test. Figure 1 The XRD pattern of the gangue-based zeolite prepared in Example 1 of the present invention was obtained by X-ray diffractometer. After comparison with the crystal structure card database, it was confirmed that the synthesized zeolite was gangue-based zeolite (analcime). Figure 1 It can be seen that the peak of the crystalline material is sharp, the crystallinity is high, and the structure is stable.

[0126] The carbon loss rate, specific surface area and pore structure of the two types of gangue-based biochar obtained in step 4 of Example 1 and Comparative Example 2 are shown in Table 2 below. Through the analysis of the data, we believe that Ca(H 2 PO4 ) 2 The carbon source retention capacity of the modified material increased by 24.55%. Ca(H 2 PO 4 ) 2 Compared with the unmodified material, the modified material showed a larger specific surface area and total pore volume, as well as a smaller average pore size. 2 PO 4 ) 2 Modification can improve the structural properties of materials.

[0127] Table 2 Comparison of carbon loss rate, specific surface area and average pore size of gangue-based biochar prepared in Example 1 and Comparative Example 2

[0128]

[0129] At the same time, the present invention conducts XPS test on the two kinds of gangue-based biochar obtained in Example 1 and Comparative Example 2, and the results are shown in Figure 2 .right Figure 2 Analysis shows that P is obtained by peak fitting of the full spectrum. 2P Peaks. From the analysis results, it can be seen that the unmodified biochar prepared in Comparative Example 2 has COP functional groups, while the gangue-based biochar prepared in Example 1 has two main peaks fitted around 134.38ev and 132.65ev, corresponding to COP and CPO respectively. 3 or C 2 PO 2 , these functional groups increased the carbon retention rate by 24.55% after phosphorus modification.

[0130] The macroscopic adsorption capacity of the microorganism-gangue-based biochar composite material was calculated by the above formula of microbial macroscopic adsorption capacity. The test results are shown in Figure 3 Through the analysis of the data, the inventors found that when the solid-liquid ratio was 1g:50ml, the maximum macroscopic adsorption amount was 86.21%, which was in line with the experimental design.

[0131] The PLFA biomarker method was used to extract the PLFA components in the microorganisms through lipids, separation of phospholipids and methanol esterification, and then n-hexane was added to dissolve the phospholipid fatty acid methyl esters after nitrogen blowing, and the PLFA biomarker amount was detected by gas chromatography. The PLFA biomarker amount of the microbial immobilization product obtained in Example 1, Comparative Example 1 and Step 6 of Example 1 of the present invention and the gangue-based biochar obtained in Step 4 of Example 1 were compared. Figure 4, through the analysis of the data, the inventors found that the order of the microbial content of the four materials from high to low is: solid waste-based microbial solidification ball > single solidification ball > microorganism-gangue-based biochar > gangue-based biochar. The reason is that biochar has a loose porous structure, which can provide rich nutrients and a good living environment for microorganisms, enhance the reproduction ability of microorganisms, but has poor retention ability for microorganisms, and is prone to desorption, which reduces the content of microorganisms; the amino group in the chitosan structure of the single solidification ball can combine with the carboxyl group in the sodium alginate molecular chain to form a polyelectrolyte membrane, thereby achieving the effect of loading microorganisms, but chitosan and sodium alginate do not contain nutrients that can be directly absorbed and utilized by microorganisms, thereby limiting the proliferation of microorganisms; and the solid waste-based microbial solidification ball contains nutrients such as phosphorus and calcium, which can provide rich nutrients and a good living environment for microorganisms, so that the growth and proliferation of microorganisms are improved. At the same time, its strong adsorption performance is also the best microbial loading material. Therefore, the best material obtained is the solid waste-based microbial solidification ball, which meets the experimental design.

[0132] The effective phosphorus concentrations of application examples 1-3, comparative application examples 1 and comparative application examples 2 were measured and the test results are shown in Figure 5 .

[0133] Figure 5 The specific test method for the effective phosphorus concentration in the medium is as follows: weigh 2.5g of degraded coal gangue into a 100ml conical flask, add 50mL of 0.5mol / L NaHCO 3 The solution was shaken for 30 minutes and then filtered. 10 ml of the filtrate was taken into a 50 mL volumetric flask, 5 mL of molybdenum antimony reagent was added, water was added to make up the volume, and the mixture was allowed to stand for 30 minutes. The colorimetry was performed using an ultraviolet spectrophotometer to obtain the effective phosphorus concentration of the degraded gangue. The calculation formula is shown below.

[0134]

[0135] Where: ρ—mass concentration of available phosphorus obtained from the spectrophotometer (μg / mL)

[0136] V—fixed volume during color development (mL);

[0137] ts—ratio of the total volume of the extract to the volume of the color developed to the volume of the absorbed extract;

[0138] m—mass of coal gangue (g);

[0139] 10 3 —Convert μg to mg.

[0140] Through Figure 5According to the analysis of the data, the inventors found that in the same environment, as the degradation time increased, the effective phosphorus concentration of each group gradually decreased, which proved that the activity of microorganisms decreased. The four degradation materials were ranked from high to low in terms of their ability to maintain microbial activity: solid waste-based microbial solidification balls > single solidification balls > microorganism-gangue-based biochar > brown ball nitrogen-fixing bacteria liquid. At 6 days of degradation, the effective phosphorus concentration of gangue degraded by each treatment was the highest. Compared with the CK treatment group, the degradation ability of solid waste-based microbial solidification balls on coal gangue increased by 94.73%, compared with single solidification balls, the degradation ability of solid waste-based microbial solidification balls on coal gangue increased by 35.39%, compared with microorganism-gangue-based biochar, the degradation ability of solid waste-based microbial solidification balls on coal gangue increased by 50.21%, and compared with brown ball nitrogen-fixing bacteria liquid, the degradation ability of solid waste-based microbial solidification balls on coal gangue increased by 59.42%. Therefore, the best material is solid waste-based microbial solidification balls, which meets the experimental design.

[0141]

[0142] Where: P 1 —Effective phosphorus concentration of solid waste-based microbial solidification ball treatment after 6 days of degradation (mg / kg)

[0143] P 2 —The effective phosphorus concentrations (mg / kg) of the four treatments (single solidified ball, microbial-gangue based biochar, brown ball nitrogen-fixing bacteria solution and CK4 treatments) at 6 days of degradation.

Claims

1. A method for fixing rhizosphere growth-promoting bacteria using decarbonized gangue-based biochar as a carrier, characterized in that: The method comprises the steps of preparing gangue-based zeolite by microwave activation and decarbonization of coal gangue; preparing gangue-based biochar by phosphorus modification; and preparing solid waste-based microbial solidification balls by adsorption-embedding-crosslinking method.

2. The method according to claim 1, characterized in that: The steps of preparing gangue-based zeolite by microwave activation and decarbonization of gangue are as follows: (1) Decarbonization of coal gangue by microwave activation: coal gangue and calcium oxide (CaO) are mixed in a proportion, ball-milled to obtain coal gangue powder; then the coal gangue powder is mixed in a proportion with a NaOH solution, the obtained mixed reaction liquid is placed in a microwave reactor, and microwave activated for 35-45 minutes at 150-170° C. under stirring conditions to obtain an active aluminum silicate ore solution dissolved from the coal gangue; the ore solution is centrifuged, and the obtained precipitate is washed, dried, and then crushed to obtain carbon slag; (2) Preparation of gangue-based zeolite: silicon dioxide (SiO2) is added to the active aluminum silicate ore solution of step (1) according to a ratio, and the atomic ratio of sodium, aluminum and silicon in the obtained mixed solution is adjusted to 1:1:2 according to the atomic ratio of the elements in the chemical formula of analcime NaAlSi2O6, and then stirred and aged at room temperature; after the aging is completed, the obtained aged solution is transferred to a reactor, and the reactor is sealed and heated to a constant temperature of 180-220°C for crystallization reaction for 8-12 hours; after the reaction is completed, the obtained product is filtered, and the crystals obtained by filtration are washed and dried to precipitate the gangue-based zeolite.

3. The method according to claim 2, characterized in that: In step (2), the mass ratio of the coal gangue to silicon dioxide is 1:(0.1-0.2).

4. The method according to claim 1, characterized in that: The steps of preparing gangue-based biochar by phosphorus modification are specifically as follows: (i) Phosphorus-modified raw materials: straw, the carbon slag, and the gangue-based zeolite are mixed according to a ratio, and then the mixed raw materials are mixed with a phosphate compound solution according to a ratio, and the mixed solution is fully stirred for 5-7 hours and then dried to obtain the phosphorus-modified raw materials; (ii) Preparation of gangue-based biochar: The phosphorus-modified raw material is heated to 450-550° C. in an inert atmosphere and pyrolyzed at a constant temperature for 3-5 hours. After the pyrolysis is completed, the gangue-based biochar is obtained.

5. The method according to claim 4, characterized in that: In step (i), the mass ratio of the straw, the carbon residue and the gangue-based zeolite is 5:2:

1.

6. The method according to claim 1, characterized in that: The steps of preparing solid waste-based microbial solidification balls by adsorption-embedding-crosslinking method are as follows: (A) Preparation of bacterial solution: inoculate the rhizosphere growth-promoting bacteria agent in a nitrogen-fixing liquid culture medium, and then culture it in a shaker at 25-35°C for 90-100 hours; after the culture is completed, centrifuge, resuspend the obtained precipitate with a 0.85% NaCl solution with a suitable concentration for microbial growth, and adjust the concentration of the bacterial solution by absorbance to obtain a rhizosphere growth-promoting bacteria solution with an absorbance of 1.5-1.6; (B) Immobilizing microorganisms by adsorption method: sterilizing the gangue-based biochar, then mixing the sterilized gangue-based biochar with the rhizosphere growth-promoting bacteria solution of step (A) according to a ratio, adjusting the pH value of the obtained mixed bacterial solution to 8, and placing it in a 30° C. shaker for adsorption for 30-40 hours to obtain a microorganism-gangue-based biochar composite material; (C) Preparation of embedding agent and cross-linking agent Dissolve sodium alginate in distilled water according to the ratio, mix well, and obtain the embedding agent; Dissolving chitosan in an acetic acid solution according to a ratio to obtain a chitosan-acetic acid solution; then adding calcium chloride (CaCl2) to the chitosan-acetic acid solution according to a ratio to fully dissolve the chitosan-acetic acid solution, and adjusting the pH value of the resulting liquid to 7 to obtain a cross-linking agent; (D) Preparation of biochar-microbial solidification balls The microorganism-gangue-based biochar composite material of step (B) and the embedding agent of step (C) are mixed according to a ratio and stirred evenly; then the cross-linking agent of step (C) is added to the obtained mixed solution according to a ratio, and the mixture is reacted at room temperature for 11-13 hours; after the reaction is completed, the obtained product is washed and vacuum-freeze-dried to obtain the solid waste-based microorganism solidified ball.

7. The method according to claim 6, characterized in that: In step (B), the solid-to-liquid ratio of the sterilized gangue-based biochar to the rhizosphere growth-promoting bacteria solution is 1 g: (40-90) mL.

8. The method according to claim 6, characterized in that: The dosage ratio of the microorganism-gangue-based biochar to the embedding agent is 1g:100ml; the dosage ratio of the microorganism-gangue-based biochar to the cross-linking agent is 1g:100ml.

9. The solid waste-based microbial solidification ball prepared by the method according to any one of claims 1 to 8.

10. Use of the solid waste-based microbial solidification balls prepared by the method according to any one of claims 1 to 8 in degrading insoluble phosphorus in coal gangue.

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