Magnetic carbon-loaded slow-release microbial agent as well as preparation method and application thereof

Abstract

The invention discloses a magnetic carbon-loaded slow-release microbial agent which comprises an activated carbon carrier containing ferroferric oxide nanoparticles and a compound microbial bacterium composed of bacillus megatherium, bacillus mucilaginosus, white-rot fungi and pseudomonas, and the mass ratio of the compound microbial bacterium to the activated carbon carrier is (3-4): 30. The invention further discloses a preparation method and application of the magnetic carbon-loaded slow-release microbial agent. According to the magnetic carbon-loaded slow-release microbial agent disclosed by the invention, the activated carbon containing the ferroferric oxide nano-particles is used as a carrier to load the compound bacteria, so that the stability of the microbial bacteria is improved, the microbial bacteria can better resist the impact of the environment, the service life of the microbial bacteria is prolonged, and the slow-release effect of the microbial agent in the remediation of vegetable planting soil is achieved; the nutrient absorption of the vegetables is promoted.

Description

Technical Field

[0001] The invention relates to the technical field of microbial agents, and in particular to a magnetically adsorbed carbon-supported slow-release microbial agent and a preparation method and application thereof. Background Art

[0002] Soil degradation is a common problem in vegetable cultivation. This is mainly caused by long-term continuous cropping, excessive use of fertilizers and pesticides, and unreasonable irrigation methods. Soil degradation is manifested in problems such as decreased soil organic matter content, soil structure damage, soil salinization, soil pH imbalance, and soil pollution. These problems seriously affect the growth and quality of vegetables, and reduce vegetable yields and economic benefits.

[0003] At present, soil remediation mainly focuses on: 1. Physical remediation: deep plowing or deep tillage to improve soil aeration and water permeability, promote the activity of soil microorganisms, and accelerate the decomposition and transformation of pollutants; 2. Chemical remediation: using chemical reagents such as calcium-containing substances or acidic substances to adjust the pH of the soil, reduce the salt content in the soil, and improve the soil environment; 3. Microbial remediation: using the metabolic activities of microorganisms, plants and other organisms to degrade and transform pollutants in the soil and improve soil fertility and biological activity.

[0004] Research has found that the use of microbial agents for remediation has great research value and can improve soil, treat heavy metal-contaminated soil, improve crop quality and yield, increase fertilizer utilization, and increase farmers' production and income. However, in actual applications, microbial agents are still unstable in the soil, easily affected by environmental fluctuations, and have poor impact resistance. Summary of the invention

[0005] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0006] Another object of the present invention is to provide a method for preparing a magnetic carbon-loaded slow-release microbial agent and a magnetic carbon-loaded slow-release microbial agent, which uses activated carbon with ferroferric oxide nanoparticles as a carrier to load the composite bacteria, thereby improving the stability of the microbial bacteria, enabling them to better resist environmental impacts, extending their service life, and achieving a slow-release effect in soil remediation for vegetable planting, thereby promoting the absorption of nutrients by vegetables.

[0007] Still another object of the present invention is to provide a method for preparing a magnetic carbon-loaded slow-release microbial agent, which optimizes the preparation process, improves the specific surface area and porosity of activated carbon, controls the loading of ferroferric oxide nanoparticles, and allows the ferroferric oxide nanoparticles to be fully adsorbed on the surface and voids of the activated carbon, thereby forming a stable magnetic carbon-loaded slow-release microbial agent with good soil remediation performance.

[0008] According to these purposes and other advantages of the present invention, a magnetic carbon-supported slow-release microbial agent is provided, which includes: an activated carbon carrier containing ferroferric oxide nanoparticles, and a composite microbial bacteria composed of Bacillus megaterium, Bacillus mucilaginosus, white rot fungi and Pseudomonas, wherein the mass ratio of the composite microbial bacteria to the activated carbon carrier is 3 to 4:30.

[0009] The object of the present invention can also be further achieved by a method for preparing the magnetic carbon-supported slow-release microbial agent. The method comprises the following steps: Step 1, preparation of activated carbon: crush rice husk or straw and pass through a 20-mesh sieve to obtain biomass powder, pyrolyze the biomass powder in a vacuum tube high-temperature furnace to form biochar, and activate the biochar to form activated carbon; Step 2: magnetizing the activated carbon: magnetizing the activated carbon using ferroferric oxide nanoparticles to obtain magnetized activated carbon; Step 3: Preparation of magnetic carbon-supported slow-release microbial agent: Bacillus megaterium, Bacillus colloidus, white rot fungi and Pseudomonas were inoculated into liquid culture medium and activated for 1 to 5 days, so that the number of microorganisms was 10 8 CFU / mL~10 9 CFU / mL, the bacterial liquid formed by Bacillus megaterium, Bacillus mucilaginosus, white rot fungi and Pseudomonas were mixed in a volume ratio of 12:12:1:12 to form a mixed bacterial liquid, the magnetized activated carbon was added to the conical flask containing the mixed bacterial liquid, and the mixture was shaken and cultured in a constant temperature shaking incubator at 25 ℃ and 100 rpm for 4 h, and then allowed to stand for 30 min, the supernatant was removed, the precipitate was collected, washed with sterile saline, and placed in a refrigerator at 4 ℃ to obtain the magnetic carbon-loaded slow-release microbial agent.

[0010] Preferably, in step 1, activating the biochar specifically comprises: S11, phosphoric acid impregnation pretreatment: biochar was mixed with a phosphoric acid solution with a mass fraction of 73% in a mass ratio of 1:2, and impregnated at room temperature for 24 h, and the impregnated biochar was filtered; S12, carbon dioxide activation: placing the impregnated biochar in a vacuum tube high temperature furnace, switching the vacuum to a carbon dioxide gas atmosphere, controlling the flow rate of carbon dioxide to 100 mL / min, and performing carbon dioxide activation at 400°C for 2 h to obtain primary activated carbon; S13, heat treatment: keep the primary activated carbon in a vacuum tube high temperature furnace, switch the carbon dioxide atmosphere to a nitrogen atmosphere, increase the temperature to 800°C, and keep it for 1 hour to obtain secondary activated carbon; S14. Post-treatment: Cool the secondary activated carbon to room temperature, wash repeatedly with deionized water until the pH of the washing solution is 6.8-7.2, and dry in an oven at 108° C. for 20 h to obtain activated carbon.

[0011] Preferably, in step 2, magnetizing specifically includes: S21, dissolving ferric chloride hexahydrate and ferrous chloride tetrahydrate in deionized water at a molar ratio of 2:1 to prepare a solution with a total iron ion concentration of 0.1 mol / L; S22, adding activated carbon to a 0.1 mol / L solution, and stirring the mixture ultrasonically at room temperature at an ultrasonic power of 500 to 800 W and an ultrasonic frequency of 40 to 50 kHz for 1 to 2 h to form a mixed solution, wherein the mixing ratio of activated carbon to solution is 1 g:20 mL; S23, dropwise add ammonia water with a mass fraction of 25% to 28% to the mixed solution until the pH value of the mixed solution is 9 to 10, and mechanically stir at a stirring speed of 600 to 800 r / min for 1 to 2 h, wherein the dropping speed of the ammonia water is 1 to 2 mL / min; S24, filtering to obtain a precipitate, washing the precipitate with deionized water for multiple times until the pH of the washing liquid is 6.8-7.2, and post-treating to obtain magnetized activated carbon.

[0012] Preferably, in step S22, the ultrasonic power is 600 W, the ultrasonic frequency is 40 kHz, and the ultrasonic stirring time is 1.5 h.

[0013] Preferably, in step S24, the post-treatment specifically includes the steps of: loading the washed precipitate into a quartz boat of a vacuum tube high-temperature furnace, sealing it, introducing nitrogen to exhaust the air in the furnace, raising the temperature in the furnace to 600°C at a heating rate of 5°C / min, maintaining it for 2 h, continuing to heat it to 800°C at a heating rate of 8°C / min, maintaining it for 3 h, and cooling it to room temperature at a cooling rate of 2°C / min to obtain magnetized activated carbon.

[0014] Preferably, in step S23, the stirring speed is 750 r / min and the stirring time is 1.5 h.

[0015] Preferably, in step three, Bacillus megaterium is inoculated in LB liquid culture medium and activated by shaking culture at 30°C and 220 r / min for 1 day, Bacillus mucilaginosus is inoculated in silicate bacterial culture medium and activated by shaking culture at 32°C and 170 r / min for 2 days, white rot fungi are inoculated in potato glucose liquid culture medium and activated by shaking culture at 32°C and 150 r / min for 5 days, and Pseudomonas is inoculated in KB culture medium and activated by shaking culture at 30°C and 200 r / min for 1 day.

[0016] Preferably, in step one, the pyrolysis in a vacuum tube high temperature furnace specifically includes: loading the biomass powder into a quartz boat of the vacuum tube high temperature furnace, using high-purity 99.999% nitrogen as a carrier gas and a protective gas, the nitrogen flow rate is 100 mL / min, the heating rate is 20°C / min, the pyrolysis final temperature is 600°C and maintained for 2 h.

[0017] The purpose of the present invention can be further achieved by using the magnetic carbon-loaded slow-release microbial agent in repairing vegetable planting soil. The present invention has at least the following beneficial effects: 1. The magnetic carbon-loaded slow-release microbial agent of the present invention uses activated carbon containing ferroferric oxide nanoparticles as a carrier to load the composite bacteria, thereby improving the stability of the microbial bacteria, enabling them to better resist environmental impacts, extending their service life, and achieving a slow-release effect in soil repair for vegetable planting, thereby promoting the absorption of nutrients by vegetables.

[0018] 2. The method for preparing a magnetically adsorbed carbon-supported slow-release microbial agent of the present invention activates biochar by combining phosphoric acid impregnation with carbon dioxide activation, and accurately controls the pore size and pore distribution of the activated carbon. At the same time, the synergistic effect of phosphoric acid and carbon dioxide can change the charge distribution and properties on the surface of the activated carbon, so that the surface of the activated carbon has a certain positive or negative charge, which helps it to undergo electrostatic adsorption with substances with opposite charges, and can more effectively adsorb and remove pollutants with different charges in the soil.

[0019] 3. The method for preparing a magnetically adsorbed carbon-supported sustained-release microbial agent of the present invention forms ferroferric oxide nanoparticles in situ in activated carbon, thereby limiting the movement and aggregation of the formed ferroferric oxide nanoparticles, allowing the ferroferric oxide nanoparticles to exist more stably in the pores of the activated carbon, thereby improving the structural stability of the carrier and enabling it to maintain good performance under different environmental conditions.

[0020] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. DETAILED DESCRIPTION

[0022] The present invention is described in further detail below so that those skilled in the art can implement it according to the description.

[0023] It should be understood that terms such as “having”, “including” and “comprising” used herein do not specify the existence or addition of one or more other elements or combinations thereof.

[0024] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0025] The raw materials used in the following examples are all common commercially available products. Vacuum tube high temperature furnace (GL-1200F, Siyang Precision Equipment (Shanghai) Co., Ltd.), LB liquid culture medium (containing 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride per liter, pH = 7.0 ± 0.1 (25 ° C), BL1056A, White Shark Biotechnology Co., Ltd.), silicate bacterial culture medium (containing 5.0 g of sucrose, 0.5 g of magnesium sulfate, 0.1 g of calcium sulfate, 2.0 g of disodium hydrogen phosphate, 0.005 g of ferric chloride, 1.0 g of glass powder, 15.0 g of agar per liter) g, pH = 7.0 ± 0.2 (25 ° C), HB8548-1, Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd.), potato glucose liquid medium (purity 99%, Shanghai Huzhen Industrial Co., Ltd.), KB medium (ORCCM0131, Auresa Biotechnology (Shanghai) Co., Ltd.), Bacillus megaterium (effective viable count ≥ 10 billion / g, Weifang Yihao Biotechnology Co., Ltd.), Bacillus mucilaginosus (effective viable count ≥ 20 billion / g, Weifang Yihao Biotechnology Co., Ltd.) White rot fungi (effective viable count ≥ 20 billion / g, Noan Gene Technology (Wuhan) Co., Ltd.), Pseudomonas (effective viable count ≥ 20 billion / g, Shanghai Xuanke Biotechnology Co., Ltd.). <Example 1> A magnetically attracted carbon-supported slow-release microbial agent, comprising a magnetized activated carbon carrier of ferroferric oxide nanoparticles and a composite microorganism composed of Bacillus megaterium, Bacillus colloids, white rot fungi and Pseudomonas, wherein the mass ratio of the composite microorganism to the activated carbon carrier is 3.7:30, and in the composite microorganism, the mass ratio of Bacillus megaterium, Bacillus colloids, white rot fungi and Pseudomonas is 12:12:1:12.

[0026] The preparation method of the magnetic carbon-supported slow-release microbial agent comprises the following steps: Step 1, preparation of activated carbon: crush rice husk and pass it through a 20-mesh sieve to obtain biomass powder, put the biomass powder into a quartz boat of a vacuum tube high-temperature furnace, use high-purity 99.999% nitrogen as carrier gas and protective gas, the nitrogen flow rate is 100 mL / min, the heating rate is 20°C / min, the pyrolysis final temperature is 600°C and maintained for 2 h to form biochar; Step 2: Activate biochar to form activated carbon: Phosphoric acid impregnation pretreatment: biochar was mixed with 73% phosphoric acid solution at a mass ratio of 1:2, and impregnated at room temperature for 24 h. The impregnated biochar was filtered. Carbon dioxide activation: The impregnated biochar was placed in a vacuum tube high-temperature furnace, the vacuum was switched to a carbon dioxide gas atmosphere, the flow rate of carbon dioxide was controlled to be 100 mL / min, and carbon dioxide activation was performed at 400 °C for 2 h to obtain primary activated carbon; Heat treatment: keep the primary activated carbon in a vacuum tube high temperature furnace, switch the carbon dioxide atmosphere to a nitrogen atmosphere, raise the temperature to 800°C, and keep it for 1 hour to obtain secondary activated carbon; Post-treatment: The secondary activated carbon was cooled to room temperature, washed repeatedly with deionized water until the pH of the washing solution was 6.8-7.2, and dried in an oven at 108°C for 20 h to obtain activated carbon; Step 3: Magnetization of activated carbon: S31, impregnation: dissolving ferric chloride hexahydrate and ferrous chloride tetrahydrate in deionized water at a molar ratio of 2:1 to prepare a solution with a total iron ion concentration of 0.1 mol / L; S32, adding activated carbon to a 0.1 mol / L solution, and stirring the mixture for 1.5 h at room temperature under an ultrasonic power of 600 W and an ultrasonic frequency of 40 kHz to form a mixed solution, wherein the mixing ratio of activated carbon to solution is 1 g:20 mL; S33, adding 28% ammonia water by mass to the mixed solution until the pH of the mixed solution is 9, and mechanically stirring at a stirring speed of 750 r / min for 1.5 h, wherein the dropping speed of the ammonia water is 1 mL / min; S34, filtering to obtain a precipitate, washing the precipitate with deionized water for multiple times until the pH of the washing solution is 6.8-7.2, placing the washed precipitate into a quartz boat of a vacuum tube high temperature furnace, sealing it, introducing nitrogen to exhaust the air in the furnace, raising the temperature in the furnace to 600°C at a heating rate of 5°C / min, maintaining it for 2 h, further raising the temperature to 800°C at a heating rate of 8°C / min, maintaining it for 3 h, and cooling it to room temperature at a cooling rate of 2°C / min to obtain magnetized activated carbon; Step 4: Preparation of magnetic carbon-supported slow-release microbial agent: Bacillus megaterium was inoculated into LB liquid culture medium, and activated by shaking culture at 30°C and 220 r / min for 1 day; Bacillus colloidus was inoculated into silicate bacterial culture medium, and activated by shaking culture at 32°C and 170 r / min for 2 days; white rot fungi were inoculated into potato glucose liquid culture medium, and activated by shaking culture at 32°C and 150 r / min for 5 days; Pseudomonas was inoculated into KB culture medium, and activated by shaking culture at 30°C and 200 r / min for 1 day, so that the number of microorganisms was 10 8 CFU / mL~10 9CFU / mL, the bacterial liquid formed by Bacillus megaterium, Bacillus mucilaginosus, white rot fungi and Pseudomonas were mixed in a volume ratio of 12:12:1:12 to form a mixed bacterial liquid, the magnetized activated carbon was added to the conical flask containing the mixed bacterial liquid, and the mixture was shaken and cultured in a constant temperature shaking incubator at 25 ℃ and 100 rpm for 4 h, and then allowed to stand for 30 min, the supernatant was removed, the precipitate was collected, washed with sterile saline, and placed in a refrigerator at 4 ℃ to obtain the magnetic carbon-loaded slow-release microbial agent.

[0027] Biochar was prepared into activated carbon through an optimized preparation process. The specific surface area increased from 200.0 m² / g of the original biochar to 1200.5 m² / g, the micropore volume increased from 0.05 cm³ / g of biochar to 0.35 cm³ / g, the mesopore volume increased from 0.1 cm³ / g to 0.4 cm³ / g, and the total pore volume reached 0.75 cm³ / g. The pore size distribution was more uniform and concentrated in the micropore and mesopore areas, which was conducive to the adsorption of molecules of different sizes.

[0028] <Example 2> The difference between the embodiment 1 and the embodiment 1 is that the rice husk in step 1 is replaced by corn stalks.

[0029] <Example 3> The difference between the embodiment 1 and the embodiment 1 is that the rice husk in step 1 is replaced by rice straw.

[0030] <Example 4> The difference between it and Example 1 is that: Step 3: Magnetization of activated carbon: S31, impregnation: dissolving ferric chloride hexahydrate and ferrous chloride tetrahydrate in deionized water at a molar ratio of 2:1 to prepare a solution with a total iron ion concentration of 0.1 mol / L; S32, adding activated carbon to a 0.1 mol / L solution, and stirring ultrasonically at room temperature at an ultrasonic power of 500 W and an ultrasonic frequency of 50 kHz for 2 h to form a mixed solution, wherein the mixing ratio of activated carbon to solution is 1 g:20 mL; S33, adding 25% ammonia water by mass to the mixed solution until the pH value of the mixed solution is 9, and stirring mechanically at a stirring speed of 600 r / min for 2 h, wherein the dropping speed of the ammonia water is 1 mL / min; S34, filter the precipitate, wash the precipitate with deionized water for several times until the pH of the washing liquid is 6.8-7.2, put the washed precipitate into a quartz boat of a vacuum tube high-temperature furnace, seal it, pass nitrogen to exhaust the air in the furnace, increase the temperature in the furnace to 600°C at a heating rate of 5°C / min, keep it for 2 h, continue to increase the temperature to 800°C at a heating rate of 8°C / min, keep it for 3 h, and cool it to room temperature at a cooling rate of 2°C / min to obtain magnetized activated carbon.

[0031] <Example 5> The difference between it and Example 1 is that: Step 3: Magnetization of activated carbon: S31, impregnation: dissolving ferric chloride hexahydrate and ferrous chloride tetrahydrate in deionized water at a molar ratio of 2:1 to prepare a solution with a total iron ion concentration of 0.1 mol / L; S32, adding activated carbon to a 0.1 mol / L solution, and stirring the mixture for 1 h at room temperature under an ultrasonic power of 800 W and an ultrasonic frequency of 40 kHz to form a mixed solution, wherein the mixing ratio of activated carbon to solution is 1 g:20 mL; S33, adding 25% ammonia water by mass to the mixed solution until the pH value of the mixed solution is 9, and mechanically stirring at a stirring speed of 800 r / min for 1 h, wherein the dropping speed of the ammonia water is 1 mL / min; S34, filter the precipitate, wash the precipitate with deionized water for several times until the pH of the washing liquid is 6.8-7.2, put the washed precipitate into a quartz boat of a vacuum tube high-temperature furnace, seal it, pass nitrogen to exhaust the air in the furnace, increase the temperature in the furnace to 600°C at a heating rate of 5°C / min, keep it for 2 h, continue to increase the temperature to 800°C at a heating rate of 8°C / min, keep it for 3 h, and cool it to room temperature at a cooling rate of 2°C / min to obtain magnetized activated carbon.

[0032] Comparative Example 1 The difference between the embodiment 1 and the embodiment 1 is that there is no phosphoric acid impregnation pretreatment in step 2, and step 2 specifically includes: Carbon dioxide activation: The biochar was placed in a vacuum tube high-temperature furnace, the vacuum was switched to a carbon dioxide gas atmosphere, the flow rate of carbon dioxide was controlled to be 100 mL / min, and carbon dioxide activation was performed at 400 °C for 2 h to obtain primary activated carbon; Heat treatment: keep the primary activated carbon in a vacuum tube high temperature furnace, switch the carbon dioxide atmosphere to a nitrogen atmosphere, raise the temperature to 800°C, and keep it for 1 hour to obtain secondary activated carbon; Post-treatment: The secondary activated carbon was cooled to room temperature, washed repeatedly with deionized water until the pH of the washing solution was 6.8-7.2, and dried in an oven at 108°C for 20 h to obtain activated carbon; Comparative Example 2 The difference between the embodiment 1 and the embodiment 1 is that: in step 2, the carbon dioxide activation is as follows: the biochar is placed in a vacuum tube high temperature furnace, the vacuum is switched to a carbon dioxide gas atmosphere, the flow rate of the carbon dioxide is controlled to be 100 mL / min, and the carbon dioxide activation is performed at 300°C for 2 h to obtain primary activated carbon; Comparative Example 3 The difference between the embodiment 1 and the embodiment 1 is that there is no heat treatment step in step 2. Step 2 specifically includes: Phosphoric acid impregnation pretreatment: biochar was mixed with 73% phosphoric acid solution at a mass ratio of 1:2, and impregnated at room temperature for 24 h. The impregnated biochar was filtered. Carbon dioxide activation: The impregnated biochar was placed in a vacuum tube high-temperature furnace, the vacuum was switched to a carbon dioxide gas atmosphere, the flow rate of carbon dioxide was controlled to be 100 mL / min, and carbon dioxide activation was performed at 400 °C for 2 h to obtain primary activated carbon; Post-treatment: The secondary activated carbon was cooled to room temperature, washed repeatedly with deionized water until the pH of the washing solution was 6.8-7.2, and dried in an oven at 108°C for 20 h to obtain activated carbon; Comparative Example 4 The difference between it and Example 1 is that: Step 3: Magnetization of activated carbon: Mix activated carbon and ferroferric oxide nanoparticles in a mass ratio of 1:1, stir ultrasonically for 1.5 h at an ultrasonic power of 600 W and an ultrasonic frequency of 40 kHz, wash the precipitate three times with deionized water, and dry it in an oven at 60°C for 12 h to obtain magnetized activated carbon.

[0033] Comparative Example 5 The difference between the embodiment 1 and the embodiment 1 is that in step S31, the molar ratio of ferric chloride hexahydrate to ferrous chloride tetrahydrate is 1:1.

[0034] Comparative Example 6 The difference between the embodiment 1 and the embodiment 1 is as follows: S32, adding activated carbon to a 0.1 mol / L solution, and stirring the mixture for 1.5 h at room temperature with an ultrasonic power of 300 W and an ultrasonic frequency of 30 kHz to form a mixed solution, wherein the mixing ratio of the activated carbon to the solution is 1 g:20 mL; Comparative Example 7 The difference between the above method and Example 1 is that: S33, 28% ammonia water is added dropwise to the mixed solution until the pH value of the mixed solution is 9, and the mixture is mechanically stirred at a stirring speed of 400 r / min for 1.5 h, wherein the dropping speed of the ammonia water is 5 mL / min; Comparative Example 8 The difference between the above method and Example 1 is that: S32, adding activated carbon to a 0.1 mol / L solution, stirring at room temperature at a stirring speed of 600 r / min for 3 h to form a mixed solution, wherein the mixing ratio of activated carbon to solution is 1 g:20 mL; Performance Test: 1. Taking the magnetic carbon-supported slow-release microbial agent prepared in Example 1 as an example, a performance test was carried out.

[0035] (1) Determination of stability performance: Physical stability test: Appearance observation: Observe the appearance of the inoculant daily, record the color, whether there is agglomeration, stratification, etc., and continue to observe for 30 days.

[0036] Particle size analysis: On the 0th, 10th, 20th and 30th days, a laser particle size analyzer was used to measure the average particle size and particle size distribution of the inoculant particles. Each measurement was repeated 3 times and the average value was taken.

[0037] Magnetic property test: Also at the above time point, use a magnetometer to detect the magnetic strength of the bacterial agent in Gauss (Gs), repeat the measurement 3 times and take the average value.

[0038] Test results: Appearance observation results: Day 1-10: The appearance of the bacterial agent is black and uniform powder, without agglomeration or stratification. Day 11-20: The color becomes slightly darker, and it is still a uniform powder without agglomeration or stratification. Day 21-30: The appearance basically remains a black and uniform powder, without obvious changes. Particle size analysis results: Day 0: Average particle size / μm: 50.2±2.1, particle size distribution range (D10-D90) 20-80, Day 10: Average particle size / μm: 51.5±2.3, particle size distribution range (D10-D90) 21-82, Day 20: Average particle size / μm: 52.0±2.5, particle size distribution range (D10-D90) 20-83, Day 30: Average particle size / μm: 53.0±2.8, particle size distribution range (D10-D90) 22-85. Magnetic properties test results: Day 0: magnetic intensity / Gs: 30.5±1.2, Day 10: magnetic intensity / Gs: 30.0±1.5, Day 20: magnetic intensity / Gs: 29.8±1.3, Day 30: magnetic intensity / Gs: 29.5±1.0.

[0039] The appearance was basically stable during the 30-day observation period, with no obvious agglomeration or stratification; the particle size analysis showed that the average particle size and particle size distribution had slight changes, but the amplitude was small and did not affect the overall performance of the microbial agent; the magnetic performance test showed that the magnetic strength decreased slightly, but still maintained at a certain level, which can ensure the normal function of the magnetic absorption function. The physical stability is good.

[0040] Chemical stability test: pH value determination: Take a small amount of bacterial agent every 3 days and measure its pH value with a pH meter, for a total of 10 measurements.

[0041] Detection of active ingredient content: High performance liquid chromatography was used to determine the content of specific enzymes (such as amylase, cellulase, etc.) produced by microbial metabolism in the inoculant on the 0th, 15th and 30th days. The content was expressed as U / mL and the measurement was repeated 3 times to take the average value.

[0042] Measurement results: pH measurement results: 1st time (day 0): pH=7.2±0.1, 2nd time (day 3): pH=7.3±0.1, 3rd time (day 6): pH=7.4±0.1, 4th time (day 9): pH=7.3±0.1, 5th time (day 12): pH=7.4±0.1, 6th time (day 15): pH=7.3±0.1, 7th time (day 18): pH=7.4±0.1, 8th time (day 21): pH=7.3±0.1, 9th time (day 24): pH=7.4±0.1, 10th time (day 27): pH=7.3±0.1. Results of active ingredient content test: On day 0, amylase content (U / mL): 120.5±5.2, cellulase content (U / mL): 80.3±3.5, on day 15, amylase content (U / mL): 118.0±4.8, cellulase content (U / mL): 78.5±3.2, on day 30, amylase content (U / mL): 115.0±4.5, cellulase content (U / mL): 76.0±3.0.

[0043] The pH value fluctuated very little during the entire test period and was maintained in a neutral range suitable for the growth of microorganisms; although the content of active ingredients decreased over time, the decrease did not exceed 5%, indicating that the chemical properties were relatively stable and could provide a relatively stable living environment for microorganisms, ensuring the continued performance of their metabolic functions.

[0044] Microbiological stability test: Live bacteria count: On the 0th, 7th, 14th, 21st and 28th days, the bacterial agent was diluted graded and inoculated onto the corresponding selective culture medium using the dilution spreading plate method. The culture was cultured at an appropriate temperature for 2-3 days, and the live bacteria count of each functional bacteria was counted and converted into CFU / mL. The counts were repeated on 3 plates for each bacterium and the average value was taken.

[0045] Strain identification and purity testing: On the 0th day and the 30th day, the 16S rRNA gene sequencing technology was used to identify the microorganisms in the inoculum, and the single colony morphology was observed by the plate streak method to detect the purity of the strain.

[0046] Test results: Viable bacteria count (CFU / mL) Results: Day 0, Bacillus megaterium: 5.0×10 8 ±2.0×10 7 , Bacillus mucilaginosus: 4.8×10 8 ±1.8×10 7 , white rot fungi: 2.0×10 7 ±5.0×10 6 , Pseudomonas: 4.5×10 8 ±1.5×10 7 , Day 7, Bacillus megaterium: 4.5×10 8 ±1.5×10 7 , Bacillus mucilaginosus: 4.2×10 8 ±1.2×10 7 , white rot fungi: 1.8×10 7 ±4.0×10 6 , Pseudomonas: 4.05×10 8 ±1.0×10 7 , Day 14, Bacillus megaterium: 4.0×10 8 ±1.0×10 7 , Bacillus mucilaginosus: 3.8×10 8 ±8.0×10 6 , white rot fungi: 1.5×10 7 ±3.0×10 6 , Pseudomonas: 3.5×10 8 ±8.0×10 6 , Day 21, Bacillus megaterium: 3.8×10 8 ±8.0×10 6 , Bacillus mucilaginosus: 3.2×10 8 ±6.0×10 6 , white rot fungi: 1.2×10 7 ±2.0×10 6 , Pseudomonas: 3.0×10 8 ±6.0×10 6 , Day 28, Bacillus megaterium: 3.0×10 8 ±6.0×10 6 , Bacillus mucilaginosus: 2.5×10 8 ±5.0×10 6 , white rot fungi: 1.0×10 7 ±1.5×10 6 , Pseudomonas: 2.5×10 8 ±5.0×10 6. Results of strain identification and purity test: Day 0: Through 16S rRNA gene sequencing and plate streaking method, it was identified that the target Bacillus megaterium, Bacillus colloids, white rot fungi and Pseudomonas were contained in the inoculum, and the single colony morphology was typical, without contamination by foreign bacteria, and the purity was 100%. Day 30: After re-identification, the strain did not change and was still the target strain. The single colony morphology was normal and the purity remained above 98%. Only a very small number of suspected foreign bacteria colonies appeared on individual plates.

[0047] The number of viable bacteria decreased gradually over time, but the number of each functional bacteria remained at 10 within 28 days. 7 - 10 8 The CFU / mL level is relatively high and can meet the needs of actual applications; the strain identification and purity testing confirmed that the strain has not mutated and has a high purity, ensuring the reliability of the bacterial agent's function.

[0048] In summary, the magnetic carbon-loaded slow-release microbial agent prepared by the present invention has good stability within 30 days and is expected to play a good role in actual vegetable planting soil remediation, agricultural production and other fields.

[0049] (2) Determination of the adsorption properties of heavy metals and organic matter: Soil samples containing different concentrations of heavy metals (lead, cadmium, copper) were prepared. Heavy metals were added in the form of corresponding soluble salts (such as lead nitrate, cadmium chloride, copper sulfate) so that the concentration of heavy metal ions in the soil reached a gradient of 50 mg / kg, 100 mg / kg, and 200 mg / kg, respectively.

[0050] Soil samples containing different types and concentrations of organic matter (such as phenol, toluene, polycyclic aromatic hydrocarbons, etc.) were prepared. The organic matter was dissolved in pure form and evenly mixed into the soil to achieve a concentration gradient of 100 mg / kg, 200 mg / kg, and 500 mg / kg.

[0051] Heavy metal adsorption: Take three 250mL stoppered Erlenmeyer flasks, add 100mL of simulated soil suspension with different heavy metal pollution concentrations respectively, and the solid-liquid ratio of the soil suspension is 1:5 (g / mL); add magnetic carbon-loaded slow-release microbial agent to each Erlenmeyer flask, and the addition amount of magnetic carbon-loaded slow-release microbial agent is set at 3% of the soil weight. Place the flask in a constant temperature oscillator and oscillate at 25℃ and 150r / min for 24 hours to allow the magnetic carbon-loaded slow-release microbial agent to fully contact with the soil to simulate the dynamic adsorption process in the natural environment; after the oscillation, centrifuge at 5000r / min for 10 minutes to separate the soil and the supernatant, and use atomic absorption spectrometer to determine the content of heavy metal ions in the supernatant. According to the difference in the concentration of heavy metal ions in the solution before and after adsorption, calculate the adsorption amount of heavy metals by the agent.

[0052] Organic matter adsorption: Take several 250 mL stoppered Erlenmeyer flasks, add 100 mL of simulated soil suspension with different organic matter pollution concentrations, and the solid-liquid ratio is also 1:5 (g / mL). Add the bacterial agent according to the gradient of the above-mentioned bacterial agent addition amount, place it in a constant temperature oscillator, and oscillate at 30°C and 120 r / min for 18 hours. After the oscillation, centrifuge and take the supernatant to determine the organic matter content with a gas chromatograph. The adsorption amount is calculated by the change in organic matter concentration before and after adsorption.

[0053] The measurement results are shown in Table 1 below.

[0054] Table 1 Performance parameters of magnetic carbon-supported sustained-release microbial agents prepared in Examples 1 to 5 As can be seen from Table 1, the magnetic carbon-supported slow-release microbial agent prepared by the present invention has good heavy metal adsorption capacity, and can be applied to the remediation and improvement of heavy metal-contaminated vegetable planting soil.

[0055] 2. Performance of activated carbon prepared in Example 1 and Comparative Examples 1 to 3: Specific surface area test: BET method - nitrogen adsorption and desorption method: First, pre-treat an appropriate amount of activated carbon sample under vacuum conditions to remove impurities and moisture adsorbed on the sample surface. Then put the treated sample into the sample tube of the specific surface area and pore size analyzer, and allow nitrogen to adsorb and desorb on the surface of the activated carbon at liquid nitrogen temperature. The instrument detects the adsorption of nitrogen under different relative pressures and calculates the specific surface area of ​​the activated carbon in m² / g according to the BET theoretical model.

[0056] Iodine adsorption value test: Accurately weigh a certain mass (m, g) of activated carbon sample, place it in an iodine volumetric bottle, and add an appropriate amount of iodine standard solution (V 1 , mL), shake it to make it fully contact for a certain time (30min), then titrate the excess iodine with sodium thiosulfate standard solution until the solution color changes from blue to colorless, and record the volume of sodium thiosulfate standard solution consumed (V 2 , mL). Calculate the iodine adsorption value (mg / g) according to the following formula: Iodine adsorption value = [(V 1 ×C 1 -V 2 ×C 2 )×126.9×1000] / m Among them C 1 is the concentration of iodine standard solution (mol / L), C 2 is the concentration of sodium thiosulfate standard solution (mol / L), and 126.9 is the molar mass of iodine (g / mol).

[0057] Methylene blue adsorption value test: weigh a certain amount (m 0 , g) of activated carbon sample was placed in a stoppered conical flask and a known concentration (C 0 , mg / L) and volume (V 0 , mL) of methylene blue standard solution, oscillate on an oscillator for a certain period of time (e.g. 60 min) to allow the adsorption to reach equilibrium. Then take an appropriate amount of supernatant and measure its absorbance at a specific wavelength (usually 665 nm) using a spectrophotometer. According to the pre-drawn methylene blue standard curve, the corresponding methylene blue concentration (C 1 , mg / L). The methylene blue adsorption value (mg / g) was calculated by the following formula: Methylene blue adsorption value = (C 0 ×V 0 -C 1 ×V 0 ) / m 0 The measurement results are shown in Table 2 below.

[0058] Table 2. Comparison of performance parameters of activated carbon prepared in Example 1 and Comparative Examples 1 to 3 As can be seen from Table 2, in Comparative Example 1, no phosphoric acid impregnation pretreatment was performed, and all performance indicators were significantly reduced, indicating that phosphoric acid impregnation pretreatment plays an important role in improving the pore structure and adsorption performance of activated carbon. This may be because phosphoric acid impregnation can etch biochar in advance, creating more active sites and reaction channels for subsequent carbon dioxide activation, thereby promoting the formation and development of pores. Moreover, the selection of a phosphoric acid solution with a relatively high mass fraction can strongly etch biochar, significantly increase its specific surface area and the number of pores, and make biochar have better adsorption performance, and achieve efficient adsorption of heavy metal ions, organic pollutants, etc. in soil. In Comparative Example 2, the activation temperature was reduced to 300°C, and its performance decreased compared with the activation temperature of 400°C, indicating that the carbon dioxide activation temperature has a significant effect on the performance of activated carbon. A lower activation temperature may lead to incomplete gasification reaction, and the pore structure cannot be fully formed and expanded, thereby affecting the improvement of adsorption performance and specific surface area. In Comparative Example 3, since the heat treatment step is omitted, the performance of the activated carbon prepared therefrom is still inferior to that of Example 1, indicating that the heat treatment step helps to further optimize the pore structure and surface chemical properties of the activated carbon. It may be through structural rearrangement and breaking and reorganization of chemical bonds at high temperature that the performance of the activated carbon is further improved, such as increasing the proportion of micropores, adjusting surface functional groups, etc.

[0059] III. Performance of magnetized activated carbon prepared in Example 1 and Comparative Examples 4 to 8: Saturation magnetization intensity determination: The magnetized activated carbon is gently loaded into a quartz sample tube dedicated to VSM, and gently compacted with a thin glass rod to ensure that the sample is evenly distributed in the tube and in close contact with the tube wall to avoid gaps that affect the measurement results. Fix the quartz sample tube containing the sample on the vibration table of the vibrating sample magnetometer, and check whether it is firmly fixed to prevent the sample tube from shaking during the measurement. Turn on the instrument, set the initial magnetic field strength to 0, and gradually increase the magnetic field strength at a rate of 50 Oe / s until it reaches the maximum magnetic field strength that the instrument can provide (such as 20000 Oe). During the increase in magnetic field strength, the instrument collects the electromotive force data in the detection coil in real time, records the data every 0.5 seconds, collects 10 sets of data in total, and calculates the average value.

[0060] The method for determining the iodine adsorption value and the methylene blue adsorption value is the same as above.

[0061] The measurement results are shown in Table 3 below.

[0062] Table 3. Comparison results of performance parameters of magnetized activated carbon prepared in Example 1 and Comparative Examples 4 to 8 In Comparative Example 4, the ferroferric oxide nanoparticles were not generated in situ, and their magnetism and adsorption capacity were reduced. This is because the in situ generation can cause the particles to anchor and grow in situ on the carrier surface, forming a strong chemical bond or a close physical adsorption relationship. The particles prepared non-in situ are often simply mixed and adsorbed on the carrier surface, with weak binding force, and are easy to fall off during actual operations such as stirring and shaking, which not only destroys the structural stability, but also makes it impossible for the magnetism and adsorption functions to be continuously and stably exerted, ultimately resulting in a decline in overall performance. In Comparative Example 5, the iron salt ratio is changed, and the structure and content of the generated magnetic material are changed, which affects the overall magnetism. The inappropriate iron salt ratio not only affects the magnetic properties, but also causes certain interference to the activated carbon adsorption site; in Comparative Example 6, due to the reduction of ultrasonic power and frequency, the mixing uniformity of the iron salt solution and the activated carbon deteriorates, resulting in uneven loading of the magnetic particles, which affects the final magnetism. The poor ultrasonic conditions make it impossible to fully utilize some of the pores of the activated carbon, and the magnetic particles may block some of the pores, reducing the adsorption efficiency; in Comparative Example 7, due to the rapid dropwise addition of ammonia water, the local pH of the reaction system changes dramatically, the generated magnetic particles are uneven in size and poor in crystallinity, affecting the overall magnetism, the magnetic particles are of poor quality and chaotically distributed, which not only affects their own magnetism, but also seriously damages the adsorption structure of the activated carbon; in Comparative Example 8, no ultrasonic stirring is performed, the activated carbon and the iron salt solution are mixed slowly, the magnetic particles are difficult to load evenly, and the magnetic material formed is seriously agglomerated. Since the agglomerated magnetic particles block the pores of the activated carbon in large quantities, its adsorption performance is greatly reduced.

[0063] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily realized. Therefore, without departing from the general concept defined by the claims and equivalent scope, the present invention is not limited to the specific details and the embodiments shown and described here.

Claims

1. A magnetic carbon-supported slow-release microbial agent, characterized in that: include: A magnetized activated carbon carrier containing ferroferric oxide nanoparticles and a composite microorganism composed of Bacillus megaterium, Bacillus mucilaginosus, white rot fungi and Pseudomonas, wherein the mass ratio of the composite microorganism to the activated carbon carrier is 3 to 4:

30.

2. A method for preparing the magnetic carbon-supported slow-release microbial agent as claimed in claim 1, characterized in that: The steps include: Step 1, preparation of activated carbon: crush rice husk or straw and pass through a 20-mesh sieve to obtain biomass powder, pyrolyze the biomass powder in a vacuum tube high-temperature furnace to form biochar, and activate the biochar to form activated carbon; Step 2: magnetizing the activated carbon: magnetizing the activated carbon using ferroferric oxide nanoparticles to obtain magnetized activated carbon; Step 3: Preparation of magnetic carbon-supported slow-release microbial agent: Bacillus megaterium, Bacillus colloidus, white rot fungi and Pseudomonas were inoculated into liquid culture medium and activated for 1 to 5 days, so that the number of microorganisms was 10 8 CFU / mL~10 9 CFU / mL, the bacterial liquid formed by Bacillus megaterium, Bacillus mucilaginosus, white rot fungi and Pseudomonas were mixed in a volume ratio of 12:12:1:12 to form a mixed bacterial liquid, the magnetized activated carbon was added to the conical flask containing the mixed bacterial liquid, and the mixture was shaken and cultured in a constant temperature shaking incubator at 25 ℃ and 100 rpm for 4 h, and then allowed to stand for 30 min, the supernatant was removed, the precipitate was collected, washed with sterile saline, and placed in a refrigerator at 4 ℃ to obtain the magnetic carbon-loaded slow-release microbial agent.

3. The method according to claim 2, characterized in that In step 1, the activation of biochar specifically includes: S11, phosphoric acid impregnation pretreatment: biochar was mixed with a phosphoric acid solution with a mass fraction of 73% in a mass ratio of 1:2, and impregnated at room temperature for 24 h, and the impregnated biochar was filtered; S12, carbon dioxide activation: placing the impregnated biochar in a vacuum tube high temperature furnace, switching the vacuum to a carbon dioxide gas atmosphere, controlling the flow rate of carbon dioxide to 100 mL / min, and performing carbon dioxide activation at 400°C for 2 h to obtain primary activated carbon; S13, heat treatment: keep the primary activated carbon in a vacuum tube high temperature furnace, switch the carbon dioxide atmosphere to a nitrogen atmosphere, increase the temperature to 800°C, and keep it for 1 hour to obtain secondary activated carbon; S14. Post-treatment: Cool the secondary activated carbon to room temperature, wash repeatedly with deionized water until the pH of the washing solution is 6.8-7.2, and dry in an oven at 108° C. for 20 h to obtain activated carbon.

4. The method according to claim 2, characterized in that In step 2, magnetization specifically includes: S21, dissolving ferric chloride hexahydrate and ferrous chloride tetrahydrate in deionized water at a molar ratio of 2:1 to prepare a solution with a total iron ion concentration of 0.1 mol / L; S22, adding activated carbon to a 0.1 mol / L solution, and stirring the mixture ultrasonically at room temperature at an ultrasonic power of 500 to 800 W and an ultrasonic frequency of 40 to 50 kHz for 1 to 2 h to form a mixed solution, wherein the mixing ratio of activated carbon to solution is 1 g:20 mL; S23, dropwise add ammonia water with a mass fraction of 25% to 28% to the mixed solution until the pH value of the mixed solution is 9 to 10, and mechanically stir at a stirring speed of 600 to 800 r / min for 1 to 2 h, wherein the dropping speed of the ammonia water is 1 to 2 mL / min; S24, filtering to obtain a precipitate, washing the precipitate with deionized water for multiple times until the pH of the washing liquid is 6.8-7.2, and post-treating to obtain magnetized activated carbon.

5. The method according to claim 4, characterized in that In step S22, the ultrasonic power is 600 W, the ultrasonic frequency is 40 kHz, and the ultrasonic stirring time is 1.5 h.

6. The method according to claim 4, characterized in that In step S24, the post-treatment specifically includes the steps of: loading the washed precipitate into a quartz boat of a vacuum tube high-temperature furnace, sealing it, introducing nitrogen to exhaust the air in the furnace, raising the temperature in the furnace to 600°C at a heating rate of 5°C / min, maintaining it for 2 hours, continuing to heat it to 800°C at a heating rate of 8°C / min, maintaining it for 3 hours, and cooling it to room temperature at a cooling rate of 2°C / min to obtain magnetized activated carbon.

7. The method according to claim 4, characterized in that In step S23, the stirring speed is 750 r / min and the stirring time is 1.5 h.

8. The method according to claim 2, characterized in that In step three, Bacillus megaterium was inoculated into LB liquid culture medium and activated by shaking culture at 30°C and 220 r / min for 1 day, Bacillus mucilaginosus was inoculated into silicate bacterial culture medium and activated by shaking culture at 32°C and 170 r / min for 2 days, white rot fungi were inoculated into potato glucose liquid culture medium and activated by shaking culture at 32°C and 150 r / min for 5 days, and Pseudomonas was inoculated into KB culture medium and activated by shaking culture at 30°C and 200 r / min for 1 day.

9. The method according to claim 2, characterized in that In step 1, the pyrolysis in a vacuum tube high temperature furnace specifically includes: loading the biomass powder into a quartz boat of the vacuum tube high temperature furnace, using high-purity 99.999% nitrogen as a carrier gas and a protective gas, the nitrogen flow rate is 100 mL / min, the heating rate is 20°C / min, the pyrolysis final temperature is 600°C and maintained for 2 h.

10. Use of the magnetic carbon-loaded slow-release microbial agent as claimed in claim 1 in repairing vegetable planting soil.

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