Preparation method of biochar composite material and soil remediation method
By combining biochar as a specific adsorbent material with earthworm mucus as a binder, a biochar composite material capable of specifically adsorbing and degrading polycyclic aromatic hydrocarbons (PAHs) was prepared. This solved the problem that conventional carrier materials could not efficiently enrich and degrade PAHs, thus improving soil remediation efficiency and enhancing carbon sequestration capacity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIAN COLLEGE
- Filing Date
- 2025-10-14
- Publication Date
- 2026-05-15
AI Technical Summary
In existing bioremediation technologies, conventional carrier materials cannot efficiently enrich and degrade polycyclic aromatic hydrocarbon pollutants, and carbon loss is severe during the microbial remediation process, affecting the soil remediation effect.
A composite biochar material was prepared by loading a specific adsorbent onto a biochar-specific adsorbent, which specifically recognizes polycyclic aromatic hydrocarbons through π-π stacking interactions, enhances carbon fixation capacity by combining it with earthworm mucus binder, and improves enzyme activity by co-culturing Rhodococcus roseus and Proteobacterium chrysosporium.
It achieves efficient enrichment and degradation of polycyclic aromatic hydrocarbon pollutants, improves soil remediation efficiency, and has carbon fixation capabilities during the remediation process, solving the defects of conventional carrier materials and the problem of microbial carbon loss.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of soil remediation technology, and particularly relates to a method for preparing a biochar composite material and a soil remediation method. Background Technology
[0002] Polycyclic aromatic hydrocarbons (PAHs), as typical persistent organic pollutants, mostly originate from the combustion and leakage of fossil fuels. Due to their strong carcinogenicity, mutagenicity, and bioaccumulation, they pose a serious threat to soil ecosystems. Medium and high molecular weight PAHs, represented by phenanthrene and pyrene, can have a half-life of more than ten years in soil. They not only damage the structure of microbial communities and reduce soil fertility, but can also enter the food chain through crop accumulation, ultimately endangering human health.
[0003] Currently, the remediation methods for PAH-contaminated soil include chemical remediation, physical remediation, and bioremediation. Physical remediation mainly includes methods such as leaching extraction, subcritical and supercritical extraction, which only change the location of PAHs and cannot fundamentally solve the PAH pollution problem. Chemical remediation mainly includes photocatalysis, wet oxidation, and electrochemical oxidation, which are not only costly but also prone to causing secondary pollution. Bioremediation is a remediation technology that uses the growth and metabolism of microorganisms to transform PAHs into substances with lower or no risk. It has the characteristics of sustainability, environmental friendliness, low operating costs, and wide applicability.
[0004] Currently, in the process of bioremediation, in order to avoid the inhibition of microbial strains in the soil by environmental factors (pH fluctuations, nutrient competition), which would lead to colonization failure and functional degradation, carrier materials are usually selected to load them. However, conventional carrier materials such as bentonite or activated carbon can adsorb pollutants, but they lack specific recognition capabilities and cannot efficiently enrich pollutants to the action sites of the microbial agents. This can easily cause a disconnect in the "adsorption-degradation" process. Furthermore, during the microbial remediation process, the metabolism of microbial agents releases CO2, most of which escapes into the atmosphere, resulting in carbon loss in the soil. Summary of the Invention
[0005] To address the aforementioned technical deficiencies, this invention presents a biochar composite material and its preparation method. The prepared biochar composite material can efficiently enrich and degrade polycyclic aromatic hydrocarbon pollutants in the soil, thereby remediating the soil and playing a carbon fixation role during the remediation process.
[0006] The technical solution of the present invention is as follows:
[0007] A method for preparing a biochar composite material includes the following steps:
[0008] S1: Preparation of biochar-specific adsorption materials
[0009] Phenanthrene was dissolved in a mixed solution of toluene and acetonitrile. Then, 4-vinylpyridine and straw biochar powder were added sequentially and ultrasonically dispersed evenly. After standing, a prepolymer solution was obtained. Ethylene glycol dimethacrylate and azobisisobutyronitrile were added, and after ultrasonic dispersion, oxygen was removed. The solution was sealed and subjected to a water bath reaction. The reaction solution was centrifuged, and the precipitate was collected, washed, and dried to obtain a biochar-specific adsorbent material.
[0010] S2: Preparation of Earthworm Mucus Adhesive
[0011] After the Eisenia fetuses were placed in a dark place to defecate and cleaned, they were placed in a container and electrically stimulated with a DC power supply. The earthworm secretions secreted under electrical stimulation were collected by filtering and centrifugation. The supernatant was collected to obtain earthworm mucus. The earthworm mucus was mixed with acetate buffer, and α-mannosidase and β-1,3-glucanase were added for enzymatic hydrolysis and enzyme inactivation to obtain activated mucus. Ethylenediamine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were added under a nitrogen atmosphere. After the reaction was sealed, the mixture was centrifuged and the supernatant was removed to obtain earthworm mucus adhesive.
[0012] S3: Compound granulation of biochar and microorganisms
[0013] Rhodococcus roseus and Phanerochaete chrysosporium were cultured in an inorganic salt medium containing phenanthrene and pyrene by shaking. The resulting Rhodococcus roseus suspension and Phanerochaete chrysosporium suspension were mixed and formulated into a bacterial suspension to obtain a composite bacterial agent. The biochar specific adsorbent material, the composite bacterial agent and earthworm mucus binder were mixed evenly and granulated to obtain a biochar composite material.
[0014] Further, step S1, the preparation of the biochar-specific adsorbent material, includes the following steps:
[0015] S1.1: Place 0.0178 parts by weight of phenanthrene in a container, add 35-40 parts by weight of toluene and 10-15 parts by weight of acetonitrile, stir until phenanthrene is completely dissolved, then add 0.042-0.045 parts by weight of 4-vinylpyridine, ultrasonically disperse at an ultrasonic frequency of 25-30 kHz for 10-15 min, then add 1.8-2 parts by weight of straw biochar powder, continue ultrasonic dispersion for 25-30 min, and let stand for 12-15 h to obtain a prepolymer solution;
[0016] S1.2: Add 0.29-0.3 parts by weight of ethylene glycol dimethacrylate and 0.025-0.03 parts by weight of azobisisobutyronitrile to the prepolymer solution, disperse by ultrasonication for 10-15 min, then purge with nitrogen gas to remove oxygen for 4-6 min, and then seal in a water bath shaker at 60-65℃ for 20-24 h to obtain the reaction solution;
[0017] S1.3: Place the reaction solution in a centrifuge and centrifuge at 10000-12000 rpm for 10-15 min. Collect the bottom precipitate and dry it in an oven at 60-65℃. Then, continuously wash it with methanol-acetic acid solution until no phenanthrene is found in the methanol-acetic acid solution after washing. Then, dry it in an oven at 60-65℃ until constant weight to obtain biochar specific adsorbent material.
[0018] Furthermore, the preparation of the earthworm mucus adhesive in step S2 includes the following steps:
[0019] S2.1: Place healthy, disease-free Eisenia fetuses in a dark place for 24 hours to defecate, then rinse them clean with deionized water and dry them to obtain clean earthworms. Take 300-400g of clean earthworms and place them in a container. Perform electrical stimulation under a DC power supply of 5V and 10mA for 60-75s each time, with an interval of 50-60s. Filter and collect the earthworm secretions under electrical stimulation, transfer them to a centrifuge, and centrifuge at 8000-10000rpm for 10-12min. Separate and collect the supernatant to obtain earthworm mucus.
[0020] S2.2: Mix earthworm mucus and acetate buffer with pH 5.2-5.5 at a volume ratio of 1:(4-5), then add α-mannosidase and β-1,3-glucanase and mix well. Incubate at 37℃ for 2-3 hours, then heat to 70℃ for 10 minutes to inactivate the enzymes and obtain activated mucus.
[0021] S2.3: Place the activated mucus in a container, purge with nitrogen to remove oxygen, then add ethylenediamine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Control the final concentration of ethylenediamine in the activated mucus to be 1.5-3 mmol / g, and the final concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the activated mucus to be 0.8-1 mmol / g. React under closed conditions at 30-35℃ and 300-350 rpm for 3-3.5 h. Then place it in a centrifuge and centrifuge at 7000-7500 rpm for 10-12 min. Separate and remove the supernatant to obtain the earthworm mucus adhesive.
[0022] Furthermore, step S3, the compound granulation of biochar microorganisms, includes the following steps:
[0023] S3.1: Rhodococcus roseus and Phanerochaete chrysosporium were inoculated at 8-10% into 100ml of inorganic salt medium containing phenanthrene and pyrene, and cultured at 30℃ and 150rpm for 5 days to obtain Rhodococcus roseus bacterial suspension and Phanerochaete chrysosporium bacterial suspension.
[0024] S3.2: Mix the suspension of Rhodotorula rubrum and the suspension of Phanerochaete chrysospora at a volume ratio of 1:(1-1.2) to prepare a compound bacterial suspension with OD600=1, thus obtaining a compound bacterial agent;
[0025] S3.3: Mix 45-50 parts by weight of biochar specific adsorbent material, 6-8 parts by weight of composite bacterial agent and 10-12 parts by weight of earthworm mucus binder evenly, and then place them in a disc pelletizer to make particles with a particle size of 3-4 mm to obtain biochar composite material.
[0026] Further, in step S1.1, the straw biochar powder is obtained by pyrolyzing corn straw in a tube furnace under vacuum at 700-750℃ for 2 hours, then pulverizing it and passing it through an 80-100 mesh sieve.
[0027] Further, step S1.3, the methanol-acetic acid solution is prepared by mixing methanol and acetic acid in a volume ratio of 9:1.
[0028] Furthermore, in step S2.2, α-mannosidase and β-1,3-glucanase each account for 2-3% of the mass of earthworm mucus.
[0029] Furthermore, in the inorganic salt culture medium containing phenanthrene and pyrene in step S3.1, the content of phenanthrene and pyrene is 30 mg / L, and it also contains 2 g / L of (NH4)2SO4, 2 g / L of KH2PO4, 2 g / L of K2HPO4, 0.2 g / L of CaCl2, 10 g / L of NaCl and 0.2 g / L of MgSO4∙7H2O, with the remainder being sterile water.
[0030] Another aspect of this application provides a soil remediation method, in which the biochar composite material prepared by the above preparation method is added to the soil to be remediated by turning and mixing, the amount added is 5-7 wt% of the soil to be remediated, and the moisture content of the soil to be remediated is controlled to be 45-50%.
[0031] The beneficial effects are as follows: 1. This invention uses phenanthrene as a template molecule, 4-vinylpyridine as a functional monomer, and biochar as a loading material. Through the π-π stacking interaction between phenanthrene and 4-vinylpyridine, a biochar-specific adsorbent material with the ability to specifically recognize polycyclic aromatic hydrocarbon pollutants is prepared. The biochar-specific adsorbent material is then used as a loading material for a composite microbial agent to prepare a biochar composite material. This composite material can specifically adsorb polycyclic aromatic hydrocarbon pollutants in the soil, thereby enabling the microorganisms in the composite microbial agent to continuously degrade a wide range of polycyclic aromatic hydrocarbon pollutants such as phenanthrene and pyrene, effectively remediating contaminated soil.
[0032] 2. This invention involves placing Eisenia fetida under electrical stimulation to induce the secretion of earthworm mucus. The earthworm mucus is then enzymatically modified to expose amino reaction sites, resulting in activated mucus. This activated mucus is then reacted in a system of ethylenediamine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to amination the earthworm mucus. This process not only enhances the viscosity of the earthworm mucus and improves the molding of the biochar composite material, but also enables it to absorb and fix emitted CO2 in the soil. This gives the biochar composite material excellent carbon fixation capabilities while remediating the soil. Furthermore, the earthworm mucus is rich in water, lipids, carbohydrates, electrolytes, sugars, proteins, and amino acids, which can enhance the metabolic activity of microorganisms in the compound microbial agent, allowing them to more effectively decompose polycyclic aromatic hydrocarbon pollutants, thereby improving soil remediation efficiency.
[0033] 3. This invention involves inoculating Rhodococcus roseum and Phanerochaete chrysospora into an inorganic salt culture medium containing phenanthrene and pyrene, and then acclimating and culturing them to obtain a bacterial suspension. The two are then combined to prepare a compound bacterial agent. The co-cultured bacterial community formed by the two in the soil can improve enzyme activity and stimulate the growth of the bacterial community, solving the problem of poor soil remediation effect of single bacteria. The two work synergistically to effectively degrade polycyclic aromatic hydrocarbon pollutants, thus improving the remediation effect of polycyclic aromatic hydrocarbon contaminated soil. Detailed Implementation
[0034] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0035] Example 1
[0036] A method for preparing a biochar composite material includes the following steps:
[0037] S1: Preparation of biochar-specific adsorption materials
[0038] S1.1: Place 0.0178 parts by weight of phenanthrene in a container, add 35 parts by weight of toluene and 10 parts by weight of acetonitrile, stir until phenanthrene is completely dissolved, then add 0.042 parts by weight of 4-vinylpyridine, ultrasonically disperse at an ultrasonic frequency of 25 kHz for 10 min, then add 1.8 parts by weight of straw biochar powder obtained by pyrolyzing corn straw in a tube furnace at 700 °C under vacuum for 2 h, pulverizing and passing through an 80-mesh sieve, continue ultrasonic dispersion for 25 min, and let stand for 12 h to obtain a prepolymer solution;
[0039] S1.2: Add 0.29 parts by weight of ethylene glycol dimethacrylate and 0.025 parts by weight of azobisisobutyronitrile to the prepolymer solution, disperse by ultrasonication for 10 min, then purge with nitrogen to remove oxygen for 4 min, and then seal in a water bath shaker at 60℃ for 20 h to obtain the reaction solution.
[0040] S1.3: Place the reaction solution in a centrifuge and centrifuge at 10,000 rpm for 10 min. Collect the bottom precipitate and dry it in a 60°C oven. Then, continuously wash it with a methanol-acetic acid solution prepared by methanol and acetic acid in a volume ratio of 9:1 until no phenanthrene is found in the washed methanol-acetic acid solution. Then, dry it in a 60°C oven to constant weight to obtain the biochar specific adsorbent material.
[0041] S2: Preparation of Earthworm Mucus Adhesive
[0042] S2.1: Place healthy, disease-free Eisenia fetuses in a dark place for 24 hours to defecate, then rinse them clean with deionized water and dry them to obtain clean earthworms. Take 300g of clean earthworms and place them in a container. Perform electrical stimulation under a DC power supply with a voltage of 5V and a current of 10mA for 60s each time, with a 50s interval between each time. Filter and collect the earthworm secretions under electrical stimulation, transfer them to a centrifuge, and centrifuge at 8000rpm for 10min. Separate and collect the supernatant to obtain earthworm mucus.
[0043] S2.2: Earthworm mucus and acetate buffer at pH 5.2 were mixed at a volume ratio of 1:4. Then, α-mannosidase and β-1,3-glucanase were added and mixed evenly. Both α-mannosidase and β-1,3-glucanase accounted for 2% of the mass of earthworm mucus. The mixture was enzymatically hydrolyzed at 37°C for 2 hours, and then heated to 70°C for 10 minutes for enzyme inactivation to obtain activated mucus.
[0044] S2.3: Place the activated mucus in a container, purge with nitrogen to remove oxygen, then add ethylenediamine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Control the final concentration of ethylenediamine in the activated mucus to be 1.5 mmol / g and the final concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the activated mucus to be 0.8 mmol / g. React for 3 hours under closed conditions at 30℃ and 300 rpm. Then place it in a centrifuge and centrifuge at 7000 rpm for 10 minutes. Separate and remove the supernatant to obtain the earthworm mucus adhesive.
[0045] S3: Compound granulation of biochar and microorganisms
[0046] S3.1: Rhodococcus roseum and Phanerochaete chrysosporium were inoculated at an inoculum size of 8% into 100 ml of inorganic salt medium containing phenanthrene and pyrene. The inorganic salt medium containing phenanthrene and pyrene contained 30 mg / L of both phenanthrene and pyrene, as well as 2 g / L of (NH4)2SO4, 2 g / L of KH2PO4, 2 g / L of K2HPO4, 0.2 g / L of CaCl2, 10 g / L of NaCl, and 0.2 g / L of MgSO4∙7H2O, with the remainder being sterile water. The medium was cultured at 30 °C and 150 rpm for 5 days with shaking to obtain Rhodococcus roseum bacterial suspension and Phanerochaete chrysosporium bacterial suspension.
[0047] S3.2: Mix the suspension of Rhodococcus roseus and the suspension of Phanerochaete chrysosporium in a 1:1 volume ratio to prepare a compound bacterial suspension with OD600=1, thus obtaining a compound bacterial agent;
[0048] S3.3: Mix 45 parts by weight of biochar specific adsorbent material, 6 parts by weight of composite bacterial agent and 10 parts by weight of earthworm mucus binder evenly, and then place them in a disc pelletizer to make particles with a particle size of 3 mm, thus obtaining biochar composite material.
[0049] Example 2
[0050] A method for preparing a biochar composite material includes the following steps:
[0051] S1: Preparation of biochar-specific adsorption materials
[0052] S1.1: Place 0.0178 parts by weight of phenanthrene in a container, add 38 parts by weight of toluene and 12 parts by weight of acetonitrile, stir until phenanthrene is completely dissolved, then add 0.044 parts by weight of 4-vinylpyridine, ultrasonically disperse at an ultrasonic frequency of 28 kHz for 13 min, then add 1.9 parts by weight of straw biochar powder obtained by pyrolyzing corn straw in a tube furnace at 725 °C under vacuum for 2 h, pulverizing and passing through a 90-mesh sieve, continue ultrasonic dispersion for 28 min, and let stand for 13 h to obtain a prepolymer solution;
[0053] S1.2: Add 0.29 parts by weight of ethylene glycol dimethacrylate and 0.025 parts by weight of azobisisobutyronitrile to the prepolymer solution, disperse by ultrasonication for 13 min, then purge with nitrogen gas for 5 min to remove oxygen, and then seal in a water bath shaker at 62℃ for 22 h to obtain the reaction solution;
[0054] S1.3: Place the reaction solution in a centrifuge and centrifuge at 11,000 rpm for 13 min. Collect the bottom precipitate and dry it in a 63°C oven. Then, continuously wash it with a methanol-acetic acid solution prepared by methanol and acetic acid in a volume ratio of 9:1 until no phenanthrene is found in the washed methanol-acetic acid solution. Then, dry it in a 63°C oven to constant weight to obtain the biochar specific adsorbent material.
[0055] S2: Preparation of Earthworm Mucus Adhesive
[0056] S2.1: Healthy and disease-free Eisenia fetuses were placed in the dark for 24 hours to defecate. They were then rinsed and dried with deionized water to obtain clean earthworms. 350g of clean earthworms were placed in a container and electrically stimulated under a DC power supply of 5V and 10mA for 68s each time with a 55s interval. The earthworm secretions secreted under the electrical stimulation were filtered and collected. The secretions were transferred to a centrifuge and centrifuged at 9000rpm for 11min. The supernatant was separated and collected to obtain earthworm mucus.
[0057] S2.2: Earthworm mucus and acetate buffer at pH 5.3 were mixed at a volume ratio of 1:5. Then, α-mannosidase and β-1,3-glucanase were added and mixed evenly. Both α-mannosidase and β-1,3-glucanase accounted for 2.5% of the mass of earthworm mucus. The mixture was enzymatically hydrolyzed at 37°C for 2.5 h, and then heated to 70°C for 10 min to inactivate the enzymes, thus obtaining activated mucus.
[0058] S2.3: Place the activated mucus in a container, purge with nitrogen to remove oxygen, then add ethylenediamine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Control the final concentration of ethylenediamine in the activated mucus to be 2.2 mmol / g and the final concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the activated mucus to be 0.9 mmol / g. React under closed conditions at 32℃ and 325 rpm for 3.2 h. Then place it in a centrifuge and centrifuge at 7250 rpm for 11 min. Separate and remove the supernatant to obtain earthworm mucus adhesive.
[0059] S3: Compound granulation of biochar and microorganisms
[0060] S3.1: Rhodococcus roseum and Phanerochaete chrysosporium were inoculated at a 9% inoculum into 100 ml of inorganic salt medium containing phenanthrene and pyrene. The inorganic salt medium containing phenanthrene and pyrene contained 30 mg / L of both phenanthrene and pyrene, as well as 2 g / L of (NH4)2SO4, 2 g / L of KH2PO4, 2 g / L of K2HPO4, 0.2 g / L of CaCl2, 10 g / L of NaCl, and 0.2 g / L of MgSO4∙7H2O, with the remainder being sterile water. The medium was cultured at 30 °C and 150 rpm for 5 days with shaking to obtain Rhodococcus roseum and Phanerochaete chrysosporium suspensions.
[0061] S3.2: Mix the suspension of Rhodococcus roseus and the suspension of Phanerochaete chrysosporium at a volume ratio of 1:1.1 to prepare a compound bacterial suspension with OD600=1, thus obtaining a compound bacterial agent;
[0062] S3.3: Mix 47 parts by weight of biochar specific adsorbent material, 7 parts by weight of composite bacterial agent and 11 parts by weight of earthworm mucus binder evenly, and then place them in a disc pelletizer to make particles with a particle size of 4 mm, thus obtaining biochar composite material.
[0063] Example 3
[0064] A method for preparing a biochar composite material includes the following steps:
[0065] S1: Preparation of biochar-specific adsorption materials
[0066] S1.1: Place 0.0178 parts by weight of phenanthrene in a container, add 40 parts by weight of toluene and 15 parts by weight of acetonitrile, stir until phenanthrene is completely dissolved, then add 0.045 parts by weight of 4-vinylpyridine, ultrasonically disperse at an ultrasonic frequency of 30 kHz for 15 min, then add 2 parts by weight of straw biochar powder obtained by pyrolyzing corn straw in a tube furnace at 750 °C under vacuum for 2 h, pulverizing and passing through a 100-mesh sieve, continue ultrasonic dispersion for 30 min, and let stand for 15 h to obtain a prepolymer solution;
[0067] S1.2: Add 0.3 parts by weight of ethylene glycol dimethacrylate and 0.03 parts by weight of azobisisobutyronitrile to the prepolymer solution, disperse by ultrasonication for 15 min, then purge with nitrogen to remove oxygen for 6 min, and then seal in a water bath shaker at 65℃ for 24 h to obtain the reaction solution;
[0068] S1.3: Place the reaction solution in a centrifuge and centrifuge at 12000 rpm for 15 min. Collect the bottom precipitate and dry it in a 65℃ oven. Then, continuously wash it with a methanol-acetic acid solution prepared by methanol and acetic acid in a volume ratio of 9:1 until there is no phenanthrene in the washed methanol-acetic acid solution. Then dry it in a 65℃ oven to constant weight to obtain biochar specific adsorbent material.
[0069] S2: Preparation of Earthworm Mucus Adhesive
[0070] S2.1: Healthy and disease-free Eisenia fetuses were placed in the dark for 24 hours to defecate. They were then rinsed and dried with deionized water to obtain clean earthworms. 400g of clean earthworms were placed in a container and electrically stimulated under a DC power supply of 5V and 10mA for 75s each time with a 60s interval. The earthworm secretions secreted under the electrical stimulation were filtered and collected. The secretions were transferred to a centrifuge and centrifuged at 10000rpm for 12min. The supernatant was separated and collected to obtain earthworm mucus.
[0071] S2.2: Earthworm mucus and acetate buffer at pH 5.5 were mixed at a volume ratio of 1:5. Then, α-mannosidase and β-1,3-glucanase were added and mixed evenly. Both α-mannosidase and β-1,3-glucanase accounted for 3% of the mass of earthworm mucus. The mixture was enzymatically hydrolyzed at 37°C for 3 hours and then heated to 70°C for 10 minutes to inactivate the enzymes, thus obtaining activated mucus.
[0072] S2.3: Place the activated mucus in a container, purge with nitrogen to remove oxygen, then add ethylenediamine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Control the final concentration of ethylenediamine in the activated mucus to be 3 mmol / g and the final concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the activated mucus to be 1 mmol / g. React under closed conditions at 35℃ and 350 rpm for 3.5 h. Then place it in a centrifuge and centrifuge at 7500 rpm for 12 min. Separate and remove the supernatant to obtain the earthworm mucus adhesive.
[0073] S3: Compound granulation of biochar and microorganisms
[0074] S3.1: Rhodococcus roseum and Phanerochaete chrysosporium were inoculated at 10% in 100 ml of inorganic salt medium containing phenanthrene and pyrene. The inorganic salt medium containing phenanthrene and pyrene contained 30 mg / L of both phenanthrene and pyrene, as well as 2 g / L of (NH4)2SO4, 2 g / L of KH2PO4, 2 g / L of K2HPO4, 0.2 g / L of CaCl2, 10 g / L of NaCl, and 0.2 g / L of MgSO4∙7H2O, with the remainder being sterile water. The medium was cultured at 30 °C and 150 rpm for 5 days with shaking to obtain Rhodococcus roseum and Phanerochaete chrysosporium suspensions.
[0075] S3.2: Mix the suspension of Rhodococcus roseus and the suspension of Phanerochaete chrysosporium at a volume ratio of 1:1.2 to prepare a compound bacterial suspension with OD600=1, thus obtaining a compound bacterial agent;
[0076] S3.3: Mix 50 parts by weight of biochar specific adsorbent material, 8 parts by weight of composite bacterial agent and 12 parts by weight of earthworm mucus binder evenly, and then place them in a disc pelletizer to make particles with a particle size of 4 mm, thus obtaining biochar composite material.
[0077] Comparative Example 1: A biochar composite material and its preparation method. Compared with the implementation method of Example 1, the difference is that step S1 is removed, and the biochar-specific adsorbent material in step S3.3 is replaced with an equal mass of straw biochar powder. The remaining steps are the same as those in Example 1.
[0078] Comparative Example 2: A biochar composite material and its preparation method. Compared with the implementation method of Example 1, the difference is that the earthworm mucus adhesive in step S3.3 is replaced with an equal mass of carboxymethyl cellulose hydrogel, and the remaining steps are the same as those in Example 1.
[0079] Comparative Example 3: A biochar composite material and its preparation method. Compared with the implementation method of Example 1, the difference is that the composite bacterial agent in step S3.3 is replaced with an equal mass of Rhodococcus roseus bacterial suspension with an OD600. The remaining steps are the same as those in Example 1.
[0080] Comparative Example 4: A biochar composite material and its preparation method. Compared with the implementation method of Example 1, the difference is that the composite bacterial agent in step S3.3 is replaced with an equal mass of Phanerochaete chrysosporium suspension with an OD600. The remaining steps are the same as in Example 1.
[0081] Experiment 1: Test on the ability to degrade PAHs in soil
[0082] Uncontaminated soil was crushed and sieved through a 2mm sieve to obtain the original soil. 10.5kg of the original soil was placed in a container and 630mg of compound PAHs (315mg each of phenanthrene and pyrene) was added. After thorough mixing, the contaminated soil was obtained. 21 flower pots were prepared, and 500g of contaminated soil was placed in each flower pot. The pots were then divided into 7 groups, with 3 pots in each group for the experiment.
[0083] The biochar composite materials prepared in Examples 1-3 and Comparative Examples 1-4 were used to prepare a set of flower pots. 25g of the corresponding biochar composite material was added to each flower pot in each set and mixed evenly. The moisture content of the contaminated soil was kept at 45% and the remediation was carried out for 100 days. The PAH content in the soil was measured every 20 days and the data were recorded. The average value of the data for each set was taken, as shown in Table 1.
[0084] Table 1: Changes in PAHs content
[0085]
[0086] As can be seen from the data in Examples 1-3 in Table 1, the biochar composite material prepared in this application can reduce the PAH content in contaminated soil from 30 mg / kg to below 1 mg / kg within 100 days, proving that its ability to degrade PAHs is excellent and can effectively remediate polycyclic aromatic hydrocarbon contaminated soil.
[0087] As can be seen from the data in Comparative Example 1, the biochar composite material prepared by replacing the biochar-specific adsorbent with ordinary straw biochar powder has a significantly reduced ability to degrade PAHs. This proves that the biochar composite material prepared by using the biochar-specific adsorbent as the loading material of the composite microbial agent can specifically adsorb polycyclic aromatic hydrocarbon pollutants in the soil, thereby enabling the microorganisms in the composite microbial agent to continuously degrade polycyclic aromatic hydrocarbon pollutants such as phenanthrene and pyrene over a wide range, effectively remediating the contaminated soil.
[0088] The data from Comparative Example 2 show that without the addition of earthworm slime binder to the biochar composite material, its ability to degrade PAHs was significantly reduced. This proves that earthworm slime can enhance the metabolic activity of microorganisms in the composite agent, enabling microorganisms to decompose polycyclic aromatic hydrocarbon pollutants more effectively, thereby improving soil remediation efficiency.
[0089] The data from Comparative Examples 3-4 show that when Rhodococcus roseus and Protocormia chrysospora were used alone to prepare biochar composites, their ability to degrade PAHs decreased. This demonstrates that the composite agent prepared by Rhodococcus roseus and Protocormia chrysospora can synergistically degrade polycyclic aromatic hydrocarbon pollutants and improve the remediation effect of polycyclic aromatic hydrocarbon contaminated soil.
[0090] Experiment 2: Carbon Sequestration Capacity Test
[0091] After 100 days of continuous remediation, 10 mg of soil was taken from each flowerpot in both Example 1 and Comparative Example 2 of Experiment 1. The soil samples were placed in the sample boat of the elemental analyzer and sent to the combustion furnace of the elemental analyzer. The samples were calcined at 1000°C in an oxygen stream to convert carbon into CO2. After separation by the chromatographic column, the CO2 was detected by the detector. The carbon content in the soil sample was calculated by comparing the peak area or signal intensity of the carbon dioxide detected by the detector with a standard substance of known concentration. The higher the carbon content in the soil sample, the stronger the carbon fixation capacity. The test results are shown in Table 2.
[0092] Table 2: Soil carbon content after 100 days of remediation
[0093]
[0094] As can be seen from the data in Example 1 and Comparative Example 2 in Table 2, the prepared earthworm mucus binder can absorb and fix the emitted CO2 in the soil, giving the biochar composite material excellent carbon fixation ability while repairing the soil.
[0095] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.
Claims
1. A method for preparing a biochar composite material, characterized in that, Includes the following steps: S1: Preparation of biochar-specific adsorption materials Phenanthrene was dissolved in a mixed solution of toluene and acetonitrile. Then, 4-vinylpyridine and straw biochar powder were added sequentially and ultrasonically dispersed evenly. After standing, a prepolymer solution was obtained. Ethylene glycol dimethacrylate and azobisisobutyronitrile were added, and after ultrasonic dispersion, oxygen was removed. The solution was sealed and subjected to a water bath reaction. The reaction solution was centrifuged, and the precipitate was collected, washed, and dried to obtain a biochar-specific adsorbent material. S2: Preparation of Earthworm Mucus Adhesive After the Eisenia fetuses were placed in a dark place to defecate and cleaned, they were placed in a container and electrically stimulated with a DC power supply. The earthworm secretions secreted under electrical stimulation were collected by filtering and centrifugation. The supernatant was collected to obtain earthworm mucus. The earthworm mucus was mixed with acetate buffer, and α-mannosidase and β-1,3-glucanase were added for enzymatic hydrolysis and enzyme inactivation to obtain activated mucus. Ethylenediamine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were added under a nitrogen atmosphere. After the reaction was sealed, the mixture was centrifuged and the supernatant was removed to obtain earthworm mucus adhesive. S3: Compound granulation of biochar and microorganisms Rhodococcus roseus and Phanerochaete chrysosporium were cultured in an inorganic salt medium containing phenanthrene and pyrene by shaking. The resulting Rhodococcus roseus suspension and Phanerochaete chrysosporium suspension were mixed and formulated into a bacterial suspension to obtain a composite bacterial agent. The biochar specific adsorbent material, the composite bacterial agent and earthworm mucus binder were mixed evenly and granulated to obtain a biochar composite material.
2. The method for preparing a biochar composite material according to claim 1, characterized in that, Step S1, the preparation of biochar-specific adsorbent materials, specifically includes the following steps: S1.1: Place 0.0178 parts by weight of phenanthrene in a container, add 35-40 parts by weight of toluene and 10-15 parts by weight of acetonitrile, stir until phenanthrene is completely dissolved, then add 0.042-0.045 parts by weight of 4-vinylpyridine, ultrasonically disperse at an ultrasonic frequency of 25-30 kHz for 10-15 min, then add 1.8-2 parts by weight of straw biochar powder, continue ultrasonic dispersion for 25-30 min, and let stand for 12-15 h to obtain a prepolymer solution; S1.2: Add 0.29-0.3 parts by weight of ethylene glycol dimethacrylate and 0.025-0.03 parts by weight of azobisisobutyronitrile to the prepolymer solution, disperse by ultrasonication for 10-15 min, then purge with nitrogen gas to remove oxygen for 4-6 min, and then seal in a water bath shaker at 60-65℃ for 20-24 h to obtain the reaction solution; S1.3: Place the reaction solution in a centrifuge and centrifuge at 10000-12000 rpm for 10-15 min. Collect the bottom precipitate and dry it in an oven at 60-65℃. Then, continuously wash it with methanol-acetic acid solution until no phenanthrene is found in the methanol-acetic acid solution after washing. Then, dry it in an oven at 60-65℃ until constant weight to obtain biochar specific adsorbent material.
3. The method for preparing a biochar composite material according to claim 2, characterized in that, Step S2, the preparation of the earthworm mucus adhesive, specifically includes the following steps: S2.1: Place healthy, disease-free Eisenia fetuses in a dark place for 24 hours to defecate, then rinse them clean with deionized water and dry them to obtain clean earthworms. Take 300-400g of clean earthworms and place them in a container. Perform electrical stimulation under a DC power supply of 5V and 10mA for 60-75s each time, with an interval of 50-60s. Filter and collect the earthworm secretions under electrical stimulation, transfer them to a centrifuge, and centrifuge at 8000-10000rpm for 10-12min. Separate and collect the supernatant to obtain earthworm mucus. S2.2: Mix earthworm mucus and acetate buffer with pH 5.2-5.5 at a volume ratio of 1:(4-5), then add α-mannosidase and β-1,3-glucanase and mix well. Incubate at 37℃ for 2-3 hours, then heat to 70℃ for 10 minutes to inactivate the enzymes and obtain activated mucus. S2.3: Place the activated mucus in a container, purge with nitrogen to remove oxygen, then add ethylenediamine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Control the final concentration of ethylenediamine in the activated mucus to be 1.5-3 mmol / g, and the final concentration of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the activated mucus to be 0.8-1 mmol / g. React under closed conditions at 30-35℃ and 300-350 rpm for 3-3.5 h. Then place it in a centrifuge and centrifuge at 7000-7500 rpm for 10-12 min. Separate and remove the supernatant to obtain the earthworm mucus adhesive.
4. The method for preparing a biochar composite material according to claim 3, characterized in that, Step S3, the compound granulation of biochar microorganisms, specifically includes the following steps: S3.1: Rhodococcus roseus and Phanerochaete chrysosporium were inoculated at 8-10% into 100ml of inorganic salt medium containing phenanthrene and pyrene, and cultured at 30℃ and 150rpm for 5 days to obtain Rhodococcus roseus bacterial suspension and Phanerochaete chrysosporium bacterial suspension. S3.2: Mix the suspension of Rhodotorula rubrum and the suspension of Phanerochaete chrysospora at a volume ratio of 1:(1-1.2) to prepare a compound bacterial suspension with OD600=1, thus obtaining a compound bacterial agent; S3.3: Mix 45-50 parts by weight of biochar specific adsorbent material, 6-8 parts by weight of composite bacterial agent and 10-12 parts by weight of earthworm mucus binder evenly, and then place them in a disc pelletizer to make particles with a particle size of 3-4 mm to obtain biochar composite material.
5. The method for preparing a biochar composite material according to claim 2, characterized in that, In step S1.1, the straw biochar powder is obtained by pyrolyzing corn straw in a tube furnace under vacuum at 700-750℃ for 2 hours, then pulverizing it and passing it through an 80-100 mesh sieve.
6. The method for preparing a biochar composite material according to claim 2, characterized in that, Step S1.3 The methanol-acetic acid solution is prepared by mixing methanol and acetic acid in a volume ratio of 9:
1.
7. The method for preparing a biochar composite material according to claim 3, characterized in that, In step S2.2, α-mannosidase and β-1,3-glucanase each account for 2-3% of the mass of earthworm mucus.
8. The method for preparing a biochar composite material according to claim 4, characterized in that, In step S3.1, the inorganic salt culture medium containing phenanthrene and pyrene contains 30 mg / L of both phenanthrene and pyrene, and also contains 2 g / L of (NH4)2SO4, 2 g / L of KH2PO4, 2 g / L of K2HPO4, 0.2 g / L of CaCl2, 10 g / L of NaCl, and 0.2 g / L of MgSO4∙7H2O, with the remainder being sterile water.
9. A soil remediation method, characterized in that, Includes the following steps: A biochar composite material is prepared using the method described in any one of claims 1-8. The prepared biochar composite material is added to the soil to be remediated by turning and mixing, with the addition amount being 5-7 wt% of the soil to be remediated, and the moisture content of the soil to be remediated is controlled to be 45-50%.