Preparation method of immobilized cellulase magnetic biochar and its application in tobacco stem composting fermentation

Abstract

The present invention discloses a preparation method of immobilized cellulase magnetic biochar and its application in tobacco stem composting fermentation, belonging to the field of fertilizer fermentation technology, and the steps are: carboxylation of biochar; photocatalytic activation of biochar; preparation of modified Fe3O4@SiO2‑NC nanoparticles to prepare new magnetic biochar; immobilization of cellulase. The composting fermentation method is to mix tobacco stems, cow dung, urea, corn flour, and fermentation aids as a tobacco stem compost matrix, add immobilized cellulase and mix evenly, add water, and ferment for 30 days. The thermal stability of the cellulase prepared by the present invention after being immobilized on the magnetic biochar material is improved. At 50°C, the cellulase activity is increased by 12%, and after 60°C, the cellulase activity is increased by 50%; the application of the cellulase immobilized magnetic biochar material in the tobacco stem and cow dung composting fermentation process can shorten the cycle and significantly improve the cellulose degradation rate, organic matter content, humic acid content, and tobacco seedling germination rate.

Description

Technical Field

[0001] The invention relates to the technical field of fertilizer fermentation, and in particular to a preparation method of immobilized cellulase magnetic biochar and application of the biochar in tobacco stem composting fermentation. Background Art

[0002] After composting and fermenting, cow dung produces nutrients such as crude protein, crude fat, nitrogen-free extract, crude fiber, and minerals. It can be used as an organic fertilizer or substrate, positively impacting soil quality and promoting plant growth. With growing awareness of organic food and environmental protection, demand for cow dung as a high-quality organic fertilizer continues to grow, particularly in the green food and organic vegetable sectors. The market for cow dung compost is booming. National policies supporting agriculture have also fostered a favorable environment for the cow dung composting industry, making it a crucial component of sustainable agricultural development. Therefore, composting offers a sustainable solution for organic waste management, promoting the development of a circular economy.

[0003] The composting process has four main phases: the first is the mesophilic or initial stage, with temperatures rising rapidly between 10 and 42°C. Mesophilic bacteria play a dominant role and last for 24 to 72 hours. The second is the thermophilic stage, characterized by temperatures between 45 and 70°C. The efficiency of organic compound degradation is closely related to the metabolic activity of thermophilic microorganisms and can last from several days to weeks. The third is the maturation stage, with temperatures dropping between 65 and 50°C and remaining for one to two months. The thermophilic microorganisms are reactivated, breaking down the most recalcitrant components. The final stage is the curing stage, which can last for one to four months at temperatures between 50 and 23°C. The converted organic matter steadily accumulates during these stages. The thermophilic fermentation in the second and third stages is a critical period in the composting process. Thermophiles are the dominant functional microorganisms, and their metabolic activity at high temperatures determines the efficiency of organic matter degradation, limiting the composting period. The cellulose content in cow manure ranges from approximately 30% to 50%, making microbial fiber-degrading activity central to the composting process.

[0004] Current methods for promoting straw cellulose degradation include chemical methods (alkalinization, acidification, alkaline-acid combination treatment, and oxidant treatment), physical methods (supercritical carbon dioxide fluid pretreatment, microwave pretreatment, and high-temperature pyrolysis), and biological methods (specialized bacterial strains or complex inoculants). While chemical treatment is effective, it is costly and prone to environmental pollution, making it difficult to promote. Physical methods require certain equipment, have high initial investment costs, and are limited by site conditions. Finally, biological methods have unstable degradation efficiency and poor applicability.

[0005] Therefore, providing a method for preparing immobilized cellulase magnetic biochar suitable for cow dung fermentation is a technical problem that those skilled in the art urgently need to solve. Summary of the Invention

[0006] In view of this, the present invention provides a method for immobilizing cellulase based on magnetic biochar material, and uses the cellulase-immobilized magnetic biochar material for composting.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for preparing immobilized cellulase magnetic biochar comprises the following steps:

[0009] (1) Preparation of magnetic biochar materials

[0010] (1.1) Carboxylated biochar: The tobacco stems were rinsed with clean water and dried by heating, then crushed through a 60-mesh sieve and pyrolyzed by furnace burning to obtain biochar. The biochar was modified with nitric acid to increase the carboxyl groups on its surface, and then rinsed with deionized water until the pH value was neutral and dried to obtain carboxylated biochar.

[0011] (1.2) Photocatalytically active biochar: ZnSO4·7H2O and carboxylated biochar were mixed in deionized water and stirred to disperse uniformly. NaOH solution was added dropwise under continuous magnetic stirring until the pH reached 10. The resulting mixture was transferred to a Teflon-lined stainless steel autoclave, sealed and heated, and then cooled to room temperature. The resulting product was washed with ethanol and deionized water, respectively, filtered, and dried to obtain a ZnO-modified biochar carbon material. This material can inhibit the recombination of photogenerated electron-hole pairs due to the formation of a P / N heterojunction between ZnO and biochar, and has strong photocatalytic activity.

[0012] (2) Preparation of modified Fe3O4@SiO2-NC nanoparticles

[0013] (2.1) Under a nitrogen atmosphere, FeCl3·6H2O and FeCl2·4H2O were dissolved in deionized water and stirred uniformly. Under vigorous stirring, sodium hydroxide solution was added dropwise to the solution, and then heated and mixed. The pH of the solution was adjusted to 10. The solution was heated and mixed again. The obtained nanoparticles were separated by a magnet, washed with pure water and ethanol, and impurities in the nanoparticles were removed by ultrasonication. The nanoparticles were dried to obtain Fe3O4 nanoparticles.

[0014] (2.2) The synthesized Fe3O4 nanoparticles were dispersed in deionized water. After filtration, the solution was added to ethanol and sonicated at 400 rpm for 20 min. Then, ethyl silicate was added to the solution, and the reaction mixture was vigorously stirred at room temperature under nitrogen for 6 h. Finally, the product was collected using a magnet, washed with water and ethanol, and dried under vacuum to obtain Fe3O4@SiO2-NC.

[0015] (2.3) Fe3O4@SiO2-NC was dispersed in ethanol by ultrasonication, and then APTES was added and vigorously stirred at room temperature for 2 h. The mixture was sealed in an autoclave and heated. The mixture temperature was lowered to room temperature, the product was separated using a magnet, and washed with ethanol to obtain aminosilane-modified Fe3O4@SiO2-NC.

[0016] (2.4) 1 g of TCT was dissolved in 20 mL of THF, and then aminosilane-modified Fe3O4@SiO2-NC was added to the solution and stirred at room temperature. The product was collected by magnet and the obtained modified Fe3O4@SiO2-NC was washed with deionized water.

[0017] (3) Preparation of new magnetic biochar

[0018] The ZnO-modified biochar carbon material was dispersed in deionized water and evenly dispersed through ultrasonic treatment. Then, EDC and NHS were added to the solution together and stirred evenly at room temperature. The modified Fe3O4@SiO2-NC particles were suspended in deionized water by ultrasonic treatment and then added to the biochar solution. The mixture was stirred to react, and the product was collected by magnetic separation, washed with deionized water and ethanol respectively, and dried to obtain a new type of magnetic biochar.

[0019] (4) Cellulase immobilization

[0020] The new magnetic biochar was added to water, followed by cellulase, and then added to 50 mL of acetate buffer. The mixture was cultured in an incubator shaker at room temperature. The cellulase-loaded magnetic biochar was separated with a magnet and washed with acetate buffer solution to obtain immobilized cellulase magnetic biochar.

[0021] Furthermore, the pyrolysis temperature in step (1.1) is 500° C. and the pyrolysis time is 2 h;

[0022] The nitric acid modification was as follows: 2.5 g of biochar was impregnated with 100 mL of 1+1 HNO3 at 60 °C for 12 h;

[0023] The drying temperature is 60°C.

[0024] Furthermore, the mass ratio of ZnSO4·7H2O and carboxylated biochar in step (1.2) is 2:3;

[0025] The concentration of the NaOH solution is 0.5 mol / L

[0026] The sealing temperature is 180°C and the sealing time is 12 hours;

[0027] The drying temperature is 70° C. and the drying time is 12 h.

[0028] Furthermore, in the technical solution of the present invention, the stirring rate of the vigorous stirring is 750 rpm.

[0029] Furthermore, in step (2.1), the molar ratio of FeCl3·6H2O to FeCl2·4H2O is 2:1;

[0030] The concentration of the sodium hydroxide solution is 4M, and the mass ratio of the volume of the sodium hydroxide solution to FeCl3·6H2O is 10mL:3.4g;

[0031] The heating mixing temperature is 70°C, and the secondary heating mixing temperature is 80°C.

[0032] Furthermore, the ratio of the mass of the Fe3O4 nanoparticles to the volume of deionized water to the volume of ethanol in step (2.2) is 5 g:150 mL:1500 mL;

[0033] The ratio of the mass of the Fe3O4 nanoparticles to the volume of ethyl silicate is 5 g:4 mL.

[0034] Furthermore, the mass ratio of Fe3O4@SiO2-NC to ethanol in step (2.3) is 1 g:100 mL;

[0035] The amount of APTES is 10% of the mass of Fe3O4@SiO2-NC;

[0036] The heating temperature is 90° C. and the heating time is 12 h.

[0037] Furthermore, the mass ratio of TCT to THF in step (2.4) is 1 g:20 mL;

[0038] The mass ratio of the TCT to the aminosilane-modified Fe3O4@SiO2-NC is 1:3.

[0039] Furthermore, the volume ratio of the ZnO-modified biochar carbon material to deionized water in step (3) is 100 mg:80 mL;

[0040] The mass ratio of the ZnO-modified biochar carbon material to EDC, NHS and modified Fe3O4@SiO2-NC is 100:30:20:100;

[0041] The stirring reaction temperature is 80°C and the stirring reaction time is 24h;

[0042] The drying temperature is 60° C. and the drying time is 6 hours.

[0043] Furthermore, the mass ratio of the novel magnetic biochar to the volume of cellulase in step (4) is 60 mg:8 mL;

[0044] The concentration of the cellulase is 0.25 mg / mL;

[0045] The pH value of the acetate buffer is 5.

[0046] The present invention also provides the use of the immobilized cellulase magnetic biochar prepared by the above method in tobacco stem composting fermentation, comprising the following steps:

[0047] Tobacco stalks, cow dung, urea, corn flour, and fermentation aids were mixed as a tobacco stalk compost matrix, immobilized cellulase was added and mixed evenly, water was added, and fermentation was carried out for 30 days;

[0048] The mass ratio of tobacco stems, cow dung, urea, corn flour, fermentation aids, immobilized cellulase magnetic biochar and water is 1000:300:5:5:4:5:60:250.

[0049] Furthermore, the fermentation aid is a base of Beijing Huaxia Kangyuan Technology Co., Ltd. 11 Fermenting agent.

[0050] The beneficial effects of the present invention are as follows: the thermal stability of the cellulase prepared by the present invention after being immobilized on the magnetic biochar material is improved, the cellulase activity is increased by 12% at 50°C, and the cellulase activity is increased by 50% after 60°C; the application of the cellulase-immobilized magnetic biochar material in the tobacco stalk and cow dung composting fermentation process can shorten the cycle by 30%, increase the cellulose degradation rate by 23.35%, and at the same time increase the organic matter content by 14.55%, the humic acid content by 6.38%, and the tobacco seedling germination rate by 23.35%.

[0051] The method of the present invention can enhance the thermal stability of cellulase, thereby increasing the enzyme activity by 10% to 170% under the same high temperature conditions (50-80°C). This is because the magnetic biochar is a material with a porous structure. After being connected to the cellulase at multiple points, the firmness of the enzyme's active conformation is increased, while also inhibiting the enzyme's own degradation and partially blocking the invasion of external adverse factors on the enzyme, thereby improving the enzyme's thermal stability. The present invention utilizes the formation of a P / N heterojunction between a synthetic nano ZnO semiconductor material and biochar to inhibit the recombination of photogenerated electron-hole pairs, thereby improving the photocatalytic activity of the composite material, accelerating the degradation of cellulose under sunlight irradiation, and increasing the organic matter content of the fermentation matrix. The low-concentration nano ZnO of the present invention has a growth-promoting effect on plant growth, can improve the fresh weight of the aboveground part and the fresh weight of the roots of seedlings, and can therefore be used as a source of zinc fertilizer for crop production. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a diagram of the root growth of tobacco seedlings in the tobacco stalk and cow dung compost matrix of Experimental Example 2 provided by the present invention. DETAILED DESCRIPTION

[0053] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0054] Example 1

[0055] A method for preparing immobilized cellulase magnetic biochar:

[0056] (1) Preparation of magnetic biochar materials

[0057] (1.1) Carboxylated biochar: The tobacco stems were rinsed with clean water and dried by heating, then crushed through a 60-mesh sieve and pyrolyzed at 500°C for 2 h to obtain biochar. The biochar was modified with nitric acid by soaking 2.5 g of biochar in 100 mL (1+1) HNO3 at 60°C for 12 h, then rinsed with deionized water until the pH was neutral, and dried at 60°C for 10 h to obtain carboxylated biochar.

[0058] (1.2) Photocatalytically activated biochar: 2 g of ZnSO4·7H2O and 3 g of carboxylated biochar were mixed in 50 mL of deionized water and stirred to disperse uniformly. 0.5 mol / L NaOH solution was added dropwise under continuous magnetic stirring until the pH reached 10. The obtained mixture was transferred to a Teflon-lined stainless steel autoclave, sealed, and maintained at 180°C for 12 h, then cooled to room temperature. The obtained product was washed with ethanol and deionized water, respectively, filtered, and then dried at 70°C for 12 h to obtain ZnO-modified biochar carbon material.

[0059] (2) Preparation of modified Fe3O4@SiO2-NC nanoparticles

[0060] (2.1) Under a nitrogen atmosphere, 3.4 g of FeCl₃·6H₂O and 1.25 g of FeCl₂·4H₂O (molar mass ratio 2:1) were dissolved in deionized water and stirred uniformly. 10 mL of 4 M sodium hydroxide solution was added dropwise to the solution under vigorous stirring at 750 rpm. The mixture was then mixed at 70°C and the pH of the solution was adjusted to 10. The solution was mixed again at 80°C for 30 min, and the obtained nanoparticles were separated using a magnet. The nanoparticles were washed with pure water and ethanol, and impurities in the nanoparticles were removed by ultrasonication. Finally, the mixture was dried at 70°C to obtain Fe₃O₄ nanoparticles.

[0061] (2.2) 5 g of the synthesized Fe3O4 nanoparticles were dispersed in 150 mL of deionized water, filtered, added to 1.5 L of ethanol, and ultrasonically treated at 400 rpm for 20 min. Then, 4 mL of ethyl silicate was added to the solution, and the reaction mixture was vigorously stirred at room temperature and 750 rpm under nitrogen for 6 h. Finally, the product was collected using a magnet, washed with water and ethanol, respectively, and dried under vacuum to obtain Fe3O4@SiO2-NC.

[0062] (2.3) 1 g of dried Fe3O4@SiO2-NC was dispersed in 100 mL of ethanol by ultrasonication, and then 10% APTES was added and vigorously stirred at 750 rpm for 2 h at room temperature. The mixture was sealed in an autoclave and heated at 90°C for 12 h. The mixture was cooled to room temperature, separated using a magnet, and washed with ethanol to obtain aminosilane-modified Fe3O4@SiO2-NC.

[0063] (2.4) 1 g of TCT was dissolved in 20 mL of THF, and then 3 g of aminosilane-modified Fe3O4@SiO2-NC was added to the solution. The mixture was stirred at room temperature for 2 h. The product was collected by magnet and the modified Fe3O4@SiO2-NC was washed with deionized water.

[0064] (3) Preparation of new magnetic biochar

[0065] 100 mg of ZnO-modified biochar carbon material was dispersed in 80 mL of deionized water and ultrasonically treated for 30 min. Then 30 mg of EDC and 20 mg of NHS were added to the solution together and stirred at room temperature for 1 h. 100 mg of modified Fe3O4@SiO2-NC particles were suspended in 20 mL of deionized water by ultrasonication and then added to the biochar solution. The mixture was stirred and reacted at 80 °C for 24 h. The product was collected by magnetic separation, washed with deionized water and ethanol respectively, and then dried at 60 °C for 6 h to obtain a new type of magnetic biochar.

[0066] (4) Cellulase immobilization

[0067] 60 mg of the new magnetic biochar was added to 2 mL of water, followed by 8 mL of cellulase (0.25 mg / mL), and then added to 50 mL of acetate buffer at pH 5. The mixture was placed in an incubator shaker at room temperature for 7 h. The magnetic biochar loaded with cellulase was separated with a magnet and washed with acetate buffer solution to obtain immobilized cellulase magnetic biochar.

[0068] Experimental Example 1 Determination of thermal stability of immobilized cellulase

[0069] Immobilized cellulase was prepared according to Example 1, and the activities of the immobilized cellulase and non-immobilized cellulase were determined by carboxymethyl cellulose and dinitrosalicylic acid. The reaction conditions were 30°C, 40°C, 50°C, 60°C, 70°C, and 80°C for 1 h, respectively.

[0070] A carboxymethyl cellulose solution (1% (w / v)) prepared in 50 mM acetate buffer at pH = 5 was used as a substrate, and the color developer was dinitrosalicylic acid (1 gram of dinitrosalicylic acid was dissolved in 50 milliliters of double distilled water). Then, 30 grams of potassium sodium tartrate and 20 milliliters of 2M sodium hydroxide solution were added. Distilled water was then added to make the final volume 100 mL, and the reagent was stored in a dark bottle. 1 mL of the enzyme solution from step 2 was added to a test tube containing 1 mL of carboxymethyl cellulose (1%) and placed at 50°C for 1 hour. 3 mL of dinitrosalicylic acid reagent was then added to the reaction mixture, and the sample and control were placed in boiling water for 15 minutes until a brown color appeared. The sample was then cooled and its absorbance was measured at 540 nm against the control using a glucose standard curve.

[0071] Table 1 Immobilized and free cellulase activities

[0072]

[0073]

[0074] As shown in Table 1, the enzymatic activity of immobilized cellulase was higher than that of free cellulase at temperatures ranging from 40°C to 80°C. At 50°C, the enzymatic activity of immobilized cellulase was 1.13 times that of free cellulase; at 70°C and 80°C, the enzymatic activity of immobilized cellulase was 1.93 times and 2.73 times that of free cellulase, respectively. These results indicate that immobilized cellulase prepared on modified magnetic biochar has higher thermal stability.

[0075] Example 2

[0076] 1 kg of tobacco stems (crushed and sieved with 1 mm pores), 0.3 kg of cow dung, 0.005 kg of urea, 0.005 kg of corn flour, and 0.004 kg of fermentation aid were mixed as a tobacco stem compost matrix, 0.06 kg of the immobilized cellulase magnetic biochar prepared in Example 1 was added and mixed evenly, 250 mL of clean water was added, and fermentation was carried out for 30 days, and the cellulose content was detected.

[0077] Experimental Example 2 Application of immobilized cellulase in tobacco stem composting

[0078] An experiment on the application of immobilized cellulase in tobacco stem and cow dung composting was conducted according to Example 2, and the experimental group was set up as follows.

[0079] Experimental group (T1): 1.314 kg compost matrix + 0.060 kg magnetic biochar material immobilized cellulase (the scheme of Example 2);

[0080] Control group (T2): 1.314 kg compost substrate + 0.002 kg cellulase;

[0081] Control group (T3): 1.314 kg compost matrix + 0.060 kg modified magnetic biochar material;

[0082] Control group (T4): 1.314 kg compost matrix + 2 kg sodium alginate immobilized cellulase;

[0083] Control group CK: 1.314 kg compost substrate + 0.060 kg water.

[0084] Note: The amount of cellulase immobilized on magnetic biochar and sodium alginate is based on 0.002 kg of cellulase during preparation.

[0085] Sodium alginate-immobilized cellulase was prepared by a conventional method. A mixture of sodium alginate: glutaraldehyde: cellulase: water was prepared at a ratio of 3.5:1:0.1:100, and the mixture was dropwise injected into a 2% CaCl2 solution. The gel pellets were filtered out, placed in a new CaCl2 solution, and allowed to harden in a 4°C refrigerator for 2 hours. The pellets were filtered out again, washed three times with a 0.9% NaCl solution, and then rinsed twice with distilled water to obtain sodium alginate-immobilized cellulase.

[0086] Table 2 Cellulose degradation rate after 30 days of composting tobacco stems and cow dung

[0087]

[0088] As shown in Table 2, the cellulose degradation rate in the experimental group (T1) containing cellulase immobilized on magnetic biochar was 22.74% ± 2.25%, which was higher than that in the control group (T2), the control group (T3), and the control group (T4). Compared with the control group, the cellulose degradation rate of the immobilized cellulase group increased by an average of 30%, indicating that the magnetic biochar-immobilized cellulase significantly promoted cellulose degradation.

[0089] Table 3 Physical and chemical parameters after 30 days of composting tobacco stems and cow dung

[0090]

[0091]

[0092] As shown in Table 3, the cellulose organic matter content and humic acid content of the experimental group with the addition of magnetic biochar-immobilized cellulase were significantly higher than those of T2, T3, T4 and CK, indicating that magnetic biochar-immobilized cellulase can increase the organic matter and humic acid content of compost.

[0093] Table 4 Agronomic characteristics of tobacco seedling growth after tobacco stem and cow dung composting

[0094]

[0095] The fermented compost of the experimental group and the control group was used as a substrate for tobacco seedling cultivation. The agronomic traits of the tobacco seedling roots were measured after 14 days. The results are shown in Table 4 and Figure 1 As shown in the data, the total root length of the experimental group T1 was 198.46 (±11.63) cm, and the total root surface area was 24.47 (±2.39) cm2. The two agronomic traits were significantly higher than those of the T2, T3, T4 and CK control groups, indicating that the magnetic biochar-immobilized cellulase-fermented tobacco stem and cow dung compost as a matrix can significantly promote the development of tobacco seedlings.

[0096] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for preparing immobilized cellulase magnetic biochar, characterized in that: The following steps are involved: (1) Preparation of magnetic biochar materials (1.1) Carboxylated biochar: The tobacco stems were rinsed with clean water and dried by heating, then crushed through a 60-mesh sieve and pyrolyzed by furnace calcination to obtain biochar. The biochar was modified with nitric acid, rinsed with deionized water until the pH was neutral, and dried to obtain carboxylated biochar. (1.2) Photocatalytically activated biochar: ZnSO4·7H2O and carboxylated biochar were mixed in deionized water and stirred to disperse uniformly; NaOH solution was added dropwise under continuous magnetic stirring until the pH reached 10; the obtained mixture was transferred to a Teflon-lined stainless steel autoclave, sealed and heated, and then cooled to room temperature. The obtained product was washed with ethanol and deionized water, respectively, filtered, and dried to obtain ZnO-modified biochar carbon material; (2) Preparation of modified Fe3O4@SiO2-NC nanoparticles (2.1) Under a nitrogen atmosphere, FeCl3·6H2O and FeCl2·4H2O were dissolved in deionized water and stirred uniformly. Under vigorous stirring, sodium hydroxide solution was added dropwise to the solution, and then heated and mixed. The pH of the solution was adjusted to 10. The solution was heated and mixed again. The obtained nanoparticles were separated by a magnet, washed with pure water and ethanol, and impurities in the nanoparticles were removed by ultrasonication. The nanoparticles were dried to obtain Fe3O4 nanoparticles. (2.2) The synthesized Fe3O4 nanoparticles were dispersed in deionized water. After filtration, the solution was added to ethanol and sonicated at 400 rpm for 20 min. Then, ethyl silicate was added to the solution, and the reaction mixture was vigorously stirred at room temperature under nitrogen for 6 h. Finally, the product was collected using a magnet, washed with water and ethanol, and dried under vacuum to obtain Fe3O4@SiO2-NC. (2.3) Fe3O4@SiO2-NC was dispersed in ethanol by ultrasonication, and then APTES was added and vigorously stirred at room temperature for 2 h. The mixture was sealed in an autoclave and heated. The mixture temperature was lowered to room temperature, the product was separated using a magnet, and washed with ethanol to obtain aminosilane-modified Fe3O4@SiO2-NC. (2.4) 1 g of TCT was dissolved in 20 mL of THF, and then aminosilane-modified Fe3O4@SiO2-NC was added to the solution and stirred at room temperature. The product was collected by magnet and the obtained modified Fe3O4@SiO2-NC was washed with deionized water. (3) Preparation of new magnetic biochar The ZnO-modified biochar carbon material was dispersed in deionized water and evenly dispersed by ultrasonic treatment. EDC and NHS were then added to the solution and stirred at room temperature. The modified Fe3O4@SiO2-NC particles were suspended in deionized water by ultrasonic treatment and then added to the biochar solution. The mixture was stirred for reaction. The product was collected by magnetic separation, washed with deionized water and ethanol, respectively, and dried to obtain a new type of magnetic biochar. (4) Cellulase immobilization The new magnetic biochar was added to water, followed by cellulase, and then added to 50 mL of acetate buffer. The mixture was cultured in an incubator shaker at room temperature. The cellulase-loaded magnetic biochar was separated with a magnet and washed with acetate buffer solution to obtain immobilized cellulase magnetic biochar.

2. The method for preparing immobilized cellulase magnetic biochar according to claim 1, characterized in that: The pyrolysis temperature in step (1.1) is 500°C and the pyrolysis time is 2h; The nitric acid modification was as follows: 2.5 g of biochar was impregnated with 100 mL of 1+1 HNO3 at 60 °C for 12 h; The drying temperature is 60°C.

3. The method for preparing immobilized cellulase magnetic biochar according to claim 1, characterized in that: The mass ratio of ZnSO4·7H2O to carboxylated biochar in step (1.2) is 2:3; The concentration of the NaOH solution is 0.5 mol / L The sealing temperature is 180°C and the sealing time is 12 hours; The drying temperature is 70° C. and the drying time is 12 h.

4. The method for preparing immobilized cellulase magnetic biochar according to claim 1, characterized in that: The molar ratio of FeCl3·6H2O to FeCl2·4H2O in step (2.1) is 2:1; The concentration of the sodium hydroxide solution is 4M, and the mass ratio of the volume of the sodium hydroxide solution to FeCl3·6H2O is 10mL:3.4g; The heating mixing temperature is 70°C, and the secondary heating mixing temperature is 80°C.

5. The method for preparing magnetic biochar with immobilized cellulase according to claim 1, characterized in that: The ratio of the mass of the Fe3O4 nanoparticles to the volume of deionized water and ethanol in step (2.2) is 5 g:150 mL:1500 mL; The ratio of the mass of the Fe3O4 nanoparticles to the volume of ethyl silicate is 5 g:4 mL.

6. The method for preparing immobilized cellulase magnetic biochar according to claim 1, characterized in that: The mass ratio of Fe3O4@SiO2-NC to ethanol in step (2.3) is 1 g:100 mL; The amount of APTES is 10% of the mass of Fe3O4@SiO2-NC; The heating temperature is 90° C. and the heating time is 12 h.

7. The method for preparing immobilized cellulase magnetic biochar according to claim 1, characterized in that: The mass ratio of TCT to THF in step (2.4) is 1 g:20 mL; The mass ratio of the TCT to the aminosilane-modified Fe3O4@SiO2-NC is 1:

3.

8. The method for preparing immobilized cellulase magnetic biochar according to claim 1, characterized in that: The volume ratio of the ZnO-modified biochar carbon material to deionized water in step (3) is 100 mg:80 mL; The mass ratio of the ZnO-modified biochar carbon material to EDC, NHS and modified Fe3O4@SiO2-NC is 100:30:20:100; The stirring reaction temperature is 80°C and the stirring reaction time is 24h; The drying temperature is 60° C. and the drying time is 6 hours.

9. The method for preparing magnetic biochar with immobilized cellulase according to claim 1, characterized in that: The mass ratio of the novel magnetic biochar to the volume of cellulase in step (4) is 60 mg:8 mL; The concentration of the cellulase is 0.25 mg / mL; The pH value of the acetate buffer is 5.

10. Use of immobilized cellulase magnetic biochar prepared by the method according to any one of claims 1 to 9 in tobacco stem composting fermentation, characterized in that: The following steps are involved: Tobacco stalks, cow dung, urea, corn flour, and fermentation aids were mixed as a tobacco stalk compost matrix, immobilized cellulase was added and mixed evenly, water was added, and fermentation was carried out for 30 days; The mass ratio of tobacco stems, cow dung, urea, corn flour, fermentation aids, immobilized cellulase magnetic biochar and water is 1000:300:5:5:4:5:60:250.

Images