Soil conditioner based on bamboo charcoal biochar immobilized microorganisms and preparation method thereof

Abstract

The invention relates to the field of soil conditioners, in particular to a soil conditioner based on bamboo charcoal biochar immobilized microorganisms and a preparation method of the soil conditioner, and aims at solving the problems that the soil modification effect of an existing soil improvement method is limited, but secondary damage is caused to soil due to long-term excessive use. The preparation method comprises the following steps: firstly, treating moso bamboo powder, then carbonizing with a potassium hydroxide solution, calcining and pickling to obtain bamboo charcoal biochar, then loading microorganisms on the bamboo charcoal biochar to obtain a loaded microbial agent, and then coating the loaded microbial agent to obtain the soil conditioner based on the bamboo charcoal biochar immobilized microorganisms. When the soil conditioner is added into the polluted soil, the microbial activity in the polluted soil can be maintained for a long time, the cyclic utilization of nitrogen and phosphorus in the polluted soil is increased, the structure of the polluted soil is improved, the fertility of the polluted soil is improved, and the soil conditioner has an excellent remediation effect on the polluted soil, so that the yield and quality of crops are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of soil conditioners, specifically to soil conditioners based on bamboo charcoal biochar immobilized with microorganisms and their preparation methods. Background Technology

[0002] Soil is a crucial foundation for agricultural production and the ecological environment, and its health directly impacts crop yield and quality. However, with rapid industrialization and urbanization, soil faces severe pollution problems, such as heavy metal and organic pollutant contamination, which seriously affect soil structure and ecological balance. Traditional soil improvement methods, such as the application of chemical and organic fertilizers, can improve soil quality to some extent, but long-term excessive use can lead to serious problems such as soil compaction and salinization, ultimately damaging the soil's ecological environment. Therefore, developing a soil conditioner based on bamboo charcoal-supported microorganisms and its preparation method is of great significance for achieving sustainable agricultural development.

[0003] To address the aforementioned technical shortcomings, a solution is proposed. Summary of the Invention

[0004] In order to overcome the above-mentioned technical problems, the purpose of this invention is to provide a soil conditioner based on bamboo charcoal biochar immobilized microorganisms and its preparation method, which solves the problem that the existing soil improvement methods have limited soil modification effects, but long-term excessive use will cause secondary damage to the soil.

[0005] The objective of this invention can be achieved through the following technical solutions: In a first aspect, this application provides a method for preparing a soil conditioner based on bamboo charcoal-supported microorganisms, comprising the following steps: Step 1: Add bamboo charcoal biochar to the mixed bacterial suspension and culture at 30℃ and 120r / min for 24-48h. Then, vacuum filter the mixture, wash the filter cake with distilled water 3-5 times, and freeze dry to obtain the loaded bacterial agent. Step 2: Add sodium alginate, chitosan, maleic anhydride, and physiological saline to a three-necked flask equipped with a stirrer, thermometer, and gas delivery tube. Purge with nitrogen for protection and stir for 20-30 minutes at a temperature of 20-25℃ and a stirring rate of 200-300 r / min. Then add the supported bacterial agent and continue stirring for 2-3 hours. After the reaction is complete, add the reaction product dropwise to a calcium chloride solution and let it stand for 10-20 hours. Then vacuum filter the mixture and wash the filter cake 3-5 times with distilled water. Finally, freeze dry to obtain a soil conditioner based on bamboo charcoal biochar-supported microorganisms.

[0006] In a preferred embodiment of the present invention, the ratio of bamboo charcoal biochar and mixed bacterial suspension used in step one is 1-2g:10mL.

[0007] In a preferred embodiment of the present invention, the ratio of sodium alginate, chitosan, maleic anhydride, physiological saline and loaded bacterial agent in step two is 1-9g: 2-4g: 0.5-0.9g: 90-100mL: 2g.

[0008] In a preferred embodiment of the present invention, the degree of deacetylation of the chitosan in step two is ≥95%; the mass fraction of the physiological saline is 0.9%; and the mass fraction of the calcium chloride solution is 4%.

[0009] In a preferred embodiment of the present invention, the bamboo charcoal biochar is prepared by the following steps: Step a1: Soak the bamboo powder in deionized water for 2-3 hours to remove floating matter and the clear liquid on the top. Then place it in a vacuum drying oven and dry it at 100-110℃ for 8-10 hours to obtain pretreated bamboo powder. Step a2: Add pretreated bamboo powder, potassium hydroxide, and deionized water to a reaction vessel and perform a hydrothermal reaction at 190-200℃ for 20-30 hours. After the reaction, cool the reaction product to room temperature and then vacuum filter it. Place the filter cake in a vacuum drying oven and dry it at 60-70℃ for 1-3 hours. Then place it in a tube furnace, purge it with nitrogen for protection, and calcine it at 700-800℃ for 2-3 hours at a heating rate of 5-7℃ / min. After that, cool it with the furnace and soak it in hydrochloric acid solution for 10-20 hours. Then vacuum filter it and place the filter cake in a vacuum drying oven and dry it at 60-70℃ for 5-6 hours to obtain bamboo charcoal biochar.

[0010] In a preferred embodiment of the present invention, the ratio of bamboo powder to deionized water in step a1 is 3g:30-40mL.

[0011] In a preferred embodiment of the present invention, the average particle size of the bamboo powder in step a1 is 60 mesh.

[0012] In a preferred embodiment of the present invention, the ratio of the amount of pretreated bamboo powder, potassium hydroxide and deionized water in step a2 is 5g:2-10g:100-110mL.

[0013] In a preferred embodiment of the present invention, the mass fraction of the hydrochloric acid solution in step a2 is 10-15%.

[0014] In a preferred embodiment of the present invention, the mixed bacterial suspension is prepared by the following steps: The mixed bacteria were added to LB medium and cultured at 30°C with shaking at 120 rpm for 96 h. Afterward, the culture was centrifuged at 8000 rpm for 10 min, and the precipitate was resuspended in physiological saline to obtain 10... 9 -10 10 A mixed bacterial suspension of CFU / mL.

[0015] In a preferred embodiment of the present invention, the ratio of the mixed bacteria to LB culture medium is 2g:100mL.

[0016] In a preferred embodiment of the present invention, the mixed bacteria are composed of *Azotobacter chrysogenum*, *Bacillus frostridae*, and *Alcaligenes faecalis* mixed in equal mass; the physiological saline has a mass fraction of 0.9%.

[0017] In a preferred embodiment of the present invention, the azotocin bacterium is designated CGMCC 1.824.

[0018] In a preferred embodiment of the present invention, the Penicillium oxalate strain is designated as CCTCC M2010350.

[0019] In a preferred embodiment of the present invention, the Alcaligenes fecalis is designated as CGMCC 1.767.

[0020] Secondly, this application provides a soil conditioner based on bamboo charcoal biochar immobilized microorganisms, which is prepared using the method described in the first aspect for preparing a soil conditioner based on bamboo charcoal biochar immobilized microorganisms.

[0021] Compared with the prior art, the beneficial effects of the present invention are: This invention relates to a soil conditioner based on bamboo charcoal biochar-supported microorganisms and its preparation method. First, bamboo powder is treated, then carbonized using potassium hydroxide solution, calcined, and acid-washed to obtain bamboo charcoal biochar. Next, microorganisms are loaded onto the bamboo charcoal biochar to obtain a loaded microbial agent. This loaded microbial agent is then coated to obtain a soil conditioner based on bamboo charcoal biochar-supported microorganisms. This preparation method utilizes hydrothermal reaction, potassium hydroxide activation, calcination, and acid washing to significantly increase the porosity and surface functional group density of bamboo charcoal, forming numerous mesopores. The high specific surface area and porous structure provide a sanctuary for microorganisms, facilitating efficient attachment and colonization. Simultaneously, it adsorbs nutrients (nitrogen, phosphorus, potassium), heavy metals, and organic matter from the soil, achieving slow nutrient release and pollution remediation. Finally, multiple microorganisms are combined and loaded, working synergistically to achieve nitrogen fixation. In terms of phosphorus and potassium activation and pollutant degradation, it forms a complementary function, enhances synergy through metabolic mutualism, comprehensively improves soil improvement effect, and enhances soil fertility and health. Subsequently, sodium alginate and chitosan form a composite film to coat its surface under the cross-linking of calcium ions. The presence of the film can form a protective shield, further protecting microorganisms from stresses such as drought, acid and alkali, and low temperature. It can also enable microorganisms to release nutrients slowly and synchronously, forming a stable functional community. Moreover, sodium alginate and chitosan are both natural polysaccharides, which still maintain biocompatibility after grafting modification. The degradation products can serve as carbon sources for microorganisms and crops, promoting the proliferation of microorganisms and the growth of crop roots. After reacting with maleic anhydride, a large number of carboxyl groups are introduced, which can significantly improve its water absorption capacity, providing sufficient water for microbial growth, and achieving a composite effect of "protection-slow release-carbon source supply-water supply". Adding this soil conditioner to contaminated soil can maintain microbial activity for a long time, increase the recycling of nitrogen and phosphorus in the contaminated soil, improve the structure of the contaminated soil, enhance the fertility of the contaminated soil, and have an excellent remediation effect on the contaminated soil, thereby significantly improving crop yield and quality. Detailed Implementation

[0022] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1:

[0023] This embodiment describes a method for preparing a soil conditioner based on bamboo charcoal biochar-supported microorganisms, comprising the following steps: Step S1: Add 3g of bamboo powder with an average particle size of 60 mesh to 30mL of deionized water and soak for 2h to remove floating matter and supernatant. Then place it in a vacuum drying oven and dry at 100℃ for 8h to obtain pretreated bamboo powder. Step S2: Add 5g of pretreated bamboo powder, 2g of potassium hydroxide and 100mL of deionized water to the reaction vessel and perform hydrothermal reaction at 190℃ for 20h. After the reaction is completed, cool the reaction product to room temperature and then vacuum filter it. Place the filter cake in a vacuum drying oven and dry it at 60℃ for 1h. Then place it in a tube furnace, purge it with nitrogen for protection, and calcine it at 700℃ for 2h at a heating rate of 5℃ / min. After that, cool it with the furnace and soak it in a 10% hydrochloric acid solution for 10h. Then vacuum filter it and place the filter cake in a vacuum drying oven and dry it at 60℃ for 5h to obtain bamboo charcoal biochar. Step S3: Add 2g of *Azotobacter chrysogenum*, *Bruchwegium frutescens*, and *Alcaligenes faecalis* (mixed in equal weights) to 100mL of LB medium. Incubate at 30℃ and 120r / min for 96h. Then centrifuge at 8000r / min for 10min. Resuspend the precipitate in 0.9% physiological saline to obtain 10... 9 A mixed bacterial suspension of CFU / mL; the azotobacter brownii is designated CGMCC 1.824; the Penicillium oxalate is designated CCTCC M2010350; and the Alcaligenes faecalis is designated CGMCC 1.767. Step S4: Add 1g of bamboo charcoal biochar to 10mL of mixed bacterial suspension and culture for 24h at 30℃ and 120r / min. Then, vacuum filter the mixture, wash the filter cake three times with distilled water, and freeze dry to obtain the loaded bacterial agent. Step S5: Add 1g sodium alginate, 2g chitosan with a deacetylation degree ≥95%, 0.5g maleic anhydride, and 90mL of 0.9% physiological saline to a three-necked flask equipped with a stirrer, thermometer, and gas delivery tube. Purge with nitrogen for protection and stir for 20min at 20℃ and 200r / min. Then add 2g of loaded bacterial agent and continue stirring for 2h. After the reaction is complete, add the reaction product dropwise to a 4% calcium chloride solution and let stand for 10h. Then vacuum filter, wash the filter cake three times with distilled water, and freeze dry to obtain a soil conditioner based on bamboo charcoal biochar-supported microorganisms. Example 2:

[0024] This embodiment describes a method for preparing a soil conditioner based on bamboo charcoal biochar-supported microorganisms, comprising the following steps: Step S1: Add 3g of bamboo powder with an average particle size of 60 mesh to 35mL of deionized water and soak for 2.5h to remove floating matter and supernatant. Then place it in a vacuum drying oven and dry it at 105℃ for 9h to obtain pretreated bamboo powder. Step S2: Add 5g of pretreated bamboo powder, 6g of potassium hydroxide and 105mL of deionized water to the reaction vessel and hydrothermally react at 195℃ for 25h. After the reaction is completed, cool the reaction product to room temperature and then vacuum filter it. Place the filter cake in a vacuum drying oven and dry it at 65℃ for 2h. Then place it in a tube furnace, purge it with nitrogen for protection, and calcine it at 750℃ for 2.5h at a heating rate of 6℃ / min. After that, cool it with the furnace and soak it in a 12% hydrochloric acid solution for 15h. Then vacuum filter it and place the filter cake in a vacuum drying oven and dry it at 65℃ for 5.5h to obtain bamboo charcoal biochar. Step S3: Add 2g of *Azotobacter chrysogenum*, *Bruchwegium frutescens*, and *Alcaligenes faecalis* (mixed in equal weights) to 100mL of LB medium. Incubate at 30℃ and 120r / min for 96h. Then centrifuge at 8000r / min for 10min. Resuspend the precipitate in 0.9% physiological saline to obtain 10... 9 A mixed bacterial suspension of CFU / mL; the azotobacter brownii is designated CGMCC 1.824; the Penicillium oxalate is designated CCTCC M2010350; and the Alcaligenes faecalis is designated CGMCC 1.767. Step S4: Add 1.5g of bamboo charcoal biochar to 10mL of mixed bacterial suspension, and culture for 36h at 30℃ and 120r / min. Then, vacuum filter, wash the filter cake 4 times with distilled water, and freeze dry to obtain the loaded bacterial agent. Step S5: Add 5g sodium alginate, 3g chitosan with a deacetylation degree ≥95%, 0.7g maleic anhydride, and 95mL of 0.9% physiological saline to a three-necked flask equipped with a stirrer, thermometer, and gas delivery tube. Purge with nitrogen for protection and stir at 22℃ and 250r / min for 25min. Then add 2g of loaded bacterial agent and continue stirring for 2.5h. After the reaction is complete, add the reaction product dropwise to a 4% calcium chloride solution and let stand for 15h. Then vacuum filter, wash the filter cake four times with distilled water, and freeze dry to obtain a soil conditioner based on bamboo charcoal biochar immobilized microorganisms. Example 3:

[0025] This embodiment describes a method for preparing a soil conditioner based on bamboo charcoal biochar-supported microorganisms, comprising the following steps: Step S1: Add 3g of bamboo powder with an average particle size of 60 mesh to 40mL of deionized water and soak for 3h to remove floating matter and supernatant. Then place it in a vacuum drying oven and dry at 110℃ for 10h to obtain pretreated bamboo powder. Step S2: Add 5g of pretreated bamboo powder, 10g of potassium hydroxide and 110mL of deionized water to the reaction vessel and perform hydrothermal reaction at 200℃ for 30h. After the reaction is completed, cool the reaction product to room temperature and then vacuum filter it. Place the filter cake in a vacuum drying oven and dry it at 70℃ for 3h. Then place it in a tube furnace, purge it with nitrogen for protection, and calcine it at 800℃ for 3h at a heating rate of 7℃ / min. After that, cool it with the furnace and soak it in a 15% hydrochloric acid solution for 20h. Then vacuum filter it and place the filter cake in a vacuum drying oven and dry it at 70℃ for 6h to obtain bamboo charcoal biochar. Step S3: Add 2g of *Azotobacter chrysogenum*, *Bruchwegium frutescens*, and *Alcaligenes faecalis* (mixed in equal weights) to 100mL of LB medium. Incubate at 30℃ and 120r / min for 96h. Then centrifuge at 8000r / min for 10min. Resuspend the precipitate in 0.9% physiological saline to obtain 10... 10 A mixed bacterial suspension of CFU / mL; the azotobacter brownii is designated CGMCC 1.824; the Penicillium oxalate is designated CCTCC M2010350; and the Alcaligenes faecalis is designated CGMCC 1.767. Step S4: Add 2g of bamboo charcoal biochar to 10mL of mixed bacterial suspension and culture at 30℃ and 120r / min for 48h. Then, vacuum filter the mixture, wash the filter cake 5 times with distilled water, and freeze dry to obtain the loaded bacterial agent. Step S5: Add 9g sodium alginate, 4g chitosan with a deacetylation degree ≥95%, 0.9g maleic anhydride, and 100mL of 0.9% physiological saline to a three-necked flask equipped with a stirrer, thermometer, and gas delivery tube. Purge with nitrogen for protection and stir at 25℃ and 300r / min for 30min. Then add 2g of loaded bacterial agent and continue stirring for 3h. After the reaction is complete, add the reaction product dropwise to a 4% calcium chloride solution and let stand for 20h. Then vacuum filter, wash the filter cake 5 times with distilled water, and freeze dry to obtain a soil conditioner based on bamboo charcoal biochar immobilized microorganisms.

[0026] Comparative Example 1: This comparative example illustrates a method for preparing a soil conditioner based on bamboo charcoal biochar-supported microorganisms, comprising the following steps: Step S1: Add 3g of bamboo powder with an average particle size of 60 mesh to 40mL of deionized water and soak for 3h to remove floating matter and supernatant. Then place it in a vacuum drying oven and dry at 110℃ for 10h to obtain pretreated bamboo powder. Step S2: Add 5g of pretreated bamboo powder, 10g of potassium hydroxide and 110mL of deionized water to the reaction vessel and perform hydrothermal reaction at 200℃ for 30h. After the reaction is completed, cool the reaction product to room temperature and then vacuum filter it. Place the filter cake in a vacuum drying oven and dry it at 70℃ for 3h. Then place it in a tube furnace, purge it with nitrogen for protection, and calcine it at 800℃ for 3h at a heating rate of 7℃ / min. After that, cool it with the furnace and soak it in a 15% hydrochloric acid solution for 20h. Then vacuum filter it and place the filter cake in a vacuum drying oven and dry it at 70℃ for 6h to obtain bamboo charcoal biochar. Step S3: Add 2g of *Azotocinobacter chrysogenum* to 100mL of LB medium and incubate at 30℃ with shaking at 120r / min for 96h. Then centrifuge at 8000r / min for 10min. Resuspend the precipitate in 0.9% physiological saline to obtain 10... 10 A suspension of *Azotobacter chrysogenum* at CFU / mL; the *Azotobacter chrysogenum* strain numbered CGMCC 1.824; the *Penicillium oxalate* strain numbered CCTCC M2010350; and the *Alcaligenes faecalis* strain numbered CGMCC 1.767. Step S4: Add 2g of bamboo charcoal biochar to 10mL of azotocinus brownii suspension and culture for 48h at 30℃ and 120r / min. Then, vacuum filter the mixture, wash the filter cake 5 times with distilled water, and freeze dry to obtain a soil conditioner based on bamboo charcoal biochar immobilized microorganisms.

[0027] Comparative Example 2: This comparative example illustrates a method for preparing a soil conditioner based on bamboo charcoal biochar-supported microorganisms, comprising the following steps: Step S1: Add 3g of bamboo powder with an average particle size of 60 mesh to 40mL of deionized water and soak for 3h to remove floating matter and supernatant. Then place it in a vacuum drying oven and dry at 110℃ for 10h to obtain pretreated bamboo powder. Step S2: Add 5g of pretreated bamboo powder, 10g of potassium hydroxide and 110mL of deionized water to the reaction vessel and perform hydrothermal reaction at 200℃ for 30h. After the reaction is completed, cool the reaction product to room temperature and then vacuum filter it. Place the filter cake in a vacuum drying oven and dry it at 70℃ for 3h. Then place it in a tube furnace, purge it with nitrogen for protection, and calcine it at 800℃ for 3h at a heating rate of 7℃ / min. After that, cool it with the furnace and soak it in a 15% hydrochloric acid solution for 20h. Then vacuum filter it and place the filter cake in a vacuum drying oven and dry it at 70℃ for 6h to obtain bamboo charcoal biochar. Step S3: Add 2g of *Azotobacter chrysogenum* and *Brachythromyces cerevisiae* (mixed in equal weight) to 100mL of LB medium and incubate at 30℃ with shaking at 120r / min for 96h. Then centrifuge at 8000r / min for 10min. Resuspend the precipitate in 0.9% physiological saline to obtain 10... 10 A mixed bacterial suspension of CFU / mL; the azotobacter brownii is designated CGMCC 1.824; the Penicillium oxalate is designated CCTCCM2010350; and the Alcaligenes faecalis is designated CGMCC 1.767. Step S4: Add 2g of bamboo charcoal biochar to 10mL of mixed bacterial suspension and culture at 30℃ and 120r / min for 48h. Then, vacuum filter the mixture, wash the filter cake 5 times with distilled water, and freeze dry to obtain a soil conditioner based on bamboo charcoal biochar immobilized microorganisms.

[0028] Comparative Example 3: This comparative example illustrates a method for preparing a soil conditioner based on bamboo charcoal biochar-supported microorganisms, comprising the following steps: Step S1: Add 3g of bamboo powder with an average particle size of 60 mesh to 40mL of deionized water and soak for 3h to remove floating matter and supernatant. Then place it in a vacuum drying oven and dry at 110℃ for 10h to obtain pretreated bamboo powder. Step S2: Add 5g of pretreated bamboo powder, 10g of potassium hydroxide and 110mL of deionized water to the reaction vessel and perform hydrothermal reaction at 200℃ for 30h. After the reaction is completed, cool the reaction product to room temperature and then vacuum filter it. Place the filter cake in a vacuum drying oven and dry it at 70℃ for 3h. Then place it in a tube furnace, purge it with nitrogen for protection, and calcine it at 800℃ for 3h at a heating rate of 7℃ / min. After that, cool it with the furnace and soak it in a 15% hydrochloric acid solution for 20h. Then vacuum filter it and place the filter cake in a vacuum drying oven and dry it at 70℃ for 6h to obtain bamboo charcoal biochar. Step S3: Add 2g of *Azotobacter chrysogenum* and *Alcaligenes faecalis* (mixed in equal weight) to 100mL of LB medium. Incubate at 30℃ and 120r / min for 96h. Then centrifuge at 8000r / min for 10min. Resuspend the precipitate in 0.9% physiological saline to obtain 10... 10 A mixed bacterial suspension of CFU / mL; the azotobacter brownii is designated CGMCC 1.824; the Penicillium oxalate is designated CCTCCM2010350; and the Alcaligenes faecalis is designated CGMCC 1.767. Step S4: Add 2g of bamboo charcoal biochar to 10mL of mixed bacterial suspension and culture at 30℃ and 120r / min for 48h. Then, vacuum filter the mixture, wash the filter cake 5 times with distilled water, and freeze dry to obtain a soil conditioner based on bamboo charcoal biochar immobilized microorganisms.

[0029] Comparative Example 4: This comparative example illustrates a method for preparing a soil conditioner based on bamboo charcoal biochar-supported microorganisms, comprising the following steps: Step S1: Add 3g of bamboo powder with an average particle size of 60 mesh to 40mL of deionized water and soak for 3h to remove floating matter and supernatant. Then place it in a vacuum drying oven and dry at 110℃ for 10h to obtain pretreated bamboo powder. Step S2: Add 5g of pretreated bamboo powder, 10g of potassium hydroxide and 110mL of deionized water to the reaction vessel and perform hydrothermal reaction at 200℃ for 30h. After the reaction is completed, cool the reaction product to room temperature and then vacuum filter it. Place the filter cake in a vacuum drying oven and dry it at 70℃ for 3h. Then place it in a tube furnace, purge it with nitrogen for protection, and calcine it at 800℃ for 3h at a heating rate of 7℃ / min. After that, cool it with the furnace and soak it in a 15% hydrochloric acid solution for 20h. Then vacuum filter it and place the filter cake in a vacuum drying oven and dry it at 70℃ for 6h to obtain bamboo charcoal biochar. Step S3: Add 2g of *Azotobacter chrysogenum*, *Bruchwegium frutescens*, and *Alcaligenes faecalis* (mixed in equal weights) to 100mL of LB medium. Incubate at 30℃ and 120r / min for 96h. Then centrifuge at 8000r / min for 10min. Resuspend the precipitate in 0.9% physiological saline to obtain 10... 10 A mixed bacterial suspension of CFU / mL; the azotobacter brownii is designated CGMCC 1.824; the Penicillium oxalate is designated CCTCC M2010350; and the Alcaligenes faecalis is designated CGMCC 1.767. Step S4: Add 2g of bamboo charcoal biochar to 10mL of mixed bacterial suspension and culture at 30℃ and 120r / min for 48h. Then, vacuum filter the mixture, wash the filter cake 5 times with distilled water, and freeze dry to obtain a soil conditioner based on bamboo charcoal biochar immobilized microorganisms.

[0030] Comparative Example 5: This comparative example illustrates a method for preparing a soil conditioner based on bamboo charcoal biochar-supported microorganisms, comprising the following steps: Step S1: Add 3g of bamboo powder with an average particle size of 60 mesh to 40mL of deionized water and soak for 3h to remove floating matter and supernatant. Then place it in a vacuum drying oven and dry at 110℃ for 10h to obtain pretreated bamboo powder. Step S2: Add 5g of pretreated bamboo powder, 10g of potassium hydroxide and 110mL of deionized water to the reaction vessel and perform hydrothermal reaction at 200℃ for 30h. After the reaction is completed, cool the reaction product to room temperature and then vacuum filter it. Place the filter cake in a vacuum drying oven and dry it at 70℃ for 3h. Then place it in a tube furnace, purge it with nitrogen for protection, and calcine it at 800℃ for 3h at a heating rate of 7℃ / min. After that, cool it with the furnace and soak it in a 15% hydrochloric acid solution for 20h. Then vacuum filter it and place the filter cake in a vacuum drying oven and dry it at 70℃ for 6h to obtain bamboo charcoal biochar. Step S3: Add 2g of *Azotobacter chrysogenum*, *Bruchwegium frutescens*, and *Alcaligenes faecalis* (mixed in equal weights) to 100mL of LB medium. Incubate at 30℃ and 120r / min for 96h. Then centrifuge at 8000r / min for 10min. Resuspend the precipitate in 0.9% physiological saline to obtain 10... 10 A mixed bacterial suspension of CFU / mL; the azotobacter brownii is designated CGMCC 1.824; the Penicillium oxalate is designated CCTCC M2010350; and the Alcaligenes faecalis is designated CGMCC 1.767. Step S4: Add 2g of bamboo charcoal biochar to 10mL of mixed bacterial suspension and culture at 30℃ and 120r / min for 48h. Then, vacuum filter the mixture, wash the filter cake 5 times with distilled water, and freeze dry to obtain the loaded bacterial agent. Step S5: Add 9g of sodium alginate and 100mL of 0.9% physiological saline to a three-necked flask equipped with a stirrer, thermometer, and gas delivery tube. Purge with nitrogen for protection and stir for 30min at 25℃ and 300r / min. Then add 2g of loaded bacterial agent and continue stirring for 3h. After the reaction is complete, add the reaction product dropwise to a 4% calcium chloride solution and let stand for 20h. Then vacuum filter, wash the filter cake 5 times with distilled water, and freeze dry to obtain a soil conditioner based on bamboo charcoal biochar immobilized microorganisms.

[0031] 5g of crude oil was added to 100g of dried and sterilized soil. Then, 1g of the soil conditioner based on bamboo charcoal biochar-supported microorganisms from Examples 1-3 and Comparative Examples 1-5 was added to the dried and sterilized soil. The petroleum degradation rate η was then tested and calculated according to the formula η=(Md-Ms) / Mc×100%; where Md is the residual crude oil mass of the blank control example (5g of crude oil added to 100g of dried and sterilized soil); Mc is the initial crude oil mass; unit, g.

[0032] The test results are shown in the table below:

[0033] Referring to the data in the table above, and based on the comparison between Examples 1-3 and Comparative Examples 1-5, it can be seen that the soil conditioner based on bamboo charcoal biochar-supported microorganisms of this application has a high petroleum degradation rate, indicating excellent soil remediation effect.

[0034] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0035] The above description is merely an example and illustration of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described or use similar methods to replace them, as long as they do not deviate from the invention or exceed the scope defined in this application, they should all fall within the protection scope of the present invention.

Claims

1. A method for preparing a soil conditioner based on bamboo charcoal biochar-supported microorganisms, characterized in that, Includes the following steps: Step 1: Add bamboo charcoal biochar to the mixed bacterial suspension and culture at 30℃ and 120r / min for 24-48h. Then, vacuum filter the mixture, wash the filter cake with distilled water 3-5 times, and freeze dry to obtain the loaded bacterial agent. Step 2: Add sodium alginate, chitosan, maleic anhydride, and physiological saline to a three-necked flask equipped with a stirrer, thermometer, and gas delivery tube. Purge with nitrogen for protection and stir for 20-30 minutes at a temperature of 20-25℃ and a stirring rate of 200-300 r / min. Then add the supported bacterial agent and continue stirring for 2-3 hours. After the reaction is complete, add the reaction product dropwise to a calcium chloride solution and let it stand for 10-20 hours. Then vacuum filter the mixture and wash the filter cake 3-5 times with distilled water. Finally, freeze dry to obtain a soil conditioner based on bamboo charcoal biochar-supported microorganisms.

2. The method for preparing a soil conditioner based on bamboo charcoal biochar-supported microorganisms according to claim 1, characterized in that, The ratio of bamboo charcoal biochar and mixed bacterial suspension used in step one is 1-2g:10mL.

3. The method for preparing a soil conditioner based on bamboo charcoal biochar-supported microorganisms according to claim 1, characterized in that, In step two, the ratio of sodium alginate, chitosan, maleic anhydride, physiological saline, and loaded bacterial agent is 1-9g: 2-4g: 0.5-0.9g: 90-100mL: 2g.

4. The method for preparing a soil conditioner based on bamboo charcoal biochar-supported microorganisms according to claim 1, characterized in that, In step two, the degree of deacetylation of the chitosan is ≥95%; the mass fraction of the physiological saline is 0.9%; and the mass fraction of the calcium chloride solution is 4%.

5. The method for preparing a soil conditioner based on bamboo charcoal biochar-supported microorganisms according to claim 1, characterized in that, The bamboo charcoal biochar is prepared by the following steps: Step a1: Soak the bamboo powder in deionized water for 2-3 hours to remove floating matter and the clear liquid on the top. Then place it in a vacuum drying oven and dry it at 100-110℃ for 8-10 hours to obtain pretreated bamboo powder. Step a2: Add pretreated bamboo powder, potassium hydroxide, and deionized water to a reaction vessel and perform a hydrothermal reaction at 190-200℃ for 20-30 hours. After the reaction, cool the reaction product to room temperature and then vacuum filter it. Place the filter cake in a vacuum drying oven and dry it at 60-70℃ for 1-3 hours. Then place it in a tube furnace, purge it with nitrogen for protection, and calcine it at 700-800℃ for 2-3 hours at a heating rate of 5-7℃ / min. After that, cool it with the furnace and soak it in hydrochloric acid solution for 10-20 hours. Then vacuum filter it and place the filter cake in a vacuum drying oven and dry it at 60-70℃ for 5-6 hours to obtain bamboo charcoal biochar.

6. The method for preparing a soil conditioner based on bamboo charcoal biochar-supported microorganisms according to claim 5, characterized in that, The ratio of bamboo powder to deionized water in step a1 is 3g:30-40mL; the average particle size of the bamboo powder is 60 mesh.

7. The method for preparing a soil conditioner based on bamboo charcoal biochar-supported microorganisms according to claim 5, characterized in that, In step a2, the ratio of the pretreated bamboo powder, potassium hydroxide, and deionized water is 5g:2-10g:100-110mL; the mass fraction of the hydrochloric acid solution is 10-15%.

8. The method for preparing a soil conditioner based on bamboo charcoal biochar-supported microorganisms according to claim 1, characterized in that, The mixed bacterial suspension was prepared by the following steps: The mixed bacteria were added to LB medium and cultured at 30°C with shaking at 120 rpm for 96 h. Afterward, the culture was centrifuged at 8000 rpm for 10 min, and the precipitate was resuspended in physiological saline to obtain 10... 9 -10 10 A mixed bacterial suspension of CFU / mL.

9. The method for preparing a soil conditioner based on bamboo charcoal biochar-supported microorganisms according to claim 8, characterized in that, The ratio of the mixed bacteria to LB medium is 2g:100mL; The mixed bacteria consist of equal masses of *Azotobacter chrysogenum*, *Bacillus frostridae*, and *Alcaligenes faecalis*; the physiological saline solution has a mass fraction of 0.9%.

10. A soil conditioner based on bamboo charcoal biochar immobilized microorganisms, characterized in that, The soil conditioner was prepared using the method described in any one of claims 1-9, which is based on bamboo charcoal biochar-supported microorganisms.

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