Sugarcane leaf in-situ decomposition microbial agent and preparation method thereof

Abstract

The invention discloses a sugarcane leaf in-situ decomposition microbial agent, which is prepared from the following active ingredients in parts by weight: 25 to 35 parts of bacillus subtilis powder, 10 to 20 parts of aspergillus niger powder, 15 to 25 parts of trichoderma reesei powder and 5 to 15 parts of pseudomonas fluorescens powder; the microbial agent further comprises 40-50 parts of a modified carrier agent and 5-10 parts of an additive agent. According to the decomposition microbial agent, lignin peroxidase and manganese peroxidase secreted by trichoderma reesei in the lignin and cellulose synchronous decomposition microbial agent are adopted, the lignin phenylpropane structure can be destroyed, and the lignin peroxidase and the manganese peroxidase form a synergistic effect with cellulase generated by aspergillus niger.

Description

Technical Field

[0001] This invention relates to the field of microbial inoculants, specifically to a microbial inoculant for the in-situ decomposition of sugarcane leaves and its preparation method. Background Technology

[0002] Guangxi is my country's largest sugarcane production base. Sugarcane leaves are rich in nutrients such as nitrogen, phosphorus, and potassium. Rational utilization can improve soil fertility and reduce the use of chemical fertilizers. However, leaving large amounts of sugarcane leaves in the field hinders the growth of ratooned sugarcane plants, increases the risk of pests and diseases, and leads to higher production costs, creating a dual contradiction between ecology and production. Existing methods for handling sugarcane leaves have significant limitations: combined harvesting and crushing for returning to the field only covers 3% of the planting area, and centralized bundling and recycling for power generation or feed requires high labor and transportation costs, making large-scale promotion difficult. In-situ decomposition, as the optimal technical path, relies heavily on the degradation efficiency of microbial agents. However, existing agents generally suffer from the following problems: limited degradation capabilities: most agents only target cellulose or hemicellulose, with weak lignin degradation capabilities, resulting in long decomposition cycles and low efficiency for sugarcane leaves (especially hard parts like sugarcane stalks and stem nodes); poor environmental adaptability: the soil in Guangxi's sugarcane-growing areas is mostly acidic, and the activity of existing agents is easily inhibited in low pH environments, making it difficult to maintain stable performance. Therefore, this invention provides a microbial agent for in-situ decomposition of sugarcane leaves and its preparation method. Summary of the Invention

[0003] In view of the deficiencies of the prior art, the purpose of this invention is to provide a sugarcane leaf in-situ decomposition microbial agent and its preparation method, so as to solve the problems mentioned in the background art.

[0004] The present invention solves the technical problem by adopting the following technical solution:

[0005] This invention provides a microbial inoculant for in-situ decomposition of sugarcane leaves. By weight, the active ingredients include: 25-35 parts of Bacillus subtilis powder, 10-20 parts of Aspergillus niger powder, 15-25 parts of Trichoderma reesei powder, and 5-15 parts of Pseudomonas fluorescens powder; the inoculant also includes 40-50 parts of a modified carrier agent and 5-10 parts of an additive.

[0006] Preferably, the viable counts of Bacillus subtilis, Aspergillus niger, Trichoderma reesei, and Pseudomonas fluorescens are not less than [amount not specified]. , , , .

[0007] Preferably, the modified carrier agent is prepared by:

[0008] S01: Preheat carbon nanotubes at 55-60℃ for 1 hour to obtain preheated carbon nanotubes. Mix the preheated carbon nanotubes and the modification liquid at a weight ratio of 5:(11-14) and stir to modify the mixture. After stirring, the modified carbon nanotube liquid is obtained.

[0009] Preparation of the modified liquid:

[0010] S01a: Stir montmorillonite in a sodium lignosulfonate solution that is 3-5 times the total volume of montmorillonite to obtain montmorillonite solution;

[0011] S01b: Add 3-5 parts silicon carbide and 2-5 parts titanium dioxide to 10-15 parts of sodium citrate solution with a mass fraction of 8%, then add 1-2 parts of silane coupling agent KH550 and stir evenly to obtain the modifier; finally, stir the montmorillonite solution and the modifier at a weight ratio of (7-11):5 to obtain the modified solution.

[0012] S02: Modified carbon nanotube liquid and carrier material are ball-milled at a weight ratio of (7-9):4, with a ball milling speed of 1000-1500 r / min for 2 hours. After ball milling, the mixture is filtered and dried to obtain the modified carrier agent.

[0013] Preferably, the stirring speed for the stirring modification treatment is 350-450 r / min, and the stirring time is 1 h; the mass fraction of the sodium lignosulfonate solution is 10-15%.

[0014] Preferably, the method for preparing the carrier material is as follows:

[0015] S02a: β-cyclodextrin, sodium silicate solution and zeolite powder are mixed evenly in a weight ratio of (3-5):7:(2-3) to obtain β-cyclodextrin solution;

[0016] 4-7 parts of hydroxyapatite, 2-5 parts of lanthanum oxide and 3-5 parts of β-cyclodextrin were thoroughly mixed to obtain hydroxyapatite-β-cyclodextrin agent;

[0017] SO2b: 5-8 parts of hydroxyapatite-β-cyclodextrin, 3-5 parts of boron nitride and 2-4 parts of calcium sulfate whiskers were mixed and ball-milled at a speed of 1000-1500 r / min for 2 h. After ball milling, the mixture was filtered and dried to obtain the carrier material.

[0018] Preferably, the sodium silicate solution has a mass fraction of 5-8%.

[0019] Preferably, the additive is prepared by mixing 3-5 parts of EDTA-chelated calcium, 3-5 parts of amino acids, 4-7 parts of trace elements, 3-5 parts of wood ash, and 10-15 parts of a 35-40% sodium humate aqueous solution thoroughly to obtain the additive.

[0020] Preferably, the trace element is one of zinc, boron, manganese, and iron.

[0021] This invention also provides a method for preparing an in-situ decomposing microbial agent for sugarcane leaves, characterized by comprising the following steps:

[0022] Step 1: Inoculate Bacillus subtilis, Aspergillus niger, Trichoderma reesei, and Pseudomonas fluorescens into their respective seed culture media and culture at 30-32℃ with shaking for 18-24 hours to obtain seed culture. Inoculate each seed culture into fermentation medium and ferment at 30-35℃ with an aeration rate of 0.8-1.2 vvm for 48-72 hours to obtain the fermentation broth of each single strain.

[0023] Step 2: Concentrate each single-strain fermentation broth through plate and frame filtration, mix it with the composite carrier and additive in a mass ratio of 1:3:1, dry it at a low temperature of 50-55℃ until the moisture content is ≤10%, pulverize it through a 100-mesh sieve, and obtain the microbial inoculant.

[0024] Preferably, in step one, the Bacillus subtilis fermentation medium is: glucose 15-20 g / L, peptone 10-15 g / L, yeast extract 3-5 g / L, NaCl 4-5 g / L, pH 7.0-7.2;

[0025] The fermentation medium for Aspergillus niger and Trichoderma reesei consisted of: wheat bran juice 30-35 g / L, sucrose 20-25 g / L, and ammonium nitrate 3-5 g / L. pH 5.5-6.0;

[0026] The fermentation medium for *Pseudomonas fluorescens* was: 10-15 g / L beef, 10-15 g / L peptone, 3-5 g / L NaCl, pH 7.0-7.5.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] This invention utilizes lignin peroxidase and manganese peroxidase secreted by *Trichoderma reesei* in a lignin-cellulose synchronous decomposing agent. These enzymes can destroy the phenylpropane structure of lignin and form a synergistic effect with cellulase produced by *Aspergillus niger*. A complex enzyme system enhances the decomposition of β-glucanase secreted by *Bacillus subtilis* and laccase produced by *Pseudomonas fluorescens*, which can decompose hemicellulose into monosaccharides such as xylose. The carrier material, modified with a pH buffering mechanism, uses montmorillonite, which, after modification with sodium lignin sulfonate, allows for the exchange of cations between its layers (such as...). , The release of microbial agents is achieved by maintaining their stability at pH 4.5-5.5. The modified carrier's carbon nanotube-montmorillonite composite structure boasts excellent specific surface area and adsorption capacity, effectively immobilizing the microorganisms and delaying release. β-cyclodextrin encapsulation technology loads hydroxyapatite and lanthanum oxide into the cyclodextrin cavity, forming a pH-responsive sustained-release system. The release period of trace elements (Zn, B, etc.) is prolonged. The sodium humate additive regulates the release by chelating aluminum ions (…). ) and regulate soil redox potential, promote lignin-degrading enzyme gene expression, enhance enzyme activity, EDTA-chelated calcium enhancement mechanism: calcium ions ( It binds to the cell wall of microorganisms, enhancing the cell's resistance to osmotic pressure. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to specific examples. 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.

[0030] This embodiment of a sugarcane leaf in-situ decomposition microbial agent comprises, by weight, the active ingredients as follows: 25-35 parts of Bacillus subtilis powder, 10-20 parts of Aspergillus niger powder, 15-25 parts of Trichoderma reesei powder, and 5-15 parts of Pseudomonas fluorescens powder; the agent also includes 40-50 parts of modified carrier agent and 5-10 parts of additive agent.

[0031] In this embodiment, the viable counts of Bacillus subtilis, Aspergillus niger, Trichoderma reesei, and Pseudomonas fluorescens are respectively no less than , , , .

[0032] The preparation method of the modified carrier agent in this embodiment is as follows:

[0033] S01: Preheat carbon nanotubes at 55-60℃ for 1 hour to obtain preheated carbon nanotubes. Mix the preheated carbon nanotubes and the modification liquid at a weight ratio of 5:(11-14) and stir to modify the mixture. After stirring, the modified carbon nanotube liquid is obtained.

[0034] Preparation of the modified liquid:

[0035] S01a: Stir montmorillonite in a sodium lignosulfonate solution that is 3-5 times the total volume of montmorillonite to obtain montmorillonite solution;

[0036] S01b: Add 3-5 parts silicon carbide and 2-5 parts titanium dioxide to 10-15 parts of sodium citrate solution with a mass fraction of 8%, then add 1-2 parts of silane coupling agent KH550 and stir evenly to obtain the modifier; finally, stir the montmorillonite solution and the modifier at a weight ratio of (7-11):5 to obtain the modified solution.

[0037] S02: Modified carbon nanotube liquid and carrier material are ball-milled at a weight ratio of (7-9):4, with a ball milling speed of 1000-1500 r / min for 2 hours. After ball milling, the mixture is filtered and dried to obtain the modified carrier agent.

[0038] In this embodiment, the stirring speed for the stirring modification treatment is 350-450 r / min, and the stirring time is 1 h; the mass fraction of the sodium lignosulfonate solution is 10-15%.

[0039] The method for preparing the carrier material in this embodiment is as follows:

[0040] S02a: β-cyclodextrin, sodium silicate solution and zeolite powder are mixed evenly in a weight ratio of (3-5):7:(2-3) to obtain β-cyclodextrin solution;

[0041] 4-7 parts of hydroxyapatite, 2-5 parts of lanthanum oxide and 3-5 parts of β-cyclodextrin were thoroughly mixed to obtain hydroxyapatite-β-cyclodextrin agent;

[0042] SO2b: 5-8 parts of hydroxyapatite-β-cyclodextrin, 3-5 parts of boron nitride and 2-4 parts of calcium sulfate whiskers were mixed and ball-milled at a speed of 1000-1500 r / min for 2 h. After ball milling, the mixture was filtered and dried to obtain the carrier material.

[0043] The sodium silicate solution in this embodiment has a mass fraction of 5-8%.

[0044] The preparation method of the additive in this embodiment is as follows: 3-5 parts of EDTA-chelated calcium, 3-5 parts of amino acids, 4-7 parts of trace elements, 3-5 parts of wood ash, and 10-15 parts of sodium humate aqueous solution with a mass fraction of 35-40% are thoroughly mixed to obtain the additive.

[0045] The trace element in this embodiment is one of zinc, boron, manganese, and iron.

[0046] This invention also provides a method for preparing an in-situ decomposing microbial agent for sugarcane leaves, characterized by comprising the following steps:

[0047] Step 1: Inoculate Bacillus subtilis, Aspergillus niger, Trichoderma reesei, and Pseudomonas fluorescens into their respective seed culture media and culture at 30-32℃ with shaking for 18-24 hours to obtain seed culture. Inoculate each seed culture into fermentation medium and ferment at 30-35℃ with an aeration rate of 0.8-1.2 vvm for 48-72 hours to obtain the fermentation broth of each single strain.

[0048] Step 2: Concentrate each single-strain fermentation broth through plate and frame filtration, mix it with the composite carrier and additive in a mass ratio of 1:3:1, dry it at a low temperature of 50-55℃ until the moisture content is ≤10%, pulverize it through a 100-mesh sieve, and obtain the microbial inoculant.

[0049] In step one of this embodiment, the Bacillus subtilis fermentation medium is: glucose 15-20 g / L, peptone 10-15 g / L, yeast powder 3-5 g / L, NaCl 4-5 g / L, pH 7.0-7.2;

[0050] The fermentation medium for Aspergillus niger and Trichoderma reesei consisted of: wheat bran juice 30-35 g / L, sucrose 20-25 g / L, and ammonium nitrate 3-5 g / L. pH 5.5-6.0;

[0051] The fermentation medium for *Pseudomonas fluorescens* was: 10-15 g / L beef, 10-15 g / L peptone, 3-5 g / L NaCl, pH 7.0-7.5.

[0052] Example 1.

[0053] This embodiment of a sugarcane leaf in-situ decomposition microbial agent comprises, by weight, 25 parts of Bacillus subtilis powder, 10 parts of Aspergillus niger powder, 15 parts of Trichoderma reesei powder, and 5 parts of Pseudomonas fluorescens powder; the agent also includes 40 parts of modified carrier agent and 5 parts of additive agent.

[0054] In this embodiment, the viable counts of Bacillus subtilis, Aspergillus niger, Trichoderma reesei, and Pseudomonas fluorescens are respectively no less than , , , .

[0055] The preparation method of the modified carrier agent in this embodiment is as follows:

[0056] S01: Preheat carbon nanotubes at 55℃ for 1 hour to obtain preheated carbon nanotubes. Mix the preheated carbon nanotubes and the modification liquid at a weight ratio of 5:11 and stir to modify them. After stirring, the modified carbon nanotube liquid is obtained.

[0057] Preparation of the modified liquid:

[0058] S01a: Stir montmorillonite in a sodium lignosulfonate solution with a volume of 3 times that of montmorillonite to obtain montmorillonite solution;

[0059] S01b: Add 3 parts silicon carbide and 2 parts titanium dioxide to 10 parts sodium citrate solution with a mass fraction of 8%, then add 1 part silane coupling agent KH550 and stir evenly to obtain the modifier; finally, stir the montmorillonite solution and the modifier at a weight ratio of 7:5 to obtain the modified solution.

[0060] S02: The modified carbon nanotube liquid and the carrier material were ball-milled at a weight ratio of 7:4 at a speed of 1000 r / min for 2 hours. After the ball milling was completed, the mixture was filtered and dried to obtain the modified carrier agent.

[0061] In this embodiment, the stirring speed for the stirring modification treatment was 350 r / min, and the stirring time was 1 h; the mass fraction of the sodium lignosulfonate solution was 10%.

[0062] The method for preparing the carrier material in this embodiment is as follows:

[0063] S02a: β-cyclodextrin, sodium silicate solution and zeolite powder are mixed evenly in a weight ratio of 3:7:2 to obtain β-cyclodextrin solution;

[0064] 4 parts hydroxyapatite, 2 parts lanthanum oxide and 3 parts β-cyclodextrin were thoroughly mixed to obtain hydroxyapatite-β-cyclodextrin agent;

[0065] S02b: 5 parts hydroxyapatite-β-cyclodextrin agent, 3 parts boron nitride and 2 parts calcium sulfate whiskers were mixed and ball-milled at 1000 r / min for 2 h. After ball milling, the mixture was filtered and dried to obtain the carrier material.

[0066] The sodium silicate solution in this embodiment has a mass fraction of 5%.

[0067] The preparation method of the additive in this embodiment is as follows: 3 parts of EDTA-chelated calcium, 3 parts of amino acids, 4 parts of trace elements, 3 parts of wood ash, and 10 parts of sodium humate aqueous solution with a mass fraction of 35% are mixed thoroughly to obtain the additive.

[0068] The trace element in this embodiment is zinc.

[0069] This invention also provides a method for preparing an in-situ decomposing microbial agent for sugarcane leaves, characterized by comprising the following steps:

[0070] Step 1: Inoculate Bacillus subtilis, Aspergillus niger, Trichoderma reesei, and Pseudomonas fluorescens into their respective seed culture media and culture them at 30°C with shaking for 18 hours to obtain seed liquids; Inoculate each seed liquid into a fermentation medium and ferment at 30°C with an aeration rate of 0.8 vvm for 48 hours to obtain fermentation broths of each single strain.

[0071] Step 2: Concentrate each single-strain fermentation broth through plate and frame filtration, mix it with the composite carrier and additive in a mass ratio of 1:3:1, dry it at 50℃ until the moisture content is ≤10%, pulverize it through a 100-mesh sieve, and obtain the microbial inoculant.

[0072] In step one of this embodiment, the Bacillus subtilis fermentation medium is: glucose 15g / L, peptone 10g / L, yeast powder 3g / L, NaCl 4g / L, pH 7.0;

[0073] The fermentation medium for Aspergillus niger and Trichoderma reesei consisted of: 30 g / L wheat bran juice, 20 g / L sucrose, and 3 g / L ammonium nitrate. pH 5.5;

[0074] The fermentation medium for *Pseudomonas fluorescens* was: 10 g / L beef, 10 g / L peptone, 3 g / L NaCl, pH 7.0.

[0075] Example 2.

[0076] This embodiment of a sugarcane leaf in-situ decomposition microbial agent comprises, by weight, 35 parts of Bacillus subtilis powder, 20 parts of Aspergillus niger powder, 25 parts of Trichoderma reesei powder, and 15 parts of Pseudomonas fluorescens powder; the agent also includes 50 parts of modified carrier agent and 10 parts of additive agent.

[0077] In this embodiment, the viable counts of Bacillus subtilis, Aspergillus niger, Trichoderma reesei, and Pseudomonas fluorescens are respectively no less than , , , .

[0078] The preparation method of the modified carrier agent in this embodiment is as follows:

[0079] S01: Preheat carbon nanotubes at 60℃ for 1 hour to obtain preheated carbon nanotubes. Mix the preheated carbon nanotubes and the modification liquid at a weight ratio of 5:14 to modify them. After stirring, the modified carbon nanotube liquid is obtained.

[0080] Preparation of the modified liquid:

[0081] S01a: Stir montmorillonite in a sodium lignosulfonate solution that is 5 times the total volume of montmorillonite to obtain montmorillonite solution;

[0082] S01b: Add 5 parts silicon carbide and 5 parts titanium dioxide to 15 parts sodium citrate solution with a mass fraction of 8%, then add 2 parts silane coupling agent KH550 and stir evenly to obtain the modifier; finally, stir the montmorillonite solution and the modifier at a weight ratio of 11:5 to obtain the modified solution.

[0083] S02: The modified carbon nanotube liquid and the carrier material were ball-milled at a weight ratio of 9:4 at a speed of 1500 r / min for 2 hours. After the ball milling was completed, the mixture was filtered and dried to obtain the modified carrier agent.

[0084] In this embodiment, the stirring speed for the stirring modification treatment was 450 r / min, and the stirring time was 1 h; the mass fraction of the sodium lignosulfonate solution was 15%.

[0085] The method for preparing the carrier material in this embodiment is as follows:

[0086] S02a: β-cyclodextrin, sodium silicate solution and zeolite powder are mixed evenly in a weight ratio of 5:7:3 to obtain β-cyclodextrin solution;

[0087] 7 parts hydroxyapatite, 5 parts lanthanum oxide and 5 parts β-cyclodextrin were thoroughly mixed to obtain hydroxyapatite-β-cyclodextrin agent;

[0088] S02b: 8 parts of hydroxyapatite-β-cyclodextrin, 5 parts of boron nitride and 4 parts of calcium sulfate whiskers were mixed and ball-milled at a speed of 1500 r / min for 2 h. After ball milling, the mixture was filtered and dried to obtain the carrier material.

[0089] The sodium silicate solution in this embodiment has a mass fraction of 8%.

[0090] The preparation method of the additive in this embodiment is as follows: 5 parts of EDTA-chelated calcium, 5 parts of amino acids, 7 parts of trace elements, 5 parts of wood ash, and 15 parts of sodium humate aqueous solution with a mass fraction of 40% are mixed thoroughly to obtain the additive.

[0091] The trace element in this embodiment is boron.

[0092] This invention also provides a method for preparing an in-situ decomposing microbial agent for sugarcane leaves, characterized by comprising the following steps:

[0093] Step 1: Inoculate Bacillus subtilis, Aspergillus niger, Trichoderma reesei, and Pseudomonas fluorescens into their respective seed culture media and culture at 32°C with shaking for 24 hours to obtain seed liquid; Inoculate each seed liquid into fermentation medium and ferment at 35°C with an aeration rate of 1.2 vvm for 72 hours to obtain fermentation broth of each single strain;

[0094] Step 2: Concentrate each single-strain fermentation broth through plate and frame filtration, mix it with the composite carrier and additive in a mass ratio of 1:3:1, dry it at 55℃ until the moisture content is ≤10%, pulverize it through a 100-mesh sieve, and obtain the microbial inoculant.

[0095] In step one of this embodiment, the Bacillus subtilis fermentation medium is: glucose 20g / L, peptone 15g / L, yeast powder 5g / L, NaCl 5g / L, pH 7.2;

[0096] The fermentation medium for Aspergillus niger and Trichoderma reesei consisted of: 35 g / L wheat bran juice, 25 g / L sucrose, and 5 g / L ammonium nitrate. pH 6.0;

[0097] The fermentation medium for *Pseudomonas fluorescens* was: 15 g / L beef, 15 g / L peptone, 5 g / L NaCl, pH 7.5.

[0098] Example 3.

[0099] This embodiment of a sugarcane leaf in-situ decomposition microbial agent comprises, by weight, 30 parts of Bacillus subtilis powder, 15 parts of Aspergillus niger powder, 20 parts of Trichoderma reesei powder, and 10 parts of Pseudomonas fluorescens powder; the agent also includes 45 parts of modified carrier agent and 7.5 parts of additive agent.

[0100] In this embodiment, the viable counts of Bacillus subtilis, Aspergillus niger, Trichoderma reesei, and Pseudomonas fluorescens are respectively no less than , , , .

[0101] The preparation method of the modified carrier agent in this embodiment is as follows:

[0102] S01: Preheat carbon nanotubes at 58℃ for 1 hour to obtain preheated carbon nanotubes. Mix the preheated carbon nanotubes and the modification liquid at a weight ratio of 5:13 and stir to modify them. After stirring, the modified carbon nanotube liquid is obtained.

[0103] Preparation of the modified liquid:

[0104] S01a: Stir montmorillonite in a sodium lignosulfonate solution with a volume of 4 times that of montmorillonite to obtain montmorillonite solution;

[0105] S01b: Add 4 parts silicon carbide and 3.5 parts titanium dioxide to 12.5 parts sodium citrate solution with a mass fraction of 8%, then add 1.5 parts silane coupling agent KH550 and stir evenly to obtain the modifier; finally, stir the montmorillonite solution and the modifier at a weight ratio of 9:5 to obtain the modified solution.

[0106] S02: The modified carbon nanotube liquid and the carrier material were ball-milled at a weight ratio of 8:4 at a speed of 1250 r / min for 2 hours. After the ball milling was completed, the mixture was filtered and dried to obtain the modified carrier agent.

[0107] In this embodiment, the stirring speed for the stirring modification treatment was 400 r / min, and the stirring time was 1 h; the mass fraction of the sodium lignosulfonate solution was 12.5%.

[0108] The method for preparing the carrier material in this embodiment is as follows:

[0109] S02a: β-cyclodextrin, sodium silicate solution and zeolite powder are mixed evenly in a weight ratio of 4:7:2.5 to obtain β-cyclodextrin solution;

[0110] 5.5 parts hydroxyapatite, 3.5 parts lanthanum oxide and 4 parts β-cyclodextrin were thoroughly mixed to obtain hydroxyapatite-β-cyclodextrin agent;

[0111] SO2b: 6.5 parts of hydroxyapatite-β-cyclodextrin, 4 parts of boron nitride and 3 parts of calcium sulfate whiskers were mixed and ball-milled at 1250 r / min for 2 h. After ball milling, the mixture was filtered and dried to obtain the carrier material.

[0112] The sodium silicate solution in this embodiment has a mass fraction of 6.5%.

[0113] The preparation method of the additive in this embodiment is as follows: 4 parts of EDTA-chelated calcium, 4 parts of amino acids, 5.5 parts of trace elements, 4 parts of wood ash, and 12.5 parts of sodium humate aqueous solution with a mass fraction of 35-40% are mixed thoroughly to obtain the additive.

[0114] The trace element in this embodiment is iron.

[0115] This invention also provides a method for preparing an in-situ decomposing microbial agent for sugarcane leaves, characterized by comprising the following steps:

[0116] Step 1: Inoculate Bacillus subtilis, Aspergillus niger, Trichoderma reesei, and Pseudomonas fluorescens into their respective seed culture media and culture at 31°C with shaking for 20 hours to obtain seed liquids; Inoculate each seed liquid into fermentation medium and ferment at 32°C with an aeration rate of 1.0 vvm for 56 hours to obtain fermentation broths of each single strain.

[0117] Step 2: The fermentation broth of each single strain is concentrated by plate and frame filtration, mixed with the composite carrier and additive in a mass ratio of 1:3:1, dried at 52℃ until the moisture content is ≤10%, and then pulverized through a 100-mesh sieve to obtain the microbial inoculant.

[0118] In step one of this embodiment, the Bacillus subtilis fermentation medium is: glucose 17.5 g / L, peptone 12.5 g / L, yeast extract 4 g / L, NaCl 4.5 g / L, pH 7.1;

[0119] The fermentation medium for Aspergillus niger and Trichoderma reesei consisted of: wheat bran juice 32.5 g / L, sucrose 22.5 g / L, and ammonium nitrate 4 g / L. pH 5.7;

[0120] The fermentation medium for *Pseudomonas fluorescens* was: 12.5 g / L beef, 12.5 g / L peptone, 4 g / L NaCl, pH 7.2.

[0121] Comparative Example 1.

[0122] Unlike Example 3, no modified carrier agent was added.

[0123] Comparative Example 2.

[0124] Unlike Example 3, no modified carbon nanotube liquid was added to the modified carrier agent.

[0125] Comparative Example 3.

[0126] No modified liquid was added to the carbon nanotube liquid modified in Example 3.

[0127] Comparative Example 4.

[0128] Unlike Example 3, no modifier was added to the modified liquid.

[0129] Comparative Example 5.

[0130] Unlike Example 3, no carrier material was added to the modified carrier agent.

[0131] Comparative Example 6.

[0132] Unlike Example 3, hydroxyapatite-β-cyclodextrin was not added to the carrier material.

[0133] Comparative Example 7.

[0134] Unlike Example 3, boron nitride and calcium sulfate whiskers were not added to the carrier material.

[0135] The product performance tests of Examples 1-3 and Comparative Examples 1-7 were conducted, and the test results are as follows:

[0136] Experimental location: Baning Village, Fusui County, Chongzuo City, Guangxi (soil pH 5.2, organic matter content 1.8%).

[0137] Experimental materials: The tested sugarcane variety was Guitang 42, the tested microbial agent was the compound microbial agent prepared in this invention, and the control was water.

[0138] Experimental design: A treatment group (20 mu) and a control group (10 mu) were set up. The treatment group was diluted with bacterial agent at a ratio of 1:600, and 0.15% organosilicon spreading agent was added. The agent was applied by drone (12L / mu). The control group was sprayed with an equal amount of water.

[0139]

[0140] From Examples 1-3, Comparative Examples 1-7, and the control group, it was found that the product of the present invention can achieve coordinated improvement in the performance of sugarcane leaf decomposition rate, soil organic matter, ratoon sugarcane sapling growth rate, and yield per acre. The product did not contain a modified carrier agent, the modified carrier agent did not contain modified carbon nanotube liquid, the modified carbon nanotube liquid did not contain a modified liquid, the modified liquid did not contain a modifier, the modified carrier agent did not contain a carrier material, the carrier material did not contain hydroxyapatite-β-cyclodextrin, and the carrier material did not contain boron nitride and calcium sulfate whiskers. All of these results showed a trend of deterioration in product performance. Only the modified carrier agent raw material obtained using the method of the present invention showed the most significant performance improvement.

[0141] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0142] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A microbial inoculant for in-situ decomposition of sugarcane leaves, characterized in that, The active ingredients, by weight, include: 25-35 parts of Bacillus subtilis powder, 10-20 parts of Aspergillus niger powder, 15-25 parts of Trichoderma reesei powder, and 5-15 parts of Pseudomonas fluorescens powder; the microbial agent also includes 40-50 parts of modified carrier agent and 5-10 parts of additive agent.

2. The microbial agent according to claim 1, characterized in that: The viable counts of Bacillus subtilis, Aspergillus niger, Trichoderma reesei, and Pseudomonas fluorescens are respectively not less than , , , .

3. The microbial agent according to claim 1, characterized in that: The preparation method of the modified carrier agent is as follows: S01: Preheat carbon nanotubes at 55-60℃ for 1 hour to obtain preheated carbon nanotubes. Mix the preheated carbon nanotubes and the modification liquid at a weight ratio of 5:(11-14) and stir to modify the mixture. After stirring, the modified carbon nanotube liquid is obtained. Preparation of the modified liquid: S01a: Stir montmorillonite in a sodium lignosulfonate solution that is 3-5 times the total volume of montmorillonite to obtain montmorillonite solution; S01b: Add 3-5 parts silicon carbide and 2-5 parts titanium dioxide to 10-15 parts of sodium citrate solution with a mass fraction of 8%, then add 1-2 parts of silane coupling agent KH550 and stir evenly to obtain the modifier; finally, stir the montmorillonite solution and the modifier at a weight ratio of (7-11):5 to obtain the modified solution. S02: Modified carbon nanotube liquid and carrier material are ball-milled at a weight ratio of (7-9):4, with a ball milling speed of 1000-1500 r / min for 2 hours. After ball milling, the mixture is filtered and dried to obtain the modified carrier agent.

4. The microbial agent according to claim 3, characterized in that: The stirring speed for the stirring modification treatment is 350-450 r / min, and the stirring time is 1 h; the mass fraction of the sodium lignosulfonate solution is 10-15%.

5. The microbial agent according to claim 3, characterized in that: The method for preparing the carrier material is as follows: S02a: β-cyclodextrin, sodium silicate solution and zeolite powder are mixed evenly in a weight ratio of (3-5):7:(2-3) to obtain β-cyclodextrin solution; 4-7 parts of hydroxyapatite, 2-5 parts of lanthanum oxide and 3-5 parts of β-cyclodextrin were thoroughly mixed to obtain hydroxyapatite-β-cyclodextrin agent; SO2b: 5-8 parts of hydroxyapatite-β-cyclodextrin, 3-5 parts of boron nitride and 2-4 parts of calcium sulfate whiskers were mixed and ball-milled at a speed of 1000-1500 r / min for 2 h. After ball milling, the mixture was filtered and dried to obtain the carrier material.

6. The microbial agent according to claim 5, characterized in that: The sodium silicate solution has a mass fraction of 5-8%.

7. The microbial agent according to claim 1, characterized in that: The additive is prepared by mixing 3-5 parts of EDTA-chelated calcium, 3-5 parts of amino acids, 4-7 parts of trace elements, 3-5 parts of wood ash, and 10-15 parts of a 35-40% sodium humate aqueous solution thoroughly to obtain the additive.

8. The microbial agent according to claim 7, characterized in that: The trace element is one of zinc, boron, manganese, and iron.

9. A method for preparing a sugarcane leaf in-situ decomposing microbial inoculant as described in any one of claims 1-8, characterized in that, Includes the following steps: Step 1: Inoculate Bacillus subtilis, Aspergillus niger, Trichoderma reesei, and Pseudomonas fluorescens into their respective seed culture media and culture at 30-32℃ with shaking for 18-24 hours to obtain seed culture. Inoculate each seed culture into fermentation medium and ferment at 30-35℃ with an aeration rate of 0.8-1.2 vvm for 48-72 hours to obtain the fermentation broth of each single strain. Step 2: Concentrate each single-strain fermentation broth through plate and frame filtration, mix it with the composite carrier and additive in a mass ratio of 1:3:1, dry it at a low temperature of 50-55℃ until the moisture content is ≤10%, pulverize it through a 100-mesh sieve, and obtain the microbial inoculant.

10. The preparation method according to claim 9, characterized in that: In step one, the Bacillus subtilis fermentation medium is: glucose 15-20 g / L, peptone 10-15 g / L, yeast extract 3-5 g / L, NaCl 4-5 g / L, pH 7.0-7.2; The fermentation medium for Aspergillus niger and Trichoderma reesei consisted of: wheat bran juice 30-35 g / L, sucrose 20-25 g / L, and ammonium nitrate 3-5 g / L. pH 5.5-6.0; The fermentation medium for *Pseudomonas fluorescens* was: 10-15 g / L beef, 10-15 g / L peptone, 3-5 g / L NaCl, pH 7.0-7.5.

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