Liquid microbial fertilizer and preparation method thereof

Abstract

The invention discloses a liquid microbial fertilizer and a preparation method thereof, and belongs to the technical field of liquid fertilizers. The bacterial fertilizer is prepared from microbial multilayer microcapsules, organic matter fermentation liquor, humic acid, trehalose, xanthan gum, nanocrystalline cellulose and water. The microbial multilayer microcapsule is designed through a multilayer structure and comprises mesoporous silica as a core layer to adsorb a compound microbial agent, an inner shell is a hydrophobic breathable pH / enzyme double-response membrane formed by reacting diglycidyl ether terminated polydimethylsiloxane with N-formyl-L-histidine and then reacting with chitosan, and an outer shell is a hydrophobic breathable pH / enzyme double-response membrane formed by chitosan. The middle layer is a tannic acid-iron complexing layer, and the outer shell is formed by cross-linking N, N '-diallyl-L-tartar amide and a polymer and loading zinc-aluminum layered double hydroxides on the N, N'-diallyl-L-tartar amide and the polymer. The micro-capsule can effectively protect microorganisms, prolong the shelf life and trigger cascade release in a rhizosphere acid environment, zinc and molybdenum nutrients are released firstly, then the microorganisms are released, and precise colonization and synergistic interaction are achieved.

Description

Technical Field

[0001] This invention belongs to the field of liquid fertilizer technology, and in particular relates to a liquid microbial fertilizer and its preparation method. Background Technology

[0002] Bio-fertilizers are a crucial pillar of green agriculture. Through their rich content of beneficial microorganisms, they play a key role in improving soil ecology, stimulating crop growth, enhancing fertilizer utilization efficiency, and controlling diseases. Compared to traditional chemical fertilizers and specialized bio-fertilizers, the new generation of compound microbial fertilizers exhibits significant advantages: they typically contain more than ten highly effective and active beneficial microorganisms, suitable for various crops, and can comprehensively activate nutrients fixed in the soil, improving nutrient utilization. This broad-spectrum approach successfully overcomes the inherent weaknesses of ordinary bio-fertilizers, such as their "specificity" and "limitations," enabling their widespread application in various soil types. They can be applied wherever plants grow, achieving the goals of soil improvement, reducing reliance on chemical fertilizers, and promoting healthy crop growth.

[0003] In existing technologies, microencapsulation has become a mainstream strategy to improve the stability of liquid microbial agents. Researchers have attempted to encapsulate microorganisms with natural polymers such as sodium alginate, gelatin, and chitosan, providing a certain degree of physical protection. However, these conventional microencapsulation systems still have significant limitations: firstly, their protection is limited to a single dimension, making it difficult to simultaneously and effectively block multiple factors that cause microbial inactivation, such as moisture, oxygen, and free radicals; secondly, the encapsulation materials often behave passively in complex soil environments, lacking the ability to intelligently and precisely release the agents according to crop needs, leading to premature release or incompatibility with the rhizosphere environment, ultimately affecting their colonization effect and functional performance.

[0004] Therefore, the industry urgently needs a more sophisticated and highly integrated microcapsule technology that can not only provide protection for microorganisms throughout storage and the initial application period, but also enable release and synergistic effects after reaching the rhizosphere, thereby fundamentally improving the practical application effect of liquid microbial fertilizers. Summary of the Invention

[0005] In response to the above situation and to overcome the shortcomings of existing technologies, this invention has prepared a liquid microbial fertilizer with synergistic effects of strong protection, high integration of functions, and precise release, which effectively solves long-standing industry problems such as short shelf life of liquid microbial agents, low field colonization efficiency, and single function.

[0006] To achieve the above objectives, the following technical solution is adopted: On the one hand, the present invention provides a liquid microbial fertilizer, which is composed of the following components in parts by weight: 10-30 parts of microbial multilayer microcapsules, 30-50 parts of organic matter fermentation broth, 5-10 parts of humic acid, 1-3 parts of trehalose, 0.2-0.5 parts of xanthan gum, 0.1-0.3 parts of nanocrystalline cellulose, and 40-60 parts of water.

[0007] Furthermore, the microbial multilayer microcapsules are prepared through the following steps:

[0008] (1) Mix the composite microbial agent with mesoporous silica powder at a mass ratio of 1:5-10, and stir at low speed at 20-30℃ for 30-60 min to allow the bacteria to be fully adsorbed into the mesoporous channels and surface, thereby obtaining a core layer composed of microbial mesoporous silica composite, for later use; the composite microbial agent is a mixture of Bacillus amyloliquefaciens, Pediococcus lactis, Actinomycetes, Azotobacter spp. brownii, and Burkholderia spp., and the total number of viable bacteria is not less than 5×10 9 CFU / mL;

[0009] (2) Diglycidyl ether-terminated polydimethylsiloxane and N-formyl-L-histidine are dissolved in a first organic solvent and reacted at 60-80℃ for 2-4h to obtain a prepolymer solution; chitosan is mixed with an acetic acid solution with a mass concentration of 0.5%-2% to prepare a chitosan-acetic acid aqueous solution with a mass concentration of 1%-3%; then the prepolymer solution is mixed with the chitosan-acetic acid aqueous solution and reacted at 40-50℃ for 3-6h. After the reaction is completed, the solvent is removed and the solution is redispersed in a phosphate buffer solution with a pH of 6.0-7.0 to obtain the inner shell spraying liquid;

[0010] The obtained core layer composite was placed in a fluidized bed and fluidized under the action of airflow. The inner shell coating liquid was atomized through a nozzle and uniformly sprayed onto the fluidized core layer surface. After spraying, it was cured at 40-50℃ for 2-4 hours to form the microcapsule initial product with inner layer coating.

[0011] (3) Prepare a tannic acid aqueous solution with a mass concentration of 1%-3% and a ferric chloride aqueous solution with a mass concentration of 0.5%-1.5%; place the microcapsule pre-product with inner coating obtained in step (2) in a fluidized bed again, first atomize and spray the tannic acid aqueous solution, then spray the ferric chloride aqueous solution, and form a stable tannic acid-iron complex intermediate layer on the surface of the microcapsule through the in-situ coordination reaction of tannic acid and ferric ions; wherein the spraying amount of tannic acid aqueous solution is 5%-10% of the mass of the microcapsule pre-product, and the spraying amount of ferric chloride aqueous solution is 3%-5% of the mass of the microcapsule pre-product;

[0012] (4) Disperse the nanosheet dispersion of N,N'-diallyl-L-tartaramide, polymer monomer containing double bonds, ammonium molybdate, and zinc-aluminum layered double hydroxide in a mixed solvent of a second organic solvent and water in a volume ratio of 1:1-3, add 2-hydroxy-2-methylphenylacetone, and mix thoroughly to obtain the outer shell spraying liquid;

[0013] The microcapsules with the intermediate layer obtained in step (3) are placed in a fluidized bed, and the outer shell coating liquid is atomized and sprayed under an inert atmosphere. Then, cross-linking and curing are carried out under ultraviolet light to form the final multilayer microcapsules.

[0014] Furthermore, the molar ratio of the diglycidyl ether-terminated polydimethylsiloxane to N-formyl-L-histidine is 1:1-1.2; the mass ratio of the prepolymer solution to the chitosan acetate aqueous solution is 1:0.5-1.

[0015] Furthermore, the polymer monomer containing double bonds is at least one of hydroxyethyl methacrylate and N-isopropylacrylamide.

[0016] Furthermore, the amount of 2-hydroxy-2-methylphenylacetone added is 1%-3% of the total mass of N,N'-diallyl-L-tartrate and the polymer monomer containing double bonds.

[0017] Furthermore, the zinc-aluminum layered double hydroxide nanosheet dispersion is prepared by liquid-phase exfoliation of zinc-aluminum layered double hydroxide bulk material in formamide, and its solid content is 10-20 mg / mL.

[0018] Furthermore, the mass ratio of the N,N'-diallyl-L-tartaramide, the polymer monomer containing double bonds, ammonium molybdate, and zinc-aluminum layered double hydroxide nanosheet dispersion is 1:5-10:0.5-1:0.2-0.5, and the total mass concentration of the diglycidyl ether-terminated polydimethylsiloxane and N-formyl-L-histidine in the first organic solvent is 10%-20%.

[0019] Furthermore, the first organic solvent is selected from N,N-dimethylformamide, dimethyl sulfoxide, and N-methylpyrrolidone.

[0020] Furthermore, the second organic solvent is selected from one of ethanol, isopropanol, and acetone.

[0021] On the other hand, the present invention also provides a method for preparing the liquid microbial fertilizer, comprising the following steps: mixing straw, mushroom cultivation waste, poultry and livestock manure, and kitchen waste in a mass ratio of 3-5:2-4:4-6:1-3, crushing and then adding water to make a slurry, wherein the mass-volume ratio of the mixture to water is 1:3-5 g / mL; subsequently adding 1.5%-2.5% of a compound fermentation liquid according to the total mass of the mixture, anaerobic fermenting at 40-45℃ for 2-4 days, followed by oxygenation at 55-65℃ for 4-6 days, and finally cooling to room temperature and standing for 3-5 days, filtering to remove residue to obtain the product; wherein the compound fermentation liquid contains viable bacteria with a count of not less than 1×10⁻⁶. 9 It is a mixture of Bacillus subtilis solution (CFU / mL), Bacillus megaterium, Bacillus mucilaginosus, and Trichoderma in a mass ratio of 2:1:1:1.

[0022] The beneficial effects of this invention are:

[0023] The liquid microbial fertilizer prepared by this invention is also eco-friendly. The entire system is constructed based on biodegradable or environmentally friendly materials. Its release mechanism is triggered entirely by the natural acidic environment and enzyme activity in the rhizosphere, ultimately producing a synergistic technical effect with strong protective effect, highly integrated functions, and precise release. It effectively solves long-standing industry problems such as short shelf life of liquid microbial agents, low field colonization efficiency, and single function.

[0024] The multilayered microcapsules prepared in this invention provide ultimate protection for the internal functional microorganisms, thereby significantly improving product shelf life and field stability. The core layer utilizes mesoporous silica to physically anchor and immobilize the microbial cells. A prepolymer is generated by the reaction of diglycidyl ether-terminated polydimethylsiloxane and N-formyl-L-histidine, producing new hydroxyl groups. The prepolymer then reacts with chitosan via a Schiff base reaction to form the inner shell. The polydimethylsiloxane segments impart excellent hydrophobicity and low surface energy to the shell, effectively... It blocks the penetration of harmful substances such as dissolved oxygen and free radicals in the aqueous phase, significantly slows down bacterial metabolism, and induces them to enter a deep dormant state. At the same time, the inherent flexibility and air permeability of this chain segment ensure limited gas exchange while providing physical buffering, preventing bacterial suffocation and death. N-formyl-L-histidine not only serves as a key cross-linking point, but its imidazole group can also bind heavy metal ions and play a pH buffering role. Combined with the inherent enzyme responsiveness of chitosan, the inner shell has hydrophobic protection, air permeable buffering, and pH / enzyme dual-response properties.

[0025] Secondly, the microcapsules achieve a cascade response and precise release of nutrients and microbial agents. The tannic acid-iron complex intermediate layer between the inner and outer layers greatly enhances the interlayer binding force through strong multiple bonding, preventing the system from stratifying in a liquid suspension state. This complex bond is extremely sensitive to acidic conditions. When the microcapsules reach the acidic rhizosphere environment, this intermediate layer dissociates, thereby actively triggering the disintegration of the inner shell. The multiple hydroxyl groups in tannic acid can form hydrogen bonds between the inner and outer shells, improving the binding force. The outer shell is composed of N,N'-diallyl-L-tartaramide and polymer crosslinked and loaded with zinc-aluminum layered double hydroxides. The layered double hydroxides themselves act as a pH-responsive framework, dissolving under acidic conditions and releasing the nutrient element zinc. At the same time, the complex of the chiral tartaramide derivative and molybdate also dissociates, releasing molybdenum. This achieves a sequential release of zinc and molybdenum nutrients first, creating a favorable soil chemical environment for microorganisms, before releasing the microorganisms themselves. Attached Figure Description

[0026] Figure 1 The results of shelf-life stability experiments on the liquid microbial fertilizers prepared in the various embodiments and comparative examples of this invention;

[0027] Figure 2 The results are experimental findings on the rhizosphere response release characteristics of the liquid microbial fertilizers prepared in various embodiments and comparative examples of the present invention.

[0028] Figure 3 These are the experimental results of the soil colonization effect of the liquid microbial fertilizers prepared in the various embodiments and comparative examples of the present invention.

[0029] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. Detailed Implementation

[0030] 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. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the experimental materials used in the following examples are all purchased from commercial channels.

[0033] Among them, the *Bacillus amyloliquefaciens*, with accession number CCTCC M2019667, is deposited at the China Center for Type Culture Collection; the *Pediococcus acidilactici*, with accession number CICC 20249, is deposited at the China Industrial Microbiological Culture Collection Center; the actinomycete *Streptomyces microflavus*, with accession number CGMCC 4.1188, is deposited at the China General Microbiological Culture Collection Center; the *Azotobacter chroococcum*, with accession number ACCC 03101, is deposited at the China Agricultural Microbiological Culture Collection Center; and the *Burkholderia cepacia*, with accession number CICC... The *Bacillus subtilis* (CGMCC 1.88) is deposited at the China General Microbiological Culture Collection Center (CGMCC); the *Bacillus megaterium* (CGMCC 10343) is deposited at the CGMCC; the *Bacillus mucilaginosus* (ACCC 10012) is deposited at the China Agricultural Microbiological Culture Collection Center (CAMP); and the *Trichoderma* species *Trichoderma harzianum* (CGMCC 3.17423) is deposited at the CGMCC.

[0034] Example 1:

[0035] A liquid microbial fertilizer is composed of the following components in parts by weight: 10 parts of multilayer microcapsules of microorganisms, 30 parts of organic matter fermentation broth, 5 parts of humic acid, 1 part of trehalose, 0.2 parts of xanthan gum, 0.1 parts of nanocrystalline cellulose, and 40 parts of water.

[0036] The microbial multilayer microcapsules are prepared through the following steps:

[0037] (1) The composite microbial agent and mesoporous silica powder are mixed at a mass ratio of 1:5 and stirred at low speed at 20°C for 30 min to allow the bacteria to be fully adsorbed into the mesoporous channels and surface, thus obtaining a core layer composed of microbial mesoporous silica composite, for later use; the composite microbial agent is a mixture of Bacillus amyloliquefaciens, Pediococcus lactis, Actinomycetes, Azotobacter spp. brownii, and Burkholderia spp., and the total number of viable bacteria is not less than 5 × 10⁻⁶. 9 CFU / mL;

[0038] (2) Diglycidyl ether-terminated polydimethylsiloxane and N-formyl-L-histidine were dissolved in a first organic solvent and reacted at 60°C for 2 h to obtain a prepolymer solution; chitosan was mixed with a 0.5% acetic acid solution to prepare a 1% chitosan-acetic acid aqueous solution, and then the prepolymer solution was mixed with the chitosan-acetic acid aqueous solution and reacted at 40°C for 3 h. After the reaction was completed, the solvent was removed and the solution was redispersed in a phosphate buffer solution with a pH of 6.0 to obtain the inner shell spraying liquid;

[0039] The obtained core layer composite was placed in a fluidized bed and fluidized under the action of airflow. The inner shell coating liquid was atomized through a nozzle and uniformly sprayed onto the fluidized core layer surface. After spraying, it was cured at 40°C for 2 hours to form the microcapsule initial product with inner layer coating.

[0040] (3) Prepare a 1% tannic acid aqueous solution and a 0.5% ferric chloride aqueous solution; place the microcapsule pre-product with inner coating obtained in step (2) in a fluidized bed again, first atomize and spray the tannic acid aqueous solution, then spray the ferric chloride aqueous solution, and form a stable tannic acid-iron complex intermediate layer on the surface of the microcapsule through the in-situ coordination reaction of tannic acid and ferric ions; wherein the amount of tannic acid aqueous solution sprayed is 5% of the mass of the microcapsule pre-product, and the amount of ferric chloride aqueous solution sprayed is 3% of the mass of the microcapsule pre-product;

[0041] (4) N,N'-diallyl-L-tartaramide, polymer monomers containing double bonds, ammonium molybdate, and zinc-aluminum layered double hydroxide nanosheet dispersions are dispersed together in a mixed solvent of a second organic solvent and water in a volume ratio of 1:1. 2-hydroxy-2-methylphenylacetone is added and the mixture is thoroughly mixed to obtain the outer shell spraying liquid.

[0042] The microcapsules with the intermediate layer obtained in step (3) are placed in a fluidized bed, and the outer shell coating liquid is atomized and sprayed under an inert atmosphere. Then, cross-linking and curing are carried out under ultraviolet light to form the final multilayer microcapsules.

[0043] The molar ratio of the diglycidyl ether-terminated polydimethylsiloxane to N-formyl-L-histidine is 1:1; the mass ratio of the prepolymer solution to the chitosan acetate aqueous solution is 1:0.5.

[0044] The polymer monomer containing double bonds is hydroxyethyl methacrylate; the amount of 2-hydroxy-2-methylphenylacetone added is 1% of the total mass of N,N'-diallyl-L-tartrate and the polymer monomer containing double bonds.

[0045] The zinc-aluminum layered double hydroxide nanosheet dispersion was prepared by liquid-phase exfoliation of zinc-aluminum layered double hydroxide bulk material in formamide, and its solid content was 10 mg / mL.

[0046] The mass ratio of the N,N'-diallyl-L-tartaramide, the double-bonded polymer monomer, ammonium molybdate, and zinc-aluminum layered double hydroxide nanosheet dispersion is 1:5:0.5:0.2, and the total mass concentration of the diglycidyl ether-terminated polydimethylsiloxane and N-formyl-L-histidine in the first organic solvent is 10%.

[0047] The first organic solvent is N,N-dimethylformamide; the second organic solvent is ethanol.

[0048] A method for preparing the liquid microbial fertilizer includes the following steps: mixing straw, mushroom cultivation waste, poultry and livestock manure, and kitchen waste in a mass ratio of 3:2:4:1, crushing the mixture, adding water, and pulping it, wherein the mass-to-volume ratio of the mixture to water is 1:3 g / mL; then adding 1.5% of a compound fermentation liquid by mass of the mixture, anaerobic fermenting at 40℃ for 2 days, followed by aerobic reaction at 55℃ for 4 days, and finally cooling to room temperature and standing for 3 days, followed by filtration to remove residue; the compound fermentation liquid contains at least 1×10⁻⁶ viable bacteria. 9 It is a mixture of Bacillus subtilis solution (CFU / mL), Bacillus megaterium, Bacillus mucilaginosus, and Trichoderma in a mass ratio of 2:1:1:1.

[0049] Example 2:

[0050] A liquid microbial fertilizer is composed of the following components in parts by weight: 30 parts of multilayer microcapsules of microorganisms, 50 parts of organic matter fermentation broth, 10 parts of humic acid, 3 parts of trehalose, 0.5 parts of xanthan gum, 0.3 parts of nanocrystalline cellulose, and 60 parts of water.

[0051] The microbial multilayer microcapsules are prepared through the following steps:

[0052] (1) The composite microbial agent and mesoporous silica powder are mixed at a mass ratio of 1:10 and stirred at low speed at 30°C for 60 min to allow the bacteria to be fully adsorbed into the mesoporous channels and surface, thus obtaining a core layer composed of microbial mesoporous silica composite, for later use; the composite microbial agent is a mixture of Bacillus amyloliquefaciens, Pediococcus lactis, Actinomycetes, Azotobacter spp. brownii, and Burkholderia spp., and the total number of viable bacteria is not less than 5 × 10⁻⁶. 9 CFU / mL;

[0053] (2) Diglycidyl ether-terminated polydimethylsiloxane and N-formyl-L-histidine were dissolved in a first organic solvent and reacted at 80°C for 4 h to obtain a prepolymer solution; chitosan was mixed with a 2% acetic acid solution to prepare a 3% chitosan-acetic acid aqueous solution, and then the prepolymer solution was mixed with the chitosan-acetic acid aqueous solution and reacted at 50°C for 6 h. After the reaction was completed, the solvent was removed and the solution was redispersed in a phosphate buffer solution with a pH of 7.0 to obtain the inner shell spraying liquid;

[0054] The obtained core layer composite was placed in a fluidized bed and fluidized under the action of airflow. The inner shell coating liquid was atomized through a nozzle and uniformly sprayed onto the fluidized core layer surface. After spraying, it was cured at 50°C for 4 hours to form the microcapsule initial product with inner layer coating.

[0055] (3) Prepare a 3% tannic acid aqueous solution and a 1.5% ferric chloride aqueous solution; place the microcapsule pre-product with inner coating obtained in step (2) in a fluidized bed again, first atomize the tannic acid aqueous solution, then spray the ferric chloride aqueous solution, and form a stable tannic acid-iron complex intermediate layer on the surface of the microcapsule through the in-situ coordination reaction of tannic acid and ferric ions; wherein the amount of tannic acid aqueous solution sprayed is 10% of the mass of the microcapsule pre-product, and the amount of ferric chloride aqueous solution sprayed is 5% of the mass of the microcapsule pre-product;

[0056] (4) N,N'-diallyl-L-tartaramide, polymer monomers containing double bonds, ammonium molybdate, and zinc-aluminum layered double hydroxide nanosheet dispersions are dispersed together in a mixed solvent of a second organic solvent and water in a volume ratio of 1:3. 2-hydroxy-2-methylphenylacetone is added and the mixture is thoroughly mixed to obtain the outer shell spraying liquid.

[0057] The microcapsules with the intermediate layer obtained in step (3) are placed in a fluidized bed, and the outer shell coating liquid is atomized and sprayed under an inert atmosphere. Then, cross-linking and curing are carried out under ultraviolet light to form the final multilayer microcapsules.

[0058] The molar ratio of the diglycidyl ether-terminated polydimethylsiloxane to N-formyl-L-histidine is 1:1.2; the mass ratio of the prepolymer solution to the chitosan acetate aqueous solution is 1:1.

[0059] The polymer monomer containing double bonds is N-isopropylacrylamide; the amount of 2-hydroxy-2-methylphenylacetone added is 3% of the total mass of N,N'-diallyl-L-tartrate and the polymer monomer containing double bonds.

[0060] The zinc-aluminum layered double hydroxide nanosheet dispersion was prepared by liquid-phase exfoliation of zinc-aluminum layered double hydroxide bulk material in formamide, and its solid content was 20 mg / mL.

[0061] The mass ratio of the N,N'-diallyl-L-tartaramide, the double-bonded polymer monomer, ammonium molybdate, and zinc-aluminum layered double hydroxide nanosheet dispersion is 1:10:1:0.5, and the total mass concentration of the diglycidyl ether-terminated polydimethylsiloxane and N-formyl-L-histidine in the first organic solvent is 20%.

[0062] The first organic solvent is dimethyl sulfoxide; the second organic solvent is isopropanol.

[0063] A method for preparing the liquid microbial fertilizer includes the following steps: mixing straw, mushroom cultivation waste, poultry and livestock manure, and kitchen waste in a mass ratio of 5:4:6:3, crushing the mixture, adding water, and pulping it, wherein the mass-to-volume ratio of the mixture to water is 1:5 g / mL; then adding 2.5% of a compound fermentation liquid by mass of the mixture, anaerobic fermenting at 45°C for 4 days, followed by aerobic reaction at 65°C for 6 days, and finally cooling to room temperature and standing for 5 days, followed by filtration to remove residue; the compound fermentation liquid contains at least 1×10⁻⁶ viable bacteria. 9 It is a mixture of Bacillus subtilis solution (CFU / mL), Bacillus megaterium, Bacillus mucilaginosus, and Trichoderma in a mass ratio of 2:1:1:1.

[0064] Example 3:

[0065] A liquid microbial fertilizer is composed of the following components in parts by weight: 20 parts of multilayer microcapsules of microorganisms, 40 parts of organic matter fermentation broth, 7 parts of humic acid, 2 parts of trehalose, 0.35 parts of xanthan gum, 0.2 parts of nanocrystalline cellulose, and 50 parts of water.

[0066] The microbial multilayer microcapsules are prepared through the following steps:

[0067] (1) The composite microbial agent and mesoporous silica powder are mixed at a mass ratio of 1:7 and stirred at low speed at 25°C for 45 min to allow the bacteria to be fully adsorbed into the mesoporous channels and surface, thus obtaining a core layer composed of microbial mesoporous silica composite, for later use; the composite microbial agent is a mixture of Bacillus amyloliquefaciens, Pediococcus lactis, Actinomycetes, Azotobacter spp. brownii, and Burkholderia spp., and the total number of viable bacteria is not less than 5 × 10⁻⁶. 9 CFU / mL;

[0068] (2) Diglycidyl ether-terminated polydimethylsiloxane and N-formyl-L-histidine were dissolved in a first organic solvent and reacted at 70°C for 3 h to obtain a prepolymer solution; chitosan was mixed with a 1% acetic acid solution to prepare a 2% chitosan-acetic acid aqueous solution, and then the prepolymer solution was mixed with the chitosan-acetic acid aqueous solution and reacted at 45°C for 4.5 h. After the reaction was completed, the solvent was removed and the solution was redispersed in a phosphate buffer solution with a pH of 6.5 to obtain the inner shell spraying liquid;

[0069] The obtained core layer composite was placed in a fluidized bed and fluidized under the action of airflow. The inner shell coating liquid was atomized through a nozzle and uniformly sprayed onto the fluidized core layer surface. After spraying, it was cured at 45°C for 3 hours to form the initial microcapsule product with inner layer coating.

[0070] (3) Prepare a 2% tannic acid aqueous solution and a 1% ferric chloride aqueous solution; place the microcapsule pre-product with inner coating obtained in step (2) in a fluidized bed again, first atomize the tannic acid aqueous solution, then spray the ferric chloride aqueous solution, and form a stable tannic acid-iron complex intermediate layer on the surface of the microcapsule through the in-situ coordination reaction of tannic acid and ferric ions; wherein the amount of tannic acid aqueous solution sprayed is 7% of the mass of the microcapsule pre-product, and the amount of ferric chloride aqueous solution sprayed is 4% of the mass of the microcapsule pre-product;

[0071] (4) N,N'-diallyl-L-tartaramide, polymer monomers containing double bonds, ammonium molybdate, and zinc-aluminum layered double hydroxide nanosheet dispersions are dispersed together in a mixed solvent of a second organic solvent and water at a volume ratio of 1:2. 2-hydroxy-2-methylphenylacetone is added and the mixture is thoroughly mixed to obtain the outer shell spraying liquid.

[0072] The microcapsules with the intermediate layer obtained in step (3) are placed in a fluidized bed, and the outer shell coating liquid is atomized and sprayed under an inert atmosphere. Then, cross-linking and curing are carried out under ultraviolet light to form the final multilayer microcapsules.

[0073] The molar ratio of the diglycidyl ether-terminated polydimethylsiloxane to N-formyl-L-histidine is 1:1.1; the mass ratio of the prepolymer solution to the chitosan acetate aqueous solution is 1:0.7.

[0074] The polymer monomer containing double bonds is a combination of hydroxyethyl methacrylate and N-isopropylacrylamide in a 1:1 mass ratio.

[0075] The amount of 2-hydroxy-2-methylphenylacetone added is 2% of the total mass of N,N'-diallyl-L-tartrate and the polymer monomer containing double bonds.

[0076] The zinc-aluminum layered double hydroxide nanosheet dispersion was prepared by liquid-phase exfoliation of zinc-aluminum layered double hydroxide bulk material in formamide, and its solid content was 15 mg / mL.

[0077] The mass ratio of the N,N'-diallyl-L-tartaramide, the double-bonded polymer monomer, ammonium molybdate, and zinc-aluminum layered double hydroxide nanosheet dispersion is 1:7.5:0.7:0.35, and the total mass concentration of the diglycidyl ether-terminated polydimethylsiloxane and N-formyl-L-histidine in the first organic solvent is 15%.

[0078] The first organic solvent is N-methylpyrrolidone; the second organic solvent is acetone.

[0079] A method for preparing the liquid microbial fertilizer includes the following steps: mixing straw, mushroom cultivation waste, poultry and livestock manure, and kitchen waste in a mass ratio of 4:3:5:2, crushing the mixture, adding water, and pulping it, wherein the mass-to-volume ratio of the mixture to water is 1:4 g / mL; then adding 2% of a compound fermentation bacterial solution by mass of the mixture, anaerobic fermenting at 42℃ for 3 days, followed by oxygenation at 60℃ for 5 days, and finally cooling to room temperature and standing for 4 days, followed by filtration to remove residue; the compound fermentation bacterial solution contains at least 1×10⁻⁶ viable bacteria. 9 It is a mixture of Bacillus subtilis solution (CFU / mL), Bacillus megaterium, Bacillus mucilaginosus, and Trichoderma in a mass ratio of 2:1:1:1.

[0080] Comparative Example 1:

[0081] This comparative example does not contain multilayer microcapsules of microorganisms; instead, a compound microbial agent is used directly to replace the multilayer microcapsules of microorganisms. Other components are the same as in Example 3.

[0082] Comparative Example 2:

[0083] In this comparative example, sodium alginate-embedded microbial microcapsules were used instead of multilayer microbial microcapsules, and other components were the same as in Example 3.

[0084] The microcapsule preparation method in this comparative example is as follows: A composite microbial agent was mixed with a sodium alginate solution (2% by mass) to form an agent-sodium alginate mixture. This mixture was then added dropwise to a calcium chloride solution (2% by mass) to form gel microspheres. After solidification for 30 minutes, the mixture was filtered and washed to obtain sodium alginate-embedded microbial microcapsules.

[0085] Comparative Example 3:

[0086] In this comparative example, the tannic acid-iron complex intermediate layer was omitted during the preparation of the microbial microcapsules. The outer layer was directly coated after the inner layer was coated. Other components were the same as in Example 3.

[0087] Comparative Example 4:

[0088] In this comparative example, the microbial microcapsules only contain the core layer and inner shell, lacking the outer shell; the other components are the same as in Example 3.

[0089] Results Analysis

[0090] The following experiments were conducted on the samples of Examples 1-3 and Comparative Examples 1-4 to evaluate their shelf-life stability, rhizosphere response release characteristics, soil colonization effect, and crop growth promotion capacity.

[0091] Test Example 1

[0092] The liquid microbial fertilizers prepared in each example and comparative example were sealed and stored at 25°C. Samples were taken at 0, 1, 2, and 3 months, and the viable cell count (CFU / mL) was determined using the plate count method. The viable cell retention rate was calculated using the following formula: Viable cell retention rate (%) = (Viable cell count after storage / Initial viable cell count) × 100%. Results are shown below. Figure 1 .

[0093] from Figure 1 It can be seen that the viable bacterial count retention rate of each embodiment of the present invention is higher than 85% after 3 months of storage, which is significantly better than all comparative examples, demonstrating the excellent protective performance of multilayer microcapsules against microorganisms.

[0094] Test Example 2

[0095] Take 1g of the liquid microbial fertilizer prepared in each example and comparative example, add 9mL of sterile physiological saline, freeze-mill, and determine the total viable count by dilution coating method. Then weigh out the product containing 10... 9The CFU live bacteria inoculum was added to 50 mL of phosphate buffer (pH 5.5) and placed in a 25°C shaker at 150 rpm. Samples were taken at 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h. Each sample was 1 mL, filtered through a 0.22 μm sterile filter, and the filtrate was serially diluted 10-fold. The filtrate was then spread onto mixed culture plates and incubated at 30°C for 48 h. The number of released live bacteria was then counted, and the microbial release rate was calculated using the following formula: Microbial release rate (%) = (Number of released live bacteria / Total number of live bacteria) × 100%. Results are shown below. Figure 2 .

[0096] Figure 2 The results show that each embodiment exhibits significant sustained-release and delayed-release characteristics, with near-complete release within 24 hours, while the comparative embodiment shows excessively rapid release and lacks sustained-release control effect.

[0097] Test Example 3

[0098] Fill plastic basins (15cm in diameter, 20cm in height) with sterilized soil, using 1kg of soil per basin, and assuming each gram of soil contains 10... 9 Add the CFU live bacteria to each liquid microbial fertilizer, stir well, and let stand for 24 hours. Plant one tomato seedling per pot and cultivate in a greenhouse (temperature 25±2℃, light 16h / d, humidity 60±5%), watering regularly to maintain soil moisture at 70% field capacity, without additional fertilization. At 7, 14, and 21 days after planting, carefully remove the plants, shake off loose soil from the root surface, and collect the rhizosphere soil (10g / plant) adhering closely to the roots, placing it in a sterile bag. Take 1g of rhizosphere soil, add 9mL of sterile physiological saline, shake for 30min to prepare a bacterial suspension, serially dilute 10-fold, and spread 100μL onto a selective medium for Bacillus amyloliquefaciens. Incubate at 30℃ for 48h, count the number of colonies in the clear zone, and calculate the number of live bacteria per gram of rhizosphere soil. Results are shown in […]. Figure 3 .

[0099] from Figure 3 As can be seen, the number of rhizosphere microorganisms in each example group reached its peak on day 14 and remained at a high level, indicating that the liquid microbial fertilizer prepared by the present invention exhibits excellent colonization and reproduction capabilities, which are significantly better than all comparative examples.

[0100] Test Example 4

[0101] For the crop growth promotion capacity test, each group consisted of 10 pots, with a blank control group (only basic fertilizer was applied, without microbial fertilizer). All treatment groups were managed as follows:

[0102] Base fertilizer application: Apply 1g of base fertilizer per pot and mix it evenly with the soil (same as the potting method in test example 3).

[0103] Application of microbial fertilizer: Dilute each liquid microbial fertilizer with tap water 100 times, and apply it for the first time on the 3rd day after the tomato plants are transplanted (100mL per pot, slowly pour along the edge of the pot), and then apply it once every 7 days for a total of 3 times.

[0104] Growth index measurement: The following indicators were measured 30 days after transplanting:

[0105] Plant height: The vertical distance from the base of the plant (soil surface) to the apical growing point using a ruler;

[0106] Root length: Measure the length of the longest taproot using a ruler;

[0107] Fresh weight of above-ground parts: The fresh weight of the above-ground parts (stems and leaves) of the plant is measured using an electronic balance.

[0108] The test results are shown in the table.

[0109] Table 1 Comparison of Crop Growth Promotion Capacity Test Results between Basic Fertilizer and Liquid Microbial Fertilizer

[0110] Group Plant height (cm) Root length (cm) Fresh weight of above-ground parts (g) Example 1 35.2 25.8 45.3 Example 2 36.1 26.3 46.2 Example 3 35.8 26.0 45.8 Comparative Example 1 28.5 20.1 35.6 Comparative Example 2 30.2 22.3 38.9 Comparative Example 3 31.5 23.1 40.2 Comparative Example 4 32.0 23.8 41.1 Blank control 25.3 18.5 30.1

[0111] The data in Table 1 show that tomato plants treated with the liquid microbial fertilizer of this invention are significantly better than the blank control and all comparative groups in terms of plant height, root length and biomass, proving its significant growth-promoting effect.

[0112] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0113] The present invention and its embodiments have been described above. This description is not restrictive, and the embodiments shown are only one of the embodiments of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A liquid microbial fertilizer, characterized in that: It is composed of the following components in parts by weight: 10-30 parts of microbial multilayer microcapsules, 30-50 parts of organic matter fermentation broth, 5-10 parts of humic acid, 1-3 parts of trehalose, 0.2-0.5 parts of xanthan gum, 0.1-0.3 parts of nanocrystalline cellulose, and 40-60 parts of water; The microbial multilayer microcapsules are prepared through the following steps: (1) Mix the composite microbial agent with mesoporous silica powder at a mass ratio of 1:5-10, and stir at low speed at 20-30℃ for 30-60 min to allow the bacteria to be fully adsorbed into the mesoporous channels and surface, thereby obtaining a core layer composed of microbial mesoporous silica composite, for later use; the composite microbial agent is a mixture of Bacillus amyloliquefaciens, Pediococcus lactis, Actinomycetes, Azotobacter spp. brownii, and Burkholderia spp., and the total number of viable bacteria is not less than 5×10 9 CFU / mL; (2) Diglycidyl ether-terminated polydimethylsiloxane and N-formyl-L-histidine are dissolved in a first organic solvent and reacted at 60-80℃ for 2-4h to obtain a prepolymer solution; chitosan is mixed with an acetic acid solution with a mass concentration of 0.5%-2% to prepare a chitosan-acetic acid aqueous solution with a mass concentration of 1%-3%; then the prepolymer solution is mixed with the chitosan-acetic acid aqueous solution and reacted at 40-50℃ for 3-6h. After the reaction is completed, the solvent is removed and the solution is redispersed in a phosphate buffer solution with a pH of 6.0-7.0 to obtain the inner shell spraying liquid; The obtained core layer composite was placed in a fluidized bed and fluidized under the action of airflow. The inner shell coating liquid was atomized through a nozzle and uniformly sprayed onto the fluidized core layer surface. After spraying, it was cured at 40-50℃ for 2-4 hours to form the microcapsule initial product with inner layer coating. (3) Prepare a tannic acid aqueous solution with a mass concentration of 1%-3% and a ferric chloride aqueous solution with a mass concentration of 0.5%-1.5%; place the microcapsule pre-product with inner coating obtained in step (2) in a fluidized bed again, first atomize and spray the tannic acid aqueous solution, then spray the ferric chloride aqueous solution, and form a stable tannic acid-iron complex intermediate layer on the surface of the microcapsule through the in-situ coordination reaction of tannic acid and ferric ions; wherein the spraying amount of tannic acid aqueous solution is 5%-10% of the mass of the microcapsule pre-product, and the spraying amount of ferric chloride aqueous solution is 3%-5% of the mass of the microcapsule pre-product; (4) Disperse the nanosheet dispersion of N,N'-diallyl-L-tartaramide, polymer monomer containing double bonds, ammonium molybdate, and zinc-aluminum layered double hydroxide in a mixed solvent of a second organic solvent and water in a volume ratio of 1:1-3, add 2-hydroxy-2-methylphenylacetone, and mix thoroughly to obtain the outer shell spraying liquid; The microcapsules with the intermediate layer obtained in step (3) are placed in a fluidized bed, and the outer shell coating liquid is atomized and sprayed under an inert atmosphere. Then, cross-linking and curing are carried out under ultraviolet light to form the final multilayer microcapsules.

2. The liquid microbial fertilizer according to claim 1, characterized in that: In step 2, the molar ratio of the diglycidyl ether-terminated polydimethylsiloxane to N-formyl-L-histidine is 1:1-1.2; the mass ratio of the prepolymer solution to the chitosan acetate aqueous solution is 1:0.5-1.

3. The liquid microbial fertilizer according to claim 1, characterized in that: In step 4, the polymer monomer containing double bonds is at least one of hydroxyethyl methacrylate and N-isopropylacrylamide.

4. The liquid microbial fertilizer according to claim 1, characterized in that: The amount of 2-hydroxy-2-methylphenylacetone added is 1%-3% of the total mass of N,N'-diallyl-L-tartrate and the polymer monomer containing double bonds.

5. The liquid microbial fertilizer according to claim 1, characterized in that: In step 4, the zinc-aluminum layered double hydroxide nanosheet dispersion is prepared by liquid-phase exfoliation of the zinc-aluminum layered double hydroxide bulk material in formamide, and its solid content is 10-20 mg / mL.

6. The liquid microbial fertilizer according to claim 1, characterized in that: In step 4, the mass ratio of the N,N'-diallyl-L-tartaramide, the polymer monomer containing double bonds, ammonium molybdate, and the zinc-aluminum layered double hydroxide nanosheet dispersion is 1:5-10:0.5-1:0.2-0.5, and the total mass concentration of the diglycidyl ether-terminated polydimethylsiloxane and N-formyl-L-histidine in the first organic solvent is 10%-20%.

7. The liquid microbial fertilizer according to claim 1, characterized in that: The first organic solvent is selected from one of N,N-dimethylformamide, dimethyl sulfoxide, and N-methylpyrrolidone.

8. The liquid microbial fertilizer according to claim 1, characterized in that: The second organic solvent is selected from ethanol, isopropanol, and acetone.

9. A method for preparing liquid microbial fertilizer according to any one of claims 1-8, characterized in that: Includes the following steps: Straw, mushroom cultivation waste, poultry and livestock manure, and kitchen waste are mixed in a mass ratio of 3-5:2-4:4-6:1-3, crushed, and then mixed with water to form a slurry. The mass-to-volume ratio of the mixture to water is 1:3-5 g / mL. Then, 1.5%-2.5% of a compound fermentation liquid is added to the mixture. Anaerobic fermentation is carried out at 40-45℃ for 2-4 days, followed by aerobic fermentation at 55-65℃ for 4-6 days. Finally, the mixture is cooled to room temperature and allowed to stand for 3-5 days. The residue is then filtered out to obtain the final product. The compound fermentation liquid contains at least 1×10⁻⁶ viable bacteria. 9 It is a mixture of Bacillus subtilis solution (CFU / mL), Bacillus megaterium, Bacillus mucilaginosus, and Trichoderma in a mass ratio of 2:1:1:1.

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