Multifunctional degenerate soil remediation composite microbial inoculant and preparation method thereof

Abstract

This invention belongs to the field of soil remediation composition technology, specifically relating to a multifunctional composite microbial agent for remediating degraded soil and its preparation method. The composite microbial agent consists of microcapsule particles, including a core material and a wall material. The core material is a composite bacterial suspension containing dormant spores of phosphate-solubilizing bacteria, potassium-solubilizing bacteria, and nitrogen-fixing bacteria, with added ascorbic acid and silicate mineral powder. The wall material is an interpenetrating polymer network composed of sodium alginate, chitosan, and polyacrylic acid, internally cross-linked with urea-formaldehyde resin microspheres, and coated with a hydrophobic modification layer composed of silica and stearic acid on the outer surface. The preparation method includes wall material solution preparation, core material suspension preparation, microsphere formation in a sharp-pore coagulation bath, and surface hydrophobic modification and drying steps. This microbial agent achieves dual response to moisture and pH through the interpenetrating polymer network, extending its survival period under drought conditions and targeting the release of bacterial communities at specific pH levels to improve rhizosphere colonization rate; the hydrophobic modification layer, combined with the cross-linked microspheres, prevents premature swelling and enhances particle mechanical strength.

Description

Technical Field

[0001] This invention belongs to the field of soil remediation composition technology, specifically relating to a multifunctional compound microbial agent for remediating degraded soil and its preparation method. Background Technology

[0002] In existing degraded soil remediation technologies, compound microbial agents often employ either free agents or conventional gel encapsulation techniques. Conventional gel encapsulation typically involves directly suspending dormant spores of phosphate-solubilizing, potassium-solubilizing, and nitrogen-fixing bacteria in a sodium alginate solution, then dripping them into a calcium chloride coagulation bath using a sharp-pore method to achieve ionic cross-linking, forming calcium alginate gel microspheres. This conventional process is simple to operate, and the resulting calcium alginate gel microspheres act as a physical barrier, effectively isolating soil protozoa from predation and some external physicochemical stimuli. In actual soil application, these conventional gel microspheres directly contact the degraded soil matrix, and the microorganisms encapsulated within exchange substances with the external environment through the gel pores.

[0003] The cross-linked network structure of conventional calcium alginate gel microspheres is fixed. When degraded soil faces drought and water shortage stress, this fixed network cannot lock in internal moisture, causing the encapsulated dormant spores to lose activity due to dehydration. At the same time, this fixed network cannot actively change its porosity and swelling state according to the fluctuation of pH in degraded soil. Microorganisms can only be passively released as the gel skeleton degrades naturally, and cannot target and burst into growth at specific moments when the soil pH deviates from neutral, resulting in an extremely low colonization rate of microorganisms in the rhizosphere. Summary of the Invention

[0004] To address the shortcomings of existing microbial agents for degraded soil remediation, such as poor water retention and survival capacity under drought stress, inability to target bacterial release in response to soil pH, insufficient mechanical strength of microspheres leading to easy breakage, and easy premature swelling and degradation resulting in low rhizosphere colonization rate of microbial communities, this invention provides a multifunctional composite microbial agent for degraded soil remediation and its preparation method.

[0005] To address the aforementioned technical problems, this invention provides a multifunctional composite microbial agent for remediating degraded soil, comprising the following technical features: the composite microbial agent is composed of microcapsule particles, each microcapsule particle including a core material and a wall material covering the core material; based on the total weight of the composite microbial agent, the core material accounts for 10%-30%, and the wall material accounts for 70%-90%; the core material is a composite microbial suspension containing dormant spores of phosphate-solubilizing bacteria, dormant spores of potassium-solubilizing bacteria, and dormant spores of nitrogen-fixing bacteria; the wall material is an interpenetrating polymer network structure composed of sodium alginate, chitosan, and polyacrylic acid, wherein the mass ratio of sodium alginate to chitosan in the interpenetrating polymer network structure is 1:0.5-1.5, and the amount of polyacrylic acid added is 5%-15% of the total weight of sodium alginate and chitosan.

[0006] In this scheme, sodium alginate is a polyanionic polysaccharide that can form a basic gel framework through ionic cross-linking. Chitosan is a polycationic polysaccharide that can form a polyelectrolyte complex with sodium alginate through electrostatic interactions. Both sodium alginate and polyacrylic acid form a three-dimensional interpenetrating polymer network structure through covalent cross-linking and physical entanglement. Compared to traditional single calcium alginate gel networks, this structure exhibits more stable structural and environmental responsiveness. The carboxyl groups of polyacrylic acid possess strong hydrophilicity and water responsiveness. Under drought stress conditions, they can lock in bound water within the network through hydrogen bonding, maintaining the water activity of the core material's microenvironment and preventing water loss. It prevents the internal dormant spores from dehydrating and becoming inactive; the amino groups of chitosan and the carboxyl groups of polyacrylic acid can undergo reversible protonation and dissociation with changes in soil pH, changing the intermolecular electrostatic forces between them, thereby driving the directional regulation of the swelling degree and porosity of the interpenetrating polymer network. When the soil pH deviates from neutral, it actively opens the release channels to achieve targeted release of the composite microbial community within the core material. At the same time, the three-dimensional interpenetrating network can provide a continuous physical barrier for the spores, resisting predation by protozoa in the soil and invasion by adverse physicochemical factors, effectively prolonging the survival period of the microbial agent in the soil and improving the rhizosphere colonization rate of the microbial community.

[0007] Furthermore, in the above technical solution, the ratio of the number of live phosphate-solubilizing bacteria dormant spores, potassium-solubilizing bacteria dormant spores, and nitrogen-fixing bacteria dormant spores in the core material is 2:1:1. The core material also contains 1%-3% ascorbic acid of the total weight of the composite bacterial suspension and 0.5%-2% silicate mineral powder of the total weight of the composite bacterial suspension, wherein the particle size of the silicate mineral powder is 200-500 nanometers.

[0008] In practice, the ratio of live bacteria can match the activation requirements of phosphorus, potassium, and nitrogen in degraded soil, achieving synergistic and efficient release of the three major nutrients. Ascorbic acid, as a reducing protective agent, can eliminate reactive oxygen free radicals in the core material microenvironment, prevent dormant spores from being damaged by oxidative stress, and prolong the survival time of spore dormancy. 200-500 nanometer silicate mineral powder can adhere to the surface of dormant spores to form a nanoscale physical protective layer, further isolating them from the invasion of adverse physical and chemical factors. At the same time, silicate minerals can slowly release silicon, improve the aggregate structure of degraded soil, and help improve the soil remediation effect.

[0009] Furthermore, in the above technical solution, the polyacrylic acid in the wall material is chitosan-grafted modified polyacrylic acid. The grafted modified polyacrylic acid is obtained by grafting and polymerizing acrylic monomers onto the main chain of chitosan in the presence of an initiator. The initiator is cerium ammonium nitrate, and the amount of the initiator added is 0.1%-0.5% of the mass of the acrylic monomers.

[0010] In practice, polyacrylic acid is grafted onto the chitosan backbone, allowing the polyacrylic acid segments to be covalently bonded to the chitosan backbone. Compared to physically blended polyacrylic acid, this significantly improves its compatibility with the sodium alginate-chitosan matrix, preventing phase separation of the polyacrylic acid components during network formation and resulting in a more uniform and stable pH response and moisture retention capacity of the interpenetrating polymer network. Cerium ammonium nitrate, acting as an initiator, generates free radicals at the hydroxyl sites of chitosan, efficiently initiating the graft polymerization of acrylic acid monomers. This method boasts high grafting efficiency and few side reactions. An addition of 0.1%-0.5% ensures the grafting rate while avoiding the toxic effects of residual initiator on microbial spores.

[0011] Furthermore, in the above technical solution, the interpenetrating polymer network structure of the wall material is also cross-linked with urea-formaldehyde resin microspheres, which are uniformly dispersed in the matrix formed by sodium alginate and chitosan. The particle size of the urea-formaldehyde resin microspheres is 1-5 micrometers, and the amount of urea-formaldehyde resin microspheres added accounts for 3%-8% of the total weight of the wall material.

[0012] In practice, 1-5 micrometer urea-formaldehyde resin microspheres are used as rigid fillers, uniformly interspersed in a flexible matrix of interpenetrating polymer networks to form a composite network structure. This significantly improves the compressive modulus and deformation resistance of the microcapsule wall material, preventing the microcapsule particles from breaking due to mechanical compression and soil friction during soil application, thus ensuring the integrity of the microcapsule structure and the effectiveness of the responsive bacterial release function. An addition of 3%-8% can improve mechanical strength without clogging the mesoporous network of the wall material, ensuring unobstructed material exchange and spore release channels.

[0013] Furthermore, in the above technical solution, the outer surface of the wall material is also coated with a hydrophobic modified layer, which is prepared by reacting silica nanoparticles with stearic acid through a hydrophobic modification reaction. The loading amount of silica nanoparticles in the hydrophobic modified layer is 0.5-1.5 mg / cm², and the thickness of the hydrophobic modified layer is 10-50 nm.

[0014] In practice, the long-chain alkyl groups of stearic acid can undergo esterification with the hydroxyl groups on the surface of silica nanoparticles, constructing a hydrophobic modified layer with low surface energy on the outer surface of the microcapsules. This can block the rapid intrusion of free water in the soil, preventing the microcapsules from swelling, degrading, or releasing spores prematurely under non-target moisture conditions. Water is only allowed to slowly penetrate into the core material when soil drought is alleviated and the overall moisture environment is suitable for microbial germination. Combined with the pH response mechanism of the internal interpenetrating polymer network, precise timing control of spore release is achieved. The thickness of 10-50 nanometers and the loading of 0.5-1.5 mg / cm² can ensure the hydrophobic protection effect without completely blocking the gas exchange between the wall material and the outside world, preventing the dormant spores in the core material from being inactivated due to the anaerobic environment.

[0015] Furthermore, in the above technical solution, the microcapsule particles have a core-shell structure, the average particle size of the microcapsule particles is 0.5-2 mm, the wall thickness of the microcapsule particles is 50-150 micrometers, and the wall material has an interconnected mesoporous network with a pore size of 10-50 nanometers.

[0016] In practical implementation, the average particle size of 0.5-2 mm is suitable for conventional operations such as broadcasting and furrow application in soil, ensuring uniform dispersion of the inoculant in degraded soil; the wall thickness of 50-150 micrometers can balance the mechanical protection capability and material exchange efficiency of the microcapsules, providing sufficient physical barrier for the core material spores without hindering the migration and release of spores during response release; the interconnected mesoporous network of 10-50 nanometers inside the wall material allows for the free exchange of water and small molecule nutrients under normal conditions, maintaining the dormancy activity of spores, while blocking the invasion of protozoa and harmful macromolecules in the soil. When pH triggers swelling, the mesoporous pore size can expand synchronously, providing a smooth channel for the release of dormant spores.

[0017] To prepare the above-mentioned multifunctional degraded soil remediation compound microbial agent, the present invention also provides a method for preparing the multifunctional degraded soil remediation compound microbial agent, comprising the following technical steps: Step S1: Dissolve sodium alginate and chitosan in water to form a mixed solution, add polyacrylic acid and a crosslinking agent to the mixed solution to carry out a crosslinking reaction, and obtain a wall material solution; Step S2: Prepare a core material suspension by mixing dormant spores of phosphate-solubilizing bacteria, dormant spores of potassium-solubilizing bacteria, and dormant spores of nitrogen-fixing bacteria. Step S3: Add the core material suspension to the wall material solution and mix evenly to obtain a blended liquid. Then, drop the blended liquid through a sharp orifice into a mixed coagulation bath containing calcium chloride and citric acid to solidify it into spheres. Step S4: The microcapsule particles that have been solidified into spheres are taken out, washed, filtered and dried at low temperature to obtain the composite bacterial agent.

[0018] In step S1, sodium alginate and chitosan initially form a polyelectrolyte complex in an aqueous solution through electrostatic interaction. After the addition of polyacrylic acid and a crosslinking agent, the three components form an interpenetrating polymer network precursor through covalent crosslinking and physical entanglement, laying the structural foundation for the responsive function of the subsequent microcapsule wall material. In step S3, the sharp-pore drop-addition process can precisely control the particle size uniformity of the microcapsules. Calcium chloride in the mixed coagulation bath can undergo ion chelation with the guluronic acid fragments of sodium alginate, rapidly forming a calcium alginate gel skeleton, achieving instantaneous shaping of the microcapsules. Citric acid can adjust the pH value of the coagulation bath, slow down the crosslinking reaction rate, avoid the formation of a dense skin layer on the surface of the wall material, and ensure the permeability of the mesoporous network inside the wall material. The low-temperature drying process in step S4 can avoid damage to the activity of dormant spores by high temperature, while maintaining the porous structure of the interpenetrating polymer network, ensuring the storage stability and application activity of the final bacterial agent.

[0019] Furthermore, in the above preparation method, in step S2, when preparing the core material suspension, the silicate mineral powder is first subjected to plasma surface etching treatment. The etched silicate mineral powder is then mixed with the ascorbic acid and placed in the composite bacterial suspension. The suspension is maintained at a vacuum of 0.05-0.08 MPa for 20-40 minutes, so that the silicate mineral powder adheres to the surface of the dormant spores.

[0020] In practice, plasma surface etching can introduce a large number of active polar groups such as hydroxyl and carboxyl groups on the surface of silicate mineral powder, thereby improving the surface energy and dispersibility of the powder and enhancing its bonding force with the surface of dormant spores. Vacuum negative pressure treatment can remove the air in the gap between the composite bacterial suspension and the silicate mineral powder, so that the mineral powder adheres tightly to the surface of the dormant spores under the action of pressure difference, forming a uniform and complete nano-protective layer, which further enhances the protection effect on the dormant spores, while avoiding the agglomeration and sedimentation of mineral powder in the core material suspension, and ensuring the uniformity of the core material components.

[0021] Furthermore, in the above preparation method, in step S1, before preparing the wall material solution, the chitosan-grafted modified polyacrylic acid is prepared in advance. The chitosan is dissolved in acetic acid solution, the acrylic acid monomer and the cerium ammonium nitrate initiator are added, and the mixture is heated to 60°C for 3-5 hours under nitrogen protection. The reaction product is dialyzed, freeze-dried, and then mixed with the sodium alginate.

[0022] In practice, acetic acid solution can protonate the amino groups of chitosan, achieving uniform dissolution of chitosan and providing a homogeneous reaction system for graft polymerization; nitrogen protection can isolate oxygen in the air, preventing oxygen from quenching the free radical polymerization reaction and ensuring the smooth progress of the graft polymerization reaction; the reaction temperature of 60℃ and the reaction time of 3-5 hours can balance the rate of graft polymerization and the grafting rate, avoiding excessive reaction that leads to excessively large molecular weight of the product and decreased water solubility; the dialysis freeze-drying process can remove unreacted monomers, residual initiators and other small molecule impurities, avoiding the adverse effects of impurities on subsequent wall material crosslinking reactions and microbial spore activity.

[0023] Furthermore, in the above preparation method, in step S4, before the low-temperature drying, the washed microcapsule particles are immersed in an ethanol dispersion containing the silica nanoparticles and stearic acid, and stirred at 40-50°C for 2-4 hours. After being removed, they are washed with anhydrous ethanol and transferred to a vacuum drying oven, where they are dried at 30-40°C for 12-24 hours to form the hydrophobic modified layer on the surface of the microcapsule particles.

[0024] In practice, ethanol serves as a dispersion medium, enabling uniform dispersion of silica nanoparticles and stearic acid. Simultaneously, it allows for moderate swelling of the microcapsule wall material, enhancing the adhesion of the hydrophobic modified component to the microcapsule surface. A reaction temperature of 40-50℃ promotes esterification between the carboxyl groups of stearic acid and the hydroxyl groups on the silica nanoparticle surface, as well as the active groups on the microcapsule wall material surface. This covalent bond firmly connects the hydrophobic modified layer to the wall material surface, preventing the modified layer from detaching. Vacuum drying at 30-40℃ gently removes the solvent while avoiding damage to the activity of dormant spores within the core material, ensuring uniform film formation of the hydrophobic modified layer and achieving a stable hydrophobic protective effect.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention utilizes an interpenetrating polymer network constructed from sodium alginate, chitosan, and polyacrylic acid as the wall material, altering the fixed state of conventional gel networks. Chitosan and polyacrylic acid form an interpenetrating structure under the action of a crosslinking agent. The polyacrylic acid component provides moisture-responsive properties, while the chitosan component provides pH-responsive properties. During drought and water scarcity, the interpenetrating polymer network maintains the moisture environment of the internal core material, preventing the dehydration and inactivation of dormant spores of phosphate-solubilizing, potassium-solubilizing, and nitrogen-fixing bacteria. When the soil pH deviates from the neutral range, the intermolecular forces between polyacrylic acid and chitosan change, causing the wall material to swell and open pores. This enables the targeted release of the complex microbial community within the core material under specific pH conditions, prolonging the survival period of the microbial agent in the soil and improving the rhizosphere colonization rate.

[0026] 2. Ascorbic acid added to the core material and silicate mineral powder treated with plasma etching adhere to the surface of dormant spores, providing a reducing microenvironment and physical barrier. Cross-linked urea-formaldehyde resin microspheres are interspersed within the interpenetrating polymer network matrix inside the wall material, increasing the mechanical strength of the microcapsule particles and preventing breakage caused by soil mechanical compression. The hydrophobic modified layer coated on the outer surface of the microcapsule particles, made from the reaction of silica nanoparticles and stearic acid, blocks the rapid intrusion of external free water, preventing premature swelling or degradation of the microcapsules under non-target moisture conditions. Combined with the response mechanism of the interpenetrating polymer network, this ensures the structural integrity of the particles in the initial application stage. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention. Unless otherwise specified, the raw materials, reagents and instruments used in the present invention can be obtained commercially; the operations involved, such as the preparation of dormant microbial spores, polymer synthesis, and microcapsule preparation, all employ conventional techniques in the art unless otherwise specified.

[0028] Example 1: The composite microbial agent of this example is composed of microcapsule particles. The microcapsule particles have a core-shell structure, including a core material and a wall material covering the outside of the core material. The outer surface of the wall material is coated with a hydrophobic modified layer. Based on the total weight of the composite microbial agent, the core material accounts for 20% and the wall material accounts for 80%.

[0029] The core material is a composite bacterial suspension containing dormant spores of phosphate-solubilizing bacteria, potassium-solubilizing bacteria, and nitrogen-fixing bacteria, with a viable count ratio of 2:1:1. The composite bacterial suspension also contains 2% ascorbic acid by weight and 1% silicate mineral powder by weight. The silicate mineral powder is nano-sized montmorillonite powder with a particle size of 300 nanometers. In this embodiment, the phosphate-solubilizing bacteria is *Bacillus megaterium*, the potassium-solubilizing bacteria is *Bacillus mucilaginosus*, and the nitrogen-fixing bacteria is *Azotobacter chroococcum*. The dormant spores of these strains were all prepared using a conventional solid-state fermentation-low-temperature drying method, and the viable spore count was ≥1×10^11 CFU / g.

[0030] The wall material is an interpenetrating polymer network structure composed of sodium alginate, chitosan, and chitosan-grafted modified polyacrylic acid, with urea-formaldehyde resin microspheres cross-linked within the network. The mass ratio of sodium alginate to chitosan is 1:1, and the amount of chitosan-grafted modified polyacrylic acid added is 10% of the total weight of sodium alginate and chitosan. The urea-formaldehyde resin microspheres have a particle size of 3 micrometers and account for 5% of the total weight of the wall material, uniformly dispersed within the matrix formed by sodium alginate and chitosan. The wall material contains an interconnected mesoporous network with pore sizes of 20-40 nanometers, and the microcapsule particles have an average particle size of 1 millimeter and a wall thickness of 100 micrometers.

[0031] The hydrophobic modified layer on the outer surface of the wall material is prepared by reacting silica nanoparticles with stearic acid through a hydrophobic modification reaction. The loading of silica nanoparticles in the hydrophobic modified layer is 1.0 mg / cm², and the thickness of the hydrophobic modified layer is 30 nm.

[0032] The preparation method of the compound microbial agent in this embodiment includes the following steps: Step S1, Preparation of wall material solution: S1-1, Preparation of chitosan-grafted modified polyacrylic acid: Take 10g of chitosan with a degree of deacetylation of 90%, dissolve it in 1L of 2% (v / v) acetic acid aqueous solution, stir until completely dissolved, and purge with nitrogen for 30 minutes to remove oxygen from the system; add 100g of acrylic monomer after removing the polymerization inhibitor by vacuum distillation, stir evenly, and then add 0.3g of cerium ammonium nitrate initiator (0.3% of the mass of acrylic monomer), continue to purge with nitrogen for protection, and heat to 60℃ for 4 hours; after the reaction is completed, dialyze the reaction product through a dialysis bag with a molecular weight cutoff of 8000-14000 Da for 72 hours to remove unreacted monomers, residual initiators and small molecule impurities, and freeze-dry the dialyzed product to obtain chitosan-grafted modified polyacrylic acid for later use.

[0033] S1-2, Preparation of interpenetrating network wall material solution: Take 20g of sodium alginate and 20g of chitosan, add them to 2L of deionized water, and stir at 40℃ until completely dissolved to obtain a sodium alginate-chitosan mixed solution; add 4g of chitosan-grafted modified polyacrylic acid obtained in step S1-1 and 2.1g of urea-formaldehyde resin microspheres with a particle size of 3 micrometers to the mixed solution, stir evenly, add 100mL of 0.5% (w / v) glutaraldehyde crosslinking agent, stir at 25℃ for 30 minutes to crosslink the reaction, and obtain a uniform wall material solution for later use.

[0034] Step S2, Preparation of core material suspension: Take 20g of dormant spores of Bacillus megaterium, 10g of dormant spores of Bacillus mucilaginosus, and 10g of dormant spores of Azotobacter chrysophagus, add them to 1L of sterile deionized water, and stir evenly to obtain a basic bacterial suspension with a viable count of approximately 5×10^10 CFU / mL; take 10g of nano-montmorillonite powder, place it in a plasma etching machine, and perform surface etching treatment using argon plasma at a radio frequency power of 100W, a working pressure of 10Pa, and an etching time of 15 minutes to obtain etched silicate mineral powder; add 20g of ascorbic acid and 10g of etched silicate mineral powder to the basic bacterial suspension, stir evenly, and place it in a vacuum jar. Maintain the vacuum at 0.06MPa for 30 minutes to allow the silicate mineral powder to uniformly adhere to the surface of the dormant spores, obtaining a core material suspension for later use.

[0035] Step S3: Microcapsules solidify into spheres: The core material suspension obtained in step S2 is slowly added to the wall material solution obtained in step S1, and stirred at a constant temperature of 30°C for 30 minutes to obtain a uniformly mixed solution. The mixed solution is then dripped into a mixing and coagulation bath through a sharp-orifice dripping device with a diameter of 0.8 mm and a dripping rate of 60 drops / minute. The mixing and coagulation bath is an aqueous solution containing 2% (w / v) calcium chloride and 0.5% (w / v) citric acid. The curing temperature is 25°C and the curing time is 30 minutes to obtain solidified microcapsule particles.

[0036] Step S4, Post-treatment and hydrophobic modification: After solidifying into spheres, the microcapsule particles were removed from the coagulation bath and washed three times with sterile deionized water. The surface free water was removed by filtration. The washed microcapsule particles were then immersed in 1L of anhydrous ethanol dispersion containing 20g of silica nanoparticles and 10g of stearic acid. The mixture was stirred at 45°C for 3 hours to form a hydrophobic modified layer on the surface of the microcapsules. The microcapsule particles were then removed, washed twice with anhydrous ethanol, and transferred to a vacuum drying oven. The mixture was then vacuum dried at 35°C for 18 hours to obtain the multifunctional degraded soil remediation composite microbial agent of this embodiment.

[0037] Example 2: The composite microbial agent in this example has a core material ratio of 10% and a wall material ratio of 90% based on the total weight of the composite microbial agent; all other formulations and preparation methods are exactly the same as in Example 1.

[0038] Example 3: The compound microbial agent in this example has a core material ratio of 30% and a wall material ratio of 70% based on the total weight of the compound microbial agent; all other formulations and preparation methods are exactly the same as in Example 1.

[0039] Example 4: In this example, the composite microbial agent has a mass ratio of sodium alginate to chitosan of 1:0.5 in the wall material; all other formulations and preparation conditions are exactly the same as in Example 1.

[0040] Example 5: In this example, the composite microbial agent has a mass ratio of sodium alginate to chitosan of 1:1.5 in the wall material; all other formulations and preparation conditions are exactly the same as in Example 1.

[0041] Example 6: In this example, the amount of chitosan-grafted modified polyacrylic acid added to the composite microbial agent is 5% of the total weight of sodium alginate and chitosan; all other formulations and preparation methods are exactly the same as in Example 1.

[0042] Example 7: In this example, the amount of chitosan-grafted modified polyacrylic acid added to the composite microbial agent is 15% of the total weight of sodium alginate and chitosan; all other formulations and preparation methods are exactly the same as in Example 1.

[0043] Example 8: In the composite bacterial agent of this example, the amount of ascorbic acid added to the composite bacterial suspension of the core material is 1% of the total weight of the composite bacterial suspension, the amount of silicate mineral powder added is 0.5% of the total weight of the composite bacterial suspension, and the particle size of the silicate mineral powder is 200 nanometers; all other formulations and preparation methods are exactly the same as in Example 1.

[0044] Example 9: In the composite bacterial agent of this example, the amount of ascorbic acid added to the composite bacterial suspension of the core material is 3% of the total weight of the composite bacterial suspension, the amount of silicate mineral powder added is 2% of the total weight of the composite bacterial suspension, and the particle size of the silicate mineral powder is 500 nanometers; all other formulations and preparation methods are exactly the same as in Example 1.

[0045] Example 10: The compound microbial agent of this example has the same formulation as that of Example 1; in the preparation method, the chitosan grafted modified polyacrylic acid preparation process in step S1-1 is kept at a constant temperature of 60°C for 3 hours; all other conditions of the preparation method are exactly the same as those of Example 1.

[0046] Example 11: The compound microbial agent of this example has the same formulation as that of Example 1; in the preparation method, the vacuum degree of the core material suspension preparation process in step S2 is 0.08 MPa and the maintenance time is 20 minutes; all other conditions of the preparation method are exactly the same as those of Example 1.

[0047] Example 12: The compound microbial agent of this example has the same formulation as that of Example 1; in the preparation method, the reaction temperature in step S4, the hydrophobic modification process, is 50°C and the reaction time is 2 hours; all other conditions of the preparation method are exactly the same as those of Example 1.

[0048] Comparative Example 1: The composite microbial agent of this comparative example does not contain polyacrylic acid (including chitosan-grafted modified polyacrylic acid) in the wall material. All other formulations and preparation conditions are exactly the same as those in Example 1.

[0049] Comparative Example 2: This comparative example is a conventional calcium alginate gel microsphere bacterial agent from the prior art. The formulation and preparation method are as follows: Take 20g of Bacillus megaterium dormant spores, 10g of Bacillus mucilaginosus dormant spores, and 10g of Azotobacter chrysophagus dormant spores, add them to 1L of 2% (w / v) sodium alginate aqueous solution, and stir evenly to obtain a blend; add the blend to 2% (w / v) calcium chloride aqueous solution through a sharp-hole dropper, and solidify at 25℃ for 30 minutes to obtain microcapsule particles; wash 3 times with sterile deionized water, and vacuum dry at 35℃ for 18 hours to obtain the bacterial agent of this comparative example.

[0050] Comparative Example 3: In this comparative example, the amount of chitosan-grafted modified polyacrylic acid added to the compound bacterial agent was 20% of the total weight of sodium alginate and chitosan; all other formulations and preparation methods were exactly the same as in Example 1.

[0051] Comparative Example 4: The preparation method of the composite bacterial agent in this comparative example omits the hydrophobic modification process in step S4. That is, the washed microcapsule particles are directly vacuum dried at 35°C for 18 hours without the preparation of the hydrophobic modification layer. All other formulations and preparation conditions are exactly the same as those in Example 1.

[0052] Test method: Drought stress survival rate test: The test fungal agent was placed in a constant temperature and humidity chamber with a relative humidity of 20% and a temperature of 25℃ to simulate drought stress. After 7 days, the number of viable spores in the fungal agent was detected by plate dilution coating method, and the spore survival rate was calculated (survival rate = number of viable bacteria after stress / initial number of viable bacteria × 100%).

[0053] pH response release rate test: Equal amounts of the test agent were placed in neutral phosphate buffer (pH=7.0), acidic phosphate buffer (pH=5.5), and alkaline Tris-HCl buffer (pH=8.5), respectively, and incubated at 25°C for 24 hours. The supernatant was then used to detect the number of viable bacteria released by the plate dilution method, and the release rate was calculated (release rate = number of viable bacteria in supernatant / total number of viable bacteria in the agent × 100%).

[0054] Mechanical strength test: The compression breakage rate of microcapsule particles was tested using a texture analyzer. 100 microcapsule particles were taken and compressed at a compression rate of 1 mm / min. The compression was stopped when the deformation reached 30%. The number of broken particles was counted and the breakage rate was calculated (breakage rate = number of broken particles / total number of particles × 100%).

[0055] Storage stability test: The test agent was sealed and stored in an environment with a temperature of 25℃ and a relative humidity of 60% for 3 months. The number of viable spores after storage was detected by the plate dilution coating method, and the spore survival rate was calculated.

[0056] Rhizosphere colonization test: Degraded soil was divided into planting pots, and an equal amount of the test inoculant was applied to each pot. Corn seedlings were planted and placed in a greenhouse for conventional culture for 28 days. Corn rhizosphere soil was collected, and the viable count of the target strain in the rhizosphere soil was detected by plate dilution spread method. The results are expressed as 1g CFU / g dry soil.

[0057] Test results: Table 1 Performance test results of each embodiment and comparative example

[0058] Results analysis: The spore survival rate of Examples 1-12 after 7 days of drought stress all reached over 86%, with the best Example 1 achieving a survival rate as high as 92.3%, while Comparative Example 1 only achieved 42.6% and Comparative Example 2 only 31.2%. This is because the present invention utilizes an interpenetrating polymer network constructed from sodium alginate, chitosan, and polyacrylic acid. The carboxyl groups of the polyacrylic acid can strongly lock the bound water within the network through hydrogen bonding, maintaining a stable water activity microenvironment for the core spores under drought stress and preventing spore dehydration and inactivation. Comparative Example 1 lacks the polyacrylic acid core component, completely losing its water-locking ability, while Comparative Example 2 uses a conventional single calcium alginate network without a stable water-locking structure, resulting in extremely poor spore retention.

[0059] Examples 1-12 showed a release rate of less than 14% under neutral pH=7.0 conditions, while the release rate reached more than 82% under slightly acidic pH=5.5 and slightly alkaline pH=8.5 conditions, achieving a significant acid-base responsive targeted release effect. The principle is that the chitosan amino and polyacrylic acid carboxyl groups in the interpenetrating network of this invention can undergo reversible protonation and dissociation with changes in environmental pH, changing the intermolecular electrostatic forces and driving the directional regulation of network swelling degree and porosity, opening the release channel only when the soil pH deviates from neutral. Comparative Example 1 lacks polyacrylic acid and has no pH response regulation ability, with a release rate of only about 20% under acidic and alkaline conditions. The conventional calcium alginate network of Comparative Example 2 has no pH response characteristics, with a release rate as high as 45.6% under neutral conditions, which cannot achieve targeted release, resulting in spores being released prematurely and unable to effectively colonize in the rhizosphere. Comparative Example 4 did not prepare a hydrophobic modification layer, and under neutral conditions, water rapidly invaded, causing the microcapsules to swell prematurely, with a release rate as high as 38.7%, losing the ability to control the timing of targeted release.

[0060] The compression breakage rate of Examples 1-12 was less than 5%, and the survival rate after 3 months of storage was higher than 85%, which was far superior to the comparative examples. The reason is that the cross-linked urea-formaldehyde resin microspheres in the wall material of the present invention act as rigid fillers, which significantly improves the compressive modulus and deformation resistance of the interpenetrating network and avoids microcapsule rupture caused by soil mechanical compression; the hydrophobic modified layer on the outer surface can block the rapid intrusion of external moisture and oxygen, avoid premature degradation of microcapsules and oxidative inactivation of spores, and greatly improve storage stability; the conventional calcium alginate gel microspheres of Comparative Example 2 have no rigid reinforcement structure, the breakage rate is as high as 28.7%, and the spore survival rate after 3 months of storage is only 25.4%; the amount of polyacrylic acid added in Comparative Example 3 exceeds the limit, resulting in non-uniform interpenetrating network structure and a significant decrease in mechanical strength and stability.

[0061] After 28 days of application, the number of viable bacteria colonizing the rhizosphere in Examples 1-12 all reached over 7.41g CFU / g, with the best example 1 reaching as high as 7.821g CFU / g. In contrast, the colonization numbers in Comparative Examples 1-4 were all below 61g CFU / g, with Comparative Example 2 only reaching 2.871g CFU / g. The core reason for this is that the interpenetrating network of this invention enables spore survival under drought stress and targeted release of bacteria in response to pH. Combined with the time-sequential control of the hydrophobic modification layer, spores are precisely released only in the rhizosphere microenvironment of degraded soil (pH deviating from neutral and moisture suitable), significantly improving spore germination rate and rhizosphere colonization efficiency. In contrast, each comparative example suffered from problems such as premature spore inactivation, premature non-targeted release, and microcapsule rupture, resulting in spores being unable to effectively colonize the rhizosphere and extremely poor repair effects.

[0062] In summary, this invention, through the construction of an interpenetrating polymer network of sodium alginate-chitosan-polyacrylic acid, combined with a spore protection system in the core material, a rigid reinforcement structure in the wall material, and a hydrophobic modified surface layer, overcomes the technical shortcomings of existing technologies, such as poor bacterial agent viability, lack of targeted bacterial release capability, low mechanical strength, poor storage stability, and low rhizosphere colonization rate, achieving unexpected technical effects. The parameter ranges in each embodiment all achieve stable technical effects, while the absence of core components, parameters exceeding the defined range, and the use of existing technical solutions all lead to a significant decrease in technical effects, fully demonstrating the inventiveness and necessity of the technical solution of this invention.

Claims

1. A multifunctional compound microbial agent for remediating degraded soil, characterized in that, The compound microbial agent is composed of microcapsule particles, wherein the microcapsule particles include a core material and a wall material covering the outside of the core material; Based on the total weight of the compound microbial agent, the core material accounts for 10%-30%, and the wall material accounts for 70%-90%. The core material is a composite bacterial suspension containing dormant spores of phosphate-solubilizing bacteria, dormant spores of potassium-solubilizing bacteria, and dormant spores of nitrogen-fixing bacteria; The wall material is an interpenetrating polymer network structure composed of sodium alginate, chitosan and polyacrylic acid. The mass ratio of sodium alginate to chitosan in the interpenetrating polymer network structure is 1:0.5-1.5, and the amount of polyacrylic acid added is 5%-15% of the total weight of sodium alginate and chitosan.

2. The multifunctional degraded soil remediation compound microbial agent according to claim 1, characterized in that, The core material contains a 2:1:1 ratio of live phosphate-solubilizing bacteria dormant spores, potassium-solubilizing bacteria dormant spores, and nitrogen-fixing bacteria dormant spores. The core material also contains 1%-3% ascorbic acid (based on the total weight of the composite bacterial suspension) and 0.5%-2% silicate mineral powder (based on the total weight of the composite bacterial suspension), wherein the silicate mineral powder has a particle size of 200-500 nanometers.

3. The multifunctional degraded soil remediation compound microbial agent according to claim 1, characterized in that, The polyacrylic acid in the wall material is chitosan-grafted polyacrylic acid. The grafted polyacrylic acid is obtained by grafting and polymerizing acrylic monomers onto the main chain of chitosan in the presence of an initiator. The initiator is cerium ammonium nitrate, and the amount of the initiator added is 0.1%-0.5% of the mass of the acrylic monomers.

4. A multifunctional compound microbial agent for remediating degraded soil according to claim 1 or 3, characterized in that, The interpenetrating polymer network structure of the wall material is further cross-linked with urea-formaldehyde resin microspheres, which are uniformly dispersed in the matrix formed by sodium alginate and chitosan. The particle size of the urea-formaldehyde resin microspheres is 1-5 micrometers, and the amount of urea-formaldehyde resin microspheres added accounts for 3%-8% of the total weight of the wall material.

5. The multifunctional degraded soil remediation compound microbial agent according to claim 1, characterized in that, The outer surface of the wall material is also coated with a hydrophobic modified layer, which is prepared by reacting silica nanoparticles with stearic acid through a hydrophobic modification reaction. The loading of silica nanoparticles in the hydrophobic modified layer is 0.5-1.5 mg / cm², and the thickness of the hydrophobic modified layer is 10-50 nm.

6. The multifunctional degraded soil remediation compound microbial agent according to claim 1, characterized in that, The microcapsule particles have a core-shell structure, with an average particle size of 0.5-2 mm and a wall thickness of 50-150 micrometers. The wall material has an interconnected mesoporous network with pore sizes of 10-50 nanometers.

7. A method for preparing a multifunctional degraded soil remediation compound microbial agent, used to prepare the compound microbial agent according to any one of claims 1-6, characterized in that, Includes the following steps: Step S1: Dissolve sodium alginate and chitosan in water to form a mixed solution, add polyacrylic acid and a crosslinking agent to the mixed solution to carry out a crosslinking reaction, and obtain a wall material solution; Step S2: Prepare a core material suspension by mixing dormant spores of phosphate-solubilizing bacteria, dormant spores of potassium-solubilizing bacteria, and dormant spores of nitrogen-fixing bacteria. Step S3: Add the core material suspension to the wall material solution and mix evenly to obtain a blended liquid. Then, drop the blended liquid through a sharp orifice into a mixed coagulation bath containing calcium chloride and citric acid to solidify it into spheres. Step S4: The microcapsule particles that have been solidified into spheres are taken out, washed, filtered and dried at low temperature to obtain the composite bacterial agent.

8. The preparation method of a multifunctional degraded soil remediation compound microbial agent according to claim 7, characterized in that, In step S2, when preparing the core material suspension, the silicate mineral powder is first subjected to plasma surface etching treatment. The etched silicate mineral powder is then mixed with ascorbic acid and placed in the composite bacterial suspension. The suspension is maintained at a vacuum of 0.05-0.08 MPa for 20-40 minutes, so that the silicate mineral powder adheres to the surface of the dormant spores.

9. The preparation method of a multifunctional degraded soil remediation compound microbial agent according to claim 7, characterized in that, In step S1, before preparing the wall material solution, the chitosan-grafted modified polyacrylic acid is prepared in advance. Chitosan is dissolved in acetic acid solution, and the acrylic acid monomer and the cerium ammonium nitrate initiator are added. The mixture is heated to 60°C and reacted for 3-5 hours under nitrogen protection. The reaction product is dialyzed, freeze-dried, and then mixed with the sodium alginate.

10. The preparation method of a multifunctional degraded soil remediation compound microbial agent according to claim 7, characterized in that, In step S4, before the low-temperature drying, the washed microcapsule particles are immersed in an ethanol dispersion containing the silica nanoparticles and stearic acid, and stirred at 40-50°C for 2-4 hours. After being removed, they are washed with anhydrous ethanol and transferred to a vacuum drying oven, where they are dried at 30-40°C for 12-24 hours to form the hydrophobic modified layer on the surface of the microcapsule particles.

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