Granular microbial fertilizer embedding functional bacteria with fungal mycelial pellets and preparation method thereof

Abstract

The present application discloses a granular microbial fertilizer of embedding functional bacteria in fungal mycelial balls and a preparation method thereof, and belongs to the field of agricultural microorganism technology. In view of the technical problems of low survival rate of living bacteria, short shelf life, poor stress resistance, insufficient colonization rate, inconvenient storage and transportation of liquid dosage form, weak combination of solid adsorption method, and high cost of microcapsule embedding of existing microbial fertilizers, the present application uses the spherical mycelium formed by filamentous fungus fermentation as a natural microcarrier. Through a two-stage fermentation process, bacteria with functions of promoting growth and improving soil structure are efficiently embedded in the network structure inside and surface of the mycelial ball. After separation, protective agent treatment and drying, a granular microbial fertilizer is obtained. The microbial fertilizer has low carrier cost, good biocompatibility, high number of living bacteria and long shelf life. The mycelial ball provides a physical barrier for the functional bacteria, improves the stress resistance and colonization ability of the bacteria in the soil, and increases soil organic matter after decomposition of the fungal carrier, realizing the synergistic effect of fungi and bacteria.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology. More specifically, this invention relates to a granular microbial fertilizer containing fungal mycelial balls encapsulating functional bacteria, and a method for preparing the same. Background Technology

[0002] As modern agriculture steadily advances towards a green and sustainable direction, reducing the use of chemical fertilizers and pesticides has become an inevitable trend in global agricultural development. Microbial fertilizers, as eco-friendly agricultural inputs, leverage the life activities of rhizosphere growth-promoting bacteria (PGPR) through mechanisms such as nitrogen fixation, phosphorus solubilization, potassium solubilization, and the synthesis of plant growth hormones. These mechanisms effectively improve the soil microecological environment, promote crop growth, and enhance their disease resistance. Currently, the market demand for highly active, highly stable, and easy-to-apply compound microbial agents continues to grow, especially those compound formulations that simultaneously exert the dual effects of fungi (such as biocontrol Trichoderma and Aspergillus niger) and bacteria (such as Bacillus), which have attracted significant attention due to their remarkable synergistic effects.

[0003] Existing microbial fertilizers are mainly divided into two categories: liquid and solid formulations. Although the production process of liquid microbial agents is relatively simple and does not require a complex drying process, they face many challenges in storage and transportation: microbial metabolism is active in liquid environments, which can easily lead to the depletion of nutrients and the accumulation of metabolic waste, resulting in a sharp decline in the number of viable bacteria in a short period of time and a generally short shelf life; at the same time, liquid formulations have strict requirements for packaging sealing, making them very susceptible to bottle swelling or contamination by other microorganisms, and they need to be diluted with water before application in the field, which increases the labor intensity of farmers and limits their application in large-scale mechanized sowing.

[0004] To overcome the aforementioned drawbacks of liquid formulations, most current mainstream solid microbial fertilizers are prepared using the "adsorption method." This involves using peat moss, lignite, straw powder, or porous minerals (such as vermiculite and diatomaceous earth) as carriers, onto which the fermented bacterial solution is sprayed and adsorbed. However, this physical adsorption-based binding method results in weak adhesion; the functional bacteria often simply attach to the surface of the carrier or remain in shallow pores, in a nearly exposed state. During drying, processing, and subsequent storage, transportation, and sowing, these exposed microorganisms are susceptible to environmental stresses such as ultraviolet radiation, high-temperature drying, and mechanical friction, leading to cell wall damage or even death. Ultimately, this results in low colonization rates and unstable fertilizer efficacy after the product is applied to the soil.

[0005] To improve microbial survival rates, microencapsulation technology has emerged. Current technologies often use chemical and biological polymers such as sodium alginate-calcium chloride, carrageenan, or polyvinyl alcohol as wall materials, encapsulating bacteria within gel beads through chemical cross-linking. While this method provides some protection, its industrial application faces significant bottlenecks: first, the high cost of raw materials like sodium alginate significantly increases fertilizer production costs; second, the preparation process typically requires specialized extrusion dripping or electrostatic granulation equipment, resulting in complex processes and limited production capacity; and third, the degradation rate of the chemically cross-linked gel shell in soil is difficult to precisely control, potentially leading to degradation residues or hindering timely release of microorganisms, thus affecting the fertilizer's rapid effectiveness.

[0006] During submerged fermentation in liquids, filamentous fungi, under specific hydraulic shear stress and culture medium conditions, undergo hyphae entanglement and self-flocculation to form dense, resilient spherical bodies (mycelial balls). These mycelial balls naturally possess a microcapsule-like three-dimensional network structure, making them ideal microbial carriers. However, current research on mycelial balls largely focuses on the fermentation optimization of single fungi or their adsorption applications in wastewater treatment. Existing technologies lack mature processes that can utilize the "self-assembly" characteristics of fungal mycelial ball formation to actively "capture" and encapsulate heterologous functional bacteria within the mycelial balls through precise co-cultivation timing control. Solving the nutrient competition conflict during fungal-bacterial co-cultivation and achieving high-density in-situ bacterial encapsulation has become a critical technical challenge in the preparation of novel granular microbial fertilizers. Summary of the Invention

[0007] One object of the present invention is to solve the problems existing in the prior art described above, and to provide the advantages that will be described later.

[0008] Another objective of this invention is to provide a method for preparing granular microbial fertilizer in which functional bacteria are embedded in fungal mycelial balls. This method utilizes mycelial balls with a natural three-dimensional network structure formed during the deep liquid fermentation process of spherical fungi as natural biological microcarriers. Through a precise "sequential co-cultivation" process, functional bacteria are induced to be in situ encapsulated inside the mycelial balls, achieving the "self-immobilization" of microorganisms. This method produces a low-cost, dense, highly bioactive, fungal-bacterial symbiotic structure with high survival rate, strong stress resistance, and slow-release function granular compound microbial fertilizer.

[0009] To achieve these objectives and other advantages according to the present invention, a method for preparing granular microbial fertilizer containing fungal mycelial spheres encapsulating functional bacteria is provided, comprising the following steps: S1. Strain pretreatment: Prepare fungal spore suspension and functional bacterial seed liquid separately for fungal spores and functional bacteria. S2, Core Formation Stage: Fungal spore suspension is inoculated into modified pelleting medium and cultured under a specific shear field until loose, flocculent mycelial clusters with a diameter of 0.5-1.5 mm are formed in the fermentation broth; S3, Phase Change Encapsulation Period: Functional bacterial seed liquid is added to the fermentation broth that forms loose flocculent mycelial clusters. By adjusting the dissolved oxygen and pH value of the fermentation broth, the mycelium is induced to shrink and compact inward, thereby physically capturing the functional bacteria inside the mycelial network and forming symbiotic mycelial balls with a diameter of 2.0-4.0 mm. S4. Surface strengthening and solidification: Collect the wet symbiotic mycelial balls obtained in step S3, immerse them in a composite protective agent solution for surface film formation treatment, and then use a gradient temperature drying method to dehydrate them until the moisture content is ≤8%, thus obtaining granular microbial fertilizer.

[0010] The collection and cleaning of the symbiotic mycelial balls includes: separating the fermentation broth after fermentation in step S3 by passing it through a 60-mesh sterile stainless steel screen or a plate and frame filter press to collect the retained wet symbiotic mycelial balls; then spraying or rinsing the collected wet symbiotic mycelial balls 2-3 times with sterile physiological saline or sterile water, each rinse lasting 1-2 minutes, until the number of free bacteria in the cleaning solution is ≤1.0×10⁻⁶. 3 The washing solution should be CFU / mL and the pH of the washing solution should be stable at 5.0-5.5 to ensure that there are no residual culture medium components and metabolic wastes, and to prevent residual nutrients from causing the growth of miscellaneous bacteria during subsequent drying or storage. After washing, the wet symbiotic mycelial balls should be drained of surface moisture and become loose granules, ready for surface film formation treatment.

[0011] The immersion film-forming treatment and permeation protection include: placing the drained, clean, wet symbiotic mycelial balls into a porous mesh basket, immersing the entire ball in a composite protective agent solution, ensuring all particles are completely submerged, and gently shaking the basket to eliminate air bubbles; maintaining the soaking state for 20-30 minutes, utilizing the concentration difference to allow trehalose and monosodium glutamate to penetrate into the shallow layer of the mycelial balls and the interior of the cells to replace water molecules and protect biological activity, while utilizing the film-forming properties of polyvinyl alcohol (PVA) to adsorb and form a highly tough, semi-permeable gel film on the outermost layer of the mycelial balls; after soaking, lifting the basket and allowing it to drain naturally for 5-10 minutes until no liquid drips from the surface of the particles.

[0012] Preferably, in step S2, the components of the modified pelleting medium, calculated in g / L, include: 15-30 g of carbon source, 3-8 g of nitrogen source, 1-3 g of KH₂PO₄, 0.5-1.0 g of MgSO₄·7H₂O, and a pelleting inducing factor, wherein the pelleting inducing factor is composed of 0.2-0.5 g / L CaCl₂ and 0.5-1.5 g / L sodium carboxymethyl cellulose. The carbon source is selected from one or more of corn starch, glucose, and sucrose, and the nitrogen source is selected from one or more of soybean meal, yeast powder, and tryptone.

[0013] Preferably, step S2 specifically includes: S21. Preparation of modified spheroidizing culture medium: Mix 15.0-30.0 g of carbon source, 3.0-8.0 g of nitrogen source, 1.0-3.0 g of KH2PO4, 0.5-1.0 g of MgSO4·7H2O, 0.2-0.5 g of CaCl2 and 0.5-1.5 g of sodium carboxymethyl cellulose, and bring the volume to 1000 mL with pure water. After thorough stirring and dissolution, adjust the pH to 5.5-6.0 with 0.5 mol / L dilute hydrochloric acid. S22. Sterilization and testing of modified pelleting culture medium: Place the modified pelleting fermentation medium prepared in step S21 into an autoclave and sterilize at 121℃ for 20-30 minutes; after sterilization, randomly select some samples and place them in a constant temperature incubator at 28℃-30℃ for blank culture for 24-48 hours to check the thoroughness of sterilization and ensure that no contaminating bacteria grow; S23. Pre-setting the pelleting environment: The modified pelleting fermentation medium, free of contaminants and passing the S22 test, is loaded into a fully automated fermenter equipped with temperature and speed control. The filling coefficient is controlled at 65%-70%. Sterile air is introduced to maintain the tank pressure at 0.03-0.05 MPa. The medium temperature is rapidly reduced to the optimal growth temperature of fungi (25℃-28℃) using a cooling jacket. The initial pH value is measured and recorded, typically 5.5-6.0, as the zero-point reference for fermentation. At the flame protection ring or aseptic interface, a pre-treated fungal spore suspension (spore concentration ≥1.0×10⁻⁶) is inoculated at a rate of 5%-8% (v / v). 7 (number / mL) was fed into the fermenter; S24. Control the tip linear velocity of the impeller of the fermenter to 1.2-1.8 m / s to create a hydrodynamic environment. Cultivate under this specific shear force field until loose, flocculent mycelial clusters with a diameter of 0.5-1.5 mm are formed in the fermentation broth.

[0014] Preferably, step S24 specifically includes: S241. Constructing a specific shear force field environment: In fungal fermentation, the rotational speed of the fermenter's agitator motor is adjusted. Based on the diameter D of the fermenter's impeller and the rotational speed n, the impeller tip linear velocity v is calculated using the formula: v = πDn / 60. The impeller tip linear velocity v is then kept constant within the range of 1.2-1.8 m / s. Cultivation is carried out under this specific shear force field. Under this specific shear force field, the collision probability provided by fluid turbulence and the calcium ion (Ca) are utilized. 2+ The cross-linking effect of dextran in the mycelial cell wall induces the entanglement and aggregation of filamentous hyphae; at the same time, sodium carboxymethyl cellulose (CMC-Na) increases the viscosity of the medium, buffering the instantaneous impact force to 0.3-0.5 N, preventing the mycelial clumps from being broken up. The two work together to achieve stable spherical formation and prevent the mycelial clumps from being broken up. S242. Monitoring biological and physicochemical indicators: The culture process is monitored in real time, with the fungal fermentation time T and the pH of the fermentation broth as dual judgment indicators. Samples are continuously taken for microscopic examination to observe the mycelial morphology, and pH changes are recorded every 1-2 hours. By dynamically controlling process parameters, the temperature is kept constant at 28℃ during the core construction period. Dissolved oxygen (DO) changes are closely monitored. As the mycelium grows rapidly, DO tends to decrease. By adjusting the aeration rate (0.8-1.2 vvm) and stirring speed (fine-tuning while maintaining a linear velocity ≤1.8 m / s), the DO in the fermentation broth is maintained at no less than 30%, ensuring that the mycelium has sufficient energy for framework construction and preventing mycelial autolysis or failure to form clusters due to lack of oxygen. The endpoint of framework morphology is determined by taking samples for microscopic examination every 2 hours starting from the 12th hour of fermentation. The judgment criteria are: when a large number of microscopic particles are visible to the naked eye in the fermentation broth, and these particles are observed under a microscope to have a loose flocculent structure with a "slightly dense center and radially radiating edges", with a diameter between 0.5-1.5 mm, the "core construction period" is considered to be over. At this time, the mycelial cluster is like an open sponge net, with the maximum physical capture space, and is ready to enter the next stage.

[0015] Preferably, a "germination protection" step is included before step S241, specifically as follows: Within the first 4-6 hours after inoculating the fungal spore suspension, the linear velocity of the impeller tip should be controlled at a low shear state of 0.5-0.8 m / s, and the aeration rate should be controlled at 0.5-0.8 vvm to avoid strong mechanical shear force damaging the germinating young hyphae and ensure that the spores germinate synchronously and form a primary hyphal network. When microscopic examination shows that more than 90% of the spores have germinated and grown hyphae with a length greater than 3 times the diameter of the spores, the specific shear force field environment of step S241 should be established.

[0016] Preferably, step S3 specifically includes: S31. Timing of inoculation of functional bacterial seed liquid: When the fermentation of the fungal spore suspension in step S2 has proceeded for 18-28 hours and the pH value of the fermentation liquid has naturally decreased to 4.5-5.0 under real-time monitoring, it is determined to be the endpoint of the formation of loose flocculent mycelial clusters. S32. Using a gradient strategy of "slow at first, then fast", functional bacterial seed liquid is added to the fermentation broth that has formed loose flocculent mycelial clusters. The flow rate is 5-8 mL / min for the first 5 minutes, and then increased to 10-15 mL / min. The inoculation amount of functional bacterial seed liquid is 10%-15% of the fermentation broth volume. The specific shear force field parameters are kept constant. The dissolved oxygen of the fermentation broth is adjusted to 25%-40%, and the pH is maintained at a natural weakly acidic environment of 4.5-5.0. After co-culturing for 24-36 hours, symbiotic mycelial balls with a diameter of 2.0-4.0 mm are formed.

[0017] Before the inoculation phase of the fed-bacterial seed culture, it is essential to ensure the accuracy of the fermentation system's monitoring and the activity of the inoculum. Specifically, the online monitoring probes of the fermenter must be systematically checked and calibrated to ensure that the pH electrode drift is controlled within ±0.05 and the dissolved oxygen (DO) electrode response time is less than 60 seconds, guaranteeing the real-time and accurate feedback of process parameters. Simultaneously, within 30 minutes before inoculation, a small amount of the inoculated functional bacterial seed culture is aseptically removed for rapid viability testing. Microscopic observation or optical density (OD) measurement confirms that the functional bacteria are in the late logarithmic growth phase and that the viable cell concentration is not less than 1.0 × 10⁻⁶. 9The concentration of CFU / mL was checked, and the absence of contaminating bacteria was confirmed to ensure sufficient competitiveness and population advantage for the functional bacteria after inoculation into the fermentation system. Biological and physicochemical indicators were monitored in step S242, and the fermentation process was monitored frequently. The optimal inoculation time was determined through dual verification of morphological and biochemical indicators. The "phase transition embedding window" was considered to have been entered when both of the following conditions were met: Firstly, microscopic examination confirmed that over 90% of the fungal mycelia in the field of view exhibited a loose, flocculent or spongy structure with a diameter between 0.5 and 1.5 mm. At this time, the interstices of the mycelia were fully open, and a dense hydrophobic shell had not yet formed, providing maximum physical containment space. Secondly, the pH value monitored online decreased from the initial 5.5-6.0 to the 4.5-5.0 range, indicating that the fungi had established an absolute metabolic advantage and formed a stable acid-producing environment. This was the optimal time for heterologous microbial integration. Once the monitoring system confirms that the above-mentioned optimal moment has been captured, the stirring linear velocity (1.2-1.8 m / s) set in step S24 is kept constant. The peristaltic pump is turned on, and the pre-prepared functional bacterial seed liquid is introduced into the fermenter through the deep feeding pipe. In order to prevent the local bacterial concentration from causing a sudden change in the rheological properties of the fermentation broth or the destruction of the mycelial ball structure, a gradient feeding strategy of "slow first and then fast" is adopted. That is, the flow rate is controlled at 5-8 mL / min in the first 5 minutes. After the bacteria are initially dispersed, the flow rate is increased to 10-15 mL / min until the feeding is completed. During this process, the turbulent vortex generated by the stirring blades is used to quickly entrain and disperse the high concentration of bacteria into the mesh gaps of the loose mycelial ball, so as to achieve uniform distribution and adsorption of bacteria in the three-dimensional space of the fungal skeleton. After the functional bacterial seed culture is introduced, the respiration intensity increases sharply due to the co-culture of the two bacteria, and the dissolved oxygen (DO) value of the fermentation broth will drop rapidly. At this time, it is necessary to activate the "DO cascade control mode" to balance the needs of bacterial survival and fungal pelleting: when the DO value is monitored to be below 25%, the dissolved oxygen level is increased by adjusting the air flow rate (gradually increasing from 1.0 vvm to 1.5 vvm) or supplementing with pure oxygen. The stirring speed should be avoided as much as possible to prevent excessive mechanical shear force from breaking up the not-yet-dense hyphal skeleton. At the same time, the DO level is maintained at a moderate level of 25%-40%, which can meet the basic survival needs of aerobic bacteria and induce the fungal hyphae to age, branch and entangle more rapidly through slight oxygen stress slightly below the optimal DO for fungi, thereby initiating the defensive pelleting mechanism of inward contraction.In the first 12-18 hours of co-culturing, biocontraction induction was achieved without artificially adding alkali to adjust the pH. Instead, the fermentation broth was maintained at a naturally weakly acidic environment of pH 4.5-5.0 for induction. This specific acidic environment has a dual regulatory effect: on the one hand, the weakly acidic conditions can appropriately inhibit the explosive proliferation of functional bacteria, preventing them from excessively consuming carbon sources due to rapid growth, which would lead to the disintegration of the fungal skeleton due to nutrient deficiency; on the other hand, the acidic stimulation causes the chitin and glucan in the fungal cell wall to undergo structural rearrangement and cross-linking, which macroscopically manifests as the loose, flocculent hyphal clusters beginning to shrink and densify drastically towards the center, thereby physically "locking" the functional bacteria dispersed inside the spherical core and forming a stable embedding structure. Maturation and endpoint determination of the symbiotic structure: Continue co-culturing for 24-36 hours under the above environmental conditions, closely observe the changes in the morphology and physicochemical parameters of the mycelial balls to determine the fermentation endpoint; when the fermentation broth gradually becomes clear from turbid (indicating that the free bacteria have been embedded or settled), and the diameter of the mycelial balls increases to 2.0-4.0 mm, and the surface changes from a fluffy texture to a smooth and dense membrane structure; or when the mycelial balls are cut open and microscopic examination reveals that the internal mycelial network is densely filled with rod-shaped bacteria or spores; or when the online pH value begins to show an upward trend (indicating that the readily available carbon source is depleted and the microorganisms begin to utilize organic acids), the co-culturing process is considered to be over. At this point, fermentation can be stopped, and preparation can be made to enter the post-processing stage of step S4.

[0018] Preferably, in step S4, the composite protective agent solution is composed of the following components by weight percentage: trehalose 4.0%-8.0%, monosodium glutamate 1.0%-2.0%, polyvinyl alcohol 0.5%-1.0%, and the balance being sterile water; the preparation of the composite protective agent solution includes: adding PVA to 30%-40% of the formulated amount of sterile water, stirring at 40℃-50℃ for 30-45 minutes until dissolved, cooling to below 60℃ and then adding trehalose, monosodium glutamate and the remaining sterile water, and filtering through a 0.45 μm microporous membrane for sterilization or pasteurizing at 65℃.

[0019] In this formulation, trehalose is used as a cell membrane protectant, monosodium glutamate (MSG) as a hygroscopic regulator, polyvinyl alcohol (PVA, degree of polymerization 1700, degree of hydrolysis 88%) as a film-forming agent, and the remainder is purified water. Since PVA has poor solubility in cold water, the PVA powder is first sprinkled into approximately one-third of the formulated amount of purified water to wet and swell. The solution is then heated to 85℃-90℃ and stirred at a constant temperature for 30-45 minutes until the PVA is completely dissolved, forming a clear and transparent colloidal solution. After naturally cooling to below 60℃, the remaining water, trehalose, and MSG are added, and stirring continues until all solutes are completely dissolved. Finally, the solution is filtered through a 0.45μm microporous membrane for sterilization or pasteurized at 65℃ to obtain a clean and homogeneous composite protectant solution for later use.

[0020] Preferably, step S4, the gradient temperature drying specifically includes: Low-temperature shaping and drying stage: Spread the film-forming mycelial balls evenly in a sterile drying tray, controlling the stacking thickness to not exceed 1.5 cm, and place them in a forced-air drying oven or fluidized bed dryer. Set the temperature to 25-30℃, turn on the circulating air, and air dry at a wind speed of 0.5 m / s for 3-5 hours. The core purpose of this stage is to gently remove the free water on the surface of the particles, so that the PVA-trehalose complex on the surface gradually loses water and solidifies, forming a semi-permeable protective film "soft shell" with a certain mechanical strength. This fixes the particle shape and prevents the particles from bursting or the protective film from being damaged due to the violent vaporization of internal moisture during subsequent heating.

[0021] Medium-temperature deep dehydration stage: The temperature is increased to 35-40℃ at a rate of 0.5-1.0℃ / min to enter the deep drying stage; drying continues at this temperature for 6-10 hours to promote the slow diffusion and evaporation of bound water inside the bacterial pellets through the surface semi-permeable membrane; during this period, samples are taken every 1 hour to quickly determine the moisture content or water activity (Aw). When the moisture content of the particles drops below 8% and the water activity Aw ≤ 0.6, and the particles are weighed every 1 hour, and the difference between two consecutive weighing results is less than 0.1%, that is, constant weight is reached, the drying endpoint is determined to have been reached, and heating is stopped immediately.

[0022] Cooling and Sealing Packaging: After drying, use the cold air circulation function of the drying equipment or transfer the material to a dehumidifying cooling room to quickly lower the temperature of the granular fertilizer to room temperature (below 25°C) to prevent residual heat from causing continuous thermal damage to the functional bacteria embedded inside the core. The cooled finished granules should be packaged within 30 minutes, using high-barrier aluminum foil composite bags for vacuum packaging or nitrogen-filled packaging, and heat-sealed to isolate external moisture and oxygen, ensuring the stability of the product's biological activity during its shelf life.

[0023] Preferably, in step S1, the spore-forming fungus is selected from one or more of Trichoderma harzianum, Aspergillus niger, and Penicillium, and the spore concentration of the fungal spore suspension is ≥1.0×10⁻⁶. 7 CFU / mL; the functional bacteria are selected from one or more of Bacillus subtilis, Bacillus mucilaginosa, nitrogen-fixing bacteria, and phosphate-solubilizing bacteria. Among them, the combination of Penicillium and nitrogen-fixing bacteria should be excluded (Penicillium metabolites will inhibit the activity of nitrogen-fixing bacteria) and the combination of Aspergillus niger and phosphate-solubilizing bacteria (high acid environment affects phosphate-solubilizing enzyme activity).

[0024] The present invention also provides a granular microbial fertilizer containing functional bacteria encapsulated in fungal mycelium balls prepared by the preparation method described above. The fertilizer is a symbiotic particle formed by fungal mycelium balls encapsulating functional bacteria, with a particle size of 2.0-4.0 mm and a water content of ≤8%, and the functional bacteria are encapsulated in the internal network structure of the mycelium balls.

[0025] The present invention has at least the following beneficial effects: 1. This invention's granular microbial fertilizer, containing fungal mycelial spheres encapsulating functional bacteria, pioneers a "bio-self-assembly" encapsulation mechanism, significantly improving microbial survival rate and stress resistance. It abandons the simple physical adsorption or expensive chemical cross-linking encapsulation techniques of traditional microbial fertilizers, innovatively utilizing the bio-contraction force generated by the fungal spheres during fermentation to achieve in-situ "active capture" of functional bacteria. The resulting granules possess a unique "core-shell" biological structure: the outer layer is a dense, hydrophobic mycelial membrane, forming a natural physical barrier that effectively blocks ultraviolet radiation and locks in internal moisture under extreme conditions such as drought and high temperatures; the inner layer is a loose mycelial network rich in functional bacteria, providing the bacteria with a "microcapsule-like" protective habitat. Experimental data shows that the microbial fertilizer prepared by this invention achieves a survival rate of over 85% for functional bacteria after 6 months of storage at room temperature, and its resistance to ultraviolet radiation and drying is significantly superior to traditional peat-based adsorbents, greatly solving the industry pain points of short shelf life and low colonization rate after application in microbial fertilizers. After the fungal carrier decomposes, it can increase soil organic matter. After two years of continuous application, the organic matter content of sandy loam soil increased by 81.25% compared with the initial level (from 0.8% to 1.45%).

[0026] 2. The granular microbial fertilizer of the present invention, which encapsulates functional bacteria within fungal mycelial balls, achieves a synergistic dual fertilizer effect and possesses a unique bio-slow-release function. The fertilizer granules prepared by this invention integrate the dual agricultural benefits of fungi and bacteria, producing a synergistic effect of "1+1>2". The carrier fungi (such as Trichoderma harzianum and Aspergillus niger) are themselves beneficial microorganisms, which, after being applied to the soil, can colonize and suppress diseases, improve soil aggregate structure, or decompose insoluble organic matter; while the functional bacteria (such as Bacillus) encapsulated within them play a role in nitrogen fixation, phosphorus solubilization, potassium solubilization, and growth promotion. More importantly, because the bacteria are deeply encapsulated inside the mycelial balls, their release is not explosive like traditional microbial agents, but rather relies on the natural degradation rate of the outer fungal mycelium in the soil for slow release. This bio-slow-release mechanism not only prolongs the fertilizer's effective duration but also avoids the phenomenon of functional bacteria dying due to competition and exclusion by native microorganisms in the early stages of soil entry due to their weak population, significantly improving the field application effect.

[0027] 3. The granular microbial fertilizer containing fungal mycelium balls encapsulating functional bacteria of this invention eliminates reliance on exogenous carriers, significantly reducing production costs and achieving green manufacturing. Existing microencapsulation technologies often rely on high-cost chemical raw materials such as sodium alginate and gelatin, or consume large amounts of non-renewable resources such as peat moss. This invention cleverly utilizes the fungal biomass that proliferates during liquid fermentation directly as the encapsulation carrier, eliminating the need for any exogenous inert carriers, significantly reducing raw material and material transportation costs. Furthermore, the entire production process is completed in a liquid fermentation tank, eliminating the need for complex granulation equipment (such as extrusion pellet machines) or energy-intensive granulation processes. In addition, both the fungal balls and functional bacteria are environmentally friendly organisms. After application to farmland, the mycelium ball carrier will eventually be completely degraded into organic nutrients such as amino acids and polysaccharides, which will be absorbed by crops. The selected protective agents (such as PVA and trehalose) are also easily biodegradable, ensuring the complete degradation of the mycelium ball carrier into organic nutrients for crop absorption. This invention uses PVA (polyvinyl alcohol) as a protective film-forming agent. PVA is a synthetic polymer, and its degradation rate and final products in soil must be based on evidence and fully comply with the production standards of green agriculture and organic agriculture.

[0028] 4. The method for preparing granular microbial fertilizer containing fungal mycelial spheres and functional bacteria of the present invention involves the regulation of a specific shear force field, the addition of a sphere-forming inducing factor, and the enhancement of PVA surface film formation in the post-treatment stage. The resulting granular microbial fertilizer exhibits uniform particle size (2.0-4.0 mm), a smooth surface, and excellent flowability. In particular, the granules possess high mechanical strength (single-particle compressive strength > 8N), effectively solving the problems of easy drifting, moisture absorption and clumping, and easy powdering of traditional powdered microbial agents. These excellent physical properties allow the fertilizer to be directly applied precisely in strips or holes using large-scale agricultural machinery, improving fertilization efficiency and meeting the needs of modern large-scale and mechanized agricultural operations.

[0029] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0030] Figure 1 This is a flowchart illustrating the preparation method of the granular microbial fertilizer containing fungal mycelial balls and functional bacteria according to the present invention. Figure 2 These are scanning electron microscope images of the soil 24 hours after fungal fermentation (before inoculation with functional bacteria) and after fertilizer application, as shown in Example 1 of this invention. Figure 3 The graph shows a comparison of the release rate of live bacteria in soil between the granular microbial fertilizer containing fungal mycelial balls with functional bacteria prepared in Example 1 of this invention and the ordinary physical adsorption fertilizer in Comparative Example 5. Detailed Implementation

[0031] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0032] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0033] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0034] Source of strain and reagents: mature fungi: Trichoderma harzianum ( Trichodermaharzianum The strain was purchased from Beijing Beina Chuanglian Biotechnology Research Institute, strain number BNCC 336465.

[0035] Functional bacteria: Bacillus subtilis ( Bacillus subtilis Purchased from the China Industrial Microbial Culture Collection Center (CICC), catalog number CICC 10275.

[0036] Other spherical fungi, such as Trichoderma, Aspergillus, and Penicillium, were purchased from CGMCC; other functional bacteria, such as nitrogen-fixing bacteria, phosphate-solubilizing bacteria, and biocontrol bacteria, were purchased from CICC.

[0037] Main reagents: Polyvinyl alcohol (PVA-1788): purchased from Sinopharm Chemical Reagent Co., Ltd. Trehalose and sodium carboxymethyl cellulose (CMC-Na): purchased from Aladdin Biochemical Technology Co., Ltd. Tween-80, monosodium glutamate, yeast extract, and tryptone: purchased from Sangon Biotech (Shanghai) Co., Ltd. Other inorganic salts (KH2PO4, CaCl2, etc.): all commercially available analytical grade.

[0038] Example 1 Preparation method of granular microbial fertilizer containing *Trichoderma harzianum* in situ encapsulated with *Bacillus subtilis*, such as... Figure 1 As shown, it includes the following steps: S1. Strain pretreatment: Preparation of fungal spore suspension: *Trichoderma harzianum* was inoculated onto PDA slant agar and cultured at 28°C for 7 days until abundant green spores appeared. The spores on the slant were washed with sterile water containing 0.1% Tween-80 (v / v). Residual hyphae were removed by filtration through four layers of sterile gauze. The spores were counted using a hemocytometer and the concentration adjusted to a final concentration of 2.0 × 10⁻⁶. 7A spore suspension of CFU / mL is prepared for use.

[0039] Preparation of functional bacterial seed culture: Bacillus subtilis was inoculated into LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) and cultured at 37℃ and 200 rpm for 12 hours with shaking. Samples were taken to measure the optical density. When the OD value... 600 ≈1.8 and the viable count reaches 3.5×10 9 When the concentration reaches CFU / mL, stop the culture and use it as a seed culture for functional bacteria.

[0040] S2, Core Formation Phase: Prepare 3L of modified pelleting medium: its components include 20 g / L corn starch, 5 g / L soybean meal, 2.0 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.3 g / L CaCl2, and 1.0 g / L sodium carboxymethyl cellulose (CMC-Na). Adjust the initial pH to 5.8 with 0.5 mol / L dilute hydrochloric acid. Sterilize in an autoclave at 121℃ for 20-30 minutes. After sterilization, randomly select a portion of the sample and place it in a constant temperature incubator at 28℃-30℃ for blank incubation for 24-48 hours to check the thoroughness of sterilization and ensure no growth of contaminating bacteria. The modified, contamination-free fermentation medium, which had passed testing, was cooled to 28°C and placed in a fermenter equipped with temperature and speed control. The fungal spore suspension was inoculated at a rate of 5% (v / v). For the first 6 hours, the stirring speed was set to 150 rpm (corresponding to a linear velocity of approximately 0.6 m / s), and the aeration rate was 0.8 vvm to protect spore germination and the formation of primary mycelia.

[0041] Shear field establishment: Starting from the 6th hour of fermentation, the stirring speed was increased to 350 rpm. Based on the formula πDn / 60, the tip linear velocity at this point was approximately v ≈ 1.46 m / s. This stirring speed was maintained constant thereafter to induce mycelial curling into balls.

[0042] Status monitoring: At the 24th hour of fermentation, online monitoring showed that the pH value naturally decreased from the initial 5.8 to 4.8. Microscopic examination of a sample at this point revealed a large number of particles approximately 1.0 mm in diameter in the fermentation broth, with a loose, spongy structure and radiating edges, indicating that the optimal inoculation state had been reached. Figure 2The images show that the hyphae have a loose, flocculent structure with the center not yet compacted. The left image in Figure 2 is a scanning electron microscope (SEM) image (50x magnification) taken 24 hours after fungal fermentation. It can be seen that the hyphae intertwine to form a loose, flocculent three-dimensional network structure with the center not yet compacted and the edges radiating outwards. This structure provides sufficient physical space for the subsequent encapsulation of functional bacteria. The right image is a scanning electron microscope image taken a certain time after the microbial fertilizer prepared in this example is applied to the soil. It can be clearly observed that the functional bacteria are tightly wrapped inside by the compacted fungal hyphal network, without any free or scattered phenomena. This directly confirms the stability of the "fungus-bacteria" symbiotic encapsulation structure and its continuous retention effect in the soil environment.

[0043] S3, Phase transition encapsulation period: Fed-fluid inoculation: A gradient strategy of "slow at first, then fast" was adopted to feed functional bacterial seed liquid into the fermentation broth that formed loose flocculent mycelial clusters. The flow rate was 6 mL / min for the first 5 minutes and then increased to 10 mL / min. The inoculation volume of functional bacterial seed liquid was 300 mL (about 10% of the fermentation broth volume). The specific shear force field parameters (stirring speed and temperature) were kept constant, and the bacteria were rapidly dispersed into the mycelial clusters by stirring eddies. Environmental induction: After inoculation with functional bacterial seed culture, maintain the pH of the fermentation broth between 4.5 and 5.0 (no manual adjustment required). At the same time, adjust the aeration rate from 1.0 vvm to 1.2 vvm, and maintain dissolved oxygen (DO) at around 30% through correlation control, using weak acidity and moderate oxygen stress to induce mycelial contraction.

[0044] Co-cultivation process: Continue co-cultivation for 36 hours. During this period, the fungal hyphae gradually age and become denser, locking the functional bacteria inside, and the fermentation broth gradually becomes clear from turbid.

[0045] Endpoint determination: When the total fermentation time reaches 60 hours, sampling observation shows that the particle diameter increases to 3.2 mm and the surface has a smooth and dense membrane structure, fermentation stops and symbiotic mycelial balls are formed.

[0046] S4. Surface strengthening and curing: Cleaning: Filter the fermentation broth through a 60-mesh sieve, collect the wet symbiotic mycelial balls, rinse twice with sterile physiological saline to remove residual culture medium from the surface.

[0047] The collected and cleaned wet symbiotic mycelium balls were immersed in a composite protective agent solution for surface film formation treatment, and then dehydrated using a gradient temperature drying method until the moisture content was ≤8%, resulting in granular microbial fertilizer.

[0048] The preparation of the composite protective agent solution includes: Prepare the following raw materials according to the weight-to-volume ratio (w / v): Film-forming matrix: Polyvinyl alcohol (PVA-1788, degree of alcoholysis 88%±1%, degree of polymerization approximately 1700) 0.8%; Bioprotective agents: Trehalose 6.0%, Monosodium glutamate 1.5%; Solvent: The remainder is sterile water.

[0049] Preparation process: First, add PVA-1788 to one-third of the formula volume of sterile water at 40-50℃ and stir until completely dissolved (PVA-1788 has excellent low-temperature water solubility, no boiling is required, avoiding heat damage in subsequent operations). Then add trehalose and monosodium glutamate, and finally add sterile water to make up the difference. Stir evenly and filter through a 0.45 μm microporous membrane for sterilization or pasteurize at 65℃ to obtain a transparent composite protective agent solution with appropriate viscosity.

[0050] Surface film formation treatment of wet symbiotic mycelial balls: Immediately immerse the collected and cleaned wet symbiotic mycelial balls (water content approximately 85-90%) in a composite protective agent solution and maintain immersion for 25 minutes, gently stirring during this period. Mechanism of action: Utilizing the film-forming properties of PVA-1788, a semi-permeable protective film is formed on the particle surface. Simultaneously, trehalose and monosodium glutamate penetrate into the deep layers of cells and mycelia, replacing water molecules within the cells and preventing the collapse of the cell membrane lipid bilayer structure during the drying process.

[0051] The gradient temperature drying method specifically includes: Low-temperature shaping and drying stage: After film formation treatment, drain excess liquid from the surface of the mycelial balls, lay them in a single layer in a sterile drying tray, control the stacking thickness to not exceed 1.5 cm, place them in a forced-air drying oven or fluidized bed dryer, set the temperature to 28℃, turn on the circulating air, and air dry at a wind speed of 0.5 m / s for 4 hours; The purpose of the low-temperature shaping and drying stage is to gently remove free water from the surface of the particles, so that the PVA film is initially shaped, and avoid the surface liquid film from boiling or breaking due to excessive temperature.

[0052] Medium-temperature deep dehydration stage: The temperature is increased to 38℃ at a rate of 0.5-1.0℃ / min, and drying continues at this temperature for approximately 8 hours until the particle weight no longer changes significantly and the moisture content drops to ≤7.5%. The purpose of this medium-temperature deep dehydration stage is to allow sufficient time for internal moisture diffusion due to the slow heating rate, preventing the pellets from bursting or internal bacteria from dying due to heat shock, ultimately resulting in a smooth, dense, mechanically strong, and biologically stable granular product.

[0053] Preliminary experiments revealed that if the gradual heating from 0.5 to 1.0 °C / min was omitted and the temperature was directly increased to 38 °C, the particle bursting rate increased by about 15%, confirming the important role of moderate heating in maintaining the integrity of the PVA film.

[0054] Example 2 The method for preparing granular microbial fertilizer containing Aspergillus niger in situ embedded in gelatinous Bacillus subtilis differs from Example 1 in that: 1. Microbial strain: Confluent fungi: Aspergillus niger ( Aspergillusniger ).

[0055] Functional bacteria: Bacillus jelly-like ( Paenibacillusmucilaginosus ).

[0056] 2. Adjustment of key process parameters: Culture medium adjustment: 0.5% (w / v) of 200-mesh ultrafine potassium feldspar powder was added to the modified pelleting medium as a mineral inducer and potassium source.

[0057] Adjustment of the timing of functional bacteria inoculation: Due to the extremely fast growth rate and strong acid production capacity of Aspergillus niger, functional bacteria were inoculated when the pH naturally dropped to 4.5 and the mycelial cluster diameter reached 0.8 mm at the 18th hour of fermentation.

[0058] Shear force field adjustment: Given that the Aspergillus niger hyphae are relatively thick, the stirring speed was adjusted to 380 rpm, which corresponds to a linear velocity of approximately 1.6 m / s, to prevent excessive clumping.

[0059] Drying adjustment: Since the particles contain mineral powder and have a high density, the drying temperature is increased to 40℃ in the medium-temperature deep dehydration stage of gradient temperature drying to improve drying efficiency.

[0060] Example 3 Optimization and parameter validation of composite protective agent solution formulation: To verify the necessity and optimization of the composite protective agent solution formulation and process parameters in Example 1 of this invention, the following comparative experiment was set up to verify the impact of PVA type selection and component synergy on the quality of the finished product through data.

[0061] 1. Experimental group setup Example 1 (Optimal group of the present invention): 6.0% trehalose + 1.5% monosodium glutamate + 0.8% PVA-1788.

[0062] Comparative Example 1 (Blank Control): No additives were used; the sample was simply soaked in sterile water.

[0063] Comparative Example 2 (without film-forming agent): 6.0% trehalose + 1.5% monosodium glutamate (PVA-free).

[0064] Comparative Example 3 (lacking bioprotectant): 0.8% PVA-1788 (without trehalose / monosodium glutamate).

[0065] Comparative Example 4 (reduced by half compared to Example 1): 3.0% trehalose + 0.75% monosodium glutamate + 0.4% PVA-1788.

[0066] 2. The experimental comparison results are shown in Table 1 below.

[0067] Table 1. Comparison of the effects of compound protective agent solution formulation on granular microbial fertilizers Note: Particle integrity rate refers to the percentage of particle weight that retains a complete spherical structure after 30 minutes of simulated transport vibration.

[0068] 3. Results Analysis: The PVA-1788 (degree of polymerization approximately 1700, degree of hydrolysis 88%) selected in this invention is a key parameter.

[0069] Comparing Example 1 and Comparative Example 2, it can be seen that the lack of PVA will cause the particles to "powder" after drying, and the mechanical strength will drop from 98% to 72%, making them unable to withstand transportation.

[0070] Selection criteria: If a PVA with excessively high polymerization (such as PVA-2488) is selected, the solution viscosity will be too high, making it difficult to penetrate the pores of the bacterial cells; if a fully alcoholyzed PVA (such as PVA-1799) is selected, it requires high-temperature dissolution, which can easily cause thermal damage to microorganisms. PVA-1788 balances cold water solubility with film-forming strength.

[0071] Synergistic protective effect: Comparing Example 1 and Comparative Example 3, it can be seen that when only PVA forms a film without sugar / amino acid protection, although the physical appearance is intact, a large number of bacteria inside die due to dehydration (survival rate is only 45.6%). This indicates that the role of trehalose and monosodium glutamate in replacing bound water within cells is indispensable.

[0072] Importance of the optimal ratio: Comparing Example 1 and Comparative Example 4, it can be seen that when the concentration of the protective agent is halved, none of the indicators have reached the optimal state, proving that the formulation ratio (6%+1.5%+0.8%) determined in this invention is the optimal dose-effect range after experimental optimization.

[0073] Example 4 The effect of different inoculation times on embedding efficiency (parameter optimization): To demonstrate the criticality of the timing of inoculation with functional bacterial seed solution, three parallel experiments were set up for Example 1, with only the inoculation time of functional bacterial seed solution being changed.

[0074] Experimental Group A (Premature Inoculation): Functional bacterial seed liquid was inoculated 10 hours after the fermentation of fungal spore suspension (at this time, the hyphae have just germinated and have not yet formed flocculent clumps).

[0075] Experimental Group B (Window Period of the Invention): Functional bacterial seed liquid was inoculated at the 24th hour of fermentation of fungal spore suspension (at this time, the mycelium is in the loose flocculent stage, pH 4.8).

[0076] Experimental group C (late inoculation): Functional bacterial seed liquid was inoculated 40 hours after the fermentation of fungal spore suspension (at this time, the mycelial balls had formed a dense shell, pH 3.5).

[0077] The experimental results are shown in Table 2 below.

[0078] Table 2. Effects of different process parameters on particle structure and bacterial load Note: Encapsulation rate = (Total number of bacteria measured after ultrasonic disruption - Number of free bacteria in the cleaning solution) / Total number of bacteria × 100% As shown in Table 2, the timing of inoculation is crucial. True "embedding" can only be achieved when bacteria are introduced when the fungus has formed a loose skeleton but has not yet become dense; otherwise, it is merely a simple mixing or surface adsorption.

[0079] Example 5 The performance of Example 1 of the present invention is compared with that of the traditional physical adsorption method (Comparative Example 5): Comparative Example 5: Traditional Physical Adsorption Method Preparation method: Trichoderma harzianum and Bacillus subtilis were fermented separately in liquid form. After fermentation, the two bacterial solutions were mixed in a 1:1 volume ratio.

[0080] Carrier adsorption: Sterilized peat moss is used as the adsorption carrier, and the mixed bacterial solution is sprayed at a liquid-to-solid ratio of 1:3.

[0081] Drying: Place in a 35℃ forced-air drying oven and dry until the moisture content is 10%.

[0082] 1. The results of stress resistance and shelf life comparison between Example 1 and Comparative Example 5 are shown in Table 3 below.

[0083] Table 3. Comparison of stress resistance and shelf life of the microbial fertilizers prepared in Example 1 and Comparative Example 5 of the present invention. Note: UV irradiation conditions: 30W UV lamp, 30 cm distance, irradiation for 2 hours (simulating an unobstructed farmland surface soil scenario; if the soil coverage depth is ≥1 cm, the survival rate can be increased by 15%-20%). Rain erosion resistance: After simulating moderate rain washing the soil surface for 1 hour, the residual amount of bacterial agent in the soil layer was detected.

[0084] As shown in Table 3, the granular fertilizer prepared by this invention is significantly superior to traditional physical adsorption products in terms of stress resistance (UV resistance and storage tolerance). This is because the dense outer mycelial membrane of the mycelial balls and the PVA coating construct a dual protective barrier.

[0085] 2. The results of the stress resistance and shelf life comparison of the microbial fertilizers prepared in Example 1 and Comparative Example 5 are shown in Table 4 below.

[0086] Table 4. Comparison of survival rates of microbial fertilizers prepared in Example 1 and Comparative Example 5 under extreme environmental conditions. As shown in Table 4, the "fungus-PVA-trehalose" composite encapsulation system of this invention endows functional bacteria with extremely strong stress resistance. Especially in high-temperature transportation in summer (simulating 50℃) and application scenarios in saline-alkali land, the survival rate is 3-4 times that of traditional products.

[0087] 3. The comparison results of the growth-promoting effects of the microbial fertilizers prepared in pot experiments of Examples 1, 2 and Comparative Example 5 are shown in Table 5 below.

[0088] Table 5. Comparison of growth-promoting effects of microbial fertilizers prepared in pot experiments of Examples 1, 2 and Comparative Example 5. As shown in Table 5, the microbial fertilizer prepared in this invention exhibits the best field application results. This is attributed to the synergistic effect of the fungus (Trichoderma harzianum) in preventing disease and the growth-promoting effect of the bacteria (Bacillus subtilis), while the slow-release mechanism ensures the long-lasting fertilizer effect.

[0089] 4. The cumulative effect of the microbial fertilizers prepared in Example 1 and Comparative Example 5 on soil improvement (after 2 years of continuous application) is shown in Table 6 below.

[0090] Table 6. Cumulative effect of microbial fertilizers prepared in Example 1 and Comparative Example 5 on soil improvement in a long-term field test (after 2 years of continuous application). As shown in Table 6, the soil remediation capacity is demonstrated by the long-term monitoring results. The microbial fertilizer prepared in this invention is not only a seasonal fertilizer but also a soil conditioner. Particularly in the improvement of saline-alkali land, the pH value decreased by nearly one unit after two years of continuous application. This is attributed to the continuous secretion of organic acids and extracellular polysaccharides by the mycelial balls after colonization in the soil, which promotes the formation of soil aggregates. This is impossible to achieve using traditional physical adsorption methods (physical adsorption agents have a short survival time in the soil and cannot form a cumulative effect).

[0091] like Figure 3As shown, the fungal mycelium ball-encapsulated fertilizer prepared in Example 1 exhibits a significant difference in live bacteria release behavior in soil compared to the ordinary physical adsorption fertilizer in Comparative Example 5. Figure 3 The release curves show that the functional bacteria in ordinary physically adsorbed fertilizers release rapidly within a short period (0-10 days) after application to the soil, followed by a rapid decline. After 30 days, the number of detectable viable bacteria in the soil is only less than 15% of the initial value. In contrast, the granular microbial fertilizer containing functional bacteria encapsulated in fungal mycelium balls of this invention exhibits typical biological slow-release characteristics. The release rate of viable bacteria is stable in the first 30 days, and it can still maintain more than 60% of the initial viable bacteria count after 60 days. This difference stems from the natural biological barrier effect of the mycelium balls: as the outer fungal mycelium slowly degrades in the soil, it gradually releases the encapsulated functional bacteria, preventing them from being competitively excluded by native soil microorganisms or dying due to environmental stress from short-term large-scale exposure. This effectively prolongs the fertilizer's effective duration and improves the stability of field application.

[0092] Although the technical solutions of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. A method for preparing granular microbial fertilizer containing fungal mycelial balls encapsulating functional bacteria, characterized in that, Includes the following steps: S1. Strain pretreatment: Prepare fungal spore suspension and functional bacterial seed liquid separately for fungal spores and functional bacteria. S2, Core Formation Stage: Fungal spore suspension is inoculated into modified pelleting medium and cultured under a specific shear field until loose, flocculent mycelial clusters with a diameter of 0.5-1.5 mm are formed in the fermentation broth; S3, Phase Change Encapsulation Period: Functional bacterial seed liquid is added to the fermentation broth that forms loose flocculent mycelial clusters. By adjusting the dissolved oxygen and pH value of the fermentation broth, the mycelium is induced to shrink and compact inward, thereby physically capturing the functional bacteria inside the mycelial network and forming symbiotic mycelial balls with a diameter of 2.0-4.0 mm. S4. Surface strengthening and solidification: Collect the wet symbiotic mycelial balls obtained in step S3, immerse them in a composite protective agent solution for surface film formation treatment, and then use a gradient temperature drying method to dehydrate them until the moisture content is ≤8%, thus obtaining granular microbial fertilizer.

2. The method for preparing granular microbial fertilizer containing fungal mycelial balls encapsulating functional bacteria as described in claim 1, characterized in that, In step S2, the components of the improved pelleting medium, in g / L, include: 15-30 g of carbon source, 3-8 g of nitrogen source, 1-3 g of KH2PO4, 0.5-1.0 g of MgSO4·7H2O, and pelleting inducing factor. The carbon source is selected from one or more of corn starch, glucose, and sucrose, and the nitrogen source is selected from one or more of soybean meal, yeast powder, and tryptone. The pelleting inducing factor consists of 0.2-0.5 g / L CaCl2 and 0.5-1.5 g / L sodium carboxymethyl cellulose.

3. The method for preparing granular microbial fertilizer containing fungal mycelial balls encapsulating functional bacteria as described in claim 2, characterized in that, Step S2 specifically includes: S21. Preparation of modified spheroidizing culture medium: Mix 15.0-30.0 g of carbon source, 3.0-8.0 g of nitrogen source, 1.0-3.0 g of KH2PO4, 0.5-1.0 g of MgSO4·7H2O, 0.2-0.5 g of CaCl2 and 0.5-1.5 g of sodium carboxymethyl cellulose, and bring the volume to 1000 mL with pure water. After thorough stirring and dissolution, adjust the pH to 5.5-6.0 with 0.5 mol / L dilute hydrochloric acid. S22. Sterilization and testing of modified pelleting culture medium: Place the modified pelleting fermentation medium prepared in step S21 into an autoclave and sterilize at 121℃ for 20-30 minutes; after sterilization, randomly select some samples and place them in a constant temperature incubator at 28℃-30℃ for blank culture for 24-48 hours to check the thoroughness of sterilization and ensure that no contaminating bacteria grow; S23. Preparing the pelleting environment: Cool the modified pelleting fermentation medium that has passed the S22 test and is free of contaminants to 25℃-28℃, and place it in a fermenter with temperature and speed control, and inoculate it with fungal spore suspension; S24. Control the tip linear velocity of the impeller of the fermenter to 1.2-1.8 m / s to create a hydrodynamic environment. Cultivate under this specific shear force field until loose, flocculent mycelial clusters with a diameter of 0.5-1.5 mm are formed in the fermentation broth.

4. The method for preparing granular microbial fertilizer containing fungal mycelial balls encapsulating functional bacteria as described in claim 3, characterized in that, Step S24 specifically includes: S241. Constructing a specific shear force field environment: In fungal fermentation, the rotation speed of the fermenter stirring motor is adjusted. Based on the diameter D of the fermenter stirring impeller and the rotation speed n, the linear velocity v of the stirring impeller tip is calculated using the formula: v=πDn / 60. The linear velocity v of the stirring impeller tip is set to be constant within the range of 1.2-1.8 m / s, and cultivation is carried out under this specific shear force field. S242. Monitoring biological and physicochemical indicators: The culture process is monitored in real time, with the fungal fermentation time T and the pH of the fermentation broth as dual judgment indicators. Samples are continuously taken for microscopic examination to observe the mycelial morphology, and pH changes are recorded every 1-2 hours.

5. The method for preparing granular microbial fertilizer containing fungal mycelial balls encapsulating functional bacteria as described in claim 4, characterized in that, Step S241 is preceded by a "germination protection" step, which is as follows: Within the first 4-6 hours after inoculating the fungal spore suspension, the impeller tip linear velocity is controlled at a low shear state of 0.5-0.8 m / s, and the aeration rate is controlled at 0.5-0.8 vvm to protect spore germination and primary hyphae formation. When microscopic examination shows that more than 90% of the spores have germinated and grown hyphae with a length greater than 3 times the spore diameter, the specific shear force field environment of step S241 is then established.

6. The method for preparing granular microbial fertilizer containing fungal mycelial balls encapsulating functional bacteria as described in claim 1, characterized in that, Step S3 specifically includes: S31. Timing of inoculation of functional bacterial seed liquid: When the fermentation of the fungal spore suspension in step S2 has proceeded for 18-28 hours and the pH value of the fermentation liquid has naturally decreased to 4.5-5.0 under real-time monitoring, it is determined to be the endpoint of the formation of loose flocculent mycelial clusters. S32. Using a gradient strategy of "slow at first, then fast", functional bacterial seed liquid is added to the fermentation broth that has formed loose flocculent mycelial clusters. The flow rate is 5-8 mL / min for the first 5 minutes, and then increased to 10-15 mL / min. The inoculation amount of functional bacterial seed liquid is 10%-15% of the fermentation broth volume. The specific shear force field parameters are kept constant. The dissolved oxygen of the fermentation broth is adjusted to 25%-40%, and the pH is maintained at a natural weakly acidic environment of 4.5-5.

0. After co-culturing for 24-36 hours, symbiotic mycelial balls with a diameter of 2.0-4.0 mm are formed.

7. The method for preparing granular microbial fertilizer containing fungal mycelial balls encapsulating functional bacteria as described in claim 1, characterized in that, In step S4, the composite protective agent solution is composed of the following components by weight percentage: trehalose 4.0%-8.0%, monosodium glutamate 1.0%-2.0%, polyvinyl alcohol 0.5%-1.0%, and the balance being sterile water. The preparation of the composite protective agent solution includes: adding PVA to 30%-40% of the formula amount of sterile water, stirring at 40℃-50℃ for 30-45 minutes until dissolved, cooling to below 60℃, adding trehalose, monosodium glutamate and the remaining sterile water, and filtering through a 0.45 μm microporous membrane for sterilization or pasteurizing at 65℃.

8. The method for preparing granular microbial fertilizer containing fungal mycelial balls encapsulating functional bacteria as described in claim 4, characterized in that, In step S4, gradient temperature drying specifically includes: Low-temperature shaping and drying stage: Spread the film-forming mycelial balls evenly in a sterile drying tray, control the stacking thickness to not exceed 1.5 cm, place them in a forced-air drying oven or fluidized bed dryer, set the temperature to 25-30℃, turn on the circulating air, and air dry at a wind speed of 0.5 m / s for 3-5 hours. Medium-temperature deep dehydration stage: Heat to 35-40℃ at a heating rate of 0.5-1.0℃ / min, dry to constant weight, and ensure that the finished product has a moisture content of ≤8% and a water activity Aw of ≤0.

6.

9. The method for preparing granular microbial fertilizer containing fungal mycelial balls encapsulating functional bacteria as described in claim 1, characterized in that, In step S1, the spore-forming fungi are selected from one or more of the genera *Trichoderma harzianum*, *Aspergillus niger*, and *Penicillium*, and the spore concentration of the fungal spore suspension is ≥1.0 × 10⁻⁶. 7 CFU / mL; the functional bacteria are selected from one or more of Bacillus subtilis, Bacillus mucilaginosa, nitrogen-fixing bacteria, and phosphate-solubilizing bacteria.

10. A granular microbial fertilizer containing functional bacteria encapsulated in fungal mycelium balls prepared by the preparation method according to any one of claims 1 to 9, wherein the fertilizer is a symbiotic particle formed by fungal mycelium balls encapsulating functional bacteria, the particle size is 2.0-4.0 mm, the moisture content is ≤8%, and the functional bacteria are encapsulated in the internal network structure of the mycelium balls.

Images