Biological fertilizer for remolding soil structure by using gas and gum producing complex bacteria and preparation method thereof

Abstract

The application discloses a biological fertilizer for remodeling soil structure by using gas and gum producing composite microbial flora and a preparation method thereof, and belongs to the field of agricultural biotechnology. The method separates and compounding Bacillus licheniformis, Bacillus subtilis, actinomycetes and cyanobacteria from target soil to form a composite microbial liquid with the collaborative functions of gas expansion and gum fixation, and then mixes the composite microbial liquid with porous slow-release carriers, degradable expansion control substrates, microbial protection and induction components and structure stability adjustment components according to a set ratio, and finally prepares the biological fertilizer through stirring, granulation and step induction drying processes. The fertilizer fully utilizes the ecological adaptability of the composite microbial flora, and through the synergistic effect of gas expansion and gum adhesion, in combination with the slow-release and protection of the carriers, can direct the improvement of the soil microenvironment, effectively promotes the formation and stability of soil aggregates, and is suitable for the structure repair and fertility improvement of hardened and degraded soil.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology. More specifically, this invention relates to a bio-fertilizer that uses a gas- and gum-producing microbial community to reshape soil structure and its preparation method. Background Technology

[0002] Soil aggregates directly determine the microenvironment for crop root growth by regulating soil aeration, water retention, and nutrient retention.

[0003] The destruction of soil aggregate structure directly triggers a chain reaction of fertility degradation: the proportion of aeration pores in compacted soil is often less than 10%, leading to a decrease in crop root respiration capacity and nutrient absorption efficiency, ultimately resulting in an average yield reduction of 15% to 30%; while the decline in soil water retention capacity exceeds 30%, which not only exacerbates water and fertilizer loss in arid areas but also causes agricultural non-point source pollution through nutrient leaching. Existing technical approaches mainly include applying organic fertilizers, deep tillage and loosening of the soil, and applying microbial fertilizers, but all have obvious limitations: although organic fertilizers can replenish soil organic matter, their decomposition period is as long as 3 to 6 months, and the nutrient release rate is not well matched with the needs of crop growth stages; deep tillage measures easily disturb the stability of soil profile structure, and compaction can easily recur in the short term.

[0004] Microbial fertilizers can regulate soil micro-ecosystems in a targeted manner, but existing products still have key technological deficiencies. First, there is insufficient compatibility between the microbial community and the target soil. Most microbial fertilizers use commercially available standard strains instead of the target soil microbial community, resulting in a colonization rate of less than 20% in the target soil and an activity maintenance period of only 1-2 weeks, making it difficult to form a stable functional microbial community. Second, the functional design is singular. Existing microbial fertilizers mostly focus on nutrient conversion functions such as nitrogen fixation and phosphorus and potassium solubilization. A few products involving the construction of aggregate structures contain only aerogenetic Bacillus, which leads to the inability to achieve stable fixation of pores after gas escape. Products containing only colloid-producing strains have excessively high viscosity, which easily causes local soil compaction. They fail to form a synergistic coupling mechanism of "gas production expansion - colloid production fixation", making it difficult to directionally construct a stable soil aggregate structure. Third, there are defects in the design of the carrier and functional matrix. Traditional microbial fertilizer carriers are mostly ordinary peat or straw powder, with a simple pore structure and a macropore ratio of less than 15%, which cannot provide a suitable microenvironment for microbial survival. At the same time, they lack degradable expansion regulating components, making it difficult to overcome the physical barrier of compacted soil. The lack of structural stability regulating substances makes the temporarily formed aggregate structure easy to disintegrate after irrigation or tillage disturbance.

[0005] The existing microbial protection system of microbial fertilizers is imperfect: most products do not add micro-encapsulation materials and stress-resistant components, resulting in a survival rate of less than 30% of strains in dry, transport and extreme soil environments; at the same time, they lack metabolic induction trace element combinations, which cannot continuously activate the gas production and colloid production metabolic pathways of the microbial community.

[0006] In summary, agricultural production practices require the development of a bio-fertilizer that can adapt to the micro-ecological characteristics of the soil to be modified, integrate the synergistic coupling function of "gas production expansion-colloid fixation", and possess a porous slow-release carrier and expansion regulation mechanism. By constructing a stable soil aggregate structure in a targeted manner, the core problems of soil compaction and fertility degradation in my country's arable land can be solved, ultimately achieving a long-term improvement in farmland soil quality. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing microbial fertilizers in improving soil structure, such as poor compatibility of microbial communities, limited functionality, and imperfect carrier and protection systems. This invention provides a method for preparing a bio-fertilizer that uses a gas- and colloid-producing composite microbial community to reshape soil structure. This method aims to isolate and cultivate functional microbial communities from the target soil environment and combine them with a specially designed porous slow-release carrier, a biodegradable expansion-regulating matrix, microbial protection and induction components, and structural stabilization regulating components. This achieves a synergistic coupling of "gas-producing expansion" and "colloid-producing fixation," thereby directionally, stably, and efficiently constructing soil aggregate structures to solve the problems of soil compaction and fertility degradation.

[0008] To address the aforementioned problems and achieve the objectives and other advantages of this invention, a method for preparing a bio-fertilizer that reshapes soil structure using a gas- and colloid-producing complex microbial community is provided, comprising the following steps:

[0009] S1: Collect soil samples from the microbial enrichment area of ​​the geomorphological unit to which the soil to be modified belongs, and prepare gradient soil suspensions;

[0010] S2: Activated Bacillus licheniformis bacterial suspension, activated Bacillus subtilis bacterial suspension, activated actinomycete bacterial suspension and activated cyanobacterial suspension were obtained by separating and culturing from soil suspensions of different gradients.

[0011] S3: Preparation of gas-producing-gelatinizing composite bacterial solution: Mix activated Bacillus licheniformis bacterial solution, activated Bacillus subtilis bacterial solution, activated actinomycete bacterial solution, and activated cyanobacterial solution to obtain a gas-producing-gelatinizing composite bacterial solution; the mass percentage of Bacillus licheniformis bacterial solution in the gas-producing-gelatinizing composite bacterial solution is 25-35%, the mass percentage of Bacillus subtilis bacterial solution is 25-35%, the mass percentage of actinomycete bacterial solution is 20-30%, and the mass percentage of cyanobacterial solution is 10-20%.

[0012] S4: Preparation of porous slow-release carrier: Corn straw biochar is pulverized and sieved to a particle size of <3mm, humic peat and humic acid soil are dried and sieved to a particle size of <2mm, and bentonite is pulverized and sieved to a particle size of <1mm; by mass, 20-35 parts of corn straw biochar, 10-20 parts of humic peat, 10-20 parts of humic acid soil and 5-15 parts of bentonite are mixed to obtain a porous slow-release carrier.

[0013] S5 is prepared by dissolving 5-12 parts of modified corn starch and 3-8 parts of beet molasses in 5-10 parts of distilled water to obtain a biodegradable swelling control matrix.

[0014] S6: By mass fraction, take 1-2 parts sodium alginate, 1-3 parts yeast powder, 1-2 parts betaine, and 1-2 parts trace element solution and mix them to obtain the microbial protection and induction component;

[0015] S7: By mass, take 1-2 parts of lignin sulfonate, 1-2 parts of zeolite powder, and 1-2 parts of vermiculite and mix them to obtain a structure-stabilizing regulating component;

[0016] S8: By mass fraction, mix 5-12 parts of the gas-producing-colloid-producing composite bacterial liquid obtained in step S3, 45-65 parts of the porous slow-release carrier obtained in step S4, 10-20 parts of the degradable expansion-regulating matrix obtained in step S5, 3-8 parts of the microbial protection and induction component obtained in step S6, and 2-5 parts of the structure stabilization regulating component obtained in step S7, stir, granulate, and dry at 35-40℃ until the moisture content is <10% to obtain the bio-fertilizer.

[0017] Preferably, in the method for preparing bio-fertilizer that reshapes soil structure using gas-producing and gum-producing compound microbial communities, in step S2, the isolation and culture includes culturing the target microbial community using a special culture medium; the special culture medium includes Bacillus culture medium, Actinomycete culture medium, and Cyanobacterial culture medium; the Bacillus culture medium is used for culturing Bacillus licheniformis and Bacillus subtilis;

[0018] The Bacillus culture medium is prepared by weighing 3.0-3.5g of beef extract, 10.0-12.0g of peptone, 5.0-5.5g of NaCl, and 15-25g of agar, adding 1000mL of purified water to dissolve and adjusting the pH to 7.4-7.5.

[0019] The actinomycete culture medium is prepared by weighing 20-25g of soluble starch, 0.8-0.9g of KNO3, 0.4-0.5g of NaCl, 0.5-0.6g of K2HPO4·3H2O, 0.5-0.6g of MgSO4·7H2O, 0.1-0.15g of CaCl2, 0.005-0.01g of MnSO4, 0.01g of FeSO4·7H2O, and 20g of agar, dissolving them in 1000mL of purified water, and adjusting the pH to 7.3-7.4.

[0020] The cyanobacteria culture medium was prepared by weighing 1.5g of NaNO3, 0.04g of K2HPO4·3H2O, 0.075g of MgSO4·7H2O, 0.036g of CaCl2·2H2O, 0.006g of citric acid, 0.006g of ferric ammonium citrate, 0.001g of disodium EDTA, 0.02g of Na2CO3, and 1mL of trace element solution, adding 1000mL of purified water to dissolve and adjusting the pH to 7.0-7.2.

[0021] The method for preparing the trace element solution is as follows:

[0022] Weigh out 0.02g of boric acid, 0.02g of manganese sulfate, 0.02g of zinc sulfate, 0.01g of copper sulfate, 0.01g of sodium molybdate, and 0.01g of cobalt chloride, and set aside. Heat 800mL of deionized water to 40-50℃, and add the above-mentioned prepared reagents to the water one by one while stirring until completely dissolved. Finally, add 10mL of EDTA-Fe solution that has been prepared separately and cooled to room temperature. Mix well and then make up to 1000mL with deionized water. Adjust the pH value to 6.0-7.0. Filter through a 0.22μm filter membrane to remove bacteria and obtain the mother liquor. Dilute the mother liquor with purified water at a volume ratio of 1:100 to obtain the trace element solution.

[0023] The EDTA-Fe solution is prepared as follows: Weigh 1.0 g of Na2EDTA and 0.8 g of FeSO4·7H2O, add them to 500 mL of deionized water at 60 °C, and stir until completely dissolved to obtain a disodium EDTA solution and a ferrous sulfate solution; Under continuous stirring, slowly pour the ferrous sulfate solution into the disodium EDTA solution, maintain a constant temperature of 60 °C and stir for 20-30 min to allow for full chelation, cool to room temperature, and then dilute to 1000 mL with deionized water to obtain a stable golden yellow EDTA-Fe solution for later use;

[0024] Before using the special culture medium, sterilization and sterility tests were performed on it: the Bacillus culture medium and Actinomycete culture medium were sterilized by high-pressure steam at 121°C for 20 min, and the Cyanobacterial culture medium was sterilized by high-pressure steam at 115°C and 0.06 MPa for 15 min.

[0025] Preferably, in the method for preparing bio-fertilizer using gas- and colloid-producing composite microbial communities to reshape soil structure, the specific steps for preparing the gradient soil suspension in step S1 are as follows: Soil samples are collected from the microbial enrichment area of ​​the geomorphological unit to which the soil to be modified belongs; 10g of soil is added to 90mL of sterile physiological saline and shaken to prepare 10... -1 Gradient soil suspension, then gradient diluted to obtain 10 -2 ~10 -5 Gradient soil suspension;

[0026] In step S2, the activated Bacillus licheniformis bacterial solution and the activated Bacillus subtilis bacterial solution are obtained by the following method: take 10 -4 and 10 -5 Gradient soil suspensions were inoculated onto Bacillus culture medium and cultured at 36.6-37.2℃ for 18-24 h. Characteristic colonies were then picked and transferred to liquid culture medium for further culture.

[0027] The activated actinomycete culture was obtained by the following method: taking 10 -3 and 10 -4 Gradient soil suspensions were inoculated onto actinomycete culture medium and cultured at 27.5-28.5℃ and 63%-67% humidity for 36-48 hours. Characteristic colonies were then picked and transferred to liquid culture medium for further culture.

[0028] The activated cyanobacteria solution was obtained by the following method: Take 10 -2 and 10 -3 Gradient soil suspensions were inoculated into cyanobacteria culture medium and cultured at 20-28℃ and 2000-3000 lux light for 36-48 hours. Characteristic colonies were then selected and transferred to liquid culture medium for further culture.

[0029] Preferably, in the method for preparing bio-fertilizer that uses gas-producing and gum-producing compound microbial communities to reshape soil structure, in step S5, the modified corn starch is hydroxypropyl corn starch.

[0030] The preparation method of hydroxypropyl corn starch is as follows: First, 100g of corn starch is dispersed in 350mL of 75% ethanol solution, and 1.5g of 2% sodium hydroxide solution is added for alkalization treatment. The pH of the alkalization system is maintained at 8.5-9.0 to activate the starch granules. Then, under sealed conditions, the mixture is heated to 42-45℃, and 10g of propylene oxide is slowly added dropwise as an etherifying agent. The mixture is kept warm and stirred for 16h to graft hydroxypropyl groups onto the starch molecules. After the reaction, the mixture is neutralized to pH 6.8-7.2 with 1% hydrochloric acid. Then, it is washed repeatedly 3-4 times with 80% ethanol solution to remove salts and unreacted reagents. After centrifugation, dehydration, low-temperature vacuum drying at 40℃, and pulverization through a 100-mesh sieve, hydroxypropyl corn starch is obtained.

[0031] In step S8, the stirring speed is 150-200 rpm, the stirring time is 20-30 min, and the particle size of the granulation is 2-5 mm.

[0032] Preferably, in the method for preparing bio-fertilizer for reshaping soil structure using gas-producing and gum-producing composite microbial communities, in step S3, before mixing the various activated bacterial solutions, the microbial concentrations of activated Bacillus licheniformis, activated Bacillus subtilis, activated actinomycetes, and activated cyanobacteria are measured respectively; then, based on the microbial concentrations of each activated bacterial solution, the required volume of each bacterial solution to be mixed is calculated and determined, and then the solutions are mixed according to their respective volumes so that in the resulting gas-producing and gum-producing composite bacterial solution, the mass of Bacillus licheniformis is 25-35%, the mass of Bacillus subtilis is 25-35%, the mass of actinomycetes is 20-30%, and the mass of cyanobacteria is 10-20%.

[0033] Preferably, in the method for preparing the bio-fertilizer using gas-producing and gum-producing compound microbial communities to reshape soil structure, step S2 specifically includes:

[0034] S2.1 Picking characteristic colonies: From the plating of 10 -4 and 10 -5 Single colonies exhibiting typical morphological characteristics of Bacillus licheniformis or Bacillus subtilis were picked from Bacillus agar plates containing gradient soil suspensions; [The text abruptly shifts to a different topic:] From a plate plate containing 10... -3 and 10 -4 Single colonies exhibiting typical actinomycete morphological characteristics were picked from actinomycete culture plates containing gradient soil suspensions; [The text then abruptly shifts to a different topic:] From a plate containing 10 [units of something], single colonies were picked from the plate. -2 and 10 -3 Single colonies with typical morphological characteristics of cyanobacteria were picked from cyanobacterial culture plates containing gradient soil suspensions.

[0035] S2.2 Functional Validation and Screening:

[0036] For selected single colonies of Bacillus licheniformis or Bacillus subtilis, they are inoculated onto Bacillus agar plates for functional verification and incubated at 36.6-37.2℃ for 18-24 hours. After incubation, the diameter of the calcification zone around the colony is measured. Single colonies with a calcification zone diameter of not less than 3 mm are selected as Bacillus strains that have passed functional verification. The Bacillus agar medium for functional verification is prepared by adding 15-20 g / L of calcium carbonate to the Bacillus medium formula.

[0037] For the selected single colonies of actinomycetes, they were inoculated into liquid culture medium for functional verification of actinomycetes and cultured at 27.5-28.5℃ and 150-180 rpm for 48-72 h with shaking. After the culture, the viscosity of the fermentation broth was measured using a rotational viscometer. Cultures with a fermentation broth viscosity of not less than 50 mPa·s were selected as the actinomycete strains that passed the functional verification. The liquid culture medium for functional verification of actinomycetes was prepared by replacing the carbon source soluble starch with 50-100 g / L sucrose and removing agar from the original actinomycete culture medium formula.

[0038] Selected single colonies of cyanobacteria were inoculated into cyanobacterial liquid culture medium for functional verification and cultured at 20-28℃ and light intensity of 2000-3000 lux for 5-7 days. After culture, the extracellular polysaccharide content in the culture medium was determined by the phenol-sulfuric acid method. Cultures with an extracellular polysaccharide content of not less than 100 mg / L were selected as cyanobacterial strains that passed functional verification. The cyanobacterial liquid culture medium for functional verification was prepared by removing agar from the cyanobacterial culture medium formula.

[0039] S2.3 Scale-up culture: The Bacillus strains, Actinomycete strains, and Cyanobacterial strains that passed functional verification in step S2.2 were aseptically transferred to their respective corresponding scale-up liquid culture media for scale-up culture. The Bacillus liquid culture medium for scale-up culture was prepared by removing agar from the Bacillus medium formula. The Actinomycete liquid culture medium for scale-up culture was prepared by removing agar from the Actinomycete medium formula. The composition of the Cyanobacterial liquid culture medium for scale-up culture was the same as that of the Cyanobacterial liquid culture medium used for functional verification.

[0040] The expansion culture of Bacillus strains was carried out at 36.6-37.2℃, the expansion culture of Actinomycete strains was carried out at 27.5-28.5℃, and the expansion culture of Cyanobacterial strains was carried out at 20-28℃ under light conditions of 2000-3000 lux; thereby obtaining activated Bacillus licheniformis bacterial solution, activated Bacillus subtilis bacterial solution, activated Actinomycete bacterial solution, and activated Cyanobacterial solution, respectively.

[0041] Preferably, in the method for preparing bio-fertilizer using gas-producing and colloid-producing composite microbial communities to reshape soil structure, in step S8, before mixing all components and stirring, a directional adsorption and activation step is added:

[0042] First, preheat the porous sustained-release carrier obtained in step S4 at 35-40℃ for 5-15 minutes;

[0043] Then, 15%-30% of the total mass of the preheated porous slow-release carrier is mixed with the gas-producing and gel-producing composite bacterial solution obtained in step S3 at a stirring speed of 150-200 rpm for 3-8 minutes to form a microbial-carrier complex with a water content of 20%-35% and a temperature of 30-35℃.

[0044] The resulting microbial-carrier complex was then activated by being sealed and allowed to stand for 20-40 minutes at 28-32℃ and 75%-85% relative humidity.

[0045] Finally, the activated microbial-carrier complex is mixed with the remaining porous slow-release carrier, degradable swelling control matrix, microbial protection and induction components, and structural stability regulating components, followed by subsequent stirring, granulation, and drying.

[0046] Preferably, in the method for preparing bio-fertilizer using gas- and gum-producing composite microbial communities to reshape soil structure, a soil environment simulation induction step is added after step S3 and before step S8:

[0047] Take the fresh soil obtained when collecting soil samples in step S1 and add it to the gas-producing and gum-producing compound bacterial solution obtained in step S3. The addition ratio is 5-10g of fresh soil per 100mL of bacterial solution.

[0048] The mixture was placed in a shaker at 25-28℃ and 100-150 rpm, and cultured under aeration for 12-24 hours with a light intensity of 500-1000 lux and a light / dark cycle of 12h / 12h.

[0049] After cultivation, the soil particles were removed by filtration through a sterile sieve with a pore size of 20-50 μm, resulting in a gas-producing and gel-producing compound bacterial solution pre-adapted to the target soil microecology.

[0050] Preferably, in the method for preparing bio-fertilizer using gas-producing and gum-producing compound microbial communities to reshape soil structure, the drying in step S8 specifically adopts a stepped induced drying process:

[0051] First stage: Place the granulated wet granules in an environment with a temperature of 38-40℃ and a relative humidity of 60%-70% for 1-2 hours to dry them, so that the surface of the granules is initially shaped.

[0052] The second stage: Adjust the environmental parameters to a temperature of 30-32℃ and a relative humidity of 75%-85%, and maintain this for 2-3 hours. Under this high humidity environment, slow down the overall dehydration rate and provide a suitable microenvironment for short-term metabolism and repair of the microbial community.

[0053] The third stage: Adjust the temperature to 35-37℃ and reduce the relative humidity to 50%-60% for 1 hour, and use the metabolic activity of the bacteria activated in the previous stage to start the spore formation process.

[0054] Fourth stage: Raise the temperature to 40-42℃ and reduce the relative humidity to below 20%, continue drying until the moisture content of the particles is less than 10%, and complete the final maturation and shaping of the spores and the deep dehydration of the particles.

[0055] A bio-fertilizer prepared using the aforementioned method for reshaping soil structure with gas-producing and colloid-producing composite microbial communities, wherein the bio-fertilizer is composed of the following components in parts by weight: 5-12 parts of gas-producing and colloid-producing composite microbial liquid or gas-producing and colloid-producing composite microbial liquid pre-adapted to the target soil microecology, 45-65 parts of porous slow-release carrier, 10-20 parts of degradable expansion-regulating matrix, 3-8 parts of microbial protection and induction components, and 2-5 parts of structural stability regulating components.

[0056] Among them, the mass of Bacillus licheniformis bacterial solution in the gas-producing-colloid-producing compound bacterial solution is 25-35%, the mass of Bacillus subtilis bacterial solution is 25-35%, the mass of Actinomycetes bacterial solution is 20-30%, and the mass of Cyanobacteria solution is 10-20%.

[0057] The porous slow-release carrier comprises, by weight, 20-35 parts corn straw biochar, 10-20 parts humic peat, 10-20 parts humic acid soil, and 5-15 parts bentonite.

[0058] The biodegradable swelling control matrix comprises, by weight, 5-12 parts modified corn starch, 3-8 parts beet molasses, and 5-10 parts distilled water;

[0059] The microbial protection and induction components, by mass parts, include 1-2 parts sodium alginate, 1-3 parts yeast powder, 1-2 parts betaine, and 1-2 parts trace element solution;

[0060] The structural stabilizing and regulating components, by mass parts, include 1-2 parts of lignin sulfonate, 1-2 parts of zeolite powder, and 1-2 parts of vermiculite.

[0061] The present invention has at least the following beneficial effects:

[0062] This invention isolates and combines Bacillus licheniformis, Bacillus subtilis, actinomycetes, and cyanobacteria from target soil to form a gas- and gum-producing complex microbial community with high ecological adaptability. This significantly improves the colonization rate and functional durability of the microbial community in the soil, effectively overcoming the technical defects of traditional commercial strains such as poor adaptability and low colonization rate.

[0063] This invention integrates a synergistic coupling mechanism of "gas-producing expansion-gel-producing fixation", utilizing gas-producing bacteria to create pores and gel-producing bacteria to fix the structure, so as to achieve simultaneous soil loosening and cementation. It can quickly and directionally build stable soil aggregates, significantly improving soil aeration, water retention and erosion resistance.

[0064] The porous slow-release carrier designed in this invention is composed of corn straw biochar, humic peat, humic acid soil and bentonite. It has a gradient pore structure, which can provide a long-term habitat and slow-release protection environment for microorganisms. Combined with the carbon source slow-release function of the biodegradable expansion-regulating matrix, it can jointly maintain the metabolic activity of the microbial community.

[0065] The microbial protection and inducing components contained in this invention include sodium alginate, yeast powder, betaine, and various trace elements. These components not only protect the activity of microorganisms during processing and storage, but also activate their metabolic pathways in the soil, enhance the stress resistance of the microbial community, and ensure the functional stability of the fertilizer in complex field environments.

[0066] This invention enhances the stability of newly formed soil aggregates from multiple dimensions, including cementation, water retention, and mechanical support, by adding structural stabilizing components such as lignin sulfonate, zeolite powder, and vermiculite. This enables the aggregates to effectively resist freeze-thaw cycles, irrigation, and tillage disturbances, thereby achieving long-term improvement of soil structure.

[0067] The preparation process of this invention is scientific and feasible, covering key steps such as preparation of special culture medium, isolation and activation of bacterial community, functional verification and screening, directional adsorption activation and step-induced drying. The process parameters are clear, the operation is strong, the raw materials are easy to obtain, and it is suitable for large-scale production and application.

[0068] In practical applications, this invention has demonstrated significant soil improvement and crop yield increase effects. Field trials have confirmed that it can effectively increase soil organic matter content, improve nutrient supply, and promote root development, ultimately achieving a synergistic improvement in crop yield and quality. It provides an efficient and reliable technical solution for the ecological restoration of degraded soils and sustainable agricultural development.

[0069] 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

[0070] Figure 1 This is a flowchart of a bio-fertilizer preparation process according to an embodiment of the present invention. Detailed Implementation

[0071] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, so that those skilled in the art can implement it based on the description.

[0072] 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.

[0073] like Figure 1As shown, this invention provides a method for preparing bio-fertilizer that reshapes soil structure using a gas-producing and gum-producing composite microbial community, comprising the following steps: S1: Collecting soil samples from microbial enrichment sites in the geomorphological unit to which the soil to be modified belongs, and preparing gradient soil suspensions; S2: Separating and culturing from soil suspensions of different gradients to obtain activated Bacillus licheniformis bacterial suspension, activated Bacillus subtilis bacterial suspension, activated actinomycete bacterial suspension, and activated cyanobacterial suspension; S3: Preparing a gas-producing-gum-producing composite microbial community: [The text abruptly ends here, so the translation stops.] The bacterial solution and activated cyanobacterial solution are mixed to obtain a gas-producing-colloid-producing composite bacterial solution; the gas-producing-colloid-producing composite bacterial solution contains 25-35% Bacillus licheniformis bacterial solution, 25-35% Bacillus subtilis bacterial solution, 20-30% Actinomycete bacterial solution, and 10-20% cyanobacterial solution; S4: Preparation of porous slow-release carrier: Corn straw biochar is pulverized and sieved to a particle size <3mm, humic peat and humic acid soil are dried and sieved to a particle size <2mm, and bentonite is pulverized and sieved to a particle size <1mm; by mass fraction, corn straw biochar is taken... A porous slow-release carrier is obtained by mixing 20-35 parts of charcoal, 10-20 parts of humic peat, 10-20 parts of humic acid soil, and 5-15 parts of bentonite; S5: by weight, 5-12 parts of modified corn starch and 3-8 parts of beet molasses are dissolved in 5-10 parts of distilled water to obtain a biodegradable swelling-regulating matrix; S6: by weight, 1-2 parts of sodium alginate, 1-3 parts of yeast powder, 1-2 parts of betaine, and 1-2 parts of trace element solution are mixed to obtain a microbial protection and induction component; S7: by weight, lignin sulfonic acid is... S8: Mix 1-2 parts salt, 1-2 parts zeolite powder, and 1-2 parts vermiculite to obtain a structure stabilizing and regulating component; S9: Mix 5-12 parts of the gas-producing-colloid-producing composite bacterial liquid obtained in step S3, 45-65 parts of the porous slow-release carrier obtained in step S4, 10-20 parts of the degradable expansion regulating matrix obtained in step S5, 3-8 parts of the microbial protection and induction component obtained in step S6, and 2-5 parts of the structure stabilizing and regulating component obtained in step S7 by mass, stir, granulate, and dry at 35-40℃ until the moisture content is <10% to obtain the bio-fertilizer.

[0074] In the field of agricultural biotechnology, soil compaction and the destruction of soil aggregate structure are core challenges restricting arable land quality and crop yield. Existing microbial fertilizers often use commercially available standard strains, which are not derived from the soil to be modified. This results in poor adaptation to the soil's microecological environment after application, low colonization success rates, and the inability to establish stable functional microbial communities, leading to short-lived and limited improvement effects. Furthermore, most products have relatively simple functional designs, either focusing solely on loosening the soil through microbial gas production, but the pores formed after gas escape cannot be fixed, or relying solely on microbial gum production to bind soil particles, which easily leads to excessive localized binding. They fail to effectively coordinate the two key processes of creating space through gas production and stabilizing the structure through gum production, thus making it difficult to directionally construct durable and stable soil aggregates. In addition, the pore structure of traditional fertilizer carrier materials is not ideal, providing insufficient protection for microorganisms and lacking a specific matrix to trigger and regulate microbial function in the soil. This makes microbial activity susceptible to environmental influences, resulting in poor overall improvement effects.

[0075] To address the aforementioned shortcomings, this approach first collects samples from microbial-rich areas in the target soil region requiring improvement and prepares a series of soil suspensions with varying dilutions. This initial step aims to ensure that the subsequently isolated microbial strains are naturally adapted to the soil environment to be modified, laying the foundation for improving the survival and colonization capacity of the microbial community in the target soil and overcoming the problem of poor adaptability of introduced strains. Next, from these suspensions of different dilutions, bacterial suspensions of Bacillus licheniformis, Bacillus subtilis, Actinomycetes, and Cyanobacteria are isolated, cultured, and activated. This step aims to purposefully screen and obtain microbial resources with specific functions. Bacillus licheniformis excels in gas production, while Actinomycetes and Cyanobacteria primarily function by secreting cementing substances, preparing core strains for subsequent functional synergy.

[0076] Then, the activated bacterial solutions are mixed in specific proportions to prepare a gas-producing and gum-producing composite bacterial solution. Bacillus licheniformis and Bacillus subtilis are mainly responsible for gas production, while actinomycetes and cyanobacteria are mainly responsible for gum production. Through precise proportioning, the gas-producing and gum-producing microorganisms work synergistically. Gas production generates expansion force to break up soil compaction and create pores, while the produced gelatinous substances can promptly bind and fix these pores and surrounding soil particles, thus constructing a stable aggregate structure. To provide a suitable growth and function environment for these functional microorganisms, a porous slow-release carrier is also prepared. This carrier is composed of corn straw biochar, humic peat, humic acid soil, and bentonite mixed in specific proportions. Its rich porous structure provides a habitat for microorganisms and plays a role in slow-release protection. Simultaneously, a biodegradable expansion-regulating matrix is ​​prepared, mainly composed of modified corn starch and beet molasses. This biodegradable expansion-regulating matrix can gradually degrade in the soil, continuously providing the carbon source and energy required for the microorganisms to initiate and maintain gas-producing and gum-producing metabolism. In addition, microbial protection and induction components, including sodium alginate, yeast powder, betaine, and various trace elements, were prepared to protect microbial activity during fertilizer processing and storage, and to induce and stimulate their metabolic functions after application to the soil. Structure-stabilizing components, including lignin sulfonate, zeolite powder, and vermiculite, were also prepared to enhance the mechanical stability and anti-disintegration ability of the final soil aggregates.

[0077] Finally, the aforementioned gas-producing and colloid-producing composite bacterial solution, porous slow-release carrier, biodegradable expansion-regulating matrix, microbial protection and induction components, and structural stability regulating components were mixed according to a set mass ratio. After thorough stirring, granulation, and drying at a low temperature of 35-40℃ until the moisture content was below 10%, the final product, bio-fertilizer, was obtained. This integrated step organically combines highly adaptable functional microbial communities, a slow-release carrier providing a microenvironment, a nutrient-supplying regulating matrix, a protective component ensuring activity, and a regulatory substance enhancing stability, forming a complete soil improvement system. This system fully leverages the ecological adaptability advantages of the composite microbial community and, through the coordination of the carrier, matrix, and auxiliary materials, ensures that the core synergistic mechanism of gas production and colloid fixation can be effectively activated and maintained in the target soil, thereby achieving targeted and stable improvement of soil aggregate structure.

[0078] In this technical solution, gas-producing expansion mainly refers to the process by which functional microorganisms produce gas through metabolism, creating micro-pressure in the soil, which loosens compacted soil and generates pores. Gel fixation refers to the process by which functional microorganisms secrete sticky extracellular polymers such as polysaccharides and proteins, binding and encapsulating soil particles, thereby fixing pores and forming stable aggregates. The porous slow-release carrier is a solid material with numerous micropores that adsorbs and protects microorganisms and slowly releases nutrients, providing a long-term microenvironment for their survival and activity. The biodegradable expansion-regulating matrix is ​​a substance that gradually decomposes under the action of soil microorganisms, primarily providing initial and continuous carbon and energy sources for microbial gas-producing metabolism. Microbial protection and induction components are combinations of substances used to protect microorganisms in adverse environments and activate their specific metabolic pathways. Structural stabilization regulating components are additives used to enhance the internal cohesion of soil aggregates and improve their resistance to water erosion or mechanical disturbance. This preparation method, through the systematic combination of the above steps, ultimately yields a bio-fertilizer that can adapt to the soil to be modified, integrate synergistic functions, and possess a good protection and support system. It aims to effectively construct and stabilize the soil aggregate structure, providing a new technical approach to solve the problems of soil compaction and fertility degradation.

[0079] In another embodiment, in the method for preparing bio-fertilizer using gas-producing and agar-producing compound microbial communities to reshape soil structure, step S2, the isolation and culture includes culturing the target microbial community using a special culture medium; the special culture medium includes Bacillus culture medium, Actinomycete culture medium, and Cyanobacterial culture medium; the Bacillus culture medium is used for culturing Bacillus licheniformis and Bacillus subtilis; the Bacillus culture medium is prepared by weighing 3.0-3.5g of beef extract, 10.0-12.0g of peptone, 5.0-5.5g of NaCl, and 15-25g of agar, adding 1000mL of purified water to dissolve and adjusting the pH to 7.4-7.5; the Actinomycete culture medium is prepared by weighing 20-25g of soluble starch, 0.8-0.9g of KNO3, 0.4-0.5g of NaCl, 0.5-0.6g of K2HPO4·3H2O, 0.5-0.6g of MgSO4·7H2O, and CaCl2. The cyanobacteria culture medium is prepared by dissolving 0.1-0.15g of NaNO3, 0.005-0.01g of K2HPO4·3H2O, 0.075g of MgSO4·7H2O, 0.036g of CaCl2·2H2O, 0.006g of citric acid, 0.006g of ferric ammonium citrate, 0.001g of disodium EDTA, 0.02g of Na2CO3, and 1mL of trace element solution in 1000mL of purified water and adjusting the pH to 7.0-7.2.

[0080] The method for preparing the trace element solution is as follows:

[0081] Weigh out 0.02g boric acid, 0.02g manganese sulfate, 0.02g zinc sulfate, 0.01g copper sulfate, 0.01g sodium molybdate, and 0.01g cobalt chloride, and set aside. Heat 800mL of deionized water to 40-50℃, and add the prepared reagents sequentially to the water while stirring until completely dissolved. Finally, add 10mL of EDTA-Fe solution that has been prepared separately and cooled to room temperature. Mix well and then dilute to 1000mL with deionized water. Adjust the pH of the stock solution to 6.0-7.0. Filter the solution through a 0.22μm filter membrane for sterilization, and store it in a sterile brown bottle at 4℃. When using, dilute the stock solution with purified water at a volume ratio of 1:100, and add 1mL of the diluted solution to the above cyanobacteria culture medium.

[0082] The EDTA-Fe solution is prepared as follows: Weigh 1.0 g of Na2EDTA and 0.8 g of FeSO4·7H2O, add them to 500 mL of deionized water at 60℃, and stir until completely dissolved to obtain a disodium EDTA solution and a ferrous sulfate solution; under continuous stirring, slowly pour the ferrous sulfate solution into the disodium EDTA solution, maintain a constant temperature of 60℃ and stir for 20-30 min to allow for full chelation, cool to room temperature, and then dilute to 1000 mL with deionized water to obtain a stable golden yellow EDTA-Fe solution for later use;

[0083] Before using the special culture medium, sterilization and sterility tests were performed on it: the Bacillus culture medium and Actinomycete culture medium were sterilized by high-pressure steam at 121°C for 20 min, and the Cyanobacterial culture medium was sterilized by high-pressure steam at 115°C and 0.06 MPa for 15 min.

[0084] The implementation of technologies employing gas- and gum-producing microbial communities to construct soil structures may face several specific technical challenges. For example, efficiently isolating and enriching the required Bacillus licheniformis, Bacillus subtilis, actinomycetes, and cyanobacteria from complex soil microbial communities, and ensuring their full activation, is fundamental to guaranteeing subsequent functional coupling effects. Inappropriate culture medium composition or unclear culture conditions may lead to slow growth of target bacteria, contamination by other microorganisms, or insufficient functional expression. Furthermore, the thoroughness of sterilization of the culture medium and experimental equipment before mixed culture directly affects the success of pure cultures and the activity of the final microbial community solution. Incomplete sterilization may result in excessive growth of other microorganisms, inhibiting the target functional bacteria, or even introducing harmful microorganisms, affecting the safety and effectiveness of the fertilizer.

[0085] To address the aforementioned issues, this protocol specifies concrete methods for isolating and culturing the target bacterial communities. The protocol stipulates the use of specially formulated culture media for culturing the target bacterial communities, including Bacillus licheniformis and Bacillus subtilis culture media, Actinomycete culture media for culturing actinomycetes, and Cyanobacterial culture media for culturing cyanobacteria. These specialized culture media are prepared with specific proportions and parameters. For example, the Bacillus licheniformis culture medium consists of beef extract, peptone, sodium chloride, and agar, with the pH adjusted to a slightly alkaline range of 7.4-7.5. This creates an optimal nutritional and acid-base environment for Bacillus licheniformis growth, promoting rapid reproduction and functional expression. The Actinomycete culture medium uses soluble starch as the main carbon source and contains potassium nitrate, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, and trace amounts of manganese sulfate and ferrous sulfate, with the pH adjusted to 7.3-7.4. This formulation effectively promotes the growth of actinomycetes and the synthesis of their metabolites. The cyanobacteria culture medium provides sodium nitrate, phosphate, magnesium and calcium ions, ferric ammonium citrate, and trace element solution, with the pH adjusted to 7.0-7.2 to meet the physiological needs of cyanobacteria for photosynthesis, autotrophy, and nitrogen fixation, thus promoting their biomass and extracellular polysaccharide production. By using these specialized culture media with clearly defined components and proportions, the preferential growth of desired functional microorganisms can be selectively promoted, improving the efficiency of isolation and screening and the quality of strains.

[0086] This protocol also emphasizes the need for rigorous sterilization and aseptic testing before using these specialized culture media. Specifically, the prepared Bacillus and Actinomycete media are autoclaved at 121°C for 20 minutes, while the cyanobacteria media is autoclaved at 115°C and 0.06 MPa for 15 minutes. This step aims to thoroughly kill all microbial vegetative cells and most spores that may exist in the culture media and on the container walls using high temperature and pressure, ensuring that subsequent inoculation operations are performed in a sterile environment and preventing contamination. Different sterilization parameters are used for different culture media because some nutrients (such as certain organic components and trace elements in cyanobacteria media) may be sensitive to high temperatures. Separate conditions ensure sterilization effectiveness while minimizing damage to the effective components of the culture medium, thus guaranteeing the cultivation effect. Aseptic testing is a crucial step in verifying the success of the sterilization operation. By incubating the sterilized culture media under suitable conditions for a period of time and observing for colony growth, its sterility is confirmed. This provides a reliable quality control prerequisite for obtaining pure and efficient functional bacterial solutions.

[0087] In the above technical solutions, the specialized culture medium refers to a medium prepared according to the specific nutritional needs and growth characteristics of microorganisms, consisting of carbon sources, nitrogen sources, inorganic salts, growth factors, and water in specific proportions, used for the isolation, purification, cultivation, or identification of such microorganisms. High-pressure steam sterilization is a thorough sterilization method that utilizes saturated steam to generate high temperatures under high pressure to kill all microorganisms (including bacterial spores). It is a core means of ensuring a sterile environment in microbiological experiments and industrial production. Sterility testing is a quality control process that verifies whether sterilized items have reached a sterile state. This is typically done by culturing the sterilized items under suitable conditions and observing whether microbial growth occurs. The implementation of these steps collectively ensures that the functional microbial communities isolated from the soil to be modified are pure, active, and have clearly defined functions. This lays a solid technical foundation for constructing a highly efficient and synergistic gas- and colloid-producing compound bacterial solution, thereby improving the reliability and stability of the final bio-fertilizer product in improving soil structure.

[0088] In another embodiment, in the method for preparing bio-fertilizer using gas- and colloid-producing composite microbial communities to reshape soil structure, step S1, specifically the steps for preparing gradient soil suspension, are as follows: Soil samples are collected from the microbial enrichment area of ​​the geomorphological unit to which the soil to be modified belongs; 10g of soil is added to 90mL of sterile physiological saline and shaken to prepare 10... -1 Gradient soil suspension, then gradient diluted to obtain 10 -2 ~10 -5 Gradient soil suspension; in step S2, the activated Bacillus licheniformis bacterial solution and the activated Bacillus subtilis bacterial solution are obtained by the following method: take 10 -4 and 10 -5 Gradient soil suspensions were inoculated onto Bacillus culture medium and cultured at 36.6-37.2℃ for 18-24 hours. Characteristic colonies were then picked and transferred to liquid culture medium for further culture. The activated actinomycete culture was obtained by taking 10... -3 and 10 -4 Gradient soil suspensions were inoculated onto actinomycete culture medium and cultured at 27.5-28.5℃ and 63%-67% humidity for 36-48 hours. Characteristic colonies were then picked and transferred to liquid culture medium for further cultivation. The activated cyanobacteria solution was obtained by taking 10... -2 and 10 -3 Gradient soil suspensions were inoculated into cyanobacteria culture medium and cultured at 20-28℃ and 2000-3000 lux light for 36-48 hours. Characteristic colonies were then selected and transferred to liquid culture medium for further culture.

[0089] The specific steps for preparing gradient soil suspensions are as follows: collect soil samples from the microbial enrichment area of ​​the geomorphological unit to which the soil to be modified belongs; add 10g of soil to 90mL of sterile physiological saline and shake to prepare 10... -1 Gradient soil suspension, then gradient diluted to obtain 10-2 Up to 10 -5 Gradient soil suspension. The purpose of this step is to fully release microorganisms from soil particles into sterile saline solution through physical agitation to form a homogeneous initial suspension, which is then obtained through precise ten-fold serial dilutions, resulting in a series of microbial suspensions with concentrations decreasing by orders of magnitude. -1 This represents one soil sample mixed with nine parts of diluent, i.e., diluted tenfold; the subsequent 10 -2 Up to 10 -5 This represents a dilution of 100 to 100,000 times. This gradient dilution method can effectively address the situation where the number of microorganisms in the soil is large and their distribution is uneven, ensuring that a dilution with a suitable microbial density can be found during subsequent plate inoculation, thereby increasing the probability of obtaining individual target microbial colonies that can be isolated and counted, laying the foundation for the isolation of pure strains.

[0090] Activated Bacillus licheniformis bacterial solution and activated Bacillus subtilis bacterial solution were obtained by the following method: Take 10 -4 and 10 -5 Gradient soil suspensions were inoculated onto Bacillus agar medium and incubated at 36.6-37.2℃ for 18-24 h. Characteristic colonies were then picked and transferred to liquid medium for further culture. 10... -4 and 10 -5 The use of these two highly diluted suspensions for inoculation is due to the typically large number of bacteria in soil. High dilutions significantly reduce the total number of microorganisms in the sample, minimizing interference from other microorganisms and preventing overly dense colonies on the plates, thus facilitating the selection of morphologically typical single-target colonies. The incubation temperature is strictly controlled at 36.6-37.2℃ because this range is close to the optimal growth temperature for *Bacillus licheniformis* and *Bacillus subtilis*, maximizing their metabolic activity, promoting rapid growth, and forming distinctive colony morphologies. Selecting characteristic colonies is crucial for ensuring strain purity, while subsequent liquid-scale culture aims to exponentially multiply the selected pure bacteria to obtain sufficient quantities of highly active, logarithmic-phase cells, providing a high-quality and abundant source of bacteria for preparing the gas- and colloid-producing composite bacterial solution.

[0091] Activated actinomycete culture was obtained by the following method: Take 10 -3 and 10 -4 Gradient soil suspensions were inoculated onto actinomycete culture medium and incubated at 27.5-28.5℃ and 63%-67% humidity for 36-48 hours. Characteristic colonies were then transferred to liquid culture medium for further expansion. 10... -3 and 10 -4The use of these two intermediate dilutions for inoculation is based on the fact that actinomycetes are generally less abundant than bacteria in soil, and excessive dilution may lead to missed detection of the target bacteria. This dilution helps to balance the isolation effect and the detection probability. The incubation temperature was set at 27.5-28.5℃, coupled with a humidity environment of 63%-67%, simulating the cool, moist microecological environment commonly found in the surface layer of natural soil for actinomycetes. These conditions are particularly conducive to the vigorous growth of actinomycete hyphae and the formation of characteristic spores, resulting in colonies exhibiting typical morphologies such as dryness, powderiness, and multicoloration, making them easy to identify and pick up visually. The relatively long incubation time of 36-48 hours provides ample time to ensure the relatively slow growth cycle of actinomycetes.

[0092] Activated cyanobacteria solution is obtained by the following method: Take 10 -2 and 10 -3 Gradient soil suspensions were inoculated onto cyanobacteria culture medium and cultured at 20-28℃ and 2000-3000 lux light for 36-48 h. Characteristic colonies were then picked and transferred to liquid culture medium for further expansion. A 10 -2 and 10 -3 These two lower dilutions were used for inoculation because cyanobacteria are typically far fewer in number than bacteria and actinomycetes in dryland soils. Lower dilutions ensure sufficient cyanobacterial cells are inoculated onto the plates, improving the isolation success rate. The culture conditions combined two key factors: temperature and light. A broad temperature range of 20-28℃ covers the suitable growth range for most cyanobacterial strains; a light intensity of 2000-3000 lux provides the necessary energy source for photosynthesis and photoautotrophy, a prerequisite for normal growth and the production of extracellular colloidal substances. Under these conditions, after 36-48 hours of cultivation, cyanobacteria form visible colonies, typically with a specific color (such as blue-green), facilitating collection and purification.

[0093] In another embodiment, in the method for preparing bio-fertilizer that uses gas-producing and gum-producing compound microbial communities to reshape soil structure, in step S5, the modified corn starch is hydroxypropyl corn starch.

[0094] The preparation method of hydroxypropyl corn starch is as follows: First, 100g of corn starch is dispersed in 350mL of 75% ethanol solution, and 1.5g of 2% sodium hydroxide solution is added for alkalization treatment (the pH of the alkalization system is maintained at 8.5-9.0) to activate the starch granules; then, under sealed conditions, it is heated to 42-45℃, and 10g of propylene oxide is slowly added dropwise as an etherifying agent. The reaction is carried out with stirring for 16 hours to graft hydroxypropyl groups onto the starch molecules; after the reaction, it is neutralized to pH 6.8-7.2 with 1% hydrochloric acid, and then washed repeatedly 3-4 times with 80% ethanol solution to remove salts and unreacted reagents. After centrifugation, dehydration, low-temperature vacuum drying at 40℃, and pulverization through a 100-mesh sieve, hydroxypropyl corn starch with good cold water swelling, stable viscosity, and good film-forming properties can be obtained.

[0095] In step S8, the stirring speed is 150-200 rpm, the stirring time is 20-30 min, and the particle size of the granulation is 2-5 mm.

[0096] In another embodiment, in the method for preparing bio-fertilizer that reshapes soil structure using gas-producing and gum-producing composite microbial communities, in step S3, before mixing the various activated bacterial solutions, the microbial concentrations of activated Bacillus licheniformis, activated Bacillus subtilis, activated actinomycetes, and activated cyanobacteria are measured respectively; then, based on the microbial concentrations of each activated bacterial solution, the required volume of each bacterial solution to be mixed is calculated and determined, and then the solutions are mixed according to their respective volumes so that in the resulting gas-producing and gum-producing composite bacterial solution, the mass of Bacillus licheniformis solution accounts for 25-35%, the mass of Bacillus subtilis solution accounts for 25-35%, the mass of actinomycetes solution accounts for 20-30%, and the mass of cyanobacteria solution accounts for 10-20%.

[0097] In the technical solution of reshaping soil structure using gas-producing and gum-producing compound microbial communities, a key challenge lies in ensuring that the final compound microbial community solution has a precise and stable microbial composition ratio. Even if various target strains are successfully isolated and activated, if equal volumes of microbial solutions are simply mixed during preparation, ignoring the potential differences in microbial concentration after activation, the actual biomass ratio of Bacillus licheniformis, Bacillus subtilis, actinomycetes, and cyanobacteria in the final mixture cannot be accurately controlled. This uncontrollability leads to inconsistent functional strength of the microbial community solution produced in each batch, making it impossible to guarantee the stability and predictability of the synergistic effect of "gas production expansion - gum production fixation," thereby affecting the uniformity and reliability of the final bio-fertilizer product's soil structure improvement effect.

[0098] To address the issue of precise control over the mixing ratios mentioned above, this method adds a crucial quantitative control step before mixing the various activated bacterial solutions. This step requires, before mixing, the microbial concentrations in the activated Bacillus licheniformis, Bacillus subtilis, Actinomycetes, and Cyanobacteria solutions, respectively. The purpose of this measurement is to obtain accurate data on the number of viable bacteria per unit volume of each type of bacterial solution, providing an objective basis for subsequent scientific formulation. Microbial concentration is a core indicator reflecting the activity and quantity of the bacterial solution; after measurement using plate counting or other reliable methods, the actual efficacy of each bacterial solution can be clearly determined.

[0099] After obtaining the microbial concentration data for each activated bacterial solution, the next step is to calculate the required volume of each type of bacterial solution based on this concentration data and the preset target mass percentage requirements for each bacterial species in the compound bacterial solution. The preset target proportions are: 25-35% by mass for Bacillus licheniformis bacterial solution, 25-35% by mass for Bacillus subtilis bacterial solution, 20-30% by mass for actinomycetes bacterial solution, and 10-20% by mass for cyanobacteria solution. The purpose of the calculation is to translate the target "mass percentage" into the instruction of "bacterial solution volume" in actual operation, ensuring that regardless of fluctuations in the concentration of each batch of original bacterial solution, the final mixture can achieve the predetermined biomass ratio by adjusting the added volume.

[0100] Finally, the calculated volumes of each bacterial solution are precisely measured and mixed. This operation directly standardizes the composition ratio of the gas-producing and gum-producing compound bacterial solution. It overcomes the problem of uncontrolled mixing ratios caused by differences in initial bacterial solution concentrations, ensuring that each batch of prepared gas-producing and gum-producing compound bacterial solution contains various functional microorganisms in a coordinated proportion that meets design requirements. This elevates the quality of the core functional units from "rough mixing" to "precise compounding," providing a fundamental guarantee for the final bio-fertilizer product to perform its stable and predictable soil aggregate building function. By introducing quantitative analysis and calculation, this step transforms the microbial mixing process from an empirical operation to a precisely controllable standardized production, significantly improving the reproducibility of the technical solution and the consistency of product quality.

[0101] In another embodiment, the method for preparing bio-fertilizer using gas- and colloid-producing composite microbial communities to reshape soil structure includes step S2 specifically: S2.1 Selecting characteristic colonies: from the coated 10 -4 and 10 -5 Single colonies exhibiting typical morphological characteristics of Bacillus licheniformis or Bacillus subtilis were picked from Bacillus agar plates containing gradient soil suspensions; [The text abruptly shifts to a different topic:] From a plate plate containing 10... -3 and 10 -4 Single colonies exhibiting typical actinomycete morphological characteristics were picked from actinomycete culture plates containing gradient soil suspensions; [The text then abruptly shifts to a different topic:] From a plate containing 10 [units of something], single colonies were picked from the plate.-2 and 10 -3 Single colonies exhibiting typical morphological characteristics of cyanobacteria were picked from gradient soil suspension cyanobacterial culture plates. S2.2 Functional Verification and Screening: For the selected single colonies of *Bacillus licheniformis* or *Bacillus subtilis*, they were inoculated onto *Bacillus* agar plates for functional verification and cultured at 36.6-37.2℃ for 18-24 hours. After culture, the diameter of the calcification zone surrounding the colony was measured. Single colonies with a calcification zone diameter of not less than 3 mm were selected as functionally validated *Bacillus* strains. The *Bacillus* agar for functional verification was prepared by adding 15-20 g / L calcium carbonate to the *Bacillus* culture medium formula. For the selected single colonies of actinomycetes, they were inoculated into liquid actinomycete culture medium for functional verification and cultured at 27.5-28.5℃ and 150-180 rpm with shaking for 48-72 hours. After culture, the viscosity of the fermentation broth was measured using a rotational viscometer. Cultures with a fermentation broth viscosity of not less than 50 mPa·s were selected as the functionally validated actinomycete strains. The functional validation liquid culture medium for actinomycetes was prepared by replacing the carbon source soluble starch with 50-100 g / L sucrose and removing agar from the original actinomycete culture medium formula. Selected single colonies of cyanobacteria were inoculated into the functional validation cyanobacterial liquid culture medium and cultured at 20-28℃ and a light intensity of 2000-3000 lux for 5-7 days. After culture, the extracellular polysaccharide content in the culture medium was determined using the phenol-sulfuric acid method. Cultures with an extracellular polysaccharide content of not less than 100 mg / L were selected as the functionally validated cyanobacterial strains. The functional validation cyanobacterial liquid culture medium was prepared by removing agar from the original cyanobacterial culture medium formula. S2.3 Expanded Culture: The Bacillus strains, Actinomycete strains, and Cyanobacterial strains that passed functional verification in step S2.2 were aseptically transferred to their respective corresponding expanded culture liquid media for expanded culture. The expanded culture liquid media for Bacillus was prepared by removing agar from the Bacillus culture medium formula; the expanded culture liquid media for Actinomycete was prepared by removing agar from the Actinomycete culture medium formula; the expanded culture liquid media for Cyanobacterial strains had the same composition as the Cyanobacterial liquid media used for functional verification. The expanded culture of Bacillus strains was carried out at 36.6-37.2℃, the expanded culture of Actinomycete strains was carried out at 27.5-28.5℃, and the expanded culture of Cyanobacterial strains was carried out at 20-28℃ under a light intensity of 2000-3000 lux, thereby obtaining activated Bacillus licheniformis bacterial solution, activated Bacillus subtilis bacterial solution, activated Actinomycete bacterial solution, and activated Cyanobacterial solution, respectively.

[0102] Building upon the successful isolation and quantification of mixed functional microorganisms from the soil to be modified, a key technical challenge remains: ensuring that each isolated target microorganism not only conforms to the required morphological characteristics but also possesses the core functions necessary for constructing soil aggregates. Soil may contain multiple microorganisms of the same genus but with different functional characteristics. For example, not all Bacillus species can efficiently produce gas, nor can all Actinomycetes secrete sufficient amounts of sticky substances. If strains are selected solely based on morphological characteristics, the resulting composite microbial community may contain strains with low or no function. This would severely dilute the overall functional strength of the community, preventing the synergistic effect of gas production and expansion with gelation and fixation from being fully realized, ultimately affecting the actual effectiveness of soil improvement.

[0103] To address the uncertainty of strain function, this protocol adds a systematic functional verification and screening step after selecting characteristic colonies. This step employs specific functional verification methods and quantitative standards for different types of target microorganisms. First, selected single colonies of *Bacillus licheniformis* or *Bacillus subtilis* are inoculated onto functional verification Bacillus agar plates supplemented with calcium carbonate for cultivation. After cultivation, the acid-producing capacity is assessed by measuring the diameter of the calcium-dissolving zone surrounding the colony, as organic acid production is an important accompanying characteristic of gas production processes in many Bacillus species. A calcium-dissolving zone diameter of at least 3 mm is set as a screening threshold. This aims to intuitively and rapidly screen for metabolically active strains with strong acid-producing potential, which is positively correlated with their gas-producing function, thus ensuring that the selected Bacillus species possess the expected soil-loosening ability.

[0104] Secondly, the selected single colonies of actinomycetes were inoculated into a functional verification liquid culture medium for actinomycetes using sucrose as the carbon source and cultured with shaking. After cultivation, the viscosity of the fermentation broth was measured using a rotational viscometer. Cultures with a fermentation broth viscosity of not less than 50 mPa·s were selected as strains that passed functional verification. The purpose of this step is to directly evaluate the ability of actinomycetes to secrete viscous metabolites such as extracellular polysaccharides, as the increase in fermentation broth viscosity mainly stems from the production of these substances. Setting a viscosity standard allows for the objective screening of high-gum-producing actinomycete strains, ensuring their effective role in binding soil particles in subsequent mixed microbial communities.

[0105] Finally, the selected single colonies of cyanobacteria were inoculated into cyanobacterial liquid culture medium for functional verification and cultured under light conditions for 5-7 days. After culture, the content of extracellular polysaccharides in the culture medium was determined using the phenol-sulfuric acid method, and cultures with an extracellular polysaccharide content of not less than 100 mg / L were selected as cyanobacterial strains that passed functional verification. Extracellular polysaccharides secreted by cyanobacteria are the main functional substances for cementing soil particles. Quantitative determination of polysaccharide content using chemical methods can accurately and directly assess the polysaccharide production capacity of cyanobacterial strains, thereby screening out highly efficient strains that make practical contributions to the construction of soil aggregates.

[0106] Through the targeted functional verification and screening described above, this approach elevates the microbial screening criteria from morphological conformity to functional confirmation. It ensures that every microorganism ultimately used for large-scale cultivation and mixing is an experimentally verified, highly efficient strain possessing the corresponding core functions, rather than merely exhibiting morphological similarity. This step significantly improves the functional purity and expected strength of the constructed gas-producing-gel-producing composite bacterial solution, laying a solid foundation of microbial strains for the stable and efficient performance of biofertilizer products, fundamentally enhancing the reliability of the technical solution and the consistency of the final application effect.

[0107] In another embodiment, in the preparation method of the bio-fertilizer using gas- and colloid-producing composite microbial community to reshape soil structure, in step S8, before mixing all components and stirring, a directional adsorption and activation step is added: First, the porous slow-release carrier obtained in step S4 is preheated at 35-40℃ for 5-15 minutes; then, 15%-30% of the total mass of the preheated porous slow-release carrier is mixed with the gas- and colloid-producing composite bacterial solution obtained in step S3 at a stirring speed of 150-200 rpm for 3-8 minutes to form a microbial-carrier complex with a water content of 20%-35% and a temperature of 30-35℃; then, the obtained microbial-carrier complex is sealed and statically activated at 28-32℃ and a relative humidity of 75%-85% for 20-40 minutes; finally, the activated microbial-carrier complex is mixed with the remaining porous slow-release carrier, degradable expansion-regulating matrix, microbial protection and induction components, and structural stability regulating components, followed by subsequent stirring, granulation, and drying.

[0108] A potential problem exists during the mixing of the gas-producing-colloid-producing bacterial solution with porous slow-release carriers and other solid components. If the liquid bacterial solution is directly mixed with a large amount of solid powder carriers and other components in a single, high-intensity mechanical stirring, the suspended active microbial cells may suffer physical and physiological stress due to sudden exposure to a dry or high-osmotic-pressure solid environment. Simultaneously, the intense mechanical stirring itself may cause shear damage to some microbial cells, leading to decreased activity or even death. This loss of microbial activity occurring at the initial stage of mixing weakens the effective content of functional microorganisms in the final product, affecting the initial establishment and functional activation effects of the fertilizer after application to the soil.

[0109] To mitigate the potential negative impact of the mixing process on microbial activity, this method incorporates a directional adsorption and activation step before the final mixing of all components. This step begins by preheating the prepared porous slow-release carrier at 35-40℃ for 5-15 minutes. Preheating aims to raise the temperature of the carrier material, bringing it closer to the optimal growth temperature range for microorganisms. This reduces the cold shock to the microorganisms during mixing with the bacterial solution and also facilitates the expulsion of air from the carrier pores, creating more favorable conditions for subsequent bacterial adsorption.

[0110] Then, 15%-30% of the total mass of the preheated porous slow-release carrier is mixed with all of the gas-producing-colloid-producing composite bacterial solution at a stirring speed of 150-200 rpm for 3-8 minutes. The purpose of this step is to use a small amount of carrier to mix with the bacterial solution first. Using part of the carrier instead of all of it ensures that the bacterial solution has sufficient carrier surface area for adsorption, forming a microbial carrier complex with a water content of 20%-35% and a temperature of 30-35℃. This semi-wet complex state provides the microorganisms with a moist, mild temporary microenvironment rich in adsorption sites, allowing them to smoothly transition from a liquid suspension state to a solid carrier-attached state, avoiding the stress caused by sudden water loss or direct contact with a large amount of dry matter.

[0111] Subsequently, the obtained microbial carrier complex was activated by static incubation at 28-32℃ and 75%-85% relative humidity for 20-40 minutes. The purpose of static activation is to allow the microorganisms already adsorbed on the carrier a brief period of recovery and adaptation under suitable temperature and humidity conditions. During this process, the microorganisms can initiate metabolism, repair any minor damage they may have suffered, and begin to adapt to the microenvironment on the carrier surface, preparing physiologically for subsequent drying and long-term storage. The higher humidity effectively prevents rapid moisture loss from the complex during static incubation.

[0112] Finally, the activated microbial carrier complex is mixed with the remaining porous slow-release carrier, biodegradable swelling control matrix, microbial protection and induction components, and structural stabilization regulating components, followed by subsequent stirring, granulation, and drying. The purpose of this final mixing step is to ensure that the microorganisms, after being stably attached to part of the carrier and activated and adapted, are uniformly combined with the remaining solid functional components. At this point, the microorganisms are in a more robust and stable state, better able to withstand the physical stress of subsequent granulation and drying processes, thus maximizing their activity. This stepwise, gentle mixing and activation strategy significantly improves the survival rate and physiological adaptability of functional microorganisms during fertilizer preparation, laying a more reliable foundation of microbial activity for the final product to exert its long-lasting and highly effective soil improvement function.

[0113] In another embodiment, the method for preparing bio-fertilizer using gas- and colloid-producing compound microbial communities to reshape soil structure includes an additional soil environment simulation induction step after step S3 and before step S8: Fresh soil obtained from the soil sample to be modified in step S1 is added to the gas- and colloid-producing compound microbial solution obtained in step S3, with an addition ratio of 5-10g of fresh soil per 100mL of microbial solution; the mixture is placed in a shaker at 25-28℃ and 100-150rpm, and cultured for 12-24 hours under conditions of 500-1000lux light intensity and a 12h / 12h light / dark cycle; after culture, the mixture is filtered through a sterile sieve with a pore size of 20-50μm to remove soil particles, obtaining a gas- and colloid-producing compound microbial solution pre-adapted to the target soil microecology; in step S8, without changing the dosage, the gas- and colloid-producing compound microbial solution pre-adapted to the target soil microecology is used instead of the gas- and colloid-producing compound microbial solution.

[0114] To address the issue of rapid adaptation of functional microbial communities to the target soil environment, this scheme adds a soil environment simulation induction step after obtaining the gas-producing and gum-producing compound bacterial solution and before preparing the final fertilizer product. This step begins by collecting fresh soil samples from microbial-rich areas of the geomorphological unit to which the soil to be modified belongs, and adding them to the gas-producing and gum-producing compound bacterial solution at a ratio of 5-10g of fresh soil per 100mL of bacterial solution. The purpose of this operation is to introduce real and complex microecological factors from the target soil into the microbial community culture system, including ions, organic matter, indigenous microbial communities, and their metabolites in the soil solution, providing a highly simulated pre-adaptation environment for the laboratory microbial community.

[0115] Next, the mixture of the bacterial culture and soil was placed in a shaker at 25-28℃ and 100-150 rpm, under conditions of 500-1000 lux light intensity and a 12h / 12h light / dark cycle, and cultured with aeration for 12-24 hours. The purpose of this simulated culture process is to create a dynamic environment that closely resembles some conditions of field soil. Suitable temperature and shaking provide favorable conditions for growth and material exchange, while the controlled light and dark cycle simulates the natural diurnal rhythm, particularly meeting the physiological needs of photosynthetic microorganisms such as cyanobacteria. Aeration ensures the supply of dissolved oxygen, supporting the metabolism of aerobic and facultative microorganisms. During this process, the functional microbial community can proactively contact and adapt to the physicochemical and biological characteristics of the target soil, initiating targeted physiological adjustments and metabolic responses. Some microorganisms may begin to form spores or protective structures, preparing for the real soil environment.

[0116] After cultivation, the mixture system was filtered using a sterile sieve with a pore size of 20-50 μm to remove soil particles, obtaining a gas- and colloid-producing compound bacterial solution pre-adapted to the target soil microecology. The purpose of filtration was to separate functional microbial cells that had undergone environmental induction and physiological adaptation, while removing larger soil particles that might interfere with subsequent granulation. Finally, without changing the dosage, this pre-adapted bacterial solution was used to replace the original bacterial solution in subsequent fertilizer preparation. The fundamental purpose of this replacement was to encapsulate the targeted environmentally acclimatized microorganisms as the core functional unit within the fertilizer product. The pre-adapted microorganisms exhibit stronger tolerance and faster response to abiotic stresses and nutrient competition in the target soil. When applied to the soil with fertilizer, they can recover, colonize, and activate their designed functions more quickly, significantly shortening the time window from application to effectiveness and improving the reliability and consistency of biofertilizer application under different field conditions.

[0117] In another embodiment, the preparation method of the bio-fertilizer using gas- and colloid-producing compound microbial communities to reshape soil structure specifically employs a stepped induced drying process in step S8: First stage: The granulated wet granules are placed in an environment with a temperature of 38-40℃ and a relative humidity of 60%-70% for 1-2 hours to allow the granule surface to initially stabilize; Second stage: The environmental parameters are adjusted to a temperature of 30-32℃ and a relative humidity of 75%-85%, and maintained for 2-3 hours. This higher humidity environment slows down the overall dehydration rate, providing a suitable microenvironment for short-term metabolism and repair of the microbial community; Third stage: The temperature is adjusted to 35-37℃ and the relative humidity is reduced to 50%-60%, and maintained for 1 hour. The metabolic activity of the microbial community activated in the previous stage is used to initiate the spore formation process; Fourth stage: The temperature is raised to 40-42℃ and the relative humidity is reduced to below 20%, and drying continues until the granule moisture content is less than 10%, completing the final spore maturation and deep granule dehydration.

[0118] After fertilizer granulation, the drying process is crucial in determining the final survival status of the microorganisms and the physical stability of the fertilizer. If a single-temperature rapid drying method is used, the rapid evaporation of moisture from the wet granules forces the microbial cells to dehydrate quickly, leading to cell structure damage and loss of metabolic activity. Simultaneously, excessively rapid surface setting may hinder the gradual dissipation of internal moisture, affecting granule uniformity and mechanical strength. More importantly, many functional microorganisms, especially Bacillus, require specific environmental induction to effectively form their resilient spores. Inappropriate drying conditions may fail to initiate or complete this important sporulation process, resulting in a product primarily composed of weaker vegetative cells, making it difficult to maintain sufficient activity after storage and application.

[0119] To address the negative impacts of drying processes on microbial activity and product quality, this solution employs a stepped induced drying process. This process divides the drying process into four stages with different environmental parameters. In the first stage, the granulated wet granules are placed in an environment with a temperature of 38-40℃ and a relative humidity of 60%-70% for 1-2 hours. The purpose of this stage is to utilize the relatively high initial temperature and moderate humidity to allow the surface moisture of the granules to evaporate quickly, forming a preliminary outer shell. This shell helps to fix the shape of the granules, preventing them from sticking and deforming during subsequent processing, while also preventing excessively rapid loss of internal moisture. In the second stage, the environmental parameters are adjusted to a temperature of 30-32℃ and a relative humidity of 75%-85%, and maintained for 2-3 hours. The purpose of this stage is to create a high-humidity, mild environment. Lowering the temperature and significantly increasing the ambient humidity aims to significantly slow down the overall dehydration rate of the granules. The higher humidity reduces the moisture gradient between the inside of the granules and the external environment, providing a brief, less stressful microenvironment for the microorganisms inside the granules that are still in a moist state. In this environment, microorganisms are able to partially recover from the physical stress of the previous process and carry out necessary metabolic activities, preparing physiologically for the upcoming environmental changes. The third stage adjusts the temperature to 35-37℃ and reduces the relative humidity to 50%-60%, lasting for 1 hour. The purpose of this stage is to utilize the activated metabolic activity of the microbial community from the previous stage to initiate the spore formation process. The moderate increase in temperature and decrease in humidity simulate the conditions of a natural environment transitioning from humid to dry, a change that effectively induces the spore-producing strains. Under this temperature and humidity combination, it is beneficial to initiate the spore differentiation pathway of microorganisms such as Bacillus, initiating the transformation into a more resilient dormant spore morphology. The fourth stage raises the temperature to 40-42℃ and reduces the relative humidity to below 20%, continuing drying until the particle moisture content is less than 10%. The purpose of this stage is to complete the final maturation and shaping of the spores and deep dehydration of the particles. The high temperature and extremely low humidity ensure the complete removal of residual moisture inside the particles, achieving the required dryness for product storage. At the same time, this condition also helps to promote the final maturation and solidification of the spore structure, giving it stronger heat resistance and dryness resistance, thereby ensuring the stability of microorganisms in fertilizer products during long-term storage.

[0120] This multi-stage, precisely controlled, stepped induced drying process not only achieves controllable removal of moisture from the particles, ensuring the product's physical properties, but more importantly, it creates a progressive environmental sequence for functional microorganisms, from adaptation and repair to induced sporulation. This process aims to minimize the damage of drying stress to microorganisms and actively guide them towards a highly resilient spore state, thereby significantly improving the survival rate, storage stability, and reactivation and colonization ability of effective functional microorganisms in the finished fertilizer after application to the soil.

[0121] A bio-fertilizer prepared using the aforementioned method for reshaping soil structure with gas-producing and colloid-producing composite microbial communities, wherein the bio-fertilizer is composed of the following components in parts by weight: 5-12 parts of gas-producing and colloid-producing composite microbial liquid or gas-producing and colloid-producing composite microbial liquid pre-adapted to the target soil microecology, 45-65 parts of porous slow-release carrier, 10-20 parts of degradable expansion-regulating matrix, 3-8 parts of microbial protection and induction components, and 2-5 parts of structural stability regulating components.

[0122] Among them, the mass of Bacillus licheniformis bacterial solution in the gas-producing-colloid-producing compound bacterial solution is 25-35%, the mass of Bacillus subtilis bacterial solution is 25-35%, the mass of Actinomycetes bacterial solution is 20-30%, and the mass of Cyanobacteria solution is 10-20%.

[0123] The porous slow-release carrier comprises, by weight, 20-35 parts corn straw biochar, 10-20 parts humic peat, 10-20 parts humic acid soil, and 5-15 parts bentonite.

[0124] The biodegradable swelling control matrix comprises, by weight, 5-12 parts modified corn starch, 3-8 parts beet molasses, and 5-10 parts distilled water;

[0125] The microbial protection and induction components, by mass parts, include 1-2 parts sodium alginate, 1-3 parts yeast powder, 1-2 parts betaine, and 1-2 parts trace element solution;

[0126] The structural stabilizing and regulating components, by mass parts, include 1-2 parts of lignin sulfonate, 1-2 parts of zeolite powder, and 1-2 parts of vermiculite.

[0127] This scheme ultimately defines the specific composition and proportion range of the bio-fertilizer prepared by the aforementioned method. Its aim is to provide a standardized product formula with a clearly defined composition, synergistic effects of each functional unit, and optimized proportions. The fertilizer consists of five core functional components in specific mass percentage ranges, ensuring the consistency and reliability of product performance. Specifically, the amount of the gas-producing-colloid-producing compound bacterial solution or its pre-adapted product is 5-12 parts. This quantitative range aims to provide a sufficient density of active microorganisms for the core functions while avoiding excessive liquid affecting the granulation and drying processes. The precisely set mass percentages of Bacillus licheniformis, Bacillus subtilis, actinomycetes, and cyanobacteria in the bacterial solution ensure the balance and synergy of the functions of the gas-producing and colloidal microorganisms, which is the foundation for achieving simultaneous soil loosening and structural fixation.

[0128] The porous slow-release carrier material, comprising 45-65 parts, is the largest component. This carrier is composed of corn straw biochar, humic peat, humic acid soil, and bentonite in a specific ratio. Its main purpose is to provide microorganisms with a physical space for habitat, reproduction, and protection. Biochar provides a large pore structure, humic materials provide organic matter and water retention capacity, and bentonite contributes to the formation of colloidal structures. This composite carrier not only serves as a slow-release reservoir for microorganisms but also continuously improves the physical structure and nutrient retention capacity of the soil after application. The biodegradable swelling regulator matrix, comprising 10-20 parts, is composed of modified corn starch and beet molasses. Its purpose is to provide functional microorganisms with an easily accessible initial carbon source to quickly initiate metabolism, while the slow degradation of starch provides a sustained energy supply, maintaining the continuity and stability of the gas production process, thereby achieving gradual physical loosening of compacted soil. The microbial protection and induction component, comprising 3-8 parts, includes sodium alginate, yeast powder, betaine, and various trace elements. The main function of this component is to provide multiple layers of protection for microorganisms during fertilizer processing, storage, and the initial stages of application. Sodium alginate forms a microencapsulated structure, yeast powder provides nutrition, betaine enhances stress resistance, and trace elements continuously activate specific metabolic pathways of microorganisms. The structural stabilizing component, consisting of lignin sulfonate, zeolite powder, and vermiculite, is used in amounts of 2-5 parts. Its purpose is to enhance the stability of aggregates at multiple scales: lignin sulfonate acts as a natural cementing agent, zeolite powder provides ion exchange and microporous structure, and vermiculite improves water retention and swelling properties, collectively enhancing the ability of newly formed soil aggregates to resist water erosion and mechanical disturbance.

[0129] Through the systematic integration of the aforementioned functional components in a clearly defined ratio, this product forms a complete chain of action, from ensuring microbial activity and stimulating function to building and stabilizing soil structure. Its ultimate effect is that, after the fertilizer is applied to the soil, the components are released in an orderly manner and work synergistically. The functional microbial community can quickly colonize and efficiently perform its core functions of gas production and loosening, as well as gum production and fixation. Combined with the physicochemical effects of the carrier and regulating components, it can directionally and persistently promote the formation of stable soil aggregates, thereby effectively solving the problems of soil compaction and structural degradation. This provides a functionally integrated and reliable product solution for soil ecological restoration and soil fertility improvement.

[0130] Example 1: An example of improving degraded black soil on sloping farmland in the low mountain and hilly area of ​​Changbai Mountain (soil erosion + farmland degradation type)

[0131] I. Background and Site Overview

[0132] The low mountain and hilly area of ​​Changbai Mountain is an important part of the Northeast Black Soil Region. Over 45% of the land in this area is sloping farmland, which has long faced the dual stresses of intensive farming and seasonal soil erosion. This has led to the disintegration of black soil aggregates, thinning of the black soil layer, and severe fertility degradation, becoming a core bottleneck restricting the sustainable development of agriculture in the region. The experimental site is located in Sanyuanpu Town, Liuhe County, Tonghua City, Jilin Province. It has a temperate continental monsoon climate with an average annual temperature of 5.2℃ and annual precipitation of 720-780mm. Rainfall is concentrated from June to August, accounting for over 70% of the annual precipitation, and often occurs in the form of torrential rain, exacerbating soil erosion on sloping farmland. Winter temperatures reach an extreme low of -26℃, with a freeze-thaw period from November to March of the following year, an average of 3-4 freeze-thaw cycles per year, and a maximum freezing depth of 1.0m. The experimental site has a typical dark brown soil type, originally a natural secondary forest. After reclamation, it was continuously planted with corn for 20 years without fallow or soil and water conservation measures, resulting in significant soil degradation: soil aggregates with a particle size of 0.25-5mm accounted for only 24.6%, and the soil bulk density was 1.58g / cm³. 3 The soil porosity was 7.8%, and the organic matter content was 16.2 g / kg, a decrease of more than 52% compared to the native black soil (35-40 g / kg). The soil pH was 6.8, available nitrogen was 95 mg / kg, available phosphorus was 18.7 mg / kg, available potassium was 105 mg / kg, and the field water holding capacity was 21.5%. The black soil layer thickness was only 28 cm, a decrease of 49% compared to the initial reclamation stage (55 cm), and the soil erodibility K value was 0.038 t·hm². 2 ·h / (hm 2 The area, classified as moderate to severe degraded black soil (·MJ·mm), has an average maize yield of only 520 kg / mu (approximately 0.067 hectares) over the past five years, significantly lower than the 750 kg / mu yield of high-quality black soil in the Northeast Plain. Furthermore, soil erosion is causing the black soil layer to be lost at a rate of 0.4 cm per year, exacerbating soil degradation. This experiment aims to verify the effectiveness of a bio-fertilizer utilizing a gas- and gum-producing microbial community to reshape soil structure under the dual stress of soil erosion and crop degradation on sloping farmland. It seeks to clarify the mechanism by which the bio-fertilizer affects black soil aggregate structure, erosion resistance, soil physicochemical properties, and maize growth and development, providing a scientific basis and technical support for the ecological restoration and productivity enhancement of large-scale degraded black soil in the low hilly areas of Changbai Mountain.

[0133] II. Experimental Materials and Design

[0134] 1. Preparation of experimental fertilizer

[0135] The raw materials used in the experimental fertilizer were all products that were readily available in Northeast China, ensuring that the fertilizer was suitable for the ecological and climatic characteristics of the soil to be modified.

[0136] Preparation of specialized culture media: For Bacillus culture medium, accurately weigh 3.3g beef extract, 11.0g peptone, 5.3g NaCl, and 20g agar according to the preferred components. Add 1000mL purified water and stir with a magnetic stirrer at 300rpm until completely dissolved. Adjust the pH to 7.4 with 0.1mol / L NaOH solution, dispense into sealed containers, and store for later use. For Actinomycete culture medium, accurately weigh 23g soluble starch, 0.86g KNO3, 0.46g NaCl, 0.56g K2HPO4·3H2O, 0.56g MgSO4·7H2O, 0.13g CaCl2, 0.008g MnSO4, 0.01g FeSO4·7H2O, and 20g agar according to the preferred components. Add 1000mL purified water, heat to boiling, and stir for 15min until completely dissolved. Adjust the pH to 7.4 with 0.1mol / L NaOH solution. Adjust the pH to 7.3 with HCl solution, then dispense and seal. For the cyanobacteria culture medium, accurately weigh 1.5g NaNO3, 0.04g K2HPO4·3H2O, 0.075g MgSO4·7H2O, 0.036g CaCl2·2H2O, 0.006g citric acid, 0.006g ferric ammonium citrate, 0.001g disodium EDTA, 0.02g Na2CO3, and 1mL trace element solution. Add these to 1000mL purified water and stir to dissolve. Finally, adjust the pH to 7.1 with 0.1mol / L HCl solution, then dispense and seal.

[0137] The method for preparing the trace element solution is as follows:

[0138] Weigh out 0.02g boric acid, 0.02g manganese sulfate, 0.02g zinc sulfate, 0.01g copper sulfate, 0.01g sodium molybdate, and 0.01g cobalt chloride, and set aside. Heat 800mL of deionized water to 40-50℃, and add the prepared reagents sequentially to the water while stirring until completely dissolved. Finally, add 10mL of EDTA-Fe solution that has been prepared separately and cooled to room temperature. Mix well and then dilute to 1000mL with deionized water. Adjust the pH of the stock solution to 6.0-7.0. Filter the solution through a 0.22 μm filter membrane for sterilization, and store it in a sterile brown bottle at 4℃. When using, dilute the stock solution with purified water at a volume ratio of 1:100, and add 1mL of the diluted solution to the above cyanobacteria culture medium. Ensure that the final conductivity of the cyanobacteria culture medium is below 0.5dS / m and the pH is maintained at around 7.1 to avoid adverse effects on microbial activity and soil structure.

[0139] The EDTA-Fe solution is prepared as follows: 1.0 g of Na2EDTA and 0.8 g of FeSO4·7H2O are accurately weighed and added to 500 mL of deionized water at 60 °C. The mixture is stirred until completely dissolved to obtain a disodium EDTA solution and a ferrous sulfate solution. Under continuous stirring, the ferrous sulfate solution is slowly poured into the disodium EDTA solution, and the mixture is stirred at a constant temperature of 60 °C for 20-30 minutes to ensure complete chelation. After cooling to room temperature, the solution is diluted to 1000 mL with deionized water to obtain a stable golden-yellow EDTA-Fe solution for later use.

[0140] Culture medium sterilization and sterility testing: Bacillus and actinomycete media were sterilized by autoclaving at 121℃, 0.1MPa, for 20 min, while cyanobacteria media were sterilized by autoclaving at 115℃, 0.06MPa, for 15 min. After sterilization, 30 mL of Bacillus media was poured into sterile petri dishes, and three replicates were performed. The dishes were incubated at 36℃ for 33 hours. No colonies grew in any of the three replicates after incubation, indicating thorough sterilization.

[0141] Soil sample collection and suspension preparation: 500g of soil sample was aseptically collected from microbial-rich areas 5-10cm away from the roots of maize plants and at a depth of 0-20cm in the experimental plot. The sample was stored at 4℃ and the suspension was prepared within 24 hours. 10g of soil was added to 90mL of sterile physiological saline and shaken at 180rpm for 30min to prepare a suspension. -1 Gradient suspension, successively diluted to obtain 10 -2 10 -3 10 -4 10 -5 Gradient suspensions are used, and aseptic operation is maintained throughout the process to avoid contamination.

[0142] Isolation and preparation of gas- and colloid-producing complex bacterial communities: 10g of Bacillus bacterial solution was taken. -4 10 -5 0.1 mL of each gradient suspension was spread onto culture plates and incubated at 36.9℃ for 21 h. Characteristic colonies of Bacillus licheniformis and Bacillus subtilis were picked, and after Gram staining and physiological and biochemical identification, they were transferred to liquid culture medium and cultured at 200 rpm and 36.9℃ for 18 h. The bacterial concentrations were 3.1 × 10⁻⁶. 9 CFU / mL, 3.4×10 9 CFU / mL; Take 10 ml of the actinomycete culture. -3 10 -4 Gradient suspensions were spread onto plates and incubated at 26.5℃ and 65% humidity for 42 hours. Actinomycete colonies were picked and verified under a microscope before being transferred to liquid culture medium and cultured at 180 rpm and 26.5℃ for 36 hours to achieve a bacterial concentration of 2.7 × 10⁻⁶. 8 CFU / mL; 10 ml of cyanobacterial culture was taken.-2 10 -3 Gradient suspensions were spread onto plates and incubated at 25°C and 2700 lux for 42 hours. Characteristic cyanobacterial colonies were picked and verified under a microscope before being transferred to liquid culture medium for further culture for 30 hours, with a bacterial concentration of 1.7 × 10⁻⁶. 7 CFU / mL.

[0143] Preparation of each functional component: For the porous slow-release carrier, weigh out 28 parts of corn straw biochar with a particle size <2mm, 15 parts of humic peat with a particle size <1mm, 15 parts of humic acid soil with a particle size <1mm, and 10 parts of bentonite with a particle size <0.5mm. Mix them thoroughly at 150 rpm for 15 min, achieving a total porosity of 57%. For the expansion control matrix, weigh out 8 parts of hydroxypropyl corn starch and 5 parts of beet molasses. Add 7 parts of distilled water and stir at 250 rpm for 20 min to dissolve them. For the protection and induction components, weigh out 1.5 parts of sodium alginate, 2 parts of yeast powder, 1.5 parts of betaine, and 1.5 parts of trace element solution (the same as the trace element solution used to prepare the cyanobacteria culture medium). Mix them thoroughly at 300 rpm for 10 min. For the structure stabilization regulating components, weigh out 1.5 parts of lignin sulfonate, 1.5 parts of zeolite powder, and 1.5 parts of vermiculite. Mix them thoroughly at 250 rpm for 8 min.

[0144] The preparation method of hydroxypropyl corn starch is as follows: First, 100g of corn starch is dispersed in 350mL of 75% ethanol solution, and 1.5g of 2% sodium hydroxide solution is added for alkalization treatment. The pH of the alkalization system is maintained at 8.5-9.0 to activate the starch granules. Then, under sealed conditions, the mixture is heated to 43℃, and 10g of propylene oxide is slowly added dropwise as an etherifying agent. The mixture is kept warm and stirred for 16h to graft hydroxypropyl groups onto the starch molecules. After the reaction, the mixture is neutralized to pH 7.1 with 1% hydrochloric acid. Then, it is washed repeatedly 3-4 times with 80% ethanol solution to remove salts and unreacted reagents. After centrifugation, dehydration, low-temperature vacuum drying at 40℃, and pulverization through a 100-mesh sieve, hydroxypropyl corn starch with good cold water swelling, stable viscosity, and good film-forming properties is obtained.

[0145] Fertilizer preparation: Activated Bacillus bacterial solution (mass ratio: Bacillus licheniformis: Bacillus subtilis = 1:1), actinomycete bacterial solution, and cyanobacterial solution were mixed at mass fractions of 30%, 30%, 25%, and 15%, respectively, to obtain a total bacterial concentration of 2.5 × 10⁻⁶. 9A gas-producing and gum-producing compound bacterial solution (CFU / ml) was prepared. Eight parts by weight of the compound bacterial solution, 55 parts by weight of the pretreated porous slow-release carrier, 15 parts by weight of the swelling-regulating matrix, 5 parts by weight of the protective inducing component, and 4 parts by weight of the structural stabilizing component were weighed and mixed thoroughly at 180 rpm for 27 minutes. The mixture was then granulated into raw material particles with a diameter of 3-4 mm using a granulator and dried at 38℃ until the moisture content reached 7.2%, yielding the finished fertilizer. Testing showed that the finished product had a pH of 7.1 and a total bacterial count of 2.0 × 10⁻⁶. 8 CFU / g, granule compressive strength 11.8N / granule, water absorption swelling rate 178%.

[0146] 2. Experimental Design

[0147] The experiment employed a randomized block design, with three treatment groups, each with an area of ​​30 m². 2 (5m × 6m), 3 replicates, with a plot spacing of 1m and a 2m wide protective row around the perimeter. Sloping farmland was divided into plots along contour lines to avoid inter-plot interference due to soil erosion. Treatment group (T1) received the bio-fertilizer of this invention; control group 1 (CK1) received commercially available microbial fertilizer (main component Bacillus subtilis, bacterial concentration 2.0 × 10⁻⁶). 8 CFU / g); Control group 2 (CK2) received no fertilizer. All treatments were applied as basal fertilizer, evenly broadcast one week before corn sowing on April 20th, and then tilled and mixed thoroughly with a rotary tiller to a depth of 20cm. The application rate was 300 kg / mu. The experimental crop was the locally grown maize variety "Xianyu 335", with a sowing density of 4500 plants / mu. The sowing date was April 27th, and the harvest date was October 8th. Irrigation, weeding, and pest and disease control were carried out according to local conventional agronomic practices. Irrigation was carried out once each at the jointing stage and tasseling stage, with each irrigation volume being 30 ml. 3 / acre, manual weeding, biological pesticides for pest and disease control, ensuring consistent conditions except for fertilizer.

[0148] 3. Measurement Indicators and Methods

[0149] Soil samples from the 0-20cm topsoil layer were collected on April 18 before fertilization, May 20 30 days after fertilization, June 20 60 days after fertilization, July 20 90 days after fertilization, October 10 after corn harvest, and April 12 of the following year after freeze-thaw. The “S”-shaped five-point mixed sampling method was used, with five sampling points collected from each plot. After removing stones and plant debris, the samples were divided into two parts: fresh samples were refrigerated at 4℃ for microbial index detection, and air-dried samples were used for physicochemical properties and aggregate index detection.

[0150] Measurement indicators and methods: The proportion of aggregates >0.25mm and mean weight diameter (MWD) were determined by wet sieving; soil bulk density and aeration porosity were determined by ring sieving; soil moisture content was determined by oven drying; organic matter content was determined by potassium dichromate oxidation-external heating method; available nitrogen was determined by alkaline hydrolysis diffusion method; available phosphorus was determined by molybdenum-antimony colorimetric method; available potassium was determined by flame photometry; colonization rate and total microbial count of target bacterial groups were determined by selective culture medium plate counting method; soil EPS (extracellular polymeric substances) content was determined by anthrone colorimetric method; soil gas production rate was determined by drainage method; and soil erodibility K value was calculated by EPIC-K model. Maize growth indicators were measured at the jointing stage (plant height and stem diameter) and at harvest (root dry weight, ear length, number of grains per ear, thousand-grain weight, and yield per acre). Data were analyzed using SPSS 26.0 software with one-way ANOVA and LSD multiple comparisons, with a significance level of P < 0.05. Charts were generated using Origin 2023.

[0151] III. Experimental Results and Analysis

[0152] 1. Improvement effect on soil aggregate structure

[0153] Before fertilization, there were no significant differences in soil aggregate indicators among the treatments, with an average proportion of aggregates >0.25mm at 24.5% and a mean molecular weight distribution (MWD) of 0.82mm, indicating consistent initial conditions at the experimental sites. After applying the fertilizer of this invention, the aggregate indicators of treatment T1 were significantly better than those of CK1 and CK2 at all time points, and showed a continuous improvement trend (Table 1). 30 days after fertilization, the proportion of aggregates >0.25mm in treatment T1 reached 33.8%, an increase of 22.5% compared to CK1 (27.6%) and 36.3% compared to CK2 (24.8%); the MWD was 1.15mm, an increase of 25.0% compared to CK1 (0.92mm) and 38.6% compared to CK2 (0.83mm). Sixty days after fertilization, the indicators of the T1 treatment further improved, with the proportion of aggregates >0.25mm reaching 40.2% and the MWD reaching 1.48mm, representing increases of 31.8% and 43.7% respectively compared to CK1, and increases of 62.4% and 78.3% compared to CK2. Ninety days after fertilization, the aggregate structure of the T1 treatment remained stable, with the proportion of aggregates >0.25mm reaching 42.6% and the MWD reaching 1.56mm, while the indicators of the CK1 treatment showed a slight decrease, indicating that the aggregate structure constructed by the fertilizer of this invention is more stable. After corn harvest, the proportion of aggregates >0.25mm in the T1 treatment remained at 41.9%, an increase of 70.3% compared to before the experiment (24.6%), fully demonstrating its long-term improvement effect on the aggregate structure of degraded black soil.

[0154] After experiencing the winter freeze-thaw cycle, the results measured on April 12th of the following year (Table 2) showed that the proportion of aggregates >0.25mm in the T1 treatment was 35.7%, with a MWD of 1.32mm, representing increases of 41.1% and 38.9% respectively compared to CK1 (25.3%, 0.95mm), and increases of 63.7% and 69.2% respectively compared to CK2 (21.8%, 0.78mm). The aggregate index of the T1 treatment remained significantly better than the control group after the freeze-thaw cycle. This is because the protective system formed by betaine and sodium alginate in the fertilizer effectively improved the freeze resistance of the soil microbial community to be modified. The EPS secreted by the microorganisms formed a gel network at low temperatures, buffering the mechanical stress generated by freeze-thaw cycles and reducing aggregate disintegration, thus adapting to the seasonal freeze-thaw climate characteristics of the low mountain and hilly areas of Changbai Mountain.

[0155] Table 1. Changes in soil aggregate structure indices at different time periods

[0156]

[0157] Note: The data in the table are mean ± standard deviation, n=3.

[0158] Table 2 Soil aggregate structure indices after freeze-thaw cycles

[0159]

[0160] Note: The data in the table are mean ± standard deviation, n=3.

[0161] 2. Changes in soil physicochemical properties and erosion resistance

[0162] After harvest, the soil bulk density of the T1 treatment decreased to 1.23 g / cm³. 3 Compared to CK1 (1.42 g / cm³), 3 The level decreased by 13.4% compared to CK2 (1.57 g / cm³). 3 The concentration of phosphorus in corn decreased by 21.6%, reaching the optimal range of 1.1-1.3 g / cm³ for corn growth. 3The aeration porosity increased to 16.5%, which is 52.8% higher than CK1 (10.8%) and 114.3% higher than CK2 (7.7%). The soil organic matter content increased to 22.8 g / kg, which is 31.8% higher than CK1 (17.3 g / kg) and 41.6% higher than CK2 (16.1 g / kg), with an average annual increase rate of 3.3 g / kg, which is in line with the reasonable increase law of black soil organic matter (Table 3). In terms of nutrient content, the T1 treatment had 138 mg / kg of available nitrogen, which was 27.8% higher than CK1 and 45.3% higher than CK2; 27.5 mg / kg of available phosphorus, which was 39.8% higher than CK1 and 46.4% higher than CK2; and 142 mg / kg of available potassium, which was 24.6% higher than CK1 and 35.2% higher than CK2. The increase in nutrient content was due to the decomposition of organic fertilizer raw materials and the activation of functional microbial communities. The organic acids secreted by Bacillus licheniformis and Bacillus subtilis accelerated the transformation of inert nutrients in the soil, and the nitrogen fixation of cyanobacteria increased nitrogen input, thus achieving simultaneous improvement in soil fertility and structure.

[0163] The soil erodibility K value of T1 treatment decreased to 0.024 t·hm. 2 ·h / (hm 2 ·MJ·mm), compared to CK1 (0.032t·hm) 2 ·h / (hm 2 The concentration of ·MJ·mm) decreased by 25.0%, compared to CK2 (0.037t·hm). 2 ·h / (hm 2 The reduction of 35.1% in the amount of micrograins (·MJ·mm) indicates that the improved aggregate structure significantly enhances the soil's resistance to erosion, meeting the needs of water erosion control in sloping farmland in the low hills of Changbai Mountain. Meanwhile, the soil gas production rate in treatment T1 peaked at 1.7 ml / (100 g·d) on day 7 after fertilization, subsequently remaining at 0.7-1.1 mL / (100 g·d). EPS content reached 178 mg / kg on day 30, an increase of 87.4% compared to CK1 (95 mg / kg) and 187.1% compared to CK2 (62 mg / kg), confirming the effective functioning of the synergistic mechanism of "gas production expansion-gel production fixation." The gas production process breaks down the compacted layer to form pores, while the gel production process cements soil particles into stable aggregates, significantly improving the soil's physical structure.

[0164] Table 3. Soil physicochemical properties and erosion resistance indicators after harvest

[0165]

[0166] Note: The data in the table are mean ± standard deviation, n=3.

[0167] 3. Changes in soil microbial activity

[0168] After harvest, the T1 treatment showed excellent colonization rates of target microbial communities in the soil, with Bacillus licheniformis colonization at 68.5%, Bacillus subtilis at 72.3%, actinomycetes at 65.7%, and cyanobacteria at 58.2%, all significantly higher than CK1 (no other target microbial communities were detected, with only the indigenous Bacillus subtilis colonization rate at 18.3%). The total microbial count in the T1 treatment soil reached 3.8 × 10⁻⁶. 8 CFU / g, compared to CK1 (1.2×10⁻⁶). 8 The CFU / g level increased by 216.7%, compared to CK2 (8.5×10). 7 The CFU / g of the microbial community increased by 347.1%; soil urease activity was 36.8 U / g, an increase of 63.6% compared to CK1 (22.5 U / g) and 97.8% compared to CK2 (18.6 U / g); sucrase activity was 28.5 U / g, an increase of 74.8% compared to CK1 (16.3 U / g) and 105.1% compared to CK2 (13.8 U / g) (Table 4). The significant increase in microbial activity is due to two main factors: firstly, the high adaptability of the complex microbial community, which rapidly colonizes the soil and forms a stable functional microbial community; secondly, the suitable microenvironment provided by the porous slow-release carrier. The large pores of corn straw biochar and the small pores of humus peat form a gradient pore structure, providing a habitat and breeding ground for microorganisms. At the same time, yeast powder and trace elements continuously activate the metabolism of the microbial community, ensuring the long-term performance of gas production and gum production.

[0169] Table 4. Indicators of soil microbial activity after harvest

[0170]

[0171] Note: The data in the table are mean ± standard deviation, n=3.

[0172] 4. Corn growth and yield performance

[0173] The growth indicators of maize treated with T1 were significantly better than those of the control group. The plant height at the jointing stage reached 158cm, which was 19.7% higher than that of CK1 (132cm) and 30.6% higher than that of CK2 (121cm). The stem diameter was 2.8cm, which was 21.7% higher than that of CK1 (2.3cm) and 40.0% higher than that of CK2 (2.0cm). At harvest, the maize plant length of treatment T1 was 278 cm, an increase of 16.8% compared to CK1 and 25.7% compared to CK2; the stem diameter was 3.5 cm, an increase of 16.7% compared to CK1 (3.0 cm) and 40% compared to CK2 (2.5 cm); the root dry weight was 32.6 g / plant, an increase of 38.7% compared to CK1 (23.5 g / plant) and 64.6% compared to CK2 (19.8 g / plant); the ear length was 22.5 cm, an increase of 14.3% compared to CK1 and 20.9% compared to CK2; the number of kernels per ear was 586, an increase of 18.5% compared to CK1 and 27.3% compared to CK2; and the thousand-kernel weight was 378 g, an increase of 12.6% compared to CK1 and 19.5% compared to CK2 (Table 5). The T1 treatment yielded 712 kg / mu of corn, an increase of 19.7% compared to CK1 (595 kg) and 36.9% compared to CK2 (520 kg). The yield increase was mainly attributed to the improved soil aggregate structure, which optimized the root growth environment, improved aeration and nutrient supply efficiency, enhanced crop resistance to drought and lodging, and increased accumulation of photosynthetic products, ultimately resulting in a significant increase in production capacity.

[0174] Table 5. Major agronomic traits and yield of maize

[0175]

[0176] Note: The data in the table are mean ± standard deviation, n=3.

[0177] IV. Discussion

[0178] The core problem of degraded black soil on sloping farmland in the low hills and mountains of Changbai Mountain is the disintegration of soil aggregates caused by soil erosion, thinning of the black soil layer, and structural damage caused by tillage compaction. This invention presents a method for preparing bio-fertilizers that reshapes soil structure using a gas-producing-gel-producing composite microbial community, achieving efficient improvement through multiple synergistic mechanisms. First, the targeted separation and enrichment of the microbial community in the soil to be modified ensures high adaptability. Bacillus licheniformis and Bacillus subtilis rapidly colonize the soil, utilizing beet molasses in the expansion-regulated matrix to quickly initiate gas production metabolism. During gas dissipation, the compacted layer is broken down, forming micropores. Simultaneously, modified corn starch slowly hydrolyzes, continuously providing a carbon source and maintaining a stable gas production rate, further expanding soil porosity. Second, EPS secreted by Bacillus subtilis and actinomycetes form a cementing network. The gel film secreted by cyanobacteria tightly binds soil particles, hyphae, and microbial cells, combined with the hyphal skeleton effect of actinomycetes, forming stable aggregates. The porous slow-release carrier's structure enables synergistic coupling of "gas production expansion - gel production fixation"; furthermore, the gradient pore structure of the porous slow-release carrier provides a suitable habitat for microorganisms, the lignin sulfonate in the structural stabilization regulating component enhances the cementation between particles, and the zeolite powder and vermiculite improve the mechanical stability and water retention capacity of the aggregates, significantly enhancing the soil's resistance to erosion; finally, the sodium alginate in the microbial protection and induction component forms a micro-embedding structure, betaine enhances the strain's resistance to freezing and drought, and the combination of trace elements continuously activates the microbial community's metabolic pathways, ensuring the fertilizer's long-term function under freeze-thaw cycles and seasonal drought conditions.

[0179] Experimental data showed that the proportion of aggregates >0.25mm increased from 24.6% to 41.9%, and the soil bulk density increased from 1.58 g / cm³. 3 Reduced to 1.23 g / cm³ 3 The erosibility K-value decreased by 35.1%, and corn yield increased by 36.9%, fully verifying the fertilizer's efficacy in improving degraded black soil on sloping farmland. Compared with commercially available conventional microbial fertilizers, the core advantages of this invention's fertilizer lie in its high colonization rate of over 60% and the synergistic function of "gas production-gel production." Conventional microbial fertilizers, due to the use of exogenous strains, have a colonization rate of less than 20% and lack a gas production-gel production coupling mechanism, making it difficult to form stable aggregate structures and resulting in short-lived improvement effects. Furthermore, the fertilizer raw materials, such as corn straw biochar and humic peat, are readily available resources in Northeast China, and the preparation process is simple and feasible. The application rate is comparable to that of conventional microbial fertilizers, showing good prospects for widespread application. The results of this experiment demonstrate that this bio-fertilizer can specifically address the core problems of aggregate disintegration, weak erosion resistance, and fertility degradation in black soil aggregates on sloping farmland in the low hilly areas of Changbai Mountain, providing an effective technical solution for regional black soil protection and sustainable agricultural development.

[0180] Example 2: Example of improving high-moisture black soil in wetland reclamation in the Sanjiang Plain (high-moisture anaerobic + aggregate dispersion type)

[0181] I. Background and Site Overview

[0182] The Sanjiang Plain is the largest distribution area of ​​marshy black soil in my country. In recent years, large-scale wetland reclamation has led to severe degradation of the black soil structure. This type of black soil, due to its long-term high-moisture environment, has dispersed soil particles, extremely poor aggregate stability, and an anaerobic environment that inhibits the activity of beneficial microorganisms, resulting in significant nutrient imbalances and becoming a key bottleneck restricting regional agricultural development. The experimental site is located in Qixing Farm, Fujin City, Jiamusi City, Heilongjiang Province (47°25′-47°38′N, 132°45′-132°60′E), belonging to the temperate humid monsoon climate zone. The average annual temperature is 3.8℃, and the annual precipitation is 580-650 mm, concentrated in June-August. The soil type is meadow black soil. The reclamation history is 18 years, with soybeans continuously planted for 12 years without drainage or improvement measures. The soil degradation characteristics are significant: the proportion of soil aggregates with a particle size >0.25 mm is only 21.3%, and the soil bulk density is 1.46 g / cm³. 3 The total porosity was 42.8%, of which 19.5% was ineffective water retention porosity. The soil organic matter content was 17.8 g / kg, which is more than 55% lower than that of native swamp black soil (40-50 g / kg). The soil redox potential (Eh) was -165 mV, indicating a strongly anaerobic environment. The soil pH was 6.6, available nitrogen was 92 mg / kg, available phosphorus was 12.5 mg / kg, available potassium was 118 mg / kg, and the soil erodibility K value was 0.039 t·hm². 2 ·h / (hm 2 (·MJ·mm). The average soybean yield in this region over the past five years has been only 165 kg / mu, significantly lower than the 220 kg / mu yield of high-quality black soil in the Sanjiang Plain. Furthermore, the high humidity and anaerobic conditions lead to low activity of soybean rhizobia and insufficient nitrogen fixation efficiency, creating a vicious cycle of "low yield - excessive fertilization - further degradation." This experiment aims to verify the improvement effect of the bio-fertilizer of this invention under high moisture and anaerobic conditions, clarify its impact on the aggregate structure, redox environment, nutrient cycling, and soybean yield of marshland-reclaimed black soil, and provide a scientific basis and technical solution for the improvement of wetland-reclaimed black soil in the Sanjiang Plain.

[0183] II. Experimental Materials and Design

[0184] 1. Preparation of experimental fertilizer

[0185] The experimental fertilizer was prepared according to the optimized parameters. The raw material ratio was slightly adjusted to ensure that the fertilizer could still perform its high-efficiency function under anaerobic conditions, taking into account the high humidity and anaerobic environment of the Sanjiang Plain.

[0186] Except for the following, the rest of the content (from the preparation of special culture medium to the isolation and preparation of gas- and gum-producing complex bacteria) is the same as in Example 1.

[0187] Preparation of each functional component: 30 parts by mass of corn straw biochar (particle size <2mm), 16 parts by mass of humic peat (particle size <1mm), 16 parts by mass of humic acid soil (particle size <1mm), and 12 parts by mass of bentonite (particle size <0.5mm) were weighed and mixed evenly at 150 rpm for 15 min, resulting in a total porosity of 59%, enhancing the water retention and air permeability balance; 9 parts by mass of hydroxypropyl corn starch (same as in Example 1) and 6 parts by mass of beet molasses were weighed and dissolved in 8 parts by mass of distilled water; 1.6 parts by mass of sodium alginate, 2.2 parts by mass of yeast powder, 1.6 parts by mass of betaine, and 1.6 parts by mass of trace element solution (same as in Example 1) were weighed and mixed evenly; 1.6 parts by mass of lignin sulfonate, 1.6 parts by mass of zeolite powder, and 1.6 parts by mass of vermiculite were weighed and mixed evenly.

[0188] Fertilizer preparation: Activated Bacillus bacterial solution (mass ratio: Bacillus licheniformis: Bacillus subtilis = 1:1), actinomycete bacterial solution, and cyanobacterial solution were mixed at mass fractions of 32%, 32%, 24%, and 12%, respectively, to obtain a total bacterial concentration of 2.7 × 10⁻⁶. 9 A gas- and gel-producing composite bacterial culture with a concentration of CFU / mL was prepared. Nine parts by weight of the bacterial culture, 58 parts by weight of the pretreated porous slow-release carrier, 16 parts by weight of the swelling control matrix, 6 parts by weight of the protective and inducing components, and 4 parts by weight of the structural stabilizing adjustment components were mixed thoroughly at 180 rpm for 27 minutes. The mixture was granulated to a particle size of 3-4 mm and dried at 38℃ until the moisture content reached 7.0%. The final product had a pH of 7.0 and a total bacterial count of 2.2 × 10⁻⁶. 8 CFU / g, granule compressive strength 12.2N / granule, water absorption swelling rate (24h) 182%.

[0189] 2. Experimental Design

[0190] The experiment employed a randomized block design, with three treatment groups, each with an area of ​​25 m². 2 (5m × 5m), 3 replicates, plot spacing 1.5m, with a 2m wide protective row around the perimeter, and drainage ditches 30cm deep within each plot to control soil moisture content at 65-70% of field capacity, avoiding experimental errors caused by excessive waterlogging. Treatment group (T2) received the bio-fertilizer of this invention; control group 1 (CK3) received commercially available microbial fertilizer (main component Bacillus subtilis, bacterial concentration 2.0 × 10⁻⁶). 8The control group (CK4) received no fertilizer (CFU / g). All treatments used furrow application. Fertilizer furrows, 15cm deep and 10cm wide, were dug 10 days before soybean sowing on May 1st. The fertilizer was evenly applied and then covered with soil. The application rate was 320kg / mu (approximately 667 square meters). In high humidity conditions, the application rate was appropriately increased to ensure effectiveness. The experimental crop was the locally grown soybean variety "Heinong 84," with a sowing density of 280,000 plants / hectare. Sowing date was May 10th, and harvest date was October 15th. Field management followed local conventional agronomic practices, with unified drainage control to ensure consistent conditions except for fertilizer application.

[0191] 3. Measurement Indicators and Methods

[0192] Soil samples from the 0-20cm topsoil layer were collected on April 28 (before fertilization), May 30 (30 days after fertilization), June 30 (60 days after fertilization), July 30 (90 days after fertilization), and October 17 (after soybean harvest). A five-point pooled sampling method was used. Fresh samples were used to determine microbial indicators, EPS content, and redox potential, while air-dried samples were used to determine physicochemical properties and aggregate stability. In addition to the indicators measured in Example 1, soil redox potential, methanogens, and the number of anaerobic microorganisms were added. Soybean growth indicators included the number of root nodules per plant and root nodule nitrogenase activity. Detection methods: Redox potential was measured using the platinum electrode method; the number of anaerobic microorganisms was measured using the selective culture medium plate counting method; root nodule nitrogenase activity was measured using the acetylene reduction method. Other indicators were measured using the same methods as in Example 1.

[0193] III. Experimental Results and Analysis

[0194] 1. Improvement effect on soil aggregate structure

[0195] Before fertilization, there were no significant differences in aggregate structure among the treatments, with an average proportion of aggregates >0.25mm at 21.5% and a mean molecular weight distribution (MWD) of 0.76mm, indicating consistent initial conditions at the experimental site. After applying the fertilizer of this invention, the aggregate indicators of the T2 treatment at each stage were significantly improved (Table 6). 30 days after fertilization, the proportion of aggregates >0.25mm in the T2 treatment reached 29.8%, an increase of 23.1% compared to CK3 (24.2%) and 39.3% compared to CK4 (21.4%); the MWD of the T2 treatment was 0.98mm, an increase of 21.0% compared to CK3 (0.81mm) and 30.7% compared to CK4 (0.75mm). 60 days after fertilization, the proportion of aggregates >0.25mm in the T2 treatment was 35.6%, and the MWD was 1.25mm, representing increases of 38.5% and 44.2% compared to CK3, and 66.4% and 66.7% compared to CK4, respectively. Ninety days after fertilization, the aggregate structure in the T2 treatment remained stable, with 37.2% of aggregates >0.25mm and a MWD of 1.32mm. The CK3 treatment showed no significant improvement in these indicators, while the CK4 treatment showed a slight decrease. After soybean harvest, the proportion of aggregates >0.25mm in the T2 treatment reached 36.8%, a 72.8% increase compared to before the experiment (21.3%), fully demonstrating the significant effect of fertilizer on improving aggregate structure under high humidity conditions.

[0196] During the experiment, the soil moisture content was maintained at 65-68%, with no significant difference. However, the stability of aggregates in the T2 treatment was significantly higher than that in the control group. The core reason is the synergistic effect of the complex functional microbial community under high humidity: the gas production metabolism of Bacillus licheniformis and Bacillus subtilis forms micropores in the soil, improving local ventilation conditions and providing space for aerobic microbial activities; the EPS secreted by Bacillus subtilis and actinomycetes has strong hydrophilicity and viscosity, forming a water-stable cementing layer on the surface of soil particles. The film secreted by cyanobacteria further enhances the cementing effect. Combined with the mycelial skeleton of actinomycetes, it cements the dispersed soil particles into stable aggregates, effectively resisting particle dispersion caused by high moisture.

[0197] Table 6 Changes in soil aggregate structure indices at different time periods

[0198] Note: The data in the table are mean ± standard deviation, n=3.

[0199] 2. Soil physicochemical properties and changes in redox environment

[0200] After harvest, the total porosity of soil treated with T2 reached 49.5%, an increase of 12.2% compared to CK3 (44.1%) and 16.2% compared to CK4 (42.6%). The content of macropores (≥100μm) was 15.6%, an increase of 51.5% compared to CK3 (10.3%) and 85.7% compared to CK4 (8.4%). The proportion of ineffective pores decreased to 13.2%, a decrease of 24.6% compared to CK3 (17.5%) and 31.6% compared to CK4 (19.3%) (Table 7). The improvement in pore structure was mainly attributed to the increase in effective pores and the decrease in ineffective pores after the formation of aggregates, which significantly improved soil aeration and permeability and alleviated the anaerobic environment. The soil redox potential (Eh) of the T2 treatment increased to 65 mV, which was 168.4% higher than that of CK3 (-95 mV) and 220.9% higher than that of CK4 (-158 mV), indicating a significant improvement in the strong anaerobic environment. The soil organic matter content increased to 24.5 g / kg, which was 31.7% higher than that of CK3 (18.6 g / kg) and 38.4% higher than that of CK4 (17.7 g / kg). The accumulation of organic matter was due to the decomposition of organic raw materials from fertilizers and the carbon fixation by microorganisms. At the same time, the improved aeration inhibited the excessive decomposition of organic matter by anaerobic microorganisms.

[0201] In terms of nutrient content, the T2 treatment showed an available nitrogen content of 135 mg / kg, a 28.3% increase compared to CK3 and a 46.7% increase compared to CK4; available phosphorus of 21.8 mg / kg, a 48.6% increase compared to CK3 and a 74.4% increase compared to CK4; and available potassium of 156 mg / kg, a 23.4% increase compared to CK3 and a 32.2% increase compared to CK4. The most significant increase in available phosphorus was closely related to the enhanced activity of phosphatase secreted by actinomycetes and Bacillus subtilis. Phosphatase accelerated the mineralization of soil organic phosphorus and improved phosphorus availability. The soil erodibility K value decreased to 0.025 t·hm² in the T2 treatment. 2 ·h / (hm 2 ·MJ·mm), compared to CK3 (0.033t·hm) 2 ·h / (hm 2 The concentration of MJ·mm decreased by 24.2%, compared to CK4 (0.038t·hm). 2 ·h / (hm 2 The 34.2% reduction in MJ·mm indicates that the improved soil aggregate structure significantly enhances the soil's resistance to erosion, which is suitable for the concentrated rainy season climate of the Sanjiang Plain.

[0202] Table 7. Soil physicochemical properties and redox environmental indicators after harvest.

[0203] Note: The data in the table are mean ± standard deviation, n=3.

[0204] 3. Changes in soil microbial activity and EPS content

[0205] After harvest, the T2 treatment showed excellent colonization rates of target microbial communities in the soil, with 65.3% colonization of Bacillus licheniformis, 69.7% of Bacillus subtilis, 62.5% of actinomycetes, and 55.8% of cyanobacteria, all significantly higher than the Bacillus subtilis colonization rate in the CK3 treatment. The total microbial count in the T2 treatment soil reached 3.2 × 10⁻⁶. 8 CFU / g, compared with CK3 (9.8×10 7 The CFU / g level increased by 226.5%, compared to CK4 (7.6×10). 7 The CFU / g concentration increased by 321.1%; the number of anaerobic microorganisms was 1.1 × 10⁻⁶. 4 CFU / g, compared with CK3 (3.5×10 4 The CFU / g level decreased by 68.6%, compared to CK4 (5.2×10⁻⁶). 4 The CFU / g decreased by 78.8%, indicating that the improved soil aeration significantly inhibited the reproduction of anaerobic harmful microorganisms.

[0206] The EPS content in the soil treated with T2 reached a peak of 172 mg / kg 60 days after fertilization, which was 102.4% higher than that of CK3 (85 mg / kg) and 196.6% higher than that of CK4 (58 mg / kg). The polysaccharide content in EPS was 105 mg / kg and the protein content was 67 mg / kg. The synergistic effect of polysaccharides and proteins enhanced the cementing effect. The EPS content in the soil treated with T2 reached 168 mg / kg after harvest, which was 102.4% higher than that of CK3 (83 mg / kg) and 194.7% higher than that of CK4 (57 mg / kg). The soil urease activity was 34.2 U / g, which was 64.4% higher than that of CK3 (20.8 U / g) and 103.6% higher than that of CK4 (16.8 U / g). The sucrase activity was 25.8 U / g, which was 76.7% higher than that of CK3 (14.6 U / g) and 109.8% higher than that of CK4 (12.3 U / g). The increased enzyme activity accelerated the soil nutrient cycle and provided sufficient nutrient supply for soybean growth (Table 8).

[0207] Table 8. Indicators of soil microbial activity after harvest

[0208]

[0209] Note: The data in the table are mean ± standard deviation, n=3.

[0210] IV. Discussion

[0211] The core problems in the reclamation of high-moisture black soil in the Sanjiang Plain wetlands are the dispersion of aggregates caused by high moisture, the inhibition of beneficial microbial activity by anaerobic environments, and nutrient imbalance. This invention's bio-fertilizer achieves efficient improvement through targeted design. First, the synergistic mechanism of "gas production expansion-gel production fixation" still functions efficiently under high humidity conditions. Bacillus licheniformis and Bacillus subtilis utilize the expansion-regulated matrix to rapidly produce gas, forming micropores in the soil, improving local aeration conditions, and alleviating anaerobic stress. Simultaneously, the EPS secreted by the gel-producing bacteria forms a strongly adhesive cementing network with the cyanobacterial film, combined with the actinomycete hyphae skeleton, cementing dispersed soil particles into stable aggregates, significantly improving aggregate stability. Second, the high porosity and gradient pore structure of the porous slow-release carrier improves soil aeration and permeability on the one hand, and provides a suitable microenvironment for microorganisms on the other, reducing the risk of soil degradation. High moisture content inhibits microbial activity, while zeolite powder and vermiculite in the structural stabilization regulating components further enhance the mechanical stability and water retention capacity of the aggregates. Furthermore, sodium alginate and betaine in the microbial protection and induction components enhance the strains' resistance to anaerobic conditions and stress. The micronutrient combination activates the metabolic pathways of the microbial community, ensuring the continuous functioning of gas production and gum production. Finally, the high adaptability of the microbial community in the soil to be modified ensures a high colonization rate under high-humidity anaerobic conditions, effectively inhibiting the reproduction of harmful anaerobic microorganisms, improving the soil microecological balance, and accelerating nutrient transformation through microbial metabolic activity, thereby enhancing soil fertility and promoting nitrogen fixation efficiency by soybean rhizobia.

[0212] Experimental data showed that the proportion of aggregates >0.25mm increased from 21.3% to 36.8%, the soil redox potential increased from -165mV to 65mV, and soybean yield increased by 44.2%, fully verifying the fertilizer's efficacy in improving high-humidity anaerobic black soil. Compared with conventional microbial fertilizers, the core advantage of this invention lies in the synergistic function of "gas production-gel production" and its high compatibility with the microbial community of the soil to be modified. Conventional microbial fertilizers lack gas production function, cannot improve the anaerobic environment, and have low colonization rates of exogenous strains, making it difficult to form stable functional microbial communities, thus limiting the improvement effect. At the same time, the fertilizer is applied by trenching, avoiding fertilizer loss caused by surface application and improving utilization. Appropriately increasing the application rate (320kg / mu) under high humidity conditions ensures the improvement effect, meeting the improvement needs of black soil reclamation in the Sanjiang Plain wetlands. The results of this experiment show that this bio-fertilizer can effectively solve the core problems of aggregate dispersion, anaerobic stress, and fertility degradation in high-humidity anaerobic black soil, providing a feasible technical solution for the improvement of black soil in wetland reclamation in the Sanjiang Plain, and has important value for promotion and application.

[0213] Example 3: An example of improving degraded black soil on sloping farmland in the low hilly area of ​​Changbai Mountain (comparative functional verification of screening effect)

[0214] This embodiment is based on the exact same experimental background, site conditions, experimental design, and measurement indicators and methods as Example 1. The experiment aims to compare and verify the impact of fully implementing the detailed operations of step S2 (including functional verification screening and large-scale cultivation) on the final improvement effect under completely identical field conditions.

[0215] Comparison of experimental fertilizer preparations:

[0216] Treatment group T3 fertilizer: Its preparation method is based on the process described in Example 1, with a complete refinement of step S2.

[0217] S2.1 Selecting characteristic colonies: The procedure is the same as in Example 1. Typical single colonies of Bacillus licheniformis, Bacillus subtilis, actinomycetes, and cyanobacteria are selected from the plates of the corresponding gradient.

[0218] S2.2 Functional Validation and Screening:

[0219] For the selected Bacillus single colonies, they were inoculated onto Bacillus agar plates containing 18 g / L calcium carbonate for functional verification and incubated at 36.9 °C for 22 h. After incubation, single colonies with a calcium dissolution zone diameter greater than 3.5 mm were selected as strains that passed functional verification.

[0220] For the selected single colonies of actinomycetes, they were inoculated into a functional actinomycete liquid medium with a carbon source of 75 g / L sucrose and cultured at 28.0 °C and 165 rpm for 60 h with shaking. After culture, the viscosity of the fermentation broth was measured using a rotational viscometer, and the culture with a viscosity of 58 mPa·s was selected as the strain that passed the functional verification.

[0221] Selected single colonies of cyanobacteria were inoculated into cyanobacterial liquid culture medium for functional verification and cultured for 6 days at 25°C and a light intensity of 2500 lux. After culture, the extracellular polysaccharide content in the culture medium was determined by the phenol-sulfuric acid method, and the culture with a polysaccharide content of 115 mg / L was selected as the strain that passed the functional verification.

[0222] S2.3 Scale-up Culture: The validated bacterial strains were aseptically transferred to their respective liquid media for scale-up culture. Bacillus was cultured at 36.9℃ for 18 hours, Abelianidae at 28.0℃ for 36 hours, and Cyanobacteria at 25℃ under 2500 lux light for 30 hours. This yielded highly active bacterial solutions for each strain.

[0223] The above bacterial solution was then mixed according to the following mass fractions: 30% Bacillus licheniformis, 30% Bacillus subtilis, 25% actinomycetes, and 15% cyanobacteria, to obtain a total bacterial concentration of 2.6 × 10⁻⁶. 9The compound bacterial solution was prepared at CFU / mL. The raw materials and proportions of all other functional components (8 parts of bacterial solution, 55 parts of porous slow-release carrier, 15 parts of expansion control matrix, 5 parts of protective and inducing components, and 4 parts of structural stabilization regulating components), as well as all subsequent processes such as mixing, granulation, and low-temperature drying at 38°C to a moisture content of 7.2%, were exactly the same as those used in the preparation of fertilizer T1 in Example 1.

[0224] Control group T1 fertilizer: The T1 fertilizer prepared in Example 1 was used directly as a comparative reference.

[0225] Comparative analysis of experimental results:

[0226] The measurement data are compared as follows, under exactly the same initial soil conditions, field management, and measurement time points:

[0227] 1. Changes in soil aggregate structure indices

[0228] Example 3 (T3 treatment):

[0229] 30 days after fertilization: the proportion of aggregates >0.25mm was 34.5%, and the average weight diameter (MWD) was 1.18mm.

[0230] 60 days after fertilization: the proportion of aggregates >0.25mm was 41.0%, and the MWD was 1.51mm.

[0231] 90 days after fertilization: >0.25mm aggregates accounted for 43.8%, and MWD was 1.59mm.

[0232] After harvest: >0.25mm aggregates accounted for 44.1%, and MWD was 1.58mm.

[0233] 2. Soil aggregate structure indices after freeze-thaw cycles

[0234] Example 3 (T3 treatment): >0.25mm aggregates accounted for 36.8%, and MWD was 1.35mm.

[0235] 3. Post-harvest soil physicochemical properties and erosion resistance

[0236] Example 3 (T3 treatment): Soil bulk density was 1.21 g / cm³ 3 The aeration porosity is 17.0%, the organic matter content is 23.0 g / kg, the available nitrogen is 142 mg / kg, the available phosphorus is 28.2 mg / kg, the available potassium is 145 mg / kg, and the erosibility K value is 0.023 t·hm. 2 ·h / (hm 2 ·MJ·mm).

[0237] 4. Post-harvest soil microbial activity

[0238] Example 3 (T3 treatment): Total soil microbial count was 3.9 × 10⁻⁶. 8 The CFU / g concentration was 37.5 U / g, the urease activity was 29.1 U / g, and the colonization rate of Bacillus licheniformis was 70.2%, Bacillus subtilis colonization rate was 74.8%, actinomycete colonization rate was 67.5%, and cyanobacterial colonization rate was 59.5%.

[0239] 5. Agronomic traits and yield of maize at harvest time

[0240] Example 3 (T3 treatment): Plant height was 281cm, stem diameter was 3.6cm, root dry weight was 33.2g / plant, ear length was 22.8cm, number of grains per ear was 592, thousand-grain weight was 381g, and yield was 718kg per mu.

[0241] Results: The results of this example demonstrate that, under identical application conditions, the fertilizer prepared using a complete functional validation screening and refined culture process (Example 3) systematically outperformed the fertilizer prepared using a basic separation process (Example 1) in all key measured periods and indicators. From the dynamic formation process of soil aggregate structure (30, 60, and 90 days after fertilization and post-harvest) to its stability after freeze-thaw cycles, all indicators of the T3 treatment were consistently higher than those of the T1 treatment, proving that the structure of the high-quality microbial community is more resilient and durable. The T3 treatment showed comprehensive advantages in post-harvest soil physicochemical properties, nutrient status, and erosion resistance. In terms of microbial effects, the T3 treatment exhibited higher colonization rates of functional bacteria and higher soil enzyme activity, indicating a more vibrant and competitive microbial community. Ultimately, these advantages accumulated and manifested as better maize plant growth traits and higher grain yield. The data comparisons covering all measurement dimensions consistently demonstrate that adding and rigorously implementing step S2 for functional verification and screening refinement in the method significantly enhances the overall effectiveness of functional microbial communities. This results in more significant and consistent improvements in multiple aspects, including rapid soil structure construction, long-term stability, improvement of physicochemical properties, activation of microbial communities, and increased crop yield. These results clarify the core necessity of targeted and quantitative screening of functional microorganisms in the technical system for constructing soil structure using microbial communities in the soil to be modified.

[0242] Example 4: An example of improving degraded black soil on sloping farmland in the low hilly area of ​​Changbai Mountain (comparing the effects of directional adsorption and activation steps).

[0243] This embodiment is based on the exact same experimental background, site overview, experimental design, and measurement indicators and methods as Example 1. The experiment aims to compare and verify, under completely identical field conditions, the impact of adding the refined operation of "directional adsorption and activation" in step S8 of the fertilizer preparation process on the final improvement effect, especially the survival and colonization of microorganisms.

[0244] Comparison of experimental fertilizer preparations:

[0245] Treatment group T4 fertilizer: Its preparation method is consistent with the core process of T1 fertilizer preparation in Example 1, including bacterial isolation and culture and preparation of each component. However, a directional adsorption and activation step is added before the final mixing of all components. Specifically, the prepared porous slow-release carrier is preheated at 38°C for 10 minutes. Then, 20% of the total mass of the preheated carrier is mixed with all the gas-producing and gel-producing bacterial solution at 180 rpm for 5 minutes to form a microbial-carrier complex with a water content of 28% and a temperature of 32°C. This complex is then activated by being sealed and allowed to stand at 30°C and 80% relative humidity for 30 minutes. Finally, the activated microbial-carrier complex is mixed with the remaining porous slow-release carrier, biodegradable expansion-regulating matrix, microbial protection and induction components, and structural stability regulating components, followed by subsequent stirring, granulation, and low-temperature drying at 38°C. All other raw materials, proportions, and drying processes are exactly the same as those used in the preparation of T1 fertilizer in Example 1.

[0246] Control group T1 fertilizer: The T1 fertilizer prepared in Example 1 was used directly as a comparative reference.

[0247] Comparative analysis of experimental results:

[0248] The measured data are compared below under the same initial soil conditions and field management:

[0249] 1. Changes in soil aggregate structure indicators (comparison of key nodes)

[0250] Example 4 (T4 treatment):

[0251] 60 days after fertilization: >0.25mm aggregates accounted for 40.8%, and MWD was 1.50mm.

[0252] After harvest: >0.25mm aggregates accounted for 42.5%, and MWD was 1.55mm.

[0253] 2. Soil aggregate structure indices after freeze-thaw cycles

[0254] Example 4 (T4 treatment): >0.25mm aggregates accounted for 36.5%, and MWD was 1.34mm.

[0255] 3. Post-harvest soil physicochemical properties and erosion resistance

[0256] Example 4 (T4 treatment): Soil bulk density was 1.22 g / cm³ 3The aeration porosity is 16.8%, the organic matter content is 23.0 g / kg, the alkaline available nitrogen is 140 mg / kg, the available phosphorus is 27.8 mg / kg, the available potassium is 143 mg / kg, and the corrosion efflorescence K value is 0.0235 t·hm. 2 ·h / (hm 2 ·MJ·mm).

[0257] 4. Post-harvest soil microbial activity (core comparative indicator)

[0258] Example 4 (T4 treatment): Total soil microbial count was 4.0 × 10⁻⁶. 8 The CFU / g concentration was 37.2 U / g, the urease activity was 28.8 U / g, the colonization rate of Bacillus licheniformis was 70.5%, the colonization rate of Bacillus subtilis was 74.2%, the colonization rate of actinomycetes was 67.2%, and the colonization rate of cyanobacteria was 59.3%.

[0259] 5. Agronomic traits and yield of maize at harvest time

[0260] Example 4 (T4 treatment): Plant height was 279cm, stem diameter was 3.55cm, root dry weight was 32.9g / plant, ear length was 22.6cm, number of grains per ear was 589, thousand-grain weight was 379g, and yield was 716kg per mu.

[0261] Results: The results of this embodiment demonstrate that, under the same microbial community and raw material formulation, adding a targeted adsorption and activation step before fertilizer preparation and mixing can effectively improve the application effect of the final product. Its core advantage lies in the protection and activation of functional microorganisms: the colonization rate, total microbial count, and enzyme activity of various functional bacteria in the soil treated with T4 were generally higher than those treated with T1, especially in the colonization rate of key functional bacteria (Bacillus licheniformis and Bacillus subtilis). This indicates that this step, through gentle pre-adsorption and a suitable environment for brief activation, effectively alleviates the physical and physiological stress on microbial cells caused by subsequent mixing and drying processes, improving the survival rate and physiological robustness of active microorganisms in the finished product. These more vigorous microorganisms, after being applied to the soil, exhibit stronger colonization competitiveness and faster metabolic initiation, thereby promoting the early formation and stability of soil aggregate structure (reflected in the improved structural indicators 60 days after fertilization and after harvest), ultimately leading to comprehensive improvements in soil physicochemical properties, erosion resistance, and crop yield. Together with Example 3 (Focusing on Strain Function Screening), which optimized the quality and activity assurance of the microbial community from different dimensions, they jointly demonstrated that within the framework of the method, by refining and optimizing the key preparation process steps, the efficiency of the entire chain of functional microorganisms from isolation to field application can be systematically improved. This is an important way to enhance the stability, reliability and consistency of the technical solution.

[0262] Although embodiments 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 a bio-fertilizer that reshapes soil structure using a gas-producing and gum-producing complex of microorganisms, characterized in that, Includes the following steps: S1: Collect soil samples from the microbial enrichment area of ​​the geomorphological unit to which the soil to be modified belongs, and prepare gradient soil suspensions; S2: Activated Bacillus licheniformis bacterial suspension, activated Bacillus subtilis bacterial suspension, activated actinomycete bacterial suspension and activated cyanobacterial suspension were obtained by separating and culturing from soil suspensions of different gradients. S3: Preparation of gas-producing-colloid-producing composite bacterial solution: Mix activated Bacillus licheniformis bacterial solution, activated Bacillus subtilis bacterial solution, activated actinomycete bacterial solution and activated cyanobacterial solution to obtain gas-producing-colloid-producing composite bacterial solution; In the gas-producing and gum-producing compound bacterial solution, the mass of Bacillus licheniformis bacterial solution is 25-35%, the mass of Bacillus subtilis bacterial solution is 25-35%, the mass of Actinomycetes bacterial solution is 20-30%, and the mass of Cyanobacteria solution is 10-20%. S4: Preparation of porous slow-release carrier: Corn straw biochar is pulverized and sieved to a particle size of <3mm, humic peat and humic acid soil are dried and sieved to a particle size of <2mm, and bentonite is pulverized and sieved to a particle size of <1mm; by mass, 20-35 parts of corn straw biochar, 10-20 parts of humic peat, 10-20 parts of humic acid soil and 5-15 parts of bentonite are mixed to obtain a porous slow-release carrier. S5 is prepared by dissolving 5-12 parts of modified corn starch and 3-8 parts of beet molasses in 5-10 parts of distilled water to obtain a biodegradable swelling control matrix. S6: By mass fraction, take 1-2 parts sodium alginate, 1-3 parts yeast powder, 1-2 parts betaine, and 1-2 parts trace element solution and mix them to obtain the microbial protection and induction component; S7: By mass, take 1-2 parts of lignin sulfonate, 1-2 parts of zeolite powder, and 1-2 parts of vermiculite and mix them to obtain a structure-stabilizing regulating component; S8: By mass fraction, mix 5-12 parts of the gas-producing-colloid-producing composite bacterial liquid obtained in step S3, 45-65 parts of the porous slow-release carrier obtained in step S4, 10-20 parts of the degradable expansion-regulating matrix obtained in step S5, 3-8 parts of the microbial protection and induction component obtained in step S6, and 2-5 parts of the structure stabilization regulating component obtained in step S7, stir, granulate, and dry at 35-40℃ until the moisture content is <10% to obtain the bio-fertilizer.

2. The method for preparing bio-fertilizer using gas-producing and gum-producing compound microbial communities to reshape soil structure as described in claim 1, characterized in that, In step S2, the isolation and culture includes culturing the target bacterial group using a special culture medium; the special culture medium includes Bacillus culture medium, Actinomycete culture medium and Cyanobacterial culture medium; the Bacillus culture medium is used for culturing Bacillus licheniformis and Bacillus subtilis; The Bacillus culture medium is prepared by weighing 3.0-3.5g of beef extract, 10.0-12.0g of peptone, 5.0-5.5g of NaCl, and 15-25g of agar, adding 1000mL of purified water to dissolve and adjusting the pH to 7.4-7.

5. The actinomycete culture medium is prepared by weighing 20-25g of soluble starch, 0.8-0.9g of KNO3, 0.4-0.5g of NaCl, 0.5-0.6g of K2HPO4·3H2O, 0.5-0.6g of MgSO4·7H2O, 0.1-0.15g of CaCl2, 0.005-0.01g of MnSO4, 0.01g of FeSO4·7H2O, and 20g of agar, dissolving them in 1000mL of purified water, and adjusting the pH to 7.3-7.

4. The cyanobacteria culture medium was prepared by weighing 1.5g of NaNO3, 0.04g of K2HPO4·3H2O, 0.075g of MgSO4·7H2O, 0.036g of CaCl2·2H2O, 0.006g of citric acid, 0.006g of ferric ammonium citrate, 0.001g of disodium EDTA, 0.02g of Na2CO3, and 1mL of trace element solution, adding 1000mL of purified water to dissolve and adjusting the pH to 7.0-7.

2. The method for preparing the trace element solution is as follows: Weigh out 0.02g of boric acid, 0.02g of manganese sulfate, 0.02g of zinc sulfate, 0.01g of copper sulfate, 0.01g of sodium molybdate, and 0.01g of cobalt chloride, and set aside. Heat 800mL of deionized water to 40-50℃, and add the above-mentioned prepared reagents to the water one by one while stirring until completely dissolved. Finally, add 10mL of EDTA-Fe solution that has been prepared separately and cooled to room temperature. Mix well and then make up to 1000mL with deionized water. Adjust the pH value to 6.0-7.

0. Filter through a 0.22μm filter membrane to remove bacteria and obtain the mother liquor. Dilute the mother liquor with purified water at a volume ratio of 1:100 to obtain the trace element solution. The EDTA-Fe solution is prepared as follows: Weigh 1.0 g of Na2EDTA and 0.8 g of FeSO4·7H2O, add them to 500 mL of deionized water at 60 °C, and stir until completely dissolved to obtain a disodium EDTA solution and a ferrous sulfate solution; Under continuous stirring, slowly pour the ferrous sulfate solution into the disodium EDTA solution, maintain a constant temperature of 60 °C and stir for 20-30 min to allow for full chelation, cool to room temperature, and then dilute to 1000 mL with deionized water to obtain a stable golden yellow EDTA-Fe solution for later use; Before using the special culture medium, sterilization and sterility tests were performed on it: the Bacillus culture medium and Actinomycete culture medium were sterilized by high-pressure steam at 121°C for 20 min, and the Cyanobacterial culture medium was sterilized by high-pressure steam at 115°C and 0.06 MPa for 15 min.

3. The method for preparing bio-fertilizer using gas-producing and gum-producing compound microbial communities to reshape soil structure as described in claim 2, characterized in that, In step S1, the specific steps for preparing the gradient soil suspension are as follows: Soil samples are collected from the microbial enrichment area of ​​the geomorphological unit to which the soil to be modified belongs; 10g of soil is added to 90mL of sterile physiological saline and shaken to prepare 10... -1 Gradient soil suspension, then gradient diluted to obtain 10 -2 ~10 -5 Gradient soil suspension; In step S2, the activated Bacillus licheniformis bacterial solution and the activated Bacillus subtilis bacterial solution are obtained by the following method: take 10 -4 and 10 -5 Gradient soil suspensions were inoculated onto Bacillus culture medium and cultured at 36.6-37.2℃ for 18-24 h. Characteristic colonies were then picked and transferred to liquid culture medium for further culture. The activated actinomycete culture was obtained by the following method: taking 10 -3 and 10 -4 Gradient soil suspensions were inoculated onto actinomycete culture medium and cultured at 27.5-28.5℃ and 63%-67% humidity for 36-48 hours. Characteristic colonies were then picked and transferred to liquid culture medium for further culture. The activated cyanobacteria solution was obtained by the following method: Take 10 -2 and 10 -3 Gradient soil suspensions were inoculated into cyanobacteria culture medium and cultured at 20-28℃ and 2000-3000 lux light for 36-48 hours. Characteristic colonies were then selected and transferred to liquid culture medium for further culture.

4. The method for preparing bio-fertilizer using gas-producing and gum-producing compound microbial communities to reshape soil structure as described in claim 1, characterized in that, In step S5, the modified corn starch is hydroxypropyl corn starch; The preparation method of hydroxypropyl corn starch is as follows: First, 100g of corn starch is dispersed in 350mL of 75% ethanol solution, and 1.5g of 2% sodium hydroxide solution is added for alkalization treatment. The pH of the alkalization system is maintained at 8.5-9.0 to activate the starch granules. Then, under sealed conditions, the mixture is heated to 42-45℃, and 10g of propylene oxide is slowly added dropwise as an etherifying agent. The mixture is kept warm and stirred for 16h to graft hydroxypropyl groups onto the starch molecules. After the reaction, the mixture is neutralized to pH 6.8-7.2 with 1% hydrochloric acid. Then, it is washed repeatedly 3-4 times with 80% ethanol solution to remove salts and unreacted reagents. After centrifugation, dehydration, low-temperature vacuum drying at 40℃, and pulverization through a 100-mesh sieve, hydroxypropyl corn starch is obtained. In step S8, the stirring speed is 150-200 rpm, the stirring time is 20-30 min, and the particle size of the granulation is 2-5 mm.

5. The method for preparing bio-fertilizer using gas-producing and gum-producing compound microbial communities to reshape soil structure as described in claim 1, characterized in that, In step S3, before mixing the various activated bacterial solutions, the microbial concentrations of the activated Bacillus licheniformis, Bacillus subtilis, Actinomycetes, and Cyanobacteria solutions are measured respectively. Based on the microbial concentrations of each activated bacterial solution, the required volume of each bacterial solution to be mixed is calculated and determined. Then, the solutions are mixed according to their respective volumes so that in the resulting gas-producing-colloid-producing composite bacterial solution, the mass of Bacillus licheniformis solution accounts for 25-35%, the mass of Bacillus subtilis solution accounts for 25-35%, the mass of Actinomycetes solution accounts for 20-30%, and the mass of Cyanobacteria solution accounts for 10-20%.

6. The method for preparing bio-fertilizer using gas-producing and gum-producing compound microbial communities to reshape soil structure as described in claim 3, characterized in that, Step S2 specifically includes: S2.1 Picking characteristic colonies: From the plating of 10 -4 and 10 -5 Single colonies exhibiting typical morphological characteristics of Bacillus licheniformis or Bacillus subtilis were picked from Bacillus agar plates containing gradient soil suspensions; [The text abruptly shifts to a different topic:] From a plate plate containing 10... -3 and 10 -4 Single colonies exhibiting typical actinomycete morphological characteristics were picked from actinomycete culture plates containing gradient soil suspensions; [The text then abruptly shifts to a different topic:] From a plate containing 10 [units of something], single colonies were picked from the plate. -2 and 10 -3 Single colonies with typical morphological characteristics of cyanobacteria were picked from cyanobacterial culture plates containing gradient soil suspensions. S2.2 Functional Validation and Screening: For selected single colonies of Bacillus licheniformis or Bacillus subtilis, they are inoculated onto Bacillus agar plates for functional verification and incubated at 36.6-37.2℃ for 18-24 hours. After incubation, the diameter of the calcification zone around the colony is measured. Single colonies with a calcification zone diameter of not less than 3 mm are selected as Bacillus strains that have passed functional verification. The Bacillus agar medium for functional verification is prepared by adding 15-20 g / L of calcium carbonate to the Bacillus medium formula. For the selected single colonies of actinomycetes, they were inoculated into liquid culture medium for functional verification of actinomycetes and cultured at 27.5-28.5℃ and 150-180 rpm for 48-72 h with shaking. After the culture, the viscosity of the fermentation broth was measured using a rotational viscometer. Cultures with a fermentation broth viscosity of not less than 50 mPa·s were selected as the actinomycete strains that passed the functional verification. The liquid culture medium for functional verification of actinomycetes was prepared by replacing the carbon source soluble starch with 50-100 g / L sucrose and removing agar from the original actinomycete culture medium formula. Selected single colonies of cyanobacteria were inoculated into cyanobacterial liquid culture medium for functional verification and cultured at 20-28℃ and light intensity of 2000-3000 lux for 5-7 days. After culture, the extracellular polysaccharide content in the culture medium was determined by the phenol-sulfuric acid method. Cultures with an extracellular polysaccharide content of not less than 100 mg / L were selected as cyanobacterial strains that passed functional verification. The cyanobacterial liquid culture medium for functional verification was prepared by removing agar from the cyanobacterial culture medium formula. S2.3 Scale-up culture: The Bacillus strains, Actinomycete strains, and Cyanobacterial strains that passed functional verification in step S2.2 were aseptically transferred to their respective corresponding scale-up liquid culture media for scale-up culture. The Bacillus liquid culture medium for scale-up culture was prepared by removing agar from the Bacillus medium formula. The Actinomycete liquid culture medium for scale-up culture was prepared by removing agar from the Actinomycete medium formula. The composition of the Cyanobacterial liquid culture medium for scale-up culture was the same as that of the Cyanobacterial liquid culture medium used for functional verification. The expansion culture of Bacillus strains was carried out at 36.6-37.2℃, the expansion culture of Actinomycete strains was carried out at 27.5-28.5℃, and the expansion culture of Cyanobacterial strains was carried out at 20-28℃ under light conditions of 2000-3000 lux; thereby obtaining activated Bacillus licheniformis bacterial solution, activated Bacillus subtilis bacterial solution, activated Actinomycete bacterial solution, and activated Cyanobacterial solution, respectively.

7. The method for preparing bio-fertilizer using gas-producing and gum-producing composite microbial communities to reshape soil structure as described in claim 1, characterized in that, In step S8, before mixing all components and stirring, a directional adsorption and activation step is added: First, preheat the porous sustained-release carrier obtained in step S4 at 35-40℃ for 5-15 minutes; Then, 15%-30% of the total mass of the preheated porous slow-release carrier is mixed with the gas-producing and gel-producing composite bacterial solution obtained in step S3 at a stirring speed of 150-200 rpm for 3-8 minutes to form a microbial-carrier complex with a water content of 20%-35% and a temperature of 30-35℃. The resulting microbial-carrier complex was then activated by being sealed and allowed to stand for 20-40 minutes at 28-32℃ and 75%-85% relative humidity. Finally, the activated microbial-carrier complex is mixed with the remaining porous slow-release carrier, degradable swelling control matrix, microbial protection and induction components, and structural stability regulating components, followed by subsequent stirring, granulation, and drying.

8. The method for preparing bio-fertilizer using gas-producing and gum-producing compound microbial communities to reshape soil structure as described in claim 1, characterized in that, After step S3 and before step S8, a soil environment simulation induction step is added: Take the fresh soil obtained when collecting soil samples in step S1 and add it to the gas-producing and gum-producing compound bacterial solution obtained in step S3. The addition ratio is 5-10g of fresh soil per 100mL of bacterial solution. The mixture was placed in a shaker at 25-28℃ and 100-150 rpm, and cultured for 12-24 hours under conditions of light intensity of 500-1000 lux and light / dark cycle of 12h / 12h with ventilation. After cultivation, the soil particles were removed by filtration through a sterile sieve with a pore size of 20-50 μm, resulting in a gas-producing and gel-producing compound bacterial solution pre-adapted to the target soil microecology.

9. The method for preparing bio-fertilizer using gas-producing and gum-producing compound microbial communities to reshape soil structure as described in claim 1, characterized in that, The drying process in step S8 specifically employs a stepped induced drying process: First stage: Place the granulated wet granules in an environment with a temperature of 38-40℃ and a relative humidity of 60%-70% for 1-2 hours to dry them, so that the surface of the granules is initially shaped. The second stage: Adjust the environmental parameters to a temperature of 30-32℃ and a relative humidity of 75%-85%, and maintain this for 2-3 hours. Under this high humidity environment, slow down the overall dehydration rate and provide a suitable microenvironment for short-term metabolism and repair of the microbial community. The third stage: Adjust the temperature to 35-37℃ and reduce the relative humidity to 50%-60% for 1 hour, and use the metabolic activity of the bacteria activated in the previous stage to start the spore formation process. Fourth stage: Raise the temperature to 40-42℃ and reduce the relative humidity to below 20%, continue drying until the moisture content of the particles is less than 10%, and complete the final maturation and shaping of the spores and the deep dehydration of the particles.

10. A bio-fertilizer prepared using the method for preparing bio-fertilizer with gas-producing and gum-producing compound microbial communities to reshape soil structure as described in any one of claims 1-9, characterized in that, The bio-fertilizer is made from the following components in parts by weight: 5-12 parts of gas-producing-colloid-producing compound bacterial liquid or gas-producing-colloid-producing compound bacterial liquid pre-adapted to the target soil microecology, 45-65 parts of porous slow-release carrier, 10-20 parts of degradable expansion-regulating matrix, 3-8 parts of microbial protection and induction components, and 2-5 parts of structural stability regulating components. Among them, the mass of Bacillus licheniformis bacterial solution in the gas-producing-colloid-producing compound bacterial solution is 25-35%, the mass of Bacillus subtilis bacterial solution is 25-35%, the mass of Actinomycetes bacterial solution is 20-30%, and the mass of Cyanobacteria solution is 10-20%. The porous slow-release carrier comprises, by weight, 20-35 parts corn straw biochar, 10-20 parts humic peat, 10-20 parts humic acid soil, and 5-15 parts bentonite. The biodegradable swelling control matrix comprises, by weight, 5-12 parts modified corn starch, 3-8 parts beet molasses, and 5-10 parts distilled water; The microbial protection and induction components, by mass parts, include 1-2 parts sodium alginate, 1-3 parts yeast powder, 1-2 parts betaine, and 1-2 parts trace element solution; The structural stabilizing and regulating components, by mass parts, include 1-2 parts of lignin sulfonate, 1-2 parts of zeolite powder, and 1-2 parts of vermiculite.

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