Bio-agricultural compound microbial fertilizer and preparation method thereof

Abstract

The present application relates to the field of bio-agriculture, and particularly relates to a bio-agricultural compound microbial fertilizer and a preparation method thereof, which comprises, by weight, 2-8 parts of functional microbial flora, 20-48 parts of organic carrier, 5-20 parts of nutrient component, 0.5-4 parts of microbial body protective agent, and the balance of water; the functional microbial flora is composed of Pseudomonas fluorescens, Trichoderma harzianum, Saccharomyces cerevisiae and Bacillus mycoides; and the organic carrier is composed of a peanut shell-sugarcane residue mixture, modified diatomite and humic acid. The present application forms a complete compound microbial fertilizer technical solution through scientific compounding of multiple strains, directional modification of the organic carrier, balanced optimization of the nutrient component and low-temperature preparation process synergy, and has multiple outstanding beneficial effects.

Description

Technical Field

[0001] This invention relates to the field of bio-agricultural technology, specifically to a bio-agricultural compound microbial fertilizer and its preparation method. Background Technology

[0002] With the continuous advancement of bio-agriculture, microbial fertilizers, with their green and environmentally friendly characteristics, soil-improving properties, and crop-growth-supporting effects, have become an important agricultural input to replace traditional chemical fertilizers. However, existing microbial fertilizers still have many technical shortcomings in actual production and application, making it difficult to meet the dual needs of modern agriculture for efficient planting and soil remediation.

[0003] Most microbial fertilizers use single strains or simple compound microbial groups, which are prone to antagonism between strains and lack functional synergy, making it impossible to simultaneously achieve soil microecological regulation, nutrient activation and heavy metal passivation. The organic carriers are mostly conventional agricultural and forestry wastes that have not undergone targeted modification treatment, resulting in weak adsorption and protection capabilities for microorganisms. The survival rate of live bacteria is low during storage and application, and the product stability is poor.

[0004] Some microbial fertilizers have a single nutrient composition, providing only basic nitrogen, phosphorus, and potassium nutrients, lacking the combination of micronutrients and trace elements, making it difficult to adapt to the nutrient requirements of different soil types. The preparation process often employs high-temperature granulation and drying processes, which can easily cause a large number of functional microorganisms to become inactive, weakening the core efficacy of the microbial fertilizer. Furthermore, existing microbial fertilizers have limited effects on improving acidic soils, cannot effectively reduce the activity of harmful metal ions in the soil, and do not significantly improve soil bulk density, organic matter content, and other physicochemical properties, resulting in crop yield and quality improvement effects not meeting expectations. Summary of the Invention

[0005] The primary objective of this invention is to provide a bio-agricultural compound microbial fertilizer and its preparation method.

[0006] A further objective of this invention is to provide a bio-agricultural compound microbial fertilizer, comprising, by weight: 2-8 parts functional microbial flora, 20-48 parts organic carrier, 5-20 parts nutrient components, 0.5-4 parts cell protectant, and water as the balance; the functional microbial flora consists of *Pseudomonas fluorescens*, *Trichoderma harzianum*, *Saccharomyces cerevisiae*, and *Bacillus mucilaginosus*; the organic carrier consists of a mixture of peanut shells and sugarcane bagasse, modified diatomaceous earth, and humic acid; the peanut shell and sugarcane bagasse mixture is modified with silane coupling agent KH-550 or KH-560 after being pulverized and sterilized; the modified diatomaceous earth is obtained by soaking diatomaceous earth in hydrochloric acid solution, washing with water, drying, and pulverizing; the nutrient components consist of urea, potassium dihydrogen phosphate, and zinc sulfate; and the cell protectant consists of trehalose and skim milk powder.

[0007] Preferably, the mass ratio of *Pseudomonas fluorescens*, *Trichoderma harzianum*, *Saccharomyces cerevisiae*, and *Bacillus mucilaginosus* is 1:1:0.5:0.5, and the viable count of each strain is 1×10⁻⁶. 10 cfu / g.

[0008] Preferably, the mass ratio of the peanut shell and sugarcane bagasse mixture, modified diatomaceous earth, and humic acid is 5:3:2, and the volume ratio of the peanut shell and sugarcane bagasse mixture is 1:1. The peanut shell and sugarcane bagasse mixture is pulverized to 80 mesh, sterilized at 121°C and 0.1 MPa for 30 min, and modified by stirring at 25°C for 2 h with 3% KH-550 silane coupling agent.

[0009] Preferably, the modified diatomaceous earth is obtained by soaking diatomaceous earth in a 5% hydrochloric acid solution for 2 hours, washing it with water until neutral, drying it at 80°C for 2 hours, and pulverizing it to 100 mesh.

[0010] Preferably, the mass ratio of urea, potassium dihydrogen phosphate, and zinc sulfate is 4:3:1, and the mass ratio of trehalose to skim milk powder is 1:1.

[0011] A method for preparing a bio-agricultural compound microbial fertilizer includes, in sequence, strain co-cultivation, organic carrier pretreatment, nutrient compounding and coating steps.

[0012] Preferably, the co-culture step of the strains is as follows: the seed liquids of each strain are mixed and inoculated into the co-culture medium, the inoculation amount is 5% of the volume of the co-culture medium, the initial pH is 7.0, the culture temperature is 30℃, the dissolved oxygen concentration is 5mg / L, and the culture is shaken for 48h.

[0013] Preferably, the organic carrier pretreatment step is as follows: after mixing the organic carrier, adjust the water content to 40%, add 5% of the volume of the compound microbial liquid composting agent, ferment at 55°C, ferment at 50 r / min, ferment for 72 h, and then cool to 25°C.

[0014] Preferably, the nutrient compounding and coating step is as follows: pulverize the nutrient components to 100 mesh, stir with the decomposed organic carrier at 200 r / min for 30 min, add the compound microbial liquid and cell protectant and continue stirring for 20 min, and adjust the water content of the mixture to 20%.

[0015] Preferably, the process also includes granulation and drying steps: granulation temperature is 50℃, granulation speed is 300r / min; drying temperature is 45℃, drying air velocity is 1.5m / s, and drying is carried out until the material moisture content is 10%.

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention forms a complete compound microbial fertilizer technology solution through the scientific compounding of multiple strains, the targeted modification of organic carriers, the balanced optimization of nutrient components, and the synergistic effect of low-temperature preparation process, which has multiple outstanding beneficial effects.

[0017] 2. This invention adopts a multi-strain composite system with no antagonistic effect between strains and complementary functions, which can significantly increase the number of beneficial microorganisms in the soil, optimize the soil micro-ecological structure, and enhance the soil nutrient activation capacity. The modified organic carrier has greatly improved adsorption performance and water and fertilizer retention capacity, which can provide a stable living environment for functional microorganisms, effectively prolong the survival time of live bacteria, and improve the stability of product storage and application.

[0018] 3. The nutritional components of this invention are rationally formulated, providing a balanced supply of nutrients needed for crop growth. It is suitable for various soil types, improves soil fertility, and the low-temperature preparation process maximizes the preservation of microbial activity, avoiding damage to the strains from high temperatures and ensuring the stable performance of the core functions of the microbial fertilizer. This microbial fertilizer can effectively improve soil physicochemical properties, reduce soil bulk density, increase organic matter content, and passivate harmful metal ions in the soil, reducing their harm to crops and the soil environment.

[0019] 4. After application, this invention can promote crop growth and development, increase crop yield and quality, and help improve the quality and efficiency of agriculture. The preparation process of this invention uses conventional equipment, which can realize large-scale production, and is both environmentally friendly and practical. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] All raw materials meet the relevant standards such as GB / T41729-2022 and NY / T525. The strains selected comply with the provisions of GB / T41728 and NY / T1847, are non-pathogenic, have no antagonistic effects, and have stable functional characteristics.

[0022] All the equipment involved are conventional biological fermentation and granulation equipment. Specific models can be flexibly selected according to the production scale. Those skilled in the art can adjust the parameters according to the conventional operating procedures to ensure that the technical solution can be implemented repeatedly.

[0023] Example 1: Raw material composition: By weight, 2 parts functional microbial flora, 20 parts organic carrier, 5 parts nutrient components, 0.5 parts cell protectant, and appropriate amount of water; the functional microbial flora consists of *Pseudomonas fluorescens*, *Trichoderma harzianum*, *Saccharomyces cerevisiae*, and *Bacillus mucilaginosus* in a mass ratio of 1:1:0.5:0.5. All strains were isolated and screened from farmland soil and underwent three rejuvenation treatments. The rejuvenation medium used was LB modified medium (10 g / L peptone, 5 g / L yeast extract, 5 g / L sodium chloride, 2 g / L glucose, pH 7.0). The viable count of each strain was 1 × 10⁻⁶. 10 CFU / g; The organic carrier is composed of a mixture of peanut shells and sugarcane bagasse, modified diatomaceous earth, and humic acid in a mass ratio of 5:3:2, with the peanut shells and sugarcane bagasse mixture having a volume ratio of 1:1. After being pulverized to 80 mesh, it is sterilized at 121℃ and 0.1MPa for 30 min, and then modified by stirring with 3% KH-550 silane coupling agent at 25℃ for 2 h. During the modification process, the stirring speed is controlled at 150 r / min. After modification, it is naturally cooled to room temperature for use. The modified diatomaceous earth is obtained by soaking diatomaceous earth in a 5% hydrochloric acid solution for 2 h, washing it with water until neutral (pH 6.5-7.0), drying it at 80℃ for 2 h, and pulverizing it to 100 mesh. The nutrient components are composed of urea, potassium dihydrogen phosphate, and zinc sulfate in a mass ratio of 4:3:1. The cell protectant is composed of trehalose and skim milk powder in a mass ratio of 1:1, all of which are of food grade purity.

[0024] Preparation method: Step 1, co-culture of strains: The seed culture medium formula is 8 g / L peptone, 4 g / L yeast extract, 5 g / L sodium chloride, 3 g / L sucrose, pH 7.0, sterilized at 121℃ and 0.1 MPa for 20 min and set aside; *Pseudomonas fluorescens*, *Trichoderma harzianum*, *Saccharomyces cerevisiae*, and *Bacillus mucilaginosus* were inoculated into the seed culture medium respectively, and cultured at 30℃ and 180 r / min for 24 h with shaking to obtain seed solutions of each strain. The viable count of each seed solution was not less than 5 × 10⁻⁶. 8 CFU / mL; Mix the seed cultures of each strain according to the above mass ratio, and inoculate them into the co-culture medium at an inoculation volume of 5% of the co-culture medium volume. Adjust the initial pH to 7.0, control the temperature at 30℃ and the dissolved oxygen concentration at 5 mg / L, and culture with shaking for 48 h to obtain a composite microbial culture with a viable count of 8 × 10⁻⁶. 9 cfu / mL; the co-culture medium was prepared by mixing wheat bran, rice bran and soybean meal in a mass ratio of 18:7:25, with 3% PEG6000 and 5% Tween 80 added to improve the dispersibility of the strain. After preparation, the medium was sterilized at 121℃ and 0.1MPa for 20 min for later use.

[0025] The second step is the pretreatment of the organic carrier: A mixture of peanut shells and sugarcane bagasse, modified diatomaceous earth, and humic acid is thoroughly mixed, and the moisture content is adjusted to 40%. This mixture is then placed in a fermentation tank, and a 5% (by volume) volume of a composting agent is added. The temperature is controlled at 55℃, and the fermentation tank rotation speed is 50 r / min. Fermentation lasts for 72 hours, turning the mixture every 12 hours, until the organic carrier reaches 85% compostability (sensory evaluation criteria: no odor, dark brown color, and can be formed into a clump without crumbling when squeezed). The mixture is then cooled to 25℃ to obtain the fully decomposed organic carrier. The composting agent is a mixture of Bacillus subtilis and Actinomycetes at a mass ratio of 2:1, with a viable count of 5 × 10⁻⁶. 8 The cfu / mL concentration is prepared by conventional liquid fermentation, and can be prepared by those skilled in the art according to conventional processes.

[0026] The third step is nutrient compounding and coating: After pulverizing the nutrient components to 100 mesh, add them to the decomposed organic carrier and stir at 200 r / min for 30 min until uniform. Then add the compound microbial inoculum and cell protectant, and continue stirring for 20 min. Adjust the moisture content of the mixture to 20%. Send the mixture to a low-temperature granulator, control the granulation temperature at 50℃ and the granulation speed at 300 r / min, and the granulation particle size at 2-4 mm. Then send it to a low-temperature dryer, control the drying temperature at 45℃ and the drying air velocity at 1.5 m / s, and dry it until the moisture content is 10% to obtain the compound microbial fertilizer product. The finished product is sealed in aluminum foil and stored in a cool and dry place (temperature 15-25℃, humidity ≤60%). During the storage period, check regularly to avoid moisture and mold.

[0027] Example 2: Based on Example 1, this embodiment optimizes the ratio of functional microbial communities. The composition of other raw materials and preparation steps are basically the same as in Example 1. Only the ratio of strains and corresponding process parameters are adjusted synchronously to verify the synergistic effect of strain compatibility.

[0028] Raw material composition: By weight, 8 parts functional microbial flora, 48 parts organic carrier, 20 parts nutrient components, 4 parts cell protectant, and appropriate amount of water; the functional microbial flora consists of *Pseudomonas fluorescens*, *Trichoderma harzianum*, *Saccharomyces cerevisiae*, and *Bacillus mucilaginosus* in a mass ratio of 3:3:2:2. Each strain underwent three rejuvenation treatments, using the same method as in Example 1. The viable count of each strain was 1×10⁻⁶. 10 The composition of the organic carrier, nutrient components, and cell protectant is completely consistent with that of Example 1, except that the dosage is increased synchronously according to the proportion of Example 1 to ensure the stability of the synergistic effect between each component and the optimized strain.

[0029] Preparation method: The preparation steps are consistent with those in Example 1, except for the optimization and adjustment of the corresponding process parameters for the strain ratio: During the co-culture stage, the inoculum size was adjusted to 7% of the co-culture medium volume, the initial pH was adjusted to 7.2, the temperature was controlled at 32℃ and the dissolved oxygen concentration at 6 mg / L, and the culture was shaken for 56 h to obtain a composite microbial culture solution with a viable count of 9.8 × 10⁻⁶ cells / mL. 9 cfu / mL; During the fermentation of the organic carrier, the temperature was controlled at 58℃ and the fermentation tank speed at 60r / min. Fermentation lasted for 96 hours until the degree of decomposition reached 90%, and then the temperature was cooled to 25℃. The granulation temperature was adjusted to 55℃ and the granulation speed to 350r / min. The drying temperature was adjusted to 48℃ and the temperature was dried until the moisture content was 8% to ensure the retention of activity under high strain dosage.

[0030] Example 3: Based on Example 1, this embodiment optimizes the ratio and modification parameters of the organic carrier to enhance the carrier's adsorption and protection capacity for microorganisms and the soil's water and fertilizer retention performance. The composition of the remaining raw materials and the preparation steps are basically the same as in Example 1, with only the carrier ratio and modification parameters being adjusted.

[0031] Raw material composition: By weight, 6 parts functional microbial flora, 40 parts organic carrier, 15 parts nutrient components, 3 parts cell protectant, and appropriate amount of water; the organic carrier is composed of peanut shell-sugarcane bagasse mixture, modified diatomaceous earth, and humic acid in a mass ratio of 7:2:1, and the volume ratio of peanut shell-sugarcane bagasse mixture is 1:1. After being pulverized to 80 mesh, it is sterilized at 121℃ and 0.1MPa for 30 min, and then modified by stirring with 4% KH-560 silane coupling agent at 30℃ for 2.5 h at a stirring speed of 160 r / min. After modification, it is cooled to room temperature; the modified diatomaceous earth is obtained by soaking diatomaceous earth in 6% hydrochloric acid solution for 2.5 h, washing it with water until neutral, drying it at 80℃ for 2 h, and pulverizing it to 100 mesh; the composition of functional microbial flora, nutrient components, and cell protectant is completely consistent with that of Example 1, only the dosage is adjusted synchronously according to the proportion of Example 1.

[0032] Preparation method: The preparation steps are consistent with those in Example 1, except that the corresponding process parameters are optimized and adjusted for the carrier: In the organic carrier pretreatment stage, the water content is adjusted to 45%, 8% of the volume of the compound microbial liquid is added as a composting agent, the temperature is controlled at 52℃, the fermentation tank speed is 55r / min, and fermentation is carried out for 84h, turning and stirring once every 10h until the organic carrier reaches 88% composting degree, and then cooled to 22℃; When compounding nutrients, the stirring speed is adjusted to 220r / min, and the stirring time is adjusted to 35min; The granulation particle size is adjusted to 3-5mm, the granulation speed is 280r / min, the drying temperature is adjusted to 42℃, and the moisture content is dried to 9%.

[0033] Example 4: This embodiment is based on Example 1, but optimizes the preparation process parameters to improve the viable count of the compound microbial liquid and the stability of the microbial fertilizer. The composition of the remaining raw materials is completely the same as in Example 1, only the process parameters are adjusted.

[0034] Raw material composition: The raw material composition is completely consistent with that of Example 1. Only the amount of water is adjusted according to the process parameters to ensure that the moisture content of the materials in each step meets the process requirements. At the same time, it is compatible with the strain system of Example 2 and the carrier optimization scheme of Example 3.

[0035] Preparation method: Step 1, co-culture of strains: The seed culture of each strain was inoculated into the co-culture medium at an inoculation amount of 6%, the initial pH was adjusted to 6.8, the temperature was controlled at 28℃ and the dissolved oxygen concentration at 4 mg / L, and the culture was shaken for 40 h to obtain a composite microbial culture with a viable count of 9.5 × 10⁻⁶ cells / mL. 9 cfu / mL; 2% yeast extract was added to the co-culture medium to improve the strain proliferation efficiency. After the medium was prepared, it was sterilized at 121℃ and 0.1MPa for 20 min for later use.

[0036] The second step is the pretreatment of the organic carrier: adjust the moisture content to 38%, add 6% of the volume of compound microbial inoculant to ferment, control the temperature at 50℃ and the fermentation tank speed at 45r / min, ferment for 60h, turning and stirring once every 15h until the organic carrier reaches 86% maturity, and then cool to 22℃.

[0037] The third step is nutrient compounding and coating: the stirring speed is adjusted to 180 r / min, the stirring time is adjusted to 25 min, and the moisture content of the mixture is adjusted to 18%; the granulation temperature is adjusted to 48℃, the granulation speed is adjusted to 320 r / min, and the granulation particle size is 1.5-3 mm. Then, a low-temperature vacuum drying method is adopted, controlling the drying temperature at 40℃ and the vacuum degree at 0.08 MPa, and drying is carried out until the moisture content is 7%. The finished product is sealed in aluminum foil and stored under the same conditions as in Example 1.

[0038] Example 5: Based on Example 1, this embodiment expands the types and ratios of nutrient components, improves the adaptability of microbial fertilizer to alkaline soil, and supplements trace elements to achieve a balanced supply of nutrients. The composition of other raw materials and preparation steps are basically the same as in Example 1, with only the nutrient components and corresponding process steps being adjusted.

[0039] Raw material composition: by weight, 4 parts functional microbial flora, 30 parts organic carrier, 10 parts nutrient components, 2 parts microbial cell protectant, and appropriate amount of water; the nutrient components consist of urea, potassium dihydrogen phosphate, zinc sulfate, magnesium sulfate, and borax in a mass ratio of 3:2:1:1:1, with an additional 1 part calcium oxide as a pH adjuster to adjust the pH of the microbial fertilizer to 7.5-8.0 to suit alkaline soil; the composition of the functional microbial flora, organic carrier, and microbial cell protectant is completely consistent with that of Example 1, only the dosage is adjusted synchronously according to the proportion of Example 1; the calcium oxide is food grade with a purity of not less than 98%.

[0040] Preparation method: The preparation steps are consistent with those in Example 1, except for adjustments to the corresponding process steps for the nutrient components: In the nutrient compounding stage, the nutrient components are pulverized to 100 mesh, mixed evenly with calcium oxide, and then added to the decomposed organic carrier. After stirring evenly, the compound microbial inoculum and cell protectant are added, and stirring is continued for 25 minutes. The moisture content of the mixture is adjusted to 19%. The granulation temperature is adjusted to 52℃, the granulation speed is 310 r / min, the drying temperature is adjusted to 46℃, and the product is dried until the moisture content is 8%. The finished product is sealed in aluminum foil and stored under the same conditions as in Example 1.

[0041] Comparative Example 1: In the raw material composition, the functional microbial community consists of *Pseudomonas fluorescens*, *Saccharomyces cerevisiae*, and *Bacillus mucilaginosus* in a mass ratio of 1.5:0.5:0.5, with *Trichoderma harzianum* strain missing. The remaining raw material composition is completely consistent with that of Example 1; the preparation method is completely consistent with that of Example 1.

[0042] Comparative Example 2: In the raw material composition, the organic carrier was not modified with silane coupling agent. The peanut shell-sugarcane bagasse mixture was directly crushed and mixed with unmodified diatomaceous earth and humic acid. The unmodified diatomaceous earth was only crushed and sterilized. The composition of the remaining raw materials was completely consistent with that in Example 1. The preparation method was completely consistent with that in Example 1.

[0043] Comparative Example 3: The raw material composition is completely consistent with that of Example 1; in the preparation method, the granulation temperature is adjusted to 75℃, the granulation speed is 300r / min, the drying temperature is adjusted to 65℃, and the remaining process parameters are completely consistent with those of Example 1.

[0044] Comparative Example 4: In the raw material composition, the functional microbial community is only *Pseudomonas fluorescens*, with no other strains. The composition of the remaining raw materials is completely consistent with that in Example 1. The preparation method is completely consistent with that in Example 1, and the strain culture parameters are consistent with the *Pseudomonas fluorescens* seed culture parameters in Example 1.

[0045] Comparative Example 5: No cell protectant was added to the raw material composition, and the remaining raw material composition was completely consistent with that of Example 1; the preparation method was completely consistent with that of Example 1.

[0046] Comparative Example 6: In the raw material composition, conventional wheat bran was used to replace the peanut shell-sugarcane bagasse mixture and modified diatomaceous earth as the organic carrier, and only humic acid was retained. The wheat bran was pulverized to 80 mesh and sterilized at 121°C for 30 minutes. The composition of the remaining raw materials was completely consistent with that in Example 1. The preparation method was completely consistent with that in Example 1.

[0047] The co-culture medium described in this invention is prepared by mixing wheat bran, rice bran, and soybean meal in a mass ratio of 18:7:25, with the addition of 3% PEG6000 and 5% Tween 80 to improve the dispersibility of the bacterial strains. After preparation, the medium is sterilized at 121°C and 0.1 MPa for 20 minutes before use, and is a conventionally applicable medium for microbial co-culture in this field. The composting agent is a special agent composed of Bacillus subtilis and Actinomycetes in a mass ratio of 2:1, with a viable count of not less than 5 × 10⁻⁶. 8 The cfu / mL solution is prepared using conventional liquid fermentation processes in the field and is specifically designed for high-temperature composting of organic carriers.

[0048] The modification of the peanut shell and sugarcane bagasse mixture described in this invention uses only silane coupling agents KH-550 and KH-560. The modification concentration, temperature, and stirring time are all specific parameters disclosed in the examples and are conventional operating parameters for the modification of agricultural and forestry waste carriers in this field. The diatomaceous earth modification involves soaking in a 5%-6% hydrochloric acid solution, washing with water until neutral, drying at 80°C, and pulverizing to 100 mesh. This process is a standard method for the purification and modification of diatomaceous earth.

[0049] The *Pseudomonas fluorescens*, *Trichoderma harzianum*, *Saccharomyces cerevisiae*, and *Bacillus cannabinoides* described in this invention have been verified by conventional plate confrontation method in the art to have no antagonistic effect and can be stably co-cultured. All process parameters for co-culture of strains, composting of organic carriers, nutrient compound coating, and low-temperature granulation and drying are conventional and controllable parameters for industrial production of microbial fertilizers. Those skilled in the art can directly repeat the implementation based on the disclosed content of the embodiments without creative labor.

[0050] Performance testing and results analysis: Test conditions: Experimental environment: The experiment was conducted in a standardized farmland in a temperate monsoon climate zone. During the experiment, the temperature ranged from 12℃ to 28℃, the field water holding capacity was kept stable at 60%-70%, and there were no extreme weather disturbances such as rainstorms or droughts.

[0051] Test soil: The test soil was acidic yellow soil. Before sowing, its basic physicochemical and biological indicators were: pH 4.5, exchangeable aluminum ion concentration 3.6 cmol / kg, and soil bulk density 1.50 g / cm³. 3Organic matter 10.5 g / kg, available nitrogen 50 mg / kg, available phosphorus 10 mg / kg, available potassium 70 mg / kg, cadmium content 0.4 mg / kg, soil bacteria count 2.5 × 10⁻⁶ 6 cfu / g.

[0052] The test crop was rapeseed, and a uniform high-yield conventional variety was selected, with consistent sowing density and row spacing.

[0053] Experimental Design: A randomized block design was adopted. Examples 1-5 and Comparative Examples 1-6 were all replicated three times, with a single experimental plot area of ​​20m². 2 The application rate of microbial fertilizer is uniformly 300 kg / hm. 2 After application, the fertilizer was tilled into the soil to a depth of 15-20 cm. No other fertilizers or chemical pesticides were used throughout the experiment, and field management practices such as irrigation, weeding, and green pest control were completely standardized.

[0054] Sampling nodes: Samples were collected at four fixed nodes: before sowing, 30 days after rapeseed emergence, 90 days after rapeseed emergence, and after rapeseed harvest. For each plot, a 5-point sampling method was used to collect mixed soil samples from the 0cm-20cm topsoil layer. After crop harvest, rapeseed plant and grain samples were collected.

[0055] Test method: Soil pH value: determined by potentiometric method as specified in NY / T1377-2021, with a water-to-soil ratio of 2.5:1. After stirring, the soil was allowed to stand for 30 minutes, and the pH value was measured three times using a pH meter and the average value was taken.

[0056] Soil exchangeable aluminum ions: The method was performed according to LY / T1240-2015, using 1 mol / L potassium chloride solution for extraction, and the exchangeable aluminum ion content was calculated by neutralization titration.

[0057] Soil bulk density: In accordance with NY / T1121.4-2021, a 100cm³ ring sampler was used, and the soil bulk density was calculated after drying to constant weight.

[0058] Soil organic matter: The organic matter content was determined and calculated according to NY / T1121.6-2021 using the potassium dichromate oxidation-external heating method.

[0059] Available nitrogen in soil: The available nitrogen content was determined by alkaline hydrolysis diffusion method in accordance with NY / T1121.7-2021.

[0060] Available phosphorus in soil: In accordance with NY / T1121.7-2021, it was extracted with 0.5 mol / L sodium bicarbonate solution, and the absorbance was measured by spectrophotometer using the molybdenum antimony colorimetric method to calculate the available phosphorus content.

[0061] Available potassium in soil: The method is in accordance with NY / T1121.7-2021. The potassium is extracted with 1 mol / L ammonium acetate solution and the available potassium content is calculated after being measured by a flame photometer.

[0062] Soil cadmium content: In accordance with GB / T17141-1997, the cadmium content of the samples was determined by graphite furnace atomic absorption spectrophotometry after digestion.

[0063] Soil bacterial count: Performed according to NY / T1113-2018, using the dilution plate count method, with beef extract peptone medium as the carrier, incubated at 37℃ for 48h, and the colony count was counted and the number of bacteria per gram of dry soil was calculated.

[0064] Rapeseed yield per mu: After the rapeseed in each experimental plot matured, all the rapeseed was harvested and weighed, and the yield per mu was calculated based on the area of ​​the plot.

[0065] Rapeseed oil yield: In accordance with GB / T14488.1-2008, the Soxhlet extraction method was adopted. After drying the rapeseed, it was extracted by reflux with petroleum ether, and the oil yield of the seeds was calculated.

[0066] The test results are shown in Table 1 below: Table 1:

[0067]

[0068]

[0069]

[0070]

[0071]

[0072] Results analysis: Test data confirms that the compound microbial fertilizer of this invention can systematically improve the physical and chemical properties of acidic soil, enhance soil fertility, passivate heavy metal activity, increase the number of beneficial microorganisms in the soil, and significantly increase rapeseed yield and oil extraction rate. Example 1 is the basic formula; after application, the soil pH increased to 5.0, organic matter content increased to 15.2 g / kg, available nitrogen, available phosphorus, and available potassium increased to 75 mg / kg, 18 mg / kg, and 102 mg / kg respectively, cadmium content decreased to 0.28 mg / kg, and the number of soil bacteria reached 6.5 × 10⁻⁶. 6Example 1: CFU / g, rapeseed yield 198 kg / mu, oil extraction rate 40.5%, with stable soil improvement and crop growth promotion effects. Example 2: Optimized functional microbial community ratio, enhanced synergistic effect of strains, further improved soil improvement and nutrient supply capacity, rapeseed yield 212 kg / mu, oil extraction rate 41.8%. Example 3: Adjusted organic carrier ratio and modification parameters, significantly enhanced carrier adsorption and protection capacity and soil water and fertilizer retention performance, increased soil bacteria count to 10.2 × 10⁻⁶. 6 With cfu / g, the rapeseed yield was 220 kg / mu, and the oil extraction rate was 42.2%, demonstrating optimal comprehensive application performance. Example 4 optimized the preparation process parameters, improving the viable bacteria count and product stability, resulting in excellent soil improvement and crop yield increase effects, with a rapeseed yield of 215 kg / mu and an oil extraction rate of 41.5%. Example 5 expanded the nutrient components and added a pH adjuster, improving the adaptability of the microbial fertilizer to alkaline soils, achieving balanced nutrient supply, and significantly improving soil quality and crop yield.

[0073] Each comparative example showed a clear performance difference from the actual example. Comparative example 1 lacked the *Trichoderma harzianum* strain, resulting in a significant reduction in soil improvement effect; the rapeseed yield was only 156 kg / mu, with an oil extraction rate of 36.2%, confirming that *Trichoderma harzianum* was the core functional strain of the complex microbial community. Comparative example 2 did not modify the organic carrier, reducing the carrier's protective effect on microorganisms; the rapeseed yield was 185 kg / mu, with an oil extraction rate of 38.8%, lower than the basic example. Comparative example 3 used a high-temperature granulation and drying process, resulting in a significant loss of functional microbial activity and a significant reduction in soil improvement and yield increase effects; the rapeseed yield was 160 kg / mu, with an oil extraction rate of 37.0%. Comparative example 4 used a single *Pseudomonas fluorescens* strain, lacking the synergistic effect of multiple strains; all indicators were inferior to Example 1, with a rapeseed yield of 152 kg / mu and an oil extraction rate of 36.5%. Comparative example 5 did not add a cell protectant, resulting in reduced microbial survival and weakened product application effect; the rapeseed yield was 165 kg / mu, with an oil extraction rate of 37.5%. Comparative Example 6 showed that replacing the compound organic carrier with conventional wheat bran had limited effects on soil improvement and crop yield increase, with rapeseed yield of 170 kg per mu and oil extraction rate of 38.0%.

[0074] In summary, the microbial fertilizer produced by this invention through the scientific formulation of compound microbial communities, modification of organic carriers, optimization of nutrient components, and synergistic low-temperature processes has outstanding effects in soil improvement, fertility enhancement, heavy metal passivation, and crop yield and quality improvement. Its overall performance is significantly better than that of conventional microbial fertilizers.

[0075] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A compound microbial fertilizer for bio-agriculture, characterized in that, The product comprises, by weight: 2-8 parts functional microbial flora, 20-48 parts organic carrier, 5-20 parts nutrient components, 0.5-4 parts cell protectant, and water as the balance; the functional microbial flora consists of *Pseudomonas fluorescens*, *Trichoderma harzianum*, *Saccharomyces cerevisiae*, and *Bacillus mucilaginosus*; the organic carrier consists of a mixture of peanut shells and sugarcane bagasse, modified diatomaceous earth, and humic acid; the peanut shell and sugarcane bagasse mixture is modified with silane coupling agent KH-550 or KH-560 after being pulverized and sterilized; the modified diatomaceous earth is obtained by soaking diatomaceous earth in hydrochloric acid solution, washing with water, drying, and pulverizing; the nutrient components consist of urea, potassium dihydrogen phosphate, and zinc sulfate; and the cell protectant consists of trehalose and skim milk powder.

2. The bio-agricultural compound microbial fertilizer according to claim 1, characterized in that, The mass ratio of *Pseudomonas fluorescens*, *Trichoderma harzianum*, *Saccharomyces cerevisiae*, and *Bacillus mucilaginosus* was 1:1:0.5:0.5, and the viable count of each strain was 1×10⁻⁶. 10 cfu / g.

3. The bio-agricultural compound microbial fertilizer according to claim 1, characterized in that, The mass ratio of the peanut shell and sugarcane bagasse mixture, modified diatomaceous earth, and humic acid is 5:3:2, and the volume ratio of the peanut shell and sugarcane bagasse mixture is 1:

1. The peanut shell and sugarcane bagasse mixture is pulverized to 80 mesh, sterilized at 121℃ and 0.1MPa for 30 min, and modified by stirring at 25℃ for 2 h with 3% KH-550 silane coupling agent.

4. The bio-agricultural compound microbial fertilizer according to claim 1, characterized in that, The modified diatomaceous earth was prepared by soaking diatomaceous earth in a 5% hydrochloric acid solution for 2 hours, washing it with water until neutral, drying it at 80°C for 2 hours, and then pulverizing it to 100 mesh.

5. The bio-agricultural compound microbial fertilizer according to claim 1, characterized in that, The mass ratio of urea, potassium dihydrogen phosphate, and zinc sulfate is 4:3:1, and the mass ratio of trehalose to skim milk powder is 1:

1.

6. A method for preparing a bio-agricultural compound microbial fertilizer, used to prepare the bio-agricultural compound microbial fertilizer according to any one of claims 1-5, characterized in that, The process includes, in sequence, strain co-culture, organic carrier pretreatment, nutrient compounding and coating steps.

7. The preparation method according to claim 6, characterized in that, The co-culture steps of the strains are as follows: the seed liquids of each strain are mixed and inoculated into the co-culture medium at an inoculation amount of 5% of the volume of the co-culture medium, the initial pH is 7.0, the culture temperature is 30℃, the dissolved oxygen concentration is 5mg / L, and the culture is shaken for 48h.

8. The preparation method according to claim 6, characterized in that, The organic carrier pretreatment steps are as follows: after mixing the organic carrier, adjust the water content to 40%, add 5% of the volume of the compound microbial liquid composting agent, ferment at 55℃, ferment at 50r / min, ferment for 72h, and then cool to 25℃.

9. The preparation method according to claim 6, characterized in that, The nutrient compounding and coating steps are as follows: pulverize the nutrient components to 100 mesh, stir with the decomposed organic carrier at 200 r / min for 30 min, add the compound microbial liquid and cell protectant and continue stirring for 20 min, and adjust the water content of the mixture to 20%.

10. The preparation method according to claim 9, characterized in that, It also includes granulation and drying steps: granulation temperature is 50℃, granulation speed is 300r / min; drying temperature is 45℃, drying air velocity is 1.5m / s, and drying is carried out until the material moisture content is 10%.

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