Preparation of wheat seedling microbial fertilizer and detection method thereof

Abstract

The application discloses a wheat seedling microbial fertilizer preparation and a detection method thereof, and relates to the technical field of biological fertilizer preparation.The raw materials include, by weight, 8-12 parts of a microbial compound inoculant, 5-8 parts of humic acid modifiers, 40-50 parts of mature organic fertilizer, 3-5 parts of potassium dihydrogen phosphate, 2-4 parts of urea, 1-3 parts of trace element fertilizer, 0.5-1.0 parts of polyglutamic acid, 0.3-0.6 parts of chitosan quaternary ammonium salt, 0.2-0.4 parts of trehalose and 15-20 parts of water.The preparation steps include raw material pretreatment, compound inoculant activation, modifier preparation, mixed fermentation, nutrient allocation, drying and granulation and finished product post-treatment, and definite process parameters and control conditions are set for each step.Through formula innovation and process optimization, the application improves microbial activity and nutrient utilization rate, enhances fertilizer stability and disease resistance, realizes the synergistic effect of nutrition supply, disease prevention and control and soil improvement, and fully meets the growth requirements of wheat seedlings.

Description

Technical Field

[0001] This invention relates to the field of microbial fertilizer preparation technology, specifically to a method for preparing and testing microbial fertilizer for wheat seedlings. Background Technology

[0002] As a core grain crop in my country, the healthy growth of wheat seedlings is crucial to ensuring final yield and quality. Currently, wheat cultivation faces frequent problems such as declining soil fertility, low nutrient absorption efficiency, and soil-borne diseases. Microbial fertilizers, with their dual functions of soil enrichment and biological control, have become an important direction for addressing these issues. However, existing technologies still have shortcomings such as limited functionality, insufficient synergy, and imperfect process design, making it difficult to meet the comprehensive needs of wheat seedlings for nutrient supply, disease control, and soil adaptation.

[0003] The patent application document with patent publication number CN106244490A and patent name "A compound microbial fertilizer for preventing and controlling wheat take-all disease and its preparation method" mentions the use of five strains of bacteria, including Streptomyces simulans and Bacillus curvatureis, to prepare microbial fertilizer through solid fermentation. It utilizes the synergistic effect between strains to inhibit the pathogen of wheat take-all disease and has nitrogen fixation and growth promotion effects. However, the core of this patent focuses on disease prevention and control, and does not make precise proportions of nitrogen, phosphorus, potassium and trace elements required for wheat seedling growth. It also does not use humic acid modification and other technologies to improve nutrient adsorption and slow release performance, resulting in rapid nutrient loss and failure to provide continuous and stable nutritional support for seedlings. At the same time, the temperature, humidity and aeration are not controlled in stages during the fermentation process, resulting in insufficient microbial fermentation efficiency and activity stability.

[0004] The patent application document with patent publication number CN115316207A and patent name "A cultivation method for increasing wheat yield using a novel fertilizer" mentions adjusting the fertilizer ratio through soil composition analysis and combining it with refined cultivation management to increase wheat yield. It also mentions adding modified biochar, maifanite powder and other raw materials to improve soil conditions. However, this patent focuses on optimizing the cultivation method and lacks innovation in the fertilizer formula itself. The microbial agent only contains Bacillus subtilis and Lactobacillus plantarum, with limited functional coverage and lacks targeted strains for phosphorus and potassium solubilization. Furthermore, it does not involve precise control of the microbial agent activation and fermentation process, resulting in poor retention of microbial activity and difficulty in fully leveraging the synergistic effect of microbial fertilizer. At the same time, it does not consider the differences in nutrient requirements of wheat seedlings at different growth stages.

[0005] The patent application document with patent publication number CN110981603B and patent name "A Coated Slow-Release Fertilizer for Wheat and Its Preparation Method and Application" mentions introducing hidden acidophilic bacteria into the slow-release fertilizer, combined with coating technology to slow down nutrient release and enhance wheat's resistance to take-all disease. However, the coating material of this patent has a complex composition and high cost. The slow-release rate does not match the nutrient requirements of wheat seedlings during the rapid growth stage, which can easily lead to insufficient nutrient supply in the early stage. At the same time, it lacks absorption-promoting components such as polyglutamic acid and chitosan quaternary ammonium salt, so the nutrient utilization rate needs to be improved. Furthermore, it does not involve the refined operation of raw material pretreatment, resulting in poor fertilizer mixing uniformity and affecting the overall application effect.

[0006] Furthermore, existing technologies generally suffer from insufficient protection of microbial inoculant activity, poor product stability, and incomplete post-processing procedures. Some products lack a dedicated inoculant activation step, leading to excessively rapid decline in the number of effective viable bacteria; some products lack hardness testing and ultraviolet sterilization processes, affecting product shelf life and safety; and some products fail to finely grind and sieve the organic fertilizer, resulting in uneven nutrient distribution. Therefore, there is an urgent need to develop a scientifically formulated, precisely processed microbial fertilizer that combines nutrient supply and disease control functions, and is suitable for the growth needs of wheat seedlings, along with its preparation and testing methods. Summary of the Invention

[0007] (a) Technical problems to be solved To address the shortcomings of existing technologies, such as single-function microbial fertilizer strains, poor nutrient adsorption and slow-release performance due to unmodified humic acid, insufficient microbial agent activation, lack of precise staged control in fermentation processes, poor storage stability due to lack of active protective components, imperfect post-processing procedures, and incomplete detection methods that fail to simultaneously assess effective viable cell count, nutrient content, and heavy metal detection, and the lack of precise nutrient ratios tailored to the growth needs of wheat seedlings, making it difficult to achieve synergistic effects in nutrient supply, disease control, and soil improvement, and ultimately failing to meet the comprehensive needs for healthy wheat seedling growth, this invention provides a method for preparing and detecting microbial fertilizer for wheat seedlings.

[0008] (II) Technical Solution A method for preparing microbial fertilizer for wheat seedlings, comprising the following raw materials by weight: 8-12 parts of microbial compound inoculant, 5-8 parts of humic acid modifier, 40-50 parts of well-rotted organic fertilizer, 3-5 parts of potassium dihydrogen phosphate, 2-4 parts of urea, 1-3 parts of trace element fertilizer, 0.5-1.0 parts of polyglutamic acid, 0.3-0.6 parts of chitosan quaternary ammonium salt, 0.2-0.4 parts of trehalose, and 15-20 parts of water; The humic acid-modified product is prepared by esterification of humic acid and aminotrimethylenephosphonic acid, with the reaction molecular formula C9H8O4+C3H 12 NO9P3→C 12 H17 NO 12 P3+H2O; Chitosan quaternary ammonium salt is prepared by etherification of chitosan with 3-chloro-2-hydroxypropyltrimethylammonium chloride, with the molecular formula (C6H2O). 11 NO4) n +nC6H 16 ClNO→(C 12 H 25 ClN2O5) n +nH2O; The mass ratio of nitrogen-fixing bacteria, phosphate-solubilizing bacteria, potassium-solubilizing bacteria, and growth-promoting bacteria in the microbial compound inoculant is 2:1:1:0.5, and the concentration of nitrogen-fixing bacteria is 1.0 × 10⁻⁶. 9 -5.0×10 9 CFU / mL, phosphate-solubilizing bacteria concentration is 5.0 × 10⁻⁶. 8 -2.0×10 9 CFU / mL, potassium-solubilizing bacteria concentration was 5.0 × 10⁻⁶. 8 -2.0×10 9 CFU / mL, growth-promoting bacteria concentration is 3.0 × 10⁻⁶ 8 -1.0×10 9 CFU / mL; The composted organic fertilizer is made by mixing corn stalks, livestock and poultry manure and mushroom residue in a mass ratio of 3:2:1 and composting for 45-60 days. The temperature is controlled at 55-65℃ during the composting process, and the pile is turned over once every 3 days. Polyglutamic acid has a molecular weight of 100,000-300,000 Da and a purity of ≥95%. It is prepared by Bacillus subtilis fermentation at a fermentation temperature of 30-32℃ for 48-56 hours. The degree of substitution of chitosan quaternary ammonium salt is 0.6-0.8, the molecular weight is 50,000-100,000 Da, and the purity is ≥98%. Trehalose has a purity of ≥99% and is an anhydrous crystalline form; The steps are as follows: S1. Raw material pretreatment: Place the decomposed organic fertilizer in a hot air circulating drying oven and dry it at 60-70℃ for 4-6 hours, controlling the moisture content to ≤15%. After drying, crush it through a universal pulverizer and then sieve it through an 80-100 mesh screen. The material on the sieve is returned to the pulverizer for re-crushing, and the material under the sieve is collected for later use. Grind each component of the trace element fertilizer to 120-150 mesh, mix them evenly, and then set them aside. S2. Activation of the compound microbial agent: Inoculate the compound microbial agent into the activation medium at an inoculum volume of 5-8%. The activation medium consists of 15-20 g / L glucose, 5-8 g / L peptone, 3-5 g / L yeast extract, 2-3 g / L sodium chloride, 0.5-1.0 g / L magnesium sulfate, 0.3-0.5 g / L potassium dihydrogen phosphate, and the remainder is water. Adjust the pH of the medium to 6.5-7.5. Sterilize at 121℃ and 0.1 MPa for 20-30 min. After sterilization, cool to 30-32℃ before inoculation. After inoculation, culture at 28-32℃ and pH 6.5-7.5 with shaking for 12-16 h at a shaking speed of 180-220 r / min. Monitor the pH every 4 h during the culture process. If it deviates from the range, adjust it with 1 mol / L hydrochloric acid or sodium hydroxide solution. S3. Preparation of modified product: Humic acid and aminotrimethylenephosphonic acid are mixed at a mass ratio of 3:1. Concentrated sulfuric acid, accounting for 2-3% of the total mass, is added as a catalyst. The catalyst is added dropwise at a rate of 0.5-1.0 mL / min. After the addition is complete, the mixture is reacted at 85-95℃ for 3-4 h. During the reaction, the mixture is continuously stirred at a speed of 60-80 r / min. After the reaction is completed, the mixture is cooled to room temperature, and the pH value is adjusted to 6.0-7.0 with 1 mol / L sodium hydroxide solution to obtain the humic acid modified product. S4. Mixed Fermentation: The pretreated decomposed organic fertilizer, humic acid modifier, and activated compound microbial agent are added to a double-layer constant temperature fermenter. After stirring evenly, fermentation is carried out at 30-35℃ for 48-72 hours. Sterile air is introduced during fermentation, and the aeration rate is adjusted in stages: 0.5L / (L·h) from 0-24 hours, 0.8L / (L·h) from 24-48 hours, and 1.0L / (L·h) from 48-72 hours. Stirring is performed once every 8 hours for 15-20 minutes each time at a stirring speed of 100-120 r / min. The temperature and pH of the fermentation system are monitored every 4 hours. If the temperature deviates from the range, it is adjusted through the fermenter jacket temperature control system. If the pH deviates from the range of 6.0-7.5, it is adjusted with citric acid or sodium hydroxide solution. S5. Nutrient formulation: Add potassium dihydrogen phosphate, urea, pretreated micronutrient fertilizer, polyglutamic acid, chitosan quaternary ammonium salt, and trehalose to the fermentation product. Continue stirring at 80-100 r / min for 20-30 min. During stirring, adjust the pH of the system to 6.0-7.0 using 1 mol / L citric acid solution or 1 mol / L sodium hydroxide solution. After adjustment, keep warm and let stand for 10-15 min. S6. Drying and Granulation: The prepared materials are fed into a belt dryer and dried with hot air at 50-60℃ for 8-10 hours. During the drying process, the hot air velocity is controlled at 0.8-1.2 m / s. The moisture content of the dried material is ≤8%. The dried material is then fed into an extrusion granulator with a granulation pressure of 1.0-1.5 MPa and a die temperature of 45-50℃ to obtain particles with a particle size of 2-4 mm. After granulation, the particles are cooled at room temperature for 30-60 minutes and then screened by a vibrating sieve with a screen mesh of 6-10 mesh. S7. Finished product post-processing: The material remaining on the sieve is subjected to hardness testing; the hardness is ≥2.0 kg / cm². 2 The granules are considered qualified products; unqualified granules are crushed and returned to the extrusion granulation step for reprocessing. Qualified products undergo ultraviolet sterilization treatment for 15-20 minutes at a sterilization intensity of 300-350 μW / cm². 2 After sterilization, seal and package.

[0009] Furthermore, its nitrogen-fixing capacity is ≥20 mg N / (g・d); the phosphate-solubilizing bacteria are Bacillus megaterium, with a phosphate-solubilizing capacity of ≥15 mg P2O5 / (g・d); the potassium-solubilizing bacteria are Bacillus mucilaginosus, with a potassium-solubilizing capacity of ≥10 mg K2O / (g・d); and the growth-promoting bacteria are Bacillus subtilis, with an indoleacetic acid production capacity of ≥5 μg / mL.

[0010] Furthermore, 0.1-0.2 g / L of vitamin B1 and 0.05-0.1 g / L of vitamin C are added to the activation culture medium. After the culture medium is sterilized, it is cooled to 30-32°C in an aseptic environment before inoculation. During the incubation process after inoculation, the humidity in the shaking incubator is maintained at 40-50%.

[0011] Furthermore, the micronutrient fertilizer includes 0.3-0.5 parts of zinc sulfate heptahydrate, 0.2-0.4 parts of ferrous sulfate heptahydrate, 0.2-0.3 parts of boric acid, 0.1-0.2 parts of manganese sulfate, 0.05-0.1 parts of ammonium molybdate, and 0.05-0.1 parts of copper sulfate pentahydrate; the purity of each component is ≥99%, and after grinding, it is sieved through a 120-150 mesh sieve. When mixing, it is stirred at a speed of 300-400 r / min for 10-15 min to ensure uniformity.

[0012] Furthermore, in step S4, 0.1-0.3 parts of cellulase and 0.05-0.1 parts of protease are added during the fermentation process. The cellulase activity is ≥10000U / g and the protease activity is ≥5000U / g. The addition is made 12 hours after the start of fermentation, and the mixture is stirred for 20 minutes after addition to ensure uniform dispersion.

[0013] Furthermore, the fermentation preparation process of polyglutamic acid is as follows: using Bacillus subtilis as the production strain, the fermentation medium includes glucose 20-25 g / L, corn steep liquor 10-15 g / L, ammonium sulfate 5-8 g / L, potassium dihydrogen phosphate 1-2 g / L, magnesium sulfate 0.5-1.0 g / L, calcium chloride 0.1-0.2 g / L, pH adjusted to 6.8-7.2, fermentation temperature 30-32℃, fermentation time 48-56 h, aeration rate of 0.6-0.8 L / (L・h) during fermentation, and stirring speed of 150-180 r / min; after fermentation, the polyglutamic acid product is obtained by centrifugation, ultrafiltration, and freeze drying.

[0014] Furthermore, in step S7, a continuous ultraviolet sterilization machine is used for ultraviolet sterilization. The material conveying speed during sterilization is 0.5-1.0 m / min. The moisture content of the product after sterilization is controlled at 6-8%. The packaging uses aluminum foil composite bags. Nitrogen gas is filled into the bags during packaging. The nitrogen purity is ≥99.9%, and the filling pressure is 0.1-0.15 MPa.

[0015] Furthermore, a method for detecting microbial fertilizers in wheat seedlings includes the following steps: S1. Sample Preparation: Take 5.0g of fertilizer sample, accurate to 0.001g, and place it in a sterile Erlenmeyer flask. Add 45mL of sterile water at 25-28℃. Place the Erlenmeyer flask in a constant temperature shaking incubator and shake for 30min at 200-250r / min and 25-28℃ to prepare a 1:10 sample suspension. Dilute the sample suspension stepwise with sterile water at a dilution gradient of 10. -1 10 -2 10 -3 10 -4 10 -5 10 -6 After each dilution, let it stand for 5 minutes before proceeding to the next dilution. S2. Effective viable bacteria count test: Take 10... -4 10 -5 10 -6Three dilutions of sample suspension, 0.1 mL each, were evenly spread onto selective medium plates, with three replicates for each concentration. The selective medium was prepared by mixing LB medium and inorganic phosphorus medium at a 1:1 volume ratio. The LB medium consisted of 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride. The inorganic phosphorus medium consisted of 10 g / L glucose, 0.5 g / L ammonium sulfate, 0.3 g / L sodium chloride, 0.3 g / L potassium chloride, 0.3 g / L magnesium sulfate, 0.03 g / L ferrous sulfate, and 0.03 g / L manganese sulfate. L, tricalcium phosphate 2g / L, adjust the pH of the culture medium to 6.8-7.2, sterilize at 121℃ and 0.1MPa for 20min; when the culture medium cools to 50-60℃, add 50μg / mL ampicillin as a screening agent, shake well and pour into plates; incubate the plates in a constant temperature incubator at 28-32℃ for 48-72h, and observe the colony growth once a day during the incubation period; after the incubation, select plates with colony counts between 30-300 for counting, calculate the average value and convert it into the effective viable bacteria count per gram of sample; S3. Nutrient content detection: Take 1.0g of sample, accurate to 0.001g, put it into a digestion tube, add 5mL of concentrated sulfuric acid, shake well and let stand overnight; the next day, place the digestion tube on a digestion furnace, first carbonize at low temperature (150-200℃) for 30min, then heat to 360-400℃ for 1h, cool to room temperature, slowly add 2mL of 30% hydrogen peroxide solution, continue to heat and digest for 30min. If the solution is still not clear, repeat adding hydrogen peroxide solution and digesting until the solution is clear; after cooling, transfer the digestion solution to a 100mL volumetric flask, dilute to the mark with distilled water, shake well and set aside; The Kjeldahl method was used to determine the total nitrogen content: 10 mL of the digested solution was placed in a Kjeldahl distillation apparatus, 10 mL of 40% sodium hydroxide solution was added, and distillation was carried out for 5 min. The distillate was absorbed with 2% boric acid solution, and titrated with 0.05 mol / L hydrochloric acid standard solution to the endpoint using methyl red-bromocresol green as an indicator. The volume of hydrochloric acid standard solution consumed was recorded, and the total nitrogen content was calculated. The hydrochloric acid standard solution was calibrated with borax as a reference standard, and the relative deviation of parallel samples was ≤0.2%. The total phosphorus content was determined using the molybdenum-antimony colorimetric method: 5 mL of digested solution was placed in a 50 mL volumetric flask, 10 mL of molybdenum-antimony colorimetric reagent was added, and the solution was diluted to the mark with distilled water. After shaking well, the solution was incubated at 25-30℃ for 30 min. The absorbance was measured at 700 nm using a spectrophotometer, and the total phosphorus content was calculated based on the standard curve. The molybdenum-antimony colorimetric reagent was prepared by mixing ammonium molybdate solution, sulfuric acid solution, and potassium antimony tartaric acid solution in a volume ratio of 1:2:0.5, and should be used immediately after preparation. The total potassium content was determined by flame photometry: 5 mL of digestion solution was placed in a 50 mL volumetric flask, diluted to the mark with distilled water, shaken well, and then introduced into a flame photometer. A standard curve was plotted using a potassium standard solution, the absorbance of the sample was measured, and the total potassium content was calculated. The operating current of the flame photometer was 5-10 mA, the gas pressure was 0.05-0.08 MPa, and the oxidizing gas pressure was 0.2-0.3 MPa. S4. Heavy metal detection: Take 2.0 g of sample, accurate to 0.001 g, and place it in a microwave digestion vessel. Add 8 mL of nitric acid and 2 mL of hydrogen peroxide, shake well, and let stand for 15 min. Place the digestion vessel in a microwave digester and digest according to the following procedure: heat to 120℃ for 5 min and hold for 5 min; then heat to 180℃ for 5 min and hold for 15-20 min. After digestion, cool to room temperature, transfer the digest to a 50 mL volumetric flask, dilute to the mark with distilled water, shake well, and set aside. Perform a blank test at the same time. The contents of lead, cadmium, mercury, and chromium were determined using an atomic absorption spectrophotometer: the detection wavelength for lead was 283.3 nm, the slit width was 0.5 nm, and the lamp current was 10 mA; the detection wavelength for cadmium was 228.8 nm, the slit width was 0.5 nm, and the lamp current was 8 mA; the detection of mercury was performed using cold atomic absorption spectrometry, with a detection wavelength of 253.7 nm and a lamp current of 15 mA; and the detection wavelength for chromium was 357.9 nm, the slit width was 0.5 nm, and the lamp current was 12 mA. The detection limits of the instruments were: lead ≤ 0.01 mg / kg, cadmium ≤ 0.001 mg / kg, mercury ≤ 0.0001 mg / kg, and chromium ≤ 0.005 mg / kg. S5. Result Interpretation: Effective viable bacteria count ≥ 2.0 × 10⁻⁶ 8 If the CFU / g, total nitrogen content ≥3.0%, total phosphorus content (as P2O5) ≥2.0%, total potassium content (as K2O) ≥2.0%, and heavy metal content meet the requirements of GB / T23349-2020 standard, then it is judged as a qualified product.

[0016] Furthermore, in step S2, the selective culture medium plates are prepared using the pouring method, with 15-20 mL of culture medium poured into each plate. After the culture medium solidifies, it is inverted and incubated at 37°C for 2 hours. After confirming that no colonies are growing, it is then spread. When counting colonies, a colony counter is used. Colonies with abnormal morphology are excluded during counting. The relative deviation of the colony count on the same plate between parallel samples is ≤10%.

[0017] Furthermore, in step S3, the distillation apparatus for total nitrogen detection needs to be cleaned three times with distilled water to ensure no residue; the spectrophotometer for total phosphorus detection needs to be preheated for 30 minutes, and the absorbance of the blank solution should be ≤0.005; the concentration gradient of the potassium standard solution for total potassium detection is 0, 2, 4, 6, 8, and 10 μg / mL, and the correlation coefficient R of the standard curve is...2 ≥0.999; The power of the microwave digester in step S4 is 600-800W, the digestion vessel is made of polytetrafluoroethylene, and it is soaked in nitric acid solution for 24 hours before use, and then cleaned and dried; The atomization efficiency of the atomic absorption spectrophotometer is ≥95%, and the stability is ≤1%.

[0018] (iii) Beneficial technical effects Compared with existing technologies, the beneficial effects of this invention are: The method for preparing and detecting microbial fertilizer for wheat seedlings provided by this invention has significant advantages over existing technologies in many aspects, effectively making up for the shortcomings of existing technologies. At the formulation level, it innovatively introduces humic acid modifiers and chitosan quaternary ammonium salts, optimizing raw material performance through esterification and etherification reactions, significantly enhancing nutrient adsorption and slow-release capabilities, and solving the problems of rapid nutrient loss and low utilization rate. The microbial compound agent uses a combination of nitrogen-fixing bacteria, phosphorus-solubilizing bacteria, potassium-solubilizing bacteria, and growth-promoting bacteria, covering nutrient conversion, growth regulation, and disease control. Compared with single or a few strains, the synergistic effect is better, more comprehensively meeting the growth needs of wheat seedlings, while precisely proportioning nitrogen, phosphorus, potassium, and trace elements to adapt to the nutrient supply at different growth stages of the seedlings.

[0019] In terms of process design, product quality is ensured through precise control at multiple stages. The compound microbial agent activation stage optimizes the culture medium composition, adding nutrients such as vitamin B1 and vitamin C, while simultaneously adjusting pH and humidity in real time, significantly enhancing microbial activity and overcoming the problems of insufficient activation and unstable activity. The fermentation process employs staged aeration and temperature / humidity control, combined with the addition of cellulase and protease, promoting organic matter degradation and nutrient conversion, resulting in significantly improved fermentation efficiency and product quality—a simple solid-state fermentation mode. The drying, granulation, and post-processing stages include steps such as hardness testing and ultraviolet sterilization, ensuring both the physical stability of the product and improving its safety, thus solving the problems of short shelf life and susceptibility to contamination.

[0020] In terms of detection methods, a comprehensive detection system covering viable bacterial count, nutrient content, and heavy metal content has been established. Optimization of selective culture media has improved the accuracy of viable bacterial count, while the application of microwave digestion and atomic absorption spectrophotometry has ensured the precision of heavy metal detection. Compared with single or simplified detection methods, the detection results are more reliable and have greater reference value, providing strong support for product quality control.

[0021] Overall, this invention achieves multiple benefits—nutrient supply, disease control, and soil improvement—through the synergistic effect of formula innovation and process optimization. The product's nutrient release rate is highly compatible with the wheat seedling growth cycle, exhibits stable microbial activity, and boasts high nutrient utilization, effectively enhancing seedling resistance and promoting root development and robust plant growth. The comprehensive and scientific testing methods effectively guarantee product quality. This technical solution addresses the problems of existing products, such as limited functionality, poor synergy, and imperfect processes, providing all-round support for the healthy growth of wheat seedlings and demonstrating broad application prospects. Attached Figure Description

[0022] Figure 1 This is a process flow diagram of the preparation method of microbial fertilizer for wheat seedlings; Figure 2 This is a flowchart of the detection method for microbial fertilizers in wheat seedlings; Figure 3 This is a bar chart comparing the effective viable bacteria counts of each embodiment and the comparative example; Figure 4 This is a line graph showing the changes in the viable bacteria retention rate during the storage period for each embodiment and comparative example; Figure 5 This is a comparison chart of the overall performance of radar systems in various embodiments and comparative examples. Detailed Implementation

[0023] according to Figures 1 to 5 The specific embodiments of the present invention are as follows: The technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples. All operations are carried out in a cleanroom that meets the standards for microbial fertilizer production. The cleanliness of the workshop environment reaches Class 100,000, the temperature is controlled at 25-28℃, and the relative humidity is ≤60%. All raw materials used are qualified food-grade or agricultural-grade products, and all parameters are strictly selected in accordance with the scope of the claims to ensure the repeatability and practicality of the technical solution.

[0024] General Parameters of Raw Materials Microbial compound inoculant: Azotobacter chrysophagus concentration 3.0 × 10⁻⁶ 9 CFU / mL, nitrogen fixation capacity 25 mg N / (g・d); Bacillus megaterium concentration 1.2 × 10⁻⁶ 9 CFU / mL, phosphorus solubilization capacity 18 mg P₂O₅ / (g・d); Bacillus mucilaginosus concentration 1.2 × 10⁻⁶ 9 CFU / mL, potassium solubilizing capacity 13 mg K₂O / (g・d); Bacillus subtilis concentration 6.0 × 10⁻⁶ 8 CFU / mL, indoleacetic acid production capacity 8 μg / mL.

[0025] Humic acid: purity ≥70%, moisture content ≤10%, humic acid content ≥60%; aminotrimethylenephosphonic acid purity ≥98%, pH value 1.5-2.5.

[0026] Well-rotted organic fertilizer: Corn stalks, livestock and poultry manure, and mushroom residue are mixed in a mass ratio of 3:2:1 and composted for 50 days. The temperature is maintained at 60℃ during the composting process. The compost is turned over every 3 days. The organic matter content is ≥45% and the moisture content is ≤20%.

[0027] Potassium dihydrogen phosphate purity ≥99%, potassium oxide content ≥34%, phosphorus pentoxide content ≥52%; urea purity ≥99.5%, nitrogen content ≥46.3%.

[0028] Micronutrient fertilizers: Zinc sulfate heptahydrate purity ≥99.0%, zinc content ≥22%; ferrous sulfate heptahydrate purity ≥99.0%, iron content ≥19%; boric acid purity ≥99.5%, boron content ≥17%; manganese sulfate purity ≥98.0%, manganese content ≥31%; ammonium molybdate purity ≥99.0%, molybdenum content ≥54%; copper sulfate pentahydrate purity ≥99.0%, copper content ≥25%.

[0029] Polyglutamic acid: molecular weight 200,000 Da, purity ≥95%, prepared by fermentation of Bacillus subtilis, fermentation medium containing glucose 22 g / L, corn steep liquor 12 g / L, ammonium sulfate 6 g / L, fermentation temperature 31℃, fermentation time 52 h.

[0030] Chitosan quaternary ammonium salt: degree of substitution 0.7, molecular weight 80000 Da, purity ≥98%, prepared by etherification reaction of chitosan and 3-chloro-2-hydroxypropyltrimethylammonium chloride.

[0031] Trehalose: anhydrous crystalline form, purity ≥99%, moisture content ≤0.5%.

[0032] Cellulase activity 15000 U / g, protease activity 8000 U / g; vitamin B1 purity ≥99%, vitamin C purity ≥99%.

[0033] Activation medium: glucose 18g / L, peptone 6g / L, yeast extract 4g / L, sodium chloride 2.5g / L, magnesium sulfate 0.8g / L, potassium dihydrogen phosphate 0.4g / L, vitamin B1 0.15g / L, vitamin C 0.08g / L, with the remainder being water.

[0034] Example 1 Formula composition (by weight) The ingredients are: 10 parts microbial compound inoculant, 6 parts humic acid modifier, 45 parts well-rotted organic fertilizer, 4 parts potassium dihydrogen phosphate, 3 parts urea, 2 parts trace element fertilizer, 0.8 parts polyglutamic acid, 0.4 parts chitosan quaternary ammonium salt, 0.3 parts trehalose, and 18 parts water.

[0035] The micronutrient fertilizer consists of: 0.4 parts zinc sulfate heptahydrate, 0.3 parts ferrous sulfate heptahydrate, 0.25 parts boric acid, 0.15 parts manganese sulfate, 0.08 parts ammonium molybdate, and 0.07 parts copper sulfate pentahydrate; the microbial compound inoculant contains Azotobacter chrysophagus, Bacillus megaterium, Bacillus mucilaginosus, and Bacillus subtilis in a mass ratio of 2:1:1:0.5.

[0036] Preparation process S1. Raw Material Pretreatment: The well-rotted organic fertilizer is placed in a hot air circulating drying oven and dried at 65℃ for 5 hours, controlling the moisture content to 12%. After drying, it is pulverized using a universal pulverizer, and then sieved through a 90-mesh sieve. The material exceeding the sieve is returned to the pulverizer for further pulverization, while the material passing through the sieve is collected for later use. Each component of the micronutrient fertilizer is separately ground through a 130-mesh sieve, then placed in a high-speed mixer and stirred at 350 rpm for 12 minutes until uniformly mixed.

[0037] S2. Activation of the compound microbial agent: After preparing the activation medium according to the formula ratio, adjust the pH to 7.0 and sterilize it at 121℃ and 0.1MPa for 25 min. After sterilization, cool to 31℃ and inoculate the compound microbial agent into the activation medium at an inoculation rate of 6%. After inoculation, place it in a shaking incubator and incubate at 30℃ and pH 7.0 for 14 h with shaking at a shaking speed of 200 r / min. During the incubation process, monitor the pH every 4 h. If it deviates from the range, adjust it with 1 mol / L hydrochloric acid or sodium hydroxide solution, and maintain the humidity in the shaking incubator at 45%.

[0038] S3. Preparation of the modified product: Humic acid and aminotrimethylenephosphonic acid were placed in a reaction vessel at a mass ratio of 3:1. Concentrated sulfuric acid, accounting for 2.5% of the total mass, was added as a catalyst at a rate of 0.8 mL / min. After the addition was complete, the temperature of the reaction vessel was raised to 90℃, and the mixture was continuously stirred at a rate of 70 r / min for 3.5 h. After the reaction was completed, the mixture was cooled to room temperature, and the pH was adjusted to 6.5 with 1 mol / L sodium hydroxide solution to obtain the humic acid modified product.

[0039] S4. Mixed Fermentation: The pretreated, well-rotted organic fertilizer, humic acid-modified material, and activated compound microbial agent are added to a double-layered constant-temperature fermenter. After thorough mixing, fermentation is carried out at 32℃ for 60 hours. Sterile air is introduced during fermentation, with the aeration rate adjusted in stages: 0.5 L / (L·h) in the early stage (0-24h), 0.8 L / (L·h) in the middle stage (24-48h), and 1.0 L / (L·h) in the later stage (48-72h). Stirring is performed every 8 hours for 18 minutes at a speed of 110 rpm. 12 hours after fermentation begins, 0.2 parts of cellulase and 0.08 parts of protease are added, and stirring is performed for 20 minutes to ensure uniform dispersion. The temperature and pH of the fermentation system are monitored every 4 hours. If the temperature deviates from the specified range, it is adjusted using the fermenter's jacketed temperature control system; if the pH deviates from the 6.0-7.5 range, it is adjusted using citric acid or sodium hydroxide solution.

[0040] S5. Nutrient Preparation: Add potassium dihydrogen phosphate, urea, pretreated micronutrient fertilizer, polyglutamic acid, chitosan quaternary ammonium salt, and trehalose to the fermentation product. Before adding polyglutamic acid, dissolve it in water at 32℃ to prepare an 8% solution, then add it to the fermentation product. Continue stirring at 90 rpm for 25 minutes. During stirring, adjust the pH of the system to 6.5 using a 1 mol / L citric acid solution or a 1 mol / L sodium hydroxide solution. After adjustment, keep the mixture at this temperature and let it stand for 12 minutes.

[0041] S6. Drying and Granulation: The formulated material is fed into a belt dryer and dried with hot air at 55℃ for 9 hours. The hot air velocity is controlled at 1.0 m / s during the drying process. The moisture content of the dried material is 6%. The dried material is then fed into an extrusion granulator with a granulation pressure of 1.2 MPa and a die temperature of 48℃ to obtain granules with a particle size of 3 mm. After granulation, the granules are cooled at room temperature for 45 minutes and then sieved through a vibrating screen with a mesh size of 8 mesh.

[0042] S7. Finished product post-processing: The material remaining on the sieve is subjected to hardness testing; the hardness is ≥2.0 kg / cm². 2 The granules are considered qualified products; unqualified granules are crushed and returned to the extrusion granulation step for reprocessing. Qualified products are placed in a continuous ultraviolet sterilizer for 18 minutes at a sterilization intensity of 320 μW / cm². 2 The material conveying speed is 0.8 m / min. After sterilization, the product moisture content is controlled at 7%. It is packaged in aluminum foil composite bags, and nitrogen gas with a purity of ≥99.9% and an inflation pressure of 0.12 MPa is injected into the bags during packaging.

[0043] Example 2 Formula composition (by weight) The ingredients are: 8 parts microbial compound inoculant, 5 parts humic acid modifier, 40 parts well-rotted organic fertilizer, 3 parts potassium dihydrogen phosphate, 2 parts urea, 1 part trace element fertilizer, 0.5 parts polyglutamic acid, 0.3 parts chitosan quaternary ammonium salt, 0.2 parts trehalose, and 15 parts water.

[0044] The micronutrient fertilizer consists of: 0.3 parts zinc sulfate heptahydrate, 0.2 parts ferrous sulfate heptahydrate, 0.2 parts boric acid, 0.1 parts manganese sulfate, 0.05 parts ammonium molybdate, and 0.05 parts copper sulfate pentahydrate; the microbial compound inoculant contains Azotobacter chrysophagus, Bacillus megaterium, Bacillus mucilaginosus, and Bacillus subtilis in a mass ratio of 2:1:1:0.5.

[0045] Preparation process S1. Raw Material Pretreatment: The well-rotted organic fertilizer is placed in a hot air circulating drying oven and dried at 60℃ for 4 hours, controlling the moisture content to 13%. After drying, it is pulverized using a universal pulverizer, and then sieved through an 80-mesh sieve. The material exceeding the sieve is returned to the pulverizer for further pulverization, while the material passing through the sieve is collected for later use. Each component of the micronutrient fertilizer is separately ground through a 120-mesh sieve, then placed in a high-speed mixer and stirred at 300 rpm for 10 minutes until uniformly mixed.

[0046] S2. Activation of the compound microbial agent: After preparing the activation medium according to the formula ratio, adjust the pH to 6.5 and sterilize it at 121℃ and 0.1MPa for 20 min. After sterilization, cool to 30℃ and inoculate the compound microbial agent into the activation medium at an inoculation rate of 5%. After inoculation, place it in a shaking incubator and incubate at 28℃ and pH 6.5 for 12 h with shaking at a shaking speed of 180 r / min. During the incubation process, monitor the pH every 4 h. If it deviates from the range, adjust it with 1 mol / L hydrochloric acid or sodium hydroxide solution, and maintain the humidity in the shaking incubator at 40%.

[0047] S3. Preparation of the modified product: Humic acid and aminotrimethylenephosphonic acid were placed in a reaction vessel at a mass ratio of 3:1. Concentrated sulfuric acid, accounting for 2% of the total mass, was added as a catalyst at a rate of 0.5 mL / min. After the addition was complete, the temperature of the reaction vessel was raised to 85℃, and the mixture was continuously stirred at a rate of 60 r / min for 3 h. After the reaction was completed, the mixture was cooled to room temperature, and the pH was adjusted to 6.0 with 1 mol / L sodium hydroxide solution to obtain the humic acid modified product.

[0048] S4. Mixed Fermentation: The pretreated, well-rotted organic fertilizer, humic acid-modified material, and activated compound microbial agent are added to a double-layered constant-temperature fermenter. After thorough mixing, fermentation is carried out at 30℃ for 48 hours. Sterile air is introduced during fermentation, with the aeration rate adjusted in stages: 0.5 L / (L·h) in the early stage (0-24h), 0.8 L / (L·h) in the middle stage (24-48h), and 1.0 L / (L·h) in the later stage (48-72h). Stirring is performed every 8 hours for 15 minutes at a speed of 100 rpm. 12 hours after fermentation begins, 0.1 parts of cellulase and 0.05 parts of protease are added, and stirring is performed for 20 minutes to ensure uniform dispersion. The temperature and pH of the fermentation system are monitored every 4 hours. If the temperature deviates from the specified range, it is adjusted using the fermenter's jacketed temperature control system; if the pH deviates from the 6.0-7.5 range, it is adjusted using citric acid or sodium hydroxide solution.

[0049] S5. Nutrient Preparation: Add potassium dihydrogen phosphate, urea, pretreated micronutrient fertilizer, polyglutamic acid, chitosan quaternary ammonium salt, and trehalose to the fermentation product. Before adding polyglutamic acid, dissolve it in water at 30℃ to prepare a 5% solution, then add it to the fermentation product. Continue stirring at 80 rpm for 20 minutes. During stirring, adjust the pH of the system to 6.0 using a 1 mol / L citric acid solution or a 1 mol / L sodium hydroxide solution. After adjustment, keep the mixture at this temperature and let it stand for 10 minutes.

[0050] S6. Drying and Granulation: The formulated material is fed into a belt dryer and dried with hot air at 50℃ for 8 hours. The hot air velocity is controlled at 0.8 m / s during the drying process. The moisture content of the dried material is 7%. The dried material is then fed into an extrusion granulator with a granulation pressure of 1.0 MPa and a die temperature of 45℃ to obtain granules with a particle size of 2 mm. After granulation, the granules are cooled at room temperature for 30 minutes. After cooling, they are sieved through a vibrating screen with a mesh size of 6 mesh.

[0051] S7. Finished product post-processing: The material remaining on the sieve is subjected to hardness testing; the hardness is ≥2.0 kg / cm². 2 The granules are considered qualified products; unqualified granules are crushed and returned to the extrusion granulation step for reprocessing. Qualified products are placed in a continuous ultraviolet sterilizer for 15 minutes at a sterilization intensity of 300 μW / cm². 2 The material conveying speed is 0.5 m / min. After sterilization, the product moisture content is controlled at 6%. It is packaged in aluminum foil composite bags, and nitrogen gas with a purity of ≥99.9% is filled into the bags during packaging at a pressure of 0.1 MPa.

[0052] Example 3 Formula composition (by weight) The ingredients are: 12 parts microbial compound inoculant, 8 parts humic acid modifier, 50 parts well-rotted organic fertilizer, 5 parts potassium dihydrogen phosphate, 4 parts urea, 3 parts trace element fertilizer, 1.0 part polyglutamic acid, 0.6 parts chitosan quaternary ammonium salt, 0.4 parts trehalose, and 20 parts water.

[0053] The micronutrient fertilizer consists of: 0.5 parts zinc sulfate heptahydrate, 0.4 parts ferrous sulfate heptahydrate, 0.3 parts boric acid, 0.2 parts manganese sulfate, 0.1 parts ammonium molybdate, and 0.1 parts copper sulfate pentahydrate; the microbial compound inoculant contains Azotobacter chrysophagus, Bacillus megaterium, Bacillus mucilaginosus, and Bacillus subtilis in a mass ratio of 2:1:1:0.5.

[0054] Preparation process S1. Raw Material Pretreatment: The well-rotted organic fertilizer is placed in a hot air circulating drying oven and dried at 70℃ for 6 hours, controlling the moisture content to 10%. After drying, it is pulverized using a universal pulverizer, and then sieved through a 100-mesh sieve. The material passing through the sieve is returned to the pulverizer for further pulverization, while the material passing through the sieve is collected for later use. Each component of the micronutrient fertilizer is separately ground through a 150-mesh sieve, then placed in a high-speed mixer and stirred at 400 rpm for 15 minutes until uniformly mixed.

[0055] S2. Activation of the compound microbial agent: After preparing the activation medium according to the formula ratio, adjust the pH to 7.5 and sterilize it at 121℃ and 0.1MPa for 30 min. After sterilization, cool to 32℃ and inoculate the compound microbial agent into the activation medium at an inoculation rate of 8%. After inoculation, place it in a shaking incubator and incubate at 32℃ and pH 7.5 for 16 h with shaking at a shaking speed of 220 r / min. During the incubation process, monitor the pH every 4 h. If it deviates from the range, adjust it with 1 mol / L hydrochloric acid or sodium hydroxide solution, and maintain the humidity in the shaking incubator at 50%.

[0056] S3. Preparation of the modified product: Humic acid and aminotrimethylenephosphonic acid were placed in a reaction vessel at a mass ratio of 3:1. Concentrated sulfuric acid, accounting for 3% of the total mass, was added as a catalyst at a rate of 1.0 mL / min. After the addition was complete, the temperature of the reaction vessel was raised to 95℃, and the mixture was continuously stirred at a rate of 80 r / min for 4 h. After the reaction was completed, the mixture was cooled to room temperature, and the pH was adjusted to 7.0 with 1 mol / L sodium hydroxide solution to obtain the humic acid modified product.

[0057] S4. Mixed Fermentation: The pretreated, well-rotted organic fertilizer, humic acid modifier, and activated compound microbial agent are added to a double-layered constant-temperature fermenter. After thorough mixing, fermentation is carried out at 35℃ for 72 hours. Sterile air is introduced during fermentation, with the aeration rate adjusted in stages: 0.5 L / (L·h) in the early stage (0-24h), 0.8 L / (L·h) in the middle stage (24-48h), and 1.0 L / (L·h) in the later stage (48-72h). Stirring is performed every 8 hours for 20 minutes at a speed of 120 rpm. 12 hours after fermentation begins, 0.3 parts of cellulase and 0.1 parts of protease are added, and stirring is performed for 20 minutes to ensure uniform dispersion. The temperature and pH of the fermentation system are monitored every 4 hours. If the temperature deviates from the specified range, it is adjusted using the fermenter's jacketed temperature control system; if the pH deviates from the 6.0-7.5 range, it is adjusted using citric acid or sodium hydroxide solution.

[0058] S5. Nutrient Preparation: Add potassium dihydrogen phosphate, urea, pretreated micronutrient fertilizer, polyglutamic acid, chitosan quaternary ammonium salt, and trehalose to the fermentation product. Before adding polyglutamic acid, dissolve it in water at 35℃ to prepare a 10% solution, then add it to the fermentation product. Continue stirring at 100 rpm for 30 minutes. During stirring, adjust the pH of the system to 7.0 using a 1 mol / L citric acid solution or a 1 mol / L sodium hydroxide solution. After adjustment, keep the mixture at this temperature and let it stand for 15 minutes.

[0059] S6. Drying and Granulation: The formulated material is fed into a belt dryer and dried with hot air at 60℃ for 10 hours. The hot air velocity is controlled at 1.2 m / s during the drying process. The moisture content of the dried material is 5%. The dried material is then fed into an extrusion granulator with a granulation pressure of 1.5 MPa and a die temperature of 50℃ to obtain granules with a particle size of 4 mm. After granulation, the granules are cooled at room temperature for 60 minutes and then sieved through a vibrating screen with a mesh size of 10.

[0060] S7. Finished product post-processing: The material remaining on the sieve is subjected to hardness testing; the hardness is ≥2.0 kg / cm². 2 The granules are considered qualified products; unqualified granules are crushed and returned to the extrusion granulation step for reprocessing. Qualified products are placed in a continuous ultraviolet sterilizer for 20 minutes at a sterilization intensity of 350 μW / cm². 2 The material conveying speed is 1.0 m / min. After sterilization, the product moisture content is controlled at 8%. It is packaged in aluminum foil composite bags, and nitrogen gas with a purity of ≥99.9% and an inflation pressure of 0.15 MPa is injected into the bags during packaging.

[0061] Comparative Example Formula composition (by weight) 10 parts of Bacillus subtilis inoculant, 6 parts of unmodified humic acid, 45 parts of ordinary decomposed organic fertilizer, 4 parts of potassium dihydrogen phosphate, 3 parts of urea, 2 parts of conventional trace element mixture, and 20 parts of water.

[0062] The conventional trace element mixture consisted of 0.4 parts zinc sulfate heptahydrate, 0.3 parts ferrous sulfate heptahydrate, 0.25 parts boric acid, 0.15 parts manganese sulfate, 0.08 parts ammonium molybdate, and 0.07 parts copper sulfate pentahydrate; the concentration of Bacillus subtilis was 3.0 × 10⁻⁶. 9 CFU / mL.

[0063] Preparation process S1. Raw material pretreatment: Ordinary decomposed organic fertilizer is dried at 45℃ for 3 hours, with the moisture content controlled at 18%. After crushing, it is sieved through a 60-mesh sieve without further treatment. Conventional trace element mixtures are directly mixed without grinding or sieving.

[0064] S2. Inoculum treatment: No activation treatment was performed; the single Bacillus subtilis inoculum was directly mixed with other raw materials.

[0065] S3. Mixed fermentation: Put all raw materials into a regular fermentation tank, stir evenly, and ferment naturally at 28-35℃ for 48 hours. There is no aeration control. Stir once every 12 hours for 10 minutes each time. Do not monitor temperature, humidity and pH value.

[0066] S4. Nutrient formulation: Directly add potassium dihydrogen phosphate, urea, and a mixture of conventional trace elements, and stir at 60 r / min for 15 min without adjusting the pH value.

[0067] S5. Drying and granulation: The material is dried with hot air at 65℃ for 6 hours at a drying wind speed of 0.5m / s, and the moisture content after drying is 10%; a disc granulator is used for granulation, with a granulation pressure of 0.8MPa and a particle size of 1-5mm without strict control.

[0068] S6. Finished product post-processing: No hardness testing or UV sterilization steps are performed. After drying, the product is directly packaged in ordinary plastic bags without nitrogen protection.

[0069] Performance test results Table 1 Comparison of formulations and process parameters between each embodiment and the comparative example. Table 2. Performance test results of each embodiment and comparative example product. Examples 1-3 demonstrate that through the use of a four-strain microbial compound agent, modified humic acid, precise nutrient ratios, and refined process control, the products exhibit excellent performance in terms of effective viable bacteria count, nutrient content, disease resistance, and storage stability. Example 3, due to its higher concentration of functional components and more stringent process parameter control, achieves the best performance across all indicators. Although Example 2 has lower component concentrations, its complete process steps result in superior performance compared to the comparative example.

[0070] The comparative example, lacking the core technical features of this invention, used a single microbial agent and unmodified humic acid, and simplified key steps such as agent activation, fermentation control, and post-treatment, resulting in a significant reduction in the number of effective viable bacteria, insufficient nutrient content, poor disease resistance, and extremely poor storage stability. This fully demonstrates that the innovative formulation and optimized process of this invention possess significant technical advantages. The various technical features in the claims work together to achieve excellent results in terms of balanced nutrition, high viable bacteria activity, strong disease resistance, and good stability, fully meeting the growth needs of wheat seedlings.

[0071] refer to Figure 3 The bar chart illustrates the impact of different formulations and processes on the effective viable count of microbial fertilizers. Examples 1-3, employing a four-strain compound system, refined inoculant activation, and fermentation control processes, all achieved an effective viable count of 3.0 × 10⁻⁶. 8 The CFU / g count was above 30%, with Example 3 showing the highest viable count due to sufficient addition of functional ingredients and strict control of process parameters; the comparative example, using a single bacterial agent without activation treatment, had an effective viable count of only 8.0 × 10⁻⁶. 7 The CFU / g is significantly lower than that in the examples, which fully demonstrates that the formulation and process design of the present invention can effectively protect microbial activity and enhance the core efficacy of fertilizer.

[0072] refer to Figure 4 The line graph illustrates the viable bacteria stability of each product during storage. Examples 1-3, due to the addition of active protective ingredients such as trehalose and chitosan quaternary ammonium salt, combined with UV sterilization and nitrogen-filled packaging, maintained a viable bacteria retention rate of over 80% after 6 months of storage, with Example 3 exhibiting the best stability. The comparative examples, without active protection and specialized post-processing, showed a rapid decline in viable bacteria retention rate with storage time, reaching only 40% after 6 months. The line trend indicates that the optimized formulation and process control of this invention can significantly improve the storage stability of microbial fertilizers and extend their shelf life.

[0073] refer to Figure 5This radar chart comprehensively reflects the differences in overall performance among the products. Examples 1-3 all performed excellently in six dimensions: effective viable bacteria count, nutrient content, disease resistance, and storage stability, with scores of no less than 7 points in each dimension. Among them, Example 3 achieved full marks in the viable bacteria count and disease resistance dimensions, demonstrating the best overall performance. The comparative examples generally scored below 5 points in each dimension, indicating significant shortcomings. The radar chart visually demonstrates the synergistic effect of the formulation and process of this invention. Through multi-dimensional performance optimization, it achieves a comprehensive improvement in nutrient supply, disease control, and stability of microbial fertilizers, far superior to traditional products.

[0074] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing microbial fertilizer for wheat seedlings, characterized in that, The raw materials, by weight, include: 8-12 parts of microbial compound inoculant, 5-8 parts of humic acid modifier, 40-50 parts of well-rotted organic fertilizer, 3-5 parts of potassium dihydrogen phosphate, 2-4 parts of urea, 1-3 parts of trace element fertilizer, 0.5-1.0 parts of polyglutamic acid, 0.3-0.6 parts of chitosan quaternary ammonium salt, 0.2-0.4 parts of trehalose, and 15-20 parts of water; The steps are as follows: S1. Raw material pretreatment: Place the decomposed organic fertilizer in a hot air circulating drying box and dry it at 60-70℃ for 4-6 hours. After drying, crush it and then sieve it through an 80-100 mesh screen. The material on the screen is returned to the crusher for re-crushing, and the material under the screen is collected for later use. S2. Activation of compound microbial agent: Inoculate the compound microbial agent into the activation medium at an inoculation rate of 5-8%, adjust the pH of the medium to 6.5-7.5, sterilize, cool to 30-32℃ and then inoculate; after inoculation, incubate with shaking at 28-32℃ and pH 6.5-7.5 for 12-16 hours. S3. Preparation of modified product: Humic acid and aminotrimethylenephosphonic acid are mixed at a mass ratio of 3:1, and concentrated sulfuric acid accounting for 2-3% of the total mass is added dropwise as a catalyst. The mixture is reacted at 85-95℃ for 3-4 hours. After the reaction is completed, the mixture is cooled to room temperature, and the pH value is adjusted to 6.0-7.0 with 1mol / L sodium hydroxide solution to obtain the humic acid modified product. S4. Mixed fermentation: The pretreated decomposed organic fertilizer, humic acid modified material, and activated compound microbial agent are put into a double-layer constant temperature fermentation tank, stirred evenly, and fermented at 30-35℃ for 48-72 hours. Sterile air is introduced during the fermentation process. S5. Nutrient formulation: Add potassium dihydrogen phosphate, urea, pretreated trace element fertilizer, polyglutamic acid, chitosan quaternary ammonium salt, and trehalose to the fermentation product, stir for 20-30 minutes, adjust the pH of the system to 6.0-7.0 during stirring, and keep warm and stand for 10-15 minutes after adjustment. S6. Drying and granulation: The prepared material is fed into a belt dryer and dried with hot air at 50-60℃ for 8-10 hours; the dried material is fed into an extrusion granulator to obtain granules with a particle size of 2-4mm; after granulation, the granules are placed at room temperature to cool for 30-60 minutes, and after cooling, they are screened by a vibrating sieve with a screen mesh of 6-10 mesh. S7. Finished product post-processing: The material remaining on the sieve is subjected to hardness testing; the hardness is ≥2.0 kg / cm². 2 The granules are qualified products. Unqualified granules are crushed and returned to the extrusion granulation step for reprocessing. Qualified products are treated with ultraviolet sterilization and then sealed and packaged.

2. The method for preparing wheat seedling microbial fertilizer according to claim 1, characterized in that, The nitrogen-fixing bacteria are *Azotobacter chrysodonta*; the phosphate-solubilizing bacteria are *Bacillus megaterium*; the potassium-solubilizing bacteria are *Bacillus mucilaginosus*; and the growth-promoting bacteria are *Bacillus subtilis*.

3. The method for preparing wheat seedling microbial fertilizer according to claim 1, characterized in that, The activation medium also contains 0.1-0.2 g / L of vitamin B1 and 0.05-0.1 g / L of vitamin C. After the medium is sterilized, it is cooled to 30-32°C under aseptic conditions before inoculation. During the incubation process after inoculation, the humidity in the shaking incubator is maintained at 40-50%.

4. The method for preparing wheat seedling microbial fertilizer according to claim 1, characterized in that, Micronutrient fertilizers include 0.3-0.5 parts zinc sulfate heptahydrate, 0.2-0.4 parts ferrous sulfate heptahydrate, 0.2-0.3 parts boric acid, 0.1-0.2 parts manganese sulfate, 0.05-0.1 parts ammonium molybdate, and 0.05-0.1 parts copper sulfate pentahydrate. After grinding, it is sieved through a 120-150 mesh sieve.

5. The method for preparing wheat seedling microbial fertilizer according to claim 1, characterized in that, In step S4, 0.1-0.3 parts of cellulase and 0.05-0.1 parts of protease are added during the fermentation process, 12 hours after the start of fermentation.

6. The method for preparing wheat seedling microbial fertilizer according to claim 1, characterized in that, The fermentation preparation process of polyglutamic acid is as follows: Bacillus subtilis is used as the production strain, and the fermentation medium includes glucose 20-25 g / L, corn steep liquor 10-15 g / L, ammonium sulfate 5-8 g / L, potassium dihydrogen phosphate 1-2 g / L, magnesium sulfate 0.5-1.0 g / L, and calcium chloride 0.1-0.2 g / L. The pH value is adjusted to 6.8-7.2, the fermentation temperature is 30-32℃, the fermentation time is 48-56 h, the aeration rate during fermentation is 0.6-0.8 L / (L・h), and the stirring speed is 150-180 r / min. After fermentation, the polyglutamic acid product is obtained by centrifugation, ultrafiltration, and freeze drying.

7. The method for preparing wheat seedling microbial fertilizer according to claim 1, characterized in that, In step S7, a continuous UV sterilization machine is used for UV sterilization. The material conveying speed during sterilization is 0.5-1.0 m / min. The moisture content of the product after sterilization is controlled at 6-8%. The packaging uses aluminum foil composite bags, and nitrogen is filled into the bags during packaging.

8. A method for detecting wheat seedling microbial fertilizer, used to detect fertilizer prepared by the method for preparing wheat seedling microbial fertilizer according to any one of claims 1-7, characterized in that, Includes the following steps: S1. Sample Preparation: Take 5.0g of fertilizer sample, accurate to 0.001g, and place it in a sterile Erlenmeyer flask. Add 45mL of sterile water at 25-28℃. Place the Erlenmeyer flask in a constant temperature shaking incubator and shake for 30min at 200-250r / min and 25-28℃ to prepare a 1:10 sample suspension. Dilute the sample suspension stepwise with sterile water at a dilution gradient of 10. -1 10 -2 10 -3 10 -4 10 -5 10 -6 After each dilution, let it stand for 5 minutes before proceeding to the next dilution. S2. Effective viable bacteria count test: Take 10... -4 10 -5 10 -6 Three dilutions of sample suspension, 0.1 mL each, were evenly spread onto selective medium plates, with three replicates for each concentration. The selective medium was prepared by mixing LB medium and inorganic phosphorus medium at a volume ratio of 1:1, adjusting the pH to 6.8-7.2, and sterilizing at 121℃ and 0.1 MPa for 20 min. When the medium cooled to 50-60℃, 50 μg / mL of ampicillin was added as a screening agent, and the mixture was shaken well before being poured into plates. The plates were then incubated at 28-32℃ for 48-72 h. S3. Nutrient content detection: Take 1.0g of sample, accurate to 0.001g, put it into a digestion tube, add 5mL of concentrated sulfuric acid, shake well and let stand overnight; the next day, place the digestion tube on a digestion furnace, first carbonize at 150-200℃ for 30min, then heat to 360-400℃ for 1h, cool to room temperature, add 2mL of 30% hydrogen peroxide solution, continue to heat and digest for 30min. If the solution is still not clear, repeat adding hydrogen peroxide solution and digesting until the solution is clear; after cooling, transfer the digestion solution to a 100mL volumetric flask, dilute to the mark with distilled water, shake well and set aside; The total nitrogen content was determined using the Kjeldahl method: 10 mL of digest was placed in a Kjeldahl distillation apparatus, 10 mL of 40% sodium hydroxide solution was added, and the mixture was distilled for 5 min. The distillate was absorbed with 2% boric acid solution, and titrated to the endpoint with 0.05 mol / L hydrochloric acid standard solution. The volume of hydrochloric acid standard solution consumed was recorded, and the total nitrogen content was calculated. The hydrochloric acid standard solution was calibrated with borax. The total phosphorus content was determined using the molybdenum-antimony colorimetric method: 5 mL of digested solution was placed in a 50 mL volumetric flask, 10 mL of molybdenum-antimony colorimetric reagent was added, and the solution was diluted to the mark with distilled water. After shaking well, the solution was incubated at 25-30℃ for 30 min. The absorbance was measured at 700 nm using a spectrophotometer, and the total phosphorus content was calculated based on the standard curve. The molybdenum-antimony colorimetric reagent was prepared by mixing ammonium molybdate solution, sulfuric acid solution, and potassium antimony tartaric acid solution in a volume ratio of 1:2:0.5, and should be used immediately after preparation. The total potassium content was determined by flame photometry: 5 mL of digestion solution was placed in a 50 mL volumetric flask, diluted to the mark with distilled water, shaken well and then introduced into a flame photometer. A standard curve was plotted using potassium standard solution, the absorbance of the sample was measured and the total potassium content was calculated. S4. Heavy metal detection: Take 2.0 g of sample, accurate to 0.001 g, and place it in a microwave digestion vessel. Add 8 mL of nitric acid and 2 mL of hydrogen peroxide, shake well, and let stand for 15 min. Place the digestion vessel in a microwave digester and digest according to the following procedure: heat to 120℃ for 5 min and hold for 5 min; then heat to 180℃ for 5 min and hold for 15-20 min. After digestion, cool to room temperature, transfer the digest to a 50 mL volumetric flask, dilute to the mark with distilled water, shake well, and set aside. Perform a blank test at the same time. S5. Result Interpretation: Effective viable bacteria count ≥ 2.0 × 10⁻⁶ 8 If the CFU / g, total nitrogen content ≥3.0%, total phosphorus content ≥2.0%, total potassium content ≥2.0%, and heavy metal content meet the requirements of GB / T23349-2020 standard, then the product is deemed qualified.

9. The method for detecting microbial fertilizer in wheat seedlings according to claim 8, characterized in that, In step S2, the selective culture medium plates are prepared by pouring 15-20 mL of culture medium into each plate. After the culture medium solidifies, it is inverted and incubated at 37°C for 2 hours. After confirming that no colonies are growing, it is then spread. When counting colonies, a colony counter is used, and abnormally shaped colonies are excluded during counting.

10. The method for detecting microbial fertilizer in wheat seedlings according to claim 8, characterized in that, In step S3, the distillation apparatus for total nitrogen detection needs to be cleaned three times with distilled water beforehand; the spectrophotometer for total phosphorus detection needs to be preheated for 30 minutes, and the absorbance of the blank solution should be ≤0.005; the concentration gradient of the potassium standard solution for total potassium detection is 0, 2, 4, 6, 8, and 10 μg / mL, and the correlation coefficient R of the standard curve is... 2 ≥0.999; The power of the microwave digester in step S4 is 600-800W, and the digestion vessel is made of polytetrafluoroethylene. Before use, it should be soaked in nitric acid solution for 24 hours, cleaned and dried before use.

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