Streptomyces fradiae, microbial inoculum, slow-release fertilizer, preparation method and application thereof

Abstract

The application discloses a kind of Streptomyces calvus, bacterial agent, slow-release fertilizer and its preparation method and application, belong to microbial technical field.Streptomyces calvus Strain GX2024L1 is isolated from soil, and the preservation number is CGMCC No.32902.The strain and its bacterial agent can be used for preparing slow-release fertilizer.When preparing slow-release fertilizer, silkworm sand is pretreated first, and the surface area of silkworm sand is increased by crushing, then Streptomyces calvus Strain GX2024L1 or its bacterial agent is inoculated for controlled fermentation, and the cellulose, hemicellulose and polysaccharide in silkworm sand are moderately degraded by microbial metabolism, so as to form a porous carrier and expose functional groups while maintaining the structural skeleton.The carrier is used to adsorb and load urea and urease inhibitor through hydrogen bonding and coordination, and slow-release fertilizer is prepared by granulation and coating.The slow-release fertilizer can realize slow release and continuous release of urea within 1-8 months, effectively inhibit nitrogen loss by using urease inhibitor, realize the dual benefits of silkworm sand resource utilization and precise supply of nutrients in the whole cycle of crops.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a Streptomyces nakedus, a microbial agent, a slow-release fertilizer, its preparation method, and its application. Background Technology

[0002] Nitrogen is a core nutrient for ensuring crop yield and quality, but the utilization rate of traditional nitrogen fertilizers is generally low. Nitrogen is easily lost rapidly through ammonia volatilization, nitrification-leaching, and denitrification, which not only wastes resources and increases agricultural production costs, but also exacerbates non-point source pollution and greenhouse gas emissions. Therefore, the development of efficient slow-release nitrogen fertilizers is crucial for sustainable agricultural development.

[0003] my country's main silkworm-producing areas generate a large amount of silkworm excrement annually, which is rich in organic matter and natural organic pores, making it a potential fertilizer carrier material. However, the dry basis of silkworm excrement is mainly composed of a dense lignocellulose complex consisting of cellulose (approximately 15%-30%), lignin (approximately 10%-20%), hemicellulose (approximately 10%-25%), and polysaccharides (approximately 10-15%), severely limiting its adsorption performance. Traditional fermentation processes are inefficient and unstable, making it difficult to achieve efficient nitrogen loading and controlled release. Therefore, improving the carrier performance of silkworm excrement through targeted technologies is a key breakthrough for realizing the high-value utilization of agricultural waste. Summary of the Invention

[0004] The purpose of this invention is to provide *Streptomyces nakedatus*, its inoculum, slow-release fertilizer, its preparation method, and its applications. The genome of this strain contains a complete cellulase and xylanase synthesis system, encompassing functional genes such as celA, celB, celC, xynA, and xynB. In particular, the efficient expression of the celB gene endows this strain with excellent cellulose depolymerization ability, not only efficiently hydrolyzing cellulose into cellobiose but also effectively untangling the physical entanglement of cellulose with hemicellulose in plant cell walls. This creates favorable conditions for xylanase to fully contact and degrade hemicellulose, thereby enabling the strain to possess highly efficient synergistic degradation characteristics of cellulose-hemicellulose complex substrates.

[0005] When this strain is applied to silkworm excrement treatment, its cellulase and xylanase work synergistically to achieve efficient degradation of the cellulose-hemicellulose complex substrate in the excrement, while simultaneously converting other polysaccharide components. This multi-enzyme synergistic degradation mechanism is highly compatible with the composition of silkworm excrement, which is mainly composed of cellulose, hemicellulose, and polysaccharides, and can significantly improve the porosity and specific surface area of ​​the treated carrier. The porous, highly active surface formed after degradation exposes abundant functional groups such as hydroxyl and carboxyl groups. These functional groups can strongly adsorb and fix nitrogen through coordination and electrostatic interactions, ultimately producing nitrogen fertilizer with excellent slow-release properties, fundamentally improving nitrogen utilization efficiency.

[0006] The solution of the present invention is specifically as follows:

[0007] A Streptomyces calvus strain GX2024L1, its taxonomic designation is: Streptomyces calvus GX2024L1, its Chinese taxonomic designation is: 秃裸链霉菌GX2024L1, its deposit number is CGMCC No.32902. The strain is deposited in the China General Microbiological Culture Collection Center, address: No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China. The deposit date is December 4, 2024. The base pair sequence of the strain is SEQ ID NO.1:

[0008]

[0009] The present invention also includes a bacterial agent containing Streptomyces calvus strain GX2024L1.

[0010] The present invention also includes the application of the aforementioned Streptomyces calvus strain GX2024L1 or its inoculum in the preparation of slow-release fertilizers.

[0011] The present invention also includes slow-release fertilizers containing the aforementioned Streptomyces calvus strain GX2024L1 or its inoculum.

[0012] The method for preparing the slow-release fertilizer of the present invention includes the following steps:

[0013] (1) Fresh silkworm excrement processing: collect fresh silkworm excrement, remove impurities, and dry it to obtain raw silkworm excrement for later use;

[0014] (2) Pretreatment of silkworm excrement: The original silkworm excrement is pressurized in an ultra-high pressure reactor to increase the specific surface area of ​​the original silkworm excrement and obtain pretreated silkworm excrement.

[0015] (3) Mixed fermentation: The pretreated silkworm excrement from step (2) is inoculated with Streptomyces calvus strain GX2024L1 or bacterial agent at a mass ratio of 0.5%-1.5% for fermentation. During fermentation, pay attention to stirring. Fermentation takes 15-18 days.

[0016] (4) Adsorb nitrogen fertilizer and urease inhibitor: The silkworm excrement material fermented in step (3) is crushed and a mixed solution of urease inhibitor (DMPP) and urea is added at a solid-liquid ratio of 1:4-5 and stirred for adsorption.

[0017] (5) Granulation and coating: Mix the material treated in step (4) with the binder, granulate and coat to obtain slow-release fertilizer.

[0018] Furthermore, in step (2), the pretreatment of silkworm excrement involves adding silkworm excrement to an ultra-high pressure reactor and then performing a three-stage pressurization process. First, the pressure is increased to 150-200 MPa and held for 30-60 seconds, followed by 3-4 pulse depressurizations. Then, the pressure is increased to 350-400 MPa and held for 30-60 seconds, followed by 3-4 pulse depressurizations. Finally, the pressure is increased to 550-600 MPa and held for 30-60 seconds before depressurization is performed to complete the pressurization process.

[0019] Furthermore, in step (4), the mixed solution of urease inhibitor and urea is prepared by first preparing a urea solution with a mass fraction of 20%-40%, and then adding 1% of the mass of urea urease inhibitor to the solution and mixing it evenly.

[0020] The slow-release fertilizer prepared by this invention can be used in crop cultivation.

[0021] The mechanism of action of this invention: Silkworm excrement, the digestive residue excreted by silkworms after consuming mulberry leaves, is mainly composed of lignin, cellulose, hemicellulose, and polysaccharides. These high molecular weight compounds are intertwined, resulting in fewer exposed oxygen-containing groups in the excrement, which is not conducive to forming strong adsorption forces with urea and urease inhibitors and increasing their adsorption capacity. To address the above problems, this application first uses dynamic ultra-high pressure pretreatment to pulverize the silkworm excrement from the inside out, fully exposing the outer surface of the excrement, thereby enhancing the loading of the microbial agent. Subsequently, controlled fermentation is carried out using Streptomyces calvus strain GX2024L1, a cellulase system that can target and destroy the aforementioned cellulose and polysaccharides, breaking the intertwined polymer chains, thereby exposing a large number of oxygen-containing groups in the excrement and forming diffusion channels within the excrement, thus forming an ideal carrier for urea and urease inhibitors. This carrier achieves physical retention through an interconnected microporous-mesoporous system and strongly immobilizes urea and urease inhibitors through chemical interactions such as hydrogen bonding using numerous exposed active functional groups on its surface, thus achieving a high capacity loading of urea and urease inhibitors. Furthermore, by coating the silkworm excrement carrier with a polyurethane membrane of controllable thickness, the controlled release of urea and urease inhibitors can be achieved. Moreover, the slow release of urease inhibitors into the soil effectively inhibits the conversion of ammonium nitrogen to nitrate nitrogen, thereby achieving efficient utilization of urea.

[0022] Compared with the prior art, the advantages of the present invention are:

[0023] 1. This invention utilizes the presence of five enzyme genes simultaneously in the cells of *Streptomyces calvus* strain GX2024L1: cellulase genes (celA, celB, celC) and xylanase genes (xynA, xynB). Among these, the celB gene exhibits the highest relative expression level, reaching 6.18. Liquid fermentation of this strain was performed, and its filter paper enzyme activity and xylanase activity were measured. Solid-state fermentation using silkworm excrement as a substrate was also conducted, and the degradation rates of cellulose and hemicellulose were measured. The results showed that the expression level of the celB gene in this strain was highly positively correlated with its cellulase activity and cellulose degradation rate; similarly, the expression level of the xynA gene was highly positively correlated with its xylanase activity and hemicellulose degradation rate. Both findings indicate that this strain possesses excellent enzymatic hydrolysis capabilities for cellulose and hemicellulose.

[0024] 2. This invention utilizes Streptomyces calvus strain GX2024L1 to carry out controlled fermentation of silkworm excrement. By using its secreted cellulase system, it targets and destroys the cellulose and polysaccharides in the silkworm excrement, breaking down the entangled polymer chains, thereby exposing a large number of oxygen-containing groups in the silkworm excrement and forming diffusion channels within the silkworm excrement, which serve as carriers for urea and urease inhibitors.

[0025] 3. This invention employs Dynamic Ultra-High Pressure-Pulse Decompression Technology (D-UHP) to pretreat silkworm excrement, thereby breaking it down, increasing its specific surface area and porosity, and enhancing the adsorption performance of the carrier. By utilizing the synergistic effect of urease inhibitor (DMPP) treatment and bacterial fermentation, the carrier is fundamentally modified, making the silkworm excrement material loose and porous with a high specific surface area adsorption structure. Infrared spectroscopy analysis confirmed that the characteristic peaks of active groups such as hydroxyl (-OH) in the silkworm excrement were significantly enhanced after the synergistic treatment, indicating a substantial increase in its surface chemisorption sites.

[0026] 4. The D-UHP / Streptomyces nakedus fermented silkworm excrement prepared by the present invention has a swelling rate of 4.81, which is 1.31 times higher than that of single D-UHP / silkworm excrement (3.68) and original silkworm excrement (2.43).

[0027] 5. The slow-release fertilizer prepared by this invention achieves precise and controllable nutrient release, significantly improving utilization. It solves the problems of rapid initial dissolution and insufficient supply in the later stages associated with traditional nitrogen fertilizers or simply processed silkworm excrement fertilizers. In both static water release and soil release experiments, the slow-release fertilizer of this invention exhibits a stable and sustained release curve. Its cumulative release rate throughout the entire test period is significantly lower than that of ordinary coated nitrogen fertilizers and simply treated silkworm excrement-based nitrogen fertilizers, effectively verifying its "slow initial release, continuous release in the middle and later stages" release pattern, which can match the nutrient requirements of crops throughout their entire growth period, thereby reducing loss and improving utilization.

[0028] 6. The slow-release fertilizer prepared by this invention not only provides a slow-release nitrogen source, but its urease inhibitor (DMPP) can effectively regulate soil nitrogen transformation. Adding DMPP significantly slows the conversion of ammonium nitrogen to nitrate nitrogen, which is beneficial for nitrogen retention in the soil and reduces the risk of loss and pollution. In pot experiments, sugarcane plants treated with the slow-release fertilizer of this invention showed significantly better growth than the control group, comprehensively verifying its advantages in promoting crop growth and improving agronomic efficiency. It has the dual benefits of nutrient enhancement and soil ecological regulation.

[0029] 7. Existing technologies often rely on complex microbial agents or raw material ratios, making cost control difficult. This invention uses silkworm excrement as a single raw material and can achieve efficient transformation by inoculating a single functional strain (Streptomyces calvus strain GX2024L1), providing convenience for the large-scale, standardized resource utilization of silkworm excrement. Attached Figure Description

[0030] Figure 1 This is a morphological analysis diagram of Streptomyces calvus strain GX2024L1 on culture medium; among which... Figure 1 (a) is a plate colony diagram. Figure 1 (b) is a microscopic morphology diagram.

[0031] Figure 2 This is a diagram illustrating the growth process of Streptomyces calvus strain GX2024L1; Figure 2 (a) is a graph showing the trend of OD value changes during the growth process. Figure 2 (b) is a graph showing the pH changes during the growth process.

[0032] Figure 3 These are the slow-release fertilizer granules prepared in Example 2.

[0033] Figure 4 These are infrared spectra of silkworm excrement materials treated with different methods.

[0034] Figure 5 These are infrared spectrum peak fitting diagrams (hydroxyl characteristic regions) of silkworm excrement materials treated with different methods.

[0035] Figure 6 These are scanning electron microscope (SEM) images of samples prepared at different stages in Example 2, wherein... Figure 6 (a) represents the original silkworm excrement. Figure 6 (b) is D-UHP / silkworm excrement; Figure 6 (c) D-UHP / Streptomyces nakedus fermented silkworm excrement; Figure 6 (d) D-UHP / Streptomyces baldii fermentation of silkworm excrement adsorbs nitrogen fertilizer and urease inhibitor; Figure 6 (e) Finished product of long-acting slow-release nitrogen fertilizer based on silkworm excrement.

[0036] Figure 7 This is a comparison chart of the swelling rates of silkworm excrement materials treated with different methods.

[0037] Figure 8 This is a graph showing the nutrient static water release rate of nitrogen fertilizer products obtained by different preparation methods.

[0038] Figure 9 This is a graph showing the nutrient release rate of nitrogen fertilizer products obtained by different preparation methods in the soil.

[0039] Figure 10 is a graph showing the release pattern of urea by the slow-release fertilizer prepared in Example 2 at different periods.

[0040] Figure 11 is a comparison graph of the influence of urease inhibitors on soil nitrogen forms at different periods, wherein Figure 11 (a) is Experimental Group 1, Figure 11 (b) is Experimental Group 2.

[0041] Figure 12 is a control graph of the influence of different slow-release fertilizer products on sugarcane growth.

[0042] Biological Material Deposit Information

[0043] The information of the strain deposited in the present application is as follows: Streptomyces calvus strain GX2024L1, whose taxonomic designation is Streptomyces calvus GX2024L1, and its Chinese taxonomic designation is 秃裸链霉菌GX2024L1, with the deposit number CGMCC No.32902. The strain is deposited in China General Microbiological Culture Collection Center, address: No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China. The deposit date is December 4, 2024. Detailed Description of the Embodiments

[0044] The present invention will be further described below with reference to the accompanying drawings and examples, but the scope claimed by the present invention is not limited to the scope protected by the examples.

[0045] Example 1

[0046] Isolation, screening, identification and culture of the strain, specifically comprising:

[0047] 1. Isolation of the strain

[0048] (1) From a soil sample collected from the experimental base in Laobaimian Village, Liangjiang Town, Laibin City, Guangxi Zhuang Autonomous Region, weigh 5 g of the soil sample and place it in a sterilized Erlenmeyer flask, add 50 mL of sterilized water, shake gently at 180 rpm for 30 min at room temperature, allow to stand for precipitation, take the supernatant and dilute it to 10 -5 -10 -8 fold to prepare a bacterial cell suspension.

[0049] (2) Take 0.1 mL of an appropriately diluted bacterial suspension and spread it onto a sterile solid culture medium plate. The medium consists of: 5 g / L yeast extract, 10 g / L trypsin, 1.0 g / L KNO3, 10 g / L NaCl, 0.5 g / L MgSO4, 0.5 g / L K2HPO4, 0.03 g / L FeSO4, and 20 g / L agar. The medium is prepared by autoclaving at 121°C for 20 minutes and then inverting the plate. Place the plate upside down in a 30°C incubator and incubate in the dark for 2-4 days.

[0050] (3) Select single colonies that are dry on the surface, wrinkled, and may produce white or grayish-white spores, and perform streak isolation to obtain 6 strains.

[0051] 2. Screening of strains

[0052] (1) The obtained single bacteria were inoculated into test tubes containing liquid culture medium using a disposable inoculation loop. The liquid culture medium consisted of: 5 g / L yeast extract, 10 g / L tryptone, 1.0 g / L KNO3, 10 g / L NaCl, 0.5 g / L MgSO4, 0.5 g / L K2HPO4, and 0.03 g / L FeSO4. The culture medium was dissolved in deionized water and the pH was adjusted to neutral. The culture was then autoclaved at 121°C for 20 minutes before use. The bacteria were cultured in a constant temperature shaking incubator at 30°C and 180 r / min until OD reached... 600 The range was 0.8–1.0. The cell density was adjusted to 1 × 10⁻⁶ using sterile saline. 5 Prepare a homogeneous seed solution at CFU / mL for later use.

[0053] (2) Extract genomic DNA from 1 mL of seed culture for PCR verification. Use the primers listed in Table 1 to amplify cellulase genes (celA, celB, celC) and xylanase genes (xynA, xynB), respectively. Detect specific bands by agarose gel electrophoresis to confirm the presence of functional genes.

[0054] The PCR amplification reaction system included: 0.3 μM forward primer (F), 0.3 μM reverse primer (R), 10 ng template DNA, 1×SYBR premix Ex Taq, and BSA at a final concentration of 0.1 mg / mL. The amplification reaction program was as follows: 95℃, 5 min; 95℃, 30 s; 56℃, 90 s; 72℃, 60 s (33 cycles); 72℃, 10 min; 4℃.

[0055] Table 1. Primers and sequence listing corresponding to cellulase / xylanase genes

[0056]

[0057] Six isolated bacterial strains were subjected to liquid fermentation. Total RNA was extracted from the logarithmic-phase cells and reverse transcribed into cDNA. Using 16S rDNA (primers shown in Table 1, SEQ ID NO: 12-13) as an internal control, the relative expression levels of five key enzyme genes were detected by qRT-PCR. The results are shown in Table 2.

[0058] Table 2. Relative expression levels of enzyme genes in 6 bacterial strains

[0059]

[0060] Relative expression level of pqq gene = X1 / X0

[0061] Where X1 is the expression level of a certain gene in the pqq gene cluster, and X0 is the expression level of the 16S rDNA gene.

[0062] As shown in Table 2, compared with other strains, the cells of strain GX2024L1 contain five enzyme genes: celA, celB, celC, xynA, and xynB. Among them, the relative expression level of the celB gene is the highest, reaching 6.18.

[0063] To verify the functionality of gene expression, the filter paper enzyme activity and xylanase activity of the strain were measured after liquid fermentation, and the degradation rate of cellulose and hemicellulose was measured using silkworm excrement as a substrate for solid-state fermentation. Pearson correlation analysis was performed on the above enzyme activity and degradation rate data with the relative expression levels of five key enzyme genes of each strain (Table 2) to obtain the correlation coefficient between gene expression and phenotypic function. The results are shown in Table 3.

[0064] Table 3. Correlation between relative expression levels of enzyme genes and enzyme activity and degradation capacity.

[0065]

[0066] As shown in Table 3, the expression level of the celB gene in strain GX2024L1 is highly positively correlated with its cellulase activity and cellulose degradation rate; while the expression level of the xynA gene is highly positively correlated with its xylanase activity and hemicellulose degradation rate.

[0067] Therefore, based on comprehensive judgment, the screened strain GX2024L1 possesses enzyme activity for degrading cellulose. Morphological observation of strain GX2024L1 revealed single colonies with a "dry surface, wrinkled appearance, and possible production of white or grayish-white spores," consistent with the characteristics of Streptomyces colonies: dry, rough, and producing chain-like spores. Combining the above molecular identification with 16S rDNA sequencing, strain GX2024L1 was confirmed as *Streptomyces calvus*, and named *Streptomyces calvus* strain GX2024L1.

[0068] 3. Morphology of the strain:

[0069] (1) Morphological observation: The growth status of Streptomyces calvus strain GX2024L1 was observed (e.g. Figure 1 , Figure 2 (As shown).

[0070] Figure 1 This is a morphological diagram of Streptomyces calvus strain GX2024L1 on culture medium. Figure 1 (a) is a plate colony diagram. Figure 1 (b) is a microscopic morphological image, which clearly shows the morphological characteristics of the strain: Figure 1 (a) The colonies of the strain in the plate are opaque and white. The colonies are raised, dense, dry and wrinkled, covered with white velvet, and tightly bound to the culture medium, making them difficult to pick up. Figure 1 (b) Under the microscope, the hyphae of this strain are interwoven, the hyphae are slightly loose and have fine wrinkles, and fine granular structures can be observed between the hyphae.

[0071] Figure 2 This is a diagram illustrating the growth process of Streptomyces calvus strain GX2024L1. (Source: [Original Source Name]) Figure 2 (a) OD value: The OD value rises rapidly from 0 to about 1.5 in 0-3 days (reaching its peak at 3 days), indicating that this stage is the logarithmic growth phase, the strain reproduces quickly, and the cell density continues to increase; the OD value drops slightly from its peak in 3-5 days (and eventually remains at about 1.0), indicating that the cells have entered the stationary phase / mild decline phase, the growth rate slows down, and some cells may experience a slight drop in density due to nutrient consumption and accumulation of metabolic products.

[0072] Figure 2(b) (pH): 0-2 days: pH rises rapidly from 5 to 8, synchronous with the rapid rise of OD value, indicating that during the logarithmic growth phase, Streptomyces decomposes nitrogen-containing substrates (such as proteins and amino acids) to produce ammonia-like alkaline metabolites, resulting in a significant increase in environmental pH; 2-5 days: pH continues to rise slowly to close to 9. At this time, OD has entered the stable period, indicating that although the growth of the bacteria slows down, the metabolism of nitrogen-containing substances continues, alkaline products continue to accumulate, and pH maintains an upward trend.

[0073] 4. Preservation of bacterial strains

[0074] Streptomyces calvus strain GX2024L1, isolated from soil samples from the experimental base in Laobaimian Village, Liangjiang Town, Laibin City, Guangxi Zhuang Autonomous Region, has been deposited at the China Culture Collection Center for Microbial Cultures, with accession number CGMCC No. 32902.

[0075] The base pair sequence is SEQ ID NO.1:

[0076]

[0077] 5. Preparation of microbial agents

[0078] Preparation of liquid inoculum: The preserved Streptomyces calvus GX2024L1 strain was directly inoculated into the liquid culture medium used in the screening stage for activation culture. The liquid culture medium consisted of: 5 g / L yeast extract, 10 g / L tryptone, 1.0 g / L KNO3, 10 g / L NaCl, 0.5 g / L MgSO4, 0.5 g / L K2HPO4, and 0.03 g / L FeSO4. The solution was dissolved in deionized water and the pH was adjusted to neutral. After autoclaving at 121℃ for 20 minutes, the culture was used. The culture was then incubated at 30℃ and 180 r / min for 24 h with shaking to obtain the seed culture. 1 mL of the seed culture was then inoculated into 500 mL of the above liquid culture medium and incubated at 30℃ and 180 r / min with shaking for 2-3 days to achieve a concentration of 1×10⁻⁶. 8 When the concentration of CFU / mL is above a certain level, a liquid bacterial agent containing Streptomyces calvus strain GX2024L1 is obtained.

[0079] Preparation of solid bacterial agent: Add sterile corn starch as an adsorption carrier to the above liquid bacterial agent, stir and mix thoroughly, and then place it in a vacuum freeze dryer and dry continuously at -35℃ for 72 h to obtain solid bacterial agent.

[0080] Example 2

[0081] A method for preparing slow-release fertilizer using Streptomyces calvus strain GX2024L1 includes the following steps:

[0082] 1. Raw material collection

[0083] Local silkworm farms collect fresh silkworm excrement (containing a small amount of quicklime and mulberry branch and leaf residues). First, they screen the excrement to remove visible large impurities such as mulberry branch fragments and pebbles. Then, the cleaned excrement is evenly spread on a clean cement floor, with a thickness of 5 cm, and exposed to the sun for 1 day. During this period, it is turned over 2-3 times a day to initially remove some moisture, reducing the moisture content of the excrement to 25%-30%, which is recorded as: original silkworm excrement.

[0084] 2. Pretreatment of silkworm excrement (Dynamic Ultra-High Pressure - Pulse Decompression Technology D-UHP)

[0085] The raw silkworm excrement was added to an ultra-high pressure reactor and then subjected to a three-stage pressurization process. First, the pressure was increased to 200 MPa and held for 30 seconds, followed by three pulse depressurizations at a rate of 100 MPa / s, each lasting 0.5 seconds. Then, the pressure was increased to 400 MPa and held for 30 seconds, followed by three pulse depressurizations at a rate of 100 MPa / s, each lasting 0.5 seconds. Finally, the pressure was increased to 600 MPa and held for 30 seconds before depressurization. The final product was a crushed material with a particle size of 0.5-1 mm, denoted as D-UHP / silkworm excrement.

[0086] 3. Mixed fermentation

[0087] Weigh 20kg of pretreated silkworm excrement from step (2) and transfer it to a sterile mixing device. Inoculate the cultured Streptomyces calvus strain GX2024L1 bacterial agent (i.e., 200g of bacterial agent) at a ratio of 1% of the pretreated silkworm excrement mass. Stir thoroughly for 20 minutes to ensure uniform contact between the bacterial agent and the silkworm excrement particles, forming a fermentation substrate. Then, place it in a fermentation chamber for fermentation. The specific operation is as follows:

[0088] (1) Fermentation tank filling: The inoculated fermentation substrate is filled into a controllable stainless steel fermentation tank, and the filling amount is 65% of the tank volume to avoid the material being too compacted and affecting the aeration.

[0089] (2) Environmental parameter control: The fermentation box is equipped with automatic ventilation, mechanical turning and temperature control. The initial fermentation temperature is 28℃. The mechanical turning device is started once every 24 hours, and the turning depth reaches the bottom of the material to ensure uniform oxygen supply. The pH value of the material is sampled and measured daily, and the pH fluctuation range is controlled within 5.0-6.0.

[0090] (3) Fermentation process monitoring: During the fermentation period, various parameters are recorded in real time. When the material temperature rises to 55℃, it is maintained for 3 days (high temperature composting stage). Then the temperature is gradually reduced and the fermentation continues for 15-18 days until the material temperature stabilizes at 32-35℃ and is maintained for 48 hours. The fermentation endpoint is judged as follows: when the material is uniformly dark brown, loose and easy to disperse, without hard core, without ammonia smell or odor, and the temperature drops below 35℃ and the pH value stabilizes at 5.0-6.0, the fermentation is judged to be complete and recorded as: D-UHP / Bare Streptomyces fermented silkworm excrement.

[0091] 4. Adsorbs nitrogen fertilizer and urease inhibitors

[0092] (1) Pretreatment of fermentation products: Take out the fermented material and dry it in a 60℃ oven until the moisture content is below 15% to remove impurities for later use.

[0093] (2) Preparation of urea solution containing urease inhibitor (DMPP): Weigh 5 kg of urea (prepared at a mass fraction of 30%), add 50 g of DMPP (1% of the mass of urea), add 11.6 L of deionized water, stir thoroughly to dissolve, and obtain a urea-urease inhibitor DMPP mixture.

[0094] (3) Adsorption operation: The sieved fermentation product is poured into the above mixture at a solid-liquid ratio of 1:5 (dry weight of fermentation product: volume of urea-urease inhibitor mixture). The mixture is then stirred and adsorbed for 60 minutes in a constant temperature stirrer at 30℃ and 150r / min. The nitrogen is stably loaded through the physical retention of the fermentation product through the pores and the coordination bond of the active groups. After adsorption, the mixture is allowed to stand for 30 minutes, and excess solution is removed by filtration before proceeding to the subsequent granulation and coating process.

[0095] 5. Granulation and coating

[0096] (1) Granulation: After adsorption, filter to remove excess free solution, add 5% (by mass of material) starch as binder to the loaded material, mix evenly and put into a drum granulator, adjust the speed to 35r / min, and make granules with a particle size of 2-4mm.

[0097] (2) Drying and Coating: The granules are fed into a 65℃ drying equipment and dried until the moisture content is <10%. Then, they are transferred to a fluidized bed coating machine. Polyurethane coating agent is sprayed at a ratio of 3%-5% of the material granules mass. The coating treatment lasts for 40 minutes, and the curing is completed to obtain the slow-release fertilizer product, which is named: D-UHP / Streptomyces baldus fermented silkworm excrement coated nitrogen fertilizer (e.g. Figure 3 (As shown).

[0098] Example 3

[0099] A method for preparing slow-release fertilizer using Streptomyces calvus strain GX2024L1 includes the following steps:

[0100] 1. Raw material collection

[0101] Local silkworm farms collect fresh silkworm excrement (containing a small amount of quicklime and mulberry branch and leaf residues). First, they screen the excrement to remove visible large impurities such as mulberry branch fragments and pebbles. Then, the cleaned excrement is evenly spread on a clean cement floor, with a thickness of 5 centimeters, and exposed to sunlight for one day. During this period, it is turned over 2-3 times a day to initially remove some moisture, reducing the moisture content of the excrement to about 25%, which is recorded as: original silkworm excrement.

[0102] 2. Pretreatment of silkworm excrement (Dynamic Ultra-High Pressure - Pulse Decompression Technology D-UHP)

[0103] Raw silkworm excrement was added to an ultra-high pressure reactor and then subjected to a three-stage pressurization process. First, the pressure was increased to 150 MPa and held for 60 seconds, followed by three pulse depressurizations at a rate of 100 MPa / s, each lasting 0.5 seconds. Then, the pressure was increased to 350 MPa and held for 60 seconds, followed by three pulse depressurizations at a rate of 100 MPa / s, each lasting 0.5 seconds. Finally, the pressure was increased to 550 MPa and held for 60 seconds before depressurization. The final product was a crushed material with a particle size of 0.5-1 mm, denoted as D-UHP / silkworm excrement.

[0104] 3. Mixed fermentation

[0105] Weigh 20 kg of the pretreated silkworm excrement from step (2) and transfer it to a sterile mixing device. Inoculate with 100 g of Streptomyces calvus strain GX2024L1 bacterial agent at a ratio of 0.5% of the pretreated silkworm excrement mass. Stir thoroughly for 10 minutes to ensure uniform contact between the bacterial agent and the silkworm excrement particles, forming a fermentation substrate. Then, place it in a fermentation chamber for fermentation. The specific operation is as follows:

[0106] (1) Fermentation tank filling: The inoculated fermentation substrate is filled into a controllable stainless steel fermentation tank, and the filling amount is 65% of the tank volume to avoid the material being too compacted and affecting the aeration.

[0107] (2) Environmental parameter control: The fermentation box is equipped with automatic ventilation, mechanical turning and temperature control. The initial fermentation temperature is 28℃. The mechanical turning device is started once every 24 hours, and the turning depth reaches the bottom of the material to ensure uniform oxygen supply. The pH value of the material is sampled and measured daily, and the pH fluctuation range is controlled within 5.0-6.0.

[0108] (3) Fermentation process monitoring: During the fermentation period, various parameters are recorded in real time. When the material temperature rises to 55℃, it is maintained for 3 days (high temperature composting stage). Then the temperature is gradually reduced and the fermentation continues for 15-18 days until the material temperature stabilizes at 32-35℃ and is maintained for 48 hours. The fermentation endpoint is judged as follows: when the material is uniformly dark brown, loose and easy to disperse, without hard core, without ammonia smell or odor, and the temperature drops below 35℃ and the pH value stabilizes at 5.0-6.0, the fermentation is judged to be complete and recorded as: D-UHP / Bare Streptomyces fermented silkworm excrement.

[0109] 4. Adsorbs nitrogen fertilizer and urease inhibitors

[0110] (1) Pretreatment of fermentation products: Take out the fermented material and dry it in a 60℃ oven until the moisture content is below 15% to remove impurities for later use.

[0111] (2) Preparation of urea solution containing urease inhibitor (DMPP): Weigh 5 kg of urea (prepared at a mass fraction of 20%), add 50 g of DMPP (1% of the mass of urea), add 7.7 L of deionized water, stir thoroughly to dissolve, and obtain a urea-urease inhibitor DMPP mixture.

[0112] (3) Adsorption operation: The fermented product after sieving is poured into the above mixture at a solid-liquid ratio of 1:4 (dry weight of fermentation product: volume of urea-urease inhibitor mixture). The mixture is then stirred in a constant temperature stirrer at 30℃ and 150r / min for 90 minutes to achieve stable nitrogen loading through the physical retention of the fermented product through pores and the coordination bond of active groups. After adsorption, the mixture is allowed to stand for 30 minutes, and excess solution is removed by filtration before proceeding to the subsequent granulation and coating process.

[0113] 5. Granulation and coating

[0114] (1) Granulation: After adsorption, filter to remove excess free solution, add 5% (by mass of material) starch as binder to the loaded material, mix evenly and put into a drum granulator, adjust the speed to 35r / min, and make granules with a particle size of 2-4mm.

[0115] (2) Drying and coating: The granules are sent to a 65℃ drying equipment and dried until the moisture content is <10%. Then they are transferred to a fluidized bed coating machine and polyurethane coating agent is sprayed at a ratio of 3%-5% of the material granules. The coating treatment lasts for 40 minutes and the curing is completed to obtain the slow-release fertilizer product.

[0116] Example 4

[0117] A method for preparing slow-release fertilizer using Streptomyces calvus strain GX2024L1 includes the following steps:

[0118] 1. Raw material collection

[0119] Local silkworm farms collect fresh silkworm excrement (containing a small amount of quicklime and mulberry branch and leaf residues). First, they screen the excrement to remove visible large impurities such as mulberry branch fragments and pebbles. Then, the cleaned excrement is evenly spread on a clean cement floor, with a thickness of 5 centimeters, and exposed to sunlight for one day. During this period, it is turned over 2-3 times a day to initially remove some moisture, reducing the moisture content of the excrement to about 30%, which is recorded as: original silkworm excrement.

[0120] 2. Pretreatment of silkworm excrement (Dynamic Ultra-High Pressure - Pulse Decompression Technology D-UHP)

[0121] The raw silkworm excrement was added to an ultra-high pressure reactor and then subjected to a three-stage pressurization process. First, the pressure was increased to 200 MPa and held for 30 seconds, followed by four pulse depressurizations at a rate of 100 MPa / s, each lasting 0.5 seconds. Then, the pressure was increased to 400 MPa and held for 30 seconds, followed by four pulse depressurizations at a rate of 100 MPa / s, each lasting 0.5 seconds. Finally, the pressure was increased to 600 MPa and held for 30 seconds before depressurization. The final product was a crushed material with a particle size of 0.5-1 mm, denoted as D-UHP / silkworm excrement.

[0122] 3. Mixed fermentation

[0123] Weigh 20 kg of the pretreated silkworm excrement from step (2) and transfer it to a sterile mixing device. Inoculate with 300 g of Streptomyces calvus strain GX2024L1 bacterial agent at a ratio of 1.5% of the pretreated silkworm excrement mass. Stir thoroughly for 20 minutes to ensure uniform contact between the bacterial agent and the silkworm excrement particles, forming a fermentation substrate. Then, place it in a fermentation chamber for fermentation. The specific operation is as follows:

[0124] (1) Fermentation tank filling: The inoculated fermentation substrate is filled into a controllable stainless steel fermentation tank, and the filling amount is 65% of the tank volume to avoid the material being too compacted and affecting the aeration.

[0125] (2) Environmental parameter control: The fermentation box is equipped with automatic ventilation, mechanical turning and temperature control. The initial fermentation temperature is 28℃. The mechanical turning device is started once every 24 hours, and the turning depth reaches the bottom of the material to ensure uniform oxygen supply. The pH value of the material is sampled and measured daily, and the pH fluctuation range is controlled within 5.0-6.0.

[0126] (3) Fermentation process monitoring: During the fermentation period, various parameters are recorded in real time. When the material temperature rises to 55℃, it is maintained for 3 days (high temperature composting stage). Then the temperature is gradually reduced and the fermentation continues for 15-18 days until the material temperature stabilizes at 32-35℃ and is maintained for 48 hours. The fermentation endpoint is judged as follows: when the material is uniformly dark brown, loose and easy to disperse, without hard core, without ammonia smell or odor, and the temperature drops below 35℃ and the pH value stabilizes at 5.0-6.0, the fermentation is judged to be complete and recorded as: D-UHP / Bare Streptomyces fermented silkworm excrement.

[0127] 4. Adsorbs nitrogen fertilizer and urease inhibitors

[0128] (1) Pretreatment of fermentation products: Take out the fermented material and dry it in a 60℃ oven until the moisture content is below 15% to remove impurities for later use.

[0129] (2) Preparation of urea solution containing urease inhibitor (DMPP): Weigh 5 kg of urea (prepared at a mass fraction of 40%), add 50 g of DMPP (1% of the mass of urea), add 15.5 L of deionized water, stir thoroughly to dissolve, and obtain a urea-urease inhibitor DMPP mixture.

[0130] (3) Adsorption operation: The sieved fermentation product is poured into the above mixture at a solid-liquid ratio of 1:5 (dry weight of fermentation product: volume of urea-urease inhibitor mixture). The mixture is then stirred and adsorbed for 120 minutes in a constant temperature stirrer at 30℃ and 150r / min. The nitrogen is stably loaded through the physical retention of the fermentation product through the pores and the coordination bond of the active groups. After adsorption, the mixture is allowed to stand for 30 minutes, and excess solution is removed by filtration before proceeding to the subsequent granulation and coating process.

[0131] 5. Granulation and coating

[0132] (1) Granulation: After adsorption, filter to remove excess free solution, add 5% (by mass of material) starch as binder to the loaded material, mix evenly and put into a drum granulator, adjust the speed to 35r / min, and make granules with a particle size of 2-4mm.

[0133] (2) Drying and coating: The granules are sent to a 65℃ drying equipment and dried until the moisture content is <10%. Then they are transferred to a fluidized bed coating machine and polyurethane coating agent is sprayed at a ratio of 3%-5% of the material granules. The coating treatment lasts for 40 minutes and the curing is completed to obtain the slow-release fertilizer product.

[0134] Comparative Example 1: Preparation of ordinary coated nitrogen fertilizer

[0135] 1. Raw material preparation

[0136] Weigh out commercially available granular urea fertilizer (total nitrogen content ≥46%) as the core raw material and set aside.

[0137] 2. Granulation

[0138] If the particle size of commercially available fertilizer does not meet the requirements, it should be crushed, and 5% of its mass of starch solution should be added as a binder. After mixing evenly, it should be fed into a drum granulator to produce regular granules with a particle size of 2-4mm.

[0139] 3. Coating treatment

[0140] The fertilizer granules were dried at 65°C until the moisture content was below 10%, and then transferred to a fluidized bed coating machine. A polyurethane coating agent was sprayed at 3% of the granule mass, and the coating was allowed to cure completely within 40 minutes, thus obtaining a "common coated nitrogen fertilizer" sample. This sample will serve as a control for subsequent comparative tests of various properties.

[0141] Comparative Example 2: Preparation of Fermented Silkworm Excrement-Based Coated Nitrogen Fertilizer

[0142] 1. Raw material collection and pretreatment

[0143] Collect fresh silkworm excrement, sieve it to remove large impurities such as mulberry branches, and spread it evenly to dry until the moisture content is 20%-30% to obtain raw silkworm excrement for later use. The specific operation is the same as step 1 in Example 2.

[0144] 2. Conventional fermentation (without inoculation with microbial agents)

[0145] Weigh 20 kg of the pretreated silkworm excrement from step 1 and transfer it to the fermentation equipment. Fermentation is carried out solely by the naturally occurring microbial community within the material, without inoculating with any exogenous microbial agents. During fermentation, the moisture content of the material is maintained at 60%-65% by spraying sterile water. The material is turned over every 24 hours to ensure uniform oxygen supply. The fermentation temperature is controlled to fluctuate naturally within the range of 28-55℃. The total fermentation cycle is 15 days. The fermentation endpoint is determined when the material turns dark brown, becomes loose in texture, has no obvious ammonia odor, and the temperature and pH values ​​stabilize. The resulting material is designated as fermented silkworm excrement.

[0146] 3. Adsorbs nitrogen fertilizer and urease inhibitors

[0147] The fermented material was dried in a 60℃ oven until the moisture content was below 15%, then pulverized and passed through a 20-mesh sieve. 5 kg of urea and 50 g of urease inhibitor (DMPP) were weighed and dissolved in 11.6 L of deionized water to prepare a urea-urease inhibitor mixture with a mass fraction of approximately 30%. The sieved fermentation product was added to the mixture at a solid-liquid ratio of 1:5 (based on dry material), and the mixture was stirred at 30℃ and 150 rpm for 60 minutes for adsorption. After adsorption, the mixture was allowed to stand for 30 minutes, and then filtered to remove excess solution.

[0148] 4. Granulation and Coating

[0149] Add 5% (by weight) of starch as a binder to the adsorbed wet material, mix thoroughly, and then feed it into a drum granulator to produce granules with a particle size of 2-4 mm. Dry the granules at 65℃ until the moisture content is below 10%, and then transfer them to a fluidized bed coating machine. Spray polyurethane coating agent at 3% (by weight) of the granules, and allow the coating layer to solidify for 40 minutes to obtain the "fermented silkworm excrement-based coated nitrogen fertilizer" sample.

[0150] Comparative Example 3: Preparation of D-UHP / Fermented Silkworm Excrement-Based Coated Nitrogen Fertilizer

[0151] 1. Raw material collection and pretreatment

[0152] Collect fresh silkworm excrement, sieve it to remove large impurities such as mulberry branches, and spread it evenly to dry until the moisture content is 20%-30% to obtain raw silkworm excrement for later use. The specific operation is the same as step 1 in Example 2.

[0153] 2. Dynamic ultra-high pressure - pulse depressurization pretreatment of silkworm excrement

[0154] The raw silkworm excrement was added to an ultra-high pressure reactor and subjected to the same three-stage dynamic ultra-high pressure-pulse depressurization pretreatment as step 2 in Example 2, finally obtaining crushed silkworm excrement material with a particle size of 0.5-1mm.

[0155] 3. Conventional fermentation (without inoculation with microbial agents)

[0156] Weigh 20 kg of the pretreated silkworm excrement and transfer it to the fermentation equipment. Fermentation is carried out solely by the naturally occurring microbial community within the material, without inoculating with any exogenous microbial agents. The physical parameters during fermentation, including loading volume, turning frequency, temperature, and moisture control, remain consistent with the fermentation conditions in step 3 of Example 2, and the total fermentation period is also 15 days. The fermentation endpoint is determined by the material darkening in color and becoming somewhat loose. The resulting material is denoted as: D-UHP / Fermented Silkworm Excrement.

[0157] 3. Adsorbs urea and urease inhibitors

[0158] The fermented material was dried in a 60℃ oven until the moisture content was below 15%, then pulverized and passed through a 20-mesh sieve. 5 kg of urea and 50 g of DMPP were weighed and dissolved in 11.6 L of deionized water to prepare a urea-urease inhibitor mixture with a mass fraction of approximately 30%. The sieved fermentation product was added to the mixture at a solid-liquid ratio of 1:5 (based on dry material), and the mixture was stirred at 30℃ and 150 rpm for 60 minutes for adsorption. After adsorption, the mixture was allowed to stand for 30 minutes, and then filtered to remove excess solution.

[0159] 4. Granulation and Coating

[0160] Add 5% (by weight) of starch as a binder to the adsorbed wet material, mix thoroughly, and then feed it into a drum granulator to produce granules with a particle size of 2-4 mm. Dry the granules at 65℃ until the moisture content is below 10%, and then transfer them to a fluidized bed coating machine. Spray polyurethane coating agent at 3% (by weight) of the granules, and allow the coating layer to solidify for 40 minutes to obtain the "D-UHP / fermented silkworm excrement-based coated nitrogen fertilizer" sample.

[0161] Application of slow-release fertilizer in sugarcane cultivation

[0162] A 1.1-acre sugarcane experimental field (red soil, pH 5.5) was selected. Three days before sowing sugarcane seed stalks, the amount of slow-release fertilizer (approximately 28.7 kg) was calculated based on a pure nitrogen (N) application rate of 180 kg / ha. The slow-release fertilizer prepared in Example 2 was evenly spread on the soil surface using a fertilizer spreader, and then tilled into the soil to a depth of approximately 25.5 cm using a rotary tiller (with a tillage depth adjustment knob) to ensure thorough mixing of the fertilizer with the soil. Sugarcane seed stalks (variety "Guitang 42") were sown at the local conventional planting density (8800 plants per acre). No additional nitrogen fertilizer was applied throughout the growing season. Field management was carried out in accordance with the "Guangxi Sugarcane High-Yield and High-Quality Cultivation Technical Regulations". During the seedling stage, the focus was on water retention (maintaining soil moisture at 65%), and during the elongation stage, hilling was carried out to prevent lodging. During the maturity stage, aphids and stem borers were controlled in a timely manner.

[0163] Material sustained-release performance testing

[0164] The product prepared in Example 2 of the present invention was subjected to characterization analysis and performance testing analysis.

[0165] (I) FT-IR Spectroscopic Characterization

[0166] The original silkworm excrement, the silkworm excrement pretreated with ultra-high pressure (D-UHP), the silkworm excrement fermented with D-UHP / Streptomyces nakedus, the fermented silkworm excrement in Comparative Example 2, and the D-UHP / fermented silkworm excrement in Comparative Example 3 were characterized by infrared spectroscopy.

[0167] Fourier transform infrared spectroscopy was performed using a Nicolet Nexus 670 FT-IR spectrometer (Thermo Fisher). The purified and dried sample was finely ground and compressed with potassium bromide. The scan range was set to 4000 cm⁻¹. -1 ~500cm -1 Spectral resolution of 4 cm -1 The number of scans was 32, and the data interval was 0.482 cm. -1 .

[0168] The results are as follows Figure 4 The infrared spectrum shows that the infrared spectrum is in the range of 3200-3600 cm⁻¹. -1 Within the specified range, the absorption peak of the original silkworm excrement is weak and flat; after ultra-high pressure pretreatment (D-UHP / silkworm excrement), the absorption peak is enhanced; after fermentation by the strain of this invention (D-UHP / streptomyces nakedus fermentation of silkworm excrement), the absorption peak is significantly enhanced and the peak shape is broadened.

[0169] Compared to the original silkworm excrement, the ordinary fermented silkworm excrement in Comparative Example 2, and the ordinary fermented silkworm excrement (D-UHP / fermented silkworm excrement) pretreated with ultra-high pressure in Comparative Example 3, showed higher absorption peak intensities in this region compared to the unfermented group. This indicates that the preceding pretreatment (such as ultra-high pressure pretreatment) can disrupt the dense structure of the silkworm excrement and expose hydroxyl groups, but fermentation can further promote their formation. However, the absorption peak intensities of fermented silkworm excrement and D-UHP / fermented silkworm excrement in this region are still significantly lower than those of the sample fermented using this invention (D-UHP / Streptomyces nakedus fermented silkworm excrement). This suggests that only by combining ultra-high pressure pretreatment with directional fermentation by Streptomyces nakedus can the content of active groups and hydrogen bonding ability be maximized.

[0170] At 2800-3000 cm -1 In the region, the absorption peaks of all fermented samples were stronger than those of the original and pretreated samples, indicating that the fermentation process itself can introduce more aliphatic structures.

[0171] In 1000-1800 cm -1 In the region, the absorption peak intensity of the fermented samples was generally improved, but the peak intensity of D-UHP / Streptomyces nakedus fermented silkworm excrement in the carbonyl, amide, and carbon-oxygen bond related bands was significantly higher than that of the two ordinary fermented silkworm excrement in Comparative Examples 2 and 3. This indicates that this synergistic treatment can more effectively generate substances containing key functional groups such as carboxyl and amide groups (such as organic acids and amino acids produced by microbial metabolism), thereby providing richer surface active sites for nutrient adsorption.

[0172] (II) FT-IR spectral peak fitting diagram (hydroxyl characteristic region)

[0173] Figure 5 This demonstrates the hydroxyl characteristic region (3000-3700 cm⁻¹) of silkworm excrement treated with different methods. -1 The infrared spectral peak fitting results show that the area ratio of free hydroxyl groups to hydrogen-bonded hydroxyl groups in the original silkworm excrement is 1:1.78, indicating that the surface hydroxyl groups mainly exist in the free state.

[0174] After different processing steps, the ratio showed a regular decrease: the ratio for the dynamic ultra-high pressure pretreatment (D-UHP / silkworm excrement) sample was 1:2.23; among the two comparative samples, the ratio for ordinary fermentation (fermented silkworm excrement) in Comparative Example 2 was 1:2.21, while the ratio for ordinary fermentation (D-UHP / fermented silkworm excrement) after dynamic ultra-high pressure pretreatment was 1:2.36. This indicates that the fermentation process itself can promote the conversion of hydroxyl groups to the bound state, and dynamic ultra-high pressure pretreatment provides a better physical basis for fermentation.

[0175] The sample (D-UHP / Streptomyces nakedus fermented silkworm excrement) subjected to dynamic ultra-high pressure pretreatment combined with directional fermentation by the strain of this invention achieved a minimum ratio of 1:2.51. This demonstrates that the synergistic process can most effectively convert surface hydroxyl groups from a free state to a bound state, thereby significantly enhancing the surface chemical activity of the material and its ability to adsorb nutrients through hydrogen bonds, laying a key foundation for constructing high-performance slow-release fertilizer carriers.

[0176] (III) Surface morphology of materials

[0177] The morphology of the samples at each preparation stage in Example 2 was characterized using a Hitachi S-3400N low-power scanning electron microscope (Japan), such as... Figure 6 As shown. (a) Original silkworm excrement; (b) D-UHP / silkworm excrement; (c) D-UHP / Streptomyces baldii fermented silkworm excrement; (d) D-UHP / Streptomyces baldii fermented silkworm excrement adsorbing nitrogen fertilizer and urease inhibitor; (e) Silkworm excrement-based nitrogen long-acting slow-release fertilizer product.

[0178] Depend on Figure 6 (a) It can be seen that the original silkworm excrement has a dense structure; (b) It can be seen that physical fragmentation occurs after D-UHP treatment; (c) It can be seen that after fermentation by the strain of this invention, the material becomes loose and porous, forming an ideal high specific surface area adsorption structure; (d) It can be seen that a large number of granular nitrogen fertilizers are attached to the surface of the silkworm excrement, and the pores are filled. (e) It can be seen that after adsorption granulation and coating, it forms a complete and smooth sphere, which is beneficial to the slow release of nitrogen fertilizer and product stability.

[0179] (iv) Comparison of swelling rates of raw silkworm excrement, D-UHP / silkworm excrement, and D-UHP / Streptomyces nakedus fermented silkworm excrement

[0180] Take 10g of raw silkworm excrement, D-UHP / silkworm excrement, and D-UHP / Streptomyces nakedus fermented silkworm excrement samples respectively, and dry them in an oven. After drying, the samples are swollen. Weigh an appropriate amount of material and soak it in water. After 1 hour, wipe the surface moisture with lens paper, and then weigh the swollen material. According to the formula:

[0181]

[0182] E is the swelling ratio, Ws is the weight of water after swelling, and Wd is the dry weight of the material. Figure 7 .Depend on Figure 7 It was found that the swelling ratio of silkworm excrement fermented with D-UHP / Streptomyces nakedus was significantly increased, reaching 4.81, which is 1.31 times that of D-UHP / silkworm excrement and 1.98 times that of original silkworm excrement, respectively. This indicates that the dynamic ultra-high pressure pretreatment and the synergistic fermentation treatment with the strain of this invention can maximize the improvement of the hydrophilicity, pore structure and swelling performance of the silkworm excrement carrier, thus providing a key physical structural basis for its efficient adsorption and loading of nitrogen nutrients.

[0183] (V) Static Water Release Experiment

[0184] This experiment used the water extraction method to determine the nutrient release characteristics of fertilizers under the conditions of a fertilizer-to-water ratio of 1:100 and 25℃. Three portions (2g each, accurate to 0.001g) of each of the following fertilizers were weighed: ordinary coated nitrogen fertilizer (Comparative Example 1), fermented silkworm excrement-based coated nitrogen fertilizer (Comparative Example 2), and the D-UHP / Streptomyces nakedus fermented silkworm excrement-based coated nitrogen fertilizer of this invention. These were placed in 250mL Erlenmeyer flasks, 100mL of deionized water was added, and the flasks were sealed and shaken to ensure the fertilizer particles were fully saturated. Samples were taken at 1, 3, 5, 7, 10, 14, 28, and 40 days, and release curves were fitted (e.g., ...). Figure 8 (As shown in the figure). The results showed that the initial release rate of the three fertilizers was about 15%, with significant differences in subsequent release: the ordinary coated nitrogen fertilizer had a fast release rate; the silkworm excrement-based coated nitrogen fertilizer had a release rate of about 72% on day 35; and the product of this invention had a release rate of about 60% on day 35, exhibiting the best controlled-release effect. Even after the coating ruptured, it could still continue to release slowly. After D-UHP pretreatment and fermentation, its slow-release regulation ability was further enhanced, significantly superior to that of ordinary coated nitrogen fertilizer.

[0185] (vi) Soil release experiment

[0186] This experiment used an indoor culture method to simulate fixed humidity soil conditions and compared the nutrient release characteristics of different fertilizers. 2g of each of the following fertilizers were weighed: ordinary coated nitrogen fertilizer (Comparative Example 1), fermented silkworm excrement-based coated nitrogen fertilizer (Comparative Example 2), and the D-UHP / Streptomyces nakedus fermented silkworm excrement-based coated nitrogen fertilizer of this invention. These were placed in 100-mesh nylon bags and buried in a culture column containing 600g of soil (5-8cm deep). The cells were placed in a 25℃ constant temperature incubator, and water was added periodically to maintain a soil moisture content of approximately 40%. Samples were taken on days 7, 14, 21, 35, 48, 61, 90, and 108. After separating the fertilizers, 10g of soil was extracted with 100mL of KCl. The nutrient release rate was estimated by measuring the increase in nitrate nitrogen and ammonium nitrogen using a UV spectrophotometer (e.g., nutrient release rate of nitrate nitrogen and ammonium nitrogen). Figure 9 (As shown). The results showed that ordinary coated nitrogen fertilizer exhibited a sudden nutrient release from the initial stage to around day 40 of the experiment, with a cumulative release rate of 90% over the experimental period; silkworm excrement-based coated nitrogen fertilizer had a slower controlled release, with a cumulative release rate of approximately 79%; the product of this invention had a more gradual controlled release, with a cumulative release rate of approximately 72%, indicating that silkworm excrement fermented with the microbial agent of this invention under dynamic ultra-high pressure had a better effect on regulating nitrogen fertilizer release.

[0187] (vii) Controlled-release leaching experiment

[0188] Weigh 10g of the product prepared in Example 2 and select a PVC pipe with a length of 40cm and an inner diameter of 2.65cm as a soil leaching column. To prevent soil loss with the filtrate during leaching, wrap a piece of 200-mesh gauze around the bottom of the PVC pipe. Fill the leaching column with materials in a specific order: first add 10g of quartz sand, then place 10g of the product of this invention in the middle of the sand bed, and finally add another 10g of quartz sand to cover the top, completing the soil column filling. Transfer the filled PVC pipe to a support with a glass funnel at the bottom and conduct the leaching test. First, add 40mL of distilled water to each soil column to bring the soil moisture content to the field capacity (the saturated soil moisture content is 30%), and then let it stand for 24 hours to ensure the soil system is balanced and stable. After stabilization, slowly pour 40mL of distilled water from the top of the soil column and collect all 40mL of filtrate as the sample for the first day of the experiment. Subsequent leaching tests were conducted according to the following procedure: 40 mL of distilled water was poured onto the top of the sand bed daily. After the water flowed through the sand bed, the leachate was collected in the bottom container. The bottom container was emptied and a leachate sample was collected every 5 days. The nitrogen content in the leachate was determined and calculated, and the results are as follows: Figure 10 As shown, during the 8-month release test period, the cumulative release rate of nitrogen reached 77% of its total content.

[0189] (VIII) Comparative Experiment on the Effects of Urease Inhibitors on Soil Nitrogen Species Using Fermented Silkworm Excrement Carriers

[0190] To investigate the effect of urease inhibitors on soil nitrogen forms, this experiment used simulated soil conditions to set up a control experiment: 400g of homogeneous test soil was placed in a reagent bottle, and 7g of fermented slow-release fertilizer sample was added (two groups were set up; experimental group 1 was supplemented with 1% DMPP by dry weight, and experimental group 2 was not supplemented). The background nitrate nitrogen and ammonium nitrogen contents of the test soil were measured beforehand as controls. The results are as follows: Figure 11 As shown, urease inhibitors significantly slowed down the rate of nitrogen hydrolysis, preventing the large-scale generation of ammonium nitrogen in the early stages of the experiment and its subsequent loss. Therefore, ammonium nitrogen in the soil of experimental group 1 showed a slow accumulation trend, and its content was consistently higher than that in experimental group 2 without inhibitors. Furthermore, the lower ammonium nitrogen concentration in the early stages of experimental group 1, combined with the inhibitory effect of urease inhibitors on the reproduction and activity of nitrifying bacteria, hindered the nitrification process. Figure 11 The data in (a) shows that the soil nitrate nitrogen content is significantly lower. In summary, the addition of urease inhibitors not only inhibits the nitrogen hydrolysis process, but also regulates the conversion of ammonium nitrogen to nitrate nitrogen, ultimately resulting in greater nitrogen retention in the soil.

[0191] (ix) Potted Plant Experiment

[0192] Pot experiments were conducted using different fertilizer samples. Four control groups were set up: a blank control group, a control group (comparative example 1) with ordinary coated nitrogen fertilizer, a control group (comparative example 2) with fermented silkworm excrement-based coated nitrogen fertilizer, and a control group (Example 2) with D-UHP / Streptomyces nakedus fermented silkworm excrement-based coated nitrogen fertilizer. The sugarcane seedlings used were of the Guiliu 05136 model, all approximately 50cm tall. The experimental pots used for planting had a height of 50cm, a bottom outer diameter of 38cm, an opening inner diameter of 42cm, and an opening outer diameter of 49cm. Initial soil conditions were set as follows: pH 6.41, nitrate nitrogen content 24.31mg / kg, and ammonium nitrogen content 8.49mg / kg. The fertilizer was applied in a single application, based on the sugarcane's total fertilizer requirement for the entire growth cycle (230.4kg / hm²). The sugarcane seedlings were buried approximately 18cm deep, and the fertilizer was applied to a depth of approximately 13cm, followed by watering until the soil was thoroughly moistened. In the middle stage of the experiment (4 months after planting), four representative sugarcane plants were selected (e.g., Figure 12 (As shown). By Figure 12 It can be seen that the sugarcane treated with the product of the present invention is 56 cm taller than the sugarcane treated with the blank control group; and 17-18 cm taller than the ordinary coated nitrogen fertilizer group and the fermented silkworm excrement-based coated nitrogen fertilizer group, which shows the significant advantages of the slow-release fertilizer product prepared by the present invention in promoting sugarcane growth.

[0193] The above embodiments of the present invention are merely illustrative examples to clearly illustrate the invention, and are not intended to limit the implementation of the invention. Those skilled in the art can make various changes or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a slow-release fertilizer containing Streptomyces calvus strain GX2024L1, characterized in that: The method includes the following steps: (1) Fresh silkworm excrement processing: collect fresh silkworm excrement, remove impurities, and dry it to obtain raw silkworm excrement for later use; (2) Pretreatment of silkworm excrement: The original silkworm excrement is pressurized in an ultra-high pressure reactor to increase the specific surface area of ​​the original silkworm excrement and obtain pretreated silkworm excrement. The pressurization process is a three-stage process: first, the pressure is increased to 150-200 MPa and held for 30-60 seconds, followed by 3-4 pulse depressurizations; then, the pressure is increased to 350-400 MPa and held for 30-60 seconds, followed by 3-4 pulse depressurizations; finally, the pressure is increased to 550-600 MPa and held for 30-60 seconds before depressurization, thus completing the pressurization process. (3) Mixed fermentation: The pretreated silkworm excrement from step (2) is inoculated with Streptomyces calvus strain GX2024L1 at a mass ratio of 0.5%-1.5% for fermentation. During fermentation, the excrement is stirred regularly and fermented for 15-18 days. The Streptomyces calvus strain GX2024L1 is deposited at the China Microbial Culture Collection Center, with accession number CGMCC No. 32902; (4) Adsorb nitrogen fertilizer and urease inhibitor: The silkworm excrement material fermented in step (3) is crushed and mixed with a solution of urease inhibitor and urea at a solid-liquid ratio of 1:4-5 and stirred for adsorption. The mixture of urease inhibitor and urea is prepared by first preparing a urea solution with a mass fraction of 20%-40%, then adding 1% of the urea mass of urea inhibitor DMPP to the solution, and mixing thoroughly. (5) Granulation and coating: Mix the material treated in step (4) with the binder, granulate and coat to obtain slow-release fertilizer.

2. A slow-release fertilizer prepared by the preparation method described in claim 1.

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