Compound microbial fertilizer for improving fertility of degraded grassland

Abstract

The present application relates to the technical field of compound fertilizer, and specifically discloses a compound microbial fertilizer for improving the fertility of degraded grassland, which is composed of the following raw materials in parts by mass: 82-84 parts of fermentation base substrate, 12-14 parts of compound microbial agent, 1.4-1.8 parts of compound protective agent, and 3-5 parts of slow-release nutrient additive; the present application improves the fertility of degraded grassland from three aspects: first, the compound protective agent is added, which is obtained by mixing phosphorylated chitosan, polyglutamic acid and glycerol in a specific ratio; second, the slow-release nutrient additive is added, which is obtained by mixing phosphate rock powder, potassium sulfate, amino acid raw powder and sulfonated sodium alginate aqueous solution in a specific ratio; and third, the compound microbial agent is added, which effectively improves the fertility of degraded grassland through the synergistic effect of the substances.

Description

Technical Field

[0001] This invention relates to the field of compound fertilizer technology, and more specifically, to a compound microbial fertilizer for enhancing the fertility of degraded grasslands. Background Technology

[0002] Grassland ecosystems have core functions such as water conservation, windbreak and sand fixation, and maintenance of biodiversity. However, due to factors such as unreasonable grazing, over-cultivation, and climate change, natural grasslands around the world are generally degraded. The main manifestations are soil organic matter deficiency, low content of available nitrogen, phosphorus and potassium, severe soil desertification or compaction, sharp decline in the number of beneficial microorganisms, low germination rate of forage grass and insufficient biomass, long restoration cycle and unstable effect.

[0003] In existing technologies, fertilizers used to improve the fertility of degraded grasslands are mostly single organic fertilizers or conventional microbial agents. Although organic fertilizers can replenish organic matter, their nutrient release is slow and they do not have a microbial growth-promoting effect, making it difficult to quickly improve the soil microecology. Conventional microbial agents are mostly single-function strains with low survival rates and poor colonization capabilities, which cannot meet the growth needs of local grassland vegetation, thus limiting their fertility-improving effect.

[0004] Therefore, there is an urgent need to develop a compound microbial fertilizer that is suitable for degraded grassland environments and can effectively improve the fertility of degraded grasslands, so as to achieve sustainable agricultural development. Summary of the Invention

[0005] To address the above problems, this invention provides a compound microbial fertilizer that enhances the fertility of degraded grasslands.

[0006] A compound microbial fertilizer for improving the fertility of degraded grasslands is composed of the following raw materials in parts by weight: 82-84 parts fermentation base substrate, 12-14 parts compound microbial agent, 1.4-1.8 parts compound protective agent and 3-5 parts slow-release nutrient additive.

[0007] Furthermore, the fermentation base substrate is prepared by mixing sheep manure fermentation product, corn stalk powder, humic acid, vermiculite powder and sterile water in a mass ratio of 32-34:26-28:16-18:8-10:50-70, and then sterilizing it.

[0008] Furthermore, the composite protective agent is composed of phosphorylated chitosan, polyglutamic acid, and glycerol in a mass ratio of 2-3:1:0.6-1.

[0009] Furthermore, the slow-release nutrient additive is composed of phosphate rock powder, potassium sulfate, amino acid raw powder and sulfonated sodium alginate aqueous solution in a mass ratio of 1.5-2.5:1-1.4:0.6-1:30-40.

[0010] Furthermore, the sulfonated sodium alginate aqueous solution is composed of sulfonated sodium alginate and deionized water at a mass ratio of 0.4-0.6:100.

[0011] Further, the sulfonated sodium alginate is prepared by the following steps: Sodium alginate was added to dichloromethane and stirred in an ice bath for 12-16 minutes. While stirring, a mixture of chlorosulfonic acid and dichloromethane was added dropwise. After the addition was complete, the temperature was raised to 26-30℃ and the reaction was stirred for 1.8-2 hours. The mixture was filtered, and deionized water was added to adjust the pH to neutral. The mixture was dialyzed, and anhydrous ethanol was added. The mixture was allowed to stand and dried to obtain sulfonated sodium alginate. In the above process, sodium alginate can undergo a sulfonation reaction with chlorosulfonic acid using dichloromethane as a solvent to obtain sulfonated sodium alginate.

[0012] Furthermore, the mass ratio of sodium alginate, dichloromethane, the mixture, deionized water, and anhydrous ethanol is 1:15-25:6-10:40-50:20-30, and the mass ratio of chlorosulfonic acid to dichloromethane in the mixture is 1:2-3.

[0013] Furthermore, the dialysis bag used in the dialysis process has a molecular weight cutoff of 3500 Da, and the dialysis time is 20-24 hours.

[0014] Furthermore, the phosphorylated chitosan is prepared by the following steps: Chitosan was dissolved in an aqueous acetic acid solution, and then an aqueous phosphoric acid solution was added. The mixture was stirred for 26-34 minutes and dried to obtain phosphorylated chitosan.

[0015] Furthermore, the mass ratio of the chitosan, aqueous acetic acid solution, and aqueous phosphoric acid solution is 1:20-30:6-8.

[0016] Furthermore, the mass fraction of the acetic acid aqueous solution is 1-3%.

[0017] Furthermore, the mass fraction of the phosphoric acid aqueous solution is 28-30%.

[0018] Furthermore, the compound microbial agent is prepared by the following steps: Step A1: Inoculate the Bacillus spp. seed culture, Burkholderia seed culture and Streptomyces seed culture into the compound fermentation medium and culture for 42-46 hours to obtain a mixed fermentation broth; Step A2: Inoculate the rhizobium seed liquid into the mixed fermentation broth and allow it to adapt statically for 1.8-2.2 hours. Then, inoculate the Trichoderma spore suspension into the mixed fermentation broth and continue stirring for 0.9-1.1 hours. Cultivate together for 24-32 hours to obtain the compound microbial agent.

[0019] Furthermore, the volume ratio of the gelatinous Bacillus seed solution, Burkholderia seed solution, and Streptomyces seed solution is 5-8:3-5:2-4.

[0020] Furthermore, in step A1, the specific process parameters for cultivation are: temperature 30-32℃; aeration rate 0.5-0.6 VVM; and pH maintained at 6.8-7.2 during cultivation.

[0021] Furthermore, in step A2, the specific process parameters for co-cultivation are: temperature 30-32℃; aeration rate 1-1.4 VVM; and pH maintained at 6.8-7.2 during cultivation.

[0022] Furthermore, the inoculation amount of the rhizobium seed liquid is 5-6% of the mixed fermentation liquid volume, and the inoculation amount of the Trichoderma spore suspension is 3-4% of the mixed fermentation liquid volume.

[0023] Further, the preparation method of the Bacillus jellyoid seed solution is as follows: 15-16g of glucose, 8-9g of protein, 0.8-1.0g of potassium dihydrogen phosphate, and 0.4-0.6g of magnesium sulfate heptahydrate are added to 1L of deionized water, the pH is adjusted to 7.0-7.2, sterilized at 121℃ for 20min, cooled to room temperature, and then inoculated with 1-2% (v / v) Bacillus jellyoid. The solution is cultured at 28-30℃ and 150rpm for 24-48h with shaking to obtain the Bacillus jellyoid seed solution.

[0024] Furthermore, the effective viable count of the gelatinous Bacillus seed solution is 1.6 × 10¹. 0 CFU / mL -1.96×10¹ 0 CFU / mL.

[0025] Further, the method for preparing the Burkholderia seed culture is as follows: 120-140g of glucose, 60-80g of peptone, 30-40g of yeast powder, 6-8g of potassium dihydrogen phosphate, and 3-5g of magnesium sulfate heptahydrate are added to 1L of deionized water, the pH is adjusted to 7.0-7.2, sterilized at 121℃ for 20min, cooled to room temperature, and then inoculated with 1-2% (v / v) Burkholderia. The culture is then carried out at 28-30℃ with shaking at 150rpm for 22-26h to obtain the Burkholderia seed culture.

[0026] Furthermore, the effective viable count of the Burkholderia seed culture was 5.92 × 10⁻⁶. 9 CFU / mL -7.24×10 9 CFU / mL.

[0027] Further, the preparation method of the Streptomyces seed solution is as follows: 100-120g of glucose, 50-54g of glycerol, 40-46g of peptone, 20-22g of yeast powder, and 5-7g of calcium carbonate are added to 1L of deionized water, the pH is adjusted to 7.0-7.2, sterilized by high-pressure steam at 121℃ for 20min, cooled to room temperature, and then inoculated with 1-2% (v / v) Streptomyces. The solution is cultured at 28-30℃ and 150rpm for 22-26h with shaking to obtain the Streptomyces seed solution.

[0028] Furthermore, the effective viable count of the Streptomyces seed culture is 1.84 × 10⁻⁶. 9 CFU / mL -2.24×10 9 CFU / mL.

[0029] Furthermore, the composition of the compound fermentation medium is as follows: each liter of medium contains 10g of glucose, 5g of glycerol, 5g of peptone, 3g of yeast powder, 1g of NaCl, 1g of K2HPO4, 0.5g of MgSO4•7H2O, 0.5g of CaCO3 and 1.0g of silicate mineral powder, and the pH value of the medium is controlled at 7.0-7.2.

[0030] Further, the method for preparing the rhizobium seed solution is as follows: 100-120g of mannitol, 50-54g of yeast powder, 5g of potassium dihydrogen phosphate, 2g of magnesium sulfate heptahydrate, 1g of sodium chloride, and 100g of alfalfa extract are added to 1L of deionized water, the pH is adjusted to 7.0-7.2, sterilized by high-pressure steam at 121℃ for 20min, cooled to room temperature, and then inoculated with 1-2% (v / v) rhizobium. The solution is then cultured at 28-30℃ with shaking at 150rpm for 24-30h to obtain the rhizobium seed solution.

[0031] Furthermore, the effective viable count of the rhizobium seed solution is 2.69 × 10⁻⁶. 9 CFU / mL -3.29×10 9 CFU / mL.

[0032] Furthermore, the *Trichoderma echinosporum* spore suspension is prepared by the following steps: Step B1: Use a sterile inoculation loop to pick up the standard strain of Trichoderma echinosporum and streak it in three zones on potato dextrose agar medium. Heat the medium to 28-30℃ and incubate for 44-48 hours to obtain the activated strain. Step B2: Using a sterile inoculation needle, pick a 5mm × 5mm piece of mycelial growth from the edge of the activated strain and transfer it to the center of potato dextrose agar slant medium. Heat to 28-30℃ and incubate in the dark for 7-10 days. Add sodium chloride mixture, scrape, filter, and wash to obtain Trichoderma spore suspension.

[0033] Furthermore, the effective viable count of the *Trichoderma echinosporum* spore suspension is 7.75 × 10⁻⁶. 9 CFU / mL -9.47×10 9 CFU / mL.

[0034] Furthermore, the sodium chloride mixture is composed of a 0.9% sodium chloride aqueous solution and Tween-80 mixed at a mass ratio of 1800-2000:1.

[0035] Furthermore, the preparation method of the compound microbial fertilizer for enhancing the fertility of degraded grassland includes the following steps: The fermentation base substrate, compound microbial agent and slow-release nutrient additive are mixed evenly, heated to 30-32℃, pre-fermented for 24-28 hours, then heated to 34-36℃, fermented for 36-40 hours to obtain the fermentation product, then sprayed with compound protective agent, dried, crushed and sieved to obtain compound microbial fertilizer that improves the fertility of degraded grassland.

[0036] Furthermore, the specific parameters for the spraying are: spraying temperature of 40-50℃, spraying pressure of 8-10MPa, and spraying rate of 4-5mL / min.

[0037] Compared with the prior art, the present invention has the following beneficial effects: To improve the fertility and survival rate of microorganisms in degraded grasslands, this invention addresses the issue from three aspects. First, a composite protectant is added. This composite protectant is obtained by mixing phosphorylated chitosan, polyglutamic acid, and glycerol in a specific ratio. The phosphate groups in phosphorylated chitosan can not only strongly bind with the calcium and potassium ions in the phosphate rock powder in the slow-release nutrient additive to prevent nutrient loss, but also form a physical protective film on the surface of microorganisms or fertilizer particles. Polyglutamic acid can act as a water-retaining agent, creating a good water film environment in arid soils to maintain the life activities of plants and microorganisms. Glycerol can help maintain the local humidity of fertilizer particles or soil, and under dry or low-temperature stress, it can help microbial cells maintain osmotic balance and prevent cell dehydration and rupture. Secondly, a slow-release nutrient additive was added. This additive is made by mixing phosphate rock powder, potassium sulfate, amino acid powder, and sodium sulfonate aqueous solution in a specific ratio. Phosphate rock powder not only provides a slow-release phosphorus and calcium source, but also provides a stable supply over a long period, which can improve the phosphorus deficiency in the soil. In addition, it can indirectly promote rhizosphere microorganisms by improving plant growth. Potassium sulfate provides the potassium source required for plant growth, while amino acids provide organic nitrogen and small molecule carbon sources, stimulating plant and microbial activity. The sodium sulfonate aqueous solution has water retention properties, which can not only significantly improve the soil microenvironment, but also physically cross-link with the phosphorylated chitosan contained in the composite protectant, synergistically enhancing the fertility of degraded grassland. Third, a compound microbial agent was added. The compound microbial agent contains Bacillus candidiasis, which can secrete phosphatase and organic acids, which can dissolve insoluble phosphorus in the soil and improve phosphorus availability. At the same time, it can enhance nitrogenase activity and increase nitrogen supply. Burkholderia can degrade complex organic matter such as cellulose and hemicellulose in the soil, release carbon sources and nutrients, and promote microbial metabolic cycle. Streptomyces can secrete plant hormones such as indoleacetic acid and gibberellin, which can stimulate the development of pasture roots, increase the number of root hairs, and promote nutrient absorption. Rhizobium can form root nodules with leguminous plants, fix atmospheric nitrogen, increase soil nitrogen content, and reduce dependence on chemical nitrogen fertilizers. Trichoderma echinosporum can secrete cellulase to degrade plant disease residues and reduce disease recurrence. Through the synergistic effect between the substances, the fertility of degraded grassland is effectively improved. Detailed Implementation

[0038] To make the embodiments of the present invention easier to understand, the present invention will be described in detail below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not limited to the application scope of the present invention.

[0039] The standard strain of *Trichoderma echinosporum* was purchased from Wuhan Gray Algae Biotechnology Co., Ltd.; *Bacillus mucilaginosus*, *Burkholderia*, and *Rhizobium* were all purchased from Beijing Tianyou Fukang Biotechnology Co., Ltd.; alfalfa extract was purchased from Ningxia Xiangcao Biotechnology Co., Ltd.; humic acid was purchased from (Hubei) Chemical Co., Ltd., CAS number 1415-93-6; vermiculite powder was purchased from Lingshou County Zhanxing Mineral Products Co., Ltd., with a mesh size of 200 and a density of 1.8-2.32 g / cm³. 3 Potato dextrose agar slant culture medium was purchased from Shandong Top Biotechnology Co., Ltd.; sodium alginate was purchased from Jiangsu Jiujia Biotechnology Co., Ltd., CAS No. 9005-38-3; amino acid raw powder was purchased from Shandong Kunbo Biotechnology Co., Ltd.; chitosan was purchased from Jiangsu Aofu Biotechnology Co., Ltd., CAS No. 9012-76-4; sheep manure fermentation product was purchased from Shijiazhuang Gaocheng District Fada Bio-fermentation Fertilizer Co., Ltd.; corn straw powder was purchased from Zhengyang County Yongjia Grass Industry; polyglutamic acid was purchased from Hubei Darli Chemical Co., Ltd., CAS No. 25513-46-6; phosphate rock powder was purchased from Jinan Jibin Chemical Co., Ltd.

[0040] The present invention will be further described in detail below with reference to embodiments and comparative examples.

[0041] Examples 1-3 and Comparative Examples 1-4 provide a compound microbial fertilizer for improving the fertility of degraded grasslands.

[0042] Example 1 This embodiment provides a compound microbial fertilizer for improving the fertility of degraded grasslands, composed of the following raw materials in parts by weight: 82 parts fermentation base substrate, 12 parts compound microbial agent, 1.4 parts compound protective agent, and 3 parts slow-release nutrient additive. The fermentation base substrate is prepared by mixing sheep manure fermentation product, corn straw powder, humic acid, vermiculite powder, and sterile water in a mass ratio of 32:26:16:8:50 at 60 rpm for 16 minutes until homogeneous, and then sterilizing at 121℃ for 1.6 hours. The compound protective agent is composed of phosphorylated chitosan, polyglutamic acid, and glycerol in a mass ratio of 2:1:0.6. The slow-release nutrient additive is composed of phosphate rock powder, potassium sulfate, and amino acid raw powder in a mass ratio of 1.5:1:0.6. The sulfonated sodium alginate aqueous solution is composed of sulfonated sodium alginate and deionized water in a mass ratio of 30:100. The method for preparing the compound microbial fertilizer for enhancing the fertility of degraded grassland includes the following steps: The fermentation base substrate, compound microbial agent, compound protective agent and slow-release nutrient additive were stirred at 20 rpm for 15 min until uniform, heated to 30℃ and pre-fermented for 24 h, then heated to 34℃ and fermented for 36 h to obtain the fermentation product. The compound protective agent was then sprayed on, and the mixture was dried at 40℃ for 1.6 h, pulverized and sieved through a 60-mesh sieve to obtain the compound microbial fertilizer that improves the fertility of degraded grassland. The specific spraying parameters were: spraying temperature of 40℃, spraying pressure of 8 MPa and spraying rate of 4 mL / min. The compound microbial agent is prepared by the following steps: Step A1: Inoculate the seed cultures of Bacillus cereus, Burkholderia spp., and Streptomyces spp. into a composite fermentation medium and culture for 42 hours to obtain a mixed fermentation broth. The volume ratio of the Bacillus cereus seed culture, Burkholderia spp., and Streptomyces spp. seed culture is 5:3:2. The composition of the composite fermentation medium is as follows: per liter of medium, it contains 10g of glucose, 5g of glycerol, 5g of peptone, 3g of yeast extract, 1g of NaCl, 1g of K2HPO4, 0.5g of MgSO4•7H2O, 0.5g of CaCO3, and 1.0g of silicate mineral powder. The pH of the medium is controlled at 7.0. The specific process parameters for cultivation are: temperature 30℃; aeration rate 0.5 VVM; and pH maintained at 6.8 during cultivation. Step A2: Inoculate the rhizobium seed culture into the mixed fermentation broth, controlling the inoculation amount of the rhizobium seed culture to be 5% of the mixed fermentation broth volume, and allow it to adapt statically for 1.8 hours. Then, inoculate the Trichoderma spore suspension into the mixed fermentation broth, controlling the inoculation amount of the Trichoderma spore suspension to be 3% of the mixed fermentation broth volume, and continue stirring for 0.9 hours. Co-culture for 24 hours to obtain the compound microbial agent. The specific process parameters for co-culture are: temperature 30℃; aeration rate 1 VVM; and pH maintained at 6.8 during the culture process. The preparation method of the Bacillus jellyoid seed culture is as follows: 15g of glucose, 8g of protein, 0.8g of potassium dihydrogen phosphate, and 0.4g of magnesium sulfate heptahydrate are added to 1L of deionized water. The pH is adjusted to 7.0 with 0.03M phosphate buffer. The culture is sterilized at 121℃ for 20min, cooled to room temperature, and then inoculated with 1% (v / v) Bacillus jellyoid. The culture is then incubated at 28℃ and 150rpm for 24h with shaking to obtain the Bacillus jellyoid seed culture. The effective viable count of the Bacillus jellyoid seed culture is 1.6×10¹. 0 CFU / mL; The method for preparing the Burkholderia seed culture is as follows: 120g of glucose, 60g of peptone, 30g of yeast extract, 6g of potassium dihydrogen phosphate, and 3g of magnesium sulfate heptahydrate are added to 1L of deionized water. The pH is adjusted to 7.0 with 0.03M phosphate buffer. The culture is sterilized at 121℃ for 20min, cooled to room temperature, and then inoculated with 1% (v / v) Burkholderia. The culture is then incubated at 28℃ with shaking at 150rpm for 22h to obtain the Burkholderia seed culture. The effective viable count of the Burkholderia seed culture is 5.92×10⁻⁶. 9 CFU / mL; The preparation method of the Streptomyces seed culture is as follows: 100g glucose, 50g glycerol, 40g peptone, 20g yeast powder, and 5g calcium carbonate are added to 1L of deionized water. The pH is adjusted to 7.0 with 0.03M phosphate buffer. The mixture is then autoclaved at 121℃ for 20min, cooled to room temperature, and inoculated with 1% (v / v) Streptomyces. The mixture is then cultured at 28℃ with shaking at 150rpm for 22h to obtain the Streptomyces seed culture. The effective viable count of the Streptomyces seed culture is 1.84×10⁻⁶. 9 CFU / mL; The method for preparing the rhizobium seed culture is as follows: 100g mannitol, 50g yeast powder, 5g potassium dihydrogen phosphate, 2g magnesium sulfate heptahydrate, 1g sodium chloride, and 100g alfalfa extract are added to 1L of deionized water. The pH is adjusted to 7.0 with 0.03M phosphate buffer. The mixture is then autoclaved at 121℃ for 20min, cooled to room temperature, and inoculated with 1% (v / v) rhizobium. The mixture is then cultured at 28℃ with shaking at 150rpm for 24h to obtain the rhizobium seed culture, wherein the effective viable count of the rhizobium seed culture is 2.69 × 10⁻⁶. 9 CFU / mL; The Trichoderma spore suspension was prepared by the following steps: Step B1: Use a sterile inoculation loop to pick up the standard strain of Trichoderma echinosporum and streak it in three zones on potato dextrose agar medium. Heat the medium to 28°C and incubate for 44 hours to obtain the activated strain. Step B2: Using a sterile inoculation needle, pick a 5mm × 5mm vigorous mycelial growth block from the edge of the activated strain and transfer it to the center of a potato dextrose agar slant. Incubate at 28℃ in the dark for 7 days. Add a sodium chloride mixture, scrape and wash, filter through a 0.22μm membrane filter, and then wash three times with a 0.85% sodium chloride aqueous solution (each time the amount of sodium chloride aqueous solution is 15% of the mass of the sodium chloride mixture) to obtain a Trichoderma echinosporum spore suspension. The sodium chloride mixture consists of a 0.9% sodium chloride aqueous solution and Tween-80 at a mass ratio of 1800:1. The effective viable count of the Trichoderma echinosporum spore suspension is 7.75 × 10⁻⁶. 9 CFU / mL; The phosphorylated chitosan is prepared by the following steps: Chitosan was dissolved in a 1% (w / w) aqueous acetic acid solution, and a 28% (w / w) aqueous phosphoric acid solution was added. The mixture was stirred at 500 rpm for 26 min and dried at 55°C to constant weight to obtain phosphorylated chitosan. The mass ratio of chitosan, aqueous acetic acid solution and aqueous phosphoric acid solution was 1:20:6. The sulfonated sodium alginate is prepared by the following steps: Sodium alginate was added to dichloromethane and stirred for 12 minutes at 400-500 rpm in an ice bath. While stirring, a mixture of chlorosulfonic acid and dichloromethane was added dropwise at a rate of 3 drops / second. After the addition was complete, the temperature was raised to 26°C, and the stirring speed was maintained. The mixture was stirred for 1.8 hours, filtered, and deionized water was added. The pH was adjusted to neutral with an 8% sodium hydroxide aqueous solution. The mixture was dialyzed with deionized water for 20 hours through a dialysis bag with a molecular weight cutoff of 3500 Da. Anhydrous ethanol was added, and the mixture was allowed to stand for 0.6 hours. The mixture was then freeze-dried at -35°C to constant weight to obtain sulfonated sodium alginate. The mass ratio of sodium alginate, dichloromethane, the mixture, deionized water, and anhydrous ethanol was 1:15:6:40:20. The mass ratio of chlorosulfonic acid to dichloromethane in the mixture was 1:2.

[0043] Example 2 This embodiment provides a compound microbial fertilizer for improving the fertility of degraded grasslands, composed of the following raw materials in parts by weight: 83 parts fermentation base substrate, 13 parts compound microbial agent, 1.6 parts compound protective agent, and 4 parts slow-release nutrient additive. The fermentation base substrate is prepared by mixing sheep manure fermentation product, corn straw powder, humic acid, vermiculite powder, and sterile water in a mass ratio of 33:27:17:9:60 at 70 rpm for 18 minutes until homogeneous, and then sterilizing at 121℃ for 1.8 hours. The compound protective agent is composed of phosphorylated chitosan, polyglutamic acid, and glycerol in a mass ratio of 2.5:1:0.8. The slow-release nutrient additive is composed of phosphate rock powder, potassium sulfate, and amino acid raw powder in a mass ratio of 2.0:1.2:0.8. The sulfonated sodium alginate aqueous solution is composed of sulfonated sodium alginate and deionized water in a mass ratio of 35:100. The method for preparing the compound microbial fertilizer for enhancing the fertility of degraded grassland includes the following steps: The fermentation substrate, compound microbial agent, and slow-release nutrient additive were stirred at 25 rpm for 20 minutes until homogeneous. The mixture was then heated to 31°C and pre-fermented for 26 hours, followed by further heating to 35°C and fermentation for 38 hours to obtain the fermentation product. A compound protective agent was then sprayed onto the product, which was dried at 42°C for 1.8 hours, pulverized, and sieved through a 70-mesh screen to obtain a compound microbial fertilizer for improving the fertility of degraded grasslands. The specific spraying parameters were: spraying temperature 45°C, spraying pressure 9 MPa, and spraying rate 4.5 mL / min. The compound microbial agent was prepared using the following steps: Step A1: Inoculate the seed cultures of Bacillus cereus, Burkholderia spp., and Streptomyces spp. into a composite fermentation medium and culture for 44 hours to obtain a mixed fermentation broth. The volume ratio of the Bacillus cereus seed culture, Burkholderia spp., and Streptomyces spp. seed culture is 6.5:4:3. The composition of the composite fermentation medium is as follows: per liter of medium, it contains 10g glucose, 5g glycerol, 5g peptone, 3g yeast extract, 1g NaCl, 1g K2HPO4, 0.5g MgSO4•7H2O, 0.5g CaCO3, and 1.0g silicate mineral powder. The pH of the medium is controlled at 7.1. The specific process parameters for cultivation are: temperature 31℃; aeration rate 0.55 VVM; and pH maintained at 7 during cultivation. Step A2: Inoculate the rhizobium seed culture into the mixed fermentation broth, controlling the inoculation amount of the rhizobium seed culture to be 5.5% of the mixed fermentation broth volume. Allow it to acclimate statically for 2 hours. Then, inoculate the Trichoderma spore suspension into the mixed fermentation broth, controlling the inoculation amount of the Trichoderma spore suspension to be 3.5% of the mixed fermentation broth volume. Continue stirring for 1 hour and co-culture for 28 hours to obtain the compound microbial inoculum. The specific process parameters for co-culture are: temperature 31℃; aeration rate 1.2 VVM; and pH maintained at 7.0 during the culture process. The preparation method of the Bacillus jellyoid seed culture is as follows: 15.5g of glucose, 8.5g of protein, 0.9g of potassium dihydrogen phosphate, and 0.5g of magnesium sulfate heptahydrate are added to 1L of deionized water. The pH is adjusted to 7.1 with 0.03M phosphate buffer. The culture is sterilized at 121℃ for 20min, cooled to room temperature, and then inoculated with 1.5% (v / v) Bacillus jellyoid. The culture is carried out at 29℃ and 150rpm with shaking for 36h to obtain the Bacillus jellyoid seed culture. The effective viable count of the Bacillus jellyoid seed culture is 1.78×10¹. 0 CFU / mL; The method for preparing the Burkholderia seed culture is as follows: 130g of glucose, 70g of peptone, 35g of yeast extract, 7g of potassium dihydrogen phosphate, and 4g of magnesium sulfate heptahydrate are added to 1L of deionized water. The pH is adjusted to 7.1 with 0.03M phosphate buffer. The culture is sterilized at 121℃ for 20min, cooled to room temperature, and then inoculated with 1.5% (v / v) Burkholderia. The culture is then incubated at 29℃ with shaking at 150rpm for 24h to obtain the Burkholderia seed culture. The effective viable count of the Burkholderia seed culture is 6.58 × 10⁻⁶. 9 CFU / mL; The preparation method of the Streptomyces seed culture is as follows: 110g of glucose, 52g of glycerol, 43g of peptone, 21g of yeast powder, and 6g of calcium carbonate are added to 1L of deionized water. The pH is adjusted to 7.1 with 0.03M phosphate buffer. The mixture is then autoclaved at 121℃ for 20min, cooled to room temperature, and inoculated with 1.5% (v / v) Streptomyces. The mixture is then cultured at 29℃ and 150rpm with shaking for 24h to obtain the Streptomyces seed culture. The effective viable count of the Streptomyces seed culture is 2.04 × 10⁻⁶. 9 CFU / mL; The method for preparing the rhizobium seed culture is as follows: 110g mannitol, 52g yeast powder, 5g potassium dihydrogen phosphate, 2g magnesium sulfate heptahydrate, 1g sodium chloride, and 100g alfalfa extract are added to 1L of deionized water. The pH is adjusted to 7.1 with 0.03M phosphate buffer. The mixture is then autoclaved at 121℃ for 20min, cooled to room temperature, and inoculated with 1.5% (v / v) rhizobium. The mixture is then cultured at 29℃ and 150rpm for 27h with shaking to obtain the rhizobium seed culture, wherein the effective viable count of the rhizobium seed culture is 2.99 × 10⁻⁶. 9 CFU / mL; The Trichoderma spore suspension was prepared by the following steps: Step B1: Use a sterile inoculation loop to pick up the standard strain of Trichoderma echinosporum and streak it in three zones on potato dextrose agar medium. Heat the medium to 29°C and incubate for 46 hours to obtain the activated strain. Step B2: Using a sterile inoculation needle, pick a 5mm × 5mm vigorous mycelial growth block from the edge of the activated strain and transfer it to the center of a potato dextrose agar slant. Incubate at 29℃ in the dark for 8 days. Add a sodium chloride mixture, scrape and wash, filter through a 0.22μm membrane filter, and then wash four times with a 0.85% sodium chloride aqueous solution (each time the amount of sodium chloride aqueous solution is 15% of the mass of the sodium chloride mixture) to obtain a Trichoderma echinosporum spore suspension. The sodium chloride mixture consists of a 0.9% sodium chloride aqueous solution and Tween-80 at a mass ratio of 1900:1. The effective viable count of the Trichoderma echinosporum spore suspension is 8.61 × 10⁻⁶. 9 CFU / mL; The phosphorylated chitosan is prepared by the following steps: Chitosan was dissolved in a 2% (w / w) aqueous acetic acid solution, and a 29% (w / w) aqueous phosphoric acid solution was added. The mixture was stirred at 550 rpm for 30 min and dried at 60°C to constant weight to obtain phosphorylated chitosan. The mass ratio of chitosan, aqueous acetic acid solution and aqueous phosphoric acid solution was 1:25:7. The sulfonated sodium alginate is prepared by the following steps: Sodium alginate was added to dichloromethane and stirred at 450 rpm for 14 min in an ice bath. While stirring, a mixture of chlorosulfonic acid and dichloromethane was added dropwise at a rate of 3-5 drops / second. After the addition was complete, the temperature was raised to 28°C, and the stirring speed was maintained. The mixture was stirred for 1.9 h, filtered, and deionized water was added. The pH was adjusted to neutral with a 9% sodium hydroxide aqueous solution. The mixture was dialyzed with deionized water for 22 h through a dialysis bag with a molecular weight cutoff of 3500 Da. Anhydrous ethanol was added, and the mixture was allowed to stand for 0.7 h. The mixture was then freeze-dried at -35°C to constant weight to obtain sulfonated sodium alginate. The mass ratio of sodium alginate, dichloromethane, the mixture, deionized water, and anhydrous ethanol was 1:20:8:45:25. The mass ratio of chlorosulfonic acid to dichloromethane in the mixture was 1:2.5.

[0044] Example 3 This embodiment provides a compound microbial fertilizer for improving the fertility of degraded grasslands, composed of the following raw materials in parts by weight: 84 parts fermentation base substrate, 14 parts compound microbial agent, 1.8 parts compound protective agent, and 5 parts slow-release nutrient additive. The fermentation base substrate is prepared by mixing sheep manure fermentation product, corn straw powder, humic acid, vermiculite powder, and sterile water in a mass ratio of 34:28:18:10:70 at 80 rpm for 20 minutes until homogeneous, and then sterilizing at 121℃ for 2 hours. The compound protective agent is composed of phosphorylated chitosan, polyglutamic acid, and glycerol in a mass ratio of 3:1:1. The slow-release nutrient additive is composed of phosphate rock powder, potassium sulfate, and amino acid raw powder in a mass ratio of 2.5:1.4:1. The sulfonated sodium alginate aqueous solution is composed of sulfonated sodium alginate and deionized water in a mass ratio of 40:100. The method for preparing the compound microbial fertilizer for enhancing the fertility of degraded grassland includes the following steps: The fermentation substrate, compound microbial agent, and slow-release nutrient additive were stirred at 30 rpm for 25 minutes until homogeneous. The mixture was then heated to 32°C and pre-fermented for 28 hours, followed by further heating to 36°C and fermentation for 40 hours to obtain the fermentation product. A compound protective agent was then sprayed onto the product, which was dried at 44°C for 2.0 hours, pulverized, and sieved through an 80-mesh screen to obtain a compound microbial fertilizer for improving the fertility of degraded grasslands. The specific spraying parameters were: spraying temperature of 50°C, spraying pressure of 10 MPa, and spraying rate of 5 mL / min. The compound microbial agent was prepared using the following steps: Step A1: Inoculate the seed cultures of Bacillus cereus, Burkholderia spp., and Streptomyces spp. into a composite fermentation medium and culture for 46 hours to obtain a mixed fermentation broth. The volume ratio of the Bacillus cereus seed culture, Burkholderia spp., and Streptomyces spp. seed culture is 8:5:4. The composition of the composite fermentation medium is as follows: per liter of medium, it contains 10g glucose, 5g glycerol, 5g peptone, 3g yeast extract, 1g NaCl, 1g K2HPO4, 0.5g MgSO4•7H2O, 0.5g CaCO3, and 1.0g silicate mineral powder. The pH of the medium is controlled at 7.2. The specific process parameters for cultivation are: temperature 32℃; aeration rate 0.6 VVM; and maintaining pH at 7.2 during cultivation. Step A2: Inoculate the rhizobium seed culture into the mixed fermentation broth, controlling the inoculation amount of the rhizobium seed culture to be 6% of the mixed fermentation broth volume, and allow it to adapt statically for 2.2 hours. Then, inoculate the Trichoderma spore suspension into the mixed fermentation broth, controlling the inoculation amount of the Trichoderma spore suspension to be 4% of the mixed fermentation broth volume, and continue stirring for 1.1 hours. Co-culture for 32 hours to obtain the compound microbial inoculum. The specific process parameters for co-culture are: temperature 32℃; aeration rate 1.4 VVM; and pH maintained at 7.2 during the culture process. The preparation method of the Bacillus jellyoid seed culture is as follows: 16g of glucose, 9g of protein, 1.0g of potassium dihydrogen phosphate, and 0.6g of magnesium sulfate heptahydrate are added to 1L of deionized water, the pH is adjusted to 7.2, sterilized at 121℃ for 20min, cooled to room temperature, and then inoculated with 2% (v / v) Bacillus jellyoid. The culture is then incubated at 30℃ and 150rpm with shaking for 48h to obtain the Bacillus jellyoid seed culture. The effective viable count of the Bacillus jellyoid seed culture is 1.96×10¹. 0 CFU / mL; The method for preparing the Burkholderia seed culture is as follows: 140g of glucose, 80g of peptone, 40g of yeast powder, 8g of potassium dihydrogen phosphate, and 5g of magnesium sulfate heptahydrate are added to 1L of deionized water, the pH is adjusted to 7.2, sterilized at 121℃ for 20min, cooled to room temperature, and then inoculated with 2% (v / v) Burkholderia. The culture is then incubated at 30℃ and 150rpm with shaking for 26h to obtain the Burkholderia seed culture. The effective viable count of the Burkholderia seed culture is 7.24 × 10⁻⁶. 9 CFU / mL; The preparation method of the Streptomyces seed culture is as follows: 120g of glucose, 54g of glycerol, 46g of peptone, 22g of yeast powder, and 7g of calcium carbonate are added to 1L of deionized water, the pH is adjusted to 7.2, and the mixture is autoclaved at 121℃ for 20min. After cooling to room temperature, 2% (v / v) Streptomyces is inoculated and cultured at 30℃ and 150rpm for 26h with shaking to obtain the Streptomyces seed culture. The effective viable count of the Streptomyces seed culture is 2.24×10⁻⁶. 9CFU / mL; The method for preparing the rhizobium seed culture is as follows: 120g mannitol, 54g yeast powder, 5g potassium dihydrogen phosphate, 2g magnesium sulfate heptahydrate, 1g sodium chloride, and 100g alfalfa extract are added to 1L of deionized water. The pH is adjusted to 7.2 with 0.03M phosphate buffer. The mixture is then autoclaved at 121℃ for 20min, cooled to room temperature, and inoculated with 2% (v / v) rhizobium. The mixture is then cultured at 30℃ and 150rpm with shaking for 30h to obtain the rhizobium seed culture. The effective viable count of the rhizobium seed culture is 3.29 × 10⁻⁶. 9 CFU / mL; The Trichoderma spore suspension was prepared by the following steps: Step B1: Use a sterile inoculation loop to pick up the standard strain of Trichoderma echinosporum and streak it in three zones on potato dextrose agar medium. Heat the medium to 30°C and incubate for 48 hours to obtain the activated strain. Step B2: Using a sterile inoculation needle, pick a 5mm × 5mm vigorous mycelial growth block from the edge of the activated strain and transfer it to the center of a potato dextrose agar slant. Incubate at 30℃ in the dark for 10 days. Add a sodium chloride mixture, scrape and wash, filter through a 0.22μm membrane filter, and then wash three times with a 0.85% sodium chloride aqueous solution (each time the amount of sodium chloride aqueous solution is 15% of the mass of the sodium chloride mixture) to obtain a Trichoderma echinosporum spore suspension. The sodium chloride mixture consists of a 0.9% sodium chloride aqueous solution and Tween-80 at a mass ratio of 2000:1. The effective viable count of the Trichoderma echinosporum spore suspension is 9.47 × 10⁻⁶. 9 CFU / mL; The phosphorylated chitosan is prepared by the following steps: Chitosan was dissolved in a 3% (w / w) aqueous acetic acid solution, and a 30% (w / w) aqueous phosphoric acid solution was added. The mixture was stirred at 600 rpm for 34 min and dried at 65°C to constant weight to obtain phosphorylated chitosan. The mass ratio of chitosan, aqueous acetic acid solution and aqueous phosphoric acid solution was 1:30:8. The sulfonated sodium alginate is prepared by the following steps: Sodium alginate was added to dichloromethane and stirred at 500 rpm for 16 min in an ice bath. While stirring, a mixture of chlorosulfonic acid and dichloromethane was added dropwise at a rate of 5 drops / second. After the addition was complete, the temperature was raised to 30°C, and the stirring speed was maintained at a constant. The mixture was stirred for 2 h, filtered, and then deionized water was added. The pH was adjusted to neutral with a 10% sodium hydroxide aqueous solution. The mixture was dialyzed with deionized water for 24 h through a dialysis bag with a molecular weight cutoff of 3500 Da. Anhydrous ethanol was added, and the mixture was allowed to stand for 0.8 h. The mixture was then freeze-dried at -35°C to constant weight to obtain sulfonated sodium alginate. The mass ratio of sodium alginate, dichloromethane, the mixture, deionized water, and anhydrous ethanol was 1:25:10:50:30. The mass ratio of chlorosulfonic acid to dichloromethane in the mixture was 1:3.

[0045] Comparative Example 1 Comparative Example 1 is the same as Example 1, except that phosphorylated chitosan is replaced with an equal mass of chitosan, while the other steps and raw materials are the same as in Example 1.

[0046] Comparative Example 2 Comparative Example 2 is the same as Example 1, except that the sulfonated sodium alginate aqueous solution is replaced with an equal mass fraction of sodium alginate aqueous solution, while the remaining steps and raw materials are the same as in Example 1.

[0047] Comparative Example 3 Comparative Example 3 is the same as Example 1, except that Burkholderia seed liquid is replaced with an equal amount of Streptomyces seed liquid, while the other steps and raw materials are the same as in Example 1.

[0048] Comparative Example 4 Comparative Example 4 is the same as Example 1, except that the Streptomyces seed liquid is replaced with an equal amount of Burkholderia seed liquid, while the other steps and raw materials are the same as in Example 1.

[0049] Performance testing 1. Basic indicators of fertilizer: The test results were conducted in accordance with the Ministry of Agriculture and Rural Affairs industry standard NY / T 798-2015 "Compound Microbial Fertilizer". The test results are shown in Table 1. Table 1 Performance parameters of the compound microbial fertilizers prepared in Examples 1-3 and Comparative Examples 1-4 2. Production Increase Trial Experimental site: Degraded grassland in Fuyu County, Heilongjiang Province. The experimental site was divided into 9 experimental sites, each with an area of ​​20 mu, which were respectively designated as Example Groups 1-3, Comparative Groups 1-4, Experimental Control Group and Blank Control Group. Tested variety: Sheepgrass Zhongke No. 7; Trial period: April 15, 2024 - August 20, 2024; Experimental Design: For Example Groups 1-3, the base fertilizer was prepared using the compound microbial fertilizer (30 kg / mu) prepared in Examples 1-3, respectively. For Comparative Example Groups 1-4, the base fertilizer was prepared using the compound microbial fertilizer (30 kg / mu) prepared in Comparative Example 1-4, respectively. The experimental control group received conventional base fertilizer (conventional base fertilizer: monoammonium phosphate, 30 kg / mu), and the blank control group received no base fertilizer. Topdressing was applied at each experimental site using: monoammonium phosphate 15 kg / mu, urea 12 kg / mu, and potassium chloride 5 kg / mu. Management at each experimental site adhered to the "five unifications": unified sheepgrass variety, unified sowing time, unified fertilizer application rate, unified sowing density, and unified field management. Experimental methods: On April 15, 2024, deep tillage (25cm depth) was carried out at each experimental site using vibration. Base fertilizer was applied immediately afterward, followed by mulching. Seeds of Leymus chinensis No. 7 were sown next to the fertilizer application furrows, with an equal row spacing of 20cm and a plant spacing of 15cm, resulting in 18,000 seedlings per mu. Watering was carried out immediately after sowing, with an irrigation amount of 26 cubic meters per mu. Topdressing was applied once on May 20, 2024. On August 20, 2024, 10 plants were randomly selected for individual plant performance and yield per mu measurement. The test results are shown in Table 2.

[0050] Table 2. Characteristics and yield per mu of Leymus chinensis under different fertilizer treatments As shown in Tables 1 and 2, the compound microbial fertilizer prepared by this invention is not only significantly superior to the industry standards for compound microbial fertilizers set by the Ministry of Agriculture and Rural Affairs, but also significantly improves key growth indicators such as plant height, number of tillers, and number of leaves of Leymus chinensis in Leymus chinensis planting. At the same time, it greatly increases the aboveground biomass and yield per acre, demonstrating excellent growth-promoting and yield-increasing effects, and providing effective technical support for the ecological restoration of degraded grasslands and the efficient production of forage grass.

[0051] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A compound microbial fertilizer for enhancing the fertility of degraded grasslands, characterized in that, It is composed of the following raw materials in parts by weight: 82-84 parts fermentation base substrate, 12-14 parts compound microbial agent, 1.4-1.8 parts compound protective agent and 3-5 parts slow-release nutrient additive; The composite protective agent is composed of phosphorylated chitosan, polyglutamic acid, and glycerol in a mass ratio of 2-3:1:0.6-1; the slow-release nutrient additive is composed of phosphate rock powder, potassium sulfate, amino acid powder, and sulfonated sodium alginate aqueous solution in a mass ratio of 1.5-2.5:1-1.4:0.6-1:30-40; the sulfonated sodium alginate aqueous solution is composed of sulfonated sodium alginate and deionized water in a mass ratio of 0.4-0.6:

100.

2. The compound microbial fertilizer for enhancing the fertility of degraded grasslands according to claim 1, characterized in that, The fermentation substrate is prepared by mixing sheep manure fermentation product, corn stalk powder, humic acid, vermiculite powder and sterile water in a mass ratio of 32-34:26-28:16-18:8-10:50-70, and then sterilizing it.

3. The compound microbial fertilizer for enhancing the fertility of degraded grasslands according to claim 1, characterized in that, The compound microbial agent is prepared by the following steps: Step A1: Inoculate the Bacillus spp. seed culture, Burkholderia seed culture and Streptomyces seed culture into the compound fermentation medium and culture for 42-46 hours to obtain a mixed fermentation broth; Step A2: Inoculate the rhizobium seed liquid into the mixed fermentation broth and allow it to adapt statically for 1.8-2.2 hours. Then, inoculate the Trichoderma spore suspension into the mixed fermentation broth and continue stirring for 0.9-1.1 hours. Cultivate together for 24-32 hours to obtain the compound microbial agent.

4. The compound microbial fertilizer for enhancing the fertility of degraded grasslands according to claim 3, characterized in that, The volume ratio of the gelatinous Bacillus seed solution, Burkholderia seed solution, and Streptomyces seed solution is 5-8:3-5:2-4. In step A1, the specific process parameters for cultivation are: temperature 30-32℃; aeration rate 0.5-0.6 VVM; and pH maintained at 6.8-7.2 during cultivation.

5. The compound microbial fertilizer for enhancing the fertility of degraded grasslands according to claim 3, characterized in that, The Trichoderma spore suspension was prepared by the following steps: Step B1: Use a sterile inoculation loop to pick up the standard strain of Trichoderma echinosporum and streak it in three zones on potato dextrose agar medium. Heat the medium to 28-30℃ and incubate for 44-48 hours to obtain the activated strain. Step B2: Using a sterile inoculation needle, pick a 5mm × 5mm piece of mycelial growth from the edge of the activated strain and transfer it to the center of potato dextrose agar slant medium. Heat to 28-30℃ and incubate in the dark for 7-10 days. Add sodium chloride mixture, scrape, filter, and wash to obtain Trichoderma spore suspension.

6. The compound microbial fertilizer for enhancing the fertility of degraded grasslands according to claim 5, characterized in that, The sodium chloride mixture is composed of a 0.9% sodium chloride aqueous solution and Tween-80 mixed at a mass ratio of 1800-2000:

1.

7. The compound microbial fertilizer for enhancing the fertility of degraded grasslands according to claim 1, characterized in that, The phosphorylated chitosan is prepared by the following steps: Chitosan was dissolved in an aqueous acetic acid solution, and then an aqueous phosphoric acid solution was added. The mixture was stirred for 26-34 minutes and dried to obtain phosphorylated chitosan.

8. The compound microbial fertilizer for enhancing the fertility of degraded grasslands according to claim 7, characterized in that, The mass ratio of chitosan, aqueous acetic acid solution, and aqueous phosphoric acid solution is 1:20-30:6-8.

9. The compound microbial fertilizer for enhancing the fertility of degraded grasslands according to claim 1, characterized in that, The sulfonated sodium alginate is prepared by the following steps: Sodium alginate was added to dichloromethane and stirred in an ice bath for 12-16 minutes. While stirring, a mixture of chlorosulfonic acid and dichloromethane was added dropwise. After the addition was complete, the temperature was raised to 26-30℃ and the reaction was stirred for 1.8-2 hours. The mixture was filtered, and deionized water was added to adjust the pH to neutral. The mixture was dialyzed, and anhydrous ethanol was added. The mixture was allowed to stand and dried to obtain sulfonated sodium alginate.

10. The compound microbial fertilizer for enhancing the fertility of degraded grasslands according to claim 9, characterized in that, The mass ratio of sodium alginate, dichloromethane, the mixture, deionized water, and anhydrous ethanol is 1:15-25:6-10:40-50:20-30, and the mass ratio of chlorosulfonic acid to dichloromethane in the mixture is 1:2-3.

Images