Preparation method and application of composite microbial agent chelated with trace element fertilizer

Abstract

The application discloses a preparation method and application of a composite microbial agent chelated trace element fertilizer, relates to the technical field of microbial fertilizers, and discloses the following steps: multi-stage step-by-step composite fermentation of butyric acid clostridium, enterococcus faecium, bacillus subtilis and bacillus licheniformis, and biological chelation of trace elements by means of natural organic acid and polysaccharide generated by microbial metabolism, so that the fixed calcium, magnesium, iron, zinc, copper and boron in the soil can be activated, nutrients can be released for a long time, leaching loss can be reduced, soil organic matter and porosity can be simultaneously improved, soil salinity and conductivity can be reduced, the soil pH can be adjusted to neutral, soil degradation problems such as compaction, salinization and acidification caused by continuous cropping can be efficiently improved, and the soil nutrient structure can be balanced.

Description

Technical Field

[0001] This invention relates to the field of microbial fertilizer technology, specifically to the preparation method and application of compound microbial inoculant chelated micronutrient fertilizer. Background Technology

[0002] In modern agricultural production systems, the large-scale and routine application of chemical fertilizers and pesticides has become the mainstream practice to ensure high and stable crop yields and improve agricultural production efficiency. For a long time, farmers have relied on the large-scale application of macro-element fertilizers such as nitrogen, phosphorus, and potassium to rapidly increase crop yields, supplemented by pesticides to control field pests and diseases. This approach has effectively addressed the core needs of increasing and stabilizing grain production. However, the long-term excessive and single-variety application of chemical fertilizers and pesticides has triggered a series of chain reactions, including soil degradation, declining crop quality, and agricultural ecological imbalance, becoming a constraint on the green and sustainable development of modern agriculture. The core bottleneck of the development is that, according to data, the current utilization rate of chemical fertilizers is only about 40%, and more than 60% of nutrients continue to accumulate in the soil. This not only wastes resources but also disrupts the soil nutrient balance, inducing degradation phenomena such as compaction, salinization, and acidification. At the same time, the long-term excessive application of macronutrients leads to the continuous consumption and depletion of micronutrients such as calcium, magnesium, and iron, resulting in an increasingly unbalanced nutrient ratio. Meanwhile, the high-frequency and excessive application of pesticides causes residue accumulation, which seriously damages the soil microbial community structure, inhibits soil activity, and thus poses a safety hazard to agricultural products, seriously damaging their commercial value and food safety.

[0003] Although micronutrients are present in very low amounts in crops and soil, they are essential functional nutrients involved in key physiological processes such as photosynthesis, cell structure building, metabolism, and stress resistance. They play an irreplaceable regulatory role in crop yield and quality. However, the natural supply of micronutrients in my country is insufficient and varies significantly across regions: nationwide, 51% of arable land is deficient in zinc, 34% in boron, 21% in manganese, and 49% in selenium. Iron and manganese availability is low in calcareous soils in the north, while many southern provinces are deficient in magnesium and calcium. This leads to frequent occurrences of nutrient deficiency in crops. This leads to an increase in physiological diseases, weakened growth, reduced yield, and deterioration in quality. Even with continuous application of macronutrient fertilizers, it is difficult to reverse this trend, ultimately creating a vicious cycle of "the more fertilizer applied, the worse the growth and the lower the yield." To improve this situation, water-soluble fertilizers containing micronutrients have been introduced to the market, especially chelated fertilizers. These fertilizers form chelated nutrients by adding chelating agents to the fertilizer mix. They have advantages such as good water solubility, high root absorption and utilization rate, and are less likely to cause soil compaction. Therefore, they have become the main technical means to supplement micronutrients and improve soil.

[0004] However, existing micronutrient chelated fertilizers still have significant technical shortcomings: First, nutrients are released too quickly and are easily leached away by water, necessitating frequent topdressing. Furthermore, micronutrients are prone to antagonistic precipitation with phosphate, which not only clogs the channels but also reduces absorption efficiency, increasing planting costs without providing stable improvement effects. Second, the products have limited functions, passively supplementing micronutrients. They cannot improve the imbalance of macronutrient accumulation in the soil, nor can they activate nutrients already fixed in the soil. Moreover, the inorganic salt form of micronutrients is easily adsorbed and fixed by soil particles, resulting in low utilization. Third, these fertilizers lack pest and disease control capabilities and cannot replace chemical pesticides. Therefore, pesticide residues and soil microecological damage remain problems. In addition, commonly used chemically synthesized chelating agents (such as EDTA and DTPA) are difficult to biodegrade and pose a risk of heavy metal accumulation. Thus, they cannot meet the comprehensive needs of modern agriculture for long-term planting and green pest control.

[0005] In summary, the current technical challenges facing agricultural production include: soil nutrient imbalance, soil degradation, low fertilizer utilization, short-term effects of micronutrient supplementation, high pesticide dependence, and difficulty in ensuring the quality and safety of agricultural products. Although chelated micronutrient fertilizers have made some improvements compared to traditional inorganic salt micronutrient fertilizers, existing products still have limited functions and very limited improvement effects. They cannot yet meet the multiple needs of soil improvement, long-term fertilization, nutrient activation, green pest control, and increased yield and quality. Based on this, we now provide a method for preparing and applying compound microbial inoculant chelated micronutrient fertilizers, which can eliminate the drawbacks of existing technologies. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing and applying a compound microbial agent chelated trace element fertilizer, in order to solve the problems in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: The preparation method of compound microbial inoculant chelated trace element fertilizer includes the following steps: Step 1: Preparation of Clostridium butyricum fermentation centrifuge broth: Take the preserved Clostridium butyricum strain and activate it on a modified CM medium plate. Transfer it to the modified CM medium to prepare seed culture. Inoculate the seed culture at a volume ratio of 0.5% to 5% for fermentation. Centrifuge to obtain Clostridium butyricum centrifuge broth. Step 2: Preparation of Enterococcus faecalis fermentation broth: Enterococcus faecalis was activated using a modified MRS medium supplemented with 0.005–0.02 g / L vancomycin, then transferred to a dedicated fermentation medium and fermented with shaking to obtain Enterococcus faecalis fermentation broth; Step 3: Fermentation of Clostridium butyricum and Enterococcus faecalis compound inoculant: Add Clostridium butyricum centrifuged liquid to the fermenter, along with glucose, molasses, corn steep liquor, magnesium sulfate, manganese sulfate, dipotassium hydrogen phosphate, and sodium acetate (carbon-nitrogen nutrient salts). Inoculate with Enterococcus faecalis fermentation broth and stir at 30℃~40℃ for fermentation. After the pH drops to 3.8~4.2 for the first time, stop temperature control and allow the fermentation to continue at room temperature until the system pH ≤ 4.3, thus obtaining the Clostridium butyricum and Enterococcus faecalis compound microbial inoculant. Step 4: Preparation of mixed fermentation broth of Bacillus subtilis and Bacillus licheniformis: The two types of Bacillus were activated using broth activation medium, and then transferred to expansion medium for shaking culture to obtain the mixed fermentation broth. Step 5: High-temperature co-fermentation of four strains: Add the compound bacterial agent of Clostridium butyricum and Enterococcus faecalis into a 500L fermenter, inoculate with the mixed fermentation liquid of Bacillus subtilis and Bacillus licheniformis, and ferment at 45℃~60℃ with aeration and stirring for 12h~24h; Step Six: Chelating and Maturation of Trace Elements: After cooling to 25℃~35℃, add molasses, glucose, dipotassium hydrogen phosphate, light calcium carbonate, expanded urea, calcium nitrate, magnesium nitrate, ferric nitrate, zinc nitrate, boric acid, and copper sulfate pentahydrate in sequence. Stir evenly and then ferment for 18h~30h until the material is viscous and does not separate into layers. Cool to 25℃~35℃ to obtain the compound microbial agent chelated trace element fertilizer.

[0008] Based on the above technical solutions, the present invention also provides the following optional technical solutions: In one alternative: the seed culture conditions for Clostridium butyricum in step one are 32–38℃ for 12–18 h, and the seed culture fermentation conditions are 30–38℃ for 35–40 h. The preservation number of Clostridium butyricum is CCTCCNO.M2022151.

[0009] In one alternative scheme: the activation conditions for Enterococcus faecalis in step two are 33℃, 140r / min shaking culture for 18h, the fermentation medium formula is 5g glucose, 3g peptone, 2g yeast extract, 1g corn steep liquor powder, 0.1g magnesium sulfate heptahydrate, 1000mL purified water, pH 6.0±0.2, the fermentation parameters are 33℃, 120r / min, 30h, the inoculum size is 2.5% (v / v), and the preservation number of Enterococcus faecalis is CGMCCNO.36539.

[0010] In one alternative: the amount of each material added in step three is 50L of Clostridium butyricum centrifuged liquid combined with 1.5kg of glucose, 1kg of molasses, 0.5kg of corn steep liquor, 12.5g of MgSO4, 7.5g of MnSO4, 50g of K2HPO4, 300g of sodium acetate, and 2.5L of Enterococcus faecalis strain solution. The mixture is stirred at 100rpm at 37℃ and allowed to stand at room temperature for a total fermentation time of 96h.

[0011] In one alternative: the activation broth culture medium in step four consists of 2g tryptone, 2g NaCl, 1g yeast extract, 100mL purified water, and pH 6.8; the expansion culture medium consists of 2g glucose, 2g peptone, 1g yeast extract, 100mL purified water, and pH 6.8; both activation and expansion cultures are conducted at 30℃, 150r / min, and 48h; and the inoculum volume of the expansion culture medium is 8mL seed culture / 200mL culture medium.

[0012] In one alternative: the feeding ratio in step five is 320L of Clostridium butyricum-Enterococcus faecalis compound inoculant mixed with 50mL of Bacillus subtilis and Bacillus licheniformis mixed fermentation broth, with the stirring speed at 120r / min and continuous aeration throughout the fermentation process.

[0013] In one alternative: the amounts of trace elements and excipients added in step six are 3.5 kg of molasses, 2 kg of glucose, 0.8 kg of dipotassium hydrogen phosphate, 1.5 kg of light calcium carbonate, 0.8 kg of expanded urea, 2.2 kg of calcium nitrate, 1.0 kg of magnesium nitrate, 0.35 kg of ferric nitrate, 0.28 kg of zinc nitrate, 0.22 kg of boric acid, and 0.08 kg of copper sulfate pentahydrate. The preservation number of Bacillus subtilis is CCTCCNO.M20241168, and the preservation number of Bacillus licheniformis is CICC10037.

[0014] In one alternative approach: the standard for the effective live bacteria content of the compound microbial agent chelated with trace element fertilizer in step six is ​​Bacillus subtilis content ≥ 1.0 × 10⁻⁶. 8 CFU / mL, Bacillus licheniformis content ≥1.0×10 8 CFU / mL, lactic acid bacteria content ≥5.0×10 8 CFU / mL, Clostridium butyricum content ≥1.0×10 6 CFU / mL.

[0015] The compound microbial inoculant chelated micronutrient fertilizer is prepared using the above-mentioned method.

[0016] The application of compound microbial inoculant chelated micronutrient fertilizer: The compound microbial inoculant chelated micronutrient fertilizer is used to repair soil, inhibit soil-borne diseases, and improve crop yield and quality. The application method is to apply 20-100g / plant to the crop plants, or dilute 300 times and drip irrigate the plant roots, or apply 5L / mu with water, or dilute 300 times and spray on the leaves.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention utilizes a multi-stage, stepwise, compound fermentation process involving Clostridium butyricum, Enterococcus faecalis, Bacillus subtilis, and Bacillus licheniformis. By using the metabolic processes of these microorganisms to generate natural organic acids and polysaccharides, the micronutrients are bio-chelated. This process activates the calcium, magnesium, iron, zinc, copper, and boron that are fixed in the soil, providing a long-lasting, slow-release effect on nutrients, reducing leaching loss, and simultaneously increasing soil organic matter and porosity. It also reduces soil salinity and electrical conductivity, adjusts soil pH to neutral, and effectively improves soil degradation problems such as compaction, salinization, and acidification caused by continuous cropping, thereby balancing the soil nutrient structure.

[0018] 2. This invention produces antibacterial active substances through the symbiotic metabolism of anaerobic bacteria and aerobic Bacillus, which can significantly inhibit soil-borne diseases such as stem rot, with a disease control effect of up to 89.9%. This greatly reduces the use of chemical pesticides and lowers the risk of pesticide residues. At the same time, beneficial live bacteria can colonize the soil for a long time, reshape a healthy soil microbial community, and enhance soil biological activity.

[0019] 3. This invention supplements essential micronutrients for crops through the synergistic effect of compound microorganisms, promotes chlorophyll synthesis, and enhances photosynthetic efficiency. This can improve crop stress resistance, increase yield-related indicators related to fruit setting, and achieve significant yield increase. Moreover, the product has multiple functions such as soil remediation, nutrient activation, biological disease prevention, and quality improvement and yield increase. It can be combined with various application methods such as fertigation, drip irrigation, and foliar spraying, and is suitable for various field and cash crops. The comprehensive application effect is far superior to that of single chemical chelated micronutrient fertilizers. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the process flow of the present invention.

[0021] Figure 2 This is a schematic diagram of the process for preparing Clostridium butyricum fermentation centrifuge broth according to the present invention.

[0022] Figure 3 This is a schematic diagram of the process for preparing the Enterococcus faecalis fermentation broth according to the present invention.

[0023] Figure 4 This is a schematic diagram of the fermentation process of the compound bacterial agent of Clostridium butyricum and Enterococcus faecalis of the present invention.

[0024] Figure 5 This is a schematic diagram of the process for preparing the mixed fermentation broth of Bacillus subtilis and Bacillus licheniformis according to the present invention.

[0025] Figure 6 This is a schematic diagram of the process structure for the high-temperature co-fermentation of four strains and the chelation and maturation of trace elements according to the present invention.

[0026] Figure 7 This is a schematic diagram illustrating the ability of Clostridium butyricum to produce various enzymes according to the present invention.

[0027] Figure 8This is a schematic diagram of the live bacterial count growth curve of Clostridium butyricum according to the present invention.

[0028] Figure 9 This is a schematic diagram of Clostridium butyricum examination according to the present invention.

[0029] Figure 10 This is a schematic diagram of the Clostridium butyricum plate of the present invention.

[0030] Figure 11 This is a schematic diagram of the microscopic examination of Bacillus subtilis according to the present invention.

[0031] Figure 12 This is a schematic diagram of the Bacillus subtilis plate of the present invention.

[0032] Figure 13 This is a schematic diagram of the microscopic examination of Enterococcus faecalis according to the present invention.

[0033] Figure 14 This is a schematic diagram of the Enterococcus faecalis plate of the present invention. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Example

[0035] like Figures 1-14 As shown, the preparation method of compound microbial inoculant chelated micronutrient fertilizer includes the following steps: Step 1: Preparation of Clostridium butyricum fermentation centrifuge broth Clostridium butyricum with preservation number CCTCCNO.M2022151 was selected. The strain was activated on modified CM medium plates. The activated strain was inoculated into modified CM medium and cultured at 32-38℃ for 12-18h to obtain seed culture. The seed culture was inoculated into modified CM medium at a volume ratio of 1%-3% and fermented at 30-38℃ for 35-40h. The fermentation broth was centrifuged to obtain Clostridium butyricum centrifuged liquid. Step 2: Preparation of Enterococcus faecalis fermentation broth Enterococcus faecalis with preservation number CGMCCNO.36539 was selected and inoculated into modified MRS medium supplemented with 0.01 g / L vancomycin. Activation was completed by shaking culture at 33℃ and 140 r / min for 18 h. The culture was then transferred to fermentation medium (5 g glucose, 3 g peptone, 2 g yeast extract, 1 g corn steep liquor powder, 0.1 g magnesium sulfate heptahydrate, 1000 mL purified water, pH 6.0±0.2) at an inoculation rate of 2.5% (v / v) and cultured at 33℃ and 120 r / min for 30 h to obtain Enterococcus faecalis fermentation broth. Step 3: Fermentation with a compound inoculum of Clostridium butyricum and Enterococcus faecalis 50L of Clostridium butyricum centrifuged liquid was added to a fermenter, along with 1.5kg of glucose, 1kg of molasses, 0.5kg of corn steep liquor, 12.5g of MgSO4, 7.5g of MnSO4, 50g of K2HPO4, and 300g of sodium acetate. 2.5L of Enterococcus faecalis fermentation broth was then added. Fermentation was carried out at 37℃ and 100rpm with stirring. The pH was measured after 20 hours of fermentation. Temperature control was stopped after the pH first dropped to 4.0. Fermentation was allowed to continue at room temperature (25℃) for a total duration of 96 hours until the pH dropped below 4.3, thus obtaining a compound microbial agent of Clostridium butyricum and Enterococcus faecalis. Step 4: Preparation of mixed fermentation broth of Bacillus subtilis and Bacillus licheniformis Bacillus subtilis preservation number CCTCCNO.M20241168, Bacillus licheniformis preservation number CICC10037, activated broth medium: 2g tryptone, 2g NaCl, 1g yeast extract, 100mL purified water, pH 6.8. The two Bacillus slant cultures were inoculated into the activated medium and cultured at 30℃ and 150r / min for 48h. 8mL of the seed culture was then inoculated into 200mL expansion medium (2g glucose, 2g peptone, 1g yeast extract, 100mL purified water, pH 6.8) and cultured at 30℃ and 150r / min for 48h to obtain a mixed fermentation broth of Bacillus subtilis and Bacillus licheniformis. Step 5: Mix the four strains and co-ferment at high temperature Take 320L of Clostridium butyricum-Enterococcus faecalis compound inoculant and put it into a 500L fermenter. Add 50mL of Bacillus subtilis and Bacillus licheniformis mixed fermentation liquid. Control the fermentation temperature at 55℃. Stir at 120r / min throughout the process and continuously introduce air to keep the material turning. Ferment continuously for 16h. Step Six: Chelation and Maturation of Trace Element Compound After fermentation, the temperature is lowered to 30℃, and while stirring, 3.5kg of molasses, 2kg of glucose, 0.8kg of dipotassium hydrogen phosphate, 1.5kg of light calcium carbonate, 0.8kg of expanded urea, 2.2kg of calcium nitrate, 1.0kg of magnesium nitrate, 0.35kg of ferric nitrate, 0.28kg of zinc nitrate, 0.22kg of boric acid, and 0.08kg of copper sulfate pentahydrate are added in sequence. After all materials are added, they are stirred and mixed evenly, and then sealed for a second fermentation for 24 hours. Fermentation continues until the material becomes a viscous liquid without stratification. The temperature is then lowered to 30℃ again before discharging to obtain the finished compound microbial inoculant chelated trace element fertilizer. Viable bacteria count of finished product: Bacillus subtilis 1.6 × 10⁻⁶ 8 CFU / mL, Bacillus licheniformis 1.3×10 8 CFU / mL, lactic acid bacteria 6.2×10 8 CFU / mL, Clostridium butyricum 2.5×10 6 CFU / mL.

[0036] Comparative Example 1 The overall process steps are consistent with those in Example 1, except that: The fertilizer used is commercially available Stanley EDTA chelated calcium and magnesium water-soluble fertilizer. The calcium and magnesium content of the fertilizer is similar to that of the finished product in Example 1. It does not contain the four compound microbial strains of this invention, nor the multi-stage microbial fermentation preparation process of this invention.

[0037] Experimental Example 1 Field plots with long-term continuous cropping, compacted soil, and deficient micronutrients were selected and divided into two experimental plots, each with an area of ​​5m². 2 The plots were labeled as Example 1 and Comparative Example 1, respectively, and the initial physicochemical properties of the soil were the same (humic acid content 15.2 g / kg, calcium content 1180 mg / kg, magnesium content 370 mg / kg). In Example 1, the compound microbial agent chelated micronutrient water-soluble fertilizer prepared in Example 1 of this invention was applied at a rate of 5 kg / mu with irrigation water. Comparative Example 1 group was treated with an equal amount of commercially available conventional chemical chelated calcium magnesium water-soluble fertilizer (Stanley EDTA chelated calcium magnesium water-soluble fertilizer), and the application method was the same as that of Example 1 group. The experiment lasted for 30 days, with other field management practices remaining consistent. After the experiment, five sampling points were randomly selected, and 100g of soil from the 0-20cm topsoil layer was collected from each point. The soil physicochemical properties and the content of trace elements were tested (the testing methods are described in "Soil Agricultural Chemical Analysis Methods" (Lu Rukun, China Agricultural Science and Technology Press, 2000), "Soil Testing Part 2: Determination of Soil pH" (NY / T1121.2-2006), and "Soil Testing Part 16: Determination of Total Water-Soluble Salts in Soil" (NY / T1121.16-2006)). Table 1 As shown in Table 1, the product of Example 1 can effectively improve the soil and enhance the availability of calcium, magnesium, iron, zinc, copper, and boron. Compared with Comparative Example 1, the soil organic matter content of Example 1 increased by 120.0%, and the contents of calcium, magnesium, iron, zinc, copper, and boron increased by 17.8%, 17.9%, 206.2%, 761.5%, 197.6%, and 97.1%, respectively. The soil porosity increased by 12.9%, the soil pH value tended to be neutral, the soil electrical conductivity decreased by 69.6%, and the salinity decreased by 68.6%. It effectively improved the problems of soil compaction, salinity, and nutrient deficiency caused by long-term continuous cropping.

[0038] Experimental Example 2 Field trials were conducted during the wheat planting season, with two experimental plots, each with an area of ​​9 m², labeled as Example 1 and Comparative Example 1, respectively, while other field management practices remained consistent. In Example 1, the compound probiotic chelated trace element water-soluble fertilizer prepared in Example 1 of this invention was diluted 300 times and sprayed on the leaves. It was sprayed once each during the wheat overwintering period, jointing period, and grain filling period, for a total of 3 times. Comparative Example 1 group was treated with an equal amount of commercially available conventional chemical chelated calcium magnesium water-soluble fertilizer (Stanley EDTA chelated calcium magnesium water-soluble fertilizer), and the spraying method and cycle were the same as those of Example 1 group. During the growth process, the incidence rate, disease index, and control effect of wheat stem base rot were statistically analyzed. At the same time, chlorophyll content (SPAD value), number of ears per mu, number of grains per ear, thousand-grain weight, and yield were measured. The incidence rate of stem base rot was based on the standard statistical method (NY / T4179-2022). The diseased plant rate was statistically analyzed by randomly surveying 5 points, with 50 plants continuously surveyed at each point, for a total of 250 plants. The disease index was graded by randomly selecting 20 plants at each point, for a total of 100 plants at 5 points, and the disease level was determined. Diseased plant rate (%) = number of diseased plants ÷ total number of surveyed plants × 100. Chlorophyll content was measured using a SPAD-502 chlorophyll meter. Table 2 Table 3 As can be seen from Tables 2 and 3, the product of Example 1 has a control effect of 89.9% on wheat stem base rot, the chlorophyll content is 14.3% higher than that of Comparative Example 1, and the yield is 24.2% higher. This shows that the product of the present invention can not only supplement micronutrients, but also inhibit soil-borne diseases, enhance crop stress resistance and significantly increase yield through the synergistic effect of compound microorganisms.

[0039] The above description is merely a specific embodiment of this disclosure, but the scope of protection of this disclosure is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this disclosure should be included within the scope of protection of this disclosure. Therefore, the scope of protection of this disclosure should be determined by the scope of the claims.

Claims

1. A method for preparing compound microbial inoculant chelated micronutrient fertilizer, characterized in that, Includes the following steps: Step 1: Preparation of Clostridium butyricum fermentation centrifuge broth: Take the preserved Clostridium butyricum strain and activate it on a modified CM medium plate. Transfer it to the modified CM medium to prepare seed culture. Inoculate the seed culture at a volume ratio of 0.5% to 5% for fermentation. Centrifuge to obtain Clostridium butyricum centrifuge broth. Step 2: Preparation of Enterococcus faecalis fermentation broth: Enterococcus faecalis was activated using a modified MRS medium supplemented with 0.005–0.02 g / L vancomycin, then transferred to a dedicated fermentation medium and fermented with shaking to obtain Enterococcus faecalis fermentation broth; Step 3: Fermentation of Clostridium butyricum and Enterococcus faecalis compound inoculant: Add Clostridium butyricum centrifuged liquid to the fermenter, along with glucose, molasses, corn steep liquor, magnesium sulfate, manganese sulfate, dipotassium hydrogen phosphate, and sodium acetate (carbon-nitrogen nutrient salts). Inoculate with Enterococcus faecalis fermentation broth and stir at 30℃~40℃ for fermentation. After the pH drops to 3.8~4.2 for the first time, stop temperature control and allow the fermentation to continue at room temperature until the system pH ≤ 4.3, thus obtaining the Clostridium butyricum and Enterococcus faecalis compound microbial inoculant. Step 4: Preparation of mixed fermentation broth of Bacillus subtilis and Bacillus licheniformis: The two types of Bacillus were activated using broth activation medium, and then transferred to expansion medium for shaking culture to obtain the mixed fermentation broth. Step 5: High-temperature co-fermentation of four strains: Add the compound bacterial agent of Clostridium butyricum and Enterococcus faecalis into a 500L fermenter, inoculate with the mixed fermentation liquid of Bacillus subtilis and Bacillus licheniformis, and ferment at 45℃~60℃ with aeration and stirring for 12h~24h; Step Six: Chelating and Maturation of Trace Elements: After cooling to 25℃~35℃, add molasses, glucose, dipotassium hydrogen phosphate, light calcium carbonate, expanded urea, calcium nitrate, magnesium nitrate, ferric nitrate, zinc nitrate, boric acid, and copper sulfate pentahydrate in sequence. Stir evenly and then ferment for 18h~30h until the material is viscous and does not separate into layers. Cool to 25℃~35℃ to obtain the compound microbial agent chelated trace element fertilizer.

2. The preparation method of compound microbial agent chelated trace element fertilizer according to claim 1, characterized in that, The seed culture conditions for Clostridium butyricum in step one are 32–38℃ for 12–18 h, and the seed culture fermentation conditions are 30–38℃ for 35–40 h. The preservation number of Clostridium butyricum is CCTCCNO.M2022151.

3. The preparation method of the compound microbial agent chelated trace element fertilizer according to claim 1, characterized in that, The activation conditions for Enterococcus faecalis in step two were: 33℃, 140r / min shaking culture for 18h, and the fermentation medium formula was: 5g glucose, 3g peptone, 2g yeast extract, 1g corn steep liquor powder, 0.1g magnesium sulfate heptahydrate, 1000mL purified water, pH 6.0±0.

2. The fermentation parameters were: 33℃, 120r / min, 30h, and the inoculum size was 2.5% (v / v). The preservation number of Enterococcus faecalis was CGMCCNO.36539.

4. The method for preparing compound microbial inoculant chelated trace element fertilizer according to claim 1, characterized in that, In step three, the following amounts of materials were added: 50L of Clostridium butyricum centrifuged liquid mixed with 1.5kg of glucose, 1kg of molasses, 0.5kg of corn steep liquor, 12.5g of MgSO4, 7.5g of MnSO4, 50g of K2HPO4, 300g of sodium acetate, and 2.5L of Enterococcus faecalis strain solution. The mixture was stirred at 100rpm at 37℃ and allowed to stand at room temperature for a total fermentation time of 96h.

5. The method for preparing compound microbial inoculant chelated trace element fertilizer according to claim 1, characterized in that, The activation broth culture medium in step four consists of 2g tryptone, 2g NaCl, 1g yeast extract, 100mL purified water, and pH 6.

8. The expansion culture medium consists of 2g glucose, 2g peptone, 1g yeast extract, 100mL purified water, and pH 6.

8. Both activation and expansion cultures are conducted at 30℃, 150r / min, and for 48h. The inoculation volume of the expansion culture medium is 8mL seed culture / 200mL culture medium.

6. The method for preparing compound microbial inoculant chelated trace element fertilizer according to claim 1, characterized in that, In step five, the feeding ratio is 320L of Clostridium butyricum-Enterococcus faecalis compound inoculant mixed with 50mL of Bacillus subtilis and Bacillus licheniformis mixed fermentation broth. The stirring speed is 120r / min and aeration is continuous throughout the fermentation process.

7. The method for preparing compound microbial inoculant chelated trace element fertilizer according to claim 1, characterized in that, The amounts of trace elements and excipients added in step six are as follows: molasses 3.5 kg, glucose 2 kg, dipotassium hydrogen phosphate 0.8 kg, light calcium carbonate 1.5 kg, expanded urea 0.8 kg, calcium nitrate 2.2 kg, magnesium nitrate 1.0 kg, ferric nitrate 0.35 kg, zinc nitrate 0.28 kg, boric acid 0.22 kg, and copper sulfate pentahydrate 0.08 kg. The preservation number of Bacillus subtilis is CCTCCNO.M20241168, and the preservation number of Bacillus licheniformis is CICC10037.

8. The method for preparing compound microbial inoculant chelated trace element fertilizer according to claim 1, characterized in that, The standard for the effective live bacteria content of the compound microbial agent chelated with trace element fertilizer in step six is ​​Bacillus subtilis content ≥ 1.0 × 10⁻⁶. 8 CFU / mL, Bacillus licheniformis content ≥1.0×10 8 CFU / mL, lactic acid bacteria content ≥5.0×10 8 CFU / mL, Clostridium butyricum content ≥1.0×10 6 CFU / mL.

9. A compound microbial inoculant chelated micronutrient fertilizer, characterized in that, Prepared by the method described in any one of claims 1-8.

10. An application of the compound microbial agent chelated with trace elements fertilizer as described in claim 9, characterized in that, The compound microbial agent chelated with trace element fertilizer is used to repair soil, inhibit soil-borne diseases, and improve crop yield and quality. The application methods are as follows: apply 20-100g / plant to the crop plants, or dilute 300 times and drip irrigate the plant roots, or apply 5L / acre with water, or dilute 300 times and spray on the leaves.

Images