Preparation method of compound microbial fertilizer

Abstract

The invention discloses a preparation method of a compound microbial fertilizer, and belongs to the technical field of microbial fertilizers, the preparation method comprises the steps of starch gelatinization, strain activation, shake-flask culture, immobilization, fermentation and material mixing; the immobilization comprises the following steps: centrifuging a paenibacillus polymyxa shake-flask culture, and collecting wet thalli; mixing the wet thalli with normal saline, stirring at room temperature, adding the gelatinized starch solution, and continuously stirring to obtain a mixed bacterial solution; stirring a calcium chloride aqueous solution at 30-35 DEG C, dropwise adding the mixed bacteria solution, continuously stirring after dropwise adding, filtering, and taking filter residues to obtain immobilized paenibacillus polymyxa; the compound microbial fertilizer prepared by the invention is high in viable count, and the viable count is slightly influenced by the content of each substance in the corn steep liquor.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fertilizers, and specifically relates to a preparation method of a compound microbial fertilizer. Background Art

[0002] The corn processing industry is the main front for the processing of bulk agricultural products. Products such as starch, syrup, corn oil, and crude fiber produced are all very important food and chemical raw materials. Among them, corn starch is the main resource for producing glucose syrup and various sugar derivatives. By deeply processing corn seeds, specifically through processes such as soaking, crushing, screening, drying, concentration, washing, and extraction, corn starch can be obtained. At the same time, by-products such as crude fiber feed, corn protein powder, and corn syrup are also produced. Corn syrup is a type of high-concentration organic wastewater concentrated from the wastewater generated after soaking corn starch with sulfurous acid during wet milling processing, and belongs to agricultural product waste. Research progress on the development of biological bacterial fertilizers from agricultural product processing waste. Zhao Xinhe, He Zhuangzhuang, Li Hongjun, Zhao Yubin, Ma Chengye. Chinese Soil and Fertilizer 2019(6). It was publicly reported in December 2019 that the protein (wet basis) content is about 21.40%, the ash content is about 15.40%, the total acid content is about 7.69%, the aflatoxin content is about 2.91 μg / L, the zearalenone content is about 556.32 μg / L, and the vomitoxin content is about 1193 μg / L. Moreover, corn syrup dry powder contains rich amino acids and has high utilization value. However, as a complex multiphase system, corn syrup is composed of various soluble substances and low-solubility solids (such as insoluble calcium salts and insoluble magnesium salts), and has the characteristics of high ammonia nitrogen, high chemical oxygen demand, and high acidity, and the toxin content is very high. The toxins mainly include aflatoxins, zearalenone, vomitoxin, etc., which limit the wide application of corn syrup.

[0003] Microbial fertilizers refer to bio-fertilizers made through biotechnologies such as artificial cultivation and fermentation, with one or more beneficial microorganisms as the main functional components. According to the types of microorganisms, microbial fertilizers mainly include bacterial fertilizers, fungal fertilizers, actinomycete fertilizers, and compound microbial fertilizers. The mechanism of action of microbial fertilizers mainly includes the following aspects: First, the beneficial microorganisms in microbial fertilizers can fix nitrogen in the air, activate nutrients such as phosphorus and potassium in the soil, improve the availability of soil nutrients, and promote the absorption and utilization of nutrients by crops; Second, the beneficial microorganisms in microbial fertilizers can secrete physiologically active substances such as auxin and gibberellin, thus promoting the growth and development of crops and enhancing the stress resistance of crops; Third, the beneficial microorganisms in microbial fertilizers can also produce some antibiotics to inhibit the growth and reproduction of pathogenic bacteria in the soil, thus avoiding the occurrence of soil-borne diseases; Fourth, the beneficial microorganisms in microbial fertilizers can improve the soil aggregate structure, increase the soil organic matter content, and enhance the diversity of the soil microbial flora, thus improving the comprehensive fertility of the soil. Among microbial fertilizers, compound microbial fertilizers can increase the types and biomass of beneficial microorganisms in the soil, provide various nutrients required for crop growth, and can also achieve a balanced ratio of various inorganic elements, organic matter components, and beneficial microorganisms. It can not only reflect the good fertilizer efficiency and quick effect of inorganic chemical fertilizers, but also achieve the comprehensive effects of improving crop quality and soil environment brought by the combination of organic matter and beneficial microorganisms. Therefore, compound microbial fertilizers have broad prospects.

[0004] Applying corn steep liquor to the preparation of compound microbial fertilizers can, on the one hand, effectively utilize the nutrient components in corn steep liquor and solve the problem of serious accumulation of agricultural product waste. On the other hand, it can effectively utilize the slow-release and degradation effects of the soil on toxins, thus avoiding the direct poisoning of humans and livestock by toxins. Bacillus has the ability to degrade fats, proteins, and starches. It is a common crop growth-promoting bacterium in the soil. The spores it produces have a long lifespan, can withstand various harsh environmental conditions, and can produce peptide ester inflammatory substances with antibacterial effects. These peptide ester inflammatory substances have a detoxifying effect on mycotoxins. Using corn steep liquor as a quick-acting nitrogen source and various Bacillus as beneficial microorganisms, through symbiotic fermentation, a compound microbial fertilizer with low cost and good growth-promoting effect can be obtained.

[0005] However, when preparing a compound microbial fertilizer by symbiotic fermentation using corn steep liquor as a quick-acting nitrogen source and various Bacillus species as beneficial microorganisms, the following problems exist: First, Bacillus species are aerobic or facultative anaerobic bacteria. When using multiple Bacillus species for symbiotic fermentation, a low-oxygen environment will occur, resulting in a decrease in the number of viable bacteria during fermentation. Further, the number of viable bacteria in the prepared compound microbial fertilizer is low. Second, the phosphorus in corn steep liquor exists in the form of phytic acid, and phytic acid can form insoluble calcium salts and insoluble magnesium salts with calcium ions and magnesium ions in corn steep liquor, affecting the utilization of nutrients by Bacillus species. Third, there are differences in the growth logarithmic phases of different Bacillus species, resulting in a low number of viable bacteria in symbiotic fermentation, and it is difficult to obtain the highest number of viable bacteria.

[0006] To address the above problems, the most common method is to use other bacteria for symbiotic fermentation with aerobic Bacillus species and facultative anaerobic Bacillus species, and control the addition rate of the carbon source and change the inoculation method. Symbiotic fermentation characteristics of biotransforming corn steep liquor to produce bio-bacterial fertilizer. Ren Xiaojie, Ban Heng, He Zhuangzhuang, Wang Xiaolong, Zhao Yubin, Song Yuanda, Zhao Xinhe. Transactions of the Chinese Society of Agricultural Engineering, Vol. 38, No. 17. September 2022 published that the development of microbial bio-fertilizer was carried out using the rich quick-acting ammonia source in corn steep liquor and the growth-promoting factors of microorganisms. Through the symbiotic fermentation study of 3 plant growth-promoting rhizobacteria, namely Bacillus subtilis, Bacillus licheniformis, and Aspergillus niger, the feasibility of high-density fermentation of the 3 growth-promoting bacteria in corn steep liquor was evaluated, and the fermentation characteristics were studied. In this literature, Aspergillus niger was inoculated first. Aspergillus niger can produce various organic acids during fermentation, dissolve the insoluble calcium salts and insoluble magnesium salts in corn steep liquor, and reduce the osmotic pressure of corn steep liquor. Then, Bacillus subtilis and Bacillus licheniformis were inoculated. Bacillus subtilis is an aerobic bacterium, and Bacillus licheniformis is a facultative anaerobic bacterium. After inoculating Bacillus subtilis and Bacillus licheniformis, Bacillus subtilis will grow rapidly, and at the same time, shear the aggregated mycelia of Aspergillus niger to increase its spore number. At the same time, Bacillus licheniformis is a facultative anaerobic bacterium, avoiding the problem of a decrease in the number of viable bacteria caused by insufficient dissolved oxygen in the fermentation broth. In addition, in this literature, the method of feeding glucose in a fed-batch manner was also adopted, which can significantly improve the utilization rate of total sugar and soluble phosphorus in symbiotic fermentation, and increase the utilization rate of soluble phosphorus by nearly 50%.

[0007] However, through analysis, the methods in the above-mentioned literature still have the following deficiencies: First, the contents of protein, ash, total acid, aflatoxin, zearalenone, and vomitoxin in corn steep liquor of different batches are different. As a fungus, Aspergillus niger is greatly affected by the contents of aflatoxin, zearalenone, vomitoxin, and osmotic pressure. The effect of osmotic pressure on the mycelial morphology and glucose oxidase activity of Aspergillus niger. Ren Wenqiang, Zhang Zhensong, Li Chunzhen, Li Jie, Li Honghua. Food and Fermentation Industries. Published in December 2022, it was disclosed that as the osmotic pressure increased, the mycelial morphology of Aspergillus niger showed a transformation from granular spherical with entangled mycelia to divergent mycelial aggregates. Therefore, for corn steep liquor of different batches, when carrying out symbiotic fermentation according to the method in the above-mentioned literature, the mycelial states of Aspergillus niger are different, and the ability to produce organic acids is different. For corn steep liquor with high toxin content and high osmotic pressure, it is difficult to obtain the highest viable cell count during symbiotic fermentation, resulting in significant differences in the viable cell count of the prepared compound microbial fertilizer; Second, in the case of fed-batch addition of glucose, the cell mass of Aspergillus niger reaches the highest after 34 hours of inoculation, the cell mass of Bacillus subtilis reaches the highest after 28 hours of inoculation, and the cell mass of Bacillus licheniformis reaches the highest after 50 hours of inoculation. The time required for the cell mass of Bacillus licheniformis to reach the highest is much later than that of Aspergillus niger and Bacillus subtilis. It is difficult to obtain the highest viable cell count during symbiotic fermentation, affecting the viable cell count of the prepared compound microbial fertilizer. Summary of the Invention

[0008] Aiming at the deficiencies of the prior art, the present invention provides a preparation method of a compound microbial fertilizer, and the prepared compound microbial fertilizer has a high viable cell count, and the viable cell count is less affected by the contents of various substances in corn steep liquor.

[0009] To solve the above technical problems, the technical solutions adopted by the present invention are as follows: A preparation method of a compound microbial fertilizer, comprising: gelatinizing starch, activating strains, shake-flask culture, immobilization, fermentation, and mixing materials; For the gelatinized starch, add β-glucan and the first portion of purified water into the reaction equipment, adjust the stirring speed of the reaction equipment to 20 - 60 rpm, stir at room temperature for 10 - 20 minutes, adjust the temperature in the reaction equipment to 80 - 85 °C, stir for 10 - 20 minutes, cool the temperature in the reaction equipment to room temperature, add corn starch, stir for 20 - 30 minutes, after vacuum freeze-drying, add it and the second portion of purified water into the reaction equipment, adjust the temperature in the reaction equipment to 90 - 95 °C, adjust the stirring speed to 60 - 120 rpm, stir for 30 - 40 minutes, adjust the temperature in the reaction equipment to room temperature, add sodium humate, stir for 30 - 40 minutes to obtain a gelatinized starch solution; In the gelatinized starch, the dosage ratio of β-glucan, the first portion of purified water, corn starch, the second portion of purified water, and sodium humate is 20 - 23 g : 1500 - 1700 mL : 200 - 220 g : 2800 - 3000 mL : 20 - 24 g; The weight-average molecular weight of the β-glucan is 100 kDa; The activation of the strains includes: activating Bacillus subtilis, activating Paenibacillus polymyxa, and activating Aspergillus niger; For the activation of Bacillus subtilis, pick 2 loops of Bacillus subtilis from the slant of the test tube with an inoculation loop and inoculate it into the beef extract peptone liquid medium, and culture it at 36 - 38 °C and 140 - 160 rpm for 18 - 20 h to obtain the activated Bacillus subtilis; In the activated Bacillus subtilis, the Bacillus subtilis was obtained as a gift from Tianjin Kunhe Biotechnology Group Co., Ltd.; For the activation of Paenibacillus polymyxa, pick 2 loops of Paenibacillus polymyxa from the slant of the test tube with an inoculation loop and inoculate it into the beef extract peptone liquid medium, and culture it at 36 - 38 °C and 140 - 160 rpm for 20 - 21 h to obtain the activated Paenibacillus polymyxa; In the activated Paenibacillus polymyxa, the Paenibacillus polymyxa was obtained as a gift from Tianjin Kunhe Biotechnology Group Co., Ltd.; In the activated Bacillus subtilis and the activated Paenibacillus polymyxa, the components of the beef extract peptone liquid medium are: 3 g / L beef extract, 10 g / L peptone, 5 g / L sodium chloride, the solvent is water, and the pH value is 7; For the activation of Aspergillus niger, pick 2 loops of Aspergillus niger from the slant of the test tube with an inoculation loop and inoculate it into the modified Martin medium, and culture it at 30 - 31 °C and 140 - 160 rpm for 25 - 26 h to obtain the activated Aspergillus niger; In the activated Aspergillus niger, the Aspergillus niger was obtained as a gift from Tianjin Kunhe Biotechnology Group Co., Ltd.; The components of the modified Martin medium are: 5 g / L peptone, 1 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, 2 g / L yeast extract powder, 20 g / L glucose, and the pH value is 6.4; The shake flask culture includes: shake flask culture of Bacillus subtilis, shake flask culture of Paenibacillus polymyxa, and shake flask culture of Aspergillus niger; For the shake flask culture of Bacillus subtilis, inoculate the activated Bacillus subtilis into a 200 mL shake flask medium at an inoculation amount of 10%, and culture it at 30 - 31 °C and 140 - 160 rpm for 72 - 75 h to obtain the Bacillus subtilis shake flask culture; For the shake flask culture of Paenibacillus polymyxa, the activated Paenibacillus polymyxa was inoculated into a 200 mL shake flask medium at an inoculation amount of 10%, and cultured at 30 - 31 °C and 140 - 160 rpm for 72 - 75 h to obtain a Paenibacillus polymyxa shake flask culture; For the shake flask culture of Aspergillus niger, the activated Aspergillus niger was inoculated into a 200 mL shake flask medium at an inoculation amount of 10%, and cultured at 30 - 31 °C and 140 - 160 rpm for 72 - 75 h to obtain an Aspergillus niger shake flask culture; In the shake flask culture of Bacillus subtilis, the shake flask culture of Paenibacillus polymyxa, and the shake flask culture of Aspergillus niger, the composition of the shake flask medium is: 200 g / L corn steep liquor dry powder, 30 g / L glucose, 5 g / L peptone, 5 g / L ammonium chloride, 2 g / L betaine, 2 g / L dipotassium hydrogen phosphate, 2 g / L magnesium sulfate, and the pH is adjusted to 6.4; For the immobilization, the Paenibacillus polymyxa shake flask culture was centrifuged to collect wet bacterial cells; the wet bacterial cells and physiological saline were added to the reaction equipment, the stirring speed of the reaction equipment was adjusted to 20 - 30 rpm, and stirred at room temperature for 10 - 15 min, then pasted starch solution was added and stirring continued for 1.5 - 2 h to obtain a mixed bacterial solution; calcium chloride aqueous solution was added to the reaction equipment, the temperature in the reaction equipment was adjusted to 30 - 35 °C, the stirring speed was adjusted to 60 - 120 rpm, the mixed bacterial solution was added dropwise, and after the addition was completed, stirring continued for 1 - 1.5 h, then filtered, and the filter residue was taken to obtain immobilized Paenibacillus polymyxa; In the immobilization, the dosage ratio of wet bacterial cells to physiological saline is 1 g:9.5 - 10 mL; The dosage ratio of wet bacterial cells to pasted starch solution is 1 g:3 - 3.2 mL; The dosage ratio of the mixed bacterial solution to calcium chloride aqueous solution is 12.5 - 13.2 mL:190 - 200 mL; The mass concentration of the calcium chloride aqueous solution is 4%; The dropping rate of the mixed bacterial solution is 2 - 3 mL / min; For the fermentation, the Aspergillus niger shake flask culture was inoculated into 3000 mL of fermentation medium at an inoculation amount of 5%, the aeration rate was controlled at 0.4 - 0.5 vvm, the pH was maintained at 6.5, and cultured at 30 - 31 °C and 280 - 300 rpm for 5 h, then 30 - 31 g of immobilized Paenibacillus polymyxa was added, the aeration rate and pH were kept unchanged, and cultured at 36 - 38 °C and 280 - 300 rpm for 10 h. Then, the Bacillus subtilis shake flask culture was inoculated at an inoculation amount of 5%, the aeration rate was kept unchanged, the pH was maintained at 7, and cultured at 36 - 38 °C and 280 - 300 rpm, and at the same time, a glucose aqueous solution was started to be added dropwise at a flow rate of 15 mL / h. After 20 h, the addition was stopped, and the culture was continued until the viable cell count reached the highest, and the fermentation was ended to obtain a fermentation broth; In the fermentation, the components of the fermentation medium are: 200 g / L of corn steep liquor dry powder, 20 g / L of glucose, 5 g / L of peptone, 5 g / L of ammonium chloride, 2 g / L of betaine, 2 g / L of dipotassium hydrogen phosphate, 2 g / L of magnesium sulfate, and the pH is adjusted to 6.4; The mass concentration of the glucose aqueous solution is 40%; In the fermentation, after inoculating the Bacillus subtilis shake flask culture, the cell counts of Aspergillus niger, Paenibacillus polymyxa, and Bacillus subtilis are counted every 1 h, and the dilution plate method is used for counting; For the mixing, the fermentation broth, preservative, and trace elements are added to the reaction equipment, the stirring speed is adjusted to 60 - 120 rpm, and stirred at room temperature for 40 - 60 min to obtain the compound microbial fertilizer; In the mixing, the dosage ratio of the fermentation broth, preservative, and trace elements is 1000 mL: 1 - 1.2 g: 5 - 5.4 g; The preservative is one of sodium benzoate or potassium sorbate; The trace elements are a combination of EDTA chelated zinc, EDTA chelated iron, EDTA chelated manganese, and EDTA chelated copper. Among them, the dosage ratio of EDTA chelated zinc, EDTA chelated iron, EDTA chelated manganese, and EDTA chelated copper is 9 - 10 g: 4 - 4.5 g: 3 - 3.4 g: 0.9 - 1 g.

[0010] Compared with the prior art, the beneficial effects of the present invention are: (1) According to the problems existing in the symbiotic fermentation of existing Bacillus subtilis, Paenibacillus polymyxa, and Aspergillus niger, betaine is added to the fermentation medium. Betaine can reduce the impact of osmotic pressure on Aspergillus niger. At the same time, Paenibacillus polymyxa is immobilized, and the addition time of Paenibacillus polymyxa is changed. In the immobilization, first, a gelatinized starch solution is prepared. When preparing, β-glucan is first mixed with starch and then gelatinized. β-glucan can interact with gelatinized starch through hydrogen bonds to form a dense, continuous, and stable network structure. Thus, after mixing the gelatinized starch solution with Paenibacillus polymyxa, the immobilization effect on Paenibacillus polymyxa can be improved. In addition, sodium humate is added to the gelatinized starch solution. Sodium humate can adsorb on the surface of the network structure and has a synergistic effect with β-glucan. During fermentation, through the synergistic effect of sodium humate and β-glucan, the adsorption capacity of the network structure for aflatoxin, zearalenone, and vomitoxin can be improved, avoiding the impact of aflatoxin, zearalenone, and vomitoxin on Aspergillus niger. In the immobilization, only calcium ions are used for immobilization. Calcium ions form immobilized microspheres through chelation with hydroxyl groups. During fermentation, the immobilized microspheres can create a certain anaerobic environment, thereby promoting the growth of Paenibacillus polymyxa. Moreover, as the amylase secreted by Bacillus subtilis and Paenibacillus polymyxa increases, the gelatinized starch in the immobilized microspheres decomposes. Only using calcium ions for cross-linking is more conducive to the release of Paenibacillus polymyxa and calcium ions. Calcium ions and betaine can jointly reduce the impact of osmotic pressure on Aspergillus niger. However, at the same time, the growth of Aspergillus niger and Bacillus subtilis leads to insufficient dissolved oxygen, but it can still promote the growth of Paenibacillus polymyxa, thus ensuring that the logarithmic growth phases of Bacillus subtilis, Paenibacillus polymyxa, and Aspergillus niger coincide, ensuring that the viable count in the compound microbial fertilizer can reach the highest, and also ensuring the stability of the compound microbial fertilizer.

[0011] (2) The compound microbial fertilizer prepared by the present invention has a high viable count, and the viable count is less affected by the content of various substances in corn steep liquor. The viable count of the prepared compound microbial fertilizer can reach 2.52×10 11 -3.40×10 11 , and after being placed at 22°C for 30 days, the viable count can remain at 3.17×10 11 -3.92×10 11 ; (3) In the pepper pot experiment, the compound microbial fertilizer prepared by the present invention can increase the plant height and stem diameter of pepper seedlings. Detailed Embodiments

[0012] The present invention will be described in detail below in conjunction with specific embodiments.

[0013] The components of the culture medium used in the examples are as follows: The composition of the beef extract peptone liquid medium is as follows: 3 g / L beef extract, 10 g / L peptone, 5 g / L sodium chloride, with water as the solvent and a pH value of 7; The composition of the modified Martin medium is as follows: 5 g / L peptone, 1 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, 2 g / L yeast extract powder, 20 g / L glucose, with a pH value of 6.4; The composition of the shake flask medium is as follows: 200 g / L dry corn steep liquor, 30 g / L glucose, 5 g / L peptone, 5 g / L ammonium chloride, 2 g / L betaine, 2 g / L dipotassium hydrogen phosphate, 2 g / L magnesium sulfate, with a pH value of 6.4; The composition of the fermentation medium is as follows: 200 g / L dry corn steep liquor, 20 g / L glucose, 5 g / L peptone, 5 g / L ammonium chloride, 2 g / L betaine, 2 g / L dipotassium hydrogen phosphate, 2 g / L magnesium sulfate, with a pH value of 6.4.

[0014] The information of the strains used in the examples is as follows: Bacillus subtilis, Paenibacillus polymyxa, and Aspergillus niger were all obtained as gifts from Tianjin Kunhe Biotechnology Group Co., Ltd.

[0015] The room temperature in the examples is 22 °C.

[0016] Example 1 A preparation method of a compound microbial fertilizer is as follows: 1. Gelatinized starch: Add 20 g of β-glucan and 1500 mL of purified water to the reaction equipment, adjust the stirring speed of the reaction equipment to 20 rpm, stir at room temperature for 10 min, adjust the temperature in the reaction equipment to 80 °C, stir for 10 min, cool the temperature in the reaction equipment to room temperature, add 200 g of corn starch, stir for 20 min, after vacuum freeze-drying, add it and 2800 mL of purified water to the reaction equipment, adjust the temperature in the reaction equipment to 90 °C, adjust the stirring speed to 60 rpm, stir for 30 min, adjust the temperature in the reaction equipment to room temperature, add 20 g of sodium humate, and stir for 30 min to obtain the gelatinized starch solution; The weight-average molecular weight of the β-glucan is 100 kDa; 2. Strain activation: (1) Activation of Bacillus subtilis: Use an inoculation loop to pick 2 loops of Bacillus subtilis from the slant of the test tube and inoculate it into the beef extract peptone liquid medium, and culture it at 36 °C and 140 rpm for 18 h to obtain the activated Bacillus subtilis; (2) Activation of Paenibacillus polymyxa: Use an inoculation loop to pick 2 loops of Paenibacillus polymyxa from the slant of the test tube and inoculate it into the beef extract peptone liquid medium, and culture it at 36 °C and 140 rpm for 20 h to obtain the activated Paenibacillus polymyxa; (3)Activating Aspergillus niger: Pick 2 loops of Aspergillus niger from the slant of the test tube with an inoculation loop and inoculate it into the modified Martin medium. Culture it at 30 °C and 140 rpm for 25 h to obtain activated Aspergillus niger; 3. Flask shaking culture: (1)Flask shaking culture of Bacillus subtilis: Inoculate the activated Bacillus subtilis into a 200 mL flask medium at an inoculation amount of 10%. Culture it at 30 °C and 140 rpm for 72 h to obtain the flask shaking culture of Bacillus subtilis; (2)Flask shaking culture of Paenibacillus polymyxa: Inoculate the activated Paenibacillus polymyxa into a 200 mL flask medium at an inoculation amount of 10%. Culture it at 30 °C and 140 rpm for 72 h to obtain the flask shaking culture of Paenibacillus polymyxa; (3)Flask shaking culture of Aspergillus niger: Inoculate the activated Aspergillus niger into a 200 mL flask medium at an inoculation amount of 10%. Culture it at 30 °C and 140 rpm for 72 h to obtain the flask shaking culture of Aspergillus niger; 4. Immobilization: Centrifuge the flask shaking culture of Paenibacillus polymyxa to collect the wet bacterial cells. Add the wet bacterial cells and physiological saline to the reaction equipment at a dosage ratio of 1 g:9.5 mL. Adjust the stirring speed of the reaction equipment to 20 rpm and stir at room temperature for 10 min. Add the gelatinized starch solution at a dosage ratio of 1 g:3 mL of the wet bacterial cells and the gelatinized starch solution, and continue to stir for 1.5 h to obtain a mixed bacterial solution. Add the calcium chloride aqueous solution to the reaction equipment, adjust the temperature in the reaction equipment to 30 °C, and adjust the stirring speed to 60 rpm. Dropwise add the mixed bacterial solution at a dosage ratio of 12.5 mL:190 mL of the mixed bacterial solution and the calcium chloride aqueous solution, control the dropping speed at 2 mL / min, continue to stir for 1 h after the dropping is completed, filter, and take the filter residue to obtain immobilized Paenibacillus polymyxa; The mass concentration of the calcium chloride aqueous solution is 4%; 5. Fermentation: Inoculate the flask shaking culture of Aspergillus niger into 3000 mL of fermentation medium at an inoculation amount of 5%. Control the ventilation rate at 0.4 vvm, maintain the pH at 6.5, culture at 30 °C and 280 rpm for 5 h, add 30 g of immobilized Paenibacillus polymyxa, control the ventilation rate and pH unchanged, culture at 36 °C and 280 rpm for 10 h, inoculate the flask shaking culture of Bacillus subtilis at an inoculation amount of 5%, control the ventilation rate unchanged, maintain the pH at 7, culture at 36 °C and 280 rpm, and start to flow in the glucose aqueous solution at a flow rate of 15 mL / h. Stop flowing in after 20 h, and culture until the viable bacteria count reaches the highest to end the fermentation and obtain the fermentation broth; The mass concentration of the glucose aqueous solution is 40%; In fermentation, after inoculating the Bacillus subtilis shake flask culture, the cell counts of Aspergillus niger, Paenibacillus polymyxa, and Bacillus subtilis were statistically analyzed every 1 h. During the statistical analysis, the symbiotic fermentation characteristics of biotransforming corn steep liquor to produce bio-bacterial fertilizer were referred to. Ren Xiaojie, Ban Heng, He Zhuangzhuang, Wang Xiaolong, Zhao Yubin, Song Yuanda, Zhao Xinhe. Transactions of the Chinese Society of Agricultural Engineering, Vol. 38, No. 17. September 2022. The method disclosed in the above was adopted, and the dilution plate method was used for counting. When counting Aspergillus niger, Rose Bengal medium was used. When counting Paenibacillus polymyxa and Bacillus subtilis, nutrient agar medium was used; 6. Mixing: The fermentation broth, preservative, and trace elements were added to the reaction equipment according to a dosage ratio of 1000 mL: 1 g: 5 g. The stirring speed was adjusted to 60 rpm, and the mixture was stirred at room temperature for 40 min to obtain the compound microbial fertilizer; The preservative is sodium benzoate; The trace elements are a combination of EDTA chelated zinc, EDTA chelated iron, EDTA chelated manganese, and EDTA chelated copper. Among them, the dosage ratio of EDTA chelated zinc, EDTA chelated iron, EDTA chelated manganese, and EDTA chelated copper is 9 g: 4 g: 3 g: 0.9 g.

[0017] Example 2 A preparation method of a compound microbial fertilizer is as follows: 1. Gelatinized starch: 23 g of β-glucan and 1700 mL of purified water were added to the reaction equipment. The stirring speed of the reaction equipment was adjusted to 60 rpm, and the mixture was stirred at room temperature for 20 min. Then, the temperature in the reaction equipment was adjusted to 85 °C and stirred for 20 min. After cooling the temperature in the reaction equipment to room temperature, 220 g of corn starch was added and stirred for 30 min. After vacuum freeze-drying, it was added to the reaction equipment together with 3000 mL of purified water. The temperature in the reaction equipment was adjusted to 95 °C, the stirring speed was adjusted to 120 rpm, and it was stirred for 40 min. Then, the temperature in the reaction equipment was adjusted to room temperature, and 24 g of sodium humate was added and stirred for 40 min to obtain the gelatinized starch solution; The weight-average molecular weight of the β-glucan is 100 kDa; 2. Strain activation: (1) Activation of Bacillus subtilis: 2 loops of Bacillus subtilis were picked from the test tube slant with an inoculation loop and inoculated into the nutrient agar liquid medium, and cultured at 37 °C and 150 rpm for 20 h to obtain the activated Bacillus subtilis; (2) Activation of Paenibacillus polymyxa: 2 loops of Paenibacillus polymyxa were picked from the test tube slant with an inoculation loop and inoculated into the nutrient agar liquid medium, and cultured at 37 °C and 150 rpm for 21 h to obtain the activated Paenibacillus polymyxa; (3)Activating Aspergillus niger: Pick 2 loops of Aspergillus niger from the slant of the test tube with an inoculation loop and inoculate it into the modified Martin medium. Culture it at 30 °C and 150 rpm for 26 h to obtain activated Aspergillus niger; 3. Flask culture: (1)Flask culture of Bacillus subtilis: Inoculate the activated Bacillus subtilis into a 200 mL flask medium at an inoculation amount of 10%. Culture it at 30 °C and 150 rpm for 75 h to obtain the flask culture of Bacillus subtilis; (2)Flask culture of Paenibacillus polymyxa: Inoculate the activated Paenibacillus polymyxa into a 200 mL flask medium at an inoculation amount of 10%. Culture it at 30 °C and 150 rpm for 75 h to obtain the flask culture of Paenibacillus polymyxa; (3)Flask culture of Aspergillus niger: Inoculate the activated Aspergillus niger into a 200 mL flask medium at an inoculation amount of 10%. Culture it at 30 °C and 150 rpm for 75 h to obtain the flask culture of Aspergillus niger; 4. Immobilization: Centrifuge the flask culture of Paenibacillus polymyxa to collect the wet cells. Add the wet cells and physiological saline to the reaction equipment at a dosage ratio of 1 g:10 mL. Adjust the stirring speed of the reaction equipment to 30 rpm and stir at room temperature for 15 min. Add the gelatinized starch solution at a dosage ratio of 1 g:3.2 mL of the wet cells and the gelatinized starch solution, and continue to stir for 2 h to obtain a mixed bacterial solution. Add the calcium chloride aqueous solution to the reaction equipment, adjust the temperature in the reaction equipment to 35 °C, and adjust the stirring speed to 120 rpm. Dropwise add the mixed bacterial solution at a dosage ratio of 13.2 mL:200 mL of the mixed bacterial solution and the calcium chloride aqueous solution, control the dropping speed at 3 mL / min, continue to stir for 1.5 h after the dropping is completed, filter, and take the filter residue to obtain immobilized Paenibacillus polymyxa; The mass concentration of the calcium chloride aqueous solution is 4%; 5. Fermentation: Inoculate the flask culture of Aspergillus niger into 3000 mL of fermentation medium at an inoculation amount of 5%. Control the ventilation volume at 0.5 vvm, maintain the pH at 6.5, culture at 30 °C and 300 rpm for 5 h, add 31 g of immobilized Paenibacillus polymyxa, control the ventilation volume and pH unchanged, culture at 37 °C and 300 rpm for 10 h, inoculate the flask culture of Bacillus subtilis at an inoculation amount of 5%, control the ventilation volume unchanged, maintain the pH at 7, culture at 37 °C and 300 rpm, and start to flow in the glucose aqueous solution at a flow rate of 15 mL / h. Stop flowing in after 20 h, and culture until the viable cell count reaches the highest to end the fermentation and obtain the fermentation broth; The mass concentration of the glucose aqueous solution is 40%; In fermentation, after inoculating the Bacillus subtilis shake-flask culture, the cell counts of Aspergillus niger, Paenibacillus polymyxa, and Bacillus subtilis were started to be statistically analyzed every 1 h. When conducting the statistics, refer to the symbiotic fermentation characteristics of biotransforming corn steep liquor to produce bio-bacterial fertilizer. Ren Xiaojie, Ban Heng, He Zhuangzhuang, Wang Xiaolong, Zhao Yubin, Song Yuanda, Zhao Xinhe. Transactions of the Chinese Society of Agricultural Engineering, Vol. 38, No. 17. In September 2022, the method disclosed was adopted, and the dilution plate method was used for counting. When counting Aspergillus niger, Rose Bengal medium was used. When counting Paenibacillus polymyxa and Bacillus subtilis, nutrient agar medium was used; 6. Mixing: Add the fermentation broth, preservative, and trace elements into the reaction equipment according to the dosage ratio of 1000 mL: 1.2 g: 5.4 g, adjust the stirring speed to 120 rpm, and stir at room temperature for 60 min to obtain the compound microbial fertilizer; The preservative is potassium sorbate; The trace elements are a combination of EDTA chelated zinc, EDTA chelated iron, EDTA chelated manganese, and EDTA chelated copper. Among them, the dosage ratio of EDTA chelated zinc, EDTA chelated iron, EDTA chelated manganese, and EDTA chelated copper is 10 g: 4.5 g: 3.4 g: 1 g.

[0018] Example 3 On the basis of Example 2, in the 1. Gelatinized starch step, the addition of β-glucan was omitted, that is, the 1. Gelatinized starch step was changed to: Add 1700 mL of purified water into the reaction equipment, adjust the stirring speed of the reaction equipment to 60 rpm, add 220 g of corn starch, stir at room temperature for 30 min, after vacuum freeze-drying, add it and 3000 mL of purified water into the reaction equipment, adjust the temperature in the reaction equipment to 95 °C, adjust the stirring speed to 120 rpm, stir for 40 min, adjust the temperature in the reaction equipment to room temperature, add 24 g of sodium humate, and stir for 40 min to obtain the gelatinized starch solution.

[0019] The rest of the operations remain unchanged.

[0020] Example 4 On the basis of Example 2, in the 1. Gelatinized starch step, the addition of sodium humate was omitted, that is, the 1. Gelatinized starch step was changed to: Add 23 g of β-glucan and 1700 mL of purified water to the reaction equipment. Adjust the stirring speed of the reaction equipment to 60 rpm, stir at room temperature for 20 min, adjust the temperature in the reaction equipment to 85 °C, stir for 20 min, cool the temperature in the reaction equipment to room temperature, add 220 g of corn starch, stir for 30 min, after vacuum freeze-drying, add it and 3000 mL of purified water to the reaction equipment, adjust the temperature in the reaction equipment to 95 °C, adjust the stirring speed to 120 rpm, stir for 40 min, adjust the temperature in the reaction equipment to room temperature, and stir for 40 min to obtain a gelatinized starch solution; The weight-average molecular weight of the β-glucan is 100 kDa.

[0021] Keep the rest of the operations unchanged.

[0022] Example 5 On the basis of Example 2, in step 1. gelatinizing starch, omit the addition of β-glucan and sodium humate at the same time, that is, change step 1. gelatinizing starch to: Add 1700 mL of purified water to the reaction equipment. Adjust the stirring speed of the reaction equipment to 60 rpm, add 220 g of corn starch, stir at room temperature for 30 min, after vacuum freeze-drying, add it and 3000 mL of purified water to the reaction equipment, adjust the temperature in the reaction equipment to 95 °C, adjust the stirring speed to 120 rpm, stir for 40 min, adjust the temperature in the reaction equipment to room temperature, and stir for 40 min to obtain a gelatinized starch solution.

[0023] Keep the rest of the operations unchanged.

[0024] Example 6 On the basis of Example 2, in step 4. immobilization, change the calcium chloride aqueous solution to a mixed aqueous solution of boric acid and calcium chloride. The mass concentration of boric acid in the mixed aqueous solution of boric acid and calcium chloride is 4%, and the mass concentration of calcium chloride is 4%.

[0025] Keep the rest of the operations unchanged.

[0026] Test Example 1 In Examples 1 - 6, corn steep liquor with a protein (wet basis) content of 25.70%, an ash content of 16.23%, a total acid content of 7.81%, an aflatoxin content of 4.70 μg / L, a zearalenone content of 613.76 μg / L, a vomitoxin content of 1508 μg / L, and a water content of 45.25% was used. According to the statistical methods in Examples 1 - 6, the cell counts of Aspergillus niger, Paenibacillus polymyxa, and Bacillus subtilis were respectively counted. Then, the highest viable cell count during fermentation in Examples 1 - 6 (i.e., the sum of the cell counts of Aspergillus niger, Paenibacillus polymyxa, and Bacillus subtilis) and the time when the viable cell count reached the highest (i.e., how long it took to reach the highest viable cell count after starting to count the cell count) were statistically analyzed. The statistical results are as follows:

[0027] It can be seen from the above results that, as the comparative example of Example 2, the highest viable cell counts in Examples 3 - 6 are all lower than that in Example 2, and the time when the highest viable cell count appears is later than that in Example 2.

[0028] Test Example 2 In Examples 1 - 6, corn steep liquor with a protein (wet basis) content of 19.41%, an ash content of 9.52%, a total acid content of 8.14%, an aflatoxin content of 3.24 μg / L, a zearalenone content of 582.90 μg / L, a vomitoxin content of 1247 μg / L, and a water content of 58.02% was used. According to the statistical methods in Examples 1 - 6, the cell counts of Aspergillus niger, Paenibacillus polymyxa, and Bacillus subtilis were respectively counted. Then, the highest viable cell count during fermentation in Examples 1 - 6 (i.e., the sum of the cell counts of Aspergillus niger, Paenibacillus polymyxa, and Bacillus subtilis) and the time when the viable cell count reached the highest (i.e., how long it took to reach the highest viable cell count after starting to count the cell count) were statistically analyzed. The statistical results are as follows:

[0029] It can be seen from the above results that, as the comparative example of Example 2, the highest viable cell counts in Examples 3 - 6 are all lower than that in Example 2, and the time when the highest viable cell count appears is later than that in Example 2. Moreover, compared with Example 2, the differences between the highest viable cell counts and the times when the highest viable cell counts appear in Examples 3 - 6 in this test example and the corresponding results in Test Example 1 are greater, indicating that they are more affected by different batches of corn steep liquor.

[0030] Test Example 3 The stability of the compound microbial fertilizers prepared in Examples 1-6 was tested. Specifically, corn steep liquor with a protein (wet basis) content of 19.41%, an ash content of 9.52%, a total acid content of 8.14%, aflatoxin content of 3.24 μg / L, zearalenone content of 582.90 μg / L, vomitoxin content of 1247 μg / L, and a water content of 58.02% was used in Examples 1-6, and then the compound microbial fertilizers were prepared according to the preparation methods of Examples 1-6. After the compound microbial fertilizers prepared in Examples 1-6 were placed at 22 °C for 30 days, the viable bacteria count was detected, and the test results are as follows:

[0031] It can be seen from the above results that, as the comparative example of Example 2, the compound microbial fertilizers prepared in Examples 3-6 have worse stability.

[0032] Test Example 4 The fertilizer efficiency of the compound microbial fertilizers prepared in Examples 1-6 was tested. Specifically, corn steep liquor with a protein (wet basis) content of 19.41%, an ash content of 9.52%, a total acid content of 8.14%, aflatoxin content of 3.24 μg / L, zearalenone content of 582.90 μg / L, vomitoxin content of 1247 μg / L, and a water content of 58.02% was used in Examples 1-6, and then the compound microbial fertilizers were prepared according to the preparation methods of Examples 1-6.

[0033] Then, the compound microbial fertilizers prepared in Examples 1-6 were subjected to a pot experiment to study their growth-promoting effect on peppers. The pepper variety used was Jinfu Spicy 06. The specific test method was to prepare 7 planting pots numbered 1-7. The pepper seeds were soaked, and after the germination rate reached 90%, a seedling tray was used for seedling raising. After the seedlings grew 6 leaves, they were transplanted into the 1-7 planting pots. 10 pepper seedlings were transplanted into each planting pot. The pepper seedlings in the 1-6 planting pots were treated with the compound microbial fertilizers prepared in Examples 1-6 5 days and 10 days after transplantation. When treating, first dilute the compound microbial fertilizers prepared in Examples 1-6 by 500 times to obtain a diluted compound microbial fertilizer solution, and then use the diluted compound microbial fertilizer solution to spray the pepper seedlings. The dosage of the diluted compound microbial fertilizer solution in each planting pot was 200 mL each time. The 7th planting pot was used as a blank control. Ensure that the other water, fertilizer management and planting conditions of the 1-7 planting pots are the same.

[0034] 30 days after transplantation, the plant height and stem diameter of each pepper seedling in the 1-7 planting pots were counted, and the average values were calculated respectively. The results are as follows:

[0035] As can be seen from the above results, as a comparative example of Example 2, the growth rate of the pepper seedlings treated in Examples 3-6 is slower.

Claims

1. A preparation method of a compound microbial fertilizer, characterized in that Including: Gelatinized starch, strain activation, shake flask culture, immobilization, fermentation, mixing; For the gelatinized starch, after mixing β-glucan and the first portion of purified water, stir at room temperature, stir at 80 - 85 °C, add corn starch at room temperature, stir, vacuum freeze-dry, then mix with the second portion of purified water, stir at 90 - 95 °C, add sodium humate at room temperature, stir to obtain the gelatinized starch solution; For the immobilization, centrifuge the Paenibacillus polymyxa shake flask culture to collect the wet bacterial cells; mix the wet bacterial cells with physiological saline, stir at room temperature, add the gelatinized starch solution, continue to stir to obtain the mixed bacterial solution; stir the calcium chloride aqueous solution at 30 - 35 °C, dropwise add the mixed bacterial solution, continue to stir after the addition is complete, filter, take the filter residue to obtain immobilized Paenibacillus polymyxa; For the fermentation, inoculate the Aspergillus niger shake flask culture into 3000 mL of fermentation medium at an inoculation amount of 5%, control the aeration rate to 0.4 - 0.5 vvm, maintain the pH at 6.5, culture at 30 - 31 °C and 280 - 300 rpm for 5 h, add 30 - 31 g of immobilized Paenibacillus polymyxa, keep the aeration rate and pH unchanged, culture at 36 - 38 °C and 280 - 300 rpm for 10 h, inoculate the Bacillus subtilis shake flask culture at an inoculation amount of 5%, keep the aeration rate unchanged, maintain the pH at 7, culture at 36 - 38 °C and 280 - 300 rpm, and start to flow in the glucose aqueous solution at a flow rate of 15 mL / h, stop flowing in after 20 h, culture until the viable cell count reaches the highest, end the fermentation to obtain the fermentation broth.

2. The preparation method of the composite microbial fertilizer according to claim 1, wherein In the gelatinized starch, the dosage ratio of β-glucan, the first portion of purified water, corn starch, the second portion of purified water, and sodium humate is 20 - 23 g : 1500 - 1700 mL : 200 - 220 g : 2800 - 3000 mL : 20 - 24 g; The weight-average molecular weight of the β-glucan is 100 kDa.

3. The preparation method of the composite microbial fertilizer according to claim 1, characterized in that, The strain activation includes: activating Bacillus subtilis, activating Paenibacillus polymyxa, activating Aspergillus niger; For the activation of Bacillus subtilis, pick 2 loops of Bacillus subtilis from the test tube slant with an inoculation loop and inoculate it into the beef extract peptone liquid medium, culture at 36 - 38 °C and 140 - 160 rpm for 18 - 20 h to obtain the activated Bacillus subtilis; In the activated Bacillus subtilis, the Bacillus subtilis is obtained as a gift from Tianjin Kunhe Biotechnology Group Co., Ltd.; For the activation of Paenibacillus polymyxa, pick 2 loops of Paenibacillus polymyxa from the test tube slant with an inoculation loop and inoculate it into the beef extract peptone liquid medium, culture at 36 - 38 °C and 140 - 160 rpm for 20 - 21 h to obtain the activated Paenibacillus polymyxa; In the activated Paenibacillus polymyxa, the Paenibacillus polymyxa is obtained as a gift from Tianjin Kunhe Biotechnology Group Co., Ltd.; In the activated Bacillus subtilis and activated Paenibacillus polymyxa, the components of the beef extract peptone liquid medium are: 3 g / L beef extract, 10 g / L peptone, 5 g / L sodium chloride, the solvent is water, and the pH value is 7.

4. The preparation method of the compound microbial fertilizer according to claim 3, characterized in that, For the activated Aspergillus niger, pick 2 loops of Aspergillus niger from the slant in the test tube with an inoculation loop and inoculate it into the modified Martin medium, and culture it at 30 - 31 °C and 140 - 160 rpm for 25 - 26 h to obtain the activated Aspergillus niger; Among the activated Aspergillus niger, the Aspergillus niger was obtained as a gift from Tianjin Kunhe Biotechnology Group Co., Ltd.; The composition of the modified Martin medium is: 5 g / L peptone, 1 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate, 2 g / L yeast extract powder, 20 g / L glucose, and the pH value is 6.

4.

5. The preparation method of the composite microbial fertilizer according to claim 1, characterized in that, The shake flask culture includes: shake flask culture of Bacillus subtilis, shake flask culture of Paenibacillus polymyxa, and shake flask culture of Aspergillus niger; For the shake flask culture of Bacillus subtilis, inoculate the activated Bacillus subtilis into a 200 mL shake flask medium at an inoculation amount of 10%, and culture it at 30 - 31 °C and 140 - 160 rpm for 72 - 75 h to obtain the Bacillus subtilis shake flask culture; For the shake flask culture of Paenibacillus polymyxa, inoculate the activated Paenibacillus polymyxa into a 200 mL shake flask medium at an inoculation amount of 10%, and culture it at 30 - 31 °C and 140 - 160 rpm for 72 - 75 h to obtain the Paenibacillus polymyxa shake flask culture; For the shake flask culture of Aspergillus niger, inoculate the activated Aspergillus niger into a 200 mL shake flask medium at an inoculation amount of 10%, and culture it at 30 - 31 °C and 140 - 160 rpm for 72 - 75 h to obtain the Aspergillus niger shake flask culture.

6. The preparation method of the composite microbial fertilizer according to claim 5, characterized in that, Among the shake flask culture of Bacillus subtilis, shake flask culture of Paenibacillus polymyxa, and shake flask culture of Aspergillus niger, the composition of the shake flask medium is: 200 g / L corn steep liquor dry powder, 30 g / L glucose, 5 g / L peptone, 5 g / L ammonium chloride, 2 g / L betaine, 2 g / L dipotassium hydrogen phosphate, 2 g / L magnesium sulfate, and the pH is adjusted to 6.

4.

7. The preparation method of the composite microbial fertilizer according to claim 1, characterized in that, In the immobilization, the dosage ratio of wet bacterial cells to normal saline is 1 g:9.5 - 10 mL; The dosage ratio of wet bacterial cells to gelatinized starch solution is 1 g:3 - 3.2 mL; The dosage ratio of the mixed bacterial liquid to calcium chloride aqueous solution is 12.5 - 13.2 mL:190 - 200 mL; The mass concentration of the calcium chloride aqueous solution is 4%; The dropping rate of the mixed bacterial liquid is 2 - 3 mL / min.

8. The preparation method of the composite microbial fertilizer according to claim 1, characterized in that In the fermentation, the composition of the fermentation medium is: 200 g / L corn steep liquor dry powder, 20 g / L glucose, 5 g / L peptone, 5 g / L ammonium chloride, 2 g / L betaine, 2 g / L dipotassium hydrogen phosphate, 2 g / L magnesium sulfate, and the pH is adjusted to 6.4; The mass concentration of the glucose aqueous solution is 40%.

9. The preparation method of the composite microbial fertilizer according to claim 1, characterized in that, For the mixing, mix the fermentation broth with preservatives and trace elements, and stir at room temperature to obtain the compound microbial fertilizer.

10. The preparation method of the composite microbial fertilizer according to claim 9, characterized in that, In the mixing, the dosage ratio of the fermentation broth to preservatives and trace elements is 1000 mL:1 - 1.2 g:5 - 5.4 g; The preservative is one of sodium benzoate or potassium sorbate; The trace elements are a combination of EDTA chelated zinc, EDTA chelated iron, EDTA chelated manganese, and EDTA chelated copper. Among them, the dosage ratio of EDTA chelated zinc, EDTA chelated iron, EDTA chelated manganese, and EDTA chelated copper is 9-10 g: 4-4.5 g: 3-3.4 g: 0.9-1 g.

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