Process for preparing bio-fertilizer by using isoleucine fermentation liquor

Abstract

The invention discloses a process for preparing a biological fertilizer by using an isoleucine fermentation broth, and relates to the technical field of fertilizer preparation, the process comprises isoleucine pretreatment, magnetic field enhanced impurity removal, strain fermentation and nutrient substance blending, and the process has the advantages that the isoleucine fermentation broth is converted into a liquid biological fertilizer; the environment pollution caused by direct discharge of the isoleucine fermentation liquor in a traditional treatment mode is avoided, the liquid bio-fertilizer meets the environment-friendly requirement, harmful substances in the waste liquor are removed through fine processing treatment operation on the isoleucine fermentation liquor, potential hazards to soil, water and air are reduced, and meanwhile, the liquid bio-fertilizer can be recycled. Valuable resources can be recycled, resource waste is reduced, sustainable development is achieved, the production cost of the bio-fertilizer is reduced by using the isoleucine fermentation waste liquid to prepare the bio-fertilizer, and compared with a traditional bio-fertilizer, the production raw material cost is low.

Description

Technical Field

[0001] The invention relates to the technical field of fertilizer preparation, in particular to a process for preparing biological fertilizer by utilizing isoleucine fermentation liquid. Background Art

[0002] Isoleucine is widely used in the food, medicine, feed and other industries, and its production scale is constantly expanding. During the fermentation process of isoleucine, a large amount of fermentation waste liquid is generated. These waste liquids are rich in organic matter, such as amino acids, sugars, proteins, vitamins and various intermediate metabolites, but also contain some impurities, such as residual culture medium components, microbial cell fragments, pigments produced during the fermentation process, and various inorganic salts. At present, the treatment methods for isoleucine fermentation broth are relatively limited. Traditional treatment methods such as direct discharge will not only cause serious pollution to the environment and violate environmental protection regulations, but also waste potential valuable resources. Some conventional treatment methods such as physical and chemical treatment methods are costly and complex, and are prone to secondary pollutants during the treatment process, making it difficult to achieve ideal treatment effects. At present, there have been some attempts to use fermentation waste liquid for fertilizer preparation, but there are many defects. The existing process of using fermentation waste liquid to prepare fertilizer is often simply mixing or concentrating, without fully considering the effective conversion and utilization of nutrients in the waste liquid. For example, macromolecules such as proteins in fermentation waste liquid are difficult for plants to directly absorb and utilize, but the existing process lacks effective conversion means to decompose them into small molecules such as amino acids and other easily absorbable substances. At the same time, the allocation of nutrients is not accurate enough, resulting in unbalanced nutrients in the fertilizer, which cannot meet the needs of different crops at different growth stages. Moreover, the stability and long-term effectiveness of the fertilizer are poor, and it is easy to have problems such as rapid release of nutrients causing waste or insufficient release in the early stage affecting crop growth. In addition, the fertilizer prepared by the existing process is not effective in improving soil structure and enhancing soil fertility, and cannot effectively solve soil problems caused by long-term use of chemical fertilizers. For this reason, we propose a process for preparing biological fertilizer using isoleucine fermentation liquid. Summary of the invention

[0003] The object of the present invention is to provide a process for preparing biofertilizer by utilizing isoleucine fermentation broth.

[0004] In order to solve the problems raised in the above background technology, the present invention provides the following technical solution: a process for preparing biofertilizer using isoleucine fermentation broth, comprising isoleucine pretreatment, magnetic field enhanced impurity removal, bacterial fermentation and nutrient preparation, and the specific operating steps of the process for preparing biofertilizer using isoleucine fermentation broth are as follows: Step 1, collecting a high-yield isoleucine strain that has been genetically engineered for fermentation, and after the fermentation is completed, adding a nutrient enhancer and mixing through a pipeline mixer to obtain a mixed isoleucine fermentation liquid; Step 2, placing the isoleucine fermentation liquid in a gradient magnetic field for magnetization treatment, gradually increasing the magnetic field strength within a set range, and controlling the flow rate of the isoleucine fermentation liquid in the magnetic field, then adding a modified flocculant, stirring and mixing, and then passing through a filter press to obtain a clarified filtrate; Step 3, inoculating the clarified filtrate with a mixed bacteria of three composite strains, and transferring the mixed bacteria into a fermentation tank for fermentation under aseptic conditions, wherein a sterile environment needs to be maintained during the process, and after the fermentation is completed, an isoleucine enhanced fermentation broth is obtained; Step 4: adding the nano-selenized polysaccharide complex to the isoleucine enhanced fermentation broth, stirring and mixing, and dispersing the nano-selenized polysaccharide complex in the fermentation broth using microwave-assisted dispersion to obtain a mixed fermentation broth; Step 5: Add biostimulants and trace elements to the mixed fermentation liquid in sequence and mix them. The biostimulants are polypeptide biostimulants. Adjust the pH value of the mixed fermentation liquid to obtain liquid biofertilizer. After completing the above steps, collect and pre-treat the waste liquid generated in steps 2 and 3 and the fermentation waste liquid, and then ferment them twice through the above steps to obtain liquid biofertilizer.

[0005] As a further scheme of the present invention: in the step 1, a genetically engineered isoleucine high-yielding strain is selected for fermentation, the fermentation medium uses glucose as the carbon source, the initial glucose concentration is 30g / L-50g / L, and ammonium sulfate is used as the nitrogen source, the initial concentration is 5g / L-10g / L, and an appropriate amount of potassium dihydrogen phosphate is added, the initial concentration is 2g / L-5g / L, and 0.5g / L-1g / L of magnesium sulfate and trace elements are added, the trace elements include 0.01g / L-0.05g / L ferrous sulfate, 0.005g / L-0.02g / L manganese sulfate and 0.01g / L-0.03g / L zinc sulfate.

[0006] As a further scheme of the present invention: in the step 1, the nutritional enhancer is selected from unsaturated fatty acids extracted from deep-sea fish oil, plant protein hydrolysate extracted from bean substances and nucleotides extracted from yeast, which are stirred and mixed in a mass ratio of 3:2:1, and the amount of the nutritional enhancer added is 3%-5% of the total mass of the isoleucine fermentation broth. The addition time is after the fermentation is completed, the temperature of the isoleucine fermentation broth is reduced to 30° C.-35° C., and the nutritional enhancer is slowly added to the fermentation broth through a pipeline mixer, and stirred and mixed at a stirring rate of 60r / min-80r / min for 30min-60min, and the nutritional enhancer is evenly dispersed in the fermentation broth to obtain a mixed isoleucine fermentation broth.

[0007] As a further solution of the present invention: in the step 2, a special electromagnetic induction device is used to generate a gradient magnetic field, the magnetic field strength is set to 0.2T at the entrance, and is increased according to a gradient of 0.1T / m to reach 1T at the magnetic field outlet, and the flow rate of the fermentation liquid through the magnetic field is controlled at 0.3m / s-0.5m / s, and the mixed isoleucine fermentation liquid needs to be treated in the magnetic field environment for 30min-35min, and the temperature is maintained between 25°C and 30°C during the treatment process. During use, magnetic shielding materials are used around the equipment for protection.

[0008] As a further solution of the present invention: in the step 2, the natural polymer material chitosan is selected for modification, the chitosan is dissolved in an acetic acid solution with a volume fraction of 2%, and a chitosan solution with a mass fraction of 5% is prepared, and then a glutaraldehyde solution with a mass fraction of 25% is added dropwise to the chitosan solution, and the mass ratio of chitosan to glutaraldehyde is 8:1, and after mixing, the mixture is stirred and reacted at a temperature of 40°C-50°C for 2h-3h to obtain a modified chitosan flocculant, and the prepared modified chitosan flocculant is added to the fermentation broth treated with a magnetic field, and the modified chitosan is The amount of flocculant added is 4g-7g per liter of fermentation liquid, and the mixture is stirred at a stirring rate of 80r / min-120r / min for 20min-30min to fully mix the flocculant and the isoleucine fermentation liquid. After mixing, a plate and frame filter press is used to filter the liquid. The operating pressure of the plate and frame filter press is set at 0.3MPa-0.5MPa, and the filtration time is 2h-3h. In the initial stage of filtration, the operating pressure is controlled to rise slowly, and the clarity of the filtrate is continuously checked. When the filtrate flow rate decreases and the turbidity increases, the filter press operation is stopped to obtain a clear filtrate.

[0009] As a further scheme of the present invention: in the step 3, the composite bacteria are selected from Bacillus licheniformis, Lactobacillus plantarum and Aspergillus niger, and they are inoculated according to the total volume of the isoleucine fermentation broth, the inoculation amount of Bacillus licheniformis is 3%-5%, the inoculation amount of Lactobacillus plantarum is 4%-6%, and the inoculation amount of Aspergillus niger is 2%-4%, and the three bacterial suspensions are sequentially accessed into the clarified filtrate in the order of bacteria first and then fungi, and a sterile environment is maintained during the process, and the mixture is fully stirred and evenly mixed, and then transferred to a fermenter for fermentation. The initial fermentation time is between 0h-12h, the temperature is controlled between 30℃-32℃, the initial stirring rate is 80r / min-120r / min, and after fermentation for 12h, the temperature is adjusted to 33℃-36℃, the stirring rate is increased to 120r / min-150r / min, the pressure is maintained at 0.02MPa-0.04MPa during the fermentation process, and the fermentation time is 36h-48h.

[0010] As a further scheme of the present invention: in the step three, after the fermentation is completed, ultrasonic treatment is first performed, and the fermentation liquid is treated with ultrasound at a frequency of 20-30kHz and a power of 500-800W for 10min-15min to break and disperse the bacterial aggregates, and then a secondary centrifugation treatment is performed, and the centrifugal speed is increased to 6000r / min-8000r / min, and the centrifugation time is 20min-30min to remove the bacteria and impurities therein, and then the fermentation liquid is filtered through a two-stage filtration system, and the first-stage filtration uses a folded filter element filter with a filter element pore size of 5um to remove larger particle impurities, and the second-stage filtration uses a hollow fiber ultrafiltration membrane filter with a membrane pore size of 0.1um to perform fine filtration and sterilization to obtain a stable isoleucine enhanced fermentation liquid.

[0011] As a further scheme of the present invention: in the step 4, the nano-selenized polysaccharide complex is passed through a 200-mesh sieve and added in an amount of 1.2%-1.5% of the total volume of the isoleucine enhanced fermentation broth. After mixing for 20 minutes to 30 minutes, the mixed solution is subjected to microwave treatment using a microwave device, and the microwave frequency is set to 2450MHz±50MHz. The special microwave reaction container containing the mixed solution is placed in the microwave device, stirred and microwave treated at a stirring rate of 50r / min-90r / min, and the microwave treatment time is 8min-12min. It is divided into three stages, each stage is separated by 1min-2min, the power in the first stage is 60%-70% of the set power, the power in the second stage is increased to 80%-90%, and the power in the third stage reaches the set power. After the treatment is completed, a mixed fermentation broth is obtained.

[0012] As a further scheme of the present invention: in the step 5, the biostimulant is a polypeptide biostimulant extracted from the insect protein fermentation liquid, the silkworm pupa protein is fermented, Bacillus subtilis and Aspergillus oryzae are selected as inoculated microorganisms, the inoculation ratio is 2:1, and the total inoculation amount is 5%-8% of the total mass of the silkworm pupa protein, and the fermentation is carried out at 30°C-35°C and a ventilation volume of 0.5vvm-0.8vvm for 48h-72h, and then trace elements are added, calcium is added in the form of amino acid chelated calcium, magnesium is added in the form of magnesium citrate, and boron is added in the form of borax chelate, and the addition amounts are respectively 1:1 and 2:1 per liter of mixed solution. The fermentation liquid is 1.5g-2.5g, 0.8g-1.2g, 0.05g-0.1g and 0.01g-0.03g. After stirring and mixing, the pH value is detected. Dipotassium hydrogen phosphate and potassium dihydrogen phosphate are respectively prepared into solutions with a concentration of 0.5mol / L-1.0mol / L. The two solutions are added dropwise to the mixed fermentation liquid using an automatic titration device. The pH sensor is used to monitor the pH value change in real time. When the pH value is between 5.5 and 7.0, the titration speed is slowed down. After each titration, stirring is required for 5min-8min to ensure that the pH value is stable. After stirring, liquid biofertilizer is obtained.

[0013] By adopting the above technical solution, compared with the prior art, the beneficial effects of the present invention are: 1. The present invention uses isoleucine fermentation liquid to convert into liquid biofertilizer, thereby avoiding the pollution to the environment caused by direct discharge of isoleucine fermentation liquid in the traditional treatment method, so that the liquid biofertilizer meets the environmental protection requirements. By performing fine processing and treatment operations on the isoleucine fermentation liquid, harmful substances in the waste liquid are removed, reducing the potential harm to the soil, water and air. At the same time, valuable resources can be recycled and utilized, reducing resource waste and achieving sustainable development. By using isoleucine fermentation waste liquid to prepare biofertilizer, the production cost of biofertilizer is reduced. Compared with the traditional biofertilizer production, the cost of raw materials is lower, reducing the dependence on other expensive raw materials in the process of preparing biofertilizer, and reducing the procurement cost of the enterprise. The produced biofertilizer has a higher added value and can bring more economic benefits to the enterprise. The polypeptide biostimulant in the biofertilizer can stimulate plant growth and improve the stress resistance of crops. The biofertilizer has a remarkable effect in improving soil structure and enhancing soil fertility. At the same time, it can effectively solve the problems of soil compaction and fertility decline caused by long-term use of chemical fertilizers, improve the water and fertilizer retention capacity of the soil, and provide a good soil environment for the growth of crops. 2. The present invention can effectively remove impurities in fermentation waste liquid by adopting gradient magnetic field enhanced impurity removal technology, improve the efficiency and quality of subsequent treatment, and can not only separate impurities through magnetic field treatment, but also have a certain effect on the components in the fermentation liquid, promote the conversion and stabilization of beneficial substances, and fully decompose the organic matter in the fermentation liquid through the application of composite bacterial fermentation technology. Through the synergistic effect of converting Bacillus licheniformis, Lactobacillus plantarum and Aspergillus niger, macromolecular substances can be decomposed into easily absorbable substances such as small molecules of amino acids, thereby improving the utilization rate of nutrients in fertilizers by plants. The addition of nano-selenized polysaccharide complexes and microwave-assisted dispersion technology enhances the stability and long-term effectiveness of biofertilizers. Microwave treatment can improve the dispersion uniformity of the complex in the fermentation liquid, ensure the stability of the quality of the fertilizer, and accurately prepare nutrients for biofertilizers, including the addition of biostimulants and trace elements and the adjustment of pH value, so that the nutrients of biofertilizers are more balanced, meet the growth needs of different crops, and provide a good promotion effect for the growth of crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The figure is a schematic diagram of a specific process of preparing biofertilizer from isoleucine fermentation broth in an embodiment of the present invention. DETAILED DESCRIPTION

[0015] The specific embodiments of the present invention are further described below in conjunction with the accompanying drawings. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0016] The process for preparing biofertilizer by using isoleucine fermentation broth of the present invention comprises isoleucine pretreatment, magnetic field enhanced impurity removal, bacterial fermentation and nutrient preparation. The specific operation steps of the process for preparing biofertilizer by using isoleucine fermentation broth are as follows: Step 1, collecting a high-yield isoleucine strain that has been genetically engineered for fermentation, and after the fermentation is completed, adding a nutrient enhancer and mixing through a pipeline mixer to obtain a mixed isoleucine fermentation liquid; Step 2, placing the isoleucine fermentation liquid in a gradient magnetic field for magnetization treatment, gradually increasing the magnetic field strength within a set range, and controlling the flow rate of the isoleucine fermentation liquid in the magnetic field, then adding a modified flocculant, stirring and mixing, and then passing through a filter press to obtain a clarified filtrate; Step 3, inoculating the clarified filtrate with a mixed bacteria of three composite strains, and transferring the mixed bacteria into a fermentation tank for fermentation under aseptic conditions, wherein a sterile environment needs to be maintained during the process, and after the fermentation is completed, an isoleucine enhanced fermentation broth is obtained; Step 4: adding the nano-selenized polysaccharide complex to the isoleucine enhanced fermentation broth, stirring and mixing, and dispersing the nano-selenized polysaccharide complex in the fermentation broth using microwave-assisted dispersion to obtain a mixed fermentation broth; Step 5: Add biostimulants and trace elements to the mixed fermentation liquid in sequence and mix them. The biostimulants are polypeptide biostimulants. Adjust the pH value of the mixed fermentation liquid to obtain liquid biofertilizer. After completing the above steps, collect and pre-treat the waste liquid generated in steps 2 and 3 and the fermentation waste liquid, and then ferment them twice through the above steps to obtain liquid biofertilizer.

[0017] In one embodiment of the present invention: in step 1, a genetically engineered isoleucine high-yielding strain is selected for fermentation, the fermentation medium uses glucose as a carbon source, the initial glucose concentration is 30g / L-50g / L, and ammonium sulfate is used as a nitrogen source, the initial concentration is 5g / L-10g / L, and an appropriate amount of potassium dihydrogen phosphate is added, the initial concentration is 2g / L-5g / L, and 0.5g / L-1g / L of magnesium sulfate and trace elements are added, the trace elements include 0.01g / L-0.05g / L ferrous sulfate, 0.005g / L-0.02g / L manganese sulfate and 0.01g / L-0.03g / L zinc sulfate.

[0018] In one embodiment of the present invention: in step 1, the nutritional enhancer is selected from unsaturated fatty acids extracted from deep-sea fish oil, plant protein hydrolysate extracted from bean substances and nucleotides extracted from yeast, which are stirred and mixed in a mass ratio of 3:2:1, and the added amount of the nutritional enhancer is 3%-5% of the total mass of the isoleucine fermentation broth. The addition time is after the fermentation is completed, the temperature of the isoleucine fermentation broth is reduced to 30°C-35°C, and the nutritional enhancer is slowly added to the fermentation broth through a pipeline mixer, and stirred and mixed at a stirring rate of 60r / min-80r / min for 30min-60min, and the nutritional enhancer is evenly dispersed in the fermentation broth to obtain a mixed isoleucine fermentation broth.

[0019] In one embodiment of the present invention: in step 2, a special electromagnetic induction device is used to generate a gradient magnetic field, the magnetic field strength is set to 0.2T at the entrance, and is increased according to a gradient of 0.1T / m to reach 1T at the magnetic field outlet. The flow rate of the fermentation liquid through the magnetic field is controlled at 0.3m / s-0.5m / s, and the mixed isoleucine fermentation liquid needs to be treated in the magnetic field environment for 30min-35min. The temperature is maintained between 25°C and 30°C during the treatment process. During use, magnetic shielding materials are used around the equipment for protection.

[0020] In one embodiment of the present invention: in step 2, a natural polymer material chitosan is selected for modification, and the chitosan is dissolved in an acetic acid solution with a volume fraction of 2% to prepare a chitosan solution with a mass fraction of 5%, and then a glutaraldehyde solution with a mass fraction of 25% is added dropwise to the chitosan solution, and the mass ratio of chitosan to glutaraldehyde is 8:1. After mixing, the mixture is stirred and reacted at a temperature of 40°C-50°C for 2h-3h to obtain a modified chitosan flocculant, and the prepared modified chitosan flocculant is added to the fermentation broth treated with a magnetic field to obtain the modified chitosan. The amount of flocculant added is 4g-7g per liter of fermentation liquid, and the mixture is stirred at a stirring rate of 80r / min-120r / min for 20min-30min to fully mix the flocculant and the isoleucine fermentation liquid. After mixing, a plate and frame filter press is used to filter the liquid. The operating pressure of the plate and frame filter press is set at 0.3MPa-0.5MPa, and the filtration time is 2h-3h. In the initial stage of filtration, the operating pressure is controlled to rise slowly, and the clarity of the filtrate is continuously checked. When the filtrate flow rate decreases and the turbidity increases, the filter press operation is stopped to obtain a clear filtrate.

[0021] In one embodiment of the present invention: in step three, the composite bacteria are selected from Bacillus licheniformis, Lactobacillus plantarum and Aspergillus niger, and they are inoculated according to the total volume of the isoleucine fermentation broth, the inoculation amount of Bacillus licheniformis is 3%-5%, the inoculation amount of Lactobacillus plantarum is 4%-6%, and the inoculation amount of Aspergillus niger is 2%-4%, and the three bacterial suspensions are sequentially accessed into the clarified filtrate in the order of bacteria first and then fungi, and a sterile environment is maintained during the process, and the mixture is fully stirred and then transferred to a fermentation tank for fermentation. The initial fermentation time is between 0h-12h, the temperature is controlled between 30℃-32℃, the initial stirring rate is 80r / min-120r / min, and after 12h of fermentation, the temperature is adjusted to 33℃-36℃, the stirring rate is increased to 120r / min-150r / min, the pressure is maintained at 0.02MPa-0.04MPa during the fermentation process, and the fermentation time is 36h-48h.

[0022] In one embodiment of the present invention: in step three, after the fermentation is completed, ultrasonic treatment is first performed, and the fermentation liquid is treated with ultrasound at a frequency of 20-30kHz and a power of 500-800W for 10min-15min to break and disperse the bacterial aggregates, and then a secondary centrifugation treatment is performed, and the centrifugal speed is increased to 6000r / min-8000r / min, and the centrifugation time is 20min-30min to remove the bacteria and impurities therein, and then the fermentation liquid is filtered through a two-stage filtration system, and the first-stage filtration uses a folded filter element filter with a filter element pore size of 5um to remove larger particle impurities, and the second-stage filtration uses a hollow fiber ultrafiltration membrane filter with a membrane pore size of 0.1um to perform fine filtration and sterilization to obtain a stable isoleucine enhanced fermentation liquid.

[0023] In one embodiment of the present invention: in step 4, the nano-selenized polysaccharide complex is passed through a 200-mesh sieve, and the added amount is 1.2%-1.5% of the total volume of the isoleucine enhanced fermentation broth. After mixing for 20min-30min, the mixed solution is subjected to microwave treatment using a microwave device, and the microwave frequency is set to 2450MHz±50MHz. The special microwave reaction container containing the mixed solution is placed in the microwave device, stirred and microwave treated at a stirring rate of 50r / min-90r / min, and the microwave treatment time is 8min-12min. It is divided into three stages, and each stage is separated by 1min-2min. The power in the first stage is 60%-70% of the set power, the power in the second stage is increased to 80%-90%, and the power in the third stage reaches the set power. After the treatment is completed, a mixed fermentation broth is obtained.

[0024] In one embodiment of the present invention: in step five, the biostimulant is a polypeptide biostimulant extracted from the insect protein fermentation liquid, the silkworm pupa protein is fermented, Bacillus subtilis and Aspergillus oryzae are selected as inoculated microorganisms, the inoculation ratio is 2:1, and the total inoculation amount is 5%-8% of the total mass of the silkworm pupa protein, and the fermentation is carried out at 30°C-35°C and a ventilation volume of 0.5vvm-0.8vvm for 48h-72h, and then trace elements are added, calcium is added in the form of amino acid chelated calcium, magnesium is added in the form of magnesium citrate, and boron is added in the form of borax chelate, and the addition amount is respectively 1.5-2.0% and 1.5-2.0% per liter of mixed solution. The fermentation liquid is 1.5g-2.5g, 0.8g-1.2g, 0.05g-0.1g and 0.01g-0.03g. After stirring and mixing, the pH value is detected. Dipotassium hydrogen phosphate and potassium dihydrogen phosphate are respectively prepared into solutions with a concentration of 0.5mol / L-1.0mol / L. The two solutions are added dropwise to the mixed fermentation liquid using an automatic titration device. The pH sensor is used to monitor the pH value change in real time. When the pH value is between 5.5 and 7.0, the titration speed is slowed down. After each titration, stirring is required for 5min-8min to ensure that the pH value is stable. After stirring, liquid biofertilizer is obtained.

[0025] In one embodiment of the present invention: in step one, the fermentation temperature needs to be controlled in stages. In the early stage of fermentation (0h-12h), the temperature is controlled between 32°C-34°C, which is conducive to the rapid growth and reproduction of the strain; in the middle and late stages of fermentation (after 12h), the temperature is adjusted to 30°C-32°C, which is conducive to the accumulation of isoleucine. The pH value is maintained at 6.8-7.2 by automatically adding acid and alkali solutions during the entire fermentation process. The ventilation volume is 0.5vvm-1.0vvm in the early stage of fermentation. As the fermentation proceeds, the bacterial concentration increases. In the middle and late stages of fermentation, the ventilation volume is adjusted to 1.0vvm-1.5vvm, and the stirring speed is correspondingly adjusted from 150r / min-200r / min to 200r / min-250r / min to ensure the supply of dissolved oxygen. The fermentation time is 48h-72h. After the fermentation is completed, the isoleucine content in the fermentation broth can reach 20g / L-30g / L.

[0026] In one embodiment of the present invention: when adding the modified flocculant, a magnetic field treatment can be added for synergy, and an alternating magnetic field is combined with a static magnetic field. The isoleucine fermentation broth is first passed through an alternating magnetic field region, and the frequency in the magnetic field is 50Hz-100Hz, the alternating magnetic field strength is 0.1T-0.3T, and the duration is 10min-15min. After the impurities form agglomerates, they are transferred to a static gradient magnetic field region for flocculation and separation. Compared with a single magnetic field or flocculation treatment, this method can increase the impurity removal rate by 20%-30%.

[0027] In one embodiment of the present invention: in step 2, when a modified flocculant is used for flocculation precipitation and filtered through a plate and frame filter press, impurities that are not completely adsorbed and bound by the flocculant and intercepted by the filter cloth of the filter press but still contain a certain amount of water and a small amount of fermentation liquid components will form waste liquid when the filter cloth and the equipment are cleaned. In step 3, some metabolic wastewater will be generated during the fermentation process, which contains organic acids, alcohols and unused culture medium components produced during microbial metabolism. During the ultrasonic treatment, centrifugal treatment and two-stage filtration process after the fermentation is completed, the ultrasonic equipment, centrifuge and filtration system will produce waste liquid containing impurities and bacterial fragments during the cleaning and maintenance process. The two waste liquids generated are collected and allowed to settle in a sedimentation tank, and the temperature is maintained at 20°C-25°C for 24h-48h. Polyaluminium chloride is added during the precipitation process. After the precipitation is mixed, it is filtered using a vacuum filtration device. After the process is completed, its The nutrient content in the product, including carbon source, nitrogen source, phosphorus source and trace elements, and according to the test results, add appropriate amounts of glucose, ammonium sulfate, potassium dihydrogen phosphate and other nutrients to make it reach a concentration suitable for microbial fermentation, and then select composite microbial strains for secondary fermentation. After the fermentation, the fermentation liquid is filtered and filtered by a combination of a plate and frame filter press and an ultrafiltration membrane. The plate and frame filter press is first used for preliminary filtration at an operating pressure of 0.2MPa-0.4MPa and a filtration time of 1h-2h to remove bacteria and large molecular impurities, and then an ultrafiltration membrane is used for fine filtration with a pore size of 0.05um-0.1um to further remove small molecular impurities and residual microbial metabolites. Finally, the nutrient content, harmful substance content and other indicators of the fermentation liquid are tested, and the pH value is adjusted to 5.5-7.0 to meet the standards of liquid bio-fertilizer to obtain usable liquid bio-fertilizer.

[0028] Embodiment 1 A genetically engineered isoleucine high-yield strain was selected for fermentation. The fermentation medium used glucose as a carbon source with an initial glucose concentration of 35 g / L, and ammonium sulfate as a nitrogen source with an initial concentration of 8 g / L. Potassium dihydrogen phosphate was added with an initial concentration of 3 g / L, magnesium sulfate was 0.8 g / L, and trace elements of ferrous sulfate 0.03 g / L, manganese sulfate 0.01 g / L, and zinc sulfate 0.02 g / L. After the fermentation was completed, unsaturated fatty acids, plant protein hydrolysates, and nucleotides extracted from yeast were mixed in a mass ratio of 3:2:1 as a nutritional enhancer, and the added amount was 4% of the total mass of the isoleucine fermentation liquid. When the temperature of the fermentation liquid dropped to 32°C, the mixture was stirred and mixed at a stirring rate of 70 r / min for 45 minutes through a pipeline mixer. A gradient magnetic field was generated using a special electromagnetic induction device. The magnetic field strength was 0.2T at the inlet and increased by 0.1T / m to 1T at the outlet. The flow rate of the fermentation liquid was 0.4m / s. The solution was treated in the magnetic field for 32min and the temperature was maintained at 28°C. Chitosan was prepared into a 5% solution with 2% acetic acid, and a 25% glutaraldehyde solution (mass ratio of chitosan to glutaraldehyde was 8:1) was added dropwise and reacted at 45°C for 2.5h to obtain a modified flocculant. 5g was added per liter of fermentation liquid. After stirring and mixing at 100r / min for 25min, the clarified filtrate was filtered using a plate and frame filter press (operating pressure 0.4MPa, filtration time 2.5h). The composite bacteria are selected from Bacillus licheniformis, Lactobacillus plantarum and Aspergillus niger, which are inoculated according to the total volume of the fermentation liquid, with the inoculation amounts of 4%, 5% and 3% respectively, and are inoculated in the clarified filtrate in the steps of bacteria first and fungi later. The temperature is controlled at 31°C and the stirring rate is 100r / min during 0-12h, and the temperature is increased to 34°C and the speed is increased to 130r / min after 12h. Fermentation is performed for 42h under the condition of a pressure of 0.03MPa. After the fermentation is completed, ultrasonic treatment is performed for 12min with an ultrasonic device of a frequency of 25kHz and a power of 650W, and then centrifuged at 7000r / min for 25min, and the synergistic fermentation liquid is obtained through two-stage filtration (the pore size of the first-stage folded filter element filter is 5um, and the pore size of the second-stage hollow fiber ultrafiltration membrane filter is 0.1um); The nano-selenated polysaccharide complex was passed through a 200-mesh sieve and then added to the isoleucine enhanced fermentation broth at a volume of 1.3%. After mixing for 25 minutes, the mixture was stirred at 70 r / min and microwaved (microwave frequency 2450 MHz ± 50 MHz, time 10 minutes, divided into three stages, intervals of 1.5 minutes, and the power was set to 65%, 85% and 100% of the microwave power in sequence) to obtain a mixed fermentation broth. The polypeptide biostimulant is extracted from the insect protein fermentation liquid. The silkworm pupa protein is fermented with Bacillus subtilis and Aspergillus oryzae (inoculation ratio is 2:1, and the total inoculation amount is 6% of the silkworm pupa protein mass) at 32°C and a ventilation volume of 0.6 vvm for 60 hours. 2 g of amino acid chelated calcium, 1 g of magnesium citrate and 0.08 g of borax chelate are added to each liter of the mixed fermentation liquid. After the pH value is detected, automatic titration is performed with dipotassium hydrogen phosphate and potassium dihydrogen phosphate solutions with a concentration of 0.8 mol / L. The pH sensor is used for monitoring at the same time. The titration speed is slowed down when the pH value is 5.5-7.0. The mixture is stirred for 6 minutes after each titration. When the pH value is within the set range, the liquid biofertilizer is obtained.

[0029] Embodiment 2 A genetically engineered isoleucine high-yield strain was selected for fermentation. The fermentation medium used glucose as a carbon source with an initial glucose concentration of 40 g / L, and ammonium sulfate as a nitrogen source with an initial concentration of 9 g / L. Potassium dihydrogen phosphate was added with an initial concentration of 3.5 g / L, magnesium sulfate was 0.9 g / L, and trace elements such as ferrous sulfate of 0.035 g / L, manganese sulfate of 0.015 g / L, and zinc sulfate of 0.025 g / L. After the fermentation was completed, unsaturated fatty acids, plant protein hydrolysates, and nucleotides were mixed in a mass ratio of 3:2:1 as nutritional enhancers, and the added amount was 4.5% of the total mass of the fermentation liquid. When the temperature of the fermentation liquid dropped to 33°C, the mixture was stirred and mixed at a stirring rate of 75 r / min for 50 minutes through a pipeline mixer. A gradient magnetic field was generated using a special electromagnetic induction device. The magnetic field strength was 0.2T at the inlet and increased by 0.1T / m to 1T at the outlet. The flow rate of the fermentation liquid was 0.45m / s. The solution was treated in the magnetic field for 33min and the temperature was maintained at 29°C. Chitosan was prepared into a 5% solution with 2% acetic acid. A 25% mass fraction glutaraldehyde solution was added dropwise and reacted at 48°C for 2.8h to obtain a modified flocculant. 5.5g was added per liter of fermentation liquid. After stirring and mixing at 105r / min for 28min, the clarified filtrate was filtered using a plate and frame filter press (operating pressure 0.45MPa, filtration time 2.8h). The composite bacteria selected were Bacillus licheniformis, Lactobacillus plantarum and Aspergillus niger, which were inoculated according to the total volume of the fermentation liquid, with inoculation amounts of 4.5%, 5.5% and 3.5% respectively. The temperature was controlled at 31.5°C and the stirring rate was 105 r / min during 0-12 hours. After 12 hours, the temperature was raised to 34.5°C and the speed was increased to 135 r / min. The fermentation was carried out at a pressure of 0.035 MPa for 44 hours. After the fermentation was completed, an ultrasonic device with a frequency of 26 kHz and a power of 700 W was used for ultrasonic treatment for 13 minutes, and then centrifuged at 7500 r / min for 28 minutes. The synergistic fermentation liquid was obtained by two-stage filtration (the pore size of the first-stage folded filter element filter was 5 um, and the pore size of the second-stage hollow fiber ultrafiltration membrane filter was 0.1 um). The nano-selenized polysaccharide complex was passed through a 200-mesh sieve and added at 1.4% of the total volume of the enhanced fermentation liquid. After mixing for 28 minutes, the mixture was stirred at 75 r / min and microwaved (microwave frequency 2450 MHz ± 50 MHz, time 11 minutes, divided into three stages, intervals of 1.8 minutes, power of 68%, 88%, 100% of microwave power in sequence) to obtain a mixed fermentation liquid. Peptide biostimulants were extracted from insect protein fermentation liquid. Silkworm pupa protein was fermented with Bacillus subtilis and Aspergillus oryzae (inoculation ratio of 2:1, and the total inoculation amount was 6.5% of the silkworm pupa protein mass) at 33°C and a ventilation volume of 0.65 vvm for 62 hours. 2.2 g of amino acid chelated calcium, 1.1 g of magnesium citrate, and 0.09 g of borax chelate were added to each liter of mixed fermentation liquid. After the pH value was detected, it was automatically titrated with 0.85 mol / L dipotassium hydrogen phosphate and potassium dihydrogen phosphate solutions. A pH sensor was used for monitoring at the same time. The titration speed was slowed down when the pH value was 5.5-7.0. After each titration, stirring was carried out for 7 minutes. When the pH value was within the set range, liquid biofertilizer was obtained.

[0030] Embodiment 3 A genetically engineered isoleucine high-yield strain was selected for fermentation. The fermentation medium used glucose as a carbon source with an initial glucose concentration of 45 g / L, and ammonium sulfate as a nitrogen source with an initial concentration of 10 g / L. Potassium dihydrogen phosphate was added with an initial concentration of 4 g / L, magnesium sulfate was added with 1 g / L, and trace elements of ferrous sulfate of 0.04 g / L, manganese sulfate of 0.02 g / L, and zinc sulfate of 0.03 g / L. After the fermentation was completed, unsaturated fatty acids, plant protein hydrolysates, and nucleotides were mixed in a mass ratio of 3:2:1 as nutritional enhancers, and the added amount was 5% of the total mass of the fermentation liquid. When the temperature of the fermentation liquid dropped to 34°C, the mixture was stirred and mixed at a stirring rate of 80 r / min for 55 minutes through a pipeline mixer. A special electromagnetic induction device was used to generate a gradient magnetic field. The magnetic field strength was 0.2T at the inlet and increased by 0.1T / m to 1T at the outlet. The flow rate of the fermentation liquid was 0.5m / s. The solution was treated in the magnetic field for 34min and the temperature was maintained at 30℃. Chitosan was prepared into a 5% solution with 2% acetic acid. A 25% mass fraction glutaraldehyde solution was added dropwise and reacted at 50℃ for 3h to obtain a modified flocculant. 6g was added per liter of fermentation liquid. After stirring and mixing at 110r / min for 30min, the clarified filtrate was filtered using a plate and frame filter press (operating pressure 0.5MPa, filtration time 3h). The composite bacteria selected were Bacillus licheniformis, Lactobacillus plantarum and Aspergillus niger, which were inoculated according to the total volume of the fermentation liquid, with the inoculation amounts of 5%, 6% and 4% respectively. The temperature was controlled at 32°C and the stirring rate was 110r / min from 0 to 12h. After 12h, the temperature was raised to 35°C and the speed was increased to 140r / min. The fermentation was carried out at a pressure of 0.04MPa for 46h. After the fermentation was completed, an ultrasonic device with a frequency of 28kHz and a power of 750W was used for ultrasonic treatment for 14min, and then centrifuged at 8000r / min for 30min. The synergistic fermentation liquid was obtained by two-stage filtration (the pore size of the first-stage folded filter element filter was 5um, and the pore size of the second-stage hollow fiber ultrafiltration membrane filter was 0.1um). The nano-selenized polysaccharide complex was passed through a 200-mesh sieve and added at 1.5% of the total volume of the enhanced fermentation liquid. After mixing for 30 minutes, the mixture was stirred at 80 r / min and microwaved (microwave frequency 2450 MHz ± 50 MHz, time 12 minutes, divided into three stages, intervals of 2 minutes, and the power was 70%, 90%, and 100% of the set microwave power in sequence) to obtain a mixed fermentation liquid; Peptide biostimulants were extracted from insect protein fermentation liquid. Silkworm pupa protein was fermented with Bacillus subtilis and Aspergillus oryzae (inoculation ratio of 2:1, and the total inoculation amount was 7% of the silkworm pupa protein mass) at 34°C and a ventilation volume of 0.7 vvm for 64 hours. 2.4 g of amino acid chelated calcium, 1.2 g of magnesium citrate and 0.1 g of borax chelate were added to each liter of mixed fermentation liquid. After the pH value was detected, it was automatically titrated with 0.9 mol / L dipotassium hydrogen phosphate and potassium dihydrogen phosphate solutions. A pH sensor was used for monitoring at the same time. The titration speed was slowed down when the pH value was 5.5-7.0. After each titration, stirring was carried out for 8 minutes. When the pH value was within the set range, liquid biofertilizer was obtained.

[0031] Embodiment 4: A genetically engineered isoleucine high-yield strain was selected for fermentation. The fermentation medium used glucose as a carbon source with an initial glucose concentration of 42 g / L, and ammonium sulfate as a nitrogen source with an initial concentration of 9.5 g / L. Potassium dihydrogen phosphate was added with an initial concentration of 3.8 g / L, magnesium sulfate was 0.95 g / L, and trace elements of ferrous sulfate, manganese sulfate, and zinc sulfate were 0.038 g / L, 0.018 g / L, and 0.028 g / L. After the fermentation, unsaturated fatty acids, plant protein hydrolysates, and nucleotides were mixed in a mass ratio of 3:2:1 as nutritional enhancers, and the added amount was 4.8% of the total mass of the fermentation liquid. When the temperature of the fermentation liquid dropped to 33.5°C, the mixture was stirred and mixed at a stirring rate of 78 r / min for 52 minutes through a pipeline mixer. A special electromagnetic induction device was used to generate a gradient magnetic field. The magnetic field strength was 0.2T at the inlet and increased by 0.1T / m to 1T at the outlet. The flow rate of the fermentation liquid was 0.48m / s. The solution was treated in the magnetic field for 33.5min and the temperature was maintained at 29.5℃. Chitosan was prepared into a 5% solution with 2% acetic acid. A 25% mass fraction glutaraldehyde solution was added dropwise and reacted at 49℃ for 2.9h to obtain a modified flocculant. 5.8g was added per liter of fermentation liquid. After stirring and mixing at 108r / min for 29min, the clarified filtrate was filtered using a plate and frame filter press (operating pressure 0.48MPa, filtration time 2.9h). The composite bacteria selected were Bacillus licheniformis, Lactobacillus plantarum and Aspergillus niger, which were inoculated according to the total volume of the fermentation liquid, with inoculation amounts of 4.8%, 5.8% and 3.8% respectively. The temperature was controlled at 31.8°C and the stirring rate was 108 r / min during 0-12 h. After 12 h, the temperature was raised to 34.8°C and the speed was increased to 138 r / min. The fermentation was carried out at a pressure of 0.038 MPa for 45 h. After the fermentation was completed, the mixture was treated with ultrasound at a frequency of 27 kHz and a power of 720 W for 13.5 min, and then centrifuged at 7800 r / min for 29 min. The enhanced fermentation liquid was obtained by two-stage filtration (the pore size of the first-stage folded filter element filter was 5 um, and the pore size of the second-stage hollow fiber ultrafiltration membrane filter was 0.1 um). The nano-selenized polysaccharide complex was passed through a 200-mesh sieve and added at 1.45% of the total volume of the enhanced fermentation liquid. After mixing for 29 minutes, the mixture was stirred at 78 r / min and microwaved (microwave frequency 2450 MHz ± 50 MHz, time 11.5 minutes, divided into three stages, intervals of 1.9 minutes, and powers of 69%, 89%, and 100% of the set microwave power, respectively) to obtain a mixed fermentation liquid. Peptide biostimulants were extracted from insect protein fermentation liquid. Silkworm pupa protein was fermented with Bacillus subtilis and Aspergillus oryzae (inoculation ratio of 2:1, and the total inoculation amount was 6.8% of the silkworm pupa protein mass) at 33.5°C and a ventilation volume of 0.68 vvm for 63 hours. 2.3 g of amino acid chelated calcium, 1.15 g of magnesium citrate and 0.095 g of borax chelate were added to each liter of mixed fermentation liquid. After the pH value was detected, it was automatically titrated with dipotassium hydrogen phosphate and potassium dihydrogen phosphate solutions with a concentration of 0.88 mol / L. A pH sensor was used for monitoring at the same time. The titration speed was slowed down when the pH value was 5.5-7.0. After each titration, stirring was carried out for 7.5 minutes. When the pH value was within the set range, liquid biofertilizer was obtained.

[0032] Embodiment 5 A genetically engineered isoleucine high-yield strain was selected for fermentation. The fermentation medium used glucose as a carbon source with an initial glucose concentration of 48 g / L, and ammonium sulfate as a nitrogen source with an initial concentration of 10.5 g / L. Potassium dihydrogen phosphate was added with an initial concentration of 4.2 g / L, magnesium sulfate was 1.05 g / L, and trace elements such as ferrous sulfate 0.042 g / L, manganese sulfate 0.022 g / L, and zinc sulfate 0.032 g / L. After the fermentation was completed, unsaturated fatty acids, plant protein hydrolysates, and nucleotides were mixed in a mass ratio of 3:2:1 as a nutritional enhancer, and the added amount was 5.2% of the total mass of the fermentation liquid. When the temperature of the fermentation liquid dropped to 34.5°C, the mixture was stirred and mixed at a stirring rate of 82 r / min for 58 minutes through a pipeline mixer. A special electromagnetic induction device was used to generate a gradient magnetic field. The magnetic field strength was 0.2T at the inlet and increased by 0.1T / m to 1T at the outlet. The flow rate of the fermentation liquid was 0.52m / s. The solution was treated in the magnetic field for 34.5min and the temperature was maintained at 30.5℃. Chitosan was prepared into a 5% solution with 2% acetic acid. A 25% mass fraction glutaraldehyde solution was added dropwise and reacted at 51℃ for 3.1h to obtain a modified flocculant. 6.2g was added per liter of fermentation liquid. After stirring and mixing at 112r / min for 31min, the clarified filtrate was filtered using a plate and frame filter press (operating pressure 0.52MPa, filtration time 3.1h). The composite bacteria selected were Bacillus licheniformis, Lactobacillus plantarum and Aspergillus niger, which were inoculated according to the total volume of the fermentation liquid, with inoculation amounts of 5.2%, 6.2% and 4.2% respectively. The temperature was controlled at 32.2°C and the stirring rate was 112r / min during 0-12h. After 12h, the temperature was raised to 35.2°C and the speed was increased to 142r / min. The fermentation was carried out at a pressure of 0.042MPa for 48h. After the fermentation was completed, ultrasonic treatment was performed at a frequency of 29kHz and a power of 780W for 14.5min, and then centrifuged at 8200r / min for 31min. The synergistic fermentation liquid was obtained by two-stage filtration (the pore size of the first-stage folded filter element filter was 5um, and the pore size of the second-stage hollow fiber ultrafiltration membrane filter was 0.1um). The nano-selenized polysaccharide complex was passed through a 200-mesh sieve and added at 1.55% of the total volume of the enhanced fermentation liquid. After mixing for 31 minutes, the mixture was stirred at 82 r / min and microwaved using a microwave device (microwave frequency 2450 MHz ± 50 MHz, time 12.5 minutes, divided into three stages, intervals of 2.1 minutes, and powers of 72%, 92%, and 100% of the set microwave power, respectively) to obtain a mixed fermentation liquid; Peptide biostimulants were extracted from insect protein fermentation liquid. Silkworm pupa protein was fermented with Bacillus subtilis and Aspergillus oryzae (inoculation ratio of 2:1, and the total inoculation amount was 7.2% of the silkworm pupa protein mass) at 34.5°C and a ventilation volume of 0.72 vvm for 66 hours. 2.5 g of amino acid chelated calcium, 1.3 g of magnesium citrate, and 0.11 g of borax chelate were added to each liter of mixed fermentation liquid. After the pH value was detected, it was automatically titrated with dipotassium hydrogen phosphate and potassium dihydrogen phosphate solutions with a concentration of 0.92 mol / L. A pH sensor was used for monitoring at the same time. The titration speed was slowed down when the pH value was 5.5-7.0. After each titration, stirring was carried out for 8.5 minutes. When the pH value was within the set range, liquid biofertilizer was obtained.

[0033] The amino acid fertilizers prepared in the above five groups of embodiments were tested, and the specific testing steps were as follows: pH determination, Weigh a certain amount of biofertilizer sample, put it into a beaker, add deionized water with a mass ratio of 1:10 of amino acid fertilizer sample to deionized water, stir with a glass rod to mix it thoroughly, insert the calibrated pH meter electrode into the solution, and record the pH value after the reading stabilizes.

[0034] Nutrient content testing, Nitrogen content detection: Kjeldahl method, The biofertilizer sample is mixed with concentrated sulfuric acid and a catalyst (such as copper sulfate and potassium sulfate), heated and digested to convert organic nitrogen into ammonium nitrogen. The digested solution is distilled to release the ammonium nitrogen in the form of ammonia gas, which is then absorbed with a boric acid solution. The absorbed solution is titrated with a standard hydrochloric acid solution, and the nitrogen content is calculated based on the amount of hydrochloric acid used.

[0035] Phosphorus content detection: Molybdenum antimony anti-spectrophotometry, The fertilizer sample is made into a test solution, and molybdenum antimony anti-color developing agent is added to the test solution to make phosphorus react with it to form a colored compound. The absorbance is measured at a specific wavelength using a spectrophotometer, and the phosphorus content is calculated based on the standard curve.

[0036] Potassium content detection: flame photometry, The fertilizer sample is diluted to make a solution, which is introduced into a flame photometer to measure the intensity of light of a specific wavelength emitted by the potassium element, and the potassium content is calculated based on the standard curve.

[0037] The test data of the finished fertilizer products in the five groups of embodiments are calculated as follows: Embodiment 1

[0038] pH: 6.5; Nitrogen content: 2.52%; Phosphorus content: 0.34; Potassium content: 1.13.

[0039] Embodiment 2 pH: 6.7; Nitrogen content: 2.74; Phosphorus content: 0.37; Potassium content: 1.32.

[0040] Embodiment 3 pH: 6.6; Nitrogen content: 2.81%; Phosphorus content: 0.34; Potassium content: 1.35.

[0041] Embodiment 4: pH: 6.8; Nitrogen content: 2.89%; Phosphorus content: 0.36; Potassium content: 1.44.

[0042] Embodiment 5 pH: 6.7; Nitrogen content: 2.73%; Phosphorus content: 0.35; Potassium content: 1.27.

[0043] In summary, the feasibility and effectiveness of the process for preparing biofertilizer using isoleucine fermentation broth in the five embodiments of the present invention have important practical significance and application value.

[0044] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the scope of protection of the present invention.

[0045] In the description of the specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0046] The above contents are merely examples and explanations of the present invention. Various modifications or additions to the specific embodiments described or replacements in similar ways by technicians in the technical field shall fall within the protection scope of the present invention as long as they do not deviate from the invention or exceed the scope defined by the claims.

[0047] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A process for preparing biofertilizer using isoleucine fermentation liquid, comprising isoleucine pretreatment, magnetic field enhanced impurity removal, bacterial fermentation and nutrient preparation, characterized in that: The specific operation steps of the process for preparing biofertilizer using isoleucine fermentation broth are as follows: Step 1: collecting the fermented liquid after a specific process and using the strain for fermentation, and then adding a nutrient enhancer to mix, so as to obtain a mixed isoleucine fermentation liquid; Step 2: placing the isoleucine fermentation broth in a gradient magnetic field for magnetization treatment, adding a modified flocculant, stirring and mixing, and then passing through a filter press to obtain a clarified filtrate; Step 3, inoculating the composite bacteria into the clarified filtrate, and transferring it into a fermentation tank for fermentation under sterile conditions to obtain isoleucine enhanced fermentation broth; Step 4: adding the nano-selenized polysaccharide complex to the isoleucine enhanced fermentation broth, stirring and mixing, and dispersing the nano-selenized polysaccharide complex in the fermentation broth using microwave-assisted dispersion to obtain a mixed fermentation broth; Step 5: Add biostimulants and trace elements to the mixed fermentation liquid in sequence, adjust the pH value of the mixed fermentation liquid, and obtain liquid biofertilizer.

2. The process for preparing biofertilizer from isoleucine fermentation liquid according to claim 1, characterized in that: In the step 1, a genetically engineered isoleucine high-yielding strain is selected for fermentation, and the fermentation medium uses glucose as a carbon source with an initial glucose concentration of 30 g / L-50 g / L, uses ammonium sulfate as a nitrogen source with an initial concentration of 5 g / L-10 g / L, and adds an appropriate amount of potassium dihydrogen phosphate with an initial concentration of 2 g / L-5 g / L, and adds 0.5 g / L-1 g / L of magnesium sulfate and trace elements, wherein the trace elements include 0.01 g / L-0.05 g / L of ferrous sulfate, 0.005 g / L-0.02 g / L of manganese sulfate, and 0.01 g / L-0.03 g / L of zinc sulfate.

3. The process for preparing biofertilizer by utilizing isoleucine fermentation liquid according to claim 1, characterized in that: In the step 1, the nutritional enhancer is selected from unsaturated fatty acids extracted from deep-sea fish oil, plant protein hydrolyzate extracted from bean substances, and nucleotides extracted from yeast, which are stirred and mixed in a mass ratio of 3:2:1, and the amount of the nutritional enhancer added is 3%-5% of the total mass of the isoleucine fermentation liquid. The addition time is after the fermentation is completed, the temperature of the isoleucine fermentation liquid is reduced to 30° C.-35° C., the nutritional enhancer is slowly added to the fermentation liquid through a pipeline mixer, and the mixture is stirred and mixed at a stirring rate of 60 r / min-80 r / min for 30 min-60 min, and the nutritional enhancer is evenly dispersed in the fermentation liquid to obtain a mixed isoleucine fermentation liquid.

4. The process for preparing biofertilizer by utilizing isoleucine fermentation liquid according to claim 1, characterized in that: In the step 2, a special electromagnetic induction device is used to generate a gradient magnetic field, and the magnetic field strength is set to 0.2T at the entrance, and is increased according to a gradient of 0.1T / m to reach 1T at the magnetic field outlet. The flow rate of the fermentation liquid through the magnetic field is controlled at 0.3m / s-0.5m / s. The mixed isoleucine fermentation liquid needs to be treated in the magnetic field environment for 30min-35min, and the temperature is maintained between 25°C and 30°C during the treatment process. During use, magnetic shielding materials are used around the equipment for protection.

5. The process for preparing biofertilizer from isoleucine fermentation liquid according to claim 1, characterized in that: In the step 2, a natural polymer material chitosan is selected for modification, and the chitosan is dissolved in an acetic acid solution with a volume fraction of 2% to prepare a chitosan solution with a mass fraction of 5%, and then a glutaraldehyde solution with a mass fraction of 25% is added dropwise to the chitosan solution, and the mass ratio of chitosan to glutaraldehyde is 8:

1. After mixing, the mixture is stirred and reacted at a temperature of 40°C-50°C for 2h-3h to obtain a modified chitosan flocculant, and the prepared modified chitosan flocculant is added to the fermentation broth treated with a magnetic field, and the addition of the modified chitosan flocculant is performed. The amount is 4g-7g per liter of fermentation liquid, and the flocculant is stirred and mixed at a stirring rate of 80r / min-120r / min for 20min-30min to fully mix the isoleucine fermentation liquid. After mixing, a plate and frame filter press is used to filter it. The operating pressure of the plate and frame filter press is set at 0.3MPa-0.5MPa, and the filtration time is 2h-3h. In the initial stage of filtration, the operating pressure is controlled to rise slowly, and the clarity of the filtrate is continuously checked. When the filtrate flow rate decreases and the turbidity increases, the filtration operation is stopped to obtain a clear filtrate.

6. The process for preparing biofertilizer using isoleucine fermentation liquid according to claim 1, characterized in that: In the step 3, the composite bacteria are selected from Bacillus licheniformis, Lactobacillus plantarum and Aspergillus niger, which are inoculated according to the total volume of the isoleucine fermentation liquid, the inoculation amount of Bacillus licheniformis is 3%-5%, the inoculation amount of Lactobacillus plantarum is 4%-6%, and the inoculation amount of Aspergillus niger is 2%-4%, and the three bacterial suspensions are sequentially accessed into the clarified filtrate in the order of bacteria first and fungi later, and a sterile environment is maintained during the process, and the mixture is fully stirred and then transferred to a fermentation tank for fermentation. The initial fermentation time is between 0h-12h, the temperature is controlled between 30°C-32°C, the initial stirring rate is 80r / min-120r / min, and after fermentation for 12h, the temperature is adjusted to 33°C-36°C, the stirring rate is increased to 120r / min-150r / min, the pressure is maintained at 0.02MPa-0.04MPa during the fermentation process, and the fermentation time is 36h-48h.

7. The process for preparing biofertilizer from isoleucine fermentation liquid according to claim 1, characterized in that: In the step three, after the fermentation is completed, ultrasonic treatment is first performed, and the fermentation liquid is treated with ultrasound at a frequency of 20-30kHz and a power of 500-800W for 10min-15min to break and disperse the bacterial aggregates, and then a secondary centrifugation treatment is performed, and the centrifugal speed is increased to 6000r / min-8000r / min, and the centrifugation time is 20min-30min to remove the bacteria and impurities therein, and then the fermentation liquid is filtered through a two-stage filtration system, and the first-stage filtration uses a folded filter element filter with a filter element pore size of 5um to remove larger particle impurities, and the second-stage filtration uses a hollow fiber ultrafiltration membrane filter with a membrane pore size of 0.1um to perform fine filtration and sterilization to obtain a stable isoleucine enhanced fermentation liquid.

8. The process for preparing biofertilizer from isoleucine fermentation liquid according to claim 1, characterized in that: In the step 4, the nano-selenized polysaccharide complex is passed through a 200-mesh sieve, and the added amount is 1.2%-1.5% of the total volume of the isoleucine enhanced fermentation liquid. After mixing for 20 minutes to 30 minutes, the mixed liquid is subjected to microwave treatment using a microwave device, and the microwave frequency is set to 2450MHz±50MHz. The special microwave reaction container containing the mixed liquid is placed in the microwave device, and the mixture is stirred and microwave treated at a stirring rate of 50r / min-90r / min. The microwave treatment time is 8min-12min, and it is divided into three stages, each stage is separated by 1min-2min, the power of the first stage is 60%-70% of the set power, the power of the second stage is increased to 80%-90%, and the power of the third stage reaches the set power. After the treatment is completed, a mixed fermentation liquid is obtained.

9. The process for preparing biofertilizer from isoleucine fermentation liquid according to claim 1, characterized in that: In the step 5, the biostimulant is a polypeptide biostimulant extracted from insect protein fermentation liquid, the silkworm pupa protein is fermented, Bacillus subtilis and Aspergillus oryzae are selected as inoculated microorganisms, the inoculation ratio is 2:1, and the total inoculation amount is 5%-8% of the total mass of the silkworm pupa protein, and the fermentation is carried out at 30°C-35°C and a ventilation volume of 0.5vvm-0.8vvm for 48h-72h, and then trace elements are added, calcium is added in the form of amino acid chelated calcium, magnesium is added in the form of magnesium citrate, and boron is added in the form of borax chelate, and the addition amount is 1. 5g-2.5g, 0.8g-1.2g, 0.05g-0.1g and 0.01g-0.03g. After stirring and mixing, detect the pH value. Prepare dipotassium hydrogen phosphate and potassium dihydrogen phosphate into solutions with a concentration of 0.5mol / L-1.0mol / L respectively. Use an automatic titration device to drop the two solutions into the mixed fermentation liquid. Use a pH sensor to monitor the pH value change in real time. When the pH value is between 5.5-7.0, slow down the titration speed. Stir for 5min-8min after each titration to ensure that the pH value is stable. After stirring, liquid biofertilizer is obtained.

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