Process for preparing fertilizer from leucine fermentation liquor and nitrogen-fixing microorganisms

Abstract

The invention discloses a technology for preparing a fertilizer from leucine fermentation liquor and nitrogen-fixing microorganisms, and relates to the technical field of fertilizer preparation, and the technology comprises the steps of leucine fermentation liquor preparation, nitrogen-fixing microorganism culture and leucine fermentation liquor and nitrogen-fixing microorganism mixing. The problem that the nutritional ingredients are single and insufficient when leucine fermentation liquor is independently used can be solved, leucine can participate in plant protein synthesis, regulate the metabolic pathway and enhance the immunity and stress resistance of plants, nitrogen-fixing microorganisms can convert nitrogen in air into a nitrogen source capable of being utilized by the plants, and the nitrogen-fixing microorganisms and the nitrogen-fixing microorganisms are mixed to prepare the fertilizer. According to the present invention, with the application of the leucine fermentation broth, the more comprehensive nutrition can be provided for the plant, the synthesis of the leucine and the growth stability of the strain are further promoted by adding the novel bioactive factor, the quality and the nutritional value of the leucine fermentation broth are improved, and the activity and the breeding speed of the nitrogen-fixing microorganism are substantially improved by adding the growth promoter.

Description

Technical Field

[0001] The invention relates to the technical field of fertilizer preparation, in particular to a process for preparing fertilizer by using leucine fermentation liquid and nitrogen-fixing microorganisms. Background Art

[0002] In current agricultural production, fertilizer plays a vital role. Although traditional fertilizers can meet the needs of crop growth to a certain extent, they generally have problems such as single nutrient composition, short-term fertilizer effect, and certain pollution to the environment. With the increasing requirements for sustainable agricultural development, it has become an urgent task to find more efficient, environmentally friendly and sustainable fertilizers. As an important amino acid, leucine has potential value in promoting plant growth and improving stress resistance. Leucine can participate in plant protein synthesis, regulate metabolic pathways, and enhance plant immunity and stress resistance. At present, the production of leucine fermentation liquid is mainly obtained by microbial fermentation. However, there are some limitations in the application of leucine fermentation liquid alone. For example, the nutrients in leucine fermentation liquid are relatively single and it is difficult to meet the needs of comprehensive plant growth. In addition, the stability and long-term effectiveness of leucine fermentation liquid need to be improved. Nitrogen-fixing microorganisms play a key role in soil ecosystems. They can convert nitrogen in the air into nitrogen sources that can be used by plants, improve soil fertility, and reduce the demand for chemical nitrogen fertilizers. Nitrogen-fixing microorganisms such as new rhizobium variants and special brown nitrogen-fixing bacteria subspecies have efficient nitrogen-fixing ability and the potential to adapt to a variety of soil environments. However, the survival and function of nitrogen-fixing microorganisms in the soil are restricted by many factors, such as soil pH, temperature, humidity and other environmental conditions, as well as competition with other microorganisms, which may affect the activity and nitrogen-fixing efficiency of nitrogen-fixing microorganisms. Using nitrogen-fixing microorganisms as fertilizers alone also has problems such as unstable fertilizer effect and difficulty in exerting their effects for a long time. In view of the respective characteristics and limitations of leucine fermentation broth and nitrogen-fixing microorganisms, combining the two to prepare fertilizers has important practical significance. To this end, we proposed a process for preparing fertilizers using leucine fermentation broth and nitrogen-fixing microorganisms. Summary of the invention

[0003] The object of the present invention is to provide a process for preparing fertilizer using leucine fermentation liquid and nitrogen-fixing microorganisms.

[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 fertilizer using leucine fermentation broth and nitrogen-fixing microorganisms, comprising preparing leucine fermentation broth, culturing nitrogen-fixing microorganisms and mixing leucine fermentation broth with nitrogen-fixing microorganisms, wherein the specific operation steps of preparing fertilizer using leucine fermentation broth and nitrogen-fixing microorganisms are as follows: Step 1: Preparation of leucine fermentation broth: Select a new leucine production strain improved by genetic engineering, prepare a special fermentation medium, and add a trace amount of new biologically active factors; Step 2: inoculating the strain into a fermentation medium, and after fermentation is complete, filtering by a multi-stage membrane to separate and obtain a high-purity leucine fermentation broth; Step 3: Cultivating nitrogen-fixing microorganisms: selecting a new rhizobium variant and a special round brown nitrogen-fixing bacteria subspecies, preparing an optimized nitrogen-fixing microorganism culture medium, adding a growth promoter, and inoculating the nitrogen-fixing microorganisms into the culture medium to obtain a nitrogen-fixing microorganism culture; Step 4: mixing the leucine fermentation liquid with the nitrogen-fixing microorganisms, adding a filler thereto, stirring and mixing them evenly, drying them using a low-temperature plasma-assisted drying technology, and introducing a magnetic field-assisted device to treat the mixture, and obtaining a semi-finished fertilizer after drying; Step 5: Crush and sieve the dried material to obtain granular fertilizer products, and conduct quality and characteristic tests on them. The qualified granular fertilizers are coated, bagged and stored in a dry and ventilated place.

[0005] As a further scheme of the present invention: in the step 1, a novel leucine-producing strain is selected, and an activation treatment is performed on it, and an activation medium is prepared, wherein the activation medium includes 10 g / L glucose, 5 g / L ammonium sulfate, 0.8 g / L potassium dihydrogen phosphate and 0.3 g / L magnesium sulfate at a concentration of 10 g / L, and the novel leucine-producing strain is inoculated on the activation medium and cultured at 27° C.-30° C. and a pH value of 6.5-7.0 for 12 h-18 h.

[0006] As a further scheme of the present invention: in the step 1, the activated strain is inoculated into the fermentation medium, and the inoculation amount is 5%-10% of the total volume of the fermentation medium. During the fermentation process, the ambient temperature is controlled at 30°C-35°C, the pH value is in the range of 6.8-7.2, and sterile air is continuously introduced, and the ventilation volume is 0.5-1.0L / min per liter of culture medium. At the same time, the stirring rate is controlled at 150r / min-200r / min to ensure that the nutrients in the culture medium are evenly distributed. The fermentation time is 55h-65h. During the fermentation period, samples are taken every 6h-8h to detect the leucine content and the bacterial concentration, and the fermentation conditions are adjusted in time.

[0007] As a further solution of the present invention: in the step 2, the microfiltration treatment uses a microfiltration membrane with a pore size of 0.2um-0.4um to perform preliminary filtration on the leucine fermentation broth, and 3 / h-2.5m 3 / h flow rate through the microfiltration membrane, the operating pressure is controlled at 0.15MPa-0.25MPa, the filtration temperature is maintained between 28℃-30℃, the ultrafiltration treatment uses an ultrafiltration membrane with a pore size of 20nm-40nm for deep filtration, the operating pressure is controlled at 0.30MPa-0.45MPa, the filtration temperature is maintained between 28℃-30℃, and the nanofiltration treatment uses a nanofiltration membrane with a pore size of 1.5nm-3nm for fine separation.

[0008] As a further scheme of the present invention: in the step 2, after multi-stage filtration, it is passed into an ion exchange resin for separation, and a strong acid cation exchange resin is selected. Before the separation operation, the resin is rinsed with deionized water until the effluent is clear, and the resin is soaked in a hydrochloric acid solution with a concentration of 1mol / L-2mol / L for 4-6h. After completion, it is rinsed with deionized water to neutrality, and then soaked in a sodium hydroxide solution with a concentration of 1mol / L-2mol / L for 4h-6h. After completion, it is rinsed with deionized water to neutrality for standby use.

[0009] As a further scheme of the present invention: in the step three, a nitrogen-fixing microbial culture medium is prepared, wherein the main components include sucrose at a concentration of 14g / L-16g / L, yeast powder at a concentration of 3g / L-3.5g / L, potassium dihydrogen phosphate at a concentration of 0.7g / L-0.8g / L, magnesium sulfate at a concentration of 0.3g / L-0.4g / L, and a growth promoter at a concentration of 0.03g / L-0.05g / L. The screened and domesticated new rhizobium variants and the special round brown nitrogen-fixing bacteria subspecies are inoculated into the nitrogen-fixing microbial culture medium, the inoculation amount is 3%-5% of the total volume of the culture medium, the culture temperature is 26°C-28°C, and the pH value is set in the range of 6.5-7.5.

[0010] As a further scheme of the present invention: in the step three, intermittent ventilation is adopted during the culture process, the ventilation volume is 0.2L / h-0.4L / h per liter of fermentation medium, the ventilation time interval is 2h-3h, and a stirring device is used during the culture process to stir the fermentation medium at a speed of 80r / min-120r / min to ensure uniform distribution of nutrients. The culture time is 35h-40h. During this period, samples are taken every 5h-8h to detect the number and activity indicators of nitrogen-fixing microorganisms, and the culture conditions are adjusted according to the test results.

[0011] As a further solution of the present invention: in the step 4, the volume ratio of leucine fermentation broth to nitrogen-fixing microorganism is 1:2, and during the mixing process, the mixture is stirred at a stirring rate of 100 r / min for 30 min. Before mixing, the leucine fermentation broth needs to be sterilized by ultraviolet irradiation. After the mixing is completed, nano-vermiculite is added to the mixture, and the particle size of the added nano-vermiculite is controlled between 50nm-80nm, and the amount of nano-vermiculite added is 25% of the total mass of the mixed solution. The mixture is stirred at a stirring rate of 80 r / min for 20 min, and the filler and the mixed solution are fully mixed, and then transferred to a low-temperature plasma-assisted drying device, and the drying temperature is set to 55°C and the energy density of the plasma is set to 500 J / m 3 And the dry air flow rate is 2m 3 / h, dry for 40min-60min under the above conditions, and obtain a semi-finished fertilizer after drying.

[0012] As a further scheme of the present invention: in the step five, the dried semi-finished fertilizer product is sent to a pulverizing device for pulverization, and the smoke produced is removed by a vacuum cleaner. The pulverized semi-finished fertilizer product is sieved, and the sieve aperture is controlled between 2mm-3mm. The quality and characteristic detection includes appearance detection, effective ingredient detection, pH value detection and moisture content detection. After the finished fertilizer product is qualified, it is placed in a coating device, and the coating thickness is controlled to be 0.2mm-0.25mm, the pressure is 0.1KPa, and the coating temperature is controlled to be 40°C. After the coating is completed, it is put into a packaging bag, and the humidity of the storage environment is controlled below 60%, and the temperature is maintained between 15°C-25°C.

[0013] By adopting the above technical solution, compared with the prior art, the beneficial effects of the present invention are: 1. The present invention combines leucine fermentation liquid with nitrogen-fixing microorganisms to make up for the problem of insufficient single nutrient components of leucine fermentation liquid when used alone. Leucine in the leucine fermentation liquid can participate in plant protein synthesis, regulate metabolic pathways, and enhance plant immunity and stress resistance. Nitrogen-fixing microorganisms can convert nitrogen in the air into nitrogen sources that can be used by plants. Mixing the two into fertilizers can provide plants with more comprehensive nutrition. By adding new biologically active factors, the synthesis of leucine and the growth stability of strains are further promoted, and the quality and nutritional value of the leucine fermentation liquid are improved. By adding growth promoters, the activity and reproduction speed of nitrogen-fixing microorganisms are greatly improved, so that the nitrogen-fixing components in the fertilizer are more abundant. By multi-stage membrane filtration and ion exchange resin separation technology, high-purity leucine fermentation liquid is obtained, impurities and harmful substances are removed, and the stability of the fertilizer is improved. By adding fillers in the fertilizer preparation process, the adsorption and water retention of the fertilizer are enhanced, and the nutrients can be slowly released, the duration of fertilizer effect is extended, and the use of the prepared fertilizer is more efficient. 2. The present invention processes the mixture through low-temperature plasma assisted drying technology and magnetic field assisted device, so that the molecular arrangement inside the fertilizer particles is more orderly, the stability and biological activity of the fertilizer are improved, and the coating treatment of the finished fertilizer further controls the release rate of the fertilizer, ensuring that the plants can obtain sufficient nutrients for a long time. By using the finished fertilizer, the dependence of organisms on chemical nitrogen fertilizers can be reduced, and the probability of environmental problems such as soil pollution and eutrophication of water bodies caused by excessive use of chemical fertilizers is reduced. By adding nitrogen-fixing microorganisms to the fertilizer, nitrogen in the air is converted into a nitrogen source that can be used by plants, the recycling of resources is achieved, and energy consumption and greenhouse gas emissions are reduced. By screening and domesticating new rhizobium variants and special round brown nitrogen-fixing bacteria subspecies, they have the potential to adapt to a variety of complex soil environments. These nitrogen-fixing microorganisms can play a role in soils with different pH, temperature and humidity, which improves the application range of the fertilizer. The components in the leucine fermentation liquid can also regulate the soil environment to a certain extent, and enhance the adaptability of plants to adverse soil conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a schematic diagram of a process for preparing fertilizer in an embodiment of the present invention; Figure 2 Schematic diagram for comparing the quality characteristics of finished fertilizer products in the embodiments 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 fertilizer using leucine fermentation liquid and nitrogen-fixing microorganisms of the present invention comprises preparing leucine fermentation liquid, culturing nitrogen-fixing microorganisms and mixing leucine fermentation liquid and nitrogen-fixing microorganisms. The specific operation steps for preparing fertilizer using leucine fermentation liquid and nitrogen-fixing microorganisms are as follows: Step 1: Preparation of leucine fermentation broth: Select a new leucine production strain improved by genetic engineering, prepare a special fermentation medium, and add a trace amount of new biologically active factors; Step 2: inoculating the strain into a fermentation medium, and after fermentation is complete, filtering by a multi-stage membrane to separate and obtain a high-purity leucine fermentation broth; Step 3: Cultivating nitrogen-fixing microorganisms: selecting a new rhizobium variant and a special round brown nitrogen-fixing bacteria subspecies, preparing an optimized nitrogen-fixing microorganism culture medium, adding a growth promoter, and inoculating the nitrogen-fixing microorganisms into the culture medium to obtain a nitrogen-fixing microorganism culture; Step 4: mixing the leucine fermentation liquid with the nitrogen-fixing microorganisms, adding a filler thereto, stirring and mixing them evenly, drying them using a low-temperature plasma-assisted drying technology, and introducing a magnetic field-assisted device to treat the mixture, and obtaining a semi-finished fertilizer after drying; Step 5: Crush and sieve the dried material to obtain granular fertilizer products, and conduct quality and characteristic tests on them. The qualified granular fertilizers are coated, bagged and stored in a dry and ventilated place.

[0017] In one embodiment of the present invention: in step 1, a novel leucine-producing strain is selected, and an activation treatment is performed on it, and an activation medium is prepared, wherein the activation medium includes 10 g / L glucose, 5 g / L ammonium sulfate, 0.8 g / L potassium dihydrogen phosphate and 0.3 g / L magnesium sulfate at a concentration of 10 g / L, and the novel leucine-producing strain is inoculated on the activation medium and cultured at 27° C.-30° C. and a pH value of 6.5-7.0 for 12 h-18 h.

[0018] In one embodiment of the present invention: in step one, the activated strain is inoculated into the fermentation medium, and the inoculation amount is 5%-10% of the total volume of the fermentation medium. During the fermentation process, the ambient temperature is controlled at 30°C-35°C, the pH value is in the range of 6.8-7.2, and sterile air is continuously introduced, and the ventilation volume is 0.5-1.0L / min per liter of culture medium. At the same time, the stirring rate is controlled at 150r / min-200r / min to ensure that the nutrients in the culture medium are evenly distributed. The fermentation time is 55h-65h. During the fermentation period, samples are taken every 6h-8h to detect the leucine content and the bacterial concentration, and the fermentation conditions are adjusted in time.

[0019] In one embodiment of the present invention, in step 2, the microfiltration treatment uses a microfiltration membrane with a pore size of 0.2um-0.4um to preliminarily filter the leucine fermentation broth and filter it according to 1.5m 3 / h-2.5m 3 / h flow rate through the microfiltration membrane, the operating pressure is controlled at 0.15MPa-0.25MPa, the filtration temperature is maintained between 28℃-30℃, the ultrafiltration treatment uses an ultrafiltration membrane with a pore size of 20nm-40nm for deep filtration, the operating pressure is controlled at 0.30MPa-0.45MPa, the filtration temperature is maintained between 28℃-30℃, and the nanofiltration treatment uses a nanofiltration membrane with a pore size of 1.5nm-3nm for fine separation.

[0020] In one embodiment of the present invention: in step 2, after multi-stage filtration, it is passed into an ion exchange resin for separation, and a strong acid cation exchange resin is selected. Before the separation operation, the resin is rinsed with deionized water until the effluent is clear, and the resin is soaked in a hydrochloric acid solution with a concentration of 1mol / L-2mol / L for 4-6h. After completion, it is rinsed with deionized water to neutrality, and then soaked in a sodium hydroxide solution with a concentration of 1mol / L-2mol / L for 4h-6h. After completion, it is rinsed with deionized water to neutrality for standby use.

[0021] In one embodiment of the present invention: in step three, a nitrogen-fixing microbial culture medium is prepared, wherein the main components include sucrose at a concentration of 14g / L-16g / L, yeast powder at a concentration of 3g / L-3.5g / L, potassium dihydrogen phosphate at a concentration of 0.7g / L-0.8g / L, magnesium sulfate at a concentration of 0.3g / L-0.4g / L, and a growth promoter at a concentration of 0.03g / L-0.05g / L. The screened and domesticated new rhizobium variants and special brown nitrogen-fixing bacteria subspecies are inoculated into the nitrogen-fixing microbial culture medium, the inoculation amount is 3%-5% of the total volume of the culture medium, the culture temperature is 26°C-28°C, and the pH value is set in the range of 6.5-7.5.

[0022] In one embodiment of the present invention: in step three, intermittent ventilation is used during the culture process, the ventilation volume is 0.2L / h-0.4L / h per liter of fermentation medium, the ventilation time interval is 2h-3h, and a stirring device is used during the culture process to stir the fermentation medium at a speed of 80r / min-120r / min to ensure uniform distribution of nutrients. The culture time is 35h-40h. During this period, samples are taken every 5h-8h to detect the number and activity indicators of nitrogen-fixing microorganisms, and the culture conditions are adjusted according to the test results.

[0023] In one embodiment of the present invention: in step 4, the volume ratio of leucine fermentation broth to nitrogen-fixing microorganisms is 1:2. During the mixing process, the mixture is stirred at a stirring rate of 100 r / min for 30 min. Before mixing, the leucine fermentation broth needs to be sterilized by ultraviolet irradiation. After the mixing is completed, nano-vermiculite is added to the mixture, and the particle size of the added nano-vermiculite is controlled between 50 nm and 80 nm. The amount of nano-vermiculite added is 25% of the total mass of the mixed solution. The mixture is stirred at a stirring rate of 80 r / min for 20 min. After the filler and the mixed solution are fully mixed, the mixture is transferred to a low-temperature plasma-assisted drying device, and the drying temperature is set to 55° C. and the energy density of the plasma is set to 500 J / m 3 And the dry air flow rate is 2m 3 / h, dry for 40min-60min under the above conditions, and obtain a semi-finished fertilizer after drying.

[0024] In one embodiment of the present invention: in step five, the dried semi-finished fertilizer product is sent to a pulverizing device for pulverization, and a vacuum cleaner is used to remove the produced smoke and dust. The pulverized semi-finished fertilizer product is sieved, and the sieve aperture is controlled between 2mm-3mm. The quality and characteristic detection includes appearance detection, effective ingredient detection, pH value detection and moisture content detection. After the finished fertilizer product is qualified, it is placed in a coating device, and the coating thickness is controlled to be 0.2mm-0.25mm, the pressure is 0.1KPa, and the coating temperature is controlled at 40°C. After the coating is completed, it is put into a packaging bag, and the humidity of the storage environment is controlled below 60%, and the temperature is maintained between 15°C-25°C.

[0025] In one embodiment of the present invention: a special fermentation medium is configured, wherein the main components include glucose, ammonium sulfate, potassium dihydrogen phosphate and magnesium sulfate, glucose provides a carbon source for the growth of a new leucine-producing strain and the synthesis of leucine, ammonium sulfate as a nitrogen source promotes the growth and metabolism of bacteria, potassium dihydrogen phosphate provides phosphorus and participates in cell metabolism and energy transfer, magnesium sulfate helps to maintain enzyme activity and cell structure stability, and at the same time, adding a trace amount of a new bioactive factor can promote the synthesis of leucine and the growth stability of the strain, and by controlling the concentration of each component, it is ensured that the fermentation medium can provide the most suitable growth environment for the strain.

[0026] In one embodiment of the present invention: after the fermentation is carried out for 55h-65h, the changes in various indicators of the fermentation liquid are closely observed, and the leucine content is detected by high performance liquid chromatography. When the change rate of the leucine content at the three detection points is less than 1 and the bacterial concentration no longer increases significantly, it can be preliminarily judged that the fermentation has reached the end point. At the same time, a comprehensive judgment is made based on factors such as the appearance of the fermentation liquid and the stability of the pH value. When the leucine fermentation liquid has a uniform color and no obvious turbidity, and the pH value is within the range of 6.8-7.2, it can also be used as an auxiliary judgment basis for the completion of the fermentation.

[0027] In one embodiment of the present invention: when low-temperature plasma assisted drying is adopted, a magnetic field auxiliary device can be introduced to treat the semi-finished fertilizer product, the magnetic field strength is set to 1000 gauss, and the magnetic field action time is 30min-45min. Under the action of the magnetic field, the molecular arrangement inside the fertilizer particles can be promoted to be more orderly, thereby improving the stability and biological activity of the fertilizer.

[0028] In one embodiment of the present invention: the novel bioactive factor is a complex composed of specific organic acids, polypeptides and biological enzymes, which can effectively promote the synthesis of leucine and the growth stability of the strain through the synergistic effect of organic acids, polypeptides and biological enzymes, and improve the quality and yield of leucine fermentation broth.

[0029] In one embodiment of the present invention: the growth promoter added to the nitrogen-fixing microorganism culture medium is a substance designed for new rhizobium variants and special brown nitrogen-fixing bacteria subspecies, which can increase the activity and reproduction rate of nitrogen-fixing microorganisms. The growth promoter contains specific nutrients, signal molecules and enzyme activators, which can stimulate the metabolic activities of nitrogen-fixing microorganisms and promote cell division and growth.

[0030] In one embodiment of the present invention: the growth promoter is mainly composed of an amino acid solution, a trace element solution and a plant growth regulator solution, which are mixed and added to a nitrogen-fixing microorganism culture medium after the mixing is completed, and the growth of the nitrogen-fixing microorganisms and the nitrogen fixation efficiency are observed, and compared with the control group without the addition of the growth promoter, the activity of the growth promoter is evaluated by measuring the number of nitrogen-fixing microorganisms and the nitrogenase activity, so as to ensure that the growth promoter can effectively promote the growth and nitrogen fixation of nitrogen-fixing microorganisms.

[0031] In one embodiment of the present invention, "screening" refers to the process of selecting strains with certain specific excellent traits from a large number of rhizobia and round brown nitrogen-fixing bacteria through specific methods and standards. The screening can be carried out according to the standards of strong nitrogen fixation ability, adaptability to specific environmental conditions, good symbiotic effect with specific plants, etc. "Domestication" refers to the process of gradually adapting the screened strains to specific culture conditions or environments. By changing the temperature, pH value, nutrients, etc., the strains can gradually adapt and optimize their performance so that they can play a better role in specific application scenarios. Domestication is carried out under the above-given culture temperature, pH value, ventilation conditions and stirring speed conditions to improve their growth and nitrogen fixation efficiency in the fermentation system.

[0032] Embodiment 1 Preparation of leucine fermentation broth: A new leucine production strain improved by genetic engineering was selected, inoculated into an activation medium (glucose 10 g / L, ammonium sulfate 5 g / L, potassium dihydrogen phosphate 0.8 g / L, magnesium sulfate 0.3 g / L), and activated and cultured for 15 hours at 28°C and pH 6.8. The activated strain was inoculated into a fermentation medium (glucose 60 g / L, ammonium sulfate 12 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate 0.7 g / L, and a new bioactive factor concentration of 0.03 g / L) at an inoculation rate of 8% of the total volume of the fermentation medium. The ambient temperature was controlled at 32°C and the pH was 7.0. Sterile air was continuously introduced with a ventilation volume of 0.8 L / min per liter of culture medium, a stirring rate of 180 r / min, and a fermentation time of 60 hours. During the period, samples were taken every 7 hours to detect the leucine content and bacterial concentration. After the fermentation was completed, the leucine fermentation broth was initially filtered using a microfiltration membrane with a pore size of 0.3um, with a flow rate of 2m³ / h, an operating pressure of 0.2MPa, and a filtration temperature of 29°C. Then, an ultrafiltration membrane with a pore size of 30nm was used for deep filtration, with an operating pressure of 0.4MPa and a filtration temperature of 29°C. Finally, a nanofiltration membrane with a pore size of 2nm was used for fine separation. After separation, a strong acid cation exchange resin was passed through, and the resin was rinsed with deionized water until the effluent was clear. Then, the resin was soaked in a hydrochloric acid solution with a concentration of 1.5mol / L for 5h, and then rinsed with deionized water until neutral. Then, the resin was soaked in a sodium hydroxide solution with a concentration of 1.5mol / L for 5h, and finally rinsed with deionized water until neutral for use. Nitrogen-fixing microorganism culture: The main components of the nitrogen-fixing microorganism culture medium include 15 g / L sucrose, 3.2 g / L yeast powder, 0.75 g / L potassium dihydrogen phosphate, 0.35 g / L magnesium sulfate and growth promoters. The screened and domesticated new rhizobium variants and special round brown nitrogen-fixing bacteria subspecies are inoculated into the nitrogen-fixing microorganism culture medium at an inoculation rate of 4% of the total volume of the culture medium. The culture temperature is 27°C, the pH value is set to 7.0, and intermittent ventilation is used. The ventilation volume is 0.3 L / h per liter of fermentation medium, and the ventilation time interval is 2.5 hours. At the same time, a stirring device is used to stir the fermentation medium at a speed of 100 r / min. The culture time is 38 hours, and samples are taken every 6 hours to detect the number and activity indicators of nitrogen-fixing microorganisms; Fertilizer preparation: The leucine fermentation liquid and the nitrogen-fixing microorganism are mixed in a volume ratio of 1:2, and stirred at a stirring rate of 100 r / min for 30 minutes during the mixing process. The leucine fermentation liquid is sterilized by ultraviolet irradiation before mixing. After the mixing is completed, nano vermiculite (the particle size is controlled between 60nm-70nm) is added to the mixture, and the amount of nano vermiculite added is 25% of the total mixed liquid mass. The mixture is stirred at a stirring rate of 80 r / min for 20 minutes. The mixture is then transferred to a low-temperature plasma-assisted drying device, and the drying temperature is set to 55°C, the energy density of the plasma is 500 J / m³, and the flow rate of dry air is 2m³ / h. The mixture is dried for 50 minutes, and a magnetic field auxiliary device with a magnetic field strength of 1000 Gauss is introduced for 40 minutes. The dried material is crushed and sieved (the mesh aperture is controlled at 2.5mm), and the quality and characteristics of the fertilizer are tested. After passing the test, the material is placed in a coating device, and the coating thickness is controlled to be 0.23mm, the pressure is 0.1KPa, and the coating temperature is controlled to be 40°C to obtain a finished fertilizer product.

[0033] Embodiment 2 A new leucine production strain improved by genetic engineering was selected, inoculated into an activation medium (glucose 9.5 g / L, ammonium sulfate 4.8 g / L, potassium dihydrogen phosphate 0.78 g / L, magnesium sulfate 0.32 g / L), and activated and cultured for 16 h at 29°C and pH 6.9. The activated strain was inoculated into a fermentation medium (glucose 58 g / L, ammonium sulfate 11.5 g / L, potassium dihydrogen phosphate 1.4 g / L, magnesium sulfate 0.65 g / L, and a new bioactive factor concentration of 0.025 g / L) at an inoculum amount of 7% of the total volume of the fermentation medium. The ambient temperature was controlled at 31°C and the pH was 6.9. Sterile air was continuously introduced with a ventilation volume of 0.7 L / min per liter of culture medium, a stirring rate of 170 r / min, and a fermentation time of 62 h. Sampling was performed every 7.5 hours to detect the leucine content and bacterial concentration. After the fermentation was completed, the leucine fermentation broth was initially filtered using a microfiltration membrane with a pore size of 0.25um, with a flow rate of 1.8m³ / h, an operating pressure of 0.18MPa, and a filtration temperature of 28.5°C. Then, an ultrafiltration membrane with a pore size of 25nm was used for deep filtration, with an operating pressure of 0.35MPa and a filtration temperature of 28.5°C. Finally, a nanofiltration membrane with a pore size of 1.8nm was used for fine separation. After separation, a strong acid cation exchange resin was introduced, and the resin was rinsed with deionized water until the effluent was clear. Then, the resin was soaked in a hydrochloric acid solution with a concentration of 1.5mol / L for 5h, and then rinsed with deionized water until neutral. Then, the resin was soaked in a sodium hydroxide solution with a concentration of 1.5mol / L for 5h, and finally rinsed with deionized water until neutral for use. Nitrogen-fixing microorganism culture: The main components of the nitrogen-fixing microorganism culture medium include sucrose 14.5g / L, yeast powder 3.1g / L, potassium dihydrogen phosphate 0.72g / L, magnesium sulfate 0.32g / L and growth promoters. The new rhizobium variants and special round brown nitrogen-fixing bacteria subspecies after screening and domestication are inoculated into the nitrogen-fixing microorganism culture medium at an inoculation rate of 3.5% of the total volume of the culture medium. The culture temperature is 26.5°C, the pH value is set to 6.8, intermittent ventilation is used, the ventilation volume is 0.25L / h per liter of fermentation medium, the ventilation time interval is 2.5h, and the fermentation medium is stirred at a speed of 90r / min using a stirring device. The culture time is 37h, and samples are taken every 7h to detect the number and activity indicators of nitrogen-fixing microorganisms; Fertilizer preparation: The leucine fermentation liquid and the nitrogen-fixing microorganism are mixed in a volume ratio of 1:2, and stirred at a stirring rate of 100 r / min for 30 minutes during the mixing process. The leucine fermentation liquid is sterilized by ultraviolet irradiation before mixing. After the mixing is completed, nano vermiculite (the particle size is controlled between 55nm-75nm) is added to the mixture, and the amount of nano vermiculite added is 25% of the total mixed liquid mass. The mixture is stirred at a stirring rate of 80 r / min for 20 minutes. The mixture is then transferred to a low-temperature plasma-assisted drying device, and the drying temperature is set to 54°C, the energy density of the plasma is set to 500 J / m³, and the flow rate of dry air is 2m³ / h. The mixture is dried for 55 minutes, and a magnetic field auxiliary device with a magnetic field strength of 1000 Gauss is introduced for treatment for 35 minutes. The dried material is crushed and sieved (the mesh aperture is controlled at 2.2mm), and the quality and characteristics of the fertilizer are tested. After passing the test, the material is placed in a coating device, and the coating thickness is controlled to be 0.22mm, the pressure is 0.1KPa, and the coating temperature is controlled to be 40°C to obtain a finished fertilizer product.

[0034] Embodiment 3 A new leucine production strain improved by genetic engineering was selected, inoculated into an activation medium (glucose 10.5 g / L, ammonium sulfate 5.5 g / L, potassium dihydrogen phosphate 0.85 g / L, magnesium sulfate 0.35 g / L), and activated and cultured for 14 h at 27.5°C and pH 6.7. The activated strain was inoculated into a fermentation medium (glucose 62 g / L, ammonium sulfate 12.5 g / L, potassium dihydrogen phosphate 1.6 g / L, magnesium sulfate 0.75 g / L, and a new bioactive factor concentration of 0.035 g / L) at an inoculum amount of 9% of the total volume of the fermentation medium. The ambient temperature was controlled at 33°C and the pH was 7.1. Sterile air was continuously introduced with a ventilation volume of 0.9 L / min per liter of culture medium, a stirring rate of 190 r / min, a fermentation time of 64 h, and a period of 2-3 hr. Sampling was performed every 6.5 hours to detect the leucine content and bacterial concentration. After the fermentation was completed, the leucine fermentation broth was initially filtered using a microfiltration membrane with a pore size of 0.35um, with a flow rate of 2.2m³ / h, an operating pressure of 0.22MPa, and a filtration temperature of 29.5°C. Then, an ultrafiltration membrane with a pore size of 35nm was used for deep filtration, with an operating pressure of 0.42MPa and a filtration temperature of 29.5°C. Finally, a nanofiltration membrane with a pore size of 2.5nm was used for fine separation. After separation, a strong acid cation exchange resin was introduced, and the resin was rinsed with deionized water until the effluent was clear. Then, the resin was soaked in a hydrochloric acid solution with a concentration of 1.5mol / L for 5h, and then rinsed with deionized water until neutral. Then, the resin was soaked in a sodium hydroxide solution with a concentration of 1.5mol / L for 5h, and finally rinsed with deionized water until neutral for use. Nitrogen-fixing microorganism culture: The main components of the nitrogen-fixing microorganism culture medium include 15.5 g / L sucrose, 3.3 g / L yeast powder, 0.78 g / L potassium dihydrogen phosphate, 0.38 g / L magnesium sulfate and growth promoters. The screened and domesticated new rhizobium variants and special round brown nitrogen-fixing bacteria subspecies are inoculated into the nitrogen-fixing microorganism culture medium at an inoculation rate of 4.5% of the total volume of the culture medium. The culture temperature is 27.5°C, the pH value is set to 7.2, and intermittent ventilation is used. The ventilation volume is 0.35 L / h per liter of fermentation medium, and the ventilation time interval is 2.5 hours. At the same time, a stirring device is used to stir the fermentation medium at a speed of 110 r / min. The culture time is 39 hours, and samples are taken every 5.5 hours to detect the number and activity indicators of nitrogen-fixing microorganisms; Fertilizer preparation: The leucine fermentation liquid and the nitrogen-fixing microorganism are mixed in a volume ratio of 1:2, and stirred at a stirring rate of 100 r / min for 30 minutes during the mixing process. The leucine fermentation liquid is sterilized by ultraviolet irradiation before mixing. After the mixing is completed, nano vermiculite (the particle size is controlled between 65nm-75nm) is added to the mixture, and the amount of nano vermiculite added is 25% of the total mixed liquid mass. The mixture is stirred at a stirring rate of 80 r / min for 20 minutes. The mixture is then transferred to a low-temperature plasma-assisted drying device, and the drying temperature is set to 56°C, the energy density of the plasma is set to 500 J / m³, and the flow rate of dry air is 2m³ / h. The mixture is dried for 45 minutes, and a magnetic field auxiliary device with a magnetic field strength of 1000 Gauss is introduced for 45 minutes. The dried material is crushed and sieved (the mesh aperture is controlled at 2.8mm), and the quality and characteristics of the fertilizer are tested. After passing the test, the material is placed in a coating device, and the coating thickness is controlled to be 0.24mm, the pressure is 0.1KPa, and the coating temperature is controlled to be 40°C to obtain a finished fertilizer product.

[0035] Embodiment 4: A new leucine production strain improved by genetic engineering was selected and inoculated into an activation medium (glucose 9.2 g / L, ammonium sulfate 4.6 g / L, potassium dihydrogen phosphate 0.76 g / L, magnesium sulfate 0.31 g / L), and activated and cultured for 15.5 h at 28.2°C and pH 6.85. The activated strain was inoculated into a fermentation medium (glucose 59 g / L, ammonium sulfate 11.8 g / L, potassium dihydrogen phosphate 1.45 g / L, magnesium sulfate 0.68 g / L, and a new bioactive factor concentration of 0.028 g / L) at an inoculation rate of 7.5% of the total volume of the fermentation medium. The ambient temperature was controlled at 31.5°C and the pH was 6.95. Sterile air was continuously introduced with a ventilation volume of 0.75 L / min per liter of culture medium, a stirring rate of 175 r / min, and a fermentation time of 24 h. 63h, during which samples were taken every 7.2h to detect the leucine content and bacterial concentration. After the fermentation was completed, the leucine fermentation broth was initially filtered using a microfiltration membrane with a pore size of 0.28um, with a flow rate of 1.9m³ / h, an operating pressure of 0.19MPa, and a filtration temperature of 28.8℃. Then, an ultrafiltration membrane with a pore size of 28nm was used for deep filtration, with an operating pressure of 0.38MPa and a filtration temperature of 28.8℃. Finally, a nanofiltration membrane with a pore size of 2.2nm was used for fine separation. After separation, a strong acid cation exchange resin was passed through, and the resin was rinsed with deionized water until the effluent was clear. Then, the resin was soaked in a hydrochloric acid solution with a concentration of 1.5mol / L for 5h, and then rinsed with deionized water until neutral. Then, the resin was soaked in a sodium hydroxide solution with a concentration of 1.5mol / L for 5h, and finally rinsed with deionized water until neutral for use; Nitrogen-fixing microorganism culture: The main components of the nitrogen-fixing microorganism culture medium include 14.8 g / L sucrose, 3.15 g / L yeast powder, 0.74 g / L potassium dihydrogen phosphate, 0.33 g / L magnesium sulfate and growth promoters. The screened and domesticated new rhizobium variants and special round brown nitrogen-fixing bacteria subspecies are inoculated into the nitrogen-fixing microorganism culture medium at an inoculation rate of 3.8% of the total volume of the culture medium. The culture temperature is 26.8°C, the pH value is set to 6.9, intermittent ventilation is used, the ventilation volume is 0.28 L / h per liter of fermentation medium, the ventilation time interval is 2.5 hours, and the fermentation medium is stirred at a speed of 95 r / min using a stirring device. The culture time is 38 hours, and samples are taken every 6.5 hours to detect the number and activity indicators of nitrogen-fixing microorganisms; Fertilizer preparation: The leucine fermentation liquid and the nitrogen-fixing microorganism are mixed in a volume ratio of 1:2, and stirred at a stirring rate of 100 r / min for 30 minutes during the mixing process. The leucine fermentation liquid is sterilized by ultraviolet irradiation before mixing. After the mixing is completed, nano vermiculite (the particle size is controlled between 58nm-73nm) is added to the mixture, and the amount of nano vermiculite added is 25% of the total mixed liquid mass. The mixture is stirred at a stirring rate of 80 r / min for 20 minutes. The mixture is then transferred to a low-temperature plasma-assisted drying device, and the drying temperature is set to 54.5°C, the energy density of the plasma is 500 J / m³, and the flow rate of dry air is 2m³ / h. The mixture is dried for 53 minutes. At the same time, a magnetic field auxiliary device with a magnetic field strength of 1000 Gauss is introduced for 40 minutes. The dried material is crushed and sieved (the mesh aperture is controlled at 2.4mm), and the quality and characteristics of the fertilizer are tested. After passing the test, the material is placed in a coating device, and the coating thickness is controlled to be 0.225mm, the pressure is 0.1KPa, and the coating temperature is controlled to be 40°C to obtain a finished fertilizer product.

[0036] Embodiment 5 A new leucine-producing strain improved by genetic engineering was selected and inoculated into an activation medium (glucose 11 g / L, ammonium sulfate 5.8 g / L, potassium dihydrogen phosphate 0.88 g / L, magnesium sulfate 0.36 g / L), and activated and cultured for 14.5 h at 27.8°C and pH 6.75. The activated strain was inoculated into a fermentation medium (glucose 64 g / L, ammonium sulfate 13 g / L, potassium dihydrogen phosphate 1.7 g / L, magnesium sulfate 0.78 g / L, and a new bioactive factor concentration of 0.038 g / L) at an inoculum amount of 9.5% of the total volume of the fermentation medium. The ambient temperature was controlled at 33.5°C and the pH was 7.15. Sterile air was continuously introduced with a ventilation volume of 0.95 L / min per liter of culture medium, a stirring rate of 195 r / min, and a fermentation time of 65 h, during which samples were taken every 6.8 h to detect the leucine content and bacterial concentration. After the fermentation was completed, a microfiltration membrane with a pore size of 0.38 um was first used to perform preliminary filtration on the leucine fermentation broth, with a flow rate of 2.3 m³ / h, an operating pressure of 0.23 MPa, and a filtration temperature of 29.2°C. Then, an ultrafiltration membrane with a pore size of 38 nm was used for deep filtration, with an operating pressure of 0.43 MPa and a filtration temperature of 29.2°C. Finally, a nanofiltration membrane with a pore size of 2.8 nm was used for fine separation. After separation, a strong acid cation exchange resin was passed through, and the resin was rinsed with deionized water until the effluent was clear. Then, the resin was soaked in a hydrochloric acid solution with a concentration of 1.5 mol / L for 5 h, and then rinsed with deionized water until neutral. Then, the resin was soaked in a sodium hydroxide solution with a concentration of 1.5 mol / L for 5 h, and finally rinsed with deionized water until neutral for use; Nitrogen-fixing microorganism culture: The main components of the nitrogen-fixing microorganism culture medium include 16 g / L sucrose, 3.4 g / L yeast powder, 0.8 g / L potassium dihydrogen phosphate, 0.39 g / L magnesium sulfate and growth promoters. The screened and domesticated new rhizobium variants and special round brown nitrogen-fixing bacteria subspecies are inoculated into the nitrogen-fixing microorganism culture medium at an inoculation rate of 4.8% of the total volume of the culture medium. The culture temperature is 27.2°C, the pH value is set to 7.1, and intermittent ventilation is used. The ventilation volume is 0.38 L / h per liter of fermentation medium, and the ventilation time interval is 2.5 hours. At the same time, a stirring device is used to stir the fermentation medium at a speed of 115 r / min. The culture time is 40 hours, and samples are taken every 5.8 hours to detect the number and activity indicators of nitrogen-fixing microorganisms; Fertilizer preparation: The leucine fermentation liquid and the nitrogen-fixing microorganism are mixed in a volume ratio of 1:2, and stirred at a stirring rate of 100 r / min for 30 minutes during the mixing process. The leucine fermentation liquid is sterilized by ultraviolet irradiation before mixing. After the mixing is completed, nano vermiculite (the particle size is controlled between 68nm-78nm) is added to the mixture, and the amount of nano vermiculite added is 25% of the total mixed liquid mass. The mixture is stirred at a stirring rate of 80 r / min for 20 minutes. The mixture is then transferred to a low-temperature plasma-assisted drying device, and the drying temperature is set to 56.5°C, the energy density of the plasma is 500 J / m3, and the flow rate of dry air is 2m3 / h. The mixture is dried for 48 minutes, and a magnetic field auxiliary device with a magnetic field strength of 1000 Gauss is introduced for 42 minutes. The dried material is crushed and sieved (the mesh aperture is controlled at 2.9mm), and the quality and characteristics of the fertilizer are tested. After passing the test, it is placed in a coating device, and the coating thickness is controlled to be 0.245mm, the pressure is 0.1KPa, and the coating temperature is controlled to be 40°C to obtain a finished fertilizer product.

[0037] Quality Inspection: Appearance inspection, Randomly select a certain number of bio-fertilizer particles, observe the color, shape and particle size of the finished fertilizer products under good lighting conditions, and use a magnifying glass or microscope to check whether the surface of the fertilizer particles is smooth and uniform.

[0038] Active ingredient testing, Leucine content detection: Use high performance liquid chromatography (HPLC) and set appropriate detection conditions, specific chromatographic columns, mobile phases, detection wavelengths, etc.; Detection of nitrogen-fixing microorganisms: Use the plate count method to dilute the fertilizer sample and spread it on a specific culture medium. After cultivation, count the number of colonies.

[0039] pH value detection, A certain number of biofertilizer particles were randomly selected, dissolved in distilled water, stirred evenly, and the pH value of the solution was measured using a precision pH meter.

[0040] Moisture content detection, Weigh a certain amount of fertilizer sample, record its initial mass, place the sample in a drying oven, dry it at a specific temperature and time, take out the dried sample, cool it to room temperature, weigh it again, and calculate the moisture content based on the mass difference before and after drying.

[0041] According to the above testing steps, the quality of the finished fertilizers prepared in the five groups of embodiments was tested, and the test results are as follows: Embodiment 1

[0042] Appearance inspection: The fertilizer is dark brown in color, with regular, nearly spherical particles, a particle size between 2.3mm and 2.5mm, and a smooth particle surface; Active ingredient detection: leucine content is 1.6%, and the number of nitrogen-fixing microorganisms is 5.5×10 7 CFU / g; pH value detection: 7.05; Moisture content test: 7.5%.

[0043] Embodiment 2 Appearance inspection: dark brown particles with regular shape, particle size between 2.1mm-2.3mm, and smooth surface; Active ingredient detection: leucine content is 1.55%, and the number of nitrogen-fixing microorganisms is 5.3×10 7 CFU / g; pH value detection: 6.95; Moisture content test: 7.2%.

[0044] Embodiment 3 Appearance inspection: dark brown in color, regular in shape, with a particle size between 2.5mm and 2.8mm, and a certain degree of surface roughness; Active ingredient detection: leucine content is 1.7%, and the number of nitrogen-fixing microorganisms is 5.8×10 7 CFU / g; pH value test: 7.12; Moisture content test: 7.8%.

[0045] Embodiment 4: Dark brown, with regular particle shape, particle size between 2.2mm-2.4mm, and relatively smooth surface; Active ingredient detection: leucine content is 1.7%, and the number of nitrogen-fixing microorganisms is 5.8×10 7 CFU / g; pH value test: 7.02; Moisture content test: 7.3%.

[0046] Embodiment 5 Appearance inspection: dark brown fertilizer particles, regular shape, particle size between 2.7mm-2.9mm, good surface smoothness; Active ingredient detection: leucine content is 1.8%, and the number of nitrogen-fixing microorganisms is 6.0×10 7 CFU / g; pH value detection: 7.18; Moisture content test: 7.6%.

[0047] In summary, the five embodiments of the present invention have important practical significance and application value in terms of the feasibility and effectiveness of preparing fertilizers using leucine fermentation broth and nitrogen-fixing microorganisms.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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 fertilizer using leucine fermentation liquid and nitrogen-fixing microorganisms, comprising preparing leucine fermentation liquid, culturing nitrogen-fixing microorganisms and mixing the leucine fermentation liquid and nitrogen-fixing microorganisms, characterized in that: The specific operation steps of preparing fertilizer using leucine fermentation liquid and nitrogen-fixing microorganisms are as follows: Step 1: Preparation of leucine fermentation broth: Select a new leucine production strain improved by genetic engineering, prepare a special fermentation medium, and add a trace amount of new biologically active factors; Step 2: inoculating the strain into a fermentation medium, and after fermentation is complete, filtering by a multi-stage membrane to separate and obtain a high-purity leucine fermentation broth; Step 3: Cultivating nitrogen-fixing microorganisms: selecting a new rhizobium variant and a special round brown nitrogen-fixing bacteria subspecies, preparing an optimized nitrogen-fixing microorganism culture medium, adding a growth promoter, and inoculating the nitrogen-fixing microorganisms into the culture medium to obtain a nitrogen-fixing microorganism culture; Step 4: mixing the leucine fermentation liquid and the nitrogen-fixing microorganisms, adding a filler, drying the mixture, and introducing a magnetic field auxiliary device to treat the mixture, and obtaining a semi-finished fertilizer after drying; Step 5: crush and sieve the semi-finished fertilizer to obtain granular fertilizer products, and test their quality and characteristics. The qualified fertilizer products are coated and bagged for storage.

2. The process for preparing fertilizer using leucine fermentation liquid and nitrogen-fixing microorganisms according to claim 1, characterized in that: In the step 1, a novel leucine-producing strain is selected, and an activation treatment is performed on it. An activation medium is prepared, wherein the activation medium includes 10 g / L glucose, 5 g / L ammonium sulfate, 0.8 g / L potassium dihydrogen phosphate, and 0.3 g / L magnesium sulfate. The novel leucine-producing strain is inoculated on the activation medium and cultured at 27° C.-30° C. and a pH value of 6.5-7.0 for 12 h-18 h.

3. The process for preparing fertilizer using leucine fermentation liquid and nitrogen-fixing microorganisms according to claim 1, characterized in that: In the step 1, the activated strain is inoculated into the fermentation medium, and the inoculation amount is 5%-10% of the total volume of the fermentation medium. During the fermentation process, the ambient temperature is controlled at 30°C-35°C, the pH value is in the range of 6.8-7.2, and sterile air is continuously introduced, the ventilation volume is 0.5-1.0L / min per liter of culture medium, and the stirring rate is controlled at 150r / min-200r / min to ensure that the nutrients in the culture medium are evenly distributed. The fermentation time is 55h-65h. During the fermentation period, samples are taken every 6h-8h to detect the leucine content and bacterial concentration, and the fermentation conditions are adjusted in time.

4. The process for preparing fertilizer using leucine fermentation liquid and nitrogen-fixing microorganisms according to claim 1, characterized in that: In the step 2, the microfiltration treatment uses a microfiltration membrane with a pore size of 0.2um-0.4um to preliminarily filter the leucine fermentation broth, and 3 / h-2.5m 3 / h flow rate through the microfiltration membrane, the operating pressure is controlled at 0.15MPa-0.25MPa, the filtration temperature is maintained between 28℃-30℃, the ultrafiltration treatment uses an ultrafiltration membrane with a pore size of 20nm-40nm for deep filtration, the operating pressure is controlled at 0.30MPa-0.45MPa, the filtration temperature is maintained between 28℃-30℃, and the nanofiltration treatment uses a nanofiltration membrane with a pore size of 1.5nm-3nm for fine separation.

5. The process for preparing fertilizer using leucine fermentation liquid and nitrogen-fixing microorganisms according to claim 1, characterized in that: In the step 2, after multi-stage filtration, it is passed into an ion exchange resin for separation, and a strong acid cation exchange resin is selected. Before the separation operation, the resin is rinsed with deionized water until the effluent is clear, and the resin is soaked in a hydrochloric acid solution with a concentration of 1mol / L-2mol / L for 4-6h. After completion, it is rinsed with deionized water to neutrality, and then soaked in a sodium hydroxide solution with a concentration of 1mol / L-2mol / L for 4h-6h. After completion, it is rinsed with deionized water to neutrality for standby use.

6. The process for preparing fertilizer using leucine fermentation liquid and nitrogen-fixing microorganisms according to claim 1, characterized in that: In the step three, a nitrogen-fixing microbial culture medium is prepared, wherein the main components include sucrose at a concentration of 14g / L-16g / L, yeast powder at a concentration of 3g / L-3.5g / L, potassium dihydrogen phosphate at a concentration of 0.7g / L-0.8g / L, magnesium sulfate at a concentration of 0.3g / L-0.4g / L, and a growth promoter at a concentration of 0.03g / L-0.05g / L. The screened and domesticated new rhizobium variant and the special brown nitrogen-fixing bacteria subspecies are inoculated into the nitrogen-fixing microbial culture medium, the inoculation amount is 3%-5% of the total volume of the culture medium, the culture temperature is 26°C-28°C, and the pH value is set in the range of 6.5-7.

5.

7. The process for preparing fertilizer using leucine fermentation liquid and nitrogen-fixing microorganisms according to claim 1, characterized in that: In the step three, intermittent ventilation is used during the culture process, the ventilation volume is 0.2L / h-0.4L / h per liter of fermentation medium, the ventilation time interval is 2h-3h, and a stirring device is used during the culture process to stir the fermentation medium at a speed of 80r / min-120r / min to ensure uniform distribution of nutrients. The culture time is 35h-40h. During this period, samples are taken every 5h-8h to detect the number and activity indicators of nitrogen-fixing microorganisms, and the culture conditions are adjusted according to the test results.

8. The process for preparing fertilizer using leucine fermentation liquid and nitrogen-fixing microorganisms according to claim 1, characterized in that: In the step 4, the volume ratio of leucine fermentation broth to nitrogen-fixing microorganisms is 1:

2. During the mixing process, the mixture is stirred at a stirring rate of 100 r / min for 30 min. Before mixing, the leucine fermentation broth needs to be sterilized by ultraviolet irradiation. After the mixing is completed, nano-vermiculite is added to the mixture, and the particle size of the added nano-vermiculite is controlled between 50 nm and 80 nm. The amount of nano-vermiculite added is 25% of the total mass of the mixed solution. The mixture is stirred at a stirring rate of 80 r / min for 20 min. After the filler and the mixed solution are fully mixed, the mixture is transferred to a low-temperature plasma-assisted drying device, and the drying temperature is set to 55° C. and the energy density of the plasma is set to 500 J / m 3 And the dry air flow rate is 2m 3 / h, dry for 40min-60min under the above conditions, and obtain a semi-finished fertilizer after drying.

9. The process for preparing fertilizer using leucine fermentation liquid and nitrogen-fixing microorganisms according to claim 1, characterized in that: In the step 5, the dried semi-finished fertilizer product is sent to a pulverizing device for pulverization, and the smoke produced is removed by a vacuum cleaner. The pulverized semi-finished fertilizer product is sieved, and the sieve aperture is controlled between 2mm and 3mm. The quality and characteristic detection includes appearance detection, effective ingredient detection, pH value detection and moisture content detection. After the finished fertilizer product passes the inspection, it is placed in a coating device, and the coating thickness is controlled to be 0.2mm-0.25mm, the pressure is 0.1KPa, and the coating temperature is controlled to be 40°C. After the coating is completed, it is put into a packaging bag, and the humidity of the storage environment is controlled to be below 60%, and the temperature is maintained between 15°C and 25°C.

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