CN108192843B - Nitrogen fixation strain and microecological preparation thereof - Google Patents
Nitrogen fixation strain and microecological preparation thereof Download PDFInfo
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Abstract
The invention discloses a low-temperature self-nitrogen-fixing strain suitable for high altitude, which is a streptomyces griseus strain. The strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation number is CGMCC No.14766, and the strain is named as Streptomyces glaucescens HG101 strain (Streptomyces canus HG 101).
Description
Technical Field
The invention belongs to the technical field of agricultural microorganisms, and particularly relates to a high-altitude low-temperature self-nitrogen-fixing strain (Streptomyces canus CGMCC NO.14766) and a fermentation method for preparing a microecological preparation based on the nitrogen-fixing strain.
Background
The chemical method used by people to produce nitrogen fertilizer at present not only needs very harsh conditions of high temperature, high pressure and the like, but also wastes a large amount of raw materials, and the effective utilization rate of nitrogen molecules is very low. Azotobacter fixes about 1.5 hundred million tons of nitrogen fertilizer from air every year, which is several times of the total amount of nitrogen fertilizer produced all over the world. Therefore, the research of azotobacter and azotase thereof has become a hotspot of agricultural production application and scientific research at present. Biological nitrogen fixation is a special physiological function of nitrogen-fixing microorganisms, and nearly 50 genera of the microorganisms with nitrogen fixation are known, including bacteria, actinomycetes and cyanobacteria, which have great differences in life style and nitrogen fixation types, but have nitrogen-fixing enzymes in cells. The azotase of different azotobacteria consists of ferromolybdenum protein and ferritin. The nitrogenase must catalyze the reaction under anaerobic conditions, i.e., under low redox conditions. According to the relationship between nitrogen-fixing microorganisms and higher plants, the microorganisms can be divided into self-growing nitrogen-fixing bacteria, symbiotic nitrogen-fixing bacteria and combined nitrogen-fixing bacteria.
The symbiotic nitrogen-fixing bacteria can fix nitrogen or effectively fix nitrogen under the symbiotic condition with plants, and the nitrogen-fixing product ammonia can directly provide a nitrogen source for the symbionts. Rhizobium symbiota formed mainly by bacteria of the genus Rhizobium (Rhizobium) symbiotic with leguminous plants, and Rhizobium symbiota formed by bacteria of the genus Freund (Frankia, an actinomycete) symbiotic with non-leguminous plants; some cyanobacteria and plants are symbiotic, such as nostoc or anabaena and gymnosperm peri to peri, azolla and anabaena to azolla, etc. The rhizobia live in soil, animal and plant residues are taken as nutrients, meanwhile, the rhizobia absorbs nitrogen from air and is converted into ammonia which is utilized by leguminous plants, and the rhizobia and the leguminous plants form a symbiotic relationship, so the rhizobia is also called symbiotic nitrogen-fixing bacteria or combined nitrogen-fixing bacteria.
The combined azotobacter, some azotobacter living in the clay sheath or between cortical cells of some plant roots, without forming root nodules, but with strong specificity, such as the combination of paspalum azotobacter and punctate paspalum, living in the clay sheath of paspalum roots for fixing nitrogen; other microorganisms, such as those living in the rhizosphere of rice, sugar cane and many tropical grasses, have a strong nitrogen fixation effect due to their association with the root system of these plants.
Free living Azotobacteria (Azotobacter) is a strain which is free to live in soil or waterSome bacteria in the territory that are capable of independently performing nitrogen fixation. Reducing molecular nitrogen as nitrogen nutrition into NH3Then amino acid and protein are synthesized. Including aerobic bacteria such as azotobacter, azospirillum and a few autotrophic bacteria; facultative anaerobes, such as klebsiella; anaerobic bacteria, such as some species of the genus Clostridium. Also photosynthetic bacteria such as Rhodospirillum, Chlorella, and cyanobacteria such as Anabaena, Nostoc, etc. Among the naturally occurring nitrogen-fixing bacteria, Azotobacter chroococcum (Azotobacter chroococcum) is the most widely used. The azotobacter chroococcum has strong azotobacter fixing capacity, can secrete auxin and promote the growth of plants and the development of fruits, so that the azotobacter chroococcum is prepared into a microbial inoculum and is applied to soil, and the yield of crops can be improved.
Many reports about azotobacteria and azotase at home and abroad exist, related patent applications and patent grants exist, and related reports and the reports mainly focus on rhizobium. According to the 2012 edition of biological organic fertilizer execution standard of the Ministry of agriculture, two indexes of 'functional strain viable count' and 'quality guarantee period' are added on the basis of the original organic fertilizer standard. In particular, the effective viable count (cfu) is more than or equal to 0.20 hundred million/g. The validity period is more than or equal to 6 months. At present, the azotobacter fertilizer is basically a biological microbial inoculum added with symbiotic azotobacter or combined azotobacter, and a rhizobium microbial inoculum is taken as a main part. The biological organic fertilizer added with the azotobacter is very little. As a rhizobium additive, the rhizobium additive also has a fatal defect of extremely short shelf life in organic fertilizer commodities. The rhizobium has the optimum growth temperature of 26-30 ℃, has no spores, the number of cells can reach 10-100 hundred million/mL under the condition of artificial culture, and because of no spores, the thalli are easy to die and lose activity under the condition of artificial culture. After the slant storage at 4 ℃ for 1 month, the death rate is 95 percent, and the strains can not be basically inoculated in 3 months. In the production process of rhizobia, the death rate of live bacteria is over 90 percent in the processes of microfiltration, ultrafiltration and concentration of rhizobia, the loss of the number of the live bacteria is very large after drying (no matter freeze-drying or adsorption natural drying), the survival rate is lower than 1 percent of the original survival rate, the shelf life of the rhizobia serving as a commodity is seriously influenced, and the quality guarantee period requirement of 6 months is hardly met.
Disclosure of Invention
The invention aims to overcome the defects in the production and application aspects of the existing azotobacter, and provides a high-altitude low-temperature type azotobacter and a production process method thereof.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
the high-altitude low-temperature self-nitrogen-fixing strain is a Streptomyces nigrolanicus strain which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.14766 and is named as a Streptomyces nigrolanicus HG101 strain (Streptomyces canus HG 101).
Wherein the growth temperature of the nitrogen-fixing bacterial strain is 15-39 ℃, and the nitrogen-fixing temperature is 15-37 ℃.
Preferably, the optimum growth temperature of the nitrogen-fixing strain is 35-37 ℃, and the optimum nitrogen-fixing temperature is 25-28 ℃.
The nitrogen-fixing strain is preserved for 1 year at the temperature of 0-4 ℃, and the survival rate reaches 60-70%; the product can be preserved for 1 year at room temperature (about 13-18 deg.C) in the valley of Lhasa, and the survival rate can reach 40-45%.
The azotobacteria spore silk is spiral in shape, the spore is oval and spherical, the surface is smooth, the bacterial colony is colorless, sometimes the reverse side is yellowish, slightly polluted yellow and light brown, the pigment is impermeable, the air silk is white or sometimes slightly ash gray, and the pigment is not contained; arabinose, xylose, fructose, galactose, lactose, sucrose, maltose, rhamnose, raffinose, starch, inulin and glycerol are utilized; inositol, sorbitol, dulcitol, sodium acetate and sodium citrate are not used; without using ammonium salts as nitrogen sources in (NH)4)2SO4Inability to grow in synthetic media; can liquefy gelatin, peptonize milk, and reduce nitrate.
The nitrogen fixing strain is a self nitrogen fixing strain, has no symbiotic characteristic with crops, is harmless to the crops, has no specificity of crop varieties, and is used for fixing nitrogen in the whole soil instead of specific plants.
The nitrogen-fixing bacterial strain is used for preparing a biological fermentation organic fertilizer (including but not limited to nitrogen-fixing bacterial manure), a soil conditioner, an improver or a scarifier.
The solid fermentation method for preparing the microecological preparation by the nitrogen-fixing strain comprises the following steps:
(1) preparing a fermentation medium according to the following parts by weight: 50-65 parts of bran, 48-55 parts of tap water and 2-5 parts of bean pulp; sterilizing at 121-124 deg.C for 40-50 min to obtain fermentation culture medium, and placing into stainless steel tray of 60cm × 40cm × 5 cm;
(2) inoculating the solid seeds of the nitrogen-fixing strain to a fermentation medium according to the inoculation amount of 2-5%, fermenting for 84-144 h at 25-38 ℃, and turning over the fermented bran koji for 1-2 times during fermentation;
(3) after fermentation is finished, drying the fermented bran koji by using a fluidized bed dryer until the water content is 6-8%, wherein the drying temperature is 60-80 ℃;
(4) and crushing the dried fermented bran koji to 40-80 meshes to prepare the microecological preparation, wherein the viable count in the microecological preparation is 50-100 hundred million/g.
The liquid fermentation method for preparing the microecological preparation by the nitrogen-fixing strain comprises the following steps:
(1) preparing a fermentation medium according to the following parts by weight: 2-5 parts of starch, 95-98 parts of tap water, 0.5-8 parts of yeast extract, 6.8-7.5 of pH, and sterilizing at 121 ℃ for 25-30 min to obtain a fermentation culture medium;
(2) adding liquid seeds of nitrogen-fixing strain into fermentation culture medium according to the inoculation amount of 5-10%, fermenting at 35-37 deg.C for 72-96 h, wherein the ventilation of fermentation tank is (1.0-1.2): 1vvm, and the linear velocity of stirring paddle terminal is less than 18 m/s;
(3) after fermentation is finished, filtering the fermentation liquor by a plate frame to prepare a filter cake, and drying the filter cake by a fluidized bed dryer until the water content is 6-8%, wherein the drying temperature is 60-80 ℃;
(4) and crushing the dried fungus cake to 40-80 meshes to prepare the microecological preparation, wherein the number of viable bacteria in the microecological preparation reaches 100-500 hundred million/g.
The invention is further illustrated below:
one of the objects of the invention is to obtain a composition of matter fromAzotobacter strains: sampling soil with an altitude of more than 3600 m from a highland barley planting area in Tibet valley, carrying out air transportation and sending back, and separating as soon as possible; preparing plate screening nitrogen-free culture medium (mannitol 2g, sodium chloride 0.02g, KH)2PO40.02g，MgSO4·7H2O0.02g, agar 2.0g, distilled water 100mL, sterilizing at 121 deg.C for 25min), coating the soil sample with distilled water by ten-fold dilution method, culturing at 37 deg.C for 2-4 days, inoculating to test tube slant (slant culture medium and screening culture medium) to obtain several azotobacter per se, as shown in figure 1; shake flask fermentation and screening, and preparing nitrogen-free fermentation culture medium (soluble starch 2g, sodium chloride 0.02g, KH)2PO40.02g，MgSO4·7H20.02g of O, 2.0g of agar, 100mL of distilled water, sterilization at 121 ℃ for 25min), inoculating the screened strain, carrying out shake culture in a shaker at 35 ℃ for 2.5-4.0 days, measuring the activity of the azotase, re-screening to obtain a strain HG101 with higher azotase activity, identifying the strain as Streptomyces obscurus (Streptomyces canus), storing the strain in the China general microbiological culture collection center in 2017, 9 and 28 months, and preserving the strain in the China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 14766; the enzyme activity of the azotase is measured by an acetylene reduction method, the ethylene content is detected by a Shimadzu GC9A type gas chromatograph, and the level of the azotase activity and the azotase activity (nmol C) are represented by the amount of ethylene generated2H4/mL·h)。
The second purpose of the invention is a method for preparing a self-growing azotobacter Streptomyces canus CGMCC No.14766 microecological preparation, which is realized by the following technical scheme of solid fermentation or liquid fermentation: the solid fermentation method comprises the steps of preparing a fermentation medium by 50-65% of bran, 48-55% of tap water, 2-5% of soybean meal, naturally sterilizing at the temperature of 121-124 ℃ for 40-50 min to prepare clinker, and filling the clinker into a stainless steel tray of 60cm × 40cm × 5 cm; inoculating solid seeds or liquid seeds of the strain according to the inoculation amount of 2-5%, uniformly mixing and culturing; the fermentation temperature is 25-38 ℃; fourth, the fermentation period is 84-144 hours, and the fermentation period is 1-2 times of turning over; after the fermentation is ended, carrying out boiling drying on the fermentation starter until the water content is 6% -8%, and drying at the temperature of 60-80 ℃; sixthly, crushing the dried fermented bran koji to 40-80 meshes to prepare a microecological bactericide, wherein the number of viable bacteria reaches 50-100 hundred million/g; the liquid fermentation method comprises the following steps of preparing a fermentation medium by 2-5% of starch, 95-98% of tap water, 0.5-8% of yeast extract, naturally adjusting the pH value to 6.8-7.5, and sterilizing at 121 ℃ for 25-30 min; inoculating liquid seeds of the strain in an inoculation amount of 5% -10%; the fermentation temperature is 35-37 ℃, the fermentation period is 72-96 hours, the ventilation volume of the fermentation tank is 1.0-1.2: 1(vvm, which is characterized in that the volume of sterile air/the volume of liquid filled in the fermentation tank/min), and the linear velocity of the tail end of the stirring paddle is less than 18 m/s; fourthly, after fermentation is finished, filtering the fermentation liquor by using a plate frame to prepare a filter cake, and boiling and drying the filter cake until the water content is 6-8% and the drying temperature is 60-80 ℃; the dried bacterium cake is crushed to 40-80 meshes to prepare the microecological bactericide, wherein the number of viable bacteria reaches 100-500 hundred million/g; the Streptomyces canus CGMCC No.14766 strain has long spore survival period, can be stored for 1 year at the temperature of 0-4 ℃, has the survival rate of 70-80 percent, can be stored for 1 year at the room temperature of the valley of Lasa, has the survival rate of 40-50 percent, and has better commercial value.
The third purpose of the invention is to utilize the spore of the prepared self-growing azotobacter candida CGMCC No.14766 for the method application of biological nitrogen fixation, and the method is realized by the following technical scheme of solid fermentation or liquid fermentation: the Streptomyces canus CGMCC No.14766 strain is used for fermenting organic fertilizer, and the nitrogen content of the organic fertilizer is maintained or increased; the Streptomyces canus CGMCC No.14766 strain is used for fermenting a microecological bactericide to prepare the biological nitrogen-fixing bacterial fertilizer; the strain Streptomyces canus CGMCC No.14766 is used for fermenting the microecological bactericide to prepare a soil conditioner, an improver and a soil loosening agent.
Compared with the prior art, the invention has the beneficial effects that:
(1) compared with the common azotobacteria, especially rhizobium, the azotobacteria HG101 belongs to azotobacteria per se, the whole soil of the azotobacteria has a large action area, is suitable for the barren soil of the Tibet plateau, and has no limitation of crop species.
(2) The azotobacter HG101 has wide growth temperature range (15-39 ℃) and nitrogen fixation temperature range (15-37 ℃), is suitable for low-temperature climate of Tibet plateau, and has obvious yield increasing effect on highland barley crops.
(3) The azotobacter HG101 is suitable for fermenting the biological organic fertilizer by livestock and poultry wastes, has the nitrogen fixation and protection effects, also has the environment protection function, and is a safe strain harmless to human bodies.
(4) The azotobacter HG101 is used as azotobacter, can bear spores resistant to high temperature, has a long shelf life, and is suitable for preparing organic fertilizer inoculants and fertilizer leavens in agricultural production.
Drawings
FIG. 1 shows that the biomass of Streptomyces glaucescens HG101(Streptomyces canus) is full when it is grown on a nitrogen-free medium, suggesting that the nitrogen fixation ability is strong.
Detailed Description
Example 1 isolation of cryodiazotrophs:
sampling soil with an altitude of more than 3600 m from a highland barley planting area in Tibet valley, carrying out air transportation and sending back, and separating as soon as possible; screening by nitrogen-free medium plate (mannitol 2g, sodium chloride 0.02g, KH)2PO40.02g，MgSO4·7H20.02g of O, 2.0g of agar and 100mL of distilled water, sterilizing for 25min at 121 ℃, coating by using distilled water according to a ten-fold dilution method, culturing for 2-4 days at 37 ℃, inoculating a test tube inclined plane (an inclined plane culture medium and a screening culture medium) after a bacterial colony grows out, and obtaining 238 different types of azotobacter per se.
Example 2 shake flask rescreening of nitrogen fixation activity of nitrogen-fixing bacteria:
several liquid nitrogen-free culture media are prepared in conical flasks (mannitol 2g, sodium chloride 0.02g, KH)2PO40.02g， MgSO4·7H2O0.02g, distilled water 100mL, sterilizing at 121 deg.C for 25min), inoculating azotobacteria test tube slant strain, culturing in shaking table at 30 deg.C and 180r/m for 3 days, measuring azotobacteria activity by acetylene reduction method, selecting C with azotobacteria activity greater than 40nmol2H4the/mL. multidot.h strain was further subjected to univariate measurement, and 7 strains having a good nitrogenase activity were obtained from this example.
Example 3 optimization determination of nitrogen fixation conditions of nitrogen-fixing bacteria:
selecting azotobacter with better azotobacter activity, carrying out temperature variable measurement, and selecting a bacterial strain with azotobacter activity at 15-25 ℃ for further optimization; the embodiment obtains a strain HG101 with high low-temperature azotobacter activity, and the strain is suitable for growth at the temperature of 15-39 ℃ and the optimal growth temperature of 35-37 ℃; the strain HG101 is suitable for fixing nitrogen at the temperature of 15-37 ℃ and the optimum nitrogen fixing temperature of 25-28 ℃, and has low-temperature nitrogen fixing capacity.
Example 4 determination of physiological and biochemical characteristics and morphological characterization of azotobacter HG 101:
respectively measuring the growth conditions of the strains of the unique carbon source or the unique carbon source culture medium according to a single variable principle, observing the colony morphology, and obtaining the morphological characteristics of the azotobacter HG 101; the strain HG101 has spiral spore filament with multiple circles, spherical spore egg, smooth surface, colorless bacterial colony, yellowish, slightly dirty yellow and light brown reverse side, non-penetration pigment, white air filament or slightly grey, and no pigment; arabinose, xylose, fructose, galactose, lactose, sucrose, maltose, rhamnose, raffinose, starch, inulin and glycerol are utilized; inositol, sorbitol, dulcitol, sodium acetate and sodium citrate are not used; without using ammonium salts as nitrogen source, in (NH4)2SO4Inability to grow in synthetic media; can liquefy gelatin, peptonize milk, and reduce nitrate.
Example 5 molecular characterization of the 16S rDNA sequence of azotobacter HG 101:
the 16S rDNA sequence of azotobacteria HG101 is sequenced by Shanghai Mipu Biotechnology GmbH, and is compared with GenBank number to determine a key phylogenetic tree as Streptomyces canus, which is consistent with morphological and physiological and biochemical determination.
Example 6 azotobacter HG101 solid fermentation gas production microecologics:
preparing a solid fermentation culture medium, namely 45% of bran, 50% of tap water and 5% of bean pulp, sterilizing at the natural pH of 121 ℃ for 40min to prepare clinker, and filling the clinker into a stainless steel tray of 60cm multiplied by 40cm multiplied by 5 cm; inoculating solid seeds or liquid seeds of the strain according to the inoculation amount of 5%, uniformly mixing and culturing; fermenting at 30 deg.C for 96 hr, turning over for 2 times, drying at 60 deg.C with boiling drier to water content of 8%, pulverizing to 80 mesh, and making into microecological bactericide with viable count of 80 hundred million/g.
Example 7 liquid fermentation of azotobacter HG101 to produce a probiotic:
preparing 80L of liquid fermentation culture medium, wherein the starch content is 2%, the yeast extract content is 0.5%, the tap water content is 97.5%, the pH value is 7.5, sterilizing at 121 ℃ for 25min, inoculating liquid seeds of azotobacter HG101 in a 100L fermentation tank according to the inoculation amount of 5%, fermenting at 35 ℃ for 84 hours, wherein the ventilation rate of the fermentation tank is 1:1(vvm), filtering a fermentation liquid plate frame after the fermentation is ended to prepare a filter cake, drying by boiling at 60 ℃ until the water content is 8%, crushing the dried bacterium cake to 80 meshes to prepare the micro-ecological bacterium agent, and the number of viable bacteria reaches 200/g.
Example 8 nitrogen-fixing bacteria HG101 micro-ecological powder fermented pig manure organic fertilizer is applied to nitrogen fixation and nitrogen preservation:
mixing fresh pig manure 4 tons with the water content of about 75% with mushroom residues with the water content of about 20% according to the weight ratio of 8:1, adjusting the water content to about 60%, simultaneously mixing 2kg of a commercial fermentation decomposition agent, dividing the compost pig manure material into two ridges, adding 500g of azotobacter HG101 micro-ecological powder with the viable count of 80 hundred million/g into one ridge, taking the other ridge as a control, and fermenting and decomposing for more than 20 days, wherein the characteristic indexes of the bio-organic fertilizer are shown in Table 1:
TABLE 1 comparison of time and nutrient for fast decomposition of pig manure and mushroom residue by azotobacteria
As can be seen from Table 1, the effect of inoculating the azotobacter inoculant and the fermentation decomposition inoculant is better than that of inoculating the fermentation decomposition inoculant singly, the inoculation is slightly white, has the characteristic of earthy smell, and has obvious unpleasant smell removal effect; the fermentation time is shortened by 2 days; the nitrogen is increased by 0.2%, which indicates that the azotobacter HG101 also has the nitrogen fixing and preserving effects in the process of fermenting the pig manure.
Example 9
The azotobacter HG101 micro-ecological powder is used for soil conditioning, improvement and loosening soil to increase the agricultural production yield: in a planting area with the altitude of more than 3500m in the Tibet valley area, the Tibet 320 is planted, nitrogen-phosphorus-potassium compound fertilizer and azotobacter HG101 micro-ecological powder (200 hundred million/g) are alternately applied by adopting a contrast method, and the yield result of the highland barley is shown in the table 2:
TABLE 2 influence of azotobacteria inoculant and Compound fertilizers application on highland barley production
As can be seen from Table 2, the highland barley yield can be greatly increased by applying the compound fertilizer or the azotobacteria HG101 microbial inoculum, wherein the yield increase rate of the azotobacteria HG101 is more than 25%, and the yield increase effect is obvious.
Example 10
The result of the survival experiment of the azotobacter HG101 in the storage period is compared with that of the silver nodule bacteria ACCC13005, and is shown in Table 3: as can be seen from Table 3, the azotobacter HG101 can be used for producing dormant spores, the quality guarantee period is far shorter than that of the silver rhizobium ACCC13005, particularly, the bio-organic fertilizer solid preparation has a longer quality guarantee period, the survival rate of the solid preparation is still 40% after the solid preparation is stored for 1 year in a high-altitude low-temperature area of the Tibet valley, higher commercial value is embodied, and the defect that rhizobium is difficult to store is overcome.
TABLE 3 comparison of Azotobacter HG101 with Rhizobium aquaticum ACCC13005 for preservation
Claims (5)
1. The nitrogen-fixing strain is a Streptomyces nigricans strain (Streptomyces canus) which is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms, has the preservation number of CGMCC No.14766 and is named as a Streptomyces nigricans HG101 strain.
2. A solid fermentation process for the preparation of a probiotic based on the nitrogen-fixing strain of claim 1, characterized in that it comprises the following steps:
(1) preparing a fermentation medium according to the following parts by weight: 50-65 parts of bran, 48-55 parts of tap water and 2-5 parts of bean pulp; sterilizing at 121-124 deg.C for 40-50 min to obtain fermentation culture medium;
(2) inoculating the solid seeds of the nitrogen-fixing strain to a fermentation medium according to the inoculation amount of 2-5%, fermenting for 84-144 h at 25-38 ℃, and turning over the fermented bran koji for 1-2 times during fermentation;
(3) drying the fermented bran koji to the water content of 6-8% after the fermentation is finished, wherein the drying temperature is 60-80 ℃;
(4) and crushing the dried fermented bran koji to 40-80 meshes to prepare the microecological preparation, wherein the viable count in the microecological preparation is 50-100 hundred million/g.
3. A liquid fermentation process for the preparation of a probiotic based on the nitrogen-fixing strain of claim 1, characterized in that it comprises the following steps:
(1) preparing a fermentation medium according to the following parts by weight: 2-5 parts of starch, 95-98 parts of tap water, 0.5-8 parts of yeast extract, 6.8-7.5 of pH, and sterilizing at 121 ℃ for 25-30 min to obtain a fermentation culture medium;
(2) adding liquid seeds of the nitrogen-fixing strain into a fermentation culture medium according to the inoculation amount of 5-10%, fermenting for 72-96 h at 35-37 ℃, wherein the ventilation volume during fermentation is 1.0-1.2: 1 vvm;
(3) after fermentation is finished, filtering the fermentation liquor by a plate frame to prepare a filter cake, and drying until the water content is 6-8%, wherein the drying temperature is 60-80 ℃;
(4) and crushing the dried fungus cake to 40-80 meshes to prepare the microecological preparation, wherein the number of viable bacteria in the microecological preparation reaches 100-500 hundred million/g.
4. The use of the nitrogen-fixing strain of claim 1 in the preparation of a bio-fermented organic fertilizer.
5. Use of the nitrogen-fixing strain of claim 1 in the preparation of a soil conditioner, amendment or loosening agent.
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