Brown spherical azotobacter, microbial fertilizer, fermentation method and application thereof

Abstract

The present application relates to the technical field of microbial fermentation, in particular to a strain of Azotobacter chroococcum, a microbial fertilizer, a fermentation method and application thereof. The Azotobacter chroococcum provided by the present application has a high nitrogen fixation efficiency and is genetically stable after being screened by ARTP mutagenesis and subculture. Azotobacter chroococcum LLBG-20240108-001, and the preservation number is CGMCC No. 29641. The Azotobacter chroococcum LLBG-20240108-001 provided by the present application is screened by ARTP mutagenesis, has a high nitrogen fixation efficiency, and is genetically stable after being subcultured for multiple times. The microbial fertilizer prepared from the Azotobacter chroococcum LLBG-20240108-001 provided by the present application can effectively increase the fresh weight, dry weight and plant height of barley plants, and has an obvious growth-promoting effect. The Azotobacter chroococcum LLBG-20240108-001 provided by the present application can effectively increase the yield per mu, fresh weight per plant and dry weight per plant of Brassica campestris.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a strain of *Azotobacter chrysogenum*, microbial fertilizer, its fermentation method, and its application. Background Technology

[0002] Nitrogen is a vital element for the growth of plants and animals as well as for human life activities. In life activities, nitrogen is an essential element for the formation of nucleic acids, amino acids, and proteins, and it is also one of the important elements in chemical fertilizers.

[0003] Agricultural production leads to greenhouse gas emissions, contributing to the greenhouse effect and accelerating global warming. Excessive application of nitrogen fertilizer causes the emission of nitrous oxide (N2O) from farmland, and increased nitrogen application can multiply carbon emissions, with a warming potential far exceeding that of other greenhouse gases, approximately 298 times that of CO2. The overuse of nitrogen fertilizer has severely disrupted global biogeochemical cycles. Under natural sedimentation conditions, large amounts of nitrogen and nitrogen-containing substances in the soil seep into groundwater, causing problems such as acid-base imbalance, eutrophication, and the deposition of trace elements and minerals, seriously threatening human health. Therefore, reducing nitrogen fertilizer use in agricultural production to achieve carbon sequestration and emission reduction, thereby mitigating global warming, is crucial.

[0004] Biological nitrogen fixation plays an important role in increasing crop yields, reducing fertilizer use, reducing water and soil pollution, maintaining sustainable agricultural development, reducing energy consumption, influencing the marine nitrogen cycle, and promoting photosynthesis in marine organisms.

[0005] *Azotobacter chrysogenum* is a free-living nitrogen-fixing bacterium. Because it does not require specific plant associations, it exhibits wide adaptability and plays a crucial role in the nitrogen cycle, making it an important agricultural inoculant. Studies have confirmed that *Azotobacter chrysogenum* significantly promotes the growth of spring wheat, wolfberry, and cotton, improving nitrogen fertilizer utilization and reducing nitrogen fertilizer application. Inoculation with *Azotobacter chrysogenum* can reduce the amount of nitrogen fertilizer required for cotton growth by 50%. In addition to nitrogen fixation, *Azotobacter chrysogenum* secretes various plant growth regulators such as auxins and gibberellins, promoting root development and nutrient absorption. It also possesses the ability to solubilize phosphorus and potassium, produce iron carriers, activate insoluble mineral nutrients in the soil, and improve fertilizer utilization. Its metabolic activities can also improve soil aggregate structure, increase soil enzyme activity, optimize the rhizosphere microecological environment, and enhance crop stress resistance. *Azotobacter chrysogenum* has broad application prospects in agriculture. However, no *Azotobacter chrysogenum* strain has been reported to simultaneously promote the growth of barley and rapeseed. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to overcome the deficiency of the prior art in that there is no reported brown azotocin bacterium that can simultaneously promote the growth of barley and rapeseed, thereby providing a brown azotocin bacterium LLBG-20240108-001, a bacterial agent, a microbial fertilizer, and its fermentation method and application.

[0007] Therefore, the present invention provides the following technical solution: This invention provides a strain of *Azotobacter brownii* (… Azotobacter chroococcum LLBG-20240108-001, accession number CGMCC No. 29641. The depositary institution is the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code 100101. The deposit date is January 17, 2024.

[0008] Azotocinus brownii ( Azotobacter chroococcum LLBG-20240108-001 is a mutant strain with high nitrogen-fixing efficiency selected by ambient pressure room temperature plasma mutagenesis (ARTP) for 80 s. It exhibits good passage stability. The nitrogen-fixing efficiency of *Azotobacter chrysogenum* LLBG-20240108-001 is 94.33% higher than the original strain before mutagenesis, reaching 13.7 mg N / g sugar. mg N / g sugar is the number of milligrams of nitrogen that the microorganism can fix and synthesize for every 1 gram of sugar substrate consumed. Before mutagenesis, *Azotobacter chrysogenum* was Gram-negative, positive for methyl red and voltammetric tests, negative for starch hydrolysis and malonate tests, negative for gelatin liquefaction and citrate tests, positive for indole tests, and negative for urease tests.

[0009] The brown azotocin bacterium provided by this invention ( Azotobacter chroococcum The LLBG-20240108-001 whole genome sequence is 4575910bp in length and contains 20 nitrogenase-related genes, including nifH, nifD, nifK, nifT, nifE, nifN, nifM, nifL, and nifA. It has good nitrogen fixation effect and high effective viable bacteria count, which can effectively increase the fresh weight, dry weight, and plant height of barley plants. In field applications, it can effectively increase the yield per mu, fresh weight per plant, and dry weight per plant of rapeseed, and has a significant growth-promoting effect.

[0010] This invention provides a microbial agent comprising Azotobacter brownii (… Azotobacter chroococcum )LLBG-20240108-001.

[0011] The microbial agent comprises a wettable powder; the effective viable count of *Azotobacter brownii* LLBG-20240108-001 in the microbial agent is ≥2.0 × 10⁻⁶. 8cfu / g or ≥2.0×10 8 cfu / mL.

[0012] This invention provides a microbial fertilizer and / or wettable powder, comprising, by weight: 4-30 parts of *Azotobacter blazei* LLBG-20240108-001 inoculant, 2-5 parts of wetting agent, 1-5 parts of dispersant, and 62-70 parts of soluble carrier; Optionally, the wetting agent comprises sodium dodecyl sulfate, the dispersant comprises sodium lignosulfonate, the soluble carrier comprises a mixture of modified starch and glucose, the mass ratio of modified starch to glucose being 1:(1-2), and the effective viable count of *Azotobacter blazei* LLBG-20240108-001 in the microbial fertilizer and / or wettable powder is ≥2.0 × 10⁻⁶. 8 cfu / g or ≥2.0×10 8 cfu / mL.

[0013] Preferably, the preparation method of the *Azotobacter brownii* LLBG-20240108-001 inoculum includes: The bacterial culture of *Azotobacter chrysogenum* LLBG-20240108-001 is mixed with diatomaceous earth and dried to obtain *Azotobacter chrysogenum* LLBG-20240108-001 inoculum; preferably, centrifugation is further performed before drying; Optionally, the amount of diatomaceous earth added is 1%-5% of the mass of the *Azotobacter globosum* LLBG-20240108-001 bacterial culture. Optionally, the mixing method includes stirring and / or centrifugation, wherein the stirring speed is 100-150 rpm and the time is 1-2 h, and the centrifugation is continuous centrifugation, wherein the continuous centrifugation speed is 8000-14000 rpm and the feed rate is 200-300 L / h; Optionally, the drying temperature is 45-55°C, and the product is dried until the moisture content is 5%-10%. Optionally, the effective viable count of *Azotocinobacter globosum* LLBG-20240108-001 inoculant is ≥2.0 × 10⁻⁶. 8 cfu / g or ≥2.0×10 8 cfu / mL.

[0014] This invention provides the application of *Azotobacter brownii* LLBG-20240108-001 or its inoculum, or the aforementioned microbial fertilizer and / or wettable powder, in promoting plant growth.

[0015] Preferably, the plant includes one or more of barley, wheat, corn, and rapeseed; And / or, the growth promotion includes increasing at least one of the following: plant fresh weight, dry weight, plant height, and yield per acre.

[0016] This invention provides a fermentation method for *Azotobacter chrysogenum*, comprising: inoculating a liquid seed of *Azotobacter chrysogenum* LLBG-20240108-001 into a fermentation medium for fermentation culture to obtain a fermentation broth; Preferably, the method for preparing the liquid seed includes: activating and culturing the *Azotobacter blazei* LLBG-20240108-001 to obtain the liquid seed; optionally, the temperature of the seed culture is 30-35℃, more preferably 30±1℃; the rotation speed is 180-220 rpm; and the time is 48-72h. The components of seed culture medium and fermentation culture medium can be the same or different. Optionally, the seed culture medium comprises: 20-40 g glucose, 1-5 g yeast powder, 1-5 g CaCO3, 0.2-0.8 g KH2PO4, 0.2-0.8 g MgSO4·7H2O, 0.2-0.8 g K2SO4, 0.01-0.02 g CaCl2, 0.01-0.02 g FeCl3, 0.001-0.002 g Na2MoO4, and 1000 mL water; Optionally, the fermentation medium comprises: 2%-4% glucose, 0.1%-0.5% yeast extract, 0.1%-0.5% CaCO3, 0.02%-0.08% KH2PO4, 0.02%-0.08% MgSO4·7H2O, 0.02%-0.08% K2SO4, 0.001%-0.002% CaCl2, 0.001%-0.002% FeCl3, and 0.0001%-0.0002% Na2MoO4, with the balance being water; Preferably, the effective viable count of *Azotobacter brownii* LLBG-20240108-001 in the fermentation broth is ≥2.0 × 10⁻⁶. 9 The nitrogen fixation efficiency is as high as 20 mg N / g sugar (cfu / mL). In specific embodiments of the present invention, the nitrogen fixation efficiency is 12.4 mg N / g sugar, 16.7 mg N / g sugar, 18 mg N / g sugar, and 20 mg N / g sugar.

[0017] Optionally, the fermentation culture temperature is 30-35℃, more preferably 30±1℃; the aeration rate is 0.8-1.2 L / (L·min), the rotation speed is 105-220 rpm, the loading coefficient is 60%-70%, and the time is 3-5 days. The loading coefficient mentioned in this invention is the percentage of the culture medium volume to the total volume of the culture container.

[0018] This invention provides a method for applying microbial fertilizers and / or wettable powders, comprising: applying microbial fertilizers to plants.

[0019] Preferably, the microbial fertilizer of the present invention is applied in the field and / or in potted plants; the application rate of the microbial fertilizer and / or wettable powder of the present invention is 0.8-1 kg / mu, and the application method includes spraying, more preferably spraying after dilution. In a specific embodiment of the present invention, a backpack electric sprayer is used to spray the rapeseed to ensure uniform spraying. The first fertilization is performed at the 3-5 leaf stage, and 2-3 consecutive applications are made, with an interval of 4-8 days between each application.

[0020] The microbial fertilizer of this invention can increase the yield, fresh weight and dry weight of rapeseed, and increase income, ultimately improving the input-output ratio, and increasing the SPAD value, plant height, fresh weight of barley, and above-ground fresh weight.

[0021] Preferably, microbial fertilizers and / or wettable powders are mixed with conventional fertilizers before application.

[0022] The technical solution of this invention has the following advantages: 1. The *Azotobacter brownii* provided by this invention (… Azotobacter chroococcum LLBG-20240108-001, preservation number CGMCC No. 29641. This invention isolated a strain of *Azotobacter brownis* from tomato rhizosphere soil. Whole-genome sequencing and BLAST sequence alignment revealed 20 nitrogenase genes. After ARTP mutagenesis, *Azotobacter brownis* (…)… Azotobacter chroococcum The nitrogen fixation efficiency of LLBG-20240108-001 is 13.7 mg N / g sugar, which is 94.33% higher than that of the original strain. After multiple subcultures, it was confirmed that it is genetically stable and has high nitrogen fixation efficiency, up to 20 mg N / g sugar.

[0023] 2. The microbial agent provided by this invention has a high number of effective live bacteria and a good plant growth-promoting effect.

[0024] 3. This invention provides a wettable powder. The microbial fertilizer prepared from *Azotocinobacter chrysogenum* LLBG-20240108-001 provided by this invention can effectively increase the fresh weight, dry weight, and plant height of barley plants, exhibiting a significant growth-promoting effect. The microbial fertilizer prepared from *Azotocinobacter chrysogenum* LLBG-20240108-001 provided by this invention can effectively increase the yield per acre, fresh weight per plant, and dry weight per plant of rapeseed.

[0025] 4. The application of *Azotobacter graminearum* LLBG-20240108-001 or its inoculum, or the microbial fertilizer and / or wettable powder provided by this invention in promoting plant growth. The microbial fertilizer of this invention can increase the yield, fresh weight per plant, and dry weight per plant of rapeseed, thereby increasing income and ultimately improving the input-output ratio. It can also increase the SPAD value, plant height, fresh weight per plant, and aboveground fresh weight of barley.

[0026] In summary, this invention screened a *Azotobacter chrysogenum* strain with good nitrogen-fixing performance from facility soil, screened a mutant strain with high nitrogen-fixing efficiency through ARTP mutagenesis at ambient pressure and room temperature, completed whole-genome sequencing of the mutant strain, determined the efficient fermentation process of the strain through fermentation optimization, and prepared nitrogen-fixing bacteria agent products through processes such as adsorption, centrifugation, drying, and compounding of the fermentation broth, and conducted application tests on the nitrogen-fixing bacteria.

[0027] Biological Preservation The brown azotocin bacterium provided by this invention ( Azotobacter chroococcum LLBG-20240108-001, accession number CGMCC No. 29641. The depositary institution is the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code 100101. The deposit date is January 17, 2024. Attached Figure Description

[0028] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0029] Figure 1 This is a graph showing the lethality rate of Azotobacter brownii induced by ARTP mutagenesis. Figure 2 This is a graph showing the effect of different sampling times and treatments on the SPAD value of barley; Figure 3 This is a graph showing the effects of different treatments on barley plant height at different stages; Figure 4 This is a graph showing the effect of different treatments on the fresh weight of a single barley plant at different stages. Figure 5 This is a graph showing the effect of different treatments on the fresh weight of the aboveground parts of barley at different stages; Figure 6 This is a graph showing the effect of different treatments on the dry weight of a single barley plant at different stages. Figure 7 This is a graph showing the effect of different treatments on the aboveground dry weight of barley at different stages; Figure 8 This is a colony culture diagram of strain LLG-8 in Assumption medium without nitrogen. Figure 9 Figure showing the results of the streak test on nitrogen-free Ashub medium to inactivate Azotobacter brownii LLBG-20240108-001 microbial inoculant. In the above attached diagram, the different letters a, b, and c represent... p The difference was statistically significant at levels <0.05. Detailed Implementation

[0030] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0031] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0032] Example 1 1. Strain screening (1) Soil samples were collected from facility soil in Huairou District, Beijing. 10 g of the sample was weighed and placed in 100 mL of sterile water containing glass beads. The mixture was shaken at 200 rpm for 30 min on a shaker. 1 mL of the supernatant was added to 40 mL of nitrogen-free medium for enrichment culture for 3 days at 30℃ and 200 rpm to obtain the fermentation broth. The fermentation broth was spread on nitrogen-free medium plates using a serial dilution method and cultured at 30℃ for 3 days. The plates were repeatedly streaked to purify the strains, resulting in multiple strains.

[0033] (2) Physiological and biochemical identification and genomic analysis of strain LLG-8 Using Assumption medium without nitrogen, nitrogen fixation efficiency was determined by shake-flask screening. A strain with high nitrogen fixation efficiency was selected and named LLG-8. Strain LLG-8 has large, round, viscous colonies, initially pale yellow, turning brownish-red later. Figure 8 The edges are smooth, the cells are spherical and have no spores.

[0034] Physiological and biochemical characteristics of strain LLG-8 were tested, and the results are shown in Table 1. Physiological and biochemical identification showed that strain LLG-8 was Gram-negative, positive for methyl red and voltammetric tests, negative for starch hydrolysis, positive for malonate, negative for gelatin liquefaction, negative for citrate, positive for indole, and negative for urease.

[0035] Table 1 Physiological and biochemical characteristics of the strains

[0036] Note: + indicates positive; - indicates negative.

[0037] The 16S rRNA sequence of strain LLG-8 is shown in SEQ ID NO:1, and is as follows: .

[0038] In summary, based on the comprehensive analysis of experimental data such as cell morphology, physiological and biochemical characteristics, and 16S rRNA gene sequence of strain LLG-8, and referring to relevant research papers in Bergey's Manual of Systematic Bacteriology and the International Journal of Systematic and Evolutionary Microbiology, strain LLG-8 was identified as *Azotobacter brownii*. Azotobacter chroococcum ).

[0039] Example 2. Mutagenesis and Breeding The LLG-8 strain obtained in step (2) of Example 1 was subjected to mutagenesis and selection. The LLG-8 strain was inoculated into modified Assumption nitrogen-free liquid fermentation medium and cultured for 72 h until the fermentation broth turned brown. The bacterial suspension was then streaked onto fresh solid plates and cultured for another 72 h. Single colonies were picked to prepare a bacterial suspension, and the bacterial concentration was adjusted to 10⁻⁶. 6 ~10 8 10 cells / mL; ARTP power set to 120 W, helium carrier gas flow rate at 10 L / min. -1 The cooling water circulation machine temperature was 20℃, and the mutagenesis times were 0 s, 20 s, 40 s, 50 s, 60 s, 70 s, 80 s, 100 s, and 120 s, respectively.

[0040] The bacterial suspension was mixed with 10% (v / v) glycerol at a volume ratio of 1:1. 10 μL of the mixture was evenly spread onto a sterile stainless steel slide compatible with the mutagenesis instrument. The treatment time started at 0 s and ended at 120 s. After each sample was treated, the stainless steel slide automatically fell into a 2 mL sterile EP tube containing 1 mL of sterile physiological saline. After all samples were treated, the EP tube was removed, and the bacteria were eluted by shaking on a shaker. Lethality calculation: The number of colonies on nitrogen-free agar plates at different treatment times was counted, and the lethality was calculated. The ARTP mutagenesis lethality curves at different mutagenesis times are shown in [Figure number missing]. Figure 1 It can be seen that mutant bacteria with mutagenesis times of 40 s, 50 s, 60 s, 70 s, and 80 s were selected for screening. The obtained single-colony mutants were numbered sequentially, and 28 mutant strains were initially selected (see Table 2). A second screening was conducted based on the effective viable count, nitrogen content, and nitrogen fixation efficiency. Four mutant strains were found in the second screening: 80s9, 80s13, 80s11, and 80s16, with an effective viable count of 3.0 × 10⁻⁶. 9 cfu / mL or higher.

[0041] The method for determining the effective viable bacteria count refers to NY / T 2321-2013 "Inspection Procedures for Microbial Fertilizer Products". The methods for determining nitrogen content and nitrogen fixation efficiency refer to the People's Republic of China Agricultural Industry Standard: NY411-2000 "Nitrogen-fixing Bacterial Fertilizers".

[0042] The results of the secondary screening are shown in Table 3. It can be seen that the effective viable count of mutant strain 80s9 was approximately the same as that in the initial screening, at 3.98 × 10⁻⁶. 9 The nitrogen fixation efficiency (CFU / mL) of the mutant strain (13.7 mg N / g sugar) was 94.33% higher than that of the original strain. The effective viable counts of the other strains all decreased to varying degrees, significantly lower than the initial screening results, indicating that these strains lacked genetic stability. With increasing passage numbers, the nitrogen fixation efficiency of the 80s9 mutant remained stable at around 13.7 mg N / g sugar, indicating that the mutant strain 80s9 possesses good genetic stability.

[0043] The mutant strain 80s9 was named strain LLBG-20240108-001. Strain LLBG-20240108-001 was sent to Shenzhen BGI Genomics Service Co., Ltd. for whole-genome sequencing. The sequencing result was 4575910 bp. BLAST alignment analysis revealed that nitrogenase-related genes included 20 genes: nifH, nifD, nifK, nifT, nifE, nifN, nifX, nifU, nifS, nifV, nifM, nifB, nifL, nifA, nifF, nifQ, nifO, nifW, nifZ, and nifY. After mutagenesis, it remained a *Azotobacter chrysogenum* strain. Azotobacter chroococcum ). (The text appears to be incomplete and contains several grammatical errors. A more accurate translation would require the full context.) Azotobacter chroococcum The specimen, LLBG-20240108-001, has been deposited with accession number CGMCC No. 29641. The depositary institution is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China. The deposit date is January 17, 2024.

[0044] Table 2 Results of effective viable count of plasma-mutated strains (unit: ×10) 9 cfu / mL)

[0045] Table 3 Results of secondary screening of plasma-mutated strains

[0046] Example 3 The modified Ashube medium consists of: 30 g glucose, 5 g CaCO3, 0.2 g KH2PO4, 0.2 g MgSO4·7H2O, 0.2 g K2SO4, 0.2 g NaCl, 20 g agar, and 1000 mL water.

[0047] The shake-flask fermentation medium consisted of: 30 g glucose, 1 g yeast extract, 3 g CaCO3, 0.6 g KH2PO4, 0.6 g MgSO4·7H2O, 0.6 g K2SO4, 0.02 g CaCl2, 0.01 g FeCl3, 0.002 g NaMoO4, and 1000 mL water.

[0048] After activation on modified Assumption medium, a slant culture of strain LLBG-20240108-001 was inoculated into a 200 mL shake flask fermentation medium. The flasks were then incubated at 30°C and 200 rpm for 3 days. The fermentation broth changed from white to brownish-red, indicating the end of the fermentation. The viable cell count and nitrogen fixation efficiency of the fermentation broth were measured, and the results were 4.29 × 10⁻⁶. 9 cfu / mL and 20 mg N / g sugar.

[0049] Example 4 The modified Ashube medium consists of: 30 g glucose, 5 g CaCO3, 0.2 g KH2PO4, 0.2 g MgSO4·7H2O, 0.2 g K2SO4, 0.2 g NaCl, 20 g agar, and 1000 mL water.

[0050] The shake-flask fermentation medium consisted of: 20 g glucose, 5 g yeast extract, 1 g CaCO3, 0.2 g KH2PO4, 0.2 g MgSO4·7H2O, 0.2 g K2SO4, 0.02 g CaCl2, 0.01 g FeCl3, 0.002 g NaMoO4, and 1000 mL water.

[0051] After activation on modified Assumption medium, a slant culture of strain LLBG-20240108-001 was inoculated into a 200 mL shake flask fermentation medium. The medium was then cultured in a shaker at 35°C and 200 rpm for 3 days. The fermentation broth changed from white to brownish-red, indicating the end of the fermentation. The viable cell count and nitrogen fixation efficiency of the fermentation broth were determined, and the results were 2.5 × 10⁻⁶. 9 cfu / mL and 12.4 mg N / g sugar.

[0052] Example 5 The modified Ashube medium consists of: 30 g glucose, 5 g CaCO3, 0.2 g KH2PO4, 0.2 g MgSO4·7H2O, 0.2 g K2SO4, 0.2 g NaCl, 20 g agar, and 1000 mL water.

[0053] The shake-flask fermentation medium consisted of: 40 g glucose, 3 g yeast extract, 5 g CaCO3, 0.8 g KH2PO4, 0.8 g MgSO4·7H2O, 0.8 g K2SO4, 0.02 g CaCl2, 0.01 g FeCl3, 0.002 g NaMoO4, and 1000 mL water.

[0054] After activation on modified Assumption medium, a slant culture of strain LLBG-20240108-001 was inoculated into a 200 mL shake flask fermentation medium. The medium was then incubated at 33°C with shaking at 200 rpm for 3 days. The fermentation broth changed from white to brown, indicating the end of the fermentation process. The viable cell count and nitrogen fixation efficiency of the fermentation broth were determined, and the results were 3.02 × 10⁻⁶. 9 cfu / mL and 16.7 mg N / g sugar.

[0055] Example 6 The seed culture medium consisted of: 30 g glucose, 1 g yeast extract, 3 g CaCO3, 0.6 g KH2PO4, 0.6 g MgSO4·7H2O, 0.6 g K2SO4, 0.02 g CaCl2, 0.01 g FeCl3, 0.002 g NaMoO4, and 1000 mL water.

[0056] After the slant culture of strain LLBG-20240108-001 was activated, one loopful was inoculated into the seed culture medium with a volume of 200 mL. Seed culture was carried out on a shaker at 30℃ and 200 rpm for 3 days. The resulting seed solution changed from white to brownish-brown, and the seed culture was then completed.

[0057] The prepared liquid seed was inoculated into a 300 L fermenter at an inoculation rate of 1% of the culture medium volume for fermentation culture. The fermentation culture medium composition was as follows (by mass percentage): glucose 3%, yeast powder 0.1%, CaCO3 0.3%, KH2PO4 0.06%, MgSO4·7H2O 0.06%, K2SO4 0.06%, CaCl2 0.002%, FeCl3 0.001%, NaMoO4 0.0002%, with the balance being water.

[0058] The fermentation conditions were as follows: temperature 30℃, rotation speed 105 rpm, fill factor 60% (fermentation medium volume 300 L × 60% = 180 L), aeration rate of 0.8 L / (L·min) for the first 0-12 hours, 1.0 L / (L·min) for the first 12-48 hours, and 1.2 L / (L·min) after the 48th hour. The fermentation broth turned brownish-red after 72 hours, and was cultured for another 8 hours, for a total fermentation period of 80 hours. After fermentation, the viable cell count and nitrogen fixation efficiency were measured, with results of 2.405 × 10⁻⁶ cells / day. 9 cfu / mL and 18 mg N / g sugar.

[0059] Example 7 In Example 6, after fermentation, 1% (by mass) of diatomaceous earth was added to the fermentation broth, and the mixture was stirred for 1 hour for adsorption. The broth was then centrifuged continuously at 14000 rpm using a tubular centrifuge at a feed rate of 300 L / h. The precipitate was dried at 50±5℃, maintaining a moisture content of 10%-15%. After pulverization, the precipitate was passed through a 100-mesh standard sieve to prepare *Azotobacter brownii* LLBG-20240108-001 powder. The effective viable count was determined to be 8.26 × 10⁻⁶. 9 cfu / g.

[0060] Example 8 The concentration obtained in Example 7 was 8.26 × 10⁻⁶. 9 25 parts by weight of *Azotobacter blazei* LLBG-20240108-001 powder (cfu / g) were added to a mixer, followed by 5 parts by weight of sodium dodecyl sulfate, 4 parts by weight of sodium lignosulfonate, 33 parts by weight of modified starch, and 33 parts by weight of glucose. The mixture was thoroughly mixed to obtain LLBG-20240108-001 wettable powder with good dispersibility and suspension properties. The suspension rate of LLBG-20240108-001 wettable powder was 75%, determined according to GB / T 14825-2023 "Determination of Suspension Rate of Pesticides". The wetting time was 84 s, determined according to GB / T 5451-2001 "Determination of Wetting Time of Wettable Powders of Pesticides". The method for determining the effective viable count of wettable powders refers to NY / T 2321-2013 "Inspection Procedures for Microbial Fertilizer Products". The effective viable count of wettable powders is approximately 2.0 × 10⁻⁶. 9 cfu / g.

[0061] Example 9 The effects of nitrogen-fixing bacteria on the agronomic traits of barley seedlings.

[0062] Select healthy, plump, uniformly sized, and undamaged barley seeds. Before soaking, disinfect them by soaking in 3% (w / v) hydrogen peroxide for 10 minutes. After rinsing with distilled water, place them on damp gauze and soak for 15 minutes. Place 30 uniformly sized soaked barley seeds in each petri dish and incubate in the dark at 25℃ until they sprout. Then transplant them into cultivation pots filled with sand. On the 10th day after transplanting, perform the following treatment: P1: Pour 10mL of CK (distilled water) into each pot; P2: Under the same nitrogen input conditions, water 10 mL / pot of nutrient solution containing ammonium nitrogen and nitrate nitrogen. The nitrogen content in the ammonium nitrogen and nitrate nitrogen nutrient solution is the same as that in the nitrogen-fixing bacteria stock solution. The nitrate nitrogen content is 0.2548 mg / L and the ammonium nitrogen content is 47.91 mg / L. P3: Irrigate with nitrogen-fixing bacteria stock solution (2.0×10⁻⁶) 8 (cfu / mL, nitrate nitrogen content 0.2548 mg / L, ammonium nitrogen content 47.91 mg / L) 10 mL / pot; the preparation method of nitrogen-fixing bacteria stock solution is the same as in Example 6.

[0063] P4: The nitrogen-fixing bacteria stock solution was diluted 5 times by volume, resulting in a viable cell count of 0.4 × 10⁻⁶. 8 The concentration of cfu / mL was increased, and the dilution solution was applied at a rate of 10 mL per pot. The preparation method of the nitrogen-fixing bacteria stock solution was the same as in Example 6.

[0064] Hogland nitrogen-free nutrient solution was applied every three days, with 10 mL per pot for each treatment. The Hogland nitrogen-free nutrient solution formula was as follows: calcium chloride tetrahydrate 0.4 mmol / L, potassium chloride 1.6 mmol / L, sodium sulfate 0.5 mmol / L, potassium dihydrogen phosphate 1 mmol / L, magnesium sulfate heptahydrate 1 mmol / L, sodium chloride 0.5 mmol / L, boric acid 2.35 mmol / L, Fe-EDTA 5 μmol / L, manganese sulfate monohydrate 0.55 μmol / L, zinc sulfate heptahydrate 0.385 μmol / L, anhydrous copper sulfate 0.0165 μmol / L, and molybdic acid 0.0065 μmol / L. Samples were taken on days 10, 20, 30, 40, and 50 after treatment. The SPAD value of the barley was measured using a SPAD instrument (SPAD-502), and the fresh weight, dry weight, and plant height were measured using vernier calipers and a 0.1% balance.

[0065] The results showed that on day 20, the SPAD value, plant height, single plant fresh weight, and aboveground fresh weight of barley treated with P4 reached their highest values, with the barley SPAD value reaching a maximum of 29.77. Figures 2-7 The barley plant height in the P4 treatment (214.64 mm) was 30.80% higher than that in the P1 treatment (164.09 mm), representing a 48.70% increase compared to the P1 treatment (20.02 mm). Figure 3 The fresh weight of barley plants in treatment P4 (0.2871 g) increased by 49.29% compared to treatment P1 (0.1923 g). Figure 4 The fresh weight of barley aboveground parts was 0.1623 g in treatment P4, which was significantly increased by 48.90% compared with 0.109 g in treatment P1. Figure 5 At day 50, the P4 treatment resulted in the highest single-plant dry weight and aboveground dry weight of barley, with a single-plant dry weight of 0.1195 g, which was 68.31% higher than that of the P1 treatment (0.0710 g). Figure 6 The aboveground dry weight of barley was 0.0815 g, which was 47.64% higher than that of the P1 treatment (0.0552 g). Figure 7 ).

[0066] Example 10 Field trial of the fertilizer effect of nitrogen-fixing bacteria inoculant on rapeseed 1. Time and place 1.1 Test Time April 1, 2025 - April 23, 2025.

[0067] 1.2 Test Site The experiment was conducted at a rapeseed planting base in Zhangzhuang Village, Shaozhuang Town, Caoxian County, Heze City, Shandong Province. The rapeseed in the experimental area was cultivated in spring and autumn steel-framed greenhouses, and the entire block experiment was completed within one greenhouse. The plot was flat and uniform, with relatively uniform soil fertility and good irrigation and drainage conditions. There were no compost sites, ditches, ponds, overflows, tall buildings, or trees providing shade around the experimental site. Furthermore, no microbial fertilizers had been applied previously. During the experiment, field management was conducted according to local agricultural management standards, including regular weeding. Irrigation was done via sprinkler irrigation, following a "dry-wet cycle" (where the relative soil moisture content drops to 45%–50% of field capacity, then the soil is re-irrigated to 80% of field capacity, and this cycle is repeated).

[0068] 2. Materials and Methods 2.1 Analysis of the tested soil Before the first fertilization, 2 kg of soil samples were collected using a five-point sampling method. After the soil samples were mixed thoroughly, 500 g samples were obtained using the quartering method and sent to the Institute of Soil and Fertilizer, Academy of Agricultural and Forestry Sciences for soil nutrient testing. The tested indicators included: pH value, total nitrogen, available phosphorus, available potassium, organic matter content, and electrical conductivity (EC value). The soil test results are shown in Table 4.

[0069] Table 4 Soil Nutrient Status

[0070] 2.2 Test Fertilizers 2.2.1 Fertilizer Products The test product was the wettable powder of *Azotobacter chrysogenum* LLBG-20240108-001 prepared in Example 8. The main technical indicators of the product were: *Azotobacter chrysogenum* ≥ 800 million / gram.

[0071] 2.2.2 Matrix In this experiment, the blank control substrate was an inactivated azotocinobacter brownii microbial agent. 1000 grams of LLBG-20240108-001 wettable powder prepared in Example 8 of the test fertilizer was taken and inactivated by high pressure sterilization. After sterilization, random samples were taken for sterility testing.

[0072] (1) High pressure sterilization method: sterilize at 121°C for 25 minutes, dry at 105°C for 3 hours and then use.

[0073] (2) Sterility test: A small amount of inactivated *Azotocinobacter chrysogenum* inoculum was streaked onto Assab nitrogen-free medium, with *Azotocinobacter chrysogenum* inoculum as a control. The mixture was incubated at 37°C for 24 hours. The sterility test results are shown in the table below. Figure 9 No colonies appeared on the culture medium streaked with the inactivating agent. Figure 9 (B) Numerous colonies appeared on the uninactivated *Azotocinobacter chrysogenum* microbial inoculant; the colonies were pale yellow. Figure 9 (A)

[0074] 2.3 Test Crops and Varieties Chinese rapeseed (Brassica chinensis), variety Zhonghuaqing.

[0075] 2.4 Weather conditions The weather was good throughout the entire test, with no severe weather events.

[0076] 2.5 Conventional Fertilization Conventional fertilization was consistent with the average fertilization amount (mainly nitrogen, phosphorus, and potassium fertilizers) of the local area over the previous three years. The fertilizer varieties and fertilization methods did not interfere with the experimental results. Before sowing on April 1, 2025, apply 40 kg of 15-15-15 NPK compound fertilizer per mu; during the growing season, apply topdressing three times, each time spraying with a solution of potassium dihydrogen phosphate diluted 800 times, spraying until the leaves are moist.

[0077] 2.6 Experimental Design and Methods The experiment consisted of 3 treatments with 3 replicates, arranged in a randomized block design, for a total of 9 plots. Each plot was approximately 15 square meters (2.6 m × 5.8 m). Treatments 1-3 each had 3 plots. Each plot was sown with 10.12 g of rapeseed seeds, with a standard thousand-seed yield of 2.0 g. Planting began on April 1st. The specific experimental design is shown in Table 5.

[0078] Table 5 Experimental Treatment Design Table

[0079] 3. Results and Analysis 3.1 The impact of different treatments on yield and output value Rapeseed was harvested on April 23, 2025. All rapeseed harvested from each plot under different treatments was weighed, and the yield per mu and growth rate were calculated accordingly. The yield and comparison results are shown in Table 6-9.

[0080] Table 6. Field trial results of the fertilizer effect of *Azotobacter chrysogenum* microbial inoculant on rapeseed yield.

[0081] Table 7. Results of field plot yield variance analysis of the fertilizer effect of *Azotobacter chrysogenum* microbial inoculant on rapeseed.

[0082] Note: * represents p <0.05, ** represents p <0.01.

[0083] Table 8. Multiple comparison results of the effect of *Azotobacter chrysogenum* microbial inoculant on the LSD yield of rapeseed in small plots.

[0084] Table 9. Field trial results of the fertilizer effect of *Azotobacter chrysogenum* microbial inoculant on rapeseed (economic benefit analysis).

[0085] Note: Costs and labor costs are calculated based on a unit price of 10 yuan / kg for microbial inoculant and 30 yuan / mu for each application of labor; the unit price of small oilseed cake is 1 yuan / kg. The total cost and labor cost of 100.00 yuan / mu is the cost of microbial inoculant: 10 yuan + 30 yuan × 3 = 100 yuan / mu. Treatments 2 and 3 are control groups. If they are not used as control groups, conventional fertilization is sufficient, so the cost and labor cost for treatments 2 and 3 are 0 yuan.

[0086] Input-output ratio = 1: Increased income per mu / New input cost. Table 7 shows the results of the analysis of variance, indicating that the differences between treatments were highly significant (p<0.01) and the differences between blocks were significant (p<0.05). The significant differences between blocks indicate that there were certain differences in soil fertility or other environmental conditions at the experimental sites. The randomized block design effectively separated this part of the variation, reduced the error, and made the test of treatment effects more sensitive. Table 8 shows the results of multiple comparisons, indicating that the yield differences among treatments were all significant, with treatment 1 having the highest yield, significantly better than treatments 2 and 3.

[0087] As shown in Table 6-8, the yield of rapeseed from highest to lowest was: Treatment 1 > Treatment 2 > Treatment 3. Treatment 1 yielded 826.67 kg per mu more than Treatment 2, with a yield increase rate of 21.18%. Analysis of variance showed that the differences among the treatments were highly significant. Treatment 1 was significantly higher than Treatment 2 and Treatment 3.

[0088] As can be seen from Table 9, treatment 1 increased income by 826.67 yuan per mu compared to treatment 2, and the input-output ratio after fertilization was 1:8.27, indicating that the return on investment of *Azotobacter graminearum* microbial agent was high.

[0089] In summary, the application of *Azotobacter chrysogenum* microbial inoculant can increase the yield of rapeseed. Under conventional fertilization conditions, applying 1 kg / mu of *Azotobacter chrysogenum* microbial inoculant three times via spraying increased the yield by 826.67 kg / mu compared to the control, a yield increase rate of 21.18%, which is highly significant. This also resulted in an additional income of 826.67 yuan / mu compared to the control.

[0090] 3.2 Effects of different treatments on biological traits and quality On the day of harvest, 10 representative rapeseed plants were selected from each plot and weighed to obtain the fresh weight of each plant. They were then taken back to the laboratory for drying and weighed after all moisture was removed to obtain the dry weight of each plant. The survey results are shown in Table 10.

[0091] Table 10 Results of fertilizer effect test of Azotobacter brownii microbial inoculant on rapeseed (biological traits and quality effects)

[0092] As can be seen from Table 10, the order of fresh weight per plant from highest to lowest is: Treatment 1 > Treatment 2 > Treatment 3; the order of dry weight per plant from highest to lowest is: Treatment 1 > Treatment 2 = Treatment 3. In summary, applying the azotocinobacter erythropoietin microbial agent by spraying can effectively increase the fresh weight and dry weight per plant of rapeseed.

[0093] 3.3 Security Analysis During the experiment, no fertilizer damage was observed in the experimental fertilizer, *Azotobacter graminearum* microbial agent, on the rapeseed plants, indicating good application safety.

[0094] 4. Experimental Conclusions Applying a microbial agent of *Azotobacter graminearum* to rapeseed three times by spraying (starting from the seedling stage, with an interval of 4-8 days, for a total of 3 applications, with a dosage of 1 kg / mu each time) can effectively improve the biological traits and quality of rapeseed, increase its yield, and enhance its economic benefits.

[0095] Under the conditions of this experiment, compared with the same amount of inactivated azotocinobacter erythropoietin inoculant + conventional fertilization: the yield per mu increased by 826.67 kg, the yield increase rate reached 21.18%, and the difference reached a highly significant level; the income increased by 826.67 yuan, and the input-output ratio was 1:8.27; the fresh weight and dry weight of single plants of rapeseed were significantly improved, and the commercial quality of rapeseed was enhanced.

[0096] In summary, this invention screened a nitrogen-fixing bacterium, *Azotobacter chrysogenum*, from facility soil, and bred a strain with strong nitrogen-fixing ability through ARTP mutagenesis. *Azotobacter chrysogenum* (Brachycoccus chrysogenum) Azotobacter chroococcum LLBG-20240108-001, with preservation number CGMCC No. 29641, exhibits a nitrogen-fixing efficiency of up to 20 mg N / g sugar in its fermentation broth, demonstrating excellent nitrogen-fixing capacity. The prepared nitrogen-fixing bacterial fertilizer, within a certain concentration range, can effectively increase the fresh weight, dry weight, and plant height of barley plants, exhibiting a significant growth-promoting effect. In field applications, it can effectively increase the yield per acre, fresh weight per plant, and dry weight per plant of rapeseed.

[0097] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A strain of Azotobacter brownii ( Azotobacter chroococcum LLBG-20240108-001, characterized in that, The accession number is CGMCC No. 29641.

2. A microbial agent, characterized in that, Including the *Azotobacter brownis* as described in claim 1 ( Azotobacter chroococcum )LLBG-20240108-001.

3. The microbial agent according to claim 2, characterized in that, The microbial agent comprises a wettable powder, and the effective viable count of *Azotobacter brownii* LLBG-20240108-001 in the microbial agent is ≥2.0 × 10⁻⁶. 8 cfu / g or ≥2.0×10 8 cfu / mL.

4. A microbial fertilizer and / or wettable powder, characterized in that, By weight, it includes: 4-30 parts of *Azotobacter brownii* LLBG-20240108-001 inoculant, 2-5 parts of wetting agent, 1-5 parts of dispersant and 62-70 parts of soluble carrier; Optionally, the wetting agent comprises sodium dodecyl sulfate, the dispersant comprises sodium lignosulfonate, the soluble carrier comprises a mixture of modified starch and glucose, the mass ratio of modified starch to glucose being 1:(1-2), and the effective viable count of *Azotobacter blazei* LLBG-20240108-001 in the microbial fertilizer and / or wettable powder is ≥2.0 × 10⁻⁶. 8 cfu / g or ≥2.0×10 8 cfu / mL.

5. The microbial fertilizer and / or wettable powder according to claim 4, characterized in that, The preparation method of the *Azotobacter brownii* LLBG-20240108-001 inoculum includes: The bacterial suspension of *Azotobacter chrysogenum* LLBG-20240108-001 was mixed with diatomaceous earth and dried to obtain *Azotobacter chrysogenum* LLBG-20240108-001 bacterial agent; Optionally, the amount of diatomaceous earth added is 1%-5% of the mass of the *Azotobacter globosum* LLBG-20240108-001 bacterial culture. Optionally, the mixing method includes stirring and / or centrifugation, wherein the stirring speed is 100-150 rpm and the time is 1-2 h, and the centrifugation is continuous centrifugation, wherein the continuous centrifugation speed is 8000-14000 rpm and the feed rate is 200-300 L / h; Optionally, the drying temperature is 45-55°C, and the product is dried until the moisture content is 5%-10%. Optionally, the effective viable count of *Azotocinobacter globosum* LLBG-20240108-001 inoculant is ≥2.0 × 10⁻⁶. 8 cfu / g or ≥2.0×10 8 cfu / mL.

6. The application of the *Azotobacter globosum* LLBG-20240108-001 as described in claim 1, or the microbial agent as described in claim 2 or 3, or the microbial fertilizer and / or wettable powder as described in claim 4 or 5, in promoting plant growth.

7. The application according to claim 6, characterized in that, The plants include one or more of barley, wheat, corn, and rapeseed; And / or, the growth promotion includes increasing at least one of the following: plant fresh weight, dry weight, plant height, and yield per acre.

8. A fermentation method for *Azotobacter brownii*, characterized in that, include: The liquid seed of *Azotobacter brownis* LLBG-20240108-001 was inoculated into the fermentation medium for fermentation culture to obtain the fermentation broth; Optionally, the seed culture medium comprises: 20-40 g glucose, 1-5 g yeast powder, 1-5 g CaCO3, 0.2-0.8 g KH2PO4, 0.2-0.8 g MgSO4·7H2O, 0.2-0.8 g K2SO4, 0.01-0.02 g CaCl2, 0.01-0.02 g FeCl3, 0.001-0.002 g Na2MoO4, and 1000 mL water; Optionally, the fermentation medium comprises: 2%-4% glucose, 0.1%-0.5% yeast extract, 0.1%-0.5% CaCO3, 0.02%-0.08% KH2PO4, 0.02%-0.08% MgSO4·7H2O, 0.02%-0.08% K2SO4, 0.001%-0.002% CaCl2, 0.001%-0.002% FeCl3, and 0.0001%-0.0002% Na2MoO4, with the balance being water.

9. The fermentation method according to claim 8, characterized in that, The effective viable count of *Azotobacter brownii* LLBG-20240108-001 in the fermentation broth is ≥2.0 × 10⁻⁶. 9 cfu / mL; Optionally, the preparation method of the liquid seed of Azotobacter chrysogenum LLBG-20240108-001 includes: activating and seeding Azotobacter chrysogenum LLBG-20240108-001 to obtain liquid seed of Azotobacter chrysogenum LLBG-20240108-001; Optionally, the seed culture temperature is 30-35℃, the rotation speed is 180-220 rpm, and the time is 48-72h; Optionally, the fermentation culture temperature is 30-35℃, the ventilation rate is 0.8-1.2 L / (L·min), the rotation speed is 105-220 rpm, the loading coefficient is 60%-70%, and the time is 3-5 days.

10. A method for applying a microbial fertilizer and / or wettable powder, characterized in that, include: Apply microbial fertilizers and / or wettable powders to plants at a rate of 0.8-1 kg / mu, using methods including spraying; the first application should be made when the plants have 3-5 leaves, and the fertilizer should be applied 2-3 times consecutively, with an interval of 4-8 days between each application. Optionally, the microbial fertilizer and / or wettable powder is mixed with conventional fertilizer before application; the microbial fertilizer and / or wettable powder is the microbial fertilizer and / or wettable powder as described in claim 4 or 5.

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