Preparation and application of a bio-nitrogen-fixing bacterium and microbial inoculant

Abstract

The application belongs to the field of microorganisms and particularly relates to a biological nitrogen-fixing bacterium and preparation and application of a microbial inoculum.The biological nitrogen-fixing bacterium is named Microbacterium sp GSS16, classified and named as Microbacterium sp, and preserved in the Guangdong Microbial Culture Collection Center, No. 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100 Courtyard, on November 14, 2022, with a preservation number of GDMCC No. 62976; and can be used for plant nitrogen fixation, production of nitrogen-fixing enzyme, promotion of plant growth and improvement and fertilization of soil.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to the preparation and application of a strain of nitrogen-fixing bacteria and microbial agents. Background Technology

[0002] The nitrogen cycle is a crucial biogeochemical process, and nitrogen is the most important mineral inorganic nutrient for plants. While industrial nitrogen fertilizers have increased crop yields to some extent, they come with numerous problems, such as: the need for high-temperature, high-pressure catalytic nitrogen and hydrogen production, consuming vast amounts of energy; low utilization rates, as not all nitrogen applied to farmland is absorbed and utilized by crops, with most entering rivers, lakes, or seeping into groundwater in unsaturated areas via surface runoff, or entering the atmosphere through ammonia volatilization and denitrification. Excessive application of industrial nitrogen fertilizers leads to soil compaction, acidification, and eutrophication of water bodies, while also increasing emissions of the greenhouse gas nitrous oxide, exacerbating the global greenhouse effect.

[0003] Biological nitrogen fixation refers to the process by which nitrogen-fixing microorganisms convert N2 into NH3 using their own nitrogenases at normal temperature and pressure. German scholar Hermann Hellriegel first discovered and proposed in 1886 that soybean root nodules have the ability to convert N2 into NH3. In 1960, American scholar Carnahan demonstrated that nitrogen-fixing bacteria could still perform biological nitrogen fixation even after cell disruption, suggesting that nitrogenase plays a crucial role in biological nitrogen fixation. Compared with industrially synthesized nitrogen fertilizers, biological nitrogen fixation has advantages such as a large nitrogen fixation capacity, low energy consumption in the reaction process, and easier absorption and utilization of synthesized nitrogen by plants. Therefore, biological nitrogen fixation is more conducive to sustainable agricultural development and ecological environmental protection. Currently, microbial nitrogen fixation includes three types: autotrophic nitrogen fixation, symbiotic nitrogen fixation, and combined nitrogen fixation. Common autotrophic nitrogen-fixing bacteria include Klebsiella, Rhodospirillum, Paenibacillus polymyxa, and Bacillus macerans. Symbiotic nitrogen-fixing bacteria include Pseudomonas, Cupriavidus, Burkholderia, and Herbaspirillum. Associative nitrogen-fixing bacteria include Azoarcus and Azotobacter paspali. Summary of the Invention

[0004] The first aspect of the present invention is to provide a strain of nitrogen-fixing bacteria.

[0005] A second aspect of the present invention is to provide a culture.

[0006] The third objective of this invention is to provide a method for preparing the culture of the second aspect of this invention.

[0007] The fourth aspect of this invention is to provide a microbial agent.

[0008] The fifth aspect of this invention is to provide a method for preparing the microbial agent of the fourth aspect of this invention.

[0009] The sixth aspect of this invention aims to provide the application of the nitrogen-fixing bacteria of the first aspect, the culture of the second aspect, and the bacterial agent of the fourth aspect.

[0010] The seventh aspect of this invention aims to provide a product.

[0011] The object of the eighth aspect of the present invention is to provide a method for planting plants.

[0012] To achieve the objectives of this invention, the technical solution adopted is as follows:

[0013] In a first aspect, a nitrogen-fixing bacterium named Microbacterium sp GSS16 is provided. It is classified as Microbacterium sp and was deposited at the Guangdong Provincial Microbial Culture Collection Center on November 14, 2022, with accession number GDMCC No. 62976.

[0014] The nitrogen-fixing bacteria are Gram-negative, aerobic, rod-shaped, non-flagellated cells, with a cell size of approximately 0.4 μm to 1.8 μm, and their colonies are yellow on the culture medium.

[0015] The growth temperature range of the nitrogen-fixing bacteria is 4-45℃, with the optimal growth temperature being 30℃; the pH growth range is 5.0-9.5, with the optimal pH being 7.0; and the salt tolerance range is 0-7%, with the optimal range being 0-1%.

[0016] The nitrogen-fixing bacteria are positive for nitrate reduction, gelatin hydrolysis, and aesculin hydrolysis. They can use p-nitro-β-D-methylgalactose, methyl-D-mannose, glycogen, citric acid, and glucose as carbon sources. During fermentation, they can produce leucine aromatic aminoaminase and valine aromatic aminoaminase.

[0017] The G+C content of the nitrogen-fixing bacteria was 69.0 mol%.

[0018] A second aspect of the present invention provides a culture obtained by culturing the nitrogen-fixing bacteria of the first aspect of the present invention.

[0019] A third aspect of the present invention provides a method for preparing a culture of the second aspect of the present invention, wherein the nitrogen-fixing bacteria of the first aspect of the present invention are inoculated into a culture medium and cultured to obtain the culture.

[0020] Preferably, the culture medium comprises at least one of LB medium, Assumption nitrogen-free medium, and HM medium.

[0021] Preferably, the culture conditions are 4-45℃ and 100-300rpm / min for 16-48h.

[0022] A fourth aspect of the present invention provides a microbial agent comprising: nitrogen-fixing bacteria of the first aspect of the present invention and / or cultures of the second aspect of the present invention.

[0023] Preferably, the microbial agent is a solid microbial agent or a liquid microbial agent.

[0024] Preferably, the liquid microbial agent comprises a liquid ferment with 0.5-2% w / v protectant added.

[0025] Preferably, the protective agent is one or more of benzoate, sorbate, and polyethylene glycol.

[0026] More preferably, the protective agent is potassium sorbate.

[0027] Preferably, the solid microbial agent further comprises a carrier.

[0028] Preferably, the carrier comprises at least one mineral material and plant material.

[0029] More preferably, the mineral material comprises at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth.

[0030] More preferably, the plant material comprises at least one of corn flour, soybean flour, and wheat bran.

[0031] Preferably, the formulation of the microbial agent is liquid, powder, or granules.

[0032] A fifth aspect of the present invention is to provide a method for preparing the bacterial agent of the fourth aspect of the present invention, wherein the bacterial agent is obtained by using the nitrogen-fixing bacteria of the first aspect of the present invention and / or the culture of the second aspect of the present invention as active ingredients.

[0033] A sixth aspect of the invention is to provide the application of any one of (1)-(3) in at least one of (a)-(h);

[0034] (1) The nitrogen-fixing bacteria of the first aspect of the present invention;

[0035] (2) The culture of the second aspect of the present invention;

[0036] (3) The bacterial agent of the fourth aspect of the present invention;

[0037] (a) Nitrogen fixation by leguminous plants;

[0038] (b) Preparation of nitrogen-fixing products from leguminous plants;

[0039] (c) Production of nitrogenase;

[0040] (d) Prepare products for the production of nitrogenase;

[0041] (e) Promotes plant growth;

[0042] (f) Prepare products that promote plant growth;

[0043] (g) Improve and enrich the soil;

[0044] (h) Prepare products for improving and enriching soil.

[0045] Preferably, the plant includes legumes.

[0046] More preferably, the legume includes at least one of soybean and alfalfa.

[0047] A seventh aspect of the present invention provides a product comprising at least one of (1)-(3):

[0048] (1) The nitrogen-fixing bacteria of the first aspect of the present invention;

[0049] (2) The culture of the second aspect of the present invention;

[0050] (3) The bacterial agent of the fourth aspect of the present invention.

[0051] Preferably, the product comprises fertilizer.

[0052] An eighth aspect of the present invention provides a method for growing plants, comprising the step of treating the plants with the product of the seventh aspect of the present invention.

[0053] Preferably, the method involves planting the plants in soil and / or a substrate;

[0054] The soil and / or substrate contain the product described in the seventh aspect of this invention; and / or

[0055] The plants are soaked in the product described in the seventh aspect of this invention before planting.

[0056] The beneficial effects of this invention are:

[0057] This invention provides a nitrogen-fixing bacterium named Microbacterium sp GSS16, taxonomically classified as Microbacterium sp., which was deposited on November 14, 2022, at the Guangdong Provincial Microbial Culture Collection Center, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 62976. This strain can be used for plant nitrogen fixation (nitrogenase activity is 27.35 nmol·mL⁻¹). -1 ·h -1 It can produce nitrogenase, promote plant growth, and improve soil fertility. It can be applied to root crops such as soybeans and alfalfa. While reducing the application of chemical fertilizers, it can improve the absorption efficiency of nitrogen in crops, thereby increasing crop yield and quality.

[0058] This invention provides a microbial agent containing the aforementioned nitrogen-fixing bacteria, which can be used as a biological agent to replace part of the nitrogen fertilizer and directly applied to agricultural production, thereby reducing the amount of chemical nitrogen fertilizer applied and improving soil fertility (using this microbial agent during the soybean seedling stage can replace 71.78% of the nitrogen application; using this microbial agent in alfalfa can achieve 123.11% of the nitrogen fertilizer application effect). Attached Figure Description

[0059] Figure 1 Scanning electron microscope image of nitrogen-fixing bacteria GSS16 at 20,000x magnification.

[0060] Figure 2 Phylogenetic tree of the nitrogen-fixing bacterium GSS16. Detailed Implementation

[0061] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0062] Example 1: Cultivation and isolation of GSS16

[0063] The nitrogen-fixing bacterium GSS16 of the present invention was obtained by isolation and purification through enrichment culture of sediment soil in the mangrove ecological zone of Zhangzhou, Fujian.

[0064] The specific separation and purification method includes the following steps:

[0065] (1) Take 5.0g of sediment and inoculate it into an enrichment and separation medium with a pH of 7.2. Incubate at 30℃ and 200rpm / min with shaking. After 3 days of incubation, transfer the culture medium to another fresh enrichment and separation medium with an inoculation volume of 10% and incubate at 30℃ and 200rpm / min with shaking. Repeat this subculture 4 times.

[0066] (2) Dilute the cultured fourth-generation medium by 10 5 -10 6 Take 0.1 mL of the diluted culture medium and spread it evenly on agar solid medium. Incubate at 30°C for 48 h. Pick a single colony for isolation and purification to obtain strain GSS16.

[0067] The enrichment and separation medium was HM medium, with the following components: Na2SO4 0.25g; MgSO4·7H2O 0.18g; FeCl3 0.004g; CaCl2·2H2O 0.013g; HEPEs buffer 1.3g; yeast extract 0.25g; water 1000mL, pH 7.2; and solid agar medium was prepared by adding 20g of agar powder to the above enrichment and separation medium.

[0068] Example 2: Identification and Preservation of GSS16

[0069] 2.1 Cell morphology characteristics

[0070] Bacterial observation was performed using a standard electron microscope, and the scanning electron microscope images are as follows: Figure 1 As shown, this strain is a Gram-negative bacterium with rod-shaped, non-flagellated cells. The cell size is approximately 0.4 μm to 1.8 μm, and the colonies are yellow on the culture medium.

[0071] 2.2 Physiological and Biochemical Characteristics

[0072] The physiological and biochemical characteristics of strain GSS16 were identified, and the results are shown in Table 1.

[0073] Table 1. Results of physiological and biochemical identification of the strains

[0074]

[0075]

[0076]

[0077] In the table: + indicates positive, - indicates negative.

[0078] The identification results show that strain GSS16 is aerobic, Gram-negative, with a growth temperature range of 4-45℃ and an optimal growth temperature of 30℃; a pH growth range of 5.0-9.5 and an optimal pH of 7.0; and a salt tolerance range of 0-7% and an optimal range of 0-1%.

[0079] GSS16 showed positive results in nitrate reduction, gelatin hydrolysis, and aesculin hydrolysis. It can utilize p-nitro-β-D-methylgalactose, methyl-D-mannosin, glycogen, citric acid, and glucose as carbon sources. During fermentation, it produces leucine aromatic aminoaminase and valine aromatic aminoaminase. The G+C content is 69.0 mol%.

[0080] 2.3 Molecular biological characteristics

[0081] Total bacterial DNA was extracted using SDS-proteinase K, chloroform-isoamyl alcohol (24:1 v / v), and precipitated with 0.6 v / v of isopropanol. Bacterial 16S rRNA was amplified using universal primers F27 and R1492R, and the PCR products were recovered and sequenced. The obtained 16S rRNA sequence is SEQ ID NO.1. The obtained sequence was then subjected to a BLAST search in international nucleic acid sequence databases such as GenBank to identify the strain with the highest homology to type strains in the database or strains deposited in international microbial culture centers such as KCTC and GDMCC.

[0082] After comparison with EZBioCloud server, it was found that strain GSS16 is similar to the type strain Microbacterium shaanxiense CCNWSP60. T The 16S rRNA gene similarity was 97.55%, and it was similar to the model strain *Microbacterium esteraromaticum* DSM 8609. T The 16S rRNA gene similarity was 97.21%, and it was similar to the model strain Microbacterium murale 1-Gi-001. T The 16S rRNA gene similarity of strain GSS16 was 97.12%. The 16S rRNA gene similarity with other strains was below 97%. Furthermore, strain GSS16 showed similarity to the type strain *Microbacterium esteraromaticum* DSM 8609. TThe average nucleotide identity (ANI) and DNA hybridization homology (dDNA-DNA) were 86.30% and 29.70%, respectively, which are below the species definition thresholds of 96% and 70% (Richter M, Rosselló–Móra R (2009) Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci USA 106:19126–19131. https: / / doi.org / 10.1073 / pnas.0906412106). Therefore, strain GSS16 belongs to a species of the genus Microbacterium and is named Microbacterium sedimentimangrovi sp. nov. GSS16. T GSS16 was deposited on November 14, 2022, at the Guangdong Provincial Center for Microbial Culture Collection, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 62976. The phylogenetic tree results for GSS16 are shown below. Figure 2 .

[0083] Example 3: Preparation of microbial inoculants

[0084] (1) Shake flask culture: A loopful of bacterial growth was picked from the GSS16 slant and inoculated into an Erlenmeyer flask containing 100 mL of LB liquid medium. The flask was then shaken at 37°C and 200 rpm / min for 24 h. The LB liquid medium formula was: 5 g yeast extract, 10 g peptone, 10 g NaCl, 1000 mL water, pH 7.2. At this time, the bacteria were in the logarithmic growth phase, with a viable count of 3 x 10⁻⁶. 8 CFU / mL.

[0085] (2) Fermentation in a fermenter: The GSS16 fermentation broth obtained in step (1) was inoculated into a 10L fermenter at an inoculum rate of 2% (V / V), and fermented at 30℃ and 200rpm / min for 36h; the fermentation medium used was the liquid medium mentioned above. At this time, the viable cell count was approximately 4.68*10⁻⁶. 8 CFU / mL.

[0086] (3) Preparation of liquid bacterial agent: The above fermentation broth was prepared by adding potassium sorbate at 1% (w / v) as a protectant to obtain liquid bacterial agent.

[0087] (4) The above fermentation broth was concentrated 6 times and then added to the solid excipients. The mass ratio of the solid excipients to the fermentation broth was 2:1. The mixture was stirred evenly, dried in a centrifugal spray dryer, and packaged. The solid excipients contained 30 wt% wheat bran, 30 wt% corn flour, and 40 wt% soybean flour. The resulting microbial preparation was counted, and the total number of viable bacteria reached more than 500 million / gram.

[0088] Example 4: Determination of microbial GSS16 nitrogenase activity

[0089] The isolated bacterial strain GSS16 was inoculated into a 100 mL Erlenmeyer flask containing 20 mL of Assabeth's nitrogen-free medium, and the flask was plugged with cotton. The flask was incubated at 30 °C and 150 rpm. When the OD600 of the bacterial culture reached 0.6, the cotton was replaced with a rubber stopper, and 5 mL of gas was extracted from the flask using a syringe. 5 mL of high-purity acetylene was then injected, and the culture was continued for 24 h. The ethylene production was determined using gas chromatography, with the formula C = (hx × c × V) / (24.9 × hs × t); the concentration of C2H4 produced (nmol·mL⁻¹) was also determined. -1 ·h -1 hx - sample peak area; hs - standard C2H4 peak area; c - standard C2H4 concentration (nmol / mL); V - container volume (mL); t - incubation time (h). The nitrogenase activity of strain GSS16 was measured to be 27.35 nmol·mL⁻¹. -1 ·h -1 A potted plant experiment will be conducted subsequently.

[0090] Example 5: Pot experiments of soybeans and alfalfa inoculated with inoculants

[0091] Soybean and alfalfa seeds were cultured separately in potting soil, and transplanted when the seedlings had 3 leaves. The pots were 25cm in diameter and 30cm high, opaque. 5kg of sterilized potting soil (prepared by high-temperature and high-pressure sterilization) was added to each pot. The inoculant was the one obtained in step (2) of Example 3, with uninoculated fermentation broth (the method was the same as in Example 3, except that GSS16 was not inoculated) serving as a control. The grown soybean and alfalfa seedlings were carefully removed without damaging their roots, washed with sterile water, and then soaked in the inoculant for 30 minutes before being transplanted into pots (A1). The control group was soaked in uninoculated fermentation broth. The experiment was divided into three groups: inoculated with inoculant A1, and uninoculated with inoculants CK1 and CK2, with three replicates for each treatment. To ensure consistency in the experimental treatments for soybeans and alfalfa, 100 mL of nutrient solution was added to the flowerpots every 2 days after transplanting. A1 and CK1 were given nitrogen-free Hogland nutrient solution, while CK2 was given nitrogen-containing Hogland nutrient solution, until the end of the experiment after 30 days.

[0092] After the experiment, soybean roots were collected, washed with sterile water, chopped, and placed into 50mL Erlenmeyer flasks containing 10mL of Assabeth nitrogen-free medium. Each flask was sealed with a rubber stopper. One mL of air was drawn off using a syringe, and one mL of high-purity acetylene was injected. After culturing for 2 hours, one mL of gas was drawn off to determine the ethylene production using the same formula as in Example 4. The results are shown in Tables 2 and 3. Nitrogenase activity was detected in the roots of A1, indicating that the inoculum from Example 3 could infect soybeans and exert nitrogen-fixing activity in the roots.

[0093] Table 2 Nitrogenase activity in soybean roots (nmol·mL) -1 ·h -1 )

[0094]

[0095] Note: Different letters indicate significant differences.

[0096] Table 3 Nitrogenase activity in alfalfa roots (nmol·mL) -1 ·h -1 )

[0097]

[0098] Note: Different letters indicate significant differences.

[0099] Example 6: The effect of microbial agents on the nitrogen content of soybeans

[0100] The soybean roots, stems, and leaves from the experiment in Example 5 were rinsed, dried (3 replicates), pulverized, and their total nitrogen content was determined using the sulfuric acid digestion method. Table 4 shows that the nitrogen content of soybean roots treated with the microbial agent A1 from step (2) of Example 3 was significantly higher than that of CK1 (no nitrogen or microbial agent added). Compared to CK2 (normal nitrogen added but no microbial agent added), the nitrogen content of roots treated with the microbial agent reached 71.78% of that of CK2, indicating that the biological agent from Example 3 can replace part of the nitrogen application during the soybean seedling stage.

[0101] Table 4. Nitrogen content in soybean roots (mg / g)

[0102]

[0103] Note: Different letters indicate significant differences.

[0104] Example 7: Effect of microbial inoculants on nitrogen content in alfalfa

[0105] The alfalfa roots from the three replicates of Example 5 were rinsed, dried, pulverized, and their total nitrogen content was determined using the method described in Example 6. Table 5 shows that the nitrogen content of alfalfa roots treated with the microbial agent A1 from step (2) of Example 3 was significantly increased compared to CK1 (without nitrogen or microbial agent). Compared to CK2 (with normal nitrogen but no microbial agent), the nitrogen content of roots treated with the microbial agent reached 123.11% of CK2, indicating that the biological microbial agent from Example 3 can replace nitrogen fertilizer for alfalfa.

[0106] Table 5. Nitrogen content in alfalfa roots (mg / g)

[0107]

[0108] Note: Different letters indicate significant differences.

Claims

1. A strain of microbacterium ( Microbacterium sp GSS16, also known as Microbacterium GSS16 ( Microbacterium sp GSS16), categorized as follows Microbacterium sp It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 14, 2022, with accession number GDMCC No. 62976.

2. A culture, obtained by culturing the microbacteria of claim 1 ( Microbacterium sp GSS16 was obtained.

3. The method for preparing the culture according to claim 2, wherein the microbacteria (According to claim 1) are used. Microbacterium sp GSS16 was inoculated into the culture medium, cultured, and obtained; The culture medium includes at least one of LB medium, Assumption nitrogen-free medium, and HM medium; The culture conditions are 4-45℃ and 100-300rpm / min for 16-48h.

4. A microbial agent comprising: the microbacterium described in claim 1 (… Microbacterium sp GSS16 and / or the culture as described in claim 2.

5. The microbial agent according to claim 4, characterized in that: The microbial agent is a solid microbial agent or a liquid microbial agent; The liquid microbial agent includes a liquid ferment with 0.5-2% w / v protectant added; The protective agent is one or more of benzoate, sorbate, and polyethylene glycol; The solid microbial agent also includes a carrier; The carrier contains at least one of mineral materials and plant materials.

6. The microbial agent according to claim 5, characterized in that: The formulation of the microbial agent is liquid, powder, or granules.

7. The method for preparing the microbial agent according to any one of claims 4 to 6, characterized in that: The microbacteria described in claim 1 ( Microbacterium sp The bacterial agent is obtained by using GSS16 and / or the culture of claim 2 as the active ingredient.

8. Any one of (1)-(3) in at least one of (a)-(h); (1) The microbacteria described in claim 1 ( Microbacterium sp GSS16; (2) The culture according to claim 2; (3) The microbial agent according to any one of claims 4 to 6; (a) Nitrogen fixation in plants; (b) Preparation of nitrogen-fixing products from plants; (c) Production of nitrogenase; (d) Prepare products for the production of nitrogenase; (e) Promotes plant growth; (f) Prepare products that promote plant growth; (g) Improve and enrich the soil; (h) Prepare products for improving and enriching soil.

9. The application according to claim 8, characterized in that: The plants mentioned include legumes.

10. The application according to claim 9, characterized in that: The plant includes at least one of soybean and alfalfa.

11. A product comprising at least one of (1)-(3): (1) The nitrogen-fixing bacteria as described in claim 1; (2) The culture according to claim 2; (3) The microbial agent according to any one of claims 4 to 6.

12. The product according to claim 11, characterized in that: The product contains fertilizer.

13. A method of growing plants, comprising the step of treating the plants with the product of claim 11 or 12; The method comprises: planting plants in soil and / or substrate; wherein the soil and / or substrate contains the product of claim 11 or 12; and / or The plant is soaked in the product described in claim 11 or 12 before planting.

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