NosZ II-type mucus bacillus A14 with N2O reducing capacity and application of nosZ II-type mucus bacillus A14

Abstract

The invention discloses nosZ II type mucus bacillus A14 with N2O reducing capacity and application of the nosZ II type mucus bacillus A14, the preservation number of the nosZ II type mucus bacillus A14 is GDMCCNO: 66255, and the nosZ II type mucus bacillus A14 is preserved in Guangdong Microbial Culture Collection Center on April 30, 2025. The strain is a new species of mucus bacillus. The novel mucus bacillus A14 disclosed by the invention has relatively strong N2O reductase activity and also has N2O reducing capacity. Therefore, the novel mucus bacillus A14 disclosed by the invention has good application potential in the aspect of reducing N2O gas emission.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural microorganisms, and particularly relates to a strain of Bacillus myxobacterium A14 with N2O reduction ability and applications thereof. Background Art

[0002] As a major greenhouse gas, N2O has a global warming potential 298 times greater than CO2 and remains in the atmosphere for up to 120 years before being decomposed by ultraviolet rays. Over the past decade, N2O has increased at an annual rate of approximately 0.25%, currently reaching approximately 17 TgN yr. -1 , and it is still growing. Agricultural soil is the main anthropogenic source of N2O emissions, contributing more than 60% of global emissions. Excessive N2O emissions not only exacerbate climate change, but also destroy stratospheric ozone. Soil microorganisms play a key role in regulating nitrogen transformation and N2O emissions, among which nitrification and denitrification are the two main pathways of N2O generation and consumption. The denitrification process is the key process of N2O production and consumption, involving NO3 - Gradually reduced to NO2 - , NO, N2O and finally N2. Denitrification can be both a source and a sink of N2O, depending on the state of the denitrifying microorganisms and environmental conditions. Among them, the discovery of the nosZ gene has enabled people to understand the only biological removal pathway for N2O. nosZ encodes N2O reductase to catalyze the reduction of N2O to N2. Studies have reported that the nosZ gene contains two different branches: nosZ I and nosZ II. Microorganisms containing nosZ I usually carry nirK and nirS genes and may produce N2O during denitrification, while most microorganisms containing nosZ II are important N2O sinks and do not contribute to N2O production.

[0003] In recent years, another class of microorganisms with specialized functions has been revealed—N2O-respiring microorganisms. These microorganisms can survive under anaerobic conditions using only N2O as an electron acceptor, reducing N2O to N2 and thereby reducing N2O emissions. These microorganisms are divided into two categories: nosZ type I and nosZ type II. Early-discovered N2O-respiring microorganisms are non-denitrifying bacteria. Unlike traditional nosZ type I microorganisms, they possess nosZ II, a different branch of the nosZ gene. Currently, the majority of N2O-respiring bacteria belong to nosZ type II, but some nosZ type I bacteria can also respire N2O for growth. Current research provides limited understanding of the N2O reduction mechanisms of nosZ type II microorganisms.

[0004] Myxobacterium is a genus of Gram-negative bacteria belonging to the phylum Bacteroidetes, widely distributed in soil, aquatic environments, sediments, and plant rhizospheres. Due to its many properties, Myxobacterium has diverse applications. Some strains have the ability to decompose pectin, others promote plant growth, and most strains produce extracellular polymers, all of which have great potential for utilization. As plant-beneficial microorganisms, enriching their microbial resources, identifying and exploring their functions, and applying them to prepare microbial fertilizers and microbial agents are effective strategies for achieving sustainable and green agriculture. Summary of the Invention

[0005] The first object of the present invention is to provide a strain of Mucilaginibacter sp. A14 having N2O reduction ability, with a deposit number of GDMCC No: 66255.

[0006] The second object of the present invention is to provide the use of the above-mentioned Bacillus myxobacterium A14 in reducing N2O.

[0007] Preferably, it is the use of Bacillus myxobacterium A14 in reducing soil N2O emissions.

[0008] Preferably, it is the use of Bacillus myxobacterium A14 in improving the N2O reduction rate and N2O reductase activity.

[0009] The third object of the present invention is to provide a microbial agent comprising the above-mentioned Bacillus subtilis A14 with the deposit number GDMCC No: 66255.

[0010] The fourth object of the present invention is to provide a biological preparation, which uses the above-mentioned Bacillus myxogenes A14 or its fermentation liquid as an active ingredient.

[0011] Preferably, the biological preparation is a liquid preparation, a powder or a solid preparation.

[0012] A fifth object of the present invention is to provide a method for culturing the above-mentioned Bacillus myxogenes A14 or the above-mentioned microbial agent for use in reducing N2O gas emissions.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention uses enrichment and separation technology to screen denitrifying bacteria with high denitrification rates and strong N2O reduction capabilities from the soil. By inoculating this strain into the soil, the emission of greenhouse gases such as N2O can be reduced.

[0015] The present invention has the following advantages and effects compared to the prior art:

[0016] The present invention discovered Mucilaginibacter sp. A14 for the first time, which is a potential new species of Mucilaginibacter sp.

[0017] The present invention discovered for the first time that Myxobacterium viscosum has strong N2O reductase activity, enriching the functional research of Myxobacterium viscosum.

[0018] The present invention discovered that Myxobacterium siliflorum A14 possesses both strong reductase activity and NO reduction capability, thus achieving the dual purpose of reducing NO emissions. Therefore, Myxobacterium siliflorum A14, as a beneficial microorganism, has great potential for emission reduction and opens up new horizons for the preparation of microbial agents and organic biofertilizers.

[0019] Mucilaginibacter sp.A14 was deposited on April 30, 2025 in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, Postal Code: 510070, with the deposit number: GDMCC No: 66255. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The colony morphology of Myxobacterium spp. A14 on R2A medium.

[0021] Figure 2 This is the phylogenetic tree of Myxobacterium spp. A14.

[0022] Figure 3 is the N2O reduction rate of Bacillus myxobacterium A14. DETAILED DESCRIPTION

[0023] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions therein, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0024] Example 1: Enrichment culture and isolation of Myxobacterium spp. A14

[0025] To enrich efficient denitrifying strains, 100 mL vials were filled with 36 mL of sterile water (or DM-Nfree liquid medium) and 4 g of rhizosphere soil from the Pomelo Planting Demonstration Base in Zhangbei Village, Xihe Town, Dapu County, Meizhou City, Guangdong Province. The vial headspace was replaced with helium, followed by a helium-filled atmosphere replaced with 10% NO and 5% O. The vials were then incubated at 30°C and 150 rpm for 7 days. During the incubation period, the vials were replenished with a 10% NO and 5% O mixture every two days to the initial pressure, for a total of four enrichment cycles.

[0026] Take samples from the fourth generation enrichment solution, take 100 μL of each gradient bacterial suspension and add it into a 2 mL centrifuge tube filled with 900 μL sterile water, that is, dilute to 10 -4 , 10 -5 and 10 -6 . Absorb bacterial suspension 10 -3 , 10 -4 , 10 -5 and 10 -6 100 μL of each was spread onto R2A medium (Guangdong Huankai, Catalog No. 022029) and TSB medium (Guangdong Huankai, Catalog No. 024051), with three culture dishes spread per gradient. The culture dishes were sealed and incubated upside down in a 28°C biochemical incubator for 5 days. Colonies with different morphological characteristics were selected and purified by multiple streaking until a pure culture was obtained, thus obtaining Myxobacterium spp. A14.

[0027] The colony morphology of Bacillus mucinus A14 after culturing on R2A medium for 3 days is as follows Figure 1 As shown, the single colony is round, 1-3 mm in diameter, yellow, with a smooth, opaque surface and neat edges.

[0028] Example 2: 16S rRNA gene sequence analysis of Bacillus myxogenes A14

[0029] DNA from Bacillus muciniphila A14 was extracted using the alkaline lysis method: a small amount of cells was placed in a 200 μL centrifuge tube with 16.6 μL of alkaline lysis buffer. The cells were lysed in an alkaline environment at 95°C for 30 minutes. After cooling, 16.6 μL of neutralization buffer was added to each well, mixed, and stored in a -20°C refrigerator. Alkaline lysis buffer: 25 mM NaOH and 0.2 mM Na2-EDTA (pH = 12). Neutralization buffer: 40 mM Tris-HCl (pH = 7.5). Autoclave at 121°C for 15 minutes. The cells can be stored at 4°C for 2 months. The 16S rRNA gene sequence of the novel Myxobacterium septorianum A14 was amplified using bacterial 16S rRNA-specific primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGACTTAACCCCAA TCGC-3′). Electrophoresis analysis revealed an approximately 1500-bp amplified product, which was then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequenced sequences were assembled using DNAMAN software to obtain a 1391-bp 16S rRNA sequence (SEQ ID NO. 1). The 16S rRNA gene sequence of Myxobacterium septorianum A14 was submitted to the EzBioCloud database (www.ezbiocloud.net) for sequence homology comparison. The comparison results showed that the 16S rRNA gene sequence of Mucilaginiba cter ximonensis XM-003 (accession number, JBHUPD0000000000) had the highest similarity of 98.49%.

[0030] Example 3: Full gene sequence analysis of Bacillus myxogenes A14

[0031] The bacterial culture of the new type of myxobacterium A14 was sent to Shanghai Meiji Biotechnology Co., Ltd. for genome sequencing. The genome of the strain myxobacterium A14 was analyzed using the software QUASTv5.0.2. The results showed that the genome sequence of the new type of myxobacterium A14 contained 40 contigs, the total genome length was 4199463bp, the N50 length was 462240bp, and the G+C content of genomic DNA was 43.78%. The genomic phylogenetic tree of the new type of myxobacterium A14 and the closely related reference model strains was constructed using the software UBCGv3.0. The results are as follows Figure 2As shown, Mucilaginibacter sp. A14 is most closely related to Mucilaginibacter ximonensis and Mucilaginibacter panaciglaebae. Its ANI values were 81.02% and 79.60%, respectively, below the proposed species delimitation thresholds of 95–96%. Its dDDH values were between 31.60% and 27.30%, respectively, well below the 70% threshold for species delimitation (Table 1). Both ANI and dDDH results support the identification of Mucilaginibacter sp. A14 as a new species of the genus Mucilaginibacter. Therefore, it was designated Mucilaginibacter sp. A14. This strain has been deposited with the Guangdong Provincial Microbial Culture Collection Center (GDMCC), 5th Floor, Building 59, 100 Xianlie Middle Road, Guangzhou, Guangdong Province, China, under the GDMCC accession number 66255, dated April 30, 2025.

[0032] Table 1 shows the ANI and dDDH values of the novel myxobacterium A14 and its closely related strains

[0033]

[0034] Example 4: Analysis of N2O Reduction Capacity and N2O Reductase Activity of Novel Myxobacterium A14

[0035] Preparation of Bacillus myxogenes A14 bacterial solution:

[0036] Bacillus myxobacterium A14 was activated and cultured on an R2A plate at 28°C for 48 hours, a single colony was picked with a toothpick, inoculated into R2A liquid culture medium, and cultured at a constant temperature of 28°C and 200 rpm for about 48 hours to prepare a bacterial solution.

[0037] Determination of N2O reduction ability of novel myxobacterium A14:

[0038] A 50 mL serum bottle was filled with 25 mL of sterile DM-Nfree medium. The bottle was then capped with a rubber stopper and aluminum cap, and the cap was secured with a press. The mixed bacterial suspension was injected into the serum bottle to be added with a syringe. The headspace gas in the serum bottle was replaced with helium, and then 2.5 mL of N2O gas (approximately 99.9% purity) was added with a syringe. Three treatments were set up: N2O and bacteria (N2O-strain), N2O (control), and He and bacteria (control, He-strain), with three replicates for each treatment. The samples were incubated in a constant-temperature incubator at 30°C and 150 rpm. During the incubation period, gas samples were collected from the headspace of the serum bottle using a syringe at 0, 4, 8, 12, 16, 20, and 24 h. The N2O concentration was measured by gas chromatography, and the N2O reduction rate of the strain was calculated based on the concentration changes.

[0039] DM-N free medium: Na2HPO4·12H2O 10 g / L; KH2PO4 1.5 g / L; MgSO4·7H2O 0.1 g / L; sodium acetate 4.7 g / L; 2 mL of trace element mixture; pH ≈ 7.5, solvent is water.

[0040] The above-mentioned trace element mixed solution consists of: EDTA 50g / L; ZnSO4 2.2g / L; CaCl2 5.5g / L; MnCl2·4H2O 5.06g / L; FeSO4·7H2O 5g / L; (NH4)6Mo7O2·4H2O 1.1g / L; CuSO4·5H2O 1.57g / L; CoCl2·6H2O 1.61g / L, and the solvent is water.

[0041] Preparation of Bacillus myxogenes A14 cells: The prepared Bacillus myxogenes A14 cell suspension was centrifuged at 8000 rpm for 2 min. The precipitate was resuspended in DM-N free medium and centrifuged again at 8000 rpm for 1 min. The supernatant was discarded and the mixture was repeated three times to obtain Bacillus myxogenes A14 cells.

[0042] Determination of N2O reductase activity of novel Myxobacterium tuberculosis A14: The double antibody sandwich method was used to determine the level of nitrous oxide reductase (Nos) in the specimen. The specific operation is as follows: aspirate the pre-prepared Myxobacterium tuberculosis A14 bacterial solution, transfer it to a 2mL centrifuge tube, centrifuge and discard the supernatant, and then quantify the number of bacteria (not less than 10 5), add the extract in a ratio of 500 to 1000:1 of the extract volume to PBS. Subsequently, perform ultrasonic disruption of bacteria (under ice bath conditions, set the power to 20% or 200W, ultrasonic for 3s, interval of 10s, repeat 3 times). After the ultrasonic disruption is completed, centrifuge at 8000×g and 4°C for 10min, and take the supernatant as the sample to be tested. Pipette 10μL of the sample to be tested, add it to the enzyme-labeled coated plate, and add 40μL of sample diluent at the same time, shake gently to mix the two thoroughly, seal the plate with a sealing film, and then incubate at 37°C for 30min. After the incubation is completed, wash the enzyme-labeled coated plate 5 times with washing solution. Then add the enzyme-labeled reagent, incubate and wash again. Finally, add the color developer and terminator, and use the blank well (without sample and enzyme-labeled reagent) as a control to measure the OD with an enzyme-labeled instrument. 450 The standard in the reagent needs to be diluted, and the dilution gradient is 3U / L, 6U / L, 12U / L, 24U / L, and 48U / L. The subsequent sample addition and other operations are the same as the sample determination operation, and the OD of each diluted concentration standard is measured using a microplate reader. 450 Absorbance value, statistical data, drawing standard curve, and calculating the N2O reductase activity of the target strain.

[0043] During this process, the N2O concentration of the new type of myxobacterium A14 decreased from the initial 3044.31μmol·L-1 to 1942.39μmol·L-1, so the average rate of N2O consumption of myxobacterium A14 was 39.57μmol·L-1·h-1( Figure 3 ), demonstrating a strong ability to reduce NO. Control experiments showed that, over time, the NO concentration in the CK-N2O group and NO production in the CK-He group increased due to experimental error. Furthermore, the NO reductase activity of Myxobacterium myxogenum A14 was calculated to be 8.304 U / g (Table 2), indicating that the novel Myxobacterium myxogenum A14 strain has a high NO reduction capacity.

[0044] Table 2 Determination of N2O reductase activity of novel myxobacterium A14

[0045]

[0046] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

[0047] SEQ ID NO.1 (16S rRNA sequence of Bacillus myxobacterium A14)

[0048]

Claims

1. A strain of Mucilaginibacter sp. A14, characterized in that: The deposit number is: GDMCC No:66255.

2. Use of the myxobacterium A14 according to claim 1 in increasing the N2O reduction rate and N2O reductase activity and reducing soil N2O emissions.

3. The use according to claim 2, characterized in that The biological agent is a microbial agent or an organic biological fertilizer.

4. A biological agent, characterized in that The active ingredient is the myxobacterium A14 or its fermentation liquid according to claim 3.

5. The biological preparation according to claim 4, characterized in that The biological preparation is a liquid preparation, a powder or a solid preparation.

6. Use of the bacterial agent according to claim 2 in increasing the N2O reduction rate and reducing soil N2O emissions.

Images