NosZ II-type naulobacter spp. A17 with N2O reducing capacity and application of nosZ II-type naulobacter spp. A17

Abstract

The invention discloses nosZ II type naulobacter spp. A17 with N2O reducing capacity and application of the nosZ II type naulobacter spp. A17. The preservation number of the Jiulongibacter sp.A17 is GDMCC No: 66268, and the Jiulongibacter sp.A17 is preserved in the Guangdong Microbial Culture Collection Center on May 7, 2025, and the preservation number of the Jiulongibacter sp.A17 is CGMCC No: 66268. The bacterial strain is a new species of Jiangludwigia. The novel N2O reductase A17 disclosed by the invention has relatively strong N2O reductase activity and also has N2O reducing capacity. Therefore, the novel Jianlong River bacillus A17 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 Jiulongjiang Bacillus A17 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] Jiulongjiang Bacillus is a Gram-negative bacterial genus belonging to the phylum Bacteroidetes, widely distributed in soil, aquatic environments, sediments, and the rhizosphere of plants. Jiulongjiang Bacillus has several plant-related effects: it produces plant growth hormones, such as indoleacetic acid (IAA), which promotes root, stem, and leaf growth; it fixes nitrogen, solubilizes phosphorus, or promotes iron absorption, thereby improving plant nutrient utilization efficiency; and it decomposes organic matter, improving soil physical structure, aeration, and water retention. In summary, Jiulongjiang Bacillus, as a plant-beneficial microorganism, plays an important role in plant growth and soil health. Enriching these 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. Future research will further explore the specific mechanisms of action and potential applications of these bacteria, hoping to further enhance their role in agricultural production and plant protection. Summary of the Invention

[0005] The first object of the present invention is to provide a strain of Jiulongibacter sp. A17 having N2O reduction ability, with a deposit number of GDMCC No: 66268.

[0006] The second object of the present invention is to provide the use of the above-mentioned Jiulongjiang Bacillus A17 in reducing N2O.

[0007] Preferably, it is the use of Jiulongjiang Bacillus A17 in reducing soil N2O emissions.

[0008] Preferably, it is the use of Jiulongjiang Bacillus A17 in improving the N2O reduction rate and N2O reductase activity.

[0009] The third object of the present invention is to provide a microbial agent, which contains the Jiulongjiang Bacillus A17 with the above-mentioned deposit number GDMCC No: 66268.

[0010] The fourth object of the present invention is to provide a biological preparation, which uses the above-mentioned Jiulongjiang Bacillus A17 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 use of culturing the above-mentioned Jiulongjiang Bacillus A17 or the above-mentioned microbial agent 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 Jiulongibacter sp. A17 for the first time, which is a potential new species of Jiulongibacter sp.

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

[0018] The present invention discovered that Jiulongjiang Bacillus A17 possesses both strong reductase activity and N2O reduction capability, thus achieving the dual purpose of reducing N2O emissions. Therefore, Jiulongjiang Bacillus A17, 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] Jiulongibacter sp.A17 was deposited on May 7, 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, and the deposit number is: GDMCC No: 66268. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the colony morphology of Jiulongjiang Bacillus A17 on R2A medium.

[0021] Figure 2 is the N2O reduction rate of Jiulongjiang Bacillus A17. DETAILED DESCRIPTION

[0022] 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.

[0023] Example 1: Enrichment culture and isolation of Jiulongjiang Bacillus A17

[0024] 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.

[0025] 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 Jiulongjiang Bacillus A17.

[0026] The colony morphology of Jiulongjiang Bacillus A17 after culturing on R2A medium for 3 days is as follows Figure 1 As shown, the single colony is round, about 2 mm in diameter, orange-yellow in color, with a convex, smooth, opaque surface and neat edges.

[0027] Example 2: 16S rRNA gene sequence analysis of Jiulongjiang Bacillus A17

[0028] DNA from Jiulongjiang Bacillus A17 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 high temperature (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 strain A17 was amplified using bacterial 16S rRNA-specific primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGACTTAACCCC AATCGC-3′). Electrophoresis analysis revealed an approximately 1500-bp amplified product, which was then sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The resulting sequence was assembled using DNAMAN software to obtain a 1371-bp 16S rRNA sequence (SEQ ID NO. 1). The 16S rRNA gene sequence of A17 was submitted to the EzBio Cloud database (www.ezbiocloud.net) for sequence homology comparison. The comparison results showed that the 16S rRNA gene sequence of Jiulongibacter A17 and the model strain Jiulongibacter sediminis JN14-9(T) (accession number, LGTQ00000000) had the highest similarity of 92.26%.

[0029] Example 3: Full gene sequence analysis of Jiulongjiang Bacillus A17

[0030] The bacterial strain A17 was sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for genome sequencing. The genome of the strain A17 was analyzed using QUAST v5.0.2. The results showed that the genome sequence of the novel A17 strain consisted of 43 contigs, with a total genome length of 6,413,017 bp, an N50 length of 612,124 bp, and a G+C content of 44.76%. The similarity value of the novel A17 strain was significantly lower than the 95% threshold commonly used to delineate potential new genera. The results showed that A17 was most closely related to Jiulongibacter sediminis ERR2094171 and Jiulongibacter sp. Ww143, with ANI values of 67.70% and 67.84%, respectively, below the 95-96% threshold for species delimitation. The dDDH values were both 12.70%, well below the 70% threshold for species delimitation (Table 1). Both ANI and dDDH results support that strain Jiulongibacter sp. A17 is a new species of the genus Jiulongibacter. Therefore, it was named Jiulongibacter sp. A17. This strain has been deposited with the Guangdong Provincial Microbiological Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, China, 510070, under the GDMCC No. 66268, and on May 7, 2025.

[0031] Table 1 shows the ANI and dDDH values of the novel Jiulongjiang Bacillus A17 and its closely related strains

[0032]

[0033] Example 4: Analysis of N2O Reduction Capacity and N2O Reductase Activity of the Novel Jiulongjiang Bacillus A17

[0034] Preparation of Jiulongjiang Bacillus A17 bacterial solution:

[0035] Jiulongjiang Bacillus A17 was activated and cultured on an R2A plate at 28°C for 48 h, 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 h to prepare a bacterial solution.

[0036] Determination of N2O reduction ability of novel Jiulongjiang Bacillus A17:

[0037] 50 mL serum bottles were filled with 25 mL of sterile DM-Nfree medium. The bottles were capped with rubber stoppers and aluminum caps, and the caps were tightened securely with a capping tool. The mixed bacterial suspension was injected into the serum bottles to be added with a syringe. The headspace gas in the serum bottles 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 bottles 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 changes in concentration.

[0038] 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.

[0039] 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.

[0040] Preparation of Jiulongjiang Bacillus A17 cells: The prepared Jiulongjiang Bacillus A17 bacterial liquid was centrifuged at 8000 rpm for 2 min, the precipitate was collected and resuspended in DM-N free medium, and then centrifuged again at 8000 rpm for 1 min. The supernatant was discarded, and the process was repeated three times to finally obtain Jiulongjiang Bacillus A17.

[0041] Determination of N2O reductase activity of the novel Jiulongjiang Bacillus A17: 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 Jiulongjiang Bacillus A17 bacterial solution, transfer it to a 2mL centrifuge tube, centrifuge and discard the supernatant, and then add 100 ml of the solution according to 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.

[0042] During this process, the N2O concentration of the novel Jiulongjiang Bacillus A17 decreased from the initial 3315.38 μmol·L-1 to 2140.36 μmol·L-1, so the average rate of N2O consumption of Jiulongjiang Bacillus A17 was 18.22 μmol·L-1·h-1( Figure 2 ), demonstrating strong NO reduction capacity. Control experiments showed that, over time, NO concentrations in the CK-N2O group and NO production in the CK-He group increased due to experimental error. Furthermore, the NO reductase activity of Jiulongjiang Bacillus A17 was calculated to be 7.228 U / g (Table 2), indicating that the novel Jiulongjiang Bacillus A17 strain has a high NO reduction capacity.

[0043] Table 2 Determination of N2O reductase activity of novel Jiulongjiang Bacillus A17

[0044]

[0045] 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.

[0046] SEQ ID NO.1 (16S rRNA sequence of Jiulongjiang Bacillus A17)

[0047]

Claims

1. A strain of Jiulongibacter sp. A17, characterized in that: The deposit number is: GDMCC No:66268.

2. Use of the Jiulongjiang Bacillus A17 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 Jiulongjiang Bacillus A17 or its fermentation liquid described in claim 3 is used as the active ingredient.

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.

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