N2O-reducing nosZ type II jiulongjiangibacter a17 and application thereof

Abstract

The application discloses a nosZ type Jiulongibacter A17 with N2O reduction capacity and application thereof. The Jiulongibacter A17 has a preservation number of GDMCC No:66268 and is preserved in the Guangdong Microbial Culture Collection Center on May 7, 2025. The strain is a new species of the Jiulongibacter genus. The novel Jiulongibacter A17 has strong N2O reductase activity and N2O reduction capacity. Therefore, the novel Jiulongibacter A17 has good application potential in reducing N2O gas emission.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of agricultural microorganism technology, and particularly relates to a Jiulongjiang bacillus A17 with N2O reduction capacity and application thereof. BACKGROUND

[0002] N2O is an important greenhouse gas, and its global warming potential is 298 times that of CO2. It remains in the atmosphere for 120 years and is eventually decomposed by ultraviolet light. In the past decade, N2O has increased at a rate of about 0.25% per year, and is currently increasing to about 17TgN yr -1 . Agricultural soil is the main anthropogenic source of N2O, contributing more than 60% of global emissions. Excessive emissions of N2O not only exacerbate climate change, but also damage the stratospheric ozone layer. Soil microorganisms play a key role in regulating nitrogen transformation and N2O emission processes, and nitrification and denitrification are the two main pathways for N2O generation and consumption. The denitrification process is a key process for the generation and consumption of N2O, involving the stepwise reduction of NO3 - to NO2 - , NO, N2O, and finally to N2. Denitrification can act as both a source and a sink of N2O, depending on the state of the denitrifying microorganisms and environmental conditions. The discovery of the nosZ gene has enabled people to understand the only biological removal pathway of N2O, in which nosZ reduces N2O to N2 through N2O reductase catalysis. 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 type of special-function microorganism, N2O respiring microorganism, has been discovered. This type of microorganism can survive in an environment where N2O is the only electron acceptor under anaerobic conditions, reducing N2O to N2 and reducing N2O emissions. This type of microorganism is divided into two types: nosZ I type microorganism and nosZ II type microorganism. The early discovered N2O respiring microorganism belongs to non-denitrifying bacteria, which is different from the traditional nosZ I type microorganism. They have nosZ II, which is another branch of the nosZ gene. Most N2O respiring bacteria belong to nosZ II type bacteria, but some nosZ I type bacteria can also respirate with N2O. Current research has limited understanding of the mechanism of N2O respiring reduction of nosZ II type microorganisms.

[0004] Jiulongibacter is a genus of gram-negative bacteria belonging to the phylum Bacteroidetes, widely distributed in soil, aquatic environments, sediments, and plant rhizospheres. Jiulongibacter has several effects on plants: it can produce plant growth hormones such as indole acetic acid (IAA), promote the growth of plant roots and stems and leaves, fix nitrogen, dissolve phosphorus, or promote iron uptake, thereby improving plant nutrient utilization efficiency, and improve soil physical structure, soil aeration, and water retention capacity by decomposing organic matter. In summary, Jiulongibacter, as a beneficial microorganism for plants, plays an important role in plant growth and soil health. Riching its microbial resources, identifying and exploiting its functions, and applying it to prepare microbial fertilizers and microbial inoculants are effective strategies to achieve sustainable and green agriculture. Future research can further explore the specific mechanisms and application potential of these bacteria to play a greater role in agricultural production and plant protection. SUMMARY

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

[0006] The second object of the present application is to provide the application of Jiulongibacter sp. A17 in reducing N2O.

[0007] Preferably, the application of Jiulongibacter sp. A17 in reducing soil N2O emission.

[0008] Preferably, the application of Jiulongibacter sp. A17 in improving N2O reduction rate and N2O reductase activity.

[0009] The third object of the present application is to provide a microbial inoculant comprising Jiulongibacter sp. A17 with the preservation number of GDMCC No:66268.

[0010] The fourth object of the present application is to provide a biological agent with Jiulongibacter sp. A17 or its fermentation broth as active ingredients.

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

[0012] The fifth object of the present application is to provide the application of Jiulongibacter sp. A17 or the microbial inoculant in reducing N2O emission.

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

[0014] The application utilizes enrichment separation technology to screen denitrifying bacteria with high denitrification rate and strong N2O reduction capacity from soil.

[0015] The application has the following advantages and effects relative to the prior art:

[0016] The application first discovers Jiulongibacter sp. A17, which is a potential new species of Jiulongibacter.

[0017] The application first discovers that Jiulongibacter has strong N2O reductase activity, which enriches the functional research of Jiulongibacter.

[0018] The application discovers that Jiulongibacter A17 has strong reductase activity and N2O reduction capacity, and has a dual role of reducing N2O emission. Therefore, Jiulongibacter A17 has great development potential as beneficial microorganisms in emission reduction, and opens up a new field for the preparation of microbial agents and organic biological fertilizers.

[0019] Jiulongibacter sp. A17 was preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on May 7, 2025, and the address is No. 59 Building, 5th Floor, Guangzhou, Guangdong, China, 510070, and the preservation number is GDMCC No: 66268. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The colony morphology of Jiulongibacter A17 on R2A medium.

[0021] Figure 2 The N2O reduction rate of Jiulongibacter A17. DETAILED DESCRIPTION

[0022] The technical solutions in the application will be described clearly and completely in combination with the embodiments of the application, but the embodiments do not limit the application in any form. Unless otherwise specified, the reagents, methods and devices used in the application are conventional reagents, methods and devices in the technical field.

[0023] Example 1: enrichment culture and separation of Jiulongibacter A17

[0024] Enrichment of highly efficient denitrifying strains was achieved using 100 mL vials, with 36 mL of sterile water (or DM-Nfree liquid medium) and 4 g of Bahia grass rhizosphere soil (taken from the pomelo planting demonstration base in Zhangbei Village, Xihe Town, Dapu County, Meizhou City, Guangdong Province) added. The headspace of the vials was purged with helium, followed by purging with a 10% N2O and 5% O2 ratio. The vials were then incubated at 30℃ and 150 rpm for 7 days. During the incubation period, a 10% N2O and 5% O2 mixture was added to the vials every two days until the initial pressure was reached, for a total of four rounds of enrichment.

[0025] Samples were taken from the fourth-generation enrichment solution, and 100 μL of each gradient bacterial suspension was added sequentially to a 2 mL centrifuge tube containing 900 μL of sterile water, thus diluting to 10⁻⁶. -4 10 -5 and 10 -6 Take 10g of bacterial suspension. -3 10 -4 10 -5 and 10 -6 100 μL of each culture was spread onto R2A medium (Guangdong Huankai, catalog number 022029) and TSB medium (Guangdong Huankai, catalog number 024051), with three culture dishes for each gradient. After sealing the culture dishes, they were incubated upside down in a 28°C biochemical incubator for 5 days. Colonies with different morphological characteristics were picked and purified by streak plating multiple times until pure cultures were obtained, yielding *Jiulongjiang* A17.

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

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

[0028] DNA of Jiulongibacter A17 was extracted by alkaline lysis method: a small amount of bacterial cells were placed in a 200 μL centrifuge tube containing 16.6 μL alkaline lysis solution, and the bacterial cells were lysed in an alkaline environment at high temperature. The program was set to 95°C for 30 min. After cooling, 16.6 μL neutralization buffer was added to each well, mixed well, and stored in a -20°C refrigerator. Alkaline lysis solution: 25 mM NaOH and 0.2 mM Na2-EDTA (pH = 12). Neutralization buffer: 40 mM Tris-HCl (pH = 7.5). 121°C high pressure sterilization for 15 minutes, which can be stored at 4°C for 2 months. The 16S rRNA gene sequence of the new Jiulongibacter A17 was amplified using bacterial 16S rRNA specific primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGACTTAACCCCAATCGC-3'). The amplified product was about 1500 bp, which was sent to Beijing Qikexin Biological Technology Co., Ltd. for sequencing. The sequence obtained by sequencing was spliced by DNAMAN software to obtain the 16S rRNA sequence (SEQ ID NO. 1) with a length of 1371 bp. The 16S rRNA gene sequence of Jiulongibacter A17 was submitted to EzBio Cloud database (www.ezbiocloud.net) for sequence homology comparison. The comparison results showed that the similarity of the 16S rRNA gene sequence of Jiulongibacter A17 and the model strain Jiulongibacter sediminis JN14-9(T) (accession number, LGTQ00000000) was the highest, which was 92.26%.

[0029] Example 3: Whole genome sequence analysis of Jiulongibacter A17

[0030] The new Jiulongibacter A17 bacterial solution was sent to Shanghai Meiji Biological Medicine Technology Co., Ltd. for genome sequencing. The software QUAST v5.0.2 was used to analyze the genome of the strain Jiulongibacter A17. The results showed that the new Jiulongibacter A17 genome sequence contains 43 contigs, the total length of the genome is 6413017 bp, the length of N50 is 612124 bp, and the G+C content of the genomic DNA is 44.76%. The similarity value of the new Jiulongibacter A17 is significantly lower than the 95% threshold commonly used to describe potential new genera. The results show that Jiulongibacter A17 is most closely related to Jiulongibacter sediminis ERR2094171 and Jiulongibacter sp. Ww143, with ANI values of 67.70% and 67.84%, respectively, which are lower than the critical value of 95-96% for species division, and dDDH values are 12.70%, respectively, which are much lower than the 70% species division threshold (Table 1). The results of ANI and dDDH support that the strain Jiulongibacter A17 is a new species of Jiulongibacter genus. Therefore, it is named Jiulongibacter sp. A17. The strain has been preserved in Guangdong Microbial Culture Collection Center (GDMCC), address: No. 59, Building 5, 100, Xianlie Middle Road, Guangzhou, Guangdong Province, China, postcode: 510070, preservation number: GDMCC No: 66268, preservation date: May 7, 2025.

[0031] Table 1 is the ANI and dDDH values of the new Jiulongibacter A17 and its close relative strains

[0032]

[0033] Example 4: Analysis of N2O reduction capacity and N2O reductase activity of new Jiulongibacter A17

[0034] Preparation of Jiulongibacter A17 bacterial solution:

[0035] Jiulongibacter A17 was activated and cultured on R2A plates at 28°C for 48h, and single colonies were picked with a toothpick and inoculated into R2A liquid medium. The culture was incubated at 28°C, 200rpm for about 48h to prepare the bacterial solution.

[0036] Determination of N2O reduction capacity of new Jiulongibacter A17:

[0037] The serum bottle with a volume of 50 mL is added with 25 mL of sterilized DM-N free medium, covered with a rubber plug and an aluminum cap, and the bottle cap is pressed with a presser. The serum bottle is injected with mixed bacterial suspension by using a syringe. The headspace gas in the serum bottle is replaced by helium, and then 2.5 mL of N2O gas (purity about 99.9%) is added by using a syringe. Three treatments are set in the experiment, i.e. N2O and bacteria (N2O-bacteria), N2O (control), He and bacteria (control, He-bacteria), and each treatment has three replicates. The samples are placed in a constant temperature incubator at 30°C and 150 rpm. During the incubation, the gas samples are collected from the headspace of the serum bottle by using a syringe, and the sampling time series are 0, 4, 8, 12, 16, 20, and 24 h. The N2O concentration is determined by using a gas chromatograph, and the N2O reduction rate of the bacterial strain is calculated according to the concentration change.

[0038] The DM-N free medium is as follows: 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, and the solvent is water.

[0039] The above trace element mixture is as follows: EDTA 50 g / L; ZnSO4 2.2 g / L; CaCl2 5.5 g / L; MnCl2·4H2O 5.06 g / L; FeSO4·7H2O 5 g / L; (NH4)6Mo7O2·4H2O 1.1 g / L; CuSO4·5H2O 1.57 g / L; CoCl2·6H2O 1.61 g / L, and the solvent is water.

[0040] Preparation of bacteria of Jiulongjiangbacterium A17: The prepared Jiulongjiangbacterium A17 bacterial liquid is centrifuged at a speed of 8000 rpm for 2 min, the precipitate is taken and resuspended with DM-N free medium, and then centrifuged at a speed of 8000 rpm for 1 min again, the supernatant is discarded, and the above steps are repeated three times to obtain Jiulongjiangbacterium A17.

[0041] Determination of N2O reductase activity of novel Jiulongjiangbacterium A17: The nitrous oxide reductase (Nos) level in the sample is determined by using a double antibody sandwich method. The specific operation is as follows: the prepared Jiulongjiangbacterium A17 bacterial liquid is transferred to a 2 mL centrifuge tube, the supernatant is discarded after centrifugation, and the bacterial number (not less than 10 5The extraction solution was added to the enzyme-labeled coating plate according to the proportion of 500-1000:1 of the PBS volume of the extraction solution. Then, the ultrasonic crushing operation of the bacteria was performed (under the condition of ice bath, the power was set to 20% or 200W, ultrasonic crushing was performed for 3s, the interval was 10s, and the operation was repeated for 3 times). After the ultrasonic crushing was completed, the supernatant was obtained by centrifugation at 8000xg and 4℃ for 10min. 10ul of the sample was taken and added to the enzyme-labeled coating plate, 40ul of the sample diluent was added, and the two were fully mixed by gently shaking, the plate was sealed with a sealing film, and then was incubated at 37℃ for 30min. After the incubation was completed, the enzyme-labeled coating plate was washed with the washing solution for 5 times. Then, the enzyme-labeled reagent was added, and the incubation and washing operations were performed again. Finally, the color developing agent and the termination agent were added, the blank hole (without the sample and the enzyme-labeled reagent) was used as a control, and the OD 450 absorbance value of the sample was measured by using an enzyme-labeled instrument. The standard sample 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 adding and other operations are the same as those of the sample determination operation, the OD 450 absorbance value of each dilution concentration standard sample is measured by using an enzyme-labeled instrument, the data is counted, a standard curve is drawn, and the N2O reductase activity of the target strain is calculated.

[0042] In this process, the N2O concentration of the novel Jiulongjiangbacterium A17 decreased from 3315.38umol / L to 2140.36umol / L, so the average rate of N2O consumption of the novel Jiulongjiangbacterium A17 was 18.22umol / L / h Figure 2 ), which showed a strong ability to reduce N2O. The experimental results of the control group showed that the N2O concentration of the CK-N2O group and the N2O production in the CK-He group increased with time due to experimental errors. The N2O reductase activity of the novel Jiulongjiangbacterium A17 was 7.228U / g (Table 2), which indicated that the novel Jiulongjiangbacterium A17 was a strain with high N2O reduction ability.

[0043] Table 2 N2O reductase activity determination of the novel Jiulongjiangbacterium A17

[0044]

[0045] The above is only a preferred embodiment of the present application, and it should be pointed out that the above preferred embodiment should not be regarded as a limitation of the present application, and the protection scope of the present application should be limited by the scope defined in the claims. For ordinary skilled persons in the art, several improvements and refinements can be made without departing from the spirit and scope of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.

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

[0047]

Claims

1. A strain of *Jiulongjiang* bacillus ( Jiulongibacter sp.) A17, characterized in that, The preservation number is: GDMCC No: 66268.

2. A biological agent, characterized in that, The nine long river bacillus A17 or its fermentation liquor in claim 1 is used as the active ingredient.

3. The biological preparation of claim 2, wherein, The biological preparation is a liquid preparation or a solid preparation.

4. The biological preparation of claim 3, wherein, The solid preparation is a powder.

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