N2O-reducing nosZ type II Agrobacterium sp. A33 and application thereof

Abstract

The application discloses a Pedobacter sp. A33 with N2O reduction capability and an application thereof. The Pedobacter sp. A33 has a preservation number of GDMCC No:66269 and is preserved in the Guangdong Microbial Culture Collection Center on May 7, 2025. The strain is a new species of the Pedobacter genus. The novel Pedobacter sp. A33 has strong N2O reductase activity and N2O reduction capability. Therefore, the novel Pedobacter sp. A33 has good application potential in reducing N2O gas emission.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of agricultural microorganisms, and particularly relates to a nosZ type II Agreia bactobacteria A33 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. N2O 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 17 TgN 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 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 type of microorganism with special functions has been discovered—N2O respiring microorganisms. These microorganisms can survive in anoxic conditions with N2O as the only electron acceptor, reducing N2O to N2 and reducing N2O emissions. These microorganisms are divided into two types: nosZ I type microorganisms and nosZ II type microorganisms. The early discovered N2O respiring microorganisms belong to non-denitrifying bacteria, which are different from traditional nosZ I type microorganisms. They possess 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 respire with N2O. Current research has limited understanding of the N2O respiratory reduction mechanism of nosZ II type microorganisms.

[0004] Pedobacter is a genus of gram-negative bacteria belonging to the phylum Bacteroidetes, widely distributed in soil, water and rhizosphere of plants. Pedobacter has the following effects on plants: degrading organic matter in soil, promoting the mineralization of organic matter, thereby releasing plant available nutrients, promoting plant growth; some Pedobacter can produce plant growth hormones such as indole acetic acid (IAA), which can promote the growth and development of plant roots; some Pedobacter have nitrogen fixation ability, which can convert atmospheric nitrogen into plant available nitrogen, thereby improving soil fertility; inhibiting plant pathogenic bacteria, Pedobacter can improve the physical structure of soil, increase the aeration and water retention of soil, and thus create a more suitable growth environment for plant roots when it is active in soil. In summary, Pedobacter, as a kind of plant beneficial microorganism, plays an important role in plant growth and soil health, and enriching its microbial resources, identifying and exploiting its functions, and applying it to prepare microbial fertilizer and microbial inoculant are effective strategies to realize sustainable agriculture and green agriculture. Future research can further explore the specific mechanisms and application potential of these bacteria, in order to play a greater role in agricultural production and plant protection. SUMMARY

[0005] The first object of the present application is to provide a Pedobacter sp. A33 with N2O reduction ability, with the preservation number of GDMCC No:66269.

[0006] The second object of the present application is to provide the application of the above-mentioned Pedobacter sp. A33 in reducing N2O.

[0007] Preferably, the application of the Pedobacter sp. A33 in reducing soil N2O emission.

[0008] Preferably, the application of the Pedobacter sp. A33 in improving N2O reduction rate and N2O reductase activity

[0009] The third object of the present application is to provide a microbial inoculant comprising the above-mentioned Pedobacter sp. A33 with the preservation number of GDMCC No:66269.

[0010] The fourth object of the present application is to provide a biological preparation taking the above-mentioned Pedobacter sp. A33 or its fermentation broth as active ingredient.

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

[0012] The fifth object of the present application is to provide the application of the above-mentioned Pedobacter sp. A33 or the above-mentioned microbial inoculant in reducing N2O gas emission.

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

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

[0015] The application has the following advantages and effects compared with the prior art:

[0016] The application first discovers Pedobacter sp. A33, which is a potential new species in Pedobacter.

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

[0018] The application discovers that Pedobacter A33 has strong reductase activity and N2O reduction capacity, and has a dual effect of reducing N2O emission. Therefore, Pedobacter A33 has great development potential in emission reduction as a beneficial microorganism, and opens up a new field for the preparation of microbial agents and organic biological fertilizers.

[0019] Pedobacter sp. A33 was preserved in Guangdong Microbial Culture Collection Center (GDMCC) on May 7, 2025, and the address is No. 59 Building, 5th Floor, Guangzhou, Guangdong, China, and the postcode is 510070, and the preservation number is GDMCC No:66269. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is the colony morphology of Pedobacter A33 on R2A medium.

[0021] Figure 2 It is the phylogenetic tree of the Pedobacter A33 genome.

[0022] Figure 3 It is the N2O reduction rate of Pedobacter A33. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the application will be described below, 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.

[0024] Example 1: enrichment culture and separation of Pedobacter A33

[0025] 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.

[0026] 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 until a pure culture of *Gastrobacterium* A33 was obtained.

[0027] Colony morphology of *Geobacterium tumefaciens* A33 after 3 days of culture on R2A medium is as follows: Figure 1 As shown, a single colony is round, raised, about 1 mm in diameter, yellow, with a smooth, opaque surface and neat edges.

[0028] Example 2: 16S rRNA gene sequence analysis of *Geobacterium tumefaciens* A33

[0029] The DNA of Pedobacter A33 was extracted by alkaline lysis method: a small amount of bacterial cells was 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, with the program 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 Pedobacter A33 was amplified using bacterial 16S rRNA specific primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGACTTAACCCCAATCGC-3'), and the amplified product was about 1500 bp. The amplified product was sent to Beijing Qikexin Biotechnology 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 1315 bp. The 16S rRNA gene sequence of Pedobacter A33 was submitted to EzBioCloud database (www.ezbiocloud.net) for sequence homology comparison. The comparison results showed that the similarity of the 16S rRNA gene sequence of Pedobacter A33 and the model strain Pedobacter seoulensis THG-G12 (accession number KF150693) was the highest, which was 98.02%.

[0030] Example 3: Whole genome sequence analysis of Pedobacter A33

[0031] The new Pedobacter A33 bacterial solution was sent to Shanghai Meiji Biomedicine Technology Co., Ltd. for genome sequencing. The software QUAST v5.0.2 was used to analyze the genome of the strain Pedobacter A33. The results showed that the genome sequence of the new Pedobacter A33 contained 83 contigs, with a total genome length of 10558842 bp, an N50 length of 655985 bp, and a genomic DNA G+C content of 54.64%. The software UBCG v3.0 was used to construct a genome phylogenetic tree of the new Pedobacter A33 and the closely related reference model strains. The results are as follows: Figure 2As shown, Pedobacter sp. A33 is most closely related to Pedobacter himalayensis, Pedobacter faecalis and Pedobacter deserti. And compared with the three strains most closely related to Pedobacter sp. A33, the ANI value is 64.88-71.74%, lower than the critical value of 95-96% proposed for species division, and the dDDH value is between 12.50-13.50%, far lower than the 70% threshold for species division (Table 1). The results of ANI and dDDH both support that strain Pedobacter sp. A33 is a new species of Pedobacter genus. Therefore, it is named Pedobacter sp. A33. The strain has been preserved in Guangdong Microbial Culture Collection Center (GDMCC), address: 59, Building 5, 100, Martyrs' Road, Guangzhou, Guangdong, China, postcode: 510070, preservation number: GDMCC No:66269, preservation date: May 7, 2025.

[0032] Table 1 is the ANI and dDDH values of the new Pedobacter sp. A33 and its closely related strains

[0033]

[0034] Example 4: Analysis of N2O reduction capacity and N2O reductase activity of the new Pedobacter sp. A33

[0035] Preparation of Pedobacter sp. A33 bacterial solution:

[0036] Pedobacter sp. A33 was activated and cultured on R2A plates at 28°C for 48h, and single colonies were picked with a toothpick and inoculated into TSB liquid medium. The bacterial solution was prepared by incubating the inoculated TSB liquid medium at 28°C, 200rpm for about 48h.

[0037] Determination of N2O reduction capacity of the new Pedobacter sp. A33:

[0038] The serum bottle with a volume of 50 mL was added with 25 mL of sterilized DM-N free medium, covered with a rubber plug and an aluminum cap, and the bottle cap was pressed with a presser. The serum bottle was injected with mixed bacterial suspension by using a syringe. The headspace gas in the serum bottle was replaced by helium, and then 2.5 mL of N2O gas (purity about 99.9%) was added by using a syringe. Three treatments were set in the experiment, i.e. N2O and bacteria (N2O-bacteria), N2O (control), He and bacteria (control, He-bacteria), and each treatment had three replicates. The samples were placed in a constant temperature incubator at 30°C and 150 rpm. During the incubation, the gas samples were collected from the headspace of the serum bottle by using a syringe, and the sampling time series were 0, 4, 8, 12, 16, 20, and 24 h. The N2O concentration was determined by using a gas chromatograph, and the N2O reduction rate of the bacterial strain was calculated according to the concentration change.

[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 trace element mixture: 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, solvent is water.

[0041] Preparation of Agrobacterium tumefaciens A33 bacterial cells: the prepared Agrobacterium tumefaciens A33 bacterial liquid was centrifuged at a speed of 8000 rpm for 2 min, the precipitate was taken and resuspended with DM-N free medium, and then centrifuged at a speed of 8000 rpm for 1 min again, the supernatant was discarded, and the above operation was repeated three times to obtain the Agrobacterium tumefaciens A33 bacterial cells.

[0042] Determination of N2O reductase activity of the novel Agrobacterium tumefaciens A33: the nitrous oxide reductase (Nos) level in the sample was determined by using a double antibody sandwich method. The specific operation was as follows: the prepared Agrobacterium tumefaciens A33 bacterial liquid was transferred to a 2 mL centrifuge tube, the supernatant was discarded after centrifugation, and the bacterial number (not less than 10 5The extraction solution was added to the PBS solution in a ratio of 500-1000:1. Then, the bacteria were subjected to ultrasonic crushing (under ice bath conditions, the power was set to 20% or 200 W, ultrasonic crushing was performed for 3 s, the interval was 10 s, and the operation was repeated for 3 times). After the ultrasonic crushing was completed, the supernatant was obtained by centrifugation at 8000 x g and 4 ℃ for 10 min. 10 μL of the supernatant was added to the enzyme-labeled coating plate, 40 μL of the sample diluent was added, and the two were mixed well, the plate was sealed with a sealing film, and then incubated at 37 ℃ for 30 min. After the incubation, 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 was diluted, and the dilution gradient was 3 U / L, 6 U / L, 12 U / L, 24 U / L and 48 U / L. The subsequent sample adding and other operations were the same as those in the sample determination operation, the OD 450 absorbance value of each dilution concentration standard sample was measured by using an enzyme-labeled instrument, the data was counted, a standard curve was drawn, and the N2O reductase activity of the target strain was calculated.

[0043] From Figure 3 It can be seen that the new type of soil bacillus A33 has strong N2O reduction ability, and the experimental results of the control show that, as time goes on, due to experimental errors, the N2O concentration of the CK-N2O group and the N2O production in the CK-He group. Through calculation, the N2O reductase activity of the new type of soil bacillus A33 is 8.261 U / g (Table 2), which indicates that the new type of soil bacillus A33 is a strain with high N2O reduction ability.

[0044] Table 2 N2O reductase activity determination of the new type of soil bacillus A33

[0045]

[0046] 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.

[0047] SEQ ID NO. 1 (16S rRNA sequence of soil bacillus A33)

[0048]

Claims

1. A strain of *Germocactus* ( Pedobacter sp.)A33, characterized in that, The preservation number is GDMCC No:66269.

2. The use of Pedobacter A33 in claim 1 in reducing soil N2O emission.

3. A biological agent, characterized in that, Pedobacter A33 or a fermentation liquor thereof in claim 1 as an active ingredient.

4. The biological preparation of claim 3, wherein, The biological agent is a microbial inoculant or an organic biological fertilizer.

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

6. The biological preparation of claim 5, wherein, The solid preparation is a powder.

7. The use of the microbial inoculant in claim 4 in reducing soil N2O emission.

Images