NosZ II-type terranobacterium A33 with N2O reducing capacity and application of nosZ II-type terranobacterium A33

Abstract

The invention discloses a nosZ II type terranean bacillus A33 with N2O reducing capacity and application of the nosZ II type terranean bacillus A33. The preservation number of the Pedobacter sp. A33 is GDMCC No: 66269, and the Pedobacter sp. A33 is preserved in the Guangdong Microbial Culture Collection Center on May 7, 2025, and the preservation number of the Pedobacter sp. A33 is GDMCC No: 66269. The bacterial strain is a new species of terribacter. The novel ternobacter sp. A33 disclosed by the invention has relatively strong N2O reductase activity and also has N2O reducing capacity. Therefore, the novel ternobacter sp. A33 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 nosZ II type Agrobacterium A33 with N2O reduction ability and an application 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] Landobacter is a genus of Gram-negative bacteria belonging to the phylum Bacteroidetes, widely distributed in soil, water, and the rhizosphere of plants. Landobacter has several beneficial effects on plants: it degrades organic matter in the soil, promoting its mineralization, thereby releasing nutrients that are usable by plants and promoting plant growth. Some Landobacter species produce plant growth hormones, such as indoleacetic acid (IAA), which promote the growth and development of plant roots. Some Landobacter species have the ability to fix nitrogen, converting atmospheric nitrogen into plant-usable nitrogen, thereby improving soil fertility. Landobacter species inhibit plant pathogens. Their metabolic activities in the soil improve the soil's physical structure, increasing aeration and water retention, thereby creating a more suitable growth environment for plant roots. In summary, Landobacter, as a plant-beneficial microorganism, plays an important role in plant growth and soil health. Enriching its microbial resources, identifying and exploring its functions, and applying it to prepare microbial fertilizers and microbial agents are effective strategies for achieving sustainable and green agriculture. Future research can further explore the specific mechanisms of action and application potential of these bacteria, in order to play a greater role in agricultural production and plant protection. Summary of the Invention

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

[0006] The second object of the present invention is to provide the use of the above-mentioned Land Bacterium A33 in reducing N2O.

[0007] Preferably, it is the use of Agribacterium tumefaciens A33 in reducing soil N2O emissions.

[0008] Preferably, it is the use of A. terrestris A33 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 Landbacterium A33 with the deposit number GDMCC No: 66269.

[0010] The fourth object of the present invention is to provide a biological preparation, which uses the above-mentioned Land Bacillus A33 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 cultivating the above-mentioned Land Bacillus A33 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 Pedobacter sp. A33 for the first time, which is a potential new species among myxobacteria.

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

[0018] The present invention discovered that Geobacter A33 possesses both strong reductase activity and N2O reduction capability, thus achieving the dual purpose of reducing N2O emissions. Therefore, Geobacter A33, as a beneficial microorganism, has great potential for emission reduction and opens new horizons for the preparation of microbial agents and organic biofertilizers.

[0019] Pedobacter sp.A33 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, with the deposit number: GDMCC No: 66269. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the colony morphology of A. aestivum A33 on R2A medium.

[0021] Figure 2 This is the phylogenetic tree of A. aestivum A33.

[0022] Figure 3 is the N2O reduction rate of A. 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 Terreobacter A33

[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 Spread 100 μL of each onto R2A medium (Guangdong Huankai, Catalog No. 022029) and TSB medium (Guangdong Huankai, Catalog No. 024051), with three culture dishes spread per gradient. Seal the culture dishes and incubate them upside down in a 28°C biochemical incubator for 5 days. Select colonies with distinct morphological characteristics and purify by multiple streaking until a pure culture, i.e., Terreobacter A33, is obtained.

[0027] The colony morphology of A. truncatum A33 after culturing on R2A medium for 3 days is as follows: Figure 1 As shown, the single colony is round, convex, about 1 mm in diameter, yellow, with a smooth, opaque surface and neat edges.

[0028] Example 2: 16S rRNA gene sequence analysis of Land Bacterium A33

[0029] Extract DNA from Terreobacter A33 using the alkaline lysis method: Place a small amount of bacteria into a 200 μL centrifuge tube with 16.6 μL of alkaline lysis buffer. Lyse the bacteria in an alkaline environment at 95°C for 30 minutes. After cooling, add 16.6 μL of neutralization buffer to each well, mix thoroughly, and store 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. Store at 4°C for 2 months. The 16S rRNA gene sequence of the novel A. tumefaciens strain A33 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 resulting sequence was assembled using DNAMAN software to yield a 1315-bp 16S rRNA sequence (SEQ ID NO. 1). The 16S rRNA gene sequence of A. tumefaciens A33 was submitted to the EzBioCloud database (www.ezbiocloud.net) for sequence homology comparison. The comparison revealed that A. tumefaciens A33 shared the highest similarity with the 16S rRNA gene sequence of the model strain Pedobacter seoulensis THG-G12 (accession number, KF150693), at 98.02%.

[0030] Example 3: Analysis of the complete gene sequence of A.

[0031] The bacterial solution of the new strain of Land Bacterium A33 was sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for genome sequencing. The genome of the strain Land Bacterium A33 was analyzed using the software QUASTv5.0.2. The results showed that the genome sequence of the new strain of Land Bacterium A33 contained 83 contigs, with a total genome length of 10558842bp, an N50 length of 655985bp, and a genomic DNA G+C content of 54.64%. The genomic phylogenetic tree of the new strain of Land Bacterium A33 and closely related reference strains was constructed using the software UBCGv3.0. The results are shown in Figure 2. Figure 2As shown, Pedobacter sp. A33 is most closely related to Pedobacter himalayensis, Pedobacter faecalis, and Pedobacter deserti. Furthermore, compared with these three closest relatives, Pedobacter sp. A33 exhibited ANI values ​​of 64.88-71.74%, below the proposed species delimitation threshold of 95-96%, and dDDH values ​​of 12.50-13.50%, well below the 70% threshold for species delimitation (Table 1). Both ANI and dDDH values ​​support the identification of Pedobacter sp. A33 as a new species of the genus Pedobacter. Therefore, it was designated Pedobacter sp. A33. This strain has been deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, Postal Code: 510070, the deposit number is GDMCC No: 66269, and the deposit date is May 7, 2025.

[0032] Table 1 shows the ANI and dDDH values ​​of the new type of Land Bacterium A33 and its closely related strains

[0033]

[0034] Example 4: Analysis of N2O Reduction Capacity and N2O Reductase Activity of the New Type of Land Bacterium A33

[0035] Preparation of Terroir A33 bacterial solution:

[0036] Land bacillus A33 was activated and cultured on an R2A plate at 28°C for 48 hours, a single colony was picked with a toothpick, inoculated into TSB 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 the new strain of Land Bacterium A33:

[0038] 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.

[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 Agrobacterium A33 cells: The prepared Agrobacterium A33 bacterial solution 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 obtain Agrobacterium A33 cells.

[0042] Determination of N2O reductase activity of the new type of Land Bacterium A33: 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 Land Bacterium A33 bacterial solution, transfer it to a 2mL centrifuge tube, centrifuge and discard the supernatant, and then count 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 soil bacterium A33 decreased from the initial 3125.28μmol·L-1 to 12692.78μmol·L-1, so the average rate of N2O consumption of the soil bacterium A33 was 31.04μmol·L-1·h-1( Figure 3 ), demonstrating strong NO reduction capabilities. 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 A. aestivum A33 was calculated to be 8.261 U / g (Table 2), demonstrating that the novel A. aestivum A33 strain possesses a high NO reduction capacity.

[0044] Table 2 Determination of N2O reductase activity of the new type of Land Bacterium A33

[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 A. aestivum A33)

[0048]

Claims

1. A Pedobacter sp. A33, characterized in that: The deposit number is: GDMCC No:66269.

2. Use of the Agrobacterium A33 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 soil bacterium A33 or its fermentation liquid as claimed in 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.

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