Mucilaginibacter sp. A14 with N2O reduction ability and application thereof

Abstract

The application discloses a nosZ type Myxococcus A14 with N2O reduction capacity and application thereof, the preservation number of the strain is GDMCC No:66255, and the strain is preserved in the Guangdong Microbial Culture Collection Center on April 30, 2025. The strain is a new species of the Myxococcus genus. The new Myxococcus A14 has strong N2O reductase activity and N2O reduction capacity. Therefore, the new Myxococcus A14 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 myxobacterium A14 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 for 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 respire with N2O. Current research has limited understanding of the mechanism of N2O respiratory reduction of nosZ II type microorganisms.

[0004] Mucilaginibacter is a genus of gram-negative bacteria belonging to the phylum Bacteroidetes, which is widely distributed in soil, aquatic environment, sediment and plant rhizosphere. Mucilaginibacter has different application modes due to its many characteristics. Some strains have pectinolytic ability, some strains have plant growth promoting ability, and most strains can produce extracellular polymers, which have great potential value. As a kind of plant beneficial microorganism, enriching its microbial resources, identifying and exploring its function, and applying it to prepare microbial fertilizer and microbial inoculant are effective strategies to realize sustainable agriculture and green agriculture. SUMMARY

[0005] The first object of the present application is to provide a Mucilaginibacter sp. A14 with N2O reduction ability, the preservation number of which is GDMCC No:66255.

[0006] The second object of the present application is to provide the application of the above-mentioned Mucilaginibacter A14 in reducing N2O.

[0007] Preferably, the application of the Mucilaginibacter A14 in reducing soil N2O emission.

[0008] Preferably, the application of the Mucilaginibacter A14 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 Mucilaginibacter A14 with the preservation number of GDMCC No:66255.

[0010] The fourth object of the present application is to provide a biological preparation taking the above-mentioned Mucilaginibacter A14 or its fermentation liquor as an 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 Mucilaginibacter A14 or the above-mentioned microbial inoculant in reducing N2O gas emission.

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

[0014] The present application uses enrichment separation technology to screen denitrifying bacteria with high denitrification rate and strong N2O reduction ability from soil. By inoculating the strain into soil, the emission of N2O and other greenhouse gases can be reduced.

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

[0016] The present application first discovers Mucilaginibacter sp. A14, which is a potential new species in Mucilaginibacter.

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

[0018] The present application discovers that Mucilaginibacter A14 has strong reductase activity and N2O reduction capacity, which has a dual role in reducing N2O emissions. Therefore, Mucilaginibacter A14 as a beneficial microorganism has great development potential in emission reduction, and also opens up a new field for the preparation of microbial inoculants and organic biological fertilizers.

[0019] Mucilaginibacter sp. A14 was preserved in Guangdong Microbial Culture Collection Center (GDMCC) on April 30, 2025, at address of No. 59 Building, 5th Floor, Guangzhou Institute of Military Personnel, Guangzhou, Guangdong Province, China, postcode: 510070, preservation number: GDMCC No: 66255. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The figure is the colony morphology of Mucilaginibacter A14 on R2A medium.

[0021] Figure 2 The figure is the phylogenetic tree of the genome of Mucilaginibacter A14.

[0022] Figure 3 The figure is the N2O reduction rate of Mucilaginibacter A14. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present application will be described below, but the embodiments do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0024] Example 1: enrichment culture and separation of Mucilaginibacter A14

[0025] The enrichment of high-efficiency denitrifying strains uses a 100mL vial, adds 36mL sterile water (or DM-Nfree liquid medium) and 4g of centipeda minima rhizosphere soil (taken from the Mei City Dabu County Xihexian Zhangbei Village Honey Orange Planting Demonstration Base in Guangdong Province), replaces the headspace of the vial with helium, and then replaces the helium with 10% N2O and 5% O2. The vial is placed in a constant temperature incubator at 30℃ and 150rpm for 7d of enrichment culture. During the culture, 10% N2O and 5% O2 mixed gas is supplemented to the vial every 2 days to the initial pressure, and a total of 4 rounds of enrichment are performed.

[0026] From the fourth generation of enrichment liquid, 100 μL of each gradient bacterial suspension was taken and added to a 2 mL centrifuge tube containing 900 μL of sterile water, i.e. diluted to 10 -4 , 10 -5 and 10 -6 . 100 μL of each bacterial suspension was taken and spread on R2A medium (Guangdong Huan Kai, item number 022029) and TSB medium (Guangdong Huan Kai, item number 024051), three Petri dishes per gradient. After sealing the Petri dishes, they were incubated at 28°C for 5 days in an inverted position in a biochemical incubator. Colonies with different morphological characteristics were picked and purified by multiple streaks until pure cultures were obtained. The Myxobacterium A14 was obtained. -3 , 10 -4 , 10 -5 and 10 -6 . 100 μL of each bacterial suspension was taken and spread on R2A medium (Guangdong Huan Kai, item number 022029) and TSB medium (Guangdong Huan Kai, item number 024051), three Petri dishes per gradient. After sealing the Petri dishes, they were incubated at 28°C for 5 days in an inverted position in a biochemical incubator. Colonies with different morphological characteristics were picked and purified by multiple streaks until pure cultures were obtained. The Myxobacterium A14 was obtained.

[0027] The colony morphology of Myxobacterium A14 on R2A medium after 3 days of incubation is shown in Figure 1. The single colonies were round, 1-3 mm in diameter, yellow, smooth on the surface, opaque, and the edges were neat. Figure 1

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

[0029] ​DNA of Mucilaginibacter A14 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 Mucilaginibacter A14 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 1391 bp. The 16S rRNA gene sequence of Mucilaginibacter A14 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 Mucilaginibacter A14 and the model strain Mucilaginibacter ximonensis XM-003 (accession number, JBHUPD000000000) was the highest, which was 98.49%.

[0030] Example 3: Whole genome sequence analysis of Mucilaginibacter A14

[0031] The new Mucilaginibacter A14 bacterial solution was sent to Shanghai Meiji Biomedicine Technology Co., Ltd. for genome sequencing. Software QUAST v5.0.2 was used to analyze the genome of strain Mucilaginibacter A14. The results showed that the genome sequence of the new Mucilaginibacter A14 contained 40 contigs, the total length of the genome was 4199463 bp, the length of N50 was 462240 bp, and the G+C content of the genomic DNA was 43.78%. Software UBCG v3.0 was used to construct the genome phylogenetic tree of the new Mucilaginibacter A14 and the closely related reference model strain. The results are as follows: Figure 2As shown, Mucilaginibacter A14 is most closely related to Mucilaginibacter ximonensis and Mucilaginibacter panaciglaebae, with ANI values of 81.02% and 79.60%, respectively, which are lower than the critical value of 95-96% proposed for species delimitation, and dDDH values of 31.60% and 27.30%, respectively, which are much lower than the species division threshold of 70% (Table 1). The results of ANI and dDDH both support that strain Mucilaginibacter A14 is a new species of the genus Mucilaginibacter. Therefore, it is named Mucilaginibacter sp. A14. The strain has been preserved in Guangdong Microbial Culture Collection Center (GDMCC), address: No. 59, Building 5, 100, Martyrs' Road, Guangzhou, Guangdong, China, postcode: 510070, preservation number: GDMCC No: 66255, and preservation date: April 30, 2025.

[0032] Table 1 is the ANI and dDDH values of the new Mucilaginibacter A14 and its close relative strains

[0033]

[0034] Example 4: Analysis of N2O reduction capacity and N2O reductase activity of the new Mucilaginibacter A14

[0035] Preparation of Mucilaginibacter A14 bacterial solution:

[0036] Mucilaginibacter A14 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, which was incubated at 28°C, 200rpm for about 48h to prepare the bacterial solution.

[0037] Determination of N2O reduction capacity of the new Mucilaginibacter A14:

[0038] The serum bottle with a volume of 50 mL was added with 25 mL of sterilized DM-N free medium, covered with rubber plug and 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 gas chromatography, 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 S. myxodeum A14 bacterial strain: the prepared S. myxodeum A14 bacterial solution 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 S. myxodeum A14 bacterial strain.

[0042] Determination of N2O reductase activity of new S. myxodeum A14: the level of nitrous oxide reductase (Nos) in the sample was determined by using double antibody sandwich method. The specific operation was as follows: the prepared S. myxodeum A14 bacterial solution was transferred to a 2 mL centrifuge tube, and the supernatant was discarded after centrifugation. According to the number of bacteria (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 (power 20% or 200 W, ultrasonic crushing for 3 s, interval 10 s, repeated 3 times under ice bath conditions). After ultrasonic crushing, 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 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 incubation, the enzyme-labeled coating plate was washed 5 times with washing solution. Then, the enzyme-labeled reagent was added, and the incubation and washing operations were repeated. Finally, the color developing agent and the termination agent were added, and the OD 450 absorbance value of the blank hole (without sample and enzyme-labeled reagent) was used as a control. 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 addition and other operations were the same as those for sample determination. The OD 450 absorbance value of each dilution concentration standard sample was determined by using an enzyme-labeled instrument, the data were counted, a standard curve was drawn, and the N2O reductase activity of the target strain was calculated.

[0043] In this process, the N2O concentration of the new Myxobacterium A14 decreased from 3044.31 μmol·L-1 to 1942.39 μmol·L-1, and the average rate of N2O consumption of the Myxobacterium A14 was 39.57 μmol·L-1·h-1 Figure 3 , which showed a strong ability to reduce N2O. The control experiment showed that the N2O concentration of the CK-N2O group and the N2O production in the CK-He group increased over time due to experimental errors. The N2O reductase activity of the Myxobacterium A14 was calculated to be 8.304 U / g (Table 2), indicating that the new Myxobacterium A14 is a strain with high N2O reduction ability.

[0044] Table 2 N2O reductase activity of the new Myxobacterium A14

[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 Myxobacterium A14)

[0048]

Claims

1. A strain of myxobacterium ( Mucilaginibacter sp.) A14, characterized in that, The accession number is: GDMCC No:66255.

2. A biological agent, characterized in that, The active ingredient is Myxobacterium A14 as described in claim 1 or its fermentation broth.

3. The biological agent according to claim 2, characterized in that, The biological agent is either a liquid or a solid formulation.

4. The biological agent according to claim 3, characterized in that, The solid dosage form is a powder.

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