Methane-oxidizing nitrogen-fixing bacterium and application thereof

Abstract

The invention discloses a methane-oxidizing nitrogen-fixing bacterium and application thereof, and belongs to the technical field of agriculture. The bacterial strain is preserved in Guangdong Microbial Culture Collection Center on September 25, 2025, the preservation number is GDMCC 67018, and the bacterial strain is named as Methylocystis iwaonis in taxonomy. The bacterial strain can grow by using methane and methanol as a unique carbon source, nitrogen is used as a unique nitrogen source for nitrogen fixation, and the bacterial strain belongs to type II methane-oxidizing bacteria with methane oxidation and nitrogen fixation functions; meanwhile, the gene has a pmoA methane monooxygenase gene and a nifH nitrogenase gene cluster, and has dual metabolic potentials; a relatively high methane oxidation rate can still be kept in a low-nitrogen environment, and an excellent nitrogen self-sufficiency characteristic is shown; the method has potential rice field emission reduction and nitrogen fixation synergistic application value, can be applied to the fields of rice field methane emission reduction, biological nitrogen fertilizer research and development, low-carbon agriculture, carbon-nitrogen coupling cycle regulation and the like, can provide new microbial resources for rice field greenhouse gas emission reduction, and also provides an important theoretical and application basis for development of sustainable agriculture with low nitrogen input.

Description

Technical Field

[0001] This invention relates to the field of agricultural technology, and more specifically to a methanogenic nitrogen-fixing bacterium and its applications. Background Technology

[0002] Methane is a significant greenhouse gas with a global warming potential approximately 28 times that of carbon dioxide. Paddy fields, as major constructed wetland ecosystems, are a significant source of global methane emissions. Methanotrophs in paddy field soil play a crucial role in regulating methane emissions by oxidizing methane into carbon dioxide. However, under natural conditions, methane oxidation is often limited by nitrogen supply. Traditional agriculture typically increases yields by applying chemical nitrogen fertilizers, but excessive fertilization not only leads to low nitrogen fertilizer utilization but also exacerbates emissions of greenhouse gases such as N2O, causing serious environmental problems.

[0003] Previous studies have shown that some methanogenic bacteria possess nitrogen-fixing capabilities, fixing atmospheric nitrogen into ammonium nitrogen to provide a nitrogen source for themselves or associated plants, thereby maintaining metabolic activity in low-nitrogen environments. However, the number of nitrogen-fixing methanogenic bacteria reported so far is limited, and most originate from natural ecosystems such as lakes and wetlands. There are few reports on strains in paddy field soil that possess both methanogenic and nitrogen-fixing functions. In particular, systematic research and application examples are lacking at the culturable bacteria level.

[0004] Methylocystis It belongs to the genus *Methanogenus*, class Alphaproteobacteria, and is characterized by its use of methane or methanol as the sole carbon source, while also being able to fix carbon via the serine pathway. (Partial) Methylocystis Species were found to carry nif Gene clusters possess nitrogen-fixing capabilities, but their isolation, identification, and application development in paddy field ecosystems are still in their early stages.

[0005] Therefore, how to find and screen strains with efficient methane oxidation and nitrogen fixation functions is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a methanogenic nitrogen-fixing bacterium and its application, so as to overcome the shortcomings of the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A strain of methanogenic nitrogen-fixing bacteria, deposited on September 25, 2025 at the Guangdong Provincial Microbial Culture Collection Center with accession number GDMCC 67018, has been taxonomically named... Methylocystis iwaonis .

[0008] A methane emission reduction agent comprising the above-mentioned methane-oxidizing nitrogen-fixing bacteria.

[0009] A biological nitrogen fertilizer preparation comprising the aforementioned methanogenic nitrogen-fixing bacteria.

[0010] The present invention also claims protection for the use of the above-mentioned methanogenic nitrogen-fixing bacteria in the preparation of methane emission reduction agents, the function of which is to reduce methane emissions.

[0011] This invention also claims protection for the application of the above-mentioned methanogenic nitrogen-fixing bacteria in the preparation of bio-nitrogen fertilizer formulations, the function of which is nitrogen fixation.

[0012] The present invention also claims protection for the application of the above-mentioned methanogenic nitrogen-fixing bacteria in rice cultivation, which has the function of reducing methane emissions and fixing nitrogen.

[0013] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows: This invention screened and obtained a methanogenic nitrogen-fixing bacterium derived from paddy field soil— Methylocystis iwaonis This strain can utilize methane and methanol as the sole carbon source for growth and nitrogen gas as the sole nitrogen source for nitrogen fixation, belonging to type II methanogenic bacteria with both methane oxidation and nitrogen fixation functions; it also possesses... pmoA methane monooxygenase gene and nifH The nitrogenase gene cluster possesses dual metabolic potential; it can maintain a high methane oxidation rate even in low-nitrogen environments, exhibiting excellent nitrogen self-sufficiency characteristics; it has potential value in reducing emissions and enhancing nitrogen fixation in paddy fields, and can be applied to fields such as methane emission reduction in paddy fields, research and development of bio-nitrogen fertilizers, low-carbon agriculture and carbon-nitrogen coupled cycle regulation. It can not only provide new microbial resources for greenhouse gas emission reduction in paddy fields, but also provide an important theoretical and applied foundation for the development of sustainable agriculture with low nitrogen input. Attached Figure Description

[0014] Figure 1 Cell morphology of methanogenic nitrogen-fixing bacteria sgz302057; Figure 2 The genome ANI index of nitrogen-fixing bacterium sgz302057; Figure 3 Phylogenetic tree of 16S rRNA genes of methanogenic nitrogen-fixing bacterium sgz302057; Figure 4 To evaluate the methane oxidation capacity of the nitrogen-fixing bacterium sgz302057 under different ammonium concentrations; Figure 5 To evaluate the methane oxidation capacity of the nitrogen-fixing bacterium sgz302057 under different methane concentrations; Figure 6To evaluate the methane oxidation capacity of the nitrogen-fixing bacterium sgz302057 under different oxygen concentrations; Figure 7 The methane oxidation capacity of the nitrogen-fixing bacterium sgz302057 under anaerobic conditions of 5 mmol ferrohydrate was evaluated. Figure 8 The iron reduction capacity of the methanogenic nitrogen-fixing bacterium sgz302057 under anaerobic conditions of 5 mmol of ferrohydrate. Figure 9 for nifH The standard curve; Figure 10 The standard curve for 16S rRNA Bactcria; Figure 11 for nifH Absolute copy number of genes; Figure 12 This represents the absolute copy number of the 16S rRNA gene. Figure 13 The expression level of nitrogen-fixing genes in the methanogenic nitrogen-fixing bacterium sgz302057; Figure 14 Comparison of germination rates of rice seeds treated with water (control group) and methanogenic nitrogen-fixing bacteria sgz302057; Figure 15 Comparison of growth indicators (main stem length and germination root length) of rice seeds after treatment with water (control group) and methanogenic nitrogen-fixing bacteria sgz302057 respectively; Figure 16 Germination of rice seeds after treatment with water (control group); Figure 17 Germination of rice seeds after treatment with bacterial solution of methanogenic nitrogen-fixing bacteria sgz302057; Figure 18 Germination of rice seeds after treatment with water (control group, left) and methanogenic nitrogen-fixing bacteria sgz302057 (right). Detailed Implementation

[0015] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Example 1: Isolation, screening and identification of methanogenic nitrogen-fixing bacteria sgz302057 The method for isolating, screening, and identifying methanogenic nitrogen-fixing bacteria sgz302057 includes the following steps: 1. Material and Equipment Preparation Solid NMS plates: NMS medium is a commonly used culture medium for methanogenic bacteria. Its core components include nitrate as a nitrogen source, inorganic salts, and trace elements. Solid NMS medium formulation (1 L as an example): Inorganic nitrate minimal salts (NMS) composition (g / L) is as follows: KH₂PO₄ 0.26; Na₂HPO₄ . 7H₂O 0.72; MgSO₄ . 7H2O1.0; CaCl . 7H₂O 0.2; KNO₃ 1.0; Trace element solution 1 mL; The trace element solution was prepared separately, with the following composition (g / L): CuSO₄ . 5H₂O 0.2; FeSO₄ . 7H₂O 0.5; ZnSO₄ . 7H₂O 0.4; H₃BO₃ 0.025; CoCl₂ . 6H₂O 0.5; Na₂EDTA 0.25; MnCl₂ . 4H₂O 0.02; NiCl₂ . 6H₂O 0.01; Na₂MoO₄ . 2H₂O 0.26. Curing agent (high acyl gellan gum): 7 g / L; Gas bag: plastic gas bag with inflation port; Inoculation loop.

[0017] 2. Separation lines Take a single colony generated from the plating and streak it onto NMS medium, streaking continuously in sections. By diluting each section, a single colony is finally obtained in the last section. For environmental samples (such as soil or wetland sediments), it is recommended to first perform liquid enrichment culture (NMS liquid medium + 10%-30% CH4, 30℃ shaking culture for 7-14 days) before serial dilution and plating.

[0018] 3. Inflation culture using air bags Place the petri dish into a sterile gas bag and seal the bag. Use a syringe to remove the existing air from the bag. Inject methane gas until the bag expands. Incubate: Incubate at 30°C in the dark, replenishing the gas every 3 days. Observation and verification: Observe the colonies after 7-14 days (methane-oxidizing bacteria colonies are usually white or light yellow with neat edges).

[0019] 4. Colony identification Morphology: Colony characteristics: white / light yellow, with neat and raised edges (some strains are mucous or red, requiring molecular identification).

[0020] Microscopic observation: short rod-shaped or spherical, Gram-negative.

[0021] Molecular biology: pmoA Gene amplification: Specific primers (such as A189 / A682) are used to detect the methane monooxygenase gene.

[0022] 16S rRNA sequencing: genus and species were confirmed by comparison with the NCBI database.

[0023] Functional verification: Methane consumption rate determination: Gas chromatography (GC-FID) was used to monitor the change in CH4 concentration inside the gas bag and calculate the oxidation efficiency.

[0024] Example 2: Physiological and biochemical characteristics of methanogenic nitrogen-fixing bacteria sgz302057 like Figure 1 As shown, the methanogenic nitrogen-fixing bacterium sgz302057 is a Gram-negative bacterium, a short rod-shaped bacterium with flagella, which is mobile, and is about 1.5-2.0 μm long and 0.5-0.8 μm wide.

[0025] The methanogenic nitrogen-fixing bacterium sgz302057 showed significant growth on NMS liquid under 20% methane conditions.

[0026] On agar plates using gellan gum as a gelling agent, the bacteria form round, raised, smooth, opaque, and well-defined white colonies. The growth temperature range is 25-37℃, with an optimum of 30℃. The optimum salt concentration is 0%, and the pH growth range is 6.5-7.5. It is nitrate-reducing positive. It is catalase-positive and oxidase-negative. Other physiological indicators are shown in Tables 1-2 below. It can grow under the antibiotics rifamycin SV, but not under the antibiotics arbutin, methyltetracycline, lincomycin, or vancomycin. The main fatty acid is C. 18:1 ω 8 c and C 18:1 ω 8 t .

[0027] Table 1. Fatty acid composition of methanogenic bacteria sgz302057

[0028] Table 2 Phenotypic characteristics of methanogenic bacteria sgz302057

[0029] Example 3: Genetic information of methanogenic nitrogen-fixing bacterium sgz302057 like Figure 2-3 As shown, the most recent type strain of methanogenic nitrogen-fixing bacteria sgz302057 is... Methylocystis iwaonis SS37A-Re TThe 16S rRNA similarity was 100%, the genome ANI similarity was 96.5%, and the dDDH similarity was 79.2%, indicating that this strain is... Methylocystis iwaonis .

[0030] The completed genome map of the methanogenic nitrogen-fixing bacterium sgz302057 is 4.2 Mb in size, with a DNA G+C content of 63.35%. The genome integrity is 99.68%, and the contamination level is 0.95%. 3944 genes were annotated, including 52 tRNAs and 9 rRNAs. 2636 functional genes were annotated using KEGG.

[0031] The 16S rRNA sequence of the methanogenic nitrogen-fixing bacterium sgz302057 is shown in SEQ ID NO.1 of the sequence listing, specifically as follows:

[0032] Example 4: Methane oxidation capacity of nitrogen-fixing bacterium sgz302057 like Figure 4 As shown, with NH4 + With increasing concentration, the methane oxidation capacity of the nitrogen-fixing bacterium sgz302057 gradually increased, especially at 5 mM NH4+. + The methane oxidation capacity of the concentration is strongest at 5 days.

[0033] like Figure 5 As shown, methane concentration has a strong effect on the methane oxidation capacity of the methanogenic nitrogen-fixing bacterium sgz30205. At a methane concentration of 30%, the methane oxidation capacity of the methanogenic nitrogen-fixing bacterium sgz30205 is the strongest.

[0034] like Figure 6 As shown, the methane oxidation capacity of the methanogenic nitrogen-fixing bacterium sgz30205 gradually increases with increasing oxygen concentration, exhibiting the strongest methane oxidation capacity at 5%-10% oxygen concentration. This indicates that this strain functions effectively at low methane concentrations.

[0035] like Figure 7-8 As shown, 5mM ferrohydrate Fe 3+ Under the given conditions, methane oxidizing capacity is strongest at 10 days. Fe in the solution... 2+ The gradual increase in concentration indicates that iron reduction promoted the oxidation of methane.

[0036] Example 5: Nitrogen fixation capacity of methanogenic nitrogen-fixing bacteria sgz302057 Genome analysis showed that sgz302057 possesses a nitrogen-fixing gene cluster. nifHDK For assessing the nitrogen fixation capacity of methanogenic bacteria sgz302057, the absolute copy number of nitrogen-fixing genes in the sample can be accurately determined using qPCR absolute quantification.

[0037] 1. Construction and preparation of standards Plasmid DNA of known concentration is used as a standard. Cloning. nifH Gene fragments are transferred to plasmids, and after plasmid extraction, precise quantification is performed using a spectrophotometer. The gene copy number per microliter of plasmid solution is calculated based on the molecular weight. This is achieved by constructing a plasmid containing the target gene fragment (…). nifH A standard curve is prepared using plasmid standards of the sample, and the absolute copy number of the starting template is calculated based on the Ct value of the sample.

[0038] 2. Standard Curve Creation Establish a linear relationship between Ct value and the logarithm of copy number. Perform serial dilutions of the plasmid standard, with each dilution used for qPCR. Plot a standard curve with Ct value on the ordinate and the logarithm of copy number on the x-axis. Ideally, the curve should have R² > 0.99 and efficiency between 90% and 110%. Results are as follows... Figure 9-10 As shown.

[0039] Depend on Figure 9 It can be seen that, nifH The standard curve equation is y = 0.2946x + 11.5174, R² = 0.997.

[0040] Depend on Figure 10 It can be seen that the standard curve equation of 16S rRNA Bactcria is y=0.2942x+13.1684, R²=0.995.

[0041] 3. Sample DNA extraction and qPCR amplification 3.1 DNA Extraction Sample culture: Two groups were established: a nitrogen-containing group (10 mM) and a nitrogen-free group. Three biological replicates were performed. Methanogenic bacteria cells were collected using a bacterial DNA kit and subjected to lysis, binding, washing, and elution. The DNA was finally dissolved in the elution buffer provided in the kit or in sterile water and stored at -20°C for later use. Detection was performed using a micro-spectrophotometer. Ideal values: A260 / A280 between 1.8 and 2.0.

[0042] Dilution: Based on the concentration, dilute all sample DNA to a uniform concentration (10 ng / μL) with sterile water as qPCR template.

[0043] 3.2 Absolute Quantitative qPCR The method for absolute quantitative qPCR mainly includes the following steps: A typical reaction system volume is 20 μL, containing 10 μL of SYBR Green premix, 0.3 μL each of specific primers, 1 μL of template DNA, and sterile water. The core temperature settings for the reaction program are as follows: pre-denaturation at 95℃ for 5 min; followed by 40-45 cycles, each cycle including 15 s of denaturation at 95℃, followed by 30 s of annealing / extension at 50-55℃ with fluorescence signal acquisition; finally, run a melting curve analysis from 65℃ to 95℃ to verify specificity. A template-free control is included.

[0044] 4. Relative abundance and expression intensity of nitrogen fixation To eliminate the interference of differences in total cell count between different treatments on the absolute quantification results, this study used qPCR to measure... nifH The absolute gene copy number was standardized by dividing by the 16S rRNA gene copy number of the same sample. The final data is expressed as " nifHThe expression "gene copy number / 16S rRNA gene copy number" accurately reflects the relative abundance and expression intensity of nitrogen-fixing genes. Results are as follows... Figure 11-13 As shown.

[0045] Depend on Figure 11-13 It can be seen that under nitrogen-limited conditions, methanogenic bacteria upregulated and significantly expressed nitrogen-fixing genes, and their nitrogen-fixing system was functionally activated. This indicates that the bacteria possess biological nitrogen-fixing capabilities that are regulated by nitrogen sources.

[0046] 5. Growth-promoting ability Depend on Figure 14-18 It can be seen that the methanogenic bacteria sgz302057 has the ability to promote growth.

[0047] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of methanogenic nitrogen-fixing bacteria, characterized in that, This strain was deposited at the Guangdong Provincial Microbial Culture Collection Center on September 25, 2025, with accession number GDMCC 67018, and its taxonomic name is... Methylocystis iwaonis .

2. A methane emission reduction agent, characterized in that, It contains the methanogenic nitrogen-fixing bacteria as described in claim 1.

3. A bio-nitrogen fertilizer formulation, characterized in that, It contains the methanogenic nitrogen-fixing bacteria as described in claim 1.

4. The use of the methanogenic nitrogen-fixing bacteria as described in claim 1 in the preparation of methane emission reduction agents.

5. The application of the methanogenic nitrogen-fixing bacteria as described in claim 1 in the preparation of bio-nitrogen fertilizer formulations.

6. The application of the methanogenic nitrogen-fixing bacteria as described in claim 1 in rice cultivation.

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