Acid-resistant bradyrhizobium capable of degrading nitrous oxide and application of acid-resistant bradyrhizobium

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to acid-resistant bradyrhizobium capable of degrading nitrous oxide and application of the acid-resistant bradyrhizobium. The invention relates to acid-resistant bradyrhizobium beet, which is bradyrhizobium beet purchased in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the acid-resistant bradyrhizobium beet is CGMCC 1.15594. The acid-resistant bradyrhizobium can still keep a good growth state and physiological activity under the conditions of low pH value and no assistance of an external carbon source. After the fertilizer is applied to farmland, stable colonization can be realized, the pH value of soil can be effectively buffered and increased through the metabolic activity of the fertilizer, and the rhizosphere microenvironment of crops is improved; meanwhile, the biological immobilization capacity of soil microbial communities on nitrogen is enhanced, inorganic nitrogen can be intercepted, and a biological basis is provided for agricultural non-point source pollution prevention and control, nitrogen fertilizer loss reduction and chemical fertilizer application reduction and synergism.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to an acid-tolerant slow-growing rhizobium that degrades nitrous oxide and its applications. Background Technology

[0002] Nitrous oxide (N₂O) is a significant greenhouse gas with a global warming potential far exceeding that of carbon dioxide. Farmland soils are a major source of anthropogenic N₂O emissions. Acidic red soils are widely distributed in tropical and subtropical regions such as southern my country. Excessive application of nitrogen fertilizers in intensive agricultural production (such as sugarcane and other cash crops) has not only resulted in severe nitrogen surpluses and low utilization rates but has also further accelerated soil acidification. Soil acidification not only restricts crop growth but also significantly promotes N₂O emissions, making these areas a key focus and challenge for agricultural greenhouse gas emission reduction.

[0003] Microbial cells nosZ The gene-encoded N2O reductase is currently the only known enzyme in the biosphere capable of reducing N2O to harmless nitrogen gas (N2). However, existing research generally indicates that low pH significantly inhibits the final reduction process of N2O. The main biochemical mechanism lies in the fact that lower pH does not necessarily directly inhibit... nosZ While it can inhibit transcription, it can severely interfere with the post-transcriptional assembly and functional expression of N2O reductase in the periplasm, resulting in a large amount of N2O produced in acidic soils not being completely reduced and being released into the atmosphere.

[0004] Although weak N2O reduction has been observed in some acidic soils, very few functional microorganisms capable of independent growth under acidic conditions and possessing high N2O reduction rates have been isolated to date. Existing literature reports some pure culture strains (such as...) Rhodanobacter Although it possesses reducing ability at pH 3.7-4.0, it is essentially unable to grow at this acidity; while other co-cultures (such as...) Serratia sp. and Desulfosporosinus When reducing N2O at pH 4.5, sp. not only requires the addition of carbon sources such as pyruvic acid, but also takes a long time to completely remove N2O, making it difficult to meet the actual carbon reduction needs of farmland.

[0005] Therefore, screening out functional strains that can tolerate low pH, grow independently, and effectively reduce N2O from typical acidic farmland soils is of great significance for controlling agricultural N2O emissions.

[0006] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide an acid-resistant, slow-growing rhizobium strain that degrades nitrous oxide and its applications, in order to solve the problems existing in the prior art.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A strain of acid-tolerant slow-growing rhizobium, which was purchased from the China General Microbiological Culture Collection Center (CGC). Bradyrhizobium betae (The accession number is CGMCC 1.15594).

[0009] The present invention also provides the application of the slow-growing rhizobium in degrading nitrous oxide, and / or improving soil acidity, and / or inhibiting nitrification-functional microorganisms, and / or reducing soil microbial carbon-nitrogen ratio, or increasing the content of acid-insoluble organic nitrogen in soil.

[0010] The present invention also provides a microbial inoculant, wherein the microbial inoculant includes the acid-resistant slow-growing rhizobium.

[0011] Furthermore, the preparation method of the microbial inoculant includes the following steps: S1. The acid-resistant slow-growing rhizobium was inoculated on YMA solid medium and activated at 28°C until plump single colonies grew. S2. Select activated single colonies and transfer them to YMA liquid medium. Incubate at 28°C with shaking at 150-200 rpm until the logarithmic growth phase to obtain the fermentation broth. S3. Collect the viable cells from the fermentation broth, resuspend and adjust the broth concentration to 1×10⁻⁶. 8 -1×10¹ 0 The microbial agent is obtained by measuring CFU / mL.

[0012] Compared with the prior art, the present invention has the following beneficial effects: (1) The acid-tolerant slow-growing rhizobium of the present invention can still maintain good growth and physiological activity under low pH conditions and without the assistance of external carbon sources. After being applied to farmland, it can not only achieve stable colonization, but also effectively buffer and increase the soil pH through its own metabolic activities, thereby improving the rhizosphere microenvironment of crops.

[0013] (2) The acid-tolerant slow-growing rhizobium of the present invention has dual functions of N2O reduction and nitrification inhibition, and is suitable for comprehensive reduction of greenhouse gas emissions in farmland. It can effectively inhibit the activity of nitrifying microorganisms such as ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB), slow down the conversion of ammonium nitrogen (NH4⁺) to easily leached nitrate nitrogen (NO3⁻), and reduce the substrate for N2O production from the source; and through terminal reduction, it can effectively degrade N2O accumulated in acidic soil into harmless nitrogen gas (N2), thus achieving bidirectional control of greenhouse gases.

[0014] (3) The acid-tolerant slow-growing rhizobium of the present invention exhibits good nutrient assimilation ability, which can significantly promote the conversion of soil inorganic nitrogen into acid-insoluble organic nitrogen that is more difficult to degrade, and reduce the microbial carbon-nitrogen ratio. This characteristic enhances the soil microbial community's biological retention capacity for nitrogen, helps to retain inorganic nitrogen (especially easily leached nitrate nitrogen), and provides a biological basis for the prevention and control of agricultural non-point source pollution, reducing nitrogen fertilizer loss, and reducing fertilizer application while increasing efficiency. Attached Figure Description

[0015] Figure 1 The colony morphology of the slow-growing rhizobium G94 of this invention on YMA solid medium; Figure 2 This is an experimental setup for determining the N2O reducing capacity of bacterial strains based on headspace analysis. Figure 3 Graphs showing the N2O emission reduction effect of different tested bacterial strains on acidic soil; Figure 4 The effect of the tested bacterial strains on the pH of acidic soil; Figure 5 The graph shows the nitrification inhibition rate of the tested strains.

[0016] Additional notes: Figure 3 In the middle, a-the dynamic change curve of N2O concentration over time; b-the total N2O emission reduction at the end of the treatment period (bar chart). Detailed Implementation

[0017] The technical solution of this invention patent will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0018] 1. Materials and Methods 1.1 Test Site Laboratory 204, College of Agriculture, Guangxi University.

[0019] 1.2 Test Soil The test soil was collected from a typical acidic red soil sugarcane growing area in southern my country. After the topsoil (0-20cm) was collected and brought back to the laboratory, plant and animal remains and stones were removed, and the soil was air-dried and sieved through a 2mm sieve. To eliminate background interference from indigenous microorganisms and accurately verify the function of a single strain, the sieved soil was placed in an autoclave and sterilized twice at 121°C and 0.1MPa (30 minutes each time, 24 hours apart to completely kill spores), resulting in sterile acidic red soil for testing.

[0020] 1.3 Test Time May 2025 to October 2025.

[0021] 1.4 Activation culture of the tested strains The official preservation numbers of the slow-growing rhizobia to be screened are: CGMCC 1.11032 (G32), CGMCC 1.15035 (G35), CGMCC 1.15558 (G58), CGMCC 1.15564 (G64), CGMCC 1.15566 (G66), and CGMCC 1.15594 (G94).

[0022] The tested strains were inoculated into YMA solid medium and incubated at 28°C until colonies grew (see...). Figure 1 Then, single colonies were picked and transferred to YMA liquid medium, and cultured at 28°C and 200 rpm with shaking until the logarithmic growth phase was reached. The viable cell count in the culture medium was then adjusted to 1 × 10⁻⁶. 9 CFU / mL, for later use.

[0023] The YMA medium was yeast mannitol agar medium (purchased from Shandong Top Biotechnology Co., Ltd.), with the following formula: K2HPO4 0.8g, KH2PO4 0.2g, FeCl3 0.1g, MgSO4·7H2O 0.2g, CaSO4·2H2O 0.1g, Na2MoO4·2H2O 0.002g, yeast extract 0.5g, mannitol 20.0g, agar powder 15g (added to solid medium), and distilled / deionized water 1L; the pH was adjusted to 6.5.

[0024] 1.5 Test Treatment 1.5.1 Indoor Soil Closed Culture Experiment The aim was to evaluate the effects of various tested slow-growing rhizobia on soil modification and carbon reduction / nitrogen fixation in a completely controlled, sterile soil matrix. The experiment consisted of 7 treatment groups, with 3 replicates per group. Control group (CK): 100g sterile soil + equal volume of sterile water; Treatment group: Activated slow-growing rhizobium G32, G35, G58, G64, G66, and G94 bacterial solutions were inoculated into 100g of sterilized soil (added at a volume ratio of 2%).

[0025] 1.5.2 Nitrification Inhibition Standoff Test in Pure Bacterial Co-culture The study was conducted in a liquid-phase system, using ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), and complete nitrifying bacteria (Comammox) as targets for co-culturing. Two treatments were included: Control group: 200 μL target cell suspension (1 × 10⁻⁶) 9 (CFU / mL) + 100μL sterile culture medium + 200μL sterile water Treatment group: 200 μL target cell suspension (1 × 10⁻⁶) 9 CFU / mL) + 100 μL sterile culture medium + 200 μL slow-growing rhizobium solution (1×10⁻⁶) 9 (CFU / mL).

[0026] 1.6 Measurement Indicators and Methods 1.6.1 Determination of N2O concentration Weigh 100g of sterilized red soil and place it in a 250mL modified Schott flask (equipped with an airtight three-way valve). See the actual experimental setup for reference. Figure 2 Add the corresponding bacterial solution or sterile water and mix well to adjust the moisture content to 60% field capacity. After establishing an anaerobic environment by vacuuming and purging with N2, inject 20 mL of standard N2O gas. Incubate at 28°C in the dark. Collect 1 mL of headspace gas at 0, 24, 48, 72, 120, 144, and 168 h, and determine the N2O concentration change using a gas chromatograph. Extract 1 mL of gas sample from the headspace culture flask and immediately inject it into a gas chromatograph equipped with an electron capture detector (ECD) (such as an Agilent 7890A) for analysis, using high-purity N2 as the carrier gas. Calculate the N2O concentration in the sample by comparing it with a standard curve plotted with the standard concentration of N2O gas.

[0027] 1.6.2 Determination of soil pH value After cultivation, soil samples were collected. The procedure was strictly performed in accordance with the People's Republic of China Agricultural Industry Standard "Determination of Soil pH" (NY / T1377-2007). Using the glass electrode method, sieved and air-dried soil samples were weighed and mixed with deionized water (removed carbon dioxide) at a water-to-soil ratio of 2.5:1 (V / W). The mixture was vigorously shaken for 5 minutes and then allowed to stand for 30 minutes. The pH value of the soil suspension was then measured using a pH meter calibrated with a standard buffer solution.

[0028] 1.6.3 Determination of Soil Microbial Biomass Carbon (MBC) and Microbial Biomass Nitrogen (MBN) The chloroform fumigation-extraction (CFE) method was used. Equal volumes of fresh soil were divided into two portions. One portion was fumigated with anhydrous chloroform in a vacuum desiccator for 24 hours in the dark, while the other portion served as a control without fumigation. Both portions were then extracted with 0.5 mol / L K₂SO₄ solution by shaking. After filtration, the soluble carbon and nitrogen content in the extract was determined using a Total Organic Carbon / Total Nitrogen Analyzer (TOC-TNanalyzer). MBC and MBN were calculated by dividing the difference in carbon and nitrogen content between the fumigated and unfumigated soil extracts by the corresponding conversion factors (Kc and Kn), respectively.

[0029] 1.6.4 Determination of various forms of nitrogen in soil (inorganic and organic nitrogen components): Inorganic nitrogen (inorganic ammonium nitrogen, inorganic nitrate nitrogen): Weigh fresh soil samples, add 2 mol / L KCl solution and shake to extract. After filtration, determine the content of NH4⁺-N and NO3⁻-N in the extract using a continuous flow analyzer (CFA) or colorimetric method.

[0030] Soil organic nitrogen components (acid-hydrolyzed ammonium nitrogen, amino acid nitrogen, amino sugar nitrogen, unknown nitrogen, and acid-insoluble nitrogen): The Bremner acid hydrolysis method was used. A quantitative amount of soil sample was weighed and acid-hydrolyzed at 105°C for 12 hours by reflux with 6 mol / L HCl. After filtration and volume adjustment, the acid-hydrolyzed ammonium nitrogen, acid-hydrolyzed amino sugar nitrogen (after alkalization and heated distillation), and acid-hydrolyzed amino acid nitrogen (distillation via ninhydrin oxidation) were determined by distillation and titration, respectively. The difference between the total acid-hydrolyzed nitrogen and the known components was the unknown nitrogen. The unhydrolyzed soil residue was digested using the Kjeldahl method and determined to be extremely stable and recalcitrant acid-insoluble nitrogen.

[0031] 1.6.5 Determination of nitrification inhibition rate Ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), and complete nitrifying bacteria (Comammox) were selected as targets for co-culture experiments. Taking AOB as an example: after aerobic culture of AOB strains for 7 days, the bacterial cells were collected by centrifugation and resuspended, and the final bacterial concentration was adjusted to 1×10⁻⁶. 9 CFU / mL. In a 1.5 mL reaction tube, add 200 μL of sterile water, 100 μL of HEPES medium, and 200 μL of target cell suspension. Add equal volumes of sterile culture medium (control group) or the culture of each slow-growing rhizobium to be tested (treatment group). Incubate at 25°C for 24 hours. After the reaction, add 0.1 mM allyl thiourea (AT) to terminate the reaction. Measure the changes in nitrite nitrogen and ammonium nitrogen concentrations in the system using a spectrophotometer to calculate the nitrification inhibition rate.

[0032] 2. Experimental Results 2.1 Effects on reducing N2O emissions and improving soil acidity See results Figure 3 and Figure 4 .

[0033] Depend on Figure 3 and Figure 4 The data from the indoor closed culture experiment showed that, after completely eliminating interference from indigenous microorganisms, the N2O concentration in the control group (CK) remained stable at 162.62 ppm at the end of the 168-hour culture period, proving that no non-biodegradation occurred in the system. Figure 3 a). After inoculation with strain G94, the N2O concentration in the system showed a continuous downward trend, reaching 20.04 ppm at the end of the period, with a total emission reduction of 133.77 ppm. Figure 3 b), its reduction degradation rate was the best among all tested strains.

[0034] Meanwhile, in terms of soil acidity regulation, the soil in the CK group remained strongly acidic at a pH of 3.75, while the soil pH in the G94 strain treatment group significantly increased and stabilized at 4.10. Furthermore, the effect of G94 in regulating soil pH was significantly better than that of slow-growing rhizobia such as G32 and G58. Figure 4 This indicates that strain G94 can not only survive independently in a low pH environment, but also effectively alleviate the acidic stress of the rhizosphere microenvironment through its physiological metabolic processes.

[0035] 2.2 Significant inhibitory effect on key nitrifying bacteria The pure bacterial confrontation experiment verified the ability of the strain to inhibit the formation of greenhouse gas substrates from the perspective of its mechanism of action. The results are shown in […]. Figure 5 .

[0036] Depend on Figure 5 The results show that strain G94 exhibits significant inhibitory efficacy against a variety of key nitrifying microorganisms. In particular, targeting the rate-limiting step of nitrification—ammonia oxidation—strain G94 achieved an inhibition rate of 71.31% against target ammonia-oxidizing bacteria (AOB); simultaneously, it achieved an inhibition rate of 67.44% against complete nitrifying bacteria (Comammox) and 33.06% against nitrite-oxidizing bacteria (NOB). Notably, strain G94 ranked first among all slow-growing rhizobia in its inhibition rate against AOB. This result indicates that strain G94 can significantly reduce the conversion of substrate inorganic nitrogen into greenhouse gases at the source, and together with its terminal N2O reduction capacity, constitutes a two-way greenhouse gas control mechanism, demonstrating a significantly superior nitrification inhibition ability compared to similar reference strains.

[0037] 2.3 Effects on nitrogen form transformation and retention in acidic soils See Table 1.

[0038] Table 1. Soil nitrogen species in each experimental group (unit: mg / kg) Group amino acid nitrogen Amino sugar nitrogen Acid hydrolysis of ammonium nitrogen Unknown state nitrogen Acid-insoluble nitrogen Inorganic ammonium nitrogen Inorganic nitrate nitrogen CK 35.61±5.09ab 48.77±6.83cd 45.24±5.36b 74.04±4.25cd 847.69±41.33bc 25.95±1.04ab 13.28±0.73a G32 14.44±1.92d 77.00±0.96b 43.31±8.66abc 109.72±3.85b 753.05±10.64b 23.65±0.09b 7.51±4.00abc G35 40.43±4.81a 75.08±4.81b 58.71±0.96a 33.69±4.81e 821.35±17.05c 24.18±0.45b 9.72±0.80c G58 29.84±3.85b 119.59±2.65a 28.88±1.92c 70.98±7.94d 769.80±12.86b 24.85±1.24ab 10.23±0.61c G64 21.17±2.89c 47.16±3.85d 25.99±2.89cd 157.85±5.77a 841.44±66.32bc 25.22±1.41ab 11.60±0.43b G66 33.69±3.85ab 62.56±3.85c 25.02±1.93cd 94.32±9.63bc 829.62±48.41bc 26.92±0.79a 10.39±1.59abc G94 34.65±2.89ab 52.94±5.77cd 20.21±2.89d 111.65±1.92b 1024.28±11.29a 24.70±1.07b 10.07±1.33bc As shown in Table 1, after inoculation with strain G94, the content of acid-insoluble organic nitrogen, the most resistant and difficult to mineralize and degrade, in the soil jumped to 1024.28 mg / kg, a significant increase of 20.8% compared to the sterile control group (847.69 mg / kg). Simultaneously, echoing the results of the pure bacterial inhibition experiment, due to the inhibition of the upstream nitrification reaction, the content of easily leached inorganic nitrate nitrogen in the microcosm system decreased to 10.07 mg / kg, a decrease of 24.1% compared to the CK group (13.28 mg / kg). This speciation data indicates that strain G94 possesses excellent nutrient assimilation capabilities, able to convert active free inorganic nitrogen into an extremely stable organic nitrogen pool, thereby fundamentally reducing nitrogen loss and the risk of gaseous emissions.

[0039] 2.4 Regulation of the carbon and nitrogen allocation ratio of soil microorganisms The results are shown in Table 2.

[0040] Table 2. Statistical table of microbial biomass carbon and nitrogen in each experimental group Group MBC / MBN CK 15.79±0.37a G32 11.03±0.45cd G35 11.94±0.38bc G58 10.70±0.41de G64 11.25±1.73bcde G66 9.89±0.21e G94 12.19±0.37b The determination results of microbial biomass carbon (MBC) and nitrogen (MBN) reflect the changes in the microecological metabolic characteristics of the system after the introduction of the strain. As shown in Table 2, after inoculation with strain G94, the microbial carbon-to-nitrogen ratio (MBC / MBN) in the system significantly decreased from 15.79 in the control group to 12.19. Ecologically, a lower microbial carbon-to-nitrogen ratio indicates that the microbial community has higher nitrogen use efficiency and stronger biocapacity. This significant decrease further corroborates that the micro-ecosystem driven by the strain of this invention achieves a high level of nitrogen assimilation, providing a biological basis for the control of agricultural non-point source pollution.

[0041] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. An acid-tolerant, slow-growing rhizobium strain, characterized in that, It was purchased from the China General Microbiological Culture Collection Center as a slow-growing rhizobium of beet ( Bradyrhizobium betae (The accession number is CGMCC 1.15594).

2. The application of the slow-growing rhizobium of claim 1 in the degradation of nitrous oxide.

3. The application of the slow-growing rhizobium as described in claim 1 in improving soil acidity.

4. The application of the slow-growing rhizobium as described in claim 1 in nitrification-inhibiting microbial bacteria.

5. The application of the slow-growing rhizobium as described in claim 1 in reducing the soil microbial carbon-nitrogen ratio.

6. The application of the slow-growing rhizobium of claim 1 in the content of acid-insoluble organic nitrogen in soil.

7. A microbial inoculant, characterized in that, The microbial agent includes the acid-resistant slow-growing rhizobium as described in claim 1.

8. The microbial agent according to claim 7, characterized in that, The preparation method of the microbial inoculant includes the following steps: S1. The acid-resistant slow-growing rhizobium was inoculated on YMA solid medium and activated at 28°C until plump single colonies grew. S2. Select activated single colonies and transfer them to YMA liquid medium. Incubate at 28°C with shaking at 150-200 rpm until the logarithmic growth phase to obtain the fermentation broth. S3. Collect the viable cells from the fermentation broth, resuspend and adjust the broth concentration to 1×10⁻⁶. 8 -1×10¹ 0 The microbial agent is obtained by measuring CFU / mL.

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