Method for promoting biological nitrogen fixation in rice fields based on long-distance electron transport

Abstract

The application discloses a method for promoting paddy field biological nitrogen fixation based on long-distance electron transfer, and comprises the following steps: S1, soil pretreatment, establishing a flooding system; S2, pre-incubation to form a stable redox stratification; S3, constructing a long-distance electron transfer channel; S4, cultivation: under the conditions of flooding and a certain temperature, continue to cultivate to promote the paddy field biological nitrogen fixation in the flooding system. The application does not need an external power supply, only by arranging a graphite rod in the flooded paddy field soil, making it span the surface layer oxidation zone and the lower layer reduction zone, forming a long-distance electron transfer channel, thereby realizing the directional transport and utilization of the lower layer reduction equivalent to the surface layer, providing continuous support for the surface layer nitrogen fixation process from the electron supply end, promoting the nitrogen fixation process, and also promoting the enrichment of paddy field biological nitrogen fixation related microbial groups in the surface layer oxidation zone and changing the iron valence state distribution, promoting the transformation of Fe(III) into Fe(II), and enhancing the coupling of iron reduction and nitrogen fixation process.

Description

Technical Field

[0001] This invention relates to the technical field of agricultural ecological engineering and environmental microorganisms, and more specifically, to a method for promoting biological nitrogen fixation in paddy fields based on long-distance electron transfer. Background Technology

[0002] Nitrogen is one of the most important limiting nutrients for rice growth. To increase rice yield, large amounts of chemical nitrogen fertilizers are used in agricultural production, but long-term excessive fertilization has led to environmental problems such as soil degradation, nitrogen loss, and eutrophication of water bodies.

[0003] Under prolonged flooding conditions in paddy fields, a distinct redox stratification forms in the soil profile: the surface layer near the water-air interface is an oxidizing zone, while the lower layer is a reducing zone. Biological nitrogen fixation requires a high level of ATP and electrons. In flooded paddy fields, the surface oxidizing zone has limited electron sources, while the lower reducing zone, although possessing a large amount of reducing equivalent, is difficult for the surface layer to utilize, thus restricting the release of nitrogen fixation potential at the rice root-soil interface.

[0004] To improve the nitrogen fixation capacity of paddy fields, existing technologies mainly focus on the following directions: (1) Electrochemical or external potential enhancement of nitrogen fixation, such as CN114651548A, which achieves in-situ nitrogen fixation by inoculating electroactive bacteria in the soil, constructing a three-electrode system and applying a working potential, but requires an electrode system and an external power supply / potential control device, making field implementation and maintenance complex; (2) Rhizosphere induction and specific microbial coupling nitrogen fixation, such as CN117814103A, which induces the formation of an iron film on the root surface and uses methanogenic bacteria to synergistically enhance nitrogen fixation, but depends on seedling raising, rhizosphere induction and specific functional microorganisms, and the operation chain is long; (3) Exogenous additives or materials to activate nitrogen fixation, such as CN121286158A, which activates the activity of rhizosphere microorganisms by adding microplastics to the soil, but requires continuous addition of exogenous materials, which poses ecological risks and long-term stability problems.

[0005] Therefore, there is an urgent need for a method that is easy to operate, uses readily available materials, requires no external power source, can utilize the redox stratification of paddy fields to achieve cross-layer electron transport, and uses the interface micro-regions as the core to stably promote biological nitrogen fixation in paddy fields. Summary of the Invention

[0006] The purpose of this invention is to provide a method for promoting biological nitrogen fixation in paddy fields based on long-distance electron transfer.

[0007] According to one aspect of the present invention, a method for promoting biological nitrogen fixation in paddy fields based on long-distance electron transfer is provided, comprising the following steps: S1. Soil pretreatment: Collect paddy field soil that has not been fertilized with nitrogen, remove plant residues and stones, air dry naturally and sieve, add water to the paddy field soil to establish a flooding system; S2. Pre-incubation: The flooded system is pre-incubated at a certain temperature to form a stable redox stratification; S3. Constructing long-distance electron transport channels: Vertically insert graphite rods into paddy soil in a flooded system with redox stratification, and utilize the high conductivity of graphite rods to construct long-distance electron transport channels across redox stratification. S4. Cultivation: Continue cultivation under flooded conditions and certain temperature conditions to promote biological nitrogen fixation in paddy fields within the flooded system.

[0008] In some implementations, the unfertilized paddy soil refers to paddy soil that has not been fertilized with inorganic nitrogen fertilizer or nitrogen-containing organic fertilizer in the previous growing season or within 6-12 months.

[0009] In some embodiments, the graphite rod is 10-15cm long and 1-2cm in diameter. When the graphite rod is inserted, the top of the rod protrudes 1-3cm above the paddy field water layer, and the insertion depth is 8-12cm.

[0010] In some embodiments, the diameter of the graphite rod is preferably 1.5 cm, the length of the top of the graphite rod protruding from the paddy field water layer after insertion is preferably 2 cm, and the insertion depth into the soil is preferably 10 cm.

[0011] In some embodiments, the graphite rod undergoes surface pretreatment before insertion. Surface pretreatment includes roughening the surface of the graphite rod by sanding it with sandpaper. After surface pretreatment, the graphite rod is ultrasonically cleaned to remove residual particles on the surface and then subjected to high-pressure steam sterilization.

[0012] In some embodiments, the pre-incubation temperature in step S2 is 25-30°C, the culture temperature in step S4 is 25-30°C, and the culture time in step S4 is 7-30 days.

[0013] In some embodiments, the pre-incubation temperature in step S2 is preferably 28°C, the culture temperature in step S4 is preferably 28°C, and the culture time in step S4 is preferably 14 days.

[0014] In some embodiments, the graphite rod can enrich nitrogen-fixing-related microbial groups in the surface biofilm and the surface oxidation zone corresponding to the interfacial soil micro-regions closely attached to the rod. These microbial groups include Geobacteraceae, Rhodocyclaceae, and Xanthobacteraceae.

[0015] In some implementations, graphite rods alter the iron valence state distribution in the topsoil, promoting the conversion of Fe(III) to Fe(II) in the topsoil and enhancing the coupling between iron reduction and nitrogen fixation processes.

[0016] According to another aspect of the present invention, a method for promoting biological nitrogen fixation in paddy fields based on long-distance electron transfer is provided for application in improving the nitrogen fixation capacity of paddy soil, reducing nitrogen fertilizer application, or improving paddy soil.

[0017] The beneficial effects of the present invention are: (1) The present invention does not require an external power source. It only lays conductive medium graphite rods in the soil of flooded paddy fields to cross the surface oxidation zone and the lower reduction zone to form a long-distance electron transfer channel, thereby realizing the directional transport and utilization of the lower reduction equivalent to the surface layer. It provides continuous support for the nitrogen fixation process on the surface layer from the electron supply end to promote the nitrogen fixation process. The operation is simple and easy to promote. (2) The size and layout parameters of the graphite rods are clearly defined, and the standardized pretreatment of sanding, ultrasonic cleaning and high-pressure steam sterilization is conducive to forming a stable biofilm on the surface of the graphite rods and ensuring its stability. (3) Taking the "biofilm on the surface of the graphite rod and the soil micro-regions closely attached to the rod" as the core evaluation object can avoid signal weakening caused by volume soil sample dilution and more accurately reflect the response of nitrogen fixation and its coupling process in the interface micro-regions. (4) Graphite rods can promote the enrichment of nitrogen-fixing microbial communities in the surface oxide zone and change the iron valence state distribution, promote the conversion of Fe(III) to Fe(II), and enhance the coupling of iron reduction and nitrogen fixation processes. Attached Figure Description

[0018] Figure 1 This is a PCoA analysis diagram of the surface microorganisms of graphite rods and the control group in the open system of Example 1 of the present invention at the ASV level, where OE is the surface oxidation zone and RE is the reduction zone.

[0019] Figure 2 This is a diagram showing the difference in microbial groups on the surface of graphite rods and the control group at the scientific level in the open system of Example 1 of the present invention, where OE represents the surface oxidation zone and RE represents the reduction zone.

[0020] Figure 3 This is a comparison diagram of the abundance of the microbial nitrogen fixation gene (nifH) on the surface of graphite rods in the open system of Example 1 of the present invention with that of the control group, where OE is the surface oxidation zone and RE is the reduction zone.

[0021] Figure 4 The graphite rod surface soil and control group in the closed system of Example 2 of the present invention. 15 Comparison of N2 isotope labeling results.

[0022] Figure 5 This is a comparison of Fe(II) content in the soil of the graphite rod micro-region in the closed system of Example 2 of the present invention with that of the control group. OE represents the surface oxidation zone, and RE represents the reduction zone.

[0023] Figure 6 This is a comparison diagram of Fe(III) content in the soil of the graphite rod micro-region in the closed system of Example 2 of the present invention and the control group, where OE is the surface oxide layer and RE is the reduction zone. Detailed Implementation

[0024] The present invention is further described in detail through specific implementation examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. After reading this invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope defined by the appended claims. Unless otherwise specified, all raw materials and reagents of the present invention are commercially available.

[0025] Both Example 1 (open system) and Example 2 (closed system) used soil from the same source, namely typical paddy field black soil. After the soil was naturally air-dried, it was sieved through a 2 mm sieve. Its basic physicochemical properties are as follows: pH 8.12; organic carbon 14.53±0.06 g / kg; total nitrogen 1.392±0.02 g / kg; nitrate nitrogen 2.22±0.05 mg / kg; ammonium nitrogen 11.86±0.12 mg / kg.

[0026] Both Example 1 and Example 2 included a graphite rod treatment group and a control group (CK). Except for the absence of graphite rods, the soil pretreatment, flooding, pre-incubation, and cultivation conditions in the control group were the same as those in the graphite rod treatment group.

[0027] Example 1

[0028] A method for promoting biological nitrogen fixation in paddy fields based on long-distance electron transfer includes the following steps: S1. Soil pretreatment: Collect paddy soil that has not been fertilized with inorganic nitrogen fertilizer or nitrogen-containing organic fertilizer in the previous growing season or within 6-12 months. Remove plant residues and stones, air dry naturally and pass through a 2mm sieve. Add water to the paddy soil to make the water layer height 1cm and establish a flooding system. S2. Pre-incubation: The above-mentioned flooded system is pre-incubated at 25-30℃ for 7 days to form a stable redox stratification; S3. Constructing a long-distance electron transport channel: Using sandpaper with mesh sizes of 100, 600, and 100, a commercially available high-purity graphite rod with a length of 13cm and a diameter of 1.5cm is roughened and pre-treated by grinding. After grinding, the surface of the graphite rod is thoroughly rinsed with deionized water, and then ultrasonically cleaned to remove residual particles from the surface of the graphite rod. The ultrasonic cleaning time is 10min / time, repeated twice, and fresh deionized water is used for each cleaning. After cleaning, the graphite rod is sterilized by high-pressure steam at a temperature of 121℃ for 20min. The pre-treated graphite rod is then vertically inserted into the paddy soil of a flooded system with redox stratification. The top of the graphite rod protrudes 2cm above the water layer, and the insertion depth is 10cm. The high conductivity of the graphite rod is used to construct a long-distance electron transport channel across the redox stratification. S4. Cultivation: Continue cultivation for 14 days under flooded conditions and at 28°C to promote biological nitrogen fixation in paddy fields within the flooded system.

[0029] Sampling Procedure: After the culture in this embodiment is completed, soil samples are taken from the biofilm on the surface of the graphite rods and from the interfacial micro-regions adjacent to the rods, primarily for the evaluation of microorganisms and physicochemical indicators. During sampling, the graphite rods of the graphite rod treatment group are removed under aseptic conditions, the biofilm on the surface of the graphite rods is scraped off and collected into sterile centrifuge tubes to obtain biofilm samples. At the same time, the interfacial micro-region soil adjacent to the rods (0-2 mm) is peeled off along the outer side of the rods and collected into sterile centrifuge tubes. The reduction zone (RE) samples are preferably taken from the area near the lower end of the rods, and the surface oxidation zone (OE) samples are taken from the corresponding water-soil interface layer to obtain interfacial micro-region soil samples. The control group (CK) collected corresponding control soil samples from the same stratum (corresponding to the surface oxidation zone and the lower reduction zone, respectively).

[0030] Total DNA was extracted from the biofilm samples, interfacial micro-region soil samples, and control soil samples, and 16S rRNA amplicon sequencing (Illumina platform) was performed. Sequencing and analysis were commissioned to Shanghai Meiji Company. Figure 1 As shown, the graphite rod treatment group and the control group were clearly separated in the PCoA plot at the ASV level ( Figure 1 This indicates that the micro-regions at the graphite rod interface significantly reshaped the microbial community structure.

[0031] Further analysis of differences in scientific and technological levels can be found in [link to analysis]. Figure 2 This indicates that the graphite rod-treated group enriched groups such as Geobacteraceae, Rhodocyclaceae, and Xanthobacteraceae in the interfacial microregions of the surface oxide zone, demonstrating that the interfacial microregions constructed by graphite rods have a directional enrichment effect on microbial groups related to nitrogen fixation / electron transfer.

[0032] Metagenomic sequencing and functional annotation were performed on soil samples from the aforementioned interfacial microregions. For example... Figure 3 As shown, the abundance of the nitrogen-fixing gene nifH in the graphite rod treatment group was increased compared with the control group, indicating that the graphite rod enhances the nitrogen-fixing potential at the functional level through long-distance electron transport channels.

[0033] Example 2

[0034] Headspace filling within the anaerobic apparatus 15 N2, referring to the specific steps of a method for promoting biological nitrogen fixation in paddy fields based on long-distance electron transfer in Example 1, was cultured in a closed environment in the above-mentioned anaerobic device, with a graphite rod treatment group and a control group (CK) set up. The control group (CK) did not have graphite rods placed, and the other conditions were the same as those of the graphite rod treatment group.

[0035] After the cultivation in this embodiment was completed, samples were taken from the biofilm on the surface of the graphite rods in the graphite rod treatment group and from the interface soil at a depth of 0-2 mm adjacent to the rods, following the sampling procedure described in Example 1. Corresponding soil samples were also collected from the same stratum in the control group (CK). The soil samples were then processed... 15 Nitrogen isotope abundance determination, results are shown in Figure 4 .

[0036] The results show that the graphite rod treatment in the closed system of this embodiment... 15 The nitrogen isotope abundance was 1.444%, compared to the control group (CK). 15 The nitrogen isotope abundance was 0.455%, proving that the graphite rod treatment in this embodiment can promote nitrogen fixation in paddy fields.

[0037] Fe(II) and Fe(III) determinations were performed on stratified soil samples from the graphite rod treatment group and the control group (CK). Interfacial soil samples were preferentially selected from the soil immediately adjacent to the graphite rods. The sampling range for the interfacial soil was 0-2 mm from the outer edge of the graphite rod. Samples from the reduction zone (RE) were preferably taken from the area near the lower end of the rod, while samples from the surface oxidation zone (OE) were taken from the corresponding soil-water interface layer. The control group (CK) collected corresponding soil samples from the same stratum.

[0038] The collected soil samples were sealed and transferred under anaerobic conditions. Extractable iron components were obtained by leaching with 0.5 M hydrochloric acid, and the contents of Fe(II) and Fe(III) were determined. The results are shown below. Figure 5 and Figure 6 .

[0039] The results showed that graphite rod treatment significantly altered the iron valence state distribution in the microregions of the surface oxide zone (OE) interface: compared with the control (CK), the Fe(II) content at the OE end was significantly increased (see...). Figure 5 The Fe(III) content at the OE end was significantly reduced (see...). Figure 6In the reduction zone (RE), the differences between the two groups of Fe(II) and Fe(III) were not significant or the changes were small. These results demonstrate that the graphite rods enhanced the Fe(III) reduction process in the micro-regions at the interface of the surface oxide zone, thus providing more favorable electron supply and coupling conditions for nitrogen fixation in paddy fields.

[0040] Example 3

[0041] A method for promoting biological nitrogen fixation in paddy fields based on long-distance electron transfer includes the following steps: S1. Soil pretreatment: Collect paddy soil that has not been fertilized with inorganic nitrogen fertilizer or nitrogen-containing organic fertilizer in the previous growing season or within 6 months. Remove plant residues and stones, air dry naturally and pass through a 2mm sieve. Add water to the paddy soil to make the water layer height 1cm and establish a flooding system. S2. Pre-incubation: The above-mentioned flooded system is pre-incubated at 25°C for 3 days to form a stable redox stratification; S3. Constructing a long-distance electron transport channel: Using sandpaper with mesh sizes of 100, 600, and 100, a 10cm long and 1cm diameter commercially available high-purity graphite rod is roughened and pre-treated by progressively grinding. After grinding, it is thoroughly rinsed with deionized water, and then ultrasonically cleaned to remove residual particles from the graphite rod surface. The ultrasonic cleaning time is 5 minutes per cycle, repeated once, with fresh deionized water used for each cleaning. After cleaning, it is sterilized by high-pressure steam at a temperature of 121℃ for 20 minutes. The pre-treated graphite rod is then vertically inserted into paddy field soil with a redox stratification system. The top of the graphite rod protrudes 1cm above the water layer, and the insertion depth is 8cm. The high conductivity of the graphite rod is used to construct a long-distance electron transport channel across the redox stratification. S4. Cultivation: Continue cultivation for 7 days under flooded conditions and at 25°C to promote biological nitrogen fixation in paddy fields within the flooded system.

[0042] Example 4

[0043] A method for promoting biological nitrogen fixation in paddy fields based on long-distance electron transfer includes the following steps: S1. Soil pretreatment: Collect paddy soil that has not been fertilized with inorganic nitrogen fertilizer or nitrogen-containing organic fertilizer in the previous growing season or 12 months, remove plant residues and stones, air dry naturally and pass through a 2mm sieve, add water to the paddy soil to make the water layer height 1cm, and establish a flooding system. S2. Pre-incubation: The above-mentioned flooded system is pre-incubated at 30°C for 10 days to form a stable redox stratification; S3. Constructing a long-distance electron transport channel: Using sandpaper with mesh sizes of 100, 600, and 100, a commercially available high-purity graphite rod with a length of 16cm and a diameter of 2cm is roughened and pre-treated by progressive grinding. After grinding, it is thoroughly rinsed with deionized water, and then ultrasonically cleaned to remove residual particles from the surface of the graphite rod. The ultrasonic cleaning time is 20min / time, repeated 3 times, and fresh deionized water is used for each cleaning. After cleaning, it is sterilized by high-pressure steam at a temperature of 121℃ for 20min. The pre-treated graphite rod is vertically inserted into the paddy soil of a flooded system with redox stratification. The top of the graphite rod protrudes 3cm above the water layer and is inserted into the soil to a depth of 12cm. The high conductivity of the graphite rod is used to construct a long-distance electron transport channel across the redox stratification. S4. Cultivation: Continue cultivation for 30 days under flooded conditions and at 30℃ to promote biological nitrogen fixation in paddy fields within the flooded system.

[0044] The above descriptions are merely some embodiments of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A method for promoting biological nitrogen fixation in paddy fields based on long-distance electron transfer, characterized in that, Includes the following steps: S1. Soil pretreatment: Collect paddy field soil that has not been fertilized with nitrogen, remove plant residues and stones, air dry naturally and sieve, add water to the paddy field soil to establish a flooding system; S2. Pre-incubation: The flooded system is pre-incubated at a certain temperature to form a stable redox stratification; S3. Constructing a long-distance electron transport channel: A graphite rod is vertically inserted into the paddy soil of the flooded system that forms a redox stratification, and the high conductivity of the graphite rod is used to construct a long-distance electron transport channel across the redox stratification. S4. Cultivation: Continue cultivation under flooded conditions and at a certain temperature to promote biological nitrogen fixation in paddy fields within the flooded system.

2. The method for promoting biological nitrogen fixation in paddy fields based on long-distance electron transfer according to claim 1, characterized in that, The term "non-nitrogen fertilizer-treated paddy soil" refers to paddy soil that has not been treated with inorganic nitrogen fertilizer or nitrogen-containing organic fertilizer in the previous growing season or within 6-12 months.

3. The method for promoting biological nitrogen fixation in paddy fields based on long-distance electron transfer according to claim 1, characterized in that, The graphite rod is 10-15cm long and 1-2cm in diameter. When the graphite rod is inserted, the top of the rod protrudes 1-3cm above the paddy field water layer, and the insertion depth is 8-12cm.

4. The method for promoting biological nitrogen fixation in paddy fields based on long-distance electron transfer according to claim 3, characterized in that, The graphite rod is preferably 1.5 cm in diameter, and the length of its top protruding from the paddy field water layer after insertion is preferably 2 cm, and the insertion depth into the soil is preferably 10 cm.

5. The method for promoting biological nitrogen fixation in paddy fields based on long-distance electron transfer according to claim 1, characterized in that, The graphite rod undergoes surface pretreatment before insertion. The surface pretreatment includes sanding the surface of the graphite rod with sandpaper to roughen the surface. After surface pretreatment, the graphite rod is ultrasonically cleaned to remove residual particles on the surface and then subjected to high-pressure steam sterilization.

6. The method for promoting biological nitrogen fixation in paddy fields based on long-distance electron transfer according to claim 1, characterized in that, The pre-incubation temperature in step S2 is 25-30℃, the culture temperature in step S4 is 25-30℃, and the culture time in step S4 is 7-30 days.

7. The method for promoting biological nitrogen fixation in paddy fields based on long-distance electron transfer according to claim 6, characterized in that, The preferred temperature for pre-incubation in step S2 is 28°C, the preferred temperature for cultivation in step S4 is 28°C, and the preferred cultivation time in step S4 is 14 days.

8. The method for promoting biological nitrogen fixation in paddy fields based on long-distance electron transfer according to claim 1, characterized in that, The graphite rod can enrich nitrogen-fixing-related microbial groups in the surface biofilm and the surface oxidation zone corresponding to the interface soil micro-regions closely attached to the rod. The microbial groups include Geobacteraceae, Rhodocyclaceae, and Xanthobacteraceae.

9. The method for promoting biological nitrogen fixation in paddy fields based on long-distance electron transfer according to claim 1, characterized in that, The graphite rod alters the iron valence state distribution in the surface soil, promotes the conversion of Fe(III) to Fe(II) in the surface soil, and enhances the coupling between iron reduction and nitrogen fixation processes.

10. The application of any one of the methods for promoting biological nitrogen fixation in paddy fields based on long-distance electron transfer according to claims 1-9 in improving the nitrogen fixation capacity of paddy field soil, reducing nitrogen fertilizer application, or improving paddy field soil.

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