A method for promoting methane oxidation coupled with iron reduction and nitrogen fixation in paddy soil

Abstract

The application discloses a method for promoting methane oxidation coupling iron reduction and nitrogen fixation process in paddy field soil, selects paddy field soil which has not applied or applied less nitrogen fertilizer for a long time, depletes organic carbon and nitrogen in the soil by flooding, adds ferrihydrite, enriches methane oxidation nitrogen fixation microorganisms and iron reduction microorganisms, and application of the method in enriching the methane oxidation nitrogen fixation microorganisms and the iron reduction microorganisms and in reducing paddy field methane emission and nitrogen fertilizer application. The application provides an environment-friendly technology, can enrich iron-dependent methane oxidation coupling biological nitrogen fixation related microorganisms, is suitable for paddy field soil with low iron oxide content and less nitrogen fertilizer application, has strong actual operability, can enhance biological nitrogen fixation of the paddy field, and couples the iron reduction process, so that the use of nitrogen fertilizer is reduced and pollution to the environment is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of methane-oxidizing nitrogen-fixing microorganism enrichment, and particularly relates to a method for promoting methane oxidation coupled with iron reduction and nitrogen fixation in paddy field soil. BACKGROUND

[0002] Methane plays an important role in human production and life as an energy source, and it is also the second largest greenhouse gas after carbon dioxide. Paddy fields and natural wetland ecosystems are one of the main sources of methane emission, and it is very important to reduce methane emission.

[0003] Rice is one of the most important food crops. Since chemical nitrogen fertilizer has been applied to paddy fields, the yield of rice has been greatly improved. However, long-term or excessive nitrogen fertilizer application can lead to the release of nitrogen load, i.e. ammonia volatilization, nitrous oxide emission and nitrate leaching from paddy soil to the natural environment, which can cause global warming, water eutrophication, nitrate pollution of groundwater and other environmental problems. Reducing the use of nitrogen fertilizer is a necessary condition for environmentally friendly and sustainable rice production. Developing an environmentally friendly and operable technology to promote methane oxidation and nitrogen fixation by microorganisms provides a potential way to reduce methane emission, and at the same time achieves the purpose of reducing the amount of nitrogen fertilizer application, which is of great significance for responding to the concept of sustainable development of ecological environment and energy saving and emission reduction. SUMMARY

[0004] The present application aims to provide a method for promoting methane oxidation coupled with iron reduction and nitrogen fixation in paddy field soil.

[0005] According to one aspect of the present application, a method for promoting methane oxidation coupled with iron reduction and nitrogen fixation in paddy field soil is provided, which selects paddy field soil that has not been applied or has been applied with less nitrogen fertilizer for a long time, depletes organic carbon and nitrogen in the soil by flooding, adds ferrihydrite, and enriches methane-oxidizing nitrogen-fixing microorganisms and iron-reducing microorganisms.

[0006] In some embodiments, the method comprises the following steps:

[0007] S1. selecting paddy field soil that has not been applied or has been applied with less nitrogen fertilizer for a long time, crushing and sieving, taking a certain amount of sieved soil into a sealed container, and depleting organic carbon and nitrogen in the soil by flooding;

[0008] S2. taking the supernatant in the container, adding 300 mM ferrihydrite solution and nitrogen-free inorganic salt medium, then removing most of the oxygen in the container by nitrogen exposure, so that the volume content of headspace oxygen in the container is 0.3%, sealing the container, and adding methane to the headspace to enrich methane-oxidizing nitrogen-fixing microorganisms and iron-reducing microorganisms.

[0009] In some embodiments, the paddy field soil contains methane-oxidizing nitrogen-fixing microorganisms and iron-reducing microorganisms.

[0010] In some embodiments, the mesh size of the sieve in step S1 is 1 mm, the soil is flooded at a water to soil ratio of 1:1, and the duration of the flooding treatment is 2-3 weeks.

[0011] In some embodiments, the volume of the ferrihydrite solution in step S2 is 1:1-50 times the volume to mass ratio of the soil and water in step S1.

[0012] In some embodiments, the methane accounts for 10% of the volume of the gas in the container in step S2, and the container is placed in a normal temperature and dark condition for microbial enrichment in step S2.

[0013] In some embodiments, the methane-oxidizing nitrogen-fixing microorganisms include Methylocystis, Methylomicrobium, and Unclassified Methylophilaceae.

[0014] In some embodiments, the iron-reducing microorganisms include Methylocystis, Methylomicrobium, Unclassified Methylophilaceae, Methylophilaceae, Pseudomonas, Thiobacillus, Bradyrhizobium, Geoalkalibacter, and Anaeromyxobacter.

[0015] According to another aspect of the present application, there is provided a use of a method for promoting methane oxidation coupled with iron reduction and nitrogen fixation processes in paddy field soil in enriching methane-oxidizing nitrogen-fixing microorganisms and iron-reducing microorganisms.

[0016] According to another aspect of the present application, there is provided a use of a method for promoting methane oxidation coupled with iron reduction and nitrogen fixation processes in paddy field soil in reducing methane emissions and nitrogen fertilizer application in paddy fields.

[0017] The present application has the following beneficial effects: (1) In the present application, the addition of ferrihydrite couples the iron reduction process, reduces Fe 3+ in ferrihydrite to Fe 2+ , and enhances the methane oxidation and nitrogen fixation of microorganisms in paddy fields, thereby reducing methane emissions in paddy fields. Under the coexistence of methane, ferrihydrite, and nitrogen, the consumption of methane by microorganisms, the production of ammonia nitrogen, and the reduction rate of Fe 3+ are significantly improved, which proves that under the condition of nitrogen deficiency, the microorganisms in the soil utilize ferrihydrite to promote the methane oxidation, iron reduction, and nitrogen fixation processes;

[0018] (2) This invention provides an environmentally friendly technology that can enrich microorganisms related to methane oxidation, iron reduction and nitrogen fixation. It is suitable for paddy soil with low iron oxide content and low nitrogen fertilizer application. It is also highly operable in practice. It provides a solution for enriching methane oxidizing bacteria and iron reducing bacteria, and provides technical support for production applications that further enhance biological nitrogen fixation and iron reduction in paddy fields and reduce nitrogen fertilizer application. Attached Figure Description

[0019] Figure 1 In Example 3 of the present invention, methane, ammonia nitrogen, and Fe... 2+ The content of [a substance] changes over time.

[0020] Figure 2 The different treatments of Example 4 of the present invention on day 100, including methane, ammonia nitrogen, and Fe. 2+ and 15 Comparison chart of nitrogen isotope content.

[0021] Figure 3 In Example 5 of this invention, different treatments involving the addition of difluoromethane inhibitors were performed on methane, ammonia nitrogen, and Fe. 2+ Content changes over time.

[0022] Figure 4 This is a graph showing the copy number of pmoA, mcrA, and nifH genes and the expression level of corresponding RNA after 20 days of culture under different treatments in Example 5 of the present invention.

[0023] Figure 5 In different treatments of Example 6 of the present invention, the mud after 30 days of cultivation 15 N isotope content and 13 Comparison chart of C isotope content.

[0024] Figure 6 This is a qPCR quantitative diagram of the functional genes pmoA, mcrA, bacterial 16S rRNA, nifH, and archaea 16S rRNA in layer 24 of Example 7 of the present invention.

[0025] Figure 7 This is a sequencing diagram of the 16S rRNA gene in the heavy and light layers of Example 7 of the present invention.

[0026] Figure 8 This is a sequencing diagram of the nifH gene in the heavy and light layers of Example 7 of the present invention. Detailed Implementation

[0027] The application will be further described in detail through specific embodiments. It should be understood that these embodiments are only used to illustrate the application and not used to limit the protection scope of the application. After reading the application, those skilled in the art can make various equivalent modifications of the application, which fall within the scope defined by the appended claims. If not otherwise specified, all raw materials and reagents of the application are commercially available.

[0028] Embodiment 1

[0029] A method for promoting methane oxidation coupled with iron reduction and nitrogen fixation in paddy field soil, comprising the following steps:

[0030] 750g of paddy field soil which has not been applied or has been applied with little nitrogen fertilizer for a long time is selected and placed in a glass bottle, 750g of water is added to the paddy field soil so that the water surface is above the soil surface, and the paddy field soil is subjected to flooding treatment for 2-3 weeks to deplete the organic carbon and nitrogen in the soil;

[0031] Then, the water above the soil is drained to expose the paddy field soil, 30mL of ferrihydrite solution with a molar concentration of 300mM is added, 1000mL of nitrogen-free inorganic salt medium is added, nitrogen gas is introduced into the glass bottle to remove oxygen therein, the glass bottle is sealed, and methane is added to the headspace to enrich methane-oxidizing nitrogen-fixing microorganisms and iron-reducing microorganisms.

[0032] Embodiment 2

[0033] A method for promoting methane oxidation coupled with iron reduction and nitrogen fixation in paddy field soil, comprising the following steps:

[0034] S1. Selecting paddy field soil which has not been applied or has been applied with little nitrogen fertilizer for a long time, the paddy field soil comprising methane-oxidizing nitrogen-fixing microorganisms and iron-reducing microorganisms, i.e., Methylocystis, Methylomicrobium, Unclassified Methylophilaceae, Pseudomonas, Thiobacillus, Bradyrhizobium, Geoalkalibacter, and Anaeromyxobacter, crushing the soil through a sieve with a pore size of 1mm, and detecting the sieved soil to measure that the ammonia nitrogen content is 0.146mg / kg, the nitrate content is 0.707mg / kg, the total iron content is 1.989g / kg, the total carbon content is 33.816g / kg, and the pH is 6.91;

[0035] 750g of the sieved soil is taken and placed in a glass bottle with a volume of 2L, 750mL of ultrapure water is added to flood the soil for 2-3 weeks to deplete the organic carbon and nitrogen therein;

[0036] S2. Remove the supernatant from the glass bottle, add 30 mL of 300 mM ferrohydrate solution, add 1000 mL of nitrogen-free inorganic salt culture medium, then aerate the glass bottle with nitrogen to remove most of the oxygen, so that the headspace oxygen content is 0.3%. Seal the glass bottle, add methane to the headspace, the volume of methane being 10% of the gas volume in the sealed glass bottle, and incubate the sealed glass bottle under room temperature and dark conditions to enrich methane-oxidizing nitrogen-fixing microorganisms and iron-reducing microorganisms.

[0037] Example 3

[0038] Using the method of Example 2, every 10 days, 0.1 mL of headspace gas was taken from a sealed glass bottle for testing. The liquid phase in the sealed glass bottle was then mixed thoroughly, and 2 mL of mud slurry was taken for testing. The methane content in the taken headspace gas was determined using a Shimadzu gas chromatograph, with each bottle tested three times to correct for sampling errors. The Fe content in the taken mud slurry was determined using the o-phenanthroline colorimetric method. 2+ The content was determined by extracting inorganic nitrogen from the extracted mud using 3M KCl, and the NH4 content was determined by the indophenol blue colorimetric method. + The measurements were performed. The amount of methane remaining in the headspace was used to determine whether to add nitrogen and methane to the sealed glass vial. The amount of ferric iron reduced in the mud was used to determine whether to replace the supernatant in the sealed glass vial with nitrogen-free inorganic salt culture medium and add ferrous sulfate solution. The results are attached. Figure 1 .

[0039] Depend on Figure 1 It can be seen that during the 0-55 day incubation period, the headspace methane content in the sealed glass bottle gradually decreased, and the Fe content in the mud (i.e., soil) decreased. 2+ NH4 + The content of methane and Fe gradually increased, indicating that... 2+ NH4 + The significant correlation between the contents of the three also indicates that microorganisms reduce some of the Fe in the ferroalloy through iron reduction. 3+ Reduced to Fe 2+ This causes Fe in the mud to 2+ Increased content; microorganisms convert nitrogen gas into NH4 through nitrogen fixation. + After adding methane, 300mM ferrous sulfate, and nitrogen-free inorganic nutrient solution to the sealed glass bottle on day 55, the second culture cycle, from day 56 to day 110, begins. Figure 1 The results showed that the headspace methane content gradually decreased in the second culture cycle, and the Fe content in the mud (i.e., soil) also decreased. 2+ NH4 +The content of [something] gradually increased. The same phenomenon occurred in the third culture cycle (days 111-165), indicating that in the culture method of this embodiment, methane and Fe [something] in the soil [something] reacted [something]. 2+ NH4 + The contents of the three substances were significantly correlated.

[0040] Example 4: Methane and Fe in different treatments 2+ NH4 + Changes in content

[0041] Four treatments were set up: nitrogen (N), methane + nitrogen (CN), ferrohydrate + nitrogen (FeN), and methane + ferrohydrate + nitrogen (CFeN). Each treatment was configured with three replicates. A control treatment without soil was also included, namely methane + ferrohydrate + nitrogen + soil-free treatment (CFeNS0). All five treatments were incubated at room temperature in the dark. After 100 days of incubation, the levels of methane and Fe in the sealed glass vials of each treatment were measured. 2+ NH4 + The content of.

[0042] The specific treatment operation of nitrogen group (N) is as follows: Take 1g of the sieved soil from step S1 in Example 2, place it in a glass bottle with a volume of 60mL, expose the glass bottle to high-purity nitrogen for 30min, and then seal the bottle with a rubber stopper and an aluminum cap.

[0043] The specific treatment operation of methane + nitrogen (CN) group is as follows: Take 1g of the sieved soil from step S1 in Example 2, place it in a glass bottle with a volume of 60mL, aerate the glass bottle with high-purity nitrogen for 30min, seal the bottle with a rubber stopper and aluminum cap, add methane to the headspace inside the sealed glass bottle, and the methane accounts for 10% of the gas volume inside the sealed glass bottle.

[0044] The specific treatment operation of the ferrohydrate + nitrogen group (FeN) is as follows: Take 1g of the sieved soil from step S1 in Example 2, place it in a glass bottle with a volume of 60mL, add 1mL of 300mM ferrohydrate, and make up to 10mL with nitrogen-free inorganic salt culture medium. After aerating the glass bottle with high-purity nitrogen for 30min, seal the bottle with a rubber stopper and an aluminum cap.

[0045] The specific treatment operation of the methane + ferrous sulfate + nitrogen group (CFeN) is as follows: Take 1g of the sieved soil from step S1 in Example 2, place it in a 60mL glass bottle, add 1mL of 300mM ferrous sulfate, and make up to 10mL with nitrogen-free inorganic salt culture medium. After aerating the glass bottle with high-purity nitrogen for 30min, seal the bottle with a rubber stopper and aluminum cap, and add methane to the headspace of the sealed glass bottle. The methane accounts for 10% of the gas volume in the sealed glass bottle.

[0046] The specific treatment procedure for the methane + ferrohydrate + nitrogen + soil-free group (CFeNS0) is as follows: Add 1 mL of 300 mM ferrohydrate to a 60 mL glass bottle, bring the volume to 10 mL with nitrogen-free inorganic salt culture medium, aerate the glass bottle with high-purity nitrogen for 30 min, seal the bottle with a rubber stopper and aluminum cap, and add methane to the headspace of the sealed glass bottle, with the methane accounting for 10% of the gas volume in the sealed glass bottle;

[0047] After 100 days of cultivation, the levels of methane and Fe in the sealed glass bottles of each treatment were measured. 2+ NH4 + The content results are attached. Figure 2 .like Figure 2 As shown in Figure A, the methane content of the methane + ferrous sulfate + nitrogen group (CFeN) was lower than that of the methane + nitrogen group (CN) and the methane + ferrous sulfate + nitrogen + soilless group (CFeNS0), indicating that the addition of ferrous sulfate promotes methane oxidation by microorganisms in the soil. Figure 2 As shown in B, the Fe in the methane + ferrohydrate + nitrogen group (CFeN) 2+ The higher content compared to other treatment groups indicates that the presence of methane and ferrous sulfate promotes the microbial iron reduction process. Figure 2 As shown in C, the NH4 in the methane + ferrous oxide + nitrogen group (CFeN) + The higher content compared to other treatment groups indicates that methane and ferrous sulfate have a promoting effect on nitrogen fixation by methanotrophic bacteria.

[0048] The nitrogen in the methane + ferrohydrite + nitrogen group (CFeN), ferrohydrite + nitrogen group (FeN), methane + nitrogen group (CN), and nitrogen group (N) was replaced with isotopes. 15 N2, yielding methane + ferrohydrate + isotopic nitrogen group (CFe) 15 N), ferrohydrate + nitrogen isotope group (Fe) 15 N), methane + isotopic nitrogen group (C 15 N), isotopic nitrogen group ( 15 The above-mentioned treatment group with replaced nitrogen gas and the methane + ferrohydrate + nitrogen gas group (CFeN) were placed in the dark at room temperature for 100 days, and the concentration of nitrogen in the soil inside the sealed glass bottles of each treatment was measured. 15 The content of nitrogen (N).

[0049] like Figure 2 As shown in D, the methane + ferrohydrate + isotopic nitrogen group (CFe) 15 N) fixed in soil 15 The highest N content indicates that the methane + ferrohydrate + isotopic nitrogen group (CFe) 15 The nitrogen-fixing activity of N was the strongest. This demonstrates that, in the presence of methane and ferrous sulfate, methane-oxidizing bacteria and iron-reducing bacteria in the soil synergistically promote nitrogen fixation.

[0050] Example 5

[0051] To determine the effect of the water iron ore on the nitrogen fixation pathway of the methane-oxidizing bacteria, the slurry after 150 days of culture of the methane + water iron ore + nitrogen treatment (CFeN) group of Example 4 was taken for the following experiments, and three treatments were set up, namely: methane + nitrogen group (CN), methane + water iron ore + nitrogen group (CFeN), and difluoromethane + methane + water iron ore + nitrogen group (CFeN with CH2F2), wherein difluoromethane can inhibit the activity of methane monooxygenase (MMO), and 18 replicates were set up for each treatment.

[0052] The specific treatment operations of the methane + nitrogen group (CN) and the methane + water iron ore + nitrogen group (CFeN) in this example were carried out with reference to the specific treatment operations of the methane + nitrogen group (CN) and the methane + water iron ore + nitrogen group (CFeN) in Example 4, except that 4 ml of slurry was added in this example instead of 1 g of soil in Example 4.

[0053] The specific treatment operation of the difluoromethane + methane + water iron ore + nitrogen group (CFeN with CH2F2) was as follows: 4 mL of slurry after 150 days of culture of the CFeN group in Example 4 was taken and placed in a glass bottle with a volume of 60 mL, 1 ml of 300 mM water iron ore was added, and the volume was made up to 10 mL with a nitrogen-free inorganic salt medium. After the glass bottle was exposed to high-purity nitrogen gas for 30 min, the bottle was sealed with a rubber plug and an aluminum cap, and methane and difluoromethane were added to the headspace of the sealed glass bottle, with methane accounting for 10% of the gas volume in the sealed glass bottle, and difluoromethane accounting for 2.5% of the gas volume in the sealed glass bottle.

[0054] The three treatment groups described above were placed in a shaker at a temperature of 30°C for 50 days, and on the 0th, 10th, 20th, 30th, 40th, and 50th day, 3 replicates were randomly selected from each treatment group, and the contents of methane, Fe 2+ , NH4 + were determined, and the results are shown in the accompanying Figure 3 .

[0055] As shown in Figure 3 , the methane + water iron ore + nitrogen group (CFeN) gradually reduced the methane content in the sealed glass bottle, and gradually increased the contents of Fe 2+ , NH4 + , and the methane content in the sealed glass bottle was lower than that of the difluoromethane + methane + water iron ore + nitrogen group (CFeN with CH2F2) and the methane + nitrogen group (CN) at the same culture time, and the contents of Fe 2+ , NH4 +The content of methane in the methane + water iron ore + nitrogen group (CFeN) was higher than that in the other two treatment groups. It was shown that the methane consumption and consumption rate in the methane + water iron ore + nitrogen group (CFeN) of the present embodiment were much higher than those in the other two treatment groups, and the iron reduction amount, iron reduction rate, nitrogen fixation amount and nitrogen fixation rate were higher than those in the other two treatment groups. It was further confirmed that methane and water iron ore had a synergistic promotion effect on the methane oxidation, nitrogen fixation and iron reduction processes of methanotrophs. The addition of water iron ore can not only accelerate the consumption of methane by methanotrophs, but also couple the iron reduction process of methanotrophs in the soil, and further promote the nitrogen fixation of microorganisms in the soil, converting gaseous nitrogen into ionic nitrogen that can be utilized by plants.

[0056] In order to determine the related microbial community and metabolic mechanism in the process of methane oxidation, iron reduction and nitrogen fixation, after 20 days of culture, the slurry of each treatment group was sampled, then the total DNA of the microbial community in the slurry sample was extracted by using the Fast DNA SPIN Kit of MP Bio Company, the total RNA of the microbial community in the slurry sample was extracted by using the OMEGA soil RNA Mini kit, and the total cDNA of the soil microbial community was obtained by reverse transcription using the TaKaRa PrimeScript II 1st Strand cDNA Synthesis Kit as a template, which was used to detect the expression amount of the corresponding functional genes (pmoA, mcrA, nifH genes). The specific results are shown in the following table 1. Figure 4 .

[0057] As shown in Figure 4 A, the copy number of pmoA, mcrA and nifH genes of the methane + water iron ore + nitrogen group (CFeN) at the 20th day of culture was significantly higher than that of the methane + nitrogen group (CN) and the difluoromethane + methane + water iron ore + nitrogen group (CFeN with CH2F2). As shown in Figure 4 B, the RNA expression amount of pmoA and nifH genes of the methane + water iron ore + nitrogen group (CFeN) was significantly higher than that of the methane + nitrogen group (CN) and the difluoromethane + methane + water iron ore + nitrogen group (CFeN with CH2F2), and the expression amount of the mcrA gene was lower than that of the difluoromethane + methane + water iron ore + nitrogen group (CFeN with CH2F2) and higher than that of the methane + nitrogen group (CN). It was shown that the inhibition of methane monooxygenase by difluoromethane affected the expression of pmoA gene of methanotrophs, and the expression amount of nifH gene also decreased accordingly, which further indicated that methane oxidation and iron reduction promoted biological nitrogen fixation in a mutual promotion relationship. It was also shown that the addition of water iron ore promoted the reproduction of methanotrophs and also promoted the expression of pmoA gene and nifH gene.

[0058] Example 6

[0059] To determine the relevant microbial communities and metabolic mechanisms involved in the reduction and nitrogen fixation process using iron oxide from methane, three treatments were set up: the methane + nitrogen isotope group (…). 13 C 15 N), methane + ferrohydrate + nitrogen isotope group ( 13 CFe 15 N), difluoromethane + methane + ferrite + nitrogen isotope group ( 13 CFe 15 The specific processing operations for the three treatment groups mentioned above are the same as those for the CN group, CFeN group, and CFeN with CH2F2 group in Example 5, respectively. The difference is that in this example, isotopic nitrogen gas is used. 15 Instead of nitrogen, N2 was used, and isotope methane was employed. 13 CH4 was used instead of methane. The culture conditions were the same as in Example 5. On day 50 of culture, isotopic analysis was performed on mud samples from the three treatment groups. 15 N and isotopes 13 C content, results are attached. Figure 5 .

[0060] like Figure 5 As shown, the isotope composition of methane + ferrohydrate + nitrogen ( 13 CFe 15 Isotopes in mud (N) 13 C and isotopes 15 The N content was significantly higher than that of the methane + nitrogen isotope group. 13 C 15 N), difluoromethane + methane + ferrite + nitrogen isotope group ( 13 CFe 15 (N with CH2F2). This indicates that the addition of ferrihydrite promotes the methane oxidation and iron reduction processes, and that ferrihydrite is related to the iron reduction cycle of microorganisms.

[0061] Example 7

[0062] The methane + ferrohydrate + nitrogen isotope composition in Example 6 ( 13 CFe 15 DNA was extracted from the mud containing N), and then separated using cesium chloride density gradient centrifugation. 15 N-DNA and 14 For N-DNA analysis of microbial community structure, qPCR was used to quantify the functional genes pmoA, mcrA, 16S rRNA, nifH, and 16S rRNA Archeaea in the DNA of layer 24. Primer sequences are shown in Table 1 below. Results are attached. Figure 6 .

[0063] Table 1 primer sequence list of each functional gene

[0064]

[0065] The isolated N-DNA was then sequenced 15 N-DNA and 14 The heavy and light layers in the N-DNA were respectively sequenced for 16S rRNA and nifH functional genes to determine the corresponding microbial community structure and metabolic mechanism. The specific results are shown in the following tables. Figures 7-8 .

[0066] The results of cesium chloride density gradient centrifugation (see Figure 6 ) and 16S rRNA gene sequencing (see Figure 7 ) showed that the methane-oxidizing nitrogen-fixing microorganisms were mainly the following three methane-oxidizing bacteria Methylocystis, Methylomicrobium, and Unclassified Methylophilaceae. The results of nifH functional gene sequencing (see Figure 8 ) showed that the hydro ferrite played the role of an electron acceptor and the microorganisms involved in iron reduction were Methylocystis, Methylomicrobium, Unclassified Methylophilaceae, Pseudomonas, Thiobacillus, Bradyrhizobium, Geoalkalibacter, and Anaeromyxobacter. It was indicated that the enhancement of methane-oxidizing nitrogen fixation in the soil was related to the reduction of Fe 3+ in the hydro ferrite.

[0067] In summary, by adding hydro ferrite to introduce Fe 3+ ions in the soil, the microorganisms related to methane-oxidizing nitrogen fixation and iron reduction were enriched in the artificially controlled environment, and finally the nitrogen fixation of the microorganisms was strengthened, the methane was consumed, the emission of greenhouse gases was reduced, and the carbon sink was increased. These results show that the hydro ferrite regulation treatment is an environmentally friendly technology for methane-oxidizing nitrogen fixation and reduction of methane emission in the paddy field and the rhizosphere of flooded plants, and has great development and application value for reducing methane emission in the paddy field and reducing nitrogen fertilizer application.

[0068] The above only describes some embodiments of the present application, and those of ordinary skill in the art can make several modifications and improvements without departing from the inventive concept, and these all belong to the protection scope of the present application.

Claims

1. A method for promoting methane oxidation coupled with iron reduction and nitrogen fixation processes in paddy soil, characterized in that, The method comprises the following steps: S1. selecting paddy soil which has not been applied with nitrogen fertilizer for a long time or has been applied with little nitrogen fertilizer, crushing and sieving the soil, taking a certain amount of sieved soil into a container, and depleting organic carbon and nitrogen in the soil by flooding; S2. taking supernatant in the container, adding 300 mM ferrihydrite solution and nitrogen-free inorganic salt medium, removing most of the oxygen in the container by nitrogen exposure, so that the volume content of oxygen in the headspace of the container is 0.3%, sealing the container, and adding methane in the headspace to enrich methanotrophic nitrogen-fixing microorganisms and iron-reducing microorganisms.

2. The method of claim 1, wherein, The paddy soil contains methanotrophic nitrogen-fixing microorganisms and iron-reducing microorganisms.

3. The method of claim 1, wherein, The pore size of the sieve in step S1 is 1 mm, the soil is flooded at a water-soil ratio of 1:1, and the duration of the flooding treatment is 2-3 weeks.

4. The method of claim 1, wherein, In step S2, the volume of the ferrihydrite solution is 1:1-50 of the volume-to-mass ratio of the mass of the soil and water in step S1.

5. The method of claim 1, wherein, In step S2, the volume of methane accounts for 10% of the volume of gas in the container, and the container is placed in a normal temperature and dark condition for microbial enrichment.

6. The method of claim 1, wherein, The methanotrophic diazotrophs include Methylocystis, Methylomicrobium, Unclassified Methylophilaceae .

7. The method of claim 1, wherein, The iron-reducing microorganisms include Methylocystis, Methylomicrobium, Unclassified Methylophilaceae, Methylophilaceae, Pseudomonas, Thiobacillus, Bradyrhizobium, Geoalkalibacter, Anaeromyxobacter .

8. The method of any one of claims 1-7 for use in enriching methanotrophic nitrogen-fixing microorganisms and iron-reducing microorganisms.

9. The method of any one of claims 1-7 for use in reducing methane emissions and nitrogen fertilizer application in rice fields.

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