Iron-reducing azotobacter, method for promoting nitrogen fixation by iron reduction and application thereof

Abstract

The application provides an iron-reducing diazotroph, a method for promoting nitrogen fixation by iron reduction and application thereof. Klebsiella grimontii The iron-reducing diazotroph is Klebsiella (Klebsiella) N7, which is preserved in the China Center for Type Culture Collection on March 20, 2025, and the preservation number is CCTCC M 2025550. The nitrogen fixation method comprises the following steps: S1. selecting a strain with iron reduction function and nitrogen fixation capacity, and adding Fe 3+ under anaerobic and N2 conditions to promote nitrogen fixation; S2. after Fe 3+ is completely reduced, a certain amount of oxygen is introduced, Fe 2+ is oxidized to Fe 3+ ; S3. after Fe 3+ is completely oxidized and the oxygen is consumed, nitrogen fixation is continued, and through the repeated circulation of the abiotic oxidation and microbial reduction of iron, continuous nitrogen fixation is realized. The iron-reducing diazotroph can couple the iron reduction and nitrogen fixation processes, and promote nitrogen fixation by iron reduction, thereby improving the nitrogen fixation efficiency.

Description

Technical Field

[0001] This invention relates to the field of nitrogen fixation technology using iron-reducing nitrogen-fixing microorganisms, specifically to an iron-reducing nitrogen-fixing bacterium, a method for promoting nitrogen fixation using iron reduction, and their applications. Background Technology

[0002] Nitrogen fixation is crucial in modern agricultural production, converting atmospheric nitrogen into nitrogen compounds usable by plants, thus significantly improving soil fertility and crop yield. Current nitrogen fertilizer production primarily relies on industrial nitrogen fixation to convert nitrogen into ammonia, which is then used to produce chemical fertilizers. However, the production process of chemical fertilizers leads to substantial carbon emissions, and while the widespread use of chemical nitrogen fertilizers has increased agricultural output in the short term, it has also caused environmental pollution, the generation of the greenhouse gas nitrogen oxides, and other problems, further exacerbating global warming. Therefore, developing green and low-carbon nitrogen fixation and nitrogen fertilizer technologies, and reducing nitrogen fertilizer use, is essential for environmental friendliness and sustainable development.

[0003] Iron is a major element in nature, widely distributed. Iron redox processes play a crucial role in ecosystems, significantly impacting the cycling of various elements, the metabolic activities of organisms / microorganisms, and nutrient cycling. However, whether iron reduction processes promote nitrogen fixation and the practical application potential of this promotional effect remain unknown. Summary of the Invention

[0004] To address the problems existing in the background art, the present invention provides an iron-reducing nitrogen-fixing bacterium, a method for promoting nitrogen fixation using iron reduction, and its application. This bacterium can couple iron reduction and nitrogen fixation, and promote nitrogen fixation through iron reduction, thereby improving nitrogen fixation efficiency. The method of promoting nitrogen fixation using iron reduction can significantly improve soil fertility and plant growth, and reduce the application of nitrogen fertilizer. The technical solution of the present invention to solve the above-mentioned technical problems is as follows: This invention provides an iron-reducing nitrogen-fixing bacterium, namely Klebsiella pneumoniae ( Klebsiella grimontii N7 was deposited at the China Center for Type Culture Collection on March 20, 2025, with accession number CCTCC M 2025550.

[0005] According to the above scheme, the Klebsiella pneumoniae ( Klebsiella grimontii N7 has the ability to fix nitrogen and reduce iron, is a facultative anaerobe, and performs nitrogen fixation by coupling iron reduction.

[0006] Secondly, the present invention provides a method for promoting nitrogen fixation by iron reduction, comprising the following steps: S1. Select strains with iron reduction and nitrogen fixation capabilities, and facultative anaerobic properties, and conduct experiments under anaerobic, N2-containing conditions in the presence of Fe. 3+ Nitrogen fixation was carried out by culturing in a nitrogen-free medium.

[0007] S2. In Fe 3+ After complete reduction, oxygen is introduced into the culture medium to remove Fe. 2+ Oxidized to Fe 3+ ; S3. In Fe 3+ Nitrogen fixation continues even after complete oxidation and depletion of oxygen, through repeated cycles of abiotic oxidation of iron and microbial reduction.

[0008] According to the above scheme, the nitrogen fixation method also includes step S4: repeating steps S2 and S3 above.

[0009] Preferably, the strain possessing iron reduction and nitrogen fixation capabilities is the aforementioned Klebsiella pneumoniae (Klebsiella pneumoniae). Klebsiella grimontii N7.

[0010] According to the above scheme, Fe 3+ The source is selected from at least one of ferric citrate, ferric chloride, or ferric sulfate, Fe 3+ The concentration is 0.2 mM to 1 mM. In step S1, N2 is used to remove oxygen and create an oxygen-free, N2-containing environment. In step S2, the oxygen injection rate is 5% to 10% of the headspace volume. The injected oxygen will reduce Fe... 2+ Oxidized to Fe 3+ Once the oxygen is depleted, the bacteria will undergo another iron reduction process, thereby promoting nitrogen fixation.

[0011] According to the above scheme, the pH of the nitrogen-free culture medium is 7.0~7.2, the culture temperature is 20~40℃, the culture time in step S1 is 2~7 days, and the culture time in step S3 is 2~4 days.

[0012] Preferably, the culture temperature is 28~30℃.

[0013] Thirdly, the present invention provides the above-mentioned Klebsiella pneumoniae ( Klebsiella grimontii Any of the following applications of N7: I. Preparation of microbial inoculants; II. Soil nitrogen fixation and improvement of soil fertility; III. Improve crop yield and quality.

[0014] According to the above scheme, the iron-reducing nitrogen-fixing bacteria are applied in the following way: Klebsiella pneumoniae (… Klebsiella grimontii N7 is prepared into a microbial agent and applied to the soil along with iron fertilizer to fix nitrogen in the soil and improve soil fertility.

[0015] According to the above scheme, the iron-reducing nitrogen-fixing bacteria are applied in the following way: Klebsiella pneumoniae (… Klebsiella grimontiiN7 is prepared into a microbial agent and applied to the soil before crop planting and iron fertilizer application, and the crops are cultivated in a conventional manner to improve crop yield and quality.

[0016] Fourthly, the present invention provides a method for promoting nitrogen fixation in soil using iron reduction, wherein the above-mentioned Klebsiella pneumoniae ( Klebsiella grimontii N7 is prepared into a microbial agent, and the microbial agent and iron fertilizer are applied to the soil. Crops are planted and cultivated in a conventional manner, and nitrogen fixation is promoted by utilizing the redox fluctuations of iron in the soil.

[0017] According to the above plan, the inoculant should be applied at a rate of not less than 1.33 × 10⁻⁶ per acre. 13 The amount of live bacteria to be applied is 20-30 kg / mu for the iron fertilizer.

[0018] Following the above plan, after applying the microbial agent and iron fertilizer, use mechanical means to mix the microbial agent and iron fertilizer evenly with the topsoil, and after maintaining this mixture for 5 to 15 days, sow the seeds.

[0019] The beneficial effects of this invention are: 1) This invention screened and obtained a strain of iron-reducing nitrogen-fixing bacterium Klebsiella pneumoniae ( Klebsiella grimontii N7, which can couple iron reduction for microbial nitrogen fixation, under anaerobic conditions, this strain reduces Fe2+. 3+ Reduced to Fe 2+ During iron reduction, nitrogen is converted into bioavailable ammonia, which is then used in the microorganism's metabolism to produce amino acids or other nitrogen-containing organic matter, thus achieving nitrogen fixation. This is achieved by adding Fe to the culture medium. 3+ Afterwards, compared to not adding Fe 3+ Its nitrogen fixation efficiency increased by 26.67%; 2) The method for promoting nitrogen fixation using iron reduction provided by this invention first utilizes iron-reducing nitrogen-fixing bacteria to perform microbial reduction of iron (Fe). 3+ →Fe 2+ This promotes microbial nitrogen fixation and introduces oxygen for the abiotic oxidation of iron (Fe). 2+ →Fe 3+ The oxidized iron was then subjected to microbial reduction again under anaerobic conditions (Fe). 3+ →Fe 2+ This promotes microbial nitrogen fixation by using iron redox fluctuations (repeated cycles of abiotic oxidation and microbial reduction) to achieve continuous nitrogen fixation and improve nitrogen fixation efficiency; Klebsiella pneumoniae ( Klebsiella grimontii N7 was used in this method, with iron (Fe) added externally to the culture medium. 3+ Then, iron redox fluctuations were performed, compared to not adding Fe. 3+ Its nitrogen fixation efficiency increased by 95.58%; 3) The Klebsiella pneumoniae of the present invention ( Klebsiella grimontii N7 can be prepared into microbial inoculants and applied to the soil along with iron fertilizer. The iron reduction and nitrogen fixation of microorganisms can help increase the content of available nitrogen sources for crops in the soil, improve soil fertility, increase crop yield and quality, and reduce the application of chemical fertilizers. This can reduce the environmental problems caused by the use of chemical fertilizers (including reducing carbon emissions caused by nitrogen fertilizer production, reducing soil and water pollution, reducing nitrogen oxide emissions, thereby mitigating the impact of greenhouse gases on climate change), reduce production costs and improve economic efficiency, and achieve green and sustainable agricultural development. Attached Figure Description

[0020] Figure 1 This is the result of using iron reduction to promote nitrogen fixation in Example 2 of the present invention; Figure 2 This is the result of using iron redox fluctuations to promote nitrogen fixation in Example 2 of the present invention; Figure 3 The figures represent the fresh weight and height of the potted bok choy in Embodiment 3 of the present invention, where 3A is the fresh weight of the bok choy and 3B is the height of the bok choy. Figure 4 The total nitrogen content in the aboveground parts and roots of the potted Chinese cabbage in Example 3 of this invention; Figure 5 The aboveground and root biomass (dry weight) of Chinese cabbage in the field experiment of Example 3 of the present invention. Figure 6 The total nitrogen content in the aboveground parts and roots of Chinese cabbage in the field experiment of Example 3 of this invention. Detailed Implementation

[0021] The principles and features of the present invention are described below with reference to the accompanying drawings and specific embodiments. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0022] The nitrogen-free medium used in the examples was Burk medium, with the following components: 0.2 g / L KH₂PO₄, 0.8 g / L K₂HPO₄, 0.2 g / L MgSO₄•7H₂O, and 0.1 g / L CaSO₄. 4• 2H₂O, 20 g / L mannitol, 0.005 g / L Na₂MoO₄, 0.1 g / L FeSO₄, pH 7.2.

[0023] Example 1 This invention screened and obtained an iron-reducing nitrogen-fixing bacterium, Klebsiella pneumoniae (Klebsiella pneumoniae). Klebsiella grimontiiN7, this bacterium has iron-reducing and nitrogen-fixing functions. It was deposited on March 20, 2025, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, with accession number CCTCM 2025550.

[0024] Example 2 Klebsiella pneumoniae ( Klebsiella grimontii N7 acts as an iron-reducing nitrogen-fixing bacterium for nitrogen fixation. 1. Using iron reduction to promote nitrogen fixation Select Klebsiella pneumoniae ( Klebsiella grimontii N7 pure colonies were enriched and cultured in LB medium for 12 h, centrifuged at 8000 rpm for 6 min, the supernatant was poured off, the bacteria were rinsed with PBS buffer and mixed well, and then centrifuged again. This process was repeated 2 to 3 times.

[0025] Dispense 50 mL portions of the prepared Burk medium into 120 mL serum bottles, deoxygenate with nitrogen (N2), sterilize in an autoclave, and add 20% headspace gas using a sterile syringe. 15 N2 isotope gas was used to inoculate the washed bacterial culture into Burk medium, and 0.2 mM ferric citrate was added to the medium. The culture was incubated at 30°C and 150 rpm. The control group did not add ferric citrate to the medium. Both groups were cultured under anaerobic conditions for 7 days, and the total nitrogen concentration was measured daily. 15 N abundance, nitrogenase activity (using acetylene reduction method) and nifH The monitoring of 16S rRNA abundance yielded the following results: Figure 1 As shown.

[0026] in Figure 1 a represents the total nitrogen concentration in the culture medium during the culture process, with added Fe. 3+ The total nitrogen concentration in the Fe-N2 group decreased from 1.45 mmol N g on day 1. -1 Increased to 1.92 mmol N g on day 7. -1 On day 7, the total nitrogen concentration was the same as that without added Fe. 3+ Compared to the B-N2 group, it improved by 77.8%; Figure 1 b is 15 N abundance, without Fe addition 3+ Group B-N2 15 N abundance increased from 90 mmol Ng on day 1. -1 Increased to 120 mmol N g on day 7 -1 Add Fe 3+ The Fe-N2 group consisted of 90 mmol N g on day 1. -1 Increased to 152 mmol N g -1On day 7, the Fe-N2 group was better than the B-N2 group. 15 N abundance increased by 26.67%; Figure c shows the results of nitrogenase activity testing in the culture medium, with nitrogenase activity characterized by ethylene concentration. No Fe was added on day 7. 3+ The ethylene concentration in the B-N2 group reached 443 μM, with the addition of Fe. 3+ The Fe-N2 group showed significantly higher concentrations than the B-N2 group, reaching 798 μM; Figure d shows... nifH Changes in 16S rRNA abundance, without Fe on day 7. 3+ The B-N2 group had only 0.3%, while the group with added Fe had 0.3%. 3+ The Fe-N2 group reached 0.7%.

[0027] The above results indicate that Reiberis ( ) Klebsiella grimontii The iron reduction process of N7 can promote its nitrogen fixation, and the addition of exogenous iron can significantly promote the growth of Reiberis ( ). Klebsiella grimontii The nitrogen fixation efficiency of N7.

[0028] 2. Utilizing the redox fluctuations of iron to promote nitrogen fixation Select Klebsiella pneumoniae ( Klebsiella grimontii N7 pure colonies were enriched and cultured in LB medium for 12 h, centrifuged at 8000 rpm for 6 min, the supernatant was poured off, the bacteria were rinsed with PBS buffer and mixed well, and then centrifuged again. This process was repeated 2 to 3 times.

[0029] The prepared Burk medium was dispensed into 50 mL aliquots into 120 mL serum bottles. Two batches were deoxygenated using N2 and Ar respectively (Ar deoxygenation served as a control). Both batches were then sterilized in an autoclave. The N2 deoxygenation group was treated with headspace gas added using a sterile syringe at 20% concentration. 15 Using N2 isotope gas, the washed bacterial culture was inoculated into Burk medium, and 0.2 mM ferric citrate was added to the medium. To simulate the redox fluctuations of iron in nature, oxygen was introduced on days 4 and 8 after complete iron reduction to oxidize ferrous iron, repeating two rounds of redox fluctuations. The group using N2 for deoxygenation was designated Fe-N2, and the control group using Ar for deoxygenation was designated Fe-Ar. Two additional control groups were also set up: the control group (B-N2) had no ferric citrate added, and the blank control group (control) had no bacteria added. The B-N2 and control groups had their medium deoxygenated with N2. OD was monitored. 600 Fe in the culture medium 2+ Concentration, dissolved oxygen concentration, nitrogenase activity, 15 N abundance and amino acid concentration, results as follows Figure 2 As shown.

[0030] 2A represents the growth curve of the bacteria during the culture process; Figure 2 B represents the redox process curve of iron in the culture medium; Figure 2 C represents the dissolved oxygen concentration in the culture medium; Figure 2 D represents the nitrogenase activity in the culture medium; Figure 2 E represents the culture medium. 15 N abundance; Figure 2 F represents the amino acid concentration in the culture medium. During the anaerobic phase, Fe... 3+ Reduction significantly promotes the nitrogenase activity of nitrogen-fixing iron-reducing bacteria and 15 N2 fixation capacity. Following the initial redox fluctuation, due to the increased cell number of microorganisms, nitrogenase activity and... 15 N concentrations all increased significantly (day 6) Figure 2 D 2E). After the second redox fluctuation (days 8-16), in the Fe-N2 group 15 The nitrogen concentration increased from 235.2 μM on day 8 to 280.8 μM on day 16; while the B-N2 group without added Fe increased from 117.4 μM to 141.6 μM. On day 16, the Fe-N2 group... 15 The nitrogen concentration was 95.58% higher than that of the B-N2 group. Furthermore, amino acid secretion and... 15 The trend of N concentration change is consistent ( Figure 2 F). In the initial anaerobic stage, Fe... 3+ Reduction increased the amino acid concentration in the system by 30.2% (day 4); after the first O2 addition, the amino acid concentration in the B-N2 and Fe-N2 groups increased by 12.2% and 20.5% respectively (day 8); after the second O2 addition, the amino acid concentration in the B-N2 and Fe-N2 groups increased by 15.2% and 29.6% respectively.

[0031] The above results indicate that Klebsiella pneumoniae ( Klebsiella grimontii N7 possesses both iron reduction and nitrogen fixation functions, and its nitrogen fixation function is coupled with iron reduction; microorganisms promote their own nitrogen fixation through the iron reduction process. Specifically, Klebsiella pneumoniae ( Klebsiella grimontii N7 converts nitrogen gas (N2) into bioavailable ammonia (NH3) through iron reduction or iron redox fluctuations. Ammonia then participates in microbial metabolism to generate amino acids (such as glutamic acid) or other nitrogen-containing organic matter, thus achieving microbial nitrogen fixation.

[0032] Furthermore, in Fe 3+ Under conditions where the source is present, compared to the one-step reduction of iron, the alternation of anaerobic and aerobic conditions allows for a cycle of abiotic oxidation and microbial reduction of iron, achieving fluctuations in iron redox activity, which can further enhance the activity of Klebsiella pneumoniae (Klebsiella pneumoniae). Klebsiella grimontii The nitrogen fixation efficiency of N7.

[0033] Example 3 Klebsiella pneumoniae ( Klebsiella grimontii N7 fixes nitrogen in soil Klebsiella pneumoniae ( Klebsiella grimontii N7 was prepared into a microbial inoculant and applied to the soil in pot and field experiments, respectively. Iron fertilizer (20-30 kg per acre, which can be inorganic iron fertilizer, chelated iron, or iron fertilizer formed by combining organic matter and iron; in this example, ferric sulfate was chosen as the iron fertilizer) was also applied. The inoculant can utilize the redox reaction of iron in the soil to fix nitrogen, converting atmospheric nitrogen into ammonia, and further metabolizing and utilizing the ammonia, thereby increasing the amount of nitrogen available to crops in the soil, improving soil fertility, and increasing crop yield and total nitrogen content. The specific experimental steps are as follows: 1. Preparation of microbial inoculants (1) Bacterial expansion culture: Klebsiella pneumoniae ( Klebsiella grimontii After N7 activation, inoculate 1% of the culture medium (beef extract peptone medium: beef extract 3g / L, peptone 10g / L, NaCl 5g / L, pH 7.2-7.6) into the fermentation medium and culture for 12-18 hours to ensure a viable count of at least 10⁻⁶ cells / mL. 9 / mL; (2) Preparation of microbial inoculant: The biomass material (rice husk powder) and fermentation liquid are mixed at a solid-liquid ratio of 1:5 (mass ratio). After mixing, the inoculant is stirred every 2 hours for the first 8 hours to make the components in the mixture uniform. After 8 hours, it is allowed to stabilize naturally for 24 hours to complete the preparation of the microbial inoculant.

[0034] 2. Application of microbial inoculants in nitrogen fixation in soil I. Pot Experiment Experimental group (applied microbial agent + iron fertilizer): 0.8 kg of sieved, air-dried soil was added to the flowerpots. The microbial agent was applied to the soil in powder form, ensuring that the dosage of microorganisms added to the soil reached 133.33 kg / mu (live bacteria count of 1.33~6.67×10⁻¹¹ per mu). 13 Then, apply iron fertilizer at a rate of 20-30 kg / mu, mixing it thoroughly with the top 0-10 cm of soil using a sterile mixer to ensure uniformity of the microbial agent and soil. Place in a cool, shaded area for 5 days. Sow 9 purchased bok choy seeds in each pot. Two weeks later, remove the seedlings, leaving only three healthy seedlings. Cultivate the bok choy plants under light (25°C) for 14 hours and under darkness (20°C) for 10 hours, maintaining good ventilation. Maintain the water content of the potted plants at 50%-60% of field capacity, irrigating with ultrapure water. The entire cultivation process of the bok choy was conducted in a climate chamber for 30-40 days.

[0035] Control group (iron fertilizer applied): No microbial agent was applied, only iron fertilizer was applied, and all other aspects were the same as the experimental group; CK: No microbial agents or iron fertilizer were applied; all other aspects were the same as the experimental group. After cultivation, the height of the pak choi plants was recorded, and the fresh weight of the pak choi and the total nitrogen of the above-ground parts and roots of the pak choi were sampled and tested.

[0036] Figure 3 The fresh weight and height of pak choy in the experimental group, control group, and CK group were compared. Applying iron fertilizer alone slightly promoted the growth of pak choy, but the effect was not significant. Applying both microbial agents and iron fertilizer increased the fresh weight of pak choy from 7.14g to 8.97g, an increase of 25.63%, and the height from 4.25cm to 6.98cm, an increase of 64.24%. Figure 4 The total nitrogen content of the aboveground parts and roots of Chinese cabbage in the experimental group, control group and CK was measured. Applying iron fertilizer alone slightly increased the total nitrogen content of the aboveground parts and roots of Chinese cabbage. Applying microbial agent and iron fertilizer at the same time promoted the total nitrogen content of the aboveground parts and roots of Chinese cabbage. The total nitrogen content of the aboveground parts increased by 26.67% and the total nitrogen content of the roots increased by 21.0%, and the crop quality was improved.

[0037] II. Field Experiments A field experiment was conducted in Zhiying Town, Yongkang County, Jinhua City, Zhejiang Province. Before the experiment, the soil was tilled using a rotary tiller to ensure uniformity of soil components. Microbial inoculants were then applied at a rate of 133.33 kg / mu (with a viable count of 1.33~6.67 × 10⁻⁶ bacteria per mu). 13 Apply the inoculant at a rate of 20-30 kg / mu. After application, apply iron fertilizer at a rate of 20-30 kg / mu. After rotary tilling, cover with breathable black film for 15 days before sowing bok choy. Monitor the biomass (dry weight) and total nitrogen of the aboveground parts and roots of the mature bok choy.

[0038] Figure 5 The biomass of the aboveground parts and roots of Chinese cabbage (by dry weight) was measured. Compared with the control group (with iron fertilizer added only), the control group (with iron fertilizer added only) increased the aboveground biomass of Chinese cabbage by 5.26% and the root biomass by 52.21%. The aboveground biomass of Chinese cabbage treated with both microbial agent and iron fertilizer increased by 25.3% and the root biomass increased by 70.0%.

[0039] Figure 6 The total nitrogen content in the aboveground parts and roots of Chinese cabbage was measured. Compared with the control (CK), the addition of iron fertilizer alone slightly promoted the total nitrogen content in the aboveground parts of Chinese cabbage, increasing it by 5.04%. However, the total nitrogen content in the aboveground parts of Chinese cabbage increased by 22.2% and the total nitrogen content in the roots increased by 15.4% when both microbial agent and iron fertilizer were added.

[0040] The Klebsiella pneumoniae of the present invention ( Klebsiella grimontiiWhen N7-prepared microbial agents and iron fertilizers are applied simultaneously to the soil, the redox fluctuations of iron in the soil can promote the growth of Klebsiella pneumoniae (Klebsiella pneumoniae). Klebsiella grimontii Nitrogen fixation (N7) involves nitrogenase reducing atmospheric nitrogen (N2) to ammonia (NH3). The ammonia then combines with α-keto acids to form amino acids (such as glutamine). These amino acids can be directly secreted into the soil or released through the decomposition of dead bacteria. Crops can directly utilize the amino acids secreted or released into the soil, absorbing them into their roots and further metabolizing them into other nitrogen-containing organic matter (such as proteins and nucleic acids) for their growth, thus improving crop yield and quality.

[0041] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An iron-reducing nitrogen-fixing bacterium, characterized in that, Klebsiella pneumoniae ( Klebsiella grimontii N7 was deposited at the China Center for Type Culture Collection on March 20, 2025, with accession number CCTCC M 2025550.

2. The iron-reducing nitrogen-fixing bacteria according to claim 1, characterized in that, The Klebsiella pneumoniae ( Klebsiella grimontii N7 has the ability to fix nitrogen and reduce iron, is a facultative anaerobe, and performs nitrogen fixation by coupling iron reduction.

3. A method for promoting nitrogen fixation using iron reduction, characterized in that, Includes the following steps: S1. Select strains with iron reduction and nitrogen fixation capabilities, and facultative anaerobic properties, and conduct experiments under anaerobic, N2-containing conditions in the presence of Fe. 3+ Nitrogen fixation was carried out by culturing in a nitrogen-free medium; S2. In Fe 3+ After complete reduction, oxygen is introduced into the culture medium to remove Fe. 2+ Oxidized to Fe 3+ ; S3. Wait for Fe 3+ After being completely oxidized and oxygen is exhausted, nitrogen fixation continues. Through repeated cycles of abiotic oxidation of iron and microbial reduction, nitrogen fixation is achieved.

4. The method for promoting nitrogen fixation using iron-reducing nitrogen-fixing bacteria according to claim 3, characterized in that, It also includes the following steps: S4. Repeat steps S2 and S3 above.

5. The method for promoting nitrogen fixation using iron-reducing nitrogen-fixing bacteria according to claim 3, characterized in that, The strain possessing iron reduction and nitrogen fixation capabilities is the Klebsiella pneumoniae as described in claim 1 or 2. Klebsiella grimontii )N7.

6. The method for promoting nitrogen fixation using iron-reducing nitrogen-fixing bacteria according to any one of claims 3 to 5, characterized in that, Fe 3 + The source is selected from at least one of ferric citrate, ferric chloride, or ferric sulfate, Fe 3+ The concentration of oxygen is 0.2 mM to 1 mM. In step S1, N2 is used to remove oxygen to create an oxygen-free and N2-containing environment. In step S2, the amount of oxygen introduced is 5% to 10% of the headspace volume.

7. The method for promoting nitrogen fixation using iron-reducing nitrogen-fixing bacteria according to any one of claims 3 to 5, characterized in that, The nitrogen-free culture medium has a pH of 7.0-7.2 and a culture temperature of 20-40℃. The culture time in step S1 is 2-7 days, and the culture time in step S3 is 2-4 days.

8. Any of the following applications of the iron-reducing nitrogen-fixing bacteria according to claim 1 or 2: I. Preparation of microbial inoculants; II. Soil nitrogen fixation and improvement of soil fertility; III. Improve crop yield and quality.

9. A method for promoting nitrogen fixation in soil using iron reduction, characterized in that, The Klebsiella pneumoniae as described in claim 1 or 2 ( Klebsiella grimontii N7 is prepared into a microbial agent, and the microbial agent and iron fertilizer are applied to the soil. Crops are planted and cultivated in a conventional manner, and nitrogen fixation is promoted by utilizing the redox fluctuations of iron in the soil.

10. The method for promoting nitrogen fixation in soil by iron reduction according to claim 9, characterized in that, The inoculant is applied at a rate of not less than 1.33 × 10⁻⁶ per acre. 13 The amount of live bacteria to be applied is 20-30 kg / mu for the iron fertilizer.

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