Method for enhancing soil nitrogen fixation by using pure iron powder

Abstract

The invention belongs to the technical field of soil nitrogen fixation and environmental protection, and relates to a method for enhancing soil nitrogen fixation by using pure iron powder. A method for enhancing soil nitrogen fixation by using pure iron powder comprises the following steps: mixing rice soil and wheat straw powder to obtain a soil sample; the method comprises the following steps: mixing a soil sample with pure iron powder under a waterflooding condition; pure iron powder is added to regulate and control microbial ecology of rice soil biological nitrogen fixation, the soil iron reduction process is enhanced, the abundance of iron reducing bacteria in soil is improved, the relative contribution of iron reducing bacteria nitrogen fixation is enhanced to a certain extent, the diversity of iron reducing bacteria communities is reduced, the ecological niches of dominant groups are enhanced, and the ecological niches of the dominant groups are increased. Finally, the nitrogenase activity of the soil is enhanced, and the nitrogen fixation capacity of the rice soil is improved.

Description

Technical Field

[0001] This invention belongs to the field of soil nitrogen fixation and environmental protection technology, and relates to a method for enhancing soil nitrogen fixation using pure iron powder. Background Technology

[0002] Geobacteraceae and Anaeromyxobacteraceae, families of iron-reducing bacteria, are the core driving groups for nitrogen fixation in anaerobic environments such as paddy soil, and their nitrogen fixation capacity has been verified: genomic analysis shows that both groups carry complete nitrogen-fixing gene clusters (nifBHDKEN), with highly conserved key functional sites encoding nitrogenase; in pure culture experiments, strains of the genera *Geomonas* and *Anaeromyxobacter* can utilize N2 as the sole nitrogen source for growth; in soil microcosm experiments, the soil acetylene reduction method (ARA) nitrogenase activity reached 0.24–0.58 nmol C2H4 g after inoculation with these strains. -1 soil h -1 Furthermore, the increased copy number of the 16S rRNA gene confirms its nitrogen-fixing function in the soil environment. The nitrogen-fixing activity of iron-reducing bacteria is strictly dependent on anaerobic environments, significantly higher during the flooded period of paddy soil than during the drained period, and is related to Fe... 2+ Concentration is positively correlated; metabolically, it uses organic carbon such as acetic acid and xylan produced from straw decomposition as electron donors, and Fe as the main component. 3+ As electron acceptors, they obtain energy through the coupling of iron reduction and nitrogen fixation processes. Geobacteraceae can directly utilize xylan, while Anaeromyxobacteraceae prefers small molecule carbon sources such as acetic acid.

[0003] On a global scale, iron-reducing bacteria dominate nitrogen-fixing microbial communities. Geobacteraceae and Anaeromyxobacteraceae have relative abundances of the nifD / K gene exceeding 50% in anaerobic environments such as paddy fields and sediments, which is 3-5 times higher than that of traditional nitrogen-fixing bacteria such as cyanobacteria and alpha-proteobacteria. The detection frequency of their nitrogen-fixing functional genes in anaerobic environments is 17.6 times that in aerobic environments, and they maintain high abundance in different climatic zones (subtropical and temperate) and soil types, confirming their wide ecological distribution and functional importance. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for enhancing nitrogen fixation in paddy soil by using pure iron powder, which addresses the shortcomings of the prior art.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] This invention discloses a method for enhancing soil nitrogen fixation using pure iron powder, comprising the following steps:

[0007] (1) Soil samples were obtained by mixing paddy soil and wheat straw powder;

[0008] (2) After mixing the soil sample with pure iron powder, add water until the water reaches 2-5 cm above the soil surface.

[0009] In some embodiments, the wheat straw powder accounts for 1% of the mass of the soil sample.

[0010] The wheat straw powder has a particle size of less than 5 mm.

[0011] The paddy soil mentioned above refers to soil formed under long-term flooding conditions for rice cultivation, resulting from the combined effects of human cultivation and natural soil-forming factors.

[0012] The paddy soil used in this invention was collected from typical paddy fields in Liuhe District, Nanjing City, Jiangsu Province, and was naturally air-dried before being sieved through a 2 mm sieve.

[0013] In some embodiments, the amount of pure iron powder added is 0.050 g to 0.200 g of pure iron powder per 40 g soil sample.

[0014] In some embodiments, the amount of pure iron powder added is: 0.050 g, 0.100 g, or 0.200 g of pure iron powder per 40 g soil sample.

[0015] In some embodiments, the amount of pure iron powder added is 0.100 g or 0.200 g of pure iron powder per 40 g soil sample.

[0016] In some embodiments, the method is carried out under flooded conditions. After mixing the soil sample with pure iron powder, water is added until the water reaches 2-5 cm above the soil surface. Water is then added every 2 days to maintain the water level.

[0017] In some embodiments, the method is carried out under flooded conditions. After mixing the soil sample with pure iron powder, water is added until the water reaches 2-5 cm above the soil surface. Water is added every 2 days to maintain the water level. The treatment lasts for 15-35 days to enhance soil nitrogen fixation.

[0018] In some embodiments, the method is carried out under flooded conditions. After mixing the soil sample with pure iron powder, water is added until the water reaches 2-5 cm above the soil surface. Water is added every 2 days to maintain the water level. The treatment lasts for 25-35 days to enhance soil nitrogen fixation.

[0019] The above-mentioned method of using pure iron powder to enhance soil nitrogen fixation is also within the scope of protection of this invention in improving soil fertility in paddy fields, and / or reducing the amount of chemical nitrogen fertilizer applied, and / or promoting rice growth or restoring degraded farmland.

[0020] In some embodiments, the method enhances the soil iron reduction process, increases the abundance of soil iron-reducing bacteria, strengthens the relative contribution of iron-reducing bacteria to nitrogen fixation, promotes a decrease in the diversity of iron-reducing bacterial communities, enhances the niche of dominant groups, and thus strengthens the activity of nitrogenase in the soil and improves the soil nitrogen fixation capacity.

[0021] The technical solution of the present invention can: (1) enhance soil iron reduction and increase soil ferrous content; (2) accelerate the proliferation of iron-reducing bacteria in the short term and increase the abundance of soil iron-reducing bacteria; (3) enhance the relative contribution of iron-reducing bacteria to nitrogen fixation; (4) promote the reduction of iron-reducing bacterial community diversity and enhance the ecological niche of the dominant group of Geobacter; (5) increase soil nitrogenase activity and improve soil nitrogen fixation capacity.

[0022] Beneficial effects:

[0023] (1) The present invention utilizes pure iron powder to treat paddy soil with the highest ferrous content compared to other iron source treatments, thereby enhancing the reduction of iron in the soil.

[0024] (2) The present invention utilizes pure iron powder treatment, which can accelerate the proliferation of iron-reducing bacteria in a short period of time compared with other iron sources, and increase the relative abundance or absolute abundance of iron-reducing bacteria.

[0025] (3) The present invention utilizes pure iron powder treatment to enhance the relative contribution of iron-reducing bacteria to nitrogen fixation.

[0026] (4) The present invention utilizes pure iron powder treatment to promote the reduction of iron-reducing bacterial community diversity, enhance the ecological niche of the dominant Geobacter group, thereby increasing the nitrogen fixation activity of paddy soil and improving the nitrogen fixation capacity of paddy soil.

[0027] (5) The present invention adds pure iron powder to regulate the microbial ecology of biological nitrogen fixation in paddy soil. By strengthening the iron reduction process in the soil, it increases the abundance of iron-reducing bacteria in paddy soil and strengthens the relative contribution of iron-reducing bacteria to nitrogen fixation to a certain extent. This leads to a decrease in the diversity of iron-reducing bacterial communities, enhances the ecological niche of dominant groups of the genus Geobacter, and ultimately strengthens the nitrogenase activity of paddy soil and improves the soil's nitrogen fixation capacity. Attached Figure Description

[0028] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0029] Figure 1The graphs show the ferrous content of paddy soil treated with low, medium, and high amounts of different iron sources. Figure a shows the ferrous content of paddy soil treated with low (L) iron source addition, Figure b shows the ferrous content of paddy soil treated with different (M) iron source addition, and Figure c shows the ferrous content of paddy soil treated with different (H) iron source addition. Note: Different letters in the graphs represent significant differences.

[0030] Figure 2 Plot of relative abundance data of iron-reducing bacteria treated with low amounts (L) of different iron sources; Note: Different letters in the figure represent significant differences.

[0031] Figure 3 Plot of absolute abundance data of iron-reducing bacteria treated with different amounts (M) of iron source; Note: Different letters in the figure represent significant differences.

[0032] Figure 4 Plot of relative abundance data of iron-reducing bacteria treated with different high iron source addition levels (H); Note: Different letters in the figure represent significant differences.

[0033] Figure 5 A graph showing the relative contribution of iron-reducing bacteria to biological nitrogen fixation under different amounts of pure iron powder; Note: Different letters in the graph represent significant differences.

[0034] Figure 6 Figure 1 shows the species composition of iron-reducing bacteria at the phylum and genus levels in paddy soil under the condition of adding pure iron powder; where Figure 2a shows the species composition of iron-reducing bacteria at the phylum level and Figure 3b shows the species composition of iron-reducing bacteria at the genus level.

[0035] Figure 7 Figure 1 shows the species composition of iron-reducing bacteria at the phylum and genus levels in paddy soils with different iron sources added on day 25 under high iron addition conditions; Figure 2a shows the species composition of iron-reducing bacteria at the phylum level, and Figure 2b shows the species composition of iron-reducing bacteria at the genus level.

[0036] Figure 8 Figure 1 shows nitrogenase activity data of paddy soil treated with low, medium, and high iron source additions. Figure 2a shows nitrogenase activity of paddy soil treated with low iron source addition (L), Figure 3b shows nitrogenase activity of paddy soil treated with different iron source additions (M), and Figure 4c shows nitrogenase activity of paddy soil treated with different iron source additions (H). Note: Different letters in the figures represent significant differences.

[0037] Figure 9 Figure 1 shows nitrogenase activity data in paddy soils with different iron sources, without distinguishing between culture time and dosage; Note: Different letters in the figure represent significant differences.

[0038] Figure 10Figure 1 shows nitrogenase activity data in paddy soil treated with different amounts of pure iron powder; Note: Different letters in the figure represent significant differences. Detailed Implementation

[0039] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0040] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0041] Unless otherwise specified, in the accompanying drawings of this invention: CK represents blank control, F represents pure iron powder, FH represents hematite, FG represents goethite, FM represents magnetite, and FOH represents ferric hydroxide (Fe(OH)3). L represents low addition amount, M represents added amount, and H represents high addition amount.

[0042] The relevant testing methods are as follows:

[0043] Soil moisture content: Soil moisture content (MC) was determined using the drying method. The specific procedure is as follows: After cleaning and marking the aluminum box accordingly, weigh it and record the weight as m. l Weigh an appropriate amount of soil sample into an aluminum box and record the weight as m2. Place the aluminum box containing the sample in an oven at 105℃ for 8 hours. After removing it and cooling it to room temperature, weigh it again and record the weight as m3. The formula for calculating the moisture content is as follows:

[0044] Soil moisture content (g·kg) -1 )= × 100%.

[0045] Soil pH and redox potential (Eh) determination: Soil pH and redox potential (Eh) were determined using potentiometric and electrode methods, respectively. Specifically, 10 g of normally air-dried soil sample that had been ground and passed through a 2 mm sieve was weighed, and sterile water was added at a soil-to-water ratio of 1:2.5. The mixture was stirred with a glass rod, and the values ​​were measured and recorded using a calibrated electrode.

[0046] Soil electrical conductivity: Soil electrical conductivity (EC) was determined by the electrode method. 10 g of normally air-dried soil sample that had been ground and passed through a 2 mm sieve was weighed and added to deionized water at a ratio of 1:5 (m / V). The sample was extracted by shaking at 20℃±1℃. The conductivity of the extract was measured at 25℃±1℃ using a calibrated conductivity meter.

[0047] Soil ferrous and ferric iron (Fe2+ and Fe3+) content: Weigh 10.00 g of fresh soil sample (while simultaneously determining moisture content) into a 250 mL Erlenmeyer flask, add 200.00 mL of aluminum sulfate extractant, stopper, and shake well. After standing for 5 min, filter through slow-speed filter paper into another 250 mL Erlenmeyer flask, stopper, and immediately determine the ferrous iron content in the filtrate. Pipette 20.00 mL of the filtrate into a 50 mL volumetric flask, add 1 mL of hydroxylamine hydrochloride solution, and shake well. After standing for several minutes, add 5 mL of o-phenanthroline solution, then dilute with water to the mark and shake well. After standing for at least 30 min, measure the absorbance at 520 nm using a 1 cm absorption dish on a spectrophotometer, and find the corresponding iron content from the working curve. The soil ferrous iron (Fe2+) was obtained without the addition of hydroxylamine hydrochloride. 2+ The content of ferric iron (Fe) was calculated using the difference method. 3+ )content.

[0048] Soil total carbon and total nitrogen content: Soil total carbon (TC) and total nitrogen (TN) were determined using an elemental analyzer (VARIO EL III, ELEMENTAR, Germany). Approximately 2 mg of normally air-dried soil sample that had passed through a 200-mesh sieve was weighed using a part per million electronic balance (MX5, METTLER, Switzerland), placed in a tin boat, wrapped into a regular square shape with tweezers, and then measured and recorded on the instrument.

[0049] Real-time quantitative PCR: DNA was extracted from soil samples refrigerated at -80°C using the HiPure Soil DNA Mini Kit (Magen, China). Total DNA was detected by 1% agarose gel electrophoresis. The concentration and purity of the extracted DNA were determined using a NanoDrop ND1000UVevis spectrophotometer (Thermo Scientific, Rockwood, TN, USA). DNA extracts that met the requirements for subsequent experiments were used for real-time quantitative PCR and delivered to Guangdong Meggene Technology Co., Ltd. for high-throughput sequencing. The 16S rRNA gene, nifH gene, Anaeromyxobacter / Geobacter nifD gene, and nifD gene of universal nitrogen-fixing bacteria were amplified using real-time quantitative PCR. Primer and corresponding amplification program information is shown in Table 1.

[0050] Table 1 Primers for Real-Time Quantitative PCR

[0051]

[0052] Nitrogenase Activity Assay: Nitrogen fixation activity of soil samples from the Microcosm was measured using a standard acetylene reduction activity (ARA) assay, based on the reduction of C2H2 to C2H4 by nitrogenase (Postgate 1972). Each soil sample (8 g) collected from the Microcosm was placed in a serum bottle, sealed with a butyl rubber stopper, and then sealed with an aluminum cap. The gas phase fraction was replaced with Ar / C2H2 gas (90:10, v / v) and incubated at 30 °C for 24 h. After incubation, gas samples were collected from the gas phase fraction in the bottle, and the C2H4 concentration was measured using a gas chromatograph (GC-2014C; Shimadzu, Kyoto, Japan) equipped with a capillary column (SH-Q BOND PLOT).

[0053] High-throughput sequencing of the nifD gene from iron-reducing bacteria: The nifD gene from iron-reducing bacteria was amplified using primer pairs Anaeromyxobacter & Geobacter nifD F (5′-CCTSATYGGYGACGAYATMAAC-3′) and Anaeromyxobacter & Geobacter nifD R (5′-TAGTTCATGGAVCGTARCAGT-3′). Purified amplicon products were ligated into sequencing adapters to construct sequencing libraries, which were then sequenced using an Illumina MiSeq-PE250 platform. Original paired-end reads were assigned to each sample based on a unique barcode. Quality control and cluster analysis were performed using the DADA2 plugin unit in QIIME2 (https: / / qiime2.org / ) to obtain amplicon sequence variants (ASVs) defined by single nucleotide differences. Chimeric sequences and single ASVs (i.e., ASVs containing only one sequence in a sample) were removed, and species annotation was performed using GraftM (0.13.1) software. The specific process for species annotation is as follows: Log in to the NCBI Nucleic Acid Database (https: / / www.ncbi.nlm.nih.gov / nuccore), search for sequences based on the functional gene name, download the target functional gene sequence and species classification information, create a functional gene database package, and then run the program to annotate the species of the functional genes.

[0054] Data processing and statistical analysis: All experimental data were processed and plotted using Excel 2021 and GraphPad Prism 10.4.1 software. All data are presented as mean ± standard error of four replicates.

[0055] Example 1:

[0056] 1. Materials and Methods

[0057] 1.1 Test Soil

[0058] Soil for the microcosm experiment was collected from the topsoil (0-20 cm) of a typical paddy field in Liuhe District, Nanjing City, Jiangsu Province. The soil type was argillaceous soil, a type of paddy soil found in alluvial paddy fields in the hilly area of ​​the middle and lower reaches of the Yangtze River. It has a moderate texture, good permeability, and is suitable for both rice and wheat. Liuhe District, Nanjing City, Jiangsu Province, is located in the upper reaches of the Zaohe River, a tributary of the Chuhe River. After collection, the soil was air-dried naturally, passed through a 2 mm nylon sieve, and plant residues, roots, stones, fragments, etc. were removed. The soil was then stored at 4℃ for later use. The physicochemical properties of the tested soil are shown in Table 2.

[0059] Table 2 Physicochemical properties of the tested soils

[0060]

[0061] Note: The soil-to-water ratio used for pH measurement was 1:2.5.

[0062] 1.2 Experimental Design

[0063] Wheat straw powder (providing a carbon source) was added to the sieved test soil (1 g wheat straw powder per 100 g soil, 1 wt%) and mixed thoroughly to obtain a soil sample.

[0064] Before aliquoting soil samples into round-bottom centrifuge tubes, exogenous iron (from five different iron sources) was added and thoroughly mixed. Soil microenvironments were incubated in the dark at 30°C. No exogenous iron was added to the blank control group.

[0065] A 40 g soil sample containing an iron source was placed in a 50 mL round-bottom centrifuge tube, and then sterile distilled water was added until it reached 2–5 cm above the soil surface. Sterile distilled water was added every two days to maintain the water level. A platinum electrode was inserted daily at a depth of 3 cm to measure the soil redox potential (Eh) until the Eh of all treatments dropped below 0 mV. The treatments applied to the 40 g soil sample are shown in Table 3. All iron sources were sieved through a 100-mesh sieve before addition. Each treatment had four replicates.

[0066] When the Eh of all treated soil samples decreased to below 0 mV, destructive sampling was performed. First, using a headless plastic syringe, soil samples were collected from a depth of approximately 3 cm for soil nitrogenase activity, soil DNA extraction, and soil physicochemical property determination. Subsequently, the remaining soil samples were thoroughly mixed, and soil samples were collected and divided into two portions. One portion was placed in a cool, shaded place to dry, passed through a 2 mm sieve, and stored at 4°C for physicochemical property analysis (MC, pH, EC, Eh, NH4). + -N, NO3 - -N, TC, TN, Fe 2+ Fe 3+Another copy was refrigerated at -80°C for qPCR and high-throughput sequencing.

[0067] Table 3 Soil Microcosm Experiment Treatments

[0068]

[0069] Note: The amount added in the table refers to the mass of iron source added in a 40 g soil sample.

[0070] 1.3. Clarify the differences in ferrous content in paddy soil under the same addition level of different iron sources.

[0071] The following graph shows the ferrous content data of paddy soil treated with low, medium, and high iron source additions: Figure 1 As shown in the figure, we can see that:

[0072] (i) On day 15, under low addition conditions, the Fe content of soil treated with FOH-L and FM-L was significantly reduced. 2+ The content was significantly higher than that of CK and FL treatments (p < 0.05); under the addition amount condition, except for the FG-M treatment, the other four iron source treatments were significantly higher than CK (p < 0.05); while under the high addition amount condition, all iron source treatments were significantly higher than CK (p < 0.05).

[0073] (ii) On day 25, under low addition conditions, there were no significant differences among treatments; under addition conditions, the soil Fe in the FM and FOH-M treatments was significantly higher. 2+ The content was significantly higher than that of CK (p < 0.05); and under high addition conditions, all iron source treatments were significantly higher than CK (p < 0.05).

[0074] (iii) On day 35, under low addition conditions, the Fe content of the soil treated with FL was... 2+ The content was significantly higher than that of CK and FM-L treatments (p < 0.05). Under the addition of a certain amount, there was no significant difference among the treatments. Under the addition of a certain amount, the FH, FG-H and FM-H treatments were significantly higher than CK (p < 0.05).

[0075] Conclusion: The ferrous content in paddy soil treated with pure iron powder was the highest compared to other iron source treatments.

[0076] 1.4. Clarify the differences in the abundance of iron-reducing bacteria in paddy soil under the same addition level of different iron sources.

[0077] The relative abundance data of iron-reducing bacteria under low iron source treatments are shown in the figure below. Figure 2 As shown in the figure, under different low iron source addition conditions, the relative abundance of iron-reducing bacteria in the FL treatment was the highest on days 15, 25, and 35, reaching 1.62 × 10⁻⁶.-3 1.70×10 -3 and 8.00×10 -4 On day 15, the FL treatment was significantly higher than the CK and the other four treatments; on day 25, it was significantly higher than the CK, FG-L and FM-L treatments; and on day 35, it was significantly higher than the FH-L, FG-L, FM-L and FOH-L treatments (p < 0.05).

[0078] The absolute abundance data of iron-reducing bacteria treated with different amounts of iron source are shown in the figure below. Figure 3 As shown in the figure, under different iron source addition conditions, the absolute abundance of iron-reducing bacteria in the FM treatment was the highest on days 15, 25, and 35, reaching 5.52 × 10⁻⁶. 5 copies g -1 dry soil, 3.91×10 5 copies g -1 dry soil and 5.56×10 5 copies g -1 The dry soil treatment was significantly higher than the CK, FG-M and FM-M treatments on day 15, significantly higher than the FH-M, FG-M and FM-M treatments on day 25, and significantly higher than the CK and FH-M treatments on day 35 (p < 0.05).

[0079] The relative abundance data of iron-reducing bacteria under high iron source addition treatments are shown in the figure below. Figure 4 As shown in the figure, the relative abundance of iron-reducing bacteria in the FH treatment was highest on days 15, 25, and 35, reaching 1.80 × 10⁻⁶. -3 8.25×10 -4 and 7.00×10 -4 On day 15, the FH treatment was significantly higher than all other treatments; on day 25, it was significantly higher than the CK, FG-H, and FM-H treatments; and on day 35, it was significantly higher than the other four iron source treatments (p < 0.05).

[0080] Conclusion: Compared with other iron sources, pure iron powder can accelerate the proliferation of iron-reducing bacteria in the short term.

[0081] 1.5. Clarify the differences in the relative contribution of iron-reducing bacteria to biological nitrogen fixation under different levels of optimal iron source addition.

[0082] The relative contribution of iron-reducing bacteria to biological nitrogen fixation under different amounts of pure iron powder is shown in the figure below. Figure 5As shown in the figure, the FM treatment had the highest relative contribution to nitrogen fixation by iron-reducing bacteria on days 15 and 35, while the FH treatment had the highest contribution on day 25. Specifically, the FM treatment on days 15 and 35 was significantly higher than the other treatments (p < 0.05), while the FH treatment on day 25 was significantly higher than the CK and FM treatments (p < 0.05).

[0083] 1.6 Composition of Iron-Reducing Bacterial Community

[0084] To further investigate the response patterns of iron-reducing bacterial communities that may lead to differences in nitrogenase activity in paddy soil, high-throughput analysis was conducted on pure iron powder treatments (different addition amounts on days 15, 25, and 35) with the strongest nitrogenase activity, as well as different iron source treatments with high addition amounts on day 25, which generally showed strong nitrogenase activity. The results are as follows.

[0085] from Figure 6 It can be seen that: (i) at the phylum level, on days 15, 25 and 35, the iron-reducing bacterial communities in the CK, FL, FM and FH treatments were mainly composed of Pseudomonadota (99.34%-99.68%), with the remainder being unclassified groups.

[0086] (ii) Meanwhile, at the genus level, on day 15, the iron-reducing bacterial communities in the CK, FL, FM, and FH treatments mainly included Geobacter (77.45%-84.32%), Anaeromyxobacter (0.48%-1.59%), and Bradyrhizobium (0.01%-0.02%); on day 25, the iron-reducing bacterial communities mainly included Geobacter (89.19%-92.35%), Anaeromyxobacter (0.26%-0.40%), and Bradyrhizobium (0.02%-0.10%); and on day 35, the iron-reducing bacterial communities mainly included Geobacter (84.40%-90.83%), Anaeromyxobacter (0.22%-0.85%), and Bradyrhizobium (0.01%-0.02%).

[0087] from Figure 7 It can be seen that on day 25, under high addition levels, at the phylum level, the iron-reducing bacterial community was mainly composed of Pseudomonadota (87.11%-99.65%). At the genus level, the iron-reducing bacterial community was mainly composed of Geobacter (67.98%-92.35%).

[0088] 1.7. Clarify the differences in nitrogenase activity in paddy soil under the same addition level of different iron sources.

[0089] The test results are as follows:

[0090] (1) Nitrogenase activity in paddy soil treated with low, medium, and high iron source addition levels, such as Figure 8 As shown in the figure, we can see that:

[0091] (i) In the low iron source addition (L) treatments, no significant differences were found between treatments on days 15 and 25, but on day 35, the FOH-L treatment was found to be significantly higher than the CK and other iron source treatments (p < 0.05), by 0.11 nmol h. -1 g -1 soil.

[0092] (ii) Among the various iron source addition amounts (M), the soil nitrogenase activity was highest in the FM treatment on days 15, 25, and 35. Specifically, on day 15, the FM treatment was significantly higher than the CK, FG-M, and FM-M treatments (p < 0.05), by 0.14 nmol h⁻¹. -1 g -1 Soil; on day 25, the FM treatment was significantly higher than other treatments (p < 0.05), by 0.23 nmol h. -1 g -1 Soil; on day 35, the FM treatment was significantly higher than the CK, FG-M, FM-M, and FOH-M treatments (p < 0.05), by 0.18 nmol h. -1 g -1 soil.

[0093] (iii) Among the high iron source addition (H) treatments, the soil nitrogenase activity in the FH treatment was the highest on days 15, 25, and 35, and was significantly higher than that in the other treatments (p < 0.05), at 0.32 nmol h⁻¹. -1 g -1 soil, 0.88 nmol h -1 g -1 soil and 0.64 nmol h -1 g -1 soil.

[0094] (2) Without differentiating between culture time and amount of iron added, the nitrogenase activity data of each iron source group were summed and averaged. The data results are as follows: Figure 9 As shown in the figure, the F (pure iron powder) treatment was significantly higher than the other four iron source treatments (p < 0.05).

[0095] Conclusion: The nitrogenase activity in paddy soil treated with pure iron powder was significantly better than that in other iron source treatment groups, especially when the pure iron powder addition was at the medium level (0.100 g / 40 g soil sample) and the high level (0.200 g / 40 g soil sample).

[0096] 1.8. Clarify the differences in nitrogenase activity in paddy soil under different levels of optimal iron source addition.

[0097] Figure 1 shows the nitrogenase activity data of paddy soil treated with different amounts of pure iron powder. Figure 10 As shown in the figure, on days 15, 25 and 35, the FH treatment was significantly higher than the CK, FL and FM treatments (p < 0.05). On day 15, the FM treatment was also significantly higher than the FL and CK treatments (p < 0.05).

[0098] The above studies suggest that the microbial ecological mechanism by which iron source addition (especially the addition of pure iron powder) regulates biological nitrogen fixation in paddy soil may be that, under flooded conditions, it enhances the soil iron reduction process, increases the abundance of iron-reducing bacteria in paddy soil, and to a certain extent (under suitable addition conditions) strengthens the relative contribution of iron-reducing bacteria to nitrogen fixation, leading to a decrease in the diversity of iron-reducing bacterial communities, enhancing the ecological niche of the dominant Geobacter group, and ultimately strengthening the nitrogenase activity of paddy soil.

[0099] This invention provides a concept and method for enhancing soil nitrogen fixation using pure iron powder. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for enhancing soil nitrogen fixation using pure iron powder, characterized in that, Includes the following steps: (1) Soil samples were obtained by mixing paddy soil and wheat straw powder; (2) After mixing the soil sample with pure iron powder, add water until the water reaches 2-5 cm above the soil surface.

2. The method according to claim 1, characterized in that, The wheat straw powder constitutes 1% of the mass of the soil sample.

3. The method according to claim 1, characterized in that, The amount of pure iron powder added is 0.050 g to 0.200 g per 40 g soil sample.

4. The method according to claim 1, characterized in that, The amount of pure iron powder added is: 0.050 g, 0.100 g or 0.200 g of pure iron powder per 40 g soil sample.

5. The method according to claim 1, characterized in that, The amount of pure iron powder added is 0.100 g or 0.200 g of pure iron powder per 40 g soil sample.

6. The method according to claim 1, characterized in that, The method is carried out under flooded conditions. Soil samples are mixed with pure iron powder, and water is added until the water reaches 2-5 cm above the soil surface. Water is added every 2 days to maintain the water level.

7. The method according to claim 1, characterized in that, The method is carried out under flooded conditions. Soil samples are mixed with pure iron powder, and water is added until the water reaches 2-5 cm above the soil surface. Water is added every 2 days to maintain the water level. The treatment lasts for 15-35 days to enhance soil nitrogen fixation.

8. The method according to claim 1, characterized in that, The method is carried out under flooded conditions. Soil samples are mixed with pure iron powder, and water is added until the water reaches 2-5 cm above the soil surface. Water is added every 2 days to maintain the water level. The treatment lasts for 25-35 days to enhance soil nitrogen fixation.

9. The application of a method for enhancing soil nitrogen fixation using pure iron powder as described in any one of claims 1 to 8 in improving soil fertility in paddy fields, and / or reducing the application of chemical nitrogen fertilizer, and / or promoting rice growth or restoring degraded farmland.

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