Biological catalytic method for stimulating nitrogen fixation in upland soil and crop growth and application thereof

Abstract

The application belongs to the technical field of biology, and discloses a biological catalysis method for stimulating soil nitrogen fixation and crop growth in dry land and application. Specifically disclosed is a biological hybrid system, which comprises nitrogen-fixing microorganisms and ferroferric oxide nanoparticles loaded on the surface of the nitrogen-fixing microorganisms. The ferroferric oxide-Curtobacterium latens biological hybrid system provided by the application can improve the nitrogen fixation level of dry land soil, promote the absorption, utilization and growth and development of crops such as lettuce to nitrogen, reduce the chemical nitrogen fertilizer input, and improve the crop biomass and nutrient accumulation; the ferroferric oxide-Curtobacterium latens biological hybrid system is not dependent on light driving, is more suitable for the real farmland rhizosphere and soil dark field environment than the existing photosensitive semiconductor-microorganism system, and has good application prospect and popularization value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a biocatalytic method and its application for stimulating nitrogen fixation and crop growth in dryland soils. Background Technology

[0002] Soil serves as the core hub of the global nitrogen cycle, and biological nitrogen fixation plays a crucial role in maintaining nitrogen balance in farmland ecosystems, ensuring crop production, and reducing the application of chemical nitrogen fertilizers. Biological nitrogen fixation refers to the process by which nitrogen-fixing microorganisms reduce atmospheric nitrogen (N2) into ammonia (NH3) or ammonium nitrogen (NH4) that can be utilized by plants through nitrogenase systems. + Compared with traditional chemical nitrogen fertilizers, biological nitrogen fixation has advantages such as being green, low-carbon, and sustainable. It is one of the important technical approaches to replace part of the input of chemical nitrogen fertilizers and improve the level of green agricultural production.

[0003] Azotobacter chrysophyte ( Azotobacter chroococcum Nitrogen-fixing bacteria, as common free-living nitrogen-fixing microorganisms in soil, possess strong environmental adaptability and certain growth-promoting effects. They can promote crop growth by fixing atmospheric nitrogen, secreting active metabolites, and regulating the rhizosphere microecological environment. Therefore, nitrogen-fixing bacteria have good application prospects in dryland agriculture, greenhouse cultivation, and the development of green agricultural inputs. However, nitrogen fixation is essentially a high-energy-consuming biochemical reaction. Nitrogenase catalyzes the reduction of N2 to NH3, requiring a continuous electron supply and high energy support. Electron transfer efficiency is one of the important factors affecting nitrogen fixation efficiency. In actual soil environments, especially under conditions of no light or weak energy supply, the extracellular and intracellular electron transfer processes of nitrogen-fixing bacteria are generally slow, resulting in insufficient electron supply, which limits nitrogenase activity and thus affects nitrogen conversion efficiency and biological nitrogen fixation effect. At the same time, the soil environment is complex and variable. The colonization, survival, and continuous nitrogen-fixing function of nitrogen-fixing bacteria in the field are easily affected by factors such as nutrient conditions, oxygen concentration, soil physicochemical properties, and environmental stress. This causes fluctuations in the application effect of existing nitrogen-fixing bacterial agents in agricultural production, making it difficult to stably meet the crop's nitrogen requirements.

[0004] The effectiveness of existing nitrogen-fixing bacterial agents in dryland soils is easily affected by factors such as insufficient electron supply, oxidative stress, and poor environmental adaptability, resulting in limited nitrogenase activity and actual nitrogen fixation efficiency, making it difficult to fully realize their growth-promoting effects. In recent years, although some studies have used photosensitive semiconductor materials such as CdS to construct microbial-material hybrid systems to enhance microbial metabolism through photogenerated electrons, this technology relies on light-driven processes. However, the rhizosphere and deep soil environments of dryland crops are typically under dark or low-light conditions, making it difficult to maintain their effectiveness and thus limiting their applicability in real agricultural soils. Furthermore, there is currently a lack of a technical solution that can effectively promote electron transfer in nitrogen-fixing bacteria, increase nitrogenase activity, enhance nitrogen fixation efficiency, and further promote the growth of dryland crops under dark conditions.

[0005] In recent years, nanomaterials have been increasingly applied to the field of agricultural microbial function regulation due to their high specific surface area, good interfacial effects, and unique electrical and magnetic properties. Among them, iron(III) oxide (Fe3O4) nanoparticles, as a typical functional nanomaterial, have good electrical conductivity, biocompatibility, and electron transport mediating ability, showing certain potential in promoting extracellular electron transport in microorganisms and enhancing interfacial reactivity. Summary of the Invention

[0006] This invention aims to solve at least one of the technical problems existing in the prior art. This invention provides a biocatalytic method and application for stimulating nitrogen fixation and crop growth in dryland soils. The method uses *Azotobacter chrysophagus* as the biocatalyst host and iron(III) oxide (Fe3O4) nanoparticles as the functional material. By loading the Fe3O4 nanoparticles onto the outer membrane surface of *Azotobacter chrysophagus*, a nitrogen-fixing bacteria-Fe3O4 interface is constructed, reducing bacterial oxidative stress, thereby increasing nitrogenase activity and nitrogen fixation efficiency, enhancing biological nitrogen fixation in dryland soils, and promoting crop nitrogen absorption, utilization, growth, and development.

[0007] The first aspect of the present invention is to provide a biological hybrid system.

[0008] The second objective of this invention is to provide a method for preparing the biohybrid system of the first aspect of this invention.

[0009] A third aspect of the present invention is to provide a composition.

[0010] The fourth aspect of this invention aims to provide an application of the biological hybrid system of the first aspect of this invention.

[0011] The fifth aspect of this invention aims to provide a method.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a biohybrid system comprising nitrogen-fixing microorganisms and iron tetroxide nanoparticles loaded on the surface of the nitrogen-fixing microorganisms.

[0013] In some embodiments of the present invention, the nitrogen-fixing microorganisms include at least one of Azotobacter chrysotrichum, Azotobacter venereum, Pseudomonas schrenckii, Burkholderia spp., Azotobacter haloj, Clostridium pasteurellii, Rhodospirillum rubrum, and Azotospirillum brasiliensis.

[0014] In some preferred embodiments of the present invention, the nitrogen-fixing microorganism is *Azotobacter chrysotrichum*.

[0015] In some embodiments of the present invention, the average particle size of the iron oxide nanoparticles is 15-25 nm.

[0016] In some preferred embodiments of the present invention, the average particle size of the iron oxide nanoparticles is 18-22 nm.

[0017] A second aspect of the present invention provides a method for preparing the biohybrid system of the first aspect of the present invention, the method comprising the following steps: mixing iron oxide nanoparticles with nitrogen-fixing microorganisms and culturing them to obtain the biohybrid system.

[0018] In some embodiments of the present invention, the concentration of the nitrogen-fixing microorganisms is 1 × 10⁻⁶. 6 -1×10 9 CFU / mL.

[0019] In some preferred embodiments of the present invention, the concentration of the nitrogen-fixing microorganisms is 1 × 10⁻⁶. 7 -5×10 8 CFU / mL.

[0020] In some embodiments of the present invention, the ratio of nitrogen-fixing microorganisms to iron tetroxide nanoparticles is 10:1. 8 CFU: (1-50) μg.

[0021] In some preferred embodiments of the present invention, the ratio of nitrogen-fixing microorganisms to iron tetroxide nanoparticles is 10:1. 8 CFU: (5-25) μg.

[0022] In some embodiments of the present invention, the preparation of the iron oxide nanoparticles includes the following steps: mixing iron oleate, oleic acid and octadecene and then reacting them to obtain iron oxide nanoparticles.

[0023] In some embodiments of the present invention, the iron oxide nanoparticles are prepared by thermal decomposition reaction under nitrogen protection.

[0024] In some preferred embodiments of the present invention, the reaction conditions for preparing the iron oxide nanoparticles are: 180-250℃ for 20-40 min, and 310-330℃ for 1.5-2.5 h.

[0025] In some embodiments of the present invention, the preparation of the iron oxide nanoparticles further includes a post-reaction cooling step.

[0026] In some embodiments of the present invention, the culture conditions are as follows: no light, 25-30°C, and 180-240 rpm.

[0027] In some preferred embodiments of the present invention, the culture conditions are as follows: no light, 27-29°C, and 180-240 rpm.

[0028] In some embodiments of the present invention, the preparation method of the biohybrid system further includes a cleaning step.

[0029] In some embodiments of the present invention, the cleaning includes cleaning with a buffer solution.

[0030] In some embodiments of the present invention, the buffer solution includes PBS buffer.

[0031] A third aspect of the invention provides the application of the biohybrid system of the first aspect of the invention in any one of (1)-(4): (1) Soil nitrogen fixation; (2) Preparation of nitrogen-fixing products from soil; (3) Promotes crop growth; (4) Prepare products that promote crop growth.

[0032] In some embodiments of the present invention, the crops include leafy vegetables and grain crops.

[0033] In some embodiments of the present invention, the leafy vegetable crop includes at least one of lettuce, spinach, cabbage, leeks, celery, coriander, and amaranth.

[0034] In some embodiments of the present invention, the food crop includes at least one of wheat, corn, sorghum, millet, soybean, potato and sweet potato.

[0035] In some embodiments of the present invention, the soil includes dryland soil.

[0036] In some embodiments of the present invention, the crop also includes dryland crops.

[0037] In some preferred embodiments of the present invention, the crop is lettuce.

[0038] A fourth aspect of the present invention provides a composition comprising the biohybrid system of the first aspect of the present invention.

[0039] In some embodiments of the present invention, the composition further includes fertilizer.

[0040] In some embodiments of the present invention, the fertilizer includes at least one of calcium fertilizer, potassium fertilizer, magnesium fertilizer and phosphate fertilizer.

[0041] A fifth aspect of the invention is to provide a method comprising the step of treatment using a bio-hybrid system of the first aspect of the invention; The method is (A) or (B): (A) A method for nitrogen fixation in soil; (B) A method to promote crop growth.

[0042] In some embodiments of the present invention, the method specifically includes the step of applying the bio-hybrid system of the first aspect of the present invention to soil and / or crop rhizosphere.

[0043] The beneficial effects of this invention are: Compared with existing technologies, this invention, by constructing an interfacial hybrid system of iron tetroxide nanoparticles and *Azotobacter chrysophyllariae*, can promote efficient electron transfer to nitrogenase under light-free conditions, improve nitrogenase activity and biological nitrogen fixation efficiency, while reducing oxidative stress on the bacteria caused by reactive oxygen species, and enhancing the survival and functional stability of nitrogen-fixing bacteria in complex arid soil environments. The iron tetroxide-*Azotobacter chrysophyllariae* biohybrid system provided by this invention can improve nitrogen fixation levels in arid soils, promote nitrogen absorption and utilization and growth of crops such as lettuce, and increase crop biomass and nutrient accumulation while reducing the input of chemical nitrogen fertilizers. It is not light-driven and is more suitable for real farmland rhizosphere and dark soil environments than existing photosensitive semiconductor-microorganism systems, showing good application prospects and promotional value. Attached Figure Description

[0044] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The images show transmission electron microscopy (TEM) images and X-ray diffraction (XRD) patterns of Fe3O4 nanoparticles. A1 and A2 are TEM images of the Fe3O4 nanoparticles; B is the XRD pattern of the Fe3O4 nanoparticles.

[0045] Figure 2 for Azotobacter chroococcum The mapping analysis results of Fe3O4 nanoparticles are shown in the figure. Where A represents... Azotobacter chroococcumSuperimposed mapping diagrams of Fe, C, and N elements loaded on Fe3O4 nanoparticles; B is the mapping diagram of Fe element; C is the mapping diagram of C element.

[0046] Figure 3 The EIS results are shown for different treatments.

[0047] Figure 4 The results of cyclic voltammetry (CV) curves under different treatments are shown in the figure.

[0048] Figure 5 The figures show the results of intracellular H2O2 content (A) and bacterial antioxidant capacity (B) under different treatments. represent P <0.001.

[0049] Figure 6 This figure shows the nitrogenase activity of microorganisms under different treatments. In the figure, represent P <0.01, represent p <0.001.

[0050] Figure 7 The figures show the results of total nitrogen content (A) and total carbon / total nitrogen ratio (B) in microorganisms under different treatments. In the figures, express P <0.05, express p <0.001.

[0051] Figure 8 Images (A) show the growth of lettuce under different treatments, along with the biomass of the edible portion (B) and roots (C). In the images, a, b, c, and d represent significant differences between groups containing different letters. P <0.05), there was no significant difference between groups with the same letter.

[0052] Figure 9 The graph shows the results of total nitrogen (A) and total phosphorus (B) content in lettuce under different treatments. In the graph, a, b, c, and d represent significant differences between groups containing different letters. P <0.05), there was no significant difference between groups with the same letter. Detailed Implementation

[0053] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0054] Terminology Explanation: Hybridization: Combining living microorganisms (such as bacteria and microalgae) with inorganic functional materials (such as semiconductor nanoparticles, carbon nanotubes, and magnetic materials) through physical or chemical methods to create a new type of "half-life, half-artificial" composite life form.

[0055] IO: Iron(II,III) oxide nanomaterials.

[0056] AZ: Azotobacter chrysophyte ( Azotobacter chroococcum ).

[0057] AZ-IO: Iron(II,III) oxide-Azotobacter chrysotile biohybrid system.

[0058] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0059] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0060] Example 1 A ferric oxide-azolidinium biohybrid system was prepared by the following method: (1) Preparation of iron(III) oxide nanoparticles (IO) 10.8 g of ferric chloride hexahydrate (FeCl3·6H2O), 36.5 g of sodium oleate, 60 mL of ethanol, 40 mL of deionized water, and 70 mL of n-hexane were mixed thoroughly and reacted at 70 °C for 4 h. After cooling, the organic phase was separated, washed three times with deionized water, and n-hexane was removed by rotary evaporation to obtain reddish-brown waxy ferric oleate. Before use, it was vacuum dried at 70 °C for 9 h. 1.806 g of freshly prepared ferric oleate (ferric oleate is extremely prone to hydrolysis, agglomeration, and deterioration; after storage, it will lead to uneven particle size and poor dispersibility, and must be prepared fresh before use) and 0.372 mL of oleic acid were added to 30 mL of octadecene. After thorough mixing, the temperature was slowly increased from 200 °C to 250 °C over 30 min, and then further increased to 320 °C. The mixture was refluxed under nitrogen protection for 2 h. After the reaction was completed, it was naturally cooled to room temperature to obtain iron(III) oxide nanoparticles.

[0061] (2) Preparation of the iron tetroxide-azolidinium biohybrid system (AZ-IO) Ferric oxide nanoparticles were combined with Azotobacter chrysozoans, allowing the ferric oxide nanoparticles to be loaded onto the bacterial in vitro membrane surface, thus constructing a ferric oxide-Azotobacter chrysozoans hybrid system. Azotobacter chroococcum The standard strain was purchased from Beina Chuanglian Biotechnology Co., Ltd. (product number BNCC192292). The nitrogen-free liquid culture medium (1L) used contained 20.0g mannitol, 0.5g yeast extract, 0.2g KH₂PO₄, 0.8g K₂HPO₄, 0.2g MgSO₄·7H₂O, 0.1g CaSO₄·2H₂O, 1.0mg FeCl₃, and 1.0mg Na₂MoO₄·2H₂O. The medium was brought to a final volume of 1L with distilled water, pH 7.2, and sterilized at 121℃ for 15min. *Azotocinus chrysophagus* was inoculated into this medium and cultured in a shaker at 28℃ and 200rpm until the logarithmic growth phase to obtain a pre-culture solution. 50mL of the pre-culture solution was adjusted to a final volume of 1×10⁻⁶. 8 The concentration of CFU / mL was mixed evenly with the iron oxide nanoparticle suspension prepared in step (1) to a final system concentration of 20 mg / L. The mixture was then cultured under dark conditions at 28°C and 200 rpm until the logarithmic growth phase, allowing the iron oxide nanoparticles to fully contact and stably load the bacterial cells, thus obtaining the iron oxide-Azotobacter chrysophagus hybrid system (AZ-IO). The ratio of bacterial concentration to nanoparticle concentration in the system was: when the concentration of Azotobacter chrysophagus was 1 × 10⁻⁶... 8 At CFU / mL, the amount of iron oxide nanoparticles added is 20 mg / L, i.e., 1 × 10⁻⁶. 8 CFU corresponds to 20 μg Fe3O4.

[0062] Example 2 The iron oxide nanoparticles (IO) and the iron oxide-azolidinium chrysophylloides biohybrid system (AZ-IO) prepared in Example 1 were characterized to verify the loading of iron oxide on the bacterial cell surface.

[0063] The morphology and crystal structure of iron oxide nanoparticles were characterized using a Thermo Fisher Scientific Talos L120C transmission electron microscope (TEM) and a Bruker D8 ADVANCE X-ray diffractometer (XRD) to clarify their particle size distribution, dispersion state and crystal form characteristics.

[0064] The surface morphology of the hybridized bacteria was observed using a ZEISS Sigma360 scanning electron microscope (SEM) from Germany, and the distribution of elements such as Fe, C, and N was detected by energy dispersive spectroscopy (EDS) mapping.

[0065] The results are as follows Figure 1 As shown, the prepared nanoparticles exhibit good dispersibility and relatively uniform particle size, with an average particle size of approximately 20 nm. Figure 1 (A1), and the distributions of Fe and O elements highly overlap ( Figure 1 (A2); X-ray diffraction (XRD) patterns show characteristic diffraction peaks at crystal planes such as 311, 400, 440, and 511. Figure 1 The result (B) matches the standard card for iron oxide, indicating that the obtained material is a well-crystallized iron oxide nanoparticle that can be used to construct a hybrid system with nitrogen-fixing bacteria.

[0066] The morphology of the biological hybrid was characterized using scanning electron microscopy, combined with energy dispersive spectroscopy (EDS) elemental surface distribution analysis (mapping). The results are as follows: Figure 2 As shown, a significant Fe element signal can be detected on the surface of the hybridized bacterial cells, and the Fe element distribution has a high degree of overlap with the C and N element distribution in the corresponding regions of the bacterial cell outline. This indicates that the Fe3O4 nanoparticles have been successfully loaded onto the surface of Azotobacter chrysophyllosis cells, thereby forming a stable microbial-nano interface hybrid structure.

[0067] Example 3 A biohybrid system of iron oxide (Fe3O4) and Azotobacter chrysophyll is prepared in a manner that differs from that in Example 1 only in that the final concentration of the iron oxide nanoparticle suspension is 5 mg / L, i.e., 50 mL of pre-cultured Azotobacter chrysophyll suspension (adjusted to 1×10⁻⁶). 8 The iron oxide nanoparticle suspension synthesized in step (1) (CFU / mL) and the final concentration of IO (5 mg / L) were mixed evenly and cultured in the dark at 28°C and 200 rpm until the logarithmic growth phase, so that the iron oxide nanoparticles could be in full contact with the bacterial surface, thus obtaining the iron oxide-azolidinium chrysophyll biohybrid system (AZ-IO).

[0068] Example 4 A biohybrid system of iron oxide (Fe3O4) and Azotobacter chrysophyllosis is prepared in a manner that differs from that in Example 1 only in that the final concentration of the iron oxide nanoparticle suspension is 10 mg / L, i.e., 50 mL of pre-cultured Azotobacter chrysophyllosis culture (adjusted to 1×10⁻⁶). 8The iron oxide nanoparticle suspension synthesized in step (1) (CFU / mL) and the final concentration of IO was 10 mg / L were mixed evenly and cultured in the dark, at 28°C and 200 rpm until the logarithmic growth phase, so that the iron oxide nanoparticles could be in full contact with the bacterial surface, and the iron oxide-azolidinium chrysophylloides biohybrid system (AZ-IO) was obtained.

[0069] Comparative Example 1 The *Azotocinus chrysophagus* was cultured using the method described in Example 1. 50 mL of the pre-cultured bacterial solution was adjusted to a concentration of 1 × 10⁻⁶. 8 CFU / mL.

[0070] Example 1 The electrochemical activity and antioxidant properties of the ferric oxide-azolidinium hybrid system were determined using the following steps: (1) Determination of electrochemical activity: Two treatment groups were set up: AZ group (Comparative Example 1) and AZ-IO group (Example 1). Each treatment group was cultured to the logarithmic growth phase under dark, 28°C, and 200 rpm conditions, and then washed with PBS solution. Subsequently, the bacterial concentration was adjusted to 1×10⁻⁶. 8 The concentration was CFU / mL, and the electrolytic cell was assembled in a clean bench. Characterization tests were performed using a three-electrode quartz reaction cell connected to a CHI660E electrochemical workstation. The system used carbon cloth as the working electrode, platinum wire as the counter electrode, Ag / AgCl as the reference electrode, and 0.01M, pH 7.20 PBS buffer as the electrolyte. Electrochemical impedance spectroscopy (EIS) was performed in the frequency range of 10,000 Hz–0.1 Hz with an AC amplitude of 5 mV applied at an open-circuit potential. Cyclic voltammetry (CV) was performed with a potential scan range of -0.8 V to 0.8 V and a scan rate of 0.015 V / s.

[0071] (2) Oxidative stress assessment: Two treatments were set up: AZ group (Comparative Example 1); AZ-IO group (Example 1). After the bacteria reached the logarithmic growth phase, they were centrifuged at 4°C and 6000 rpm, and the supernatant was discarded. Subsequently, the hydrogen peroxide content and total antioxidant capacity in the bacteria were determined according to the hydrogen peroxide kit (Beijing Box Biotechnology Co., Ltd., AKAO003-2M), the total antioxidant capacity assay kit (ABTS rapid method, Shanghai Beyotime Biotechnology Co., Ltd., S0121), and the BCA protein concentration assay kit (Beijing Solarbio Science & Technology Co., Ltd., PC0020).

[0072] The results of electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) curves are as follows: Figure 3 and Figure 4 As shown, the magnitudes of the charge transfer resistances of the three groups are, in descending order: IO group > AZ group > AZ. IO group, including AZ The charge transfer resistance of the IO bio-hybrid system is significantly reduced ( Figure 3 The CV curves also showed that the AZ-IO group of biohybrid systems exhibited stronger reduced state and electroactivity than the AZ group of Azotobacter chrysophyll. Figure 4 This indicates that IO promotes more efficient electron transfer within biological hybridization systems.

[0073] The results are as follows Figure 5 As shown, compared with the AZ group, the hydrogen peroxide content per gram of protein in the AZ-IO group decreased by 63.52% ( Figure 5 (A), and the total antioxidant capacity of the bacteria increased by 104.25% ( Figure 5 (Figure B) indicates that the iron(III) oxide hybrid treatment enhances the antioxidant capacity of *Azotobacter chrysotrichum*, thereby reducing hydrogen peroxide accumulation and increasing the survival rate of *Azotobacter chrysotrichum*. Therefore, iron(III) oxide hybrid treatment can promote the electron transfer and antioxidant capacity of *Azotobacter chrysotrichum*, potentially stimulating nitrogen fixation in the soil.

[0074] Example 2 The determination of bacterial nitrogenase activity in the ferric oxide-Azotobacter chrysoprase hybrid system is performed using the following steps: Ferric oxide nanoparticles were combined with Azotobacter chrysophyllosis at different concentrations to construct biohybrid systems with different concentration gradients. Four treatment groups were established: CK group (Comparative Example 1), IO5 group (Example 3), IO10 group (Example 4), and IO20 group (Example 1). The pre-cultured Azotobacter chrysophyllosis culture in each treatment group was adjusted to OD0. 600 The bacterial culture was incubated at 0.1 oz in the dark at 28°C and 200 rpm. After the bacteria reached the logarithmic growth phase, they were washed three times with Ashby nitrogen-free medium, and the OD value was adjusted to 0.1. 600 =0.7, take 20 mL and add it to a 50 mL sealed vial. Replace the gas in the upper part of the vial with a mixed gas containing 5% oxygen, 10% acetylene and 85% argon. The total pressure is 1 atmosphere. Incubate for 4 h under dark conditions, 28℃ and 200 rpm. Then, extract 200 µL of headspace gas and inject it into the gas chromatograph. Use a gas chromatograph equipped with an FID detector and an HP-PLOT Q capillary column, with high-purity helium as the carrier gas, and quantitatively determine the content of ethylene, the acetylene reduction product, by external standard method. The amount of ethylene produced is used to characterize nitrogenase activity to evaluate nitrogenase activity under different treatment conditions. The Ashby nitrogen-free medium (1L) was prepared as follows: mannitol 10.0g, KH2PO4 0.2g, MgSO4·7H2O 0.2g, NaCl 0.2g, CaSO4·2H2O 0.1g, CaCO3 5.0g, diluted to 1L with distilled water, pH 7.0, and sterilized at 121℃ for 15min before use.

[0075] As shown in Figure 6, the nitrogenase activity in all treatment groups was improved compared with the control group after the addition of iron oxide. The IO10 and IO20 groups showed the most significant promoting effect, increasing to 143.15% and 138.81% respectively. This indicates that iron oxide nanoparticles can improve the electron transfer efficiency of Azotobacter chrysophagus under light-free conditions, thereby enhancing the catalytic activity of nitrogenase.

[0076] Example 3 The nitrogen fixation efficiency of the iron tetroxide-azolidinium hybrid system was determined using the following steps: The bacterial cultures of the four treatment groups (CK group, IO5 group, IO10 group and IO20 group) in Example 2 were cultured to the logarithmic phase and then diluted 50 times with ammonia-free water. The total nitrogen content of the iron tetroxide-azolidinium biohybrid system was determined by the alkaline potassium persulfate digestion ultraviolet spectrophotometric method (HJ 636—2012) to evaluate the overall nitrogen fixation effect of the biohybrid system under different treatment conditions.

[0077] To further determine the carbon and nitrogen accumulation characteristics of the bacteria, two additional treatments were set up: AZ group (Comparative Example 1); AZ-IO group (Example 1). After the bacteria grew to the logarithmic phase, they were washed three times with PBS solution, centrifuged to leave cell pellets, and then freeze-dried to obtain solid bacterial samples. Using a German Elementar Vario EL cube organic elemental analyzer, 5 mg of freeze-dried bacterial sample was weighed, placed in a tin capsule, compressed, and catalytically combusted at 1150 °C in CHN mode to convert carbon and nitrogen in the sample into CO2 and N2, respectively. After adsorption separation, the samples were detected by a thermal conductivity detector (TCD). The total carbon and total nitrogen mass fractions were quantitatively calculated using the external standard method to further characterize the promoting effect of the hybrid system on the nitrogen accumulation capacity of the bacteria.

[0078] The results are as follows Figure 7 As shown, the total nitrogen content in the treatment groups with added iron oxide was higher than that in the control group, and the total nitrogen accumulation generally showed an upward trend with the increase of iron oxide concentration, with the IO20 group having the highest total nitrogen content. Figure 7 (A) indicates that iron(III) oxide hybridization can improve the overall nitrogen-fixing capacity of *Azotobacter chrysoprase*. Regarding the determination of cell carbon and nitrogen elements, the results showed that compared with the AZ group, the total nitrogen content of the AZ-IO group increased by 113.27%, and the cell C / N ratio decreased by 42.90%. Figure 7 (B) indicates that the iron tetroxide hybridization treatment is beneficial to promoting nitrogen fixation and accumulation by Azotobacter chrysoprase.

[0079] Example 4 The ferric oxide-azolidinium hybrid system was applied to dryland soils to verify its promoting effects on soil nitrogen fixation and crop growth. The specific steps are as follows: Four treatment groups were set up as follows: CK group (no nitrogen fertilizer treatment, 44.4 mg superphosphate, 69.4 mg potassium sulfate were mixed with 10 mL ultrapure water and evenly applied to 250 g of dryland soil); AZ group (no nitrogen fertilizer + Azotobacter chrysotile treatment, 44.4 mg superphosphate, 69.4 mg potassium sulfate were mixed with 10 mL OD... 600 =0.2% Azotobacter chrysophagus bacterial suspension (Azotobacter chrysophagus bacterial suspension in Comparative Example 1 adjusted to OD) 600 =0.2) After mixing, apply evenly to 250g of dryland soil; AZIO group (no nitrogen fertilizer + nitrogen-fixing bacteria) The system was treated with a ferric oxide hybrid system, consisting of 44.4 mg of superphosphate, 69.4 mg of potassium sulfate, and 10 mL of OD. 600 =0.2 AZ IO hybrid bacterial culture (AZ-IO in Example 1 adjusted to OD) 600 =0.2) After mixing, it was evenly applied to 250g of dryland soil; Fer group (conventional nitrogen fertilizer treatment, 81.5mg urea, 44.4mg superphosphate, 69.4mg potassium sulfate were mixed with 10mL ultrapure water and evenly applied to 250g of dryland soil). Lettuce seedlings with uniform growth were transplanted into pre-dug pits in treatment pots containing 250g of soil. They were cultured from the seedling stage to the rosette stage, for a total culture time of 35 days. During the culture period, consistent water management and environmental conditions were maintained (27℃, natural ambient humidity, water content maintained at 50% field capacity) to ensure comparability between different treatments. At harvest, the aboveground and belowground biomass of each treatment lettuce plant was measured, and the total nitrogen and total phosphorus content in the plant tissues were further measured to evaluate the effects of the biocatalytic method of this invention on nitrogen fixation in dryland soil, crop nitrogen absorption and utilization, and growth promotion.

[0080] The results showed that both the AZ group and the AZIO group promoted lettuce growth compared with the CK group, with the AZIO group showing a more significant promoting effect. Figure 8 (A). The aboveground and root biomass of lettuce in the AZIO group were significantly higher than those in the CK group (94.67% and 126.50%) and the AZ group (26.33% and 11.85%), and the total nitrogen content of the plants was also significantly increased by 99.77% and 39.93%, respectively. Figure 8 China B and Figure 8The presence of iron tetroxide (Fe3O4) and Azotobacter chrysotile (Azotobacter chrysotile) in the AZIO group indicates that the hybrid system can enhance biological nitrogen fixation in dryland soils and improve crop nitrogen uptake. The total phosphorus content in the AZIO group also showed an increasing trend, reaching the highest level among all treatment groups, suggesting that this hybrid system, in addition to improving nitrogen supply, may also promote the absorption and utilization of other mineral nutrients by improving the rhizosphere environment and enhancing root vigor. The overall growth performance of the AZIO group was similar to that of the conventional nitrogen-fertilized Fer group (…). Figure 9 This indicates that the present invention has the potential to reduce the input of chemical nitrogen fertilizers.

[0081] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A biological hybrid system, characterized in that, The biohybrid system includes nitrogen-fixing microorganisms and iron tetroxide nanoparticles loaded on the surface of the nitrogen-fixing microorganisms.

2. The biological hybrid system according to claim 1, characterized in that, The nitrogen-fixing microorganisms include at least one of Azotobacter chrysotrichum, Azotobacter venereum, Pseudomonas schrenckii, Burkholderia spp., Azotobacter haloj, Clostridium pasteurellii, Rhodospirillum rubrum, and Azotospirillum brasiliensis.

3. The biological hybrid system according to claim 1, characterized in that, The average particle size of the iron oxide nanoparticles is 15-25 nm.

4. The method for preparing the bio-hybrid system according to any one of claims 1-3, characterized in that, The method includes the following steps: mixing iron oxide nanoparticles with nitrogen-fixing microorganisms and culturing them to obtain a bio-hybrid system.

5. The preparation method according to claim 4, characterized in that, The ratio of nitrogen-fixing microorganisms to iron tetroxide nanoparticles is 10:

1. 8 CFU: (1-50) μg.

6. The preparation method according to claim 4, characterized in that, The culture conditions are as follows: no light, 25-30℃, and 180-240 rpm.

7. The use of the biohybrid system according to any one of claims 1-3 in any one of (1)-(4): (1) Soil nitrogen fixation; (2) Preparation of nitrogen-fixing products from soil; (3) Promotes crop growth; (4) Prepare products that promote crop growth.

8. A composition, characterized in that, The composition comprises the biohybrid system according to any one of claims 1-3.

9. A method comprising the step of treatment using a bio-hybrid system according to any one of claims 1-3; The method is (A) or (B): (A) A method for nitrogen fixation in soil; (B) A method to promote crop growth.

10. The method according to claim 9, characterized in that, The method specifically includes the step of applying the bio-hybrid system of any one of claims 1-3 to the soil and / or crop rhizosphere.

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