Functionalized carrier-azotobacter compound system as well as preparation method and application thereof

Abstract

The invention discloses a functionalized carrier-azotobacter compound system as well as a preparation method and application thereof, and belongs to the technical field of biology. The invention discloses a preparation method of a functionalized carrier-azotobacter compound system. The preparation method comprises the following steps: preparing a zirconium-oxygen cluster, preparing a zirconium-based metal organic framework, preparing a copper monatomic anchored zirconium-based metal organic framework and preparing a copper monatomic anchored zirconium-based metal organic framework-brown azotobacter heterozygote. A photocatalyst with optical excitation characteristics and microorganisms with specific biological nitrogen fixation pathways are combined, a photocatalyst-microorganism heterozygote is constructed, a new strategy for regulating and optimizing the photocatalytic performance of UiO-66 through copper single atoms is provided, the electronic structure of UiO-66 is improved, the light absorption capacity is improved, and the photocatalytic performance of UiO-66 is improved through a metal-phenolic aldehyde network interface strategy. The stable anchoring of the photocatalyst on an azotobacter cell membrane is realized, the biocompatibility of the photocatalyst is enhanced, and a stable and efficient UiO-66-brown azotobacter heterozygote is constructed.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and more particularly to a functional carrier-diazotroph composite system, a preparation method and application thereof. BACKGROUND

[0002] Ammonia is one of the most important industrial raw materials in the world, playing a crucial role in nitrogen fertilizer, hydrogen storage and other chemical manufacturing, and is closely related to agriculture, food industry, energy distribution and global ecology. At present, the global production of ammonia mainly relies on the Haber-Bosch process, which must be carried out in a high-temperature and high-pressure environment (about 500℃, 100-150 bar), thereby causing great consumption of the environment and energy. For example, in 2020, the carbon dioxide emissions caused by the Haber-Bosch process accounted for 1.4% of the global total, and the energy consumption accounted for 1% of the global total. Therefore, it is urgent to develop a new process for the synthesis of ammonia in order to reduce carbon dioxide emissions and energy consumption.

[0003] Photocatalytic nitrogen fixation technology uses solar energy as a driving force to produce ammonia directly from nitrogen and water at room temperature and pressure, and has zero carbon emissions, which is one of the most promising artificial nitrogen fixation methods. However, existing photocatalytic technology is restricted by factors such as low light utilization efficiency, serious carrier recombination and difficulty in activating nitrogen, resulting in low ammonia production efficiency. Therefore, improving the ammonia production efficiency is the research focus in the field of photocatalyst nitrogen fixation. Relatively speaking, biological nitrogen fixation is a key step in the entire nitrogen cycle and plays an important role in maintaining ecological, environmental and agricultural balance. Nitrogen-fixing microorganisms are a natural resource that can be taken without depletion and used without exhaustion, and they have a rich nitrogen reduction metabolic network under environmental conditions. The nitrogen fixation process of diazotrophs depends on continuous electron supply, which limits the nitrogen fixation efficiency due to insufficient reducing power supply. The construction of a photocatalyst-microorganism hybrid system is expected to break through the bottlenecks of high cost of traditional photocatalysts and insufficient electron supply of natural nitrogen fixation pathways, and to realize the complementary advantages of solar energy capture, catalytic reaction activity and selectivity. In the photocatalyst-microorganism hybrid system, electrons are generated by photoexcitation of the catalyst, and the electrons are transported to the inside of the cell by transport proteins on the microbial membrane, providing sufficient reducing power for the natural nitrogen fixation pathway, thereby improving the nitrogen fixation efficiency using nitrogen and sunlight. Therefore, the construction of a high-efficiency photocatalyst-microorganism hybrid system for photocatalytic nitrogen fixation is of great significance for enhancing the nitrogen fixation capacity of the nitrogen-fixing microbial chassis and improving the efficiency of biological nitrogen fixation.

[0004] The selection of the photocatalyst is particularly important in the construction of a photocatalyst-microorganism hybrid deep coupling process. Metal-organic frameworks, as a new type of porous crystalline material, have attracted increasing attention in the field of photocatalysis and have shown broad application prospects in various photocatalytic systems. Among them, zirconium-based metal-organic frameworks (UiO-66) have become a photocatalyst of great concern due to their large specific surface area, rich pore structure, and tunable band structure. On this basis, introducing single atoms is one of the effective strategies to obtain efficient photocatalytic nitrogen fixation. Single atom sites act as electron-rich centers, and the introduction of single atoms into metal-organic frameworks can effectively adjust the electronic structure, charge transport, and surface adsorption capacity of the material, thereby reducing the activation energy barrier and promoting the catalytic reaction. Therefore, anchoring single atoms can further enhance the photocatalytic performance of UiO-66 and significantly improve its reactivity and stability in the photocatalytic nitrogen fixation system. However, compared with other photocatalysts, the application of single atom-modified UiO-66 in photocatalyst-microorganism hybrids is still in the exploratory stage, especially in the aspect of using it as a light capture module for photocatalytic nitrogen fixation, which still lacks systematic development.

[0005] Therefore, it is an urgent problem for those skilled in the art to provide a functional carrier-nitrogen-fixing bacteria composite system, a preparation method and application thereof. SUMMARY

[0006] Therefore, the present application provides a functional carrier-nitrogen-fixing bacteria composite system, a preparation method and application thereof, which solves the problems of energy consumption and pollution in industrial ammonia production and insufficient microbial nitrogen fixation reduction.

[0007] The present application combines photocatalysts with light excitation characteristics and microorganisms with specific biological nitrogen fixation pathways to construct a photocatalyst-microorganism hybrid, achieving complementary advantages in solar energy capture, catalytic reaction activity and selectivity.

[0008] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0009] A preparation method of a functional carrier-nitrogen-fixing bacteria composite system, the specific steps are as follows:

[0010] 1) Preparation of zirconium oxide clusters

[0011] 2g of ZrCl4 was added to a mixed solution of 3mL of glacial acetic acid and 5mL of isopropyl alcohol, and stirred at 500rpm for 60 minutes at 115-125℃; the product was collected by centrifugation at 10,000rpm, and the collected white solid was then washed twice with acetone and dried in a 60-70℃ oven to obtain zirconium oxide clusters;

[0012] 2) Preparation of zirconium-based metal-organic framework UiO-66

[0013] 75 mg of zirconium oxide clusters were dispersed in 0.5 mL of acetic acid and stirred at 600 rpm at room temperature. Then, 1.25 mL of H2O was added, and the reaction mixture was stirred until completely clear. Subsequently, 10 mL of anhydrous ethanol was added to the solution, followed immediately by 50 mg of 1,4-phthalic acid, and the mixture was stirred at room temperature for 3 hours. The resulting solution was centrifuged at 13,000 rpm for 15 minutes, and then washed twice with ethanol, centrifuged at 13,000 rpm for 15 minutes each time. The collected solid was dried in an oven at 60–70 °C to obtain UiO-66.

[0014] 3) Preparation of copper single-atom anchored zirconium-based metal-organic framework Cu1 / UiO-66

[0015] 40 mg UiO-66 and 200 mg CuCl2 were added to a microwave container, followed by 6 mL of acetonitrile. The mixture was then rapidly heated to 85 °C using a microwave and maintained at that temperature for 30 minutes. After cooling to room temperature, the solid was separated by centrifugation, washed sequentially with N,N-dimethylformamide and acetonitrile, and the collected solid was dried in an oven at 60–70 °C to obtain Cu1 / UiO-66.

[0016] 4) Preparation of copper single-atom anchored zirconium-based metal-organic framework-brown nitrogen-fixing bacteria hybrid Cu1 / UiO-66@TA / A. vinelandii

[0017] 200 mg Cu1 / UiO-66 was dispersed in 10 mL of ultrapure water to obtain a Cu1 / UiO-66 suspension. Then, 65 mg FeCl3·6H2O and 400 mg tannic acid were added to the Cu1 / UiO-66 suspension in sequence, followed by 10 mL of pH 8.0, 100 mM Tris buffer to obtain Cu1 / UiO-66@TA. Cu1 / UiO-66@TA was centrifuged at 8000 g for 5 minutes, washed 3-4 times with ultrapure water, washed with 70% ethanol and incubated for 10 minutes, and finally washed 3-4 times with ultrapure water.

[0018] OD values ​​of A. vinelandii suspension and Cu1 / UiO-66@TA solution were compared. 600 Adjust the concentrations to 2.0 and 1.0 respectively; mix equal volumes of both and vortex for 60 seconds; add Fe to a final concentration of 0.05 mg / mL. 3+ Add an equal volume of pH 7.4, 10mM PBS buffer solution to obtain Cu1 / UiO-66@TA / A.vinelandii. Wash 3-4 times with ultrapure water and centrifuge at 2000g for 2 minutes to remove free Cu1 / UiO-66, thus obtaining the functionalized carrier-nitrogen-fixing bacteria complex system.

[0019] Key points:

[0020] ①Preparation method of copper monatomic atom anchoring zirconium-based metal organic framework;

[0021] ②Construction method of biological hybrid.

[0022] Further, the functional carrier-diazotroph composite system prepared by the method.

[0023] Further, the application of the functional carrier-diazotroph composite system in improving biological nitrogen fixation efficiency.

[0024] Further, the application of the functional carrier-diazotroph composite system in improving the level of reducing power and energy.

[0025] The design concept of the present application innovatively combines monatomic catalysts with diazotrophic microorganisms to achieve efficient photocatalytic ammonia production.

[0026] Through the above technical solution, compared with the prior art, the present application provides a functional carrier-diazotroph composite system, its preparation method and application, which combines a photocatalyst with light excitation characteristics and a microorganism with a specific biological nitrogen fixation pathway, constructs a photocatalyst-microorganism hybrid, proposes a new strategy for copper monatomic atom to regulate and optimize the photocatalytic performance of UiO-66, improves its electronic structure and light absorption capacity, and realizes stable anchoring of the photocatalyst on the cell membrane of diazotrophic bacteria through a metal-phenolic network interface strategy, enhances its biocompatibility, and constructs a stable and efficient UiO-66-brown diazotrophic bacteria hybrid. The present application does not require any auxiliary agent or sacrificial agent, and can efficiently fix nitrogen only by relying on solar energy, realizing green and clean production of ammonia. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only embodiments of the present application, and those skilled in the art can obtain other drawings according to the provided drawings without creative labor.

[0028] Figure 1 X-ray diffraction pattern of zirconium oxygen cluster.

[0029] Figure 2 Transmission electron microscope image; wherein, A represents the transmission electron microscope image of UiO-66; B represents the transmission electron microscope image of Cu1 / UiO-66.

[0030] Figure 3Figure 1 is a ball difference electron microscope image; wherein A represents high angle annular dark field-scanning transmission electron microscopy (HAADF-STEM); B represents copper single atom phase; C represents energy dispersive spectroscopy.

[0031] Figure 4 Figure 1 is a ball difference electron microscope image; wherein A represents high angle annular dark field-scanning transmission electron microscopy (HAADF-STEM); B represents copper single atom phase; C represents energy dispersive spectroscopy.

[0032] Figure 5 Figure 1 is a ball difference electron microscope image; wherein A represents high angle annular dark field-scanning transmission electron microscopy (HAADF-STEM); B represents copper single atom phase; C represents energy dispersive spectroscopy.

[0033] Figure 6 Figure 1 is a ball difference electron microscope image; wherein A represents high angle annular dark field-scanning transmission electron microscopy (HAADF-STEM); B represents copper single atom phase; C represents energy dispersive spectroscopy.

[0034] Figure 7 Figure 1 is a ball difference electron microscope image; wherein A represents high angle annular dark field-scanning transmission electron microscopy (HAADF-STEM); B represents copper single atom phase; C represents energy dispersive spectroscopy.

[0035] Figure 8 Figure 1 is a ball difference electron microscope image; wherein A represents high angle annular dark field-scanning transmission electron microscopy (HAADF-STEM); B represents copper single atom phase; C represents energy dispersive spectroscopy.

[0036] Figure 9 Figure 1 is a ball difference electron microscope image; wherein A represents high angle annular dark field-scanning transmission electron microscopy (HAADF-STEM); B represents copper single atom phase; C represents energy dispersive spectroscopy.

[0037] Figure 10 Figure 1 is a ball difference electron microscope image; wherein A represents high angle annular dark field-scanning transmission electron microscopy (HAADF-STEM); B represents copper single atom phase; C represents energy dispersive spectroscopy.

[0038] Figure 11 Figure 1 is a ball difference electron microscope image; wherein A represents high angle annular dark field-scanning transmission electron microscopy (HAADF-STEM); B represents copper single atom phase; C represents energy dispersive spectroscopy.

[0039] Figure 12 Figure 1 is a ball difference electron microscope image; wherein A represents high angle annular dark field-scanning transmission electron microscopy (HAADF-STEM); B represents copper single atom phase; C represents energy dispersive spectroscopy.

[0040] Figure 13 Figure 1 is a ball difference electron microscope image; wherein A represents high angle annular dark field-scanning transmission electron microscopy (HAADF-STEM); B represents copper single atom phase; C represents energy dispersive spectroscopy.

[0041] Figure 14 Figure 1 is a ball difference electron microscope image; wherein A represents high angle annular dark field-scanning transmission electron microscopy (HAADF-STEM); B represents copper single atom phase; C represents energy dispersive spectroscopy. +ATP; wherein, A represents NADH / NAD + ; B represents ATP. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0043] Embodiment 1

[0044] A preparation method of a functionalized carrier-diazotroph composite system, the specific steps are as follows:

[0045] 1) Preparation of zirconium oxygen cluster

[0046] 2g of ZrCl4 was added to a mixed solution of 3mL of glacial acetic acid and 5mL of isopropyl alcohol, and stirred at 500rpm for 60 minutes at 120°C. The product was collected by centrifugation at 10,000rpm, and the collected white solid was then washed twice with acetone and dried in a 60°C oven to obtain the zirconium oxygen cluster.

[0047] 2) Preparation of zirconium-based metal organic framework (UiO-66)

[0048] 75mg of zirconium oxygen cluster was dispersed in 0.5mL of acetic acid and stirred at 600rpm at room temperature. Then 1.25mL of H2O was added, and the reaction mixture was stirred until it was completely clear. Then, 10mL of anhydrous ethanol was added to the solution, followed by the immediate addition of 50mg of 1,4-benzenedicarboxylic acid, and the reaction was stirred at room temperature for 3 hours. The resulting solution was centrifuged at 13000rpm for 15 minutes, and then washed twice with ethanol (13000rpm for 15 minutes). The collected solid was dried in a 60°C oven to obtain UiO-66.

[0049] 3) Preparation of copper monatomic-anchored zirconium-based metal organic framework (Cu1 / UiO-66)

[0050] 40mg of UiO-66 and 200mg of CuCl2 were added to a microwave container, followed by the addition of 6mL of acetonitrile. Then the mixture was quickly heated to 85°C using a microwave, and the temperature was maintained for 30 minutes. After cooling to room temperature, the solid was separated by centrifugation, washed with N,N-dimethylformamide and acetonitrile in turn, and the collected solid was dried in a 60°C oven to obtain Cu1 / UiO-66.

[0051] 4) Preparation of Cu single atom anchored zirconium-based metal organic framework-brown nitrogen-fixing bacteria hybrid (Cu1 / UiO-66@TA / A. vinelandii)

[0052] Cu1 / UiO-66@TA / A. vinelandii was prepared by mixing Cu1 / UiO-66@TA and A. vinelandii (QCD10473 JCM20040 = ATCC9046, purchased from Shanghai Qincheng Biotechnology Co., Ltd.) suspensions. OD values of Cu1 / UiO-66@TA and A. vinelandii were measured at 600 nm. For the assembly of Cu1 / UiO-66@TA / A. vinelandii biohybrid, OD values of A. vinelandii suspension and Cu1 / UiO-66@TA solution were adjusted to ~2.0 and ~1.0, respectively. During the assembly process, both were mixed in equal volume and vortexed for 60 seconds to facilitate the collision between Cu1 / UiO-66 and A. vinelandii cells. Particles on the cells were stabilized by adding metal ions (Fe3+, final concentration ~0.05 mg / mL) and equal volume of PBS buffer solution (pH 7.4, 10 mM). The obtained Cu1 / UiO-66@TA / A. vinelandii was washed with ultrapure water 3-4 times to remove free Cu1 / UiO-66 at 2000 g for 2 minutes to obtain the biohybrid.

[0053] Cu1 / UiO-66@TA / A. vinelandii was prepared by mixing Cu1 / UiO-66@TA and A. vinelandii (QCD10473 JCM20040 = ATCC9046, purchased from Shanghai Qincheng Biotechnology Co., Ltd.) suspensions. OD values of Cu1 / UiO-66@TA and A. vinelandii were measured at 600 nm. For the assembly of Cu1 / UiO-66@TA / A. vinelandii biohybrid, OD values of A. vinelandii suspension and Cu1 / UiO-66@TA solution were adjusted to ~2.0 and ~1.0, respectively. During the assembly process, both were mixed in equal volume and vortexed for 60 seconds to facilitate the collision between Cu1 / UiO-66 and A. vinelandii cells. Particles on the cells were stabilized by adding metal ions (Fe3+, final concentration ~0.05 mg / mL) and equal volume of PBS buffer solution (pH 7.4, 10 mM). The obtained Cu1 / UiO-66@TA / A. vinelandii was washed with ultrapure water 3-4 times to remove free Cu1 / UiO-66 at 2000 g for 2 minutes to obtain the biohybrid. 600 600 Cu1 / UiO-66@TA / A. vinelandii was prepared by mixing Cu1 / UiO-66@TA and A. vinelandii (QCD10473 JCM20040 = ATCC9046, purchased from Shanghai Qincheng Biotechnology Co., Ltd.) suspensions. OD values of Cu1 / UiO-66@TA and A. vinelandii were measured at 600 nm. For the assembly of Cu1 / UiO-66@TA / A. vinelandii biohybrid, OD values of A. vinelandii suspension and Cu1 / UiO-66@TA solution were adjusted to ~2.0 and ~1.0, respectively. During the assembly process, both were mixed in equal volume and vortexed for 60 seconds to facilitate the collision between Cu1 / UiO-66 and A. vinelandii cells. Particles on the cells were stabilized by adding metal ions (Fe3+, final concentration ~0.05 mg / mL) and equal volume of PBS buffer solution (pH 7.4, 10 mM). The obtained Cu1 / UiO-66@TA / A. vinelandii was washed with ultrapure water 3-4 times to remove free Cu1 / UiO-66 at 2000 g for 2 minutes to obtain the biohybrid. 3+

[0054] Cu1 / UiO-66@TA / A. vinelandii was prepared by mixing Cu1 / UiO-66@TA and A. vinelandii (QCD10473 JCM20040 = ATCC9046, purchased from Shanghai Qincheng Biotechnology Co., Ltd.) suspensions. OD values of Cu1 / UiO-66@TA and A. vinelandii were measured at 600 nm. For the assembly of Cu1 / UiO-66@TA / A. vinelandii biohybrid, OD values of A. vinelandii suspension and Cu1 / UiO-66@TA solution were adjusted to ~2.0 and ~1.0, respectively. During the assembly process, both were mixed in equal volume and vortexed for 60 seconds to facilitate the collision between Cu1 / UiO-66 and A. vinelandii cells. Particles on the cells were stabilized by adding metal ions (Fe3+, final concentration ~0.05 mg / mL) and equal volume of PBS buffer solution (pH 7.4, 10 mM). The obtained Cu1 / UiO-66@TA / A. vinelandii was washed with ultrapure water 3-4 times to remove free Cu1 / UiO-66 at 2000 g for 2 minutes to obtain the biohybrid.

[0055] Figure 1 XRD pattern of zirconium oxygen cluster is shown, and crystal diffraction peak appears at 9.6°. As shown in FIG. 4, the XRD pattern of Cu1 / UiO-66@TA / A. vinelandii is similar to that of Cu1 / UiO-66@TA, and the characteristic peak of A. vinelandii is not observed, indicating that the biohybrid is successfully prepared. Figure 2 ​​As shown, the dimensions of both UiO-66 and Cu1 / UiO-66 in the TEM image are approximately 50 nm. Further observation of the morphology of Cu1 / UiO-66 using spherical aberration electron microscopy reveals that it exhibits a regular octahedral (…). Figure 3 A), the bright spots appearing in the atomic phase are copper single atoms ( Figure 3 B), and the C, O, Zr, and Cu elements are evenly distributed ( Figure 3 C). For example Figure 4 As shown, the crystal structures of UiO-66 and Cu1 / UiO-66 were characterized again using XRD. Their diffraction peak positions were the same, and no copper phase was detected. Compared to the specific surface area of ​​UiO-66 (1091.88 m²), the difference is significant. 2 The specific surface area of ​​Cu1 / UiO-66 is 1020.88 m² / g. 2 The slight decrease in / g was attributed to the occupancy of copper sites. Figure 5 After anchoring with copper single atoms, the doublet of Zr3d in Cu1 / UiO-66 shifted to a higher bond energy, and a clear Cu 2p peak was detected, indicating that copper single atoms were successfully introduced into the defect sites of the zirconium-oxygen cluster in UiO-66. Figure 6 ).

[0056] The morphology of the biological hybrid was characterized by scanning electron microscopy, and the valence band and conduction band positions of UiO-66 and Cu1 / UiO-66 were analyzed by X-ray photoelectron spectroscopy and ultraviolet-visible diffuse reflectance absorption spectroscopy.

[0057] like Figure 7 As shown, obvious particulate matter appears on the surface of nitrogen-fixing bacteria, indicating that UiO-66 and Cu1 / UiO-66 were successfully assembled on the surface of nitrogen-fixing bacteria. UV-Vis diffuse reflectance absorption spectroscopy confirms that Cu1 / UiO-66 has better light absorption capacity and a narrower band gap than UiO-66, thus being more conducive to converting solar energy into photogenerated electrons and transferring them to nitrogen-fixing bacteria. Figure 8 Compared to UiO-66, Cu1 / UiO-66 has a lower valence band (). Figure 9 Calculations show that the conduction band positions of UiO-66 and Cu1 / UiO-66 are -1.06V and -2.08V, respectively. Figure 10 A more negative conduction band position is more conducive to transferring photogenerated electrons to the molybdenum-iron protein in nitrogen-fixing bacteria for nitrogen reduction. Figure 11 ).

[0058] Example 2

[0059] 1) Nitrogen fixation efficiency test of copper single-atom anchored zirconium-based metal-organic framework-brown nitrogen-fixing bacteria hybrid (Cu1 / UiO-66@TA / A.vinelandii)

[0060] The collected A. vinelandii or Cu1 / UiO-66@TA / A. vinelandii cells were suspended in PBS. The OD value of the suspension at 600 nm was adjusted to 0.3, and then continuously stirred at 200 rpm at 28 °C with or without light. The ammonia concentration in the sample was quantitatively determined by spectrophotometry using the indigo blue method. According to the series of reaction time sampling, then centrifuged at 10000 rpm for 5 minutes, 2 mL of supernatant was taken, 0.5 mL of 1 M NaOH solution containing 5 wt% salicylic acid and 5 wt% sodium citrate was added, then 0.1 mL of 0.05 M NaClO and 0.1 mL of 1 wt% Na2[Fe(CN)5NO]·2H2O was added. After 30 minutes of reaction, the absorbance was determined at 630 nm, and the ammonia concentration was calculated according to the standard curve calibrated by different concentrations of ammonium chloride.

[0061] 2) NAD + / NADH content determination

[0062] NAD + / NADH concentration in the biohybrid was determined using a commercially available WST-8 kit from Beyotime Biotechnology. Briefly, 1 x 10 6 suspended cells were added to 200 μL of extraction solution (provided in the kit), mixed well to facilitate cell lysis. The supernatant was collected by centrifugation (12000 g, 5 min) for later use. Reagent preparation and sample determination were performed according to the standard instructions of the kit.

[0063] 3) ATP content determination

[0064] The biohybrid was collected by centrifugation at 12000 g for 5 minutes, and the supernatant was discarded. Subsequently, 200 μL of lysis buffer was added to the collected cells, and vortexed appropriately to ensure complete cell disruption. The lysate was collected by centrifugation (12000 g, 5 min) to remove cell debris, and transferred to a 96-well plate. Finally, the detection solution (provided in the ATP detection kit, purchased from Beyotime Biotechnology) was added to each well, and immediately measured with an enzyme marker to quantitatively determine the ATP content.

[0065] Determination results:

[0066] The ammonia production efficiency of the biohybrid was determined by the indigo blue colorimetric method, and the reducing power and energy generation ability were studied.

[0067] As Figure 12 shown, the indigo blue colorimetric method for different concentrations of ammonia has a good linear relationship (R 2=0.999). Within 24 hours, the ammonia production efficiency of the bio-hybrid assembled with Cu1 / UiO-66 under light conditions was higher than that of other treatments, reaching its peak at the 16th hour. Figure 13 Compared to individual nitrogen-fixing bacteria, the nitrogen-fixing efficiency of the Cu1 / UiO-66 assembled biohybrid was increased by 9 times. Figure 13 ).like Figure 14 As shown, the reducing power of the Cu1 / UiO-66 assembled bio-hybrid is 1.97 times that of nitrogen-fixing bacteria, and the energy level is 3.2 times that of nitrogen-fixing bacteria.

[0068] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a functionalized carrier-diazotroph complex system, characterized in that, The specific steps are as follows: 1) Preparation of zirconium oxide clusters 2g of ZrCl4 was added to a mixed solution of 3mL of glacial acetic acid and 5mL of isopropyl alcohol, and stirred at 500rpm for 60 minutes at 115-125°C; The product was collected by centrifugation at 10,000rpm, and the collected white solid was then washed twice with acetone and dried in an oven at 60-70°C to obtain zirconium oxide clusters; 2) Preparation of zirconium-based metal organic framework UiO-66 75mg of zirconium oxide clusters was dispersed in 0.5mL of acetic acid and stirred at 600rpm at room temperature; then 1.25mL of H2O was added, and the reaction mixture was stirred until it was completely clear; then, 10mL of anhydrous ethanol was added to the solution, followed by the immediate addition of 50mg of 1,4-benzenedicarboxylic acid, and the reaction was stirred at room temperature for 3 hours; the resulting solution was centrifuged at 13000rpm for 15 minutes, and then washed twice with ethanol, each time at 13000rpm for 15 minutes; the collected solid was dried in an oven at 60-70°C to obtain UiO-66; 3) Preparation of copper monatomic-anchored zirconium-based metal organic framework Cu1 / UiO-66 40mg of UiO-66 and 200mg of CuCl2 were added to a microwave container, followed by the addition of 6mL of acetonitrile; then the mixture was rapidly heated to 85°C using a microwave, and the temperature was maintained for 30 minutes; after cooling to room temperature, the solid was separated by centrifugation, washed with N,N-dimethylformamide and acetonitrile, and the collected solid was dried in an oven at 60-70°C to obtain Cu1 / UiO-66; 4) Preparation of copper monatomic-anchored zirconium-based metal organic framework-brown nitrogen-fixing bacteria hybrid Cu1 / UiO-66@TA / A.vinelandii 200mg of Cu1 / UiO-66 was dispersed in 10mL of ultrapure water to obtain a Cu1 / UiO-66 suspension; then 65mg of FeCl3·6H2O and 400mg of tannic acid were added to the Cu1 / UiO-66 suspension in sequence, followed by the addition of 10mL of pH 8.0, 100mM Tris buffer to obtain Cu1 / UiO-66@TA; the Cu1 / UiO-66@TA was centrifuged at 8000g for 5 minutes, washed with ultrapure water for 3-4 times, washed with 70% ethanol and incubated for 10min, and finally washed with ultrapure water for 3-4 times; OD of A. vinelandii suspension and Cu1 / UiO-66@TA solution were adjusted to 2.0 and 1.0, respectively; both were mixed in equal volume and vortexed for 60 seconds; Fe 600 and equal volume of pH 7.4, 10 mM PBS buffer solution, obtaining Cu1 / UiO-66@TA / A. vinelandii, which was washed with ultrapure water for 3-4 times, and centrifuged at 2000 g for 2 minutes to remove free Cu1 / UiO-66, obtaining the functionalized carrier-azotobacter composite system. 3+ and equal volume of pH 7.4, 10 mM PBS buffer solution, obtaining Cu1 / UiO-66@TA / A. vinelandii, which was washed with ultrapure water for 3-4 times, and centrifuged at 2000 g for 2 minutes to remove free Cu1 / UiO-66, obtaining the functionalized carrier-azotobacter composite system.

2. The functionalized carrier-nitrogen-fixing bacteria composite system prepared by the method of claim 1.

3. The application of the functionalized carrier-nitrogen-fixing bacteria composite system of claim 2 in improving the efficiency of biological nitrogen fixation.

4. The application of the functionalized carrier-nitrogen-fixing bacteria composite system of claim 2 in improving the level of reducing power and energy.

Images