A sponge-like mycelium with photo-driven antimony oxidation and nitrogen fixation synergistic effect, its preparation method and application

Abstract

This invention provides a sponge-like mycelium for photo-driven antimony oxidation and nitrogen fixation, its preparation method, and its applications, belonging to the field of metal oxidation and microbial nitrogen fixation technology. The sponge-like mycelium of this invention consists of a metal oxidation zone and antimony-tolerant nitrogen-fixing bacteria. This mycelium can enhance the support of antimony oxidation for the chemo-nitrogen fixation process of the strains through light irradiation, utilizing photogenerated electrons to supply reducing power to achieve nitrogen fixation. Simultaneously, it oxidizes heavy metals through associated photogenerated holes, exhibiting excellent metal oxidation capacity and nitrogen fixation activity, with a nitrogenase activity ≥198.57 nmolC2H2 / (g·h). It can spontaneously transform highly toxic and low-nutrient habitats in mining areas. Unlike conventional chemoautotrophic nitrogen-fixing strains, the strains in this mycelium only need to possess metal tolerance to expand the chemo-nitrogen fixation pathway, showing promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of metal oxidation and microbial nitrogen fixation technology, specifically relating to a sponge-like bacterial gel with photo-driven antimony oxidation and nitrogen fixation synergistic, its preparation method, and its application. Background Technology

[0002] Ecological restoration is a practical and feasible method for transforming the soil environment and improving soil quality. For example, hyperaccumulating plants can effectively absorb heavy metals from the soil. However, mining area soil habitats have two distinct characteristics: firstly, high levels of toxic and harmful heavy metals; and secondly, a lack of nutrients such as carbon and nitrogen sources. This high toxicity and low nutrient content poses a significant challenge to the survival of plants and microorganisms in mining area soils. Artificially increasing organic matter can improve the low nutrient content of mining areas, but this leads to a sharp increase in remediation costs and is not a fundamental solution. Therefore, achieving spontaneous transformation of the high toxicity and low nutrient content of mining area soil habitats is a prerequisite for large-scale ecological restoration.

[0003] Microbial nitrogen fixation is a crucial source of nitrogen input in soil; however, most nitrogen-fixing bacteria are heterotrophic and struggle to adapt to highly toxic and oligotrophic mining areas. Currently, a class of microorganisms capable of chemoautotrophic nitrogen fixation using reduced As, Sb, and S has been reported. These microorganisms can promote metal oxidation, reducing metal toxicity while utilizing the energy released from metal oxidation to participate in the energy-intensive nitrogen fixation process. For example, patent application CN120738081A discloses an autotrophic nitrogen-fixing Klebsiella pneumoniae strain with antimony oxidation and growth-promoting capabilities. The chemoautotrophic nitrogen fixation process of these microorganisms is highly dependent on obtaining energy from oxidation reactions. However, most microorganisms face insufficient oxidation motive power, leading to low nitrogen fixation efficiency. Conductive framework materials containing photogenerating quantum dots can promote heavy metal oxidation through light-driven hole generation, potentially enhancing oxidation motive power. The generated photoelectrons provide sufficient reducing power for the microbial nitrogen fixation process. Simultaneously, the consumption of photoelectrons can improve the electron-hole pair separation efficiency problem caused by photoelectron accumulation, further enhancing the metal oxidation capacity of the conductive framework. However, there is still a need to enhance the electron transfer capability between microorganisms and photosensitive materials. Therefore, based on this innovative idea, developing a visible light-driven microbial gel to enhance metal oxidation power and thus promote microbial nitrogen fixation is essential for achieving spontaneous transformation of highly toxic and oligotrophic mining areas. Summary of the Invention

[0004] The primary objective of this invention is to provide a sponge-like mycelium that enables photo-driven antimony oxidation and nitrogen fixation. This mycelium has a novel structure, is simple to prepare, and is inexpensive. It can achieve synergistic effects of antimony oxidation and nitrogen fixation and has excellent application prospects.

[0005] The light-driven antimony oxidation and nitrogen fixation synergistic sponge-like bacterial gel is composed of a metal oxidation zone and antimony-tolerant nitrogen-fixing bacteria, wherein the antimony-tolerant nitrogen-fixing bacteria are uniformly dispersed in the metal oxidation zone.

[0006] The metal oxide region has a sponge-like double network structure, which is a porous structure formed by the topological bonding of a conductive skeleton and a sponge-supporting network.

[0007] The conductive framework is formed by the polymerization of pyrrole containing amine groups and is tightly connected to the photoelectric quantum dots through an amidation reaction;

[0008] The sponge-supporting network is formed by cross-linking sodium alginate with polyvalent metal ions.

[0009] In this application, topological bonding refers to the bonding formed by the physical entanglement of molecular chains at the interface, which is different from chemical bonding methods such as covalent bonds.

[0010] Furthermore,

[0011] The aforementioned light-driven antimony oxidation and nitrogen fixation synergistic sponge-like mycelium, wherein the conductive framework is formed by polymerization of at least one of 2-aminopyrrole and 3-aminopyrrole; the photoelectric quantum dots are obtained by electrolyzing an acid solution using a high-purity conductive graphite electrode plate, followed by dialyzing and filtering the electrolyte, and drying the product to obtain photoelectric quantum dots with carboxyl groups on the surface.

[0012] Furthermore,

[0013] The antimony-tolerant nitrogen-fixing bacteria retain nitrogen-fixing activity in systems containing 1-10 mM Sb(III), and these antimony-tolerant nitrogen-fixing bacteria include, but are not limited to, Venetian azotobacter (Venelandia diffusa). Azotobacter vinelandii D50645 strain, Bacillus polymyxa ( Paenibacillus polymyxa At least one of D16876, or a functionally equivalent strain.

[0014] The above-mentioned strains were purchased from Nuoan Gene Technology (Wuhan) Co., Ltd.

[0015] The nitrogen-fixing bacteria used in this invention have the ability to tolerate the heavy metal antimony. Although they have the potential to fix nitrogen in nutrient-poor environments, they are almost unable to complete the nitrogen fixation process due to a lack of energy sources. Their nitrogen-fixing ability can be enhanced through photogenerated electrons.

[0016] The sponge-like bacterial gel described in this invention, which combines light-driven antimony oxidation and nitrogen fixation, exhibits the ability to generate photogenerated electron-hole pairs through photogenerated quantum dots under visible light irradiation. The generated photogenerated electrons are transferred via a conductive framework to antimony-tolerant nitrogen-fixing bacteria to enhance nitrogen fixation. The consumption of photogenerated electrons improves the electron-hole pair separation efficiency problem caused by photogenerated electron accumulation, further enhancing the metal oxidation capability of the conductive framework. Therefore, it simultaneously improves nitrogen fixation activity and Sb(III) oxidation rate.

[0017] The second aspect of this invention aims to provide a method for preparing a sponge-like mycelium that synergistically induces antimony oxidation and nitrogen fixation. This method is simple to operate, low in cost, and yields a novel product structure that achieves synergistic effects of antimony oxidation and nitrogen fixation, demonstrating promising application prospects.

[0018] The above method includes dissolving amino-containing pyrrole and photoelectric quantum dots in deionized water, adding a catalyst, heating to complete the amidation reaction, cooling the solution and adding a polymerization initiator to induce pyrrole polymerization, washing the product and drying it to obtain a conductive framework and sterilizing it; then adding sterilized sodium alginate to a solution of antimony-tolerant nitrogen-fixing bacteria and stirring, followed by adding a sterilized conductive framework to obtain a uniform gel solution, dropping the gel solution into a sterilized multivalent metal solution for reaction and washing it to obtain a sponge-like bacterial gel with photo-driven antimony oxidation and nitrogen fixation synergy.

[0019] Furthermore, the specific steps include the following:

[0020] Step a: Electrolyze the acid solution at constant voltage using a high-purity conductive graphite electrode plate, then filter the electrolyte using a dialysis bag, and vacuum dry the product collected in the dialysis bag to obtain photoelectric quantum dots with carboxyl groups on the surface;

[0021] Step b: Dissolve at least one of 2-aminopyrrole and 3-aminopyrrole with the photoelectric quantum dots obtained in step a in deionized water, add a catalyst, heat in an oil bath to complete the amidation reaction, cool the solution and add a polymerization initiator to induce pyrrole polymerization, wash the product with deionized water and vacuum dry to obtain a conductive framework and sterilize it.

[0022] Step c: Add sterilized sodium alginate to the solution of antimony-tolerant nitrogen-fixing bacteria and stir evenly. Then add sterilized conductive framework to obtain a uniform gel solution. Drop the gel solution evenly into a sterilized and pre-cooled (to 4°C) multivalent metal solution. After the reaction, wash with deionized water to obtain a sponge-like bacterial gel with photo-driven antimony oxidation and nitrogen fixation synergy.

[0023] Further, the acid solution used in step a is at least one of acetic acid, citric acid, oxalic acid, and malic acid, with a concentration controlled at 0.01~0.1 M and an electrolysis voltage controlled at 5~15 V / cm. During dialysis, a 30~60 kDa dialysis bag is first used for dialysis for 60~72 h to collect the filtrate. Then, the collected filtrate is dialyzed through a 500~2000 Da dialysis bag for 3~5 days to collect the product.

[0024] This invention has experimentally discovered that adding photogenerating quantum dots is fundamental to ensuring metal oxidation. Holes generated by light irradiation in conductive framework materials containing photogenerating quantum dots can promote heavy metal oxidation and are expected to enhance oxidation kinetics. The generated photoelectrons provide sufficient energy support for the microbial nitrogen fixation process. The consumption of photoelectrons can improve the electron-hole pair separation efficiency problem caused by the accumulation of photoelectrons, and can further enhance the metal oxidation capability of the conductive framework.

[0025] Further experiments revealed that carboxylation modification of photogenerating quantum dots is also an important step in improving metal oxidation capabilities. Using an acid solution as the electrolyte results in the generated photogenerating quantum dots possessing carboxyl groups, which can undergo an amidation reaction with 2-aminopyrrole, stably binding to the conductive framework. Therefore, carboxylation modification of photogenerating quantum dots is a crucial step in enhancing metal oxidation capabilities.

[0026] Further, in step b, the mass ratio of at least one of 2-aminopyrrole and 3-aminopyrrole to the photoelectric quantum dots is controlled at 10~100:1, the total mass of both is 3~5 g, the amount of deionized water is 90~110 mL; the catalyst is 4-N,N-dimethylpyridine or 1-hydroxybenzotriazole, the amount of catalyst is 0.1~0.3 g, the oil bath temperature is controlled at 100~120℃, the oil bath reaction time is 3~5 h, the polymerization initiator includes persulfate, the mass is 5~10 g, and the polymerization reaction time is 1~2 h.

[0027] The structure and composition of the conductive framework of this invention also have a crucial impact on the final heavy metal oxidation and nitrogen fixation effects. This is because the efficiency of the conductive framework in transporting photogenerated electrons is key to stimulating and enhancing microbial nitrogen fixation, which in turn leads to the accumulation of photogenerated holes. Only when the conductive framework efficiently transports photogenerated electrons can nitrogen fixation and heavy metal oxidation be synergistically enhanced.

[0028] Further, in step c, the OD of the nitrogen-fixing bacteria solution... 600The concentration of sodium alginate is controlled at 0.1-0.5 g / 100 mL, the stirring time is controlled at 18-24 h, the amount of conductive framework added is 3-5 g / 100 mL, the polyvalent metal solution is a 3-5% mass concentration calcium chloride or ferric chloride solution, the droplet diameter is controlled at 1-3 mm, 500-1000 mL of polyvalent metal solution is added dropwise to every 100 mL of gel solution, and the reaction time after the addition is completed is 5-8 h.

[0029] In this invention, the conductive framework, sodium alginate, and multivalent metal solution are all sterilized by treating at 121°C for 15 minutes.

[0030] The sponge-retaining network of this invention is formed by cross-linking sodium alginate with polyvalent metal ions. Its primary function is to immobilize nitrogen-fixing bacteria, prevent bacterial loss, and provide a reaction microenvironment for the nitrogen-fixing bacteria. Too much sponge-retaining network leads to a decrease in the porosity of the bacterial adhesive, affecting the Sb diffusion rate and reducing the Sb oxidation rate; too little sponge-retaining network easily leads to an unstable bacterial adhesive structure, causing nitrogen-fixing bacteria to be easily lost.

[0031] The third aspect of this invention aims to provide the aforementioned sponge-like mycelium that synergistically induces antimony oxidation and nitrogen fixation, or the application of the sponge-like mycelium that synergistically induces antimony oxidation and nitrogen fixation prepared by the method, for the simultaneous realization of Sb(III) oxidation and biological nitrogen fixation under light conditions.

[0032] Furthermore, it enhances Sb(III) oxidation and biological nitrogen fixation remediation in oligotrophic soils of mining areas.

[0033] In particular, the application can increase the available nitrogen content in the soil and reduce Sb(III) biotoxicity.

[0034] The lighting conditions for this invention include natural light or artificial light. The preferred light wavelength is 300-800 nm.

[0035] The bacterial adhesive of this invention consists of a metal oxide region and antimony-tolerant nitrogen-fixing bacteria. The nitrogen-fixing bacteria are uniformly dispersed in the metal oxide region, which provides energy support for the nitrogen-fixing bacteria. The metal oxide region is a sponge-like double network structure formed by a conductive framework and a sponge-like receiving network. The conductive framework and the sponge-like receiving network are topologically bonded to form a sponge-like porous structure. The conductive framework and the photoelectric quantum dots are tightly connected through an amidation reaction, and the photoelectric quantum dots provide the light-driving site.

[0036] The mycelium of this invention can enhance the antimony oxidation process of the strain under light, thereby supporting the chemo-nitrogen fixation process. It utilizes photogenerated electrons to supply energy and reducing power to achieve nitrogen fixation, while simultaneously oxidizing heavy metals through associated photogenerated holes. It has both excellent metal oxidation ability and nitrogen fixation activity, with nitrogenase activity ≥198.57 nmolC2H2 / (g·h). It can spontaneously transform the highly toxic and low-nutrient habitat of mining areas. Unlike conventional chemo-autotrophic nitrogen-fixing strains, the strain of this mycelium only needs to have metal tolerance, which can expand the chemo-nitrogen fixation pathway and has good application prospects.

[0037] The accompanying drawings, which constitute a part of this application, are used to provide a further understanding of the invention and do not constitute an undue limitation of the invention. Attached Figure Description

[0038] Figure 1 A schematic diagram of the light-driven antimony oxidation and nitrogen fixation synergistic sponge-like mycogel structure prepared in this invention;

[0039] Figure 2 This refers to the amidation reaction process between 2-aminopyrrole and photoelectric quantum dots during the preparation of this invention;

[0040] Figure 3 The results of Sb(III) concentration changes in the heavy metal oxidation capacity determination of the products prepared in the examples and comparative examples are shown.

[0041] Figure 4 The results of Sb(V) concentration changes in the heavy metal oxidation capacity determination of the products prepared in the examples and comparative examples are shown.

[0042] Figure 5 The results of nitrogenase activity assays were performed on the nitrogen fixation capabilities of the products prepared in the examples and comparative examples. Detailed Implementation

[0043] The present invention will be described in detail below with reference to embodiments, but the present invention can be implemented in many different ways as defined and covered by the claims.

[0044] Example 1

[0045] like Figure 1 The sponge-like mycelium exhibiting photo-driven antimony oxidation and nitrogen fixation synergistic process includes the following preparation steps in this embodiment:

[0046] Step a: Electrolyze a 0.01 M acetic acid solution at a constant voltage of 5 V / cm using a high-purity conductive graphite electrode plate. Then, dialyze the electrolyte through a 30 kDa dialysis bag for 60 h. The collected filtrate is then dialyzed for 3 days using a 500 Da dialysis bag. The product collected in the dialysis bag is then vacuum dried (specific parameters: 50℃, -0.09 MPa, 24 h) to obtain photoelectric quantum dots.

[0047] Step b: Maintaining a mass ratio of 10:1 between 2-aminopyrrole and the photoelectric quantum dots obtained in step a, dissolve 3 g of the mixture in 100 mL of deionized water, add 0.1 g of 4-N,N-dimethylpyridine as a catalyst, and heat in an oil bath at 100℃ for 3 h to complete the amidation reaction. After cooling the solution to room temperature, add 5 g of ammonium persulfate as a polymerization initiator to induce pyrrole polymerization. The polymerization reaction time is 1 h. After the reaction is completed, wash the product with deionized water and vacuum dry (specific parameters: 50℃, -0.09 MPa, 24 h) to obtain the conductive framework.

[0048] Step c: To OD 600 0.1% of 100 mL of antimony-tolerant nitrogen-fixing bacteria, *Venelandia diffusa* (ZA), was used. Azotobacter vinelandii 1 g of sterilized sodium alginate was added to solution D50645 and stirred evenly for 18 h. Then, 3 g of sterilized conductive framework was added to obtain a uniform gel solution. The gel solution was evenly dropped into 500 mL of sterilized calcium chloride solution with a mass concentration of 3% and pre-cooled to 4°C. The droplet diameter was controlled at 1 mm. After reacting for 5 h, it was washed with deionized water and stored. The conductive framework, sodium alginate, and polyvalent metal solution were all sterilized at 121°C for 15 min to obtain the final sponge-like nitrogen-fixing bacterial gel with metal oxidation function.

[0049] Example 2

[0050] The difference between Example 2 and Example 1 is that:

[0051] 1. In step a, the electrolysis voltage is 10 V / cm, the acetic acid solution concentration is 0.05 M, the dialysis bag used for the first time is 45 kDa and the dialysis time is 66 h, and the dialysis bag used for the second time is 1000 Da and the dialysis time is 4 days;

[0052] 2. In step b, the mass ratio of 2-aminopyrrole to photoelectrogenerating quantum dots is 50:1, the mass of the mixture is 4 g, the oil bath reaction temperature is 110℃, the time is 4 h, the mass of ammonium persulfate added is 7 g, and the polymerization reaction time is 1.5 h.

[0053] 3. Step c: OD of nitrogen-fixing bacteria solution 600 The concentration of sodium alginate was 1.5 g / 100 mL, the stirring time was 21 h, the amount of conductive framework added was 4 g / 100 mL, the volume of calcium chloride solution was 750 mL, the mass concentration of calcium chloride solution was 4%, the droplet diameter was controlled at 2 mm, and the cross-linking reaction time was 6.5 h.

[0054] Example 3

[0055] The difference between Example 3 and Example 1 is that:

[0056] 1. In step a, the electrolysis voltage is 15 V / cm, the acetic acid solution concentration is 0.1 M, the dialysis bag used for the first time is 60 kDa and the dialysis time is 72 h, and the dialysis bag used for the second time is 2000 Da and the dialysis time is 5 days;

[0057] 2. In the steps, the mass ratio of 2-aminopyrrole to photoelectrogenerating quantum dots is 100:1, the mass of the mixture is 5 g, the oil bath reaction temperature is 120℃, the time is 5 h, the mass of ammonium persulfate added is 10 g, and the polymerization reaction time is 2 h;

[0058] 3. Step c: OD of nitrogen-fixing bacteria solution 600 The concentration of sodium alginate was 2 g / 100 mL, the stirring time was 24 h, the conductive framework was 5 g / 100 mL, the calcium chloride solution volume was 1000 mL, the calcium chloride solution mass concentration was 5%, the droplet diameter was controlled at 3 mm, and the cross-linking reaction time was 8 h.

[0059] Comparative Example 1

[0060] The difference between this comparative example and Example 1 is that no microorganisms are added during the preparation process; instead, 100 mL of sterile water is used directly to replace the microbial culture.

[0061] Comparative Example 2

[0062] The difference between this comparative example and Example 1 is that: during the preparation process, *Azotomyces brasiliensis*, a nitrogen-fixing bacterium that does not possess metal tolerance, was added. Azospirillum brasilense D50646 was purchased from Nuoan Gene Technology (Wuhan) Co., Ltd.

[0063] Comparative Example 3

[0064] The difference between this comparative example and Example 1 is that a strain of *Pseudomonas stearothermii*, which is only tolerant of antimony but does not fix nitrogen, was added during the preparation process. Pseudomonas stutzeri TS274549 was purchased from Ningbo Taiste Biotechnology Co., Ltd.

[0065] Comparative Example 4

[0066] The difference between this comparative example and Example 1 is that in step c of the preparation process of a sponge-like bacterial gel with photo-driven antimony oxidation and nitrogen fixation synergy, no conductive framework is added, that is, 100 mL of nitrogen-fixing bacteria ( Azotobacter vinelandiiAdd 1 g of sterile sodium alginate to the D50645 solution and stir evenly for 18 h. Then, drop the resulting solution evenly into a 500 mL volume of sterile, pre-cooled calcium chloride solution with a mass concentration of 3%.

[0067] Comparative Example 5

[0068] The difference between this comparative example and Example 1 is that in step b of the preparation process of a sponge-like mycelium with photo-driven antimony oxidation and nitrogen fixation synergy, non-conductive chitosan is used to replace 2-aminopyrrole to synthesize the framework. That is, in step b, the mass ratio of chitosan to photoelectric quantum dots obtained in step a is maintained at 10:1. 3 g of chitosan is dissolved in 100 mL of deionized water, and 0.1 g of 4-N,N-dimethylpyridine is added as a catalyst. The amidation reaction is completed by heating in an oil bath at 100°C for 3 h. After the reaction, the product is washed with deionized water and vacuum dried to obtain the framework.

[0069] Comparative Example 6

[0070] The difference between this comparative example and Example 1 is that in step b of the preparation process of a sponge-like mycelium with photo-driven antimony oxidation and nitrogen fixation synergy, no photoelectric quantum dots are added. That is, in step b, 3 g of 2-aminopyrrole is dissolved in 100 mL of deionized water, 0.1 g of 4-N,N-dimethylpyridine is added as a catalyst, and the mixture is heated in an oil bath at 100°C for 3 h to complete the amidation reaction. After the solution is cooled to room temperature, 5 g of ammonium persulfate is added as a polymerization initiator to induce pyrrole polymerization. The polymerization reaction time is 1 h. After the reaction is completed, the product is washed with deionized water and vacuum dried to obtain a conductive framework.

[0071] Comparative Example 7

[0072] The difference between this comparative example and Example 1 is that in step a of the preparation process of a sponge-like mycelium with photo-driven antimony oxidation and nitrogen fixation synergistic, a sodium chloride solution of the same concentration is used instead of an acid solution as the electrolyte.

[0073] Comparative Example 8

[0074] This comparative example is an autotrophic nitrogen-fixing Klebsiella pneumoniae strain with antimony oxidation and growth-promoting capabilities. Klebsiella sp. XXY0502, with accession number CCTCC NO:M 20251574, is a strain from the patent application document with publication number CN120738081A.

[0075] Comparative Example 9

[0076] The difference between this comparative example and Example 1 is that only antimony-tolerant nitrogen-fixing bacteria were used. Azotobacter vinelandii D50645 does not form bacterial gum.

[0077] In this invention, the products obtained in Examples 1-3 and Comparative Examples 1-9 are sequentially labeled as C1-C3 and D1-D9. The following effects were verified using C1-C3 and D1-D9, and the effects were implemented as follows:

[0078] I. Determination of heavy metal oxidizing capacity:

[0079] The heavy metal oxidation capacity of C1~C3 and D1~D9 was rapidly tested in a liquid environment. Specifically, 5 g of samples were taken respectively (where D8 and D9 were equal masses of bacterial suspension, and the bacterial suspension OD was...). 600 =0.1) Place in a 100 mL serum bottle, add 80 mL of basal salt medium (MSM medium) containing 1 mM Sb(III), and incubate at 25℃ for 5 days. During this period, use an 800 W xenon lamp (light source height 30 cm) to maintain light for 12 h / 12 h darkness treatment every day. After the culture is completed on the 1st, 3rd and 5th days, take samples respectively, and use ICP-OES coupled with hydrogenation method to measure the concentration of Sb(III) and Sb(V) in salt medium.

[0080] Table 1. Oxidizing power of different samples for Sb(III) (average of three results)

[0081] Combination Figure 3 , Figure 4 The following conclusions can be drawn from Table 1:

[0082] 1. C1 to C3 exhibit similar Sb(III) oxidation effects, with Sb(III) oxidation rates measured on day 5 ranging from 56.85% to 61.44%, showing close similarities. Therefore, the experimental results indicate that, within the parameter range required by this invention, the obtained light-driven antimony oxidation-nitrogen fixation synergistic sponge-like mycelium possesses stable metal oxidation capabilities.

[0083] 2. The Sb(III) oxidation capabilities of D1 and D2 are approximately 46.31% and 46.02%, respectively, lower than those of C1-C3. This is attributed to the lack of microorganisms in D1. Although the photogenerating quantum dots can generate photogenerated holes under illumination to promote the oxidation of Sb(III) to Sb(V), the continuous accumulation of photogenerated electrons inhibits the separation of electron-hole pairs, resulting in a lower oxidation effect than C1-C3. Similarly, although nitrogen-fixing bacteria were added to D2, their intolerance to antimony caused the bacteria to die, leading to a similar effect to D1. Therefore, adding tolerant microorganisms during the preparation process is an important guarantee for improving the metal oxidation capability.

[0084] 3. Compared with C1~C3, the oxidation effect of D3 is similar, at 56.94%. The experimental results further verify that the addition of metal-tolerant microorganisms (which can also consume photogenerated electrons) is an important guarantee for improving the oxidation capacity of Sb(Ⅲ).

[0085] 4. D4 and D6 exhibit almost no Sb(III) oxidation ability, with oxidation rates of only 1.09% and 3.48% respectively for 1 mM Sb(III) solution. The low oxidation efficiency of D4 and D6 is due to the lack of photoelectric quantum dots, which prevent the generation of holes and thus the generation of oxidation motive force. Therefore, adding photoelectric quantum dots is fundamental to ensuring metal oxidation.

[0086] 5. D5 exhibits general oxidation performance, with an Sb(III) oxidation rate of 30.85%, accounting for approximately 50% of the examples, significantly higher than D4 and D6. Experimental results indicate that adding photogenerating quantum dots can ensure the metal oxidation capability of the bacterial gel. However, due to the non-conductive framework, the opportunity for metal-resistant microorganisms to access photogenerated electrons is reduced, leading to the accumulation of photogenerated electrons and consequently affecting the electron-hole pair separation efficiency. Therefore, it is necessary to increase the conductive framework to improve the electron-hole pair separation effect and promote the metal oxidation capability of the bacterial gel.

[0087] 6. Compared to C1-C3, the oxidation effect of D7 was only 19.31%, accounting for approximately 33% of the effect in the examples. This is because no acid solution was used as the electrolyte during the preparation of photogenerating quantum dots, resulting in the generated photogenerating quantum dots lacking carboxyl groups. Consequently, they could not undergo an amidation reaction with 2-aminopyrrole to stably bind to the conductive framework, affecting the electron-hole pair separation effect. Therefore, carboxylation modification of photogenerating quantum dots is an important step in improving metal oxidation capabilities.

[0088] 7. D9 has almost no Sb(III) oxidation ability, with an oxidation rate of only 0.39%. This is because antimony-tolerant nitrogen-fixing microorganisms may not have enough oxidative motivation to drive antimony oxidation. Therefore, nitrogen-fixing bacteria with metal tolerance may not be able to complete metal oxidation independently.

[0089] 8. The metal oxidation effect of C1~C3 is higher than that of D8. This means that by rationally configuring the metal-tolerant nitrogen-fixing bacteria and materials, this product can achieve a synergistic effect between the two, enhance the oxidation power of microorganisms, and produce a higher metal oxidation effect compared with the actual antimony-oxidizing autotrophic nitrogen-fixing bacteria.

[0090] II. Nitrogen Fixation Capacity Test:

[0091] The nitrogen fixation efficiency of C1-C3 and D1-D9 was tested in a liquid environment. The nitrogen fixation efficiency was reflected by nitrogenase activity, which was measured using the acetylene reduction method. Specifically, 5 g of product was first taken (D8 and D9 were equal masses of bacterial suspension, and the OD of the bacterial suspension was...).600 =0.1) was placed in a 100 mL serum bottle, and 80 mL of salt medium containing 1 mM Sb(III) was added. The mixture was incubated at 25 °C for 24 h, during which an 800 W xenon lamp (light source height 30 cm) was used to maintain a 12 h light / 12 h dark treatment every day. Then, the air in the bottle was purged with argon gas, and 10% (v / v) of the headspace argon gas in the bottle was replaced with acetylene gas for 12 h. Finally, 1 mL of gas was extracted from the serum bottle, and the ethylene yield was measured using a gas chromatograph.

[0092] Combination Figure 5 The following conclusions can be drawn:

[0093] 1. C1 to C3 have similar nitrogenase activities, ranging from 198.57 to 203.24 nmolC2H2 / (g·h), indicating that all three have excellent nitrogen fixation effects. Therefore, the experimental results show that, within the parameter range required by this invention, the obtained light-driven antimony oxidation-nitrogen fixation synergistic sponge-like bacterial gel has excellent nitrogen fixation effects.

[0094] 2. Compared with C1-C3, nitrogenase activity was almost undetectable in D1 and D3, with activities less than 1 nmol C2H2 / (g·h). The experimental results indicate that the lack of nitrogen-fixing bacteria prevents effective nitrogen fixation. This means that the photogenerated electrons produced by the photoelectrons under illumination cannot directly participate in nitrogen fixation, which is related to the high energy barrier of the nitrogen fixation process.

[0095] 3. Compared to D1 and D3, the addition of antimony-intolerant nitrogen-fixing bacteria resulted in a significant nitrogen-fixing effect in D2, but it was far lower than that of C1-C3, only 22.49 nmolC2H2 / (g·h). This is because while adding nitrogen-fixing bacteria can achieve nitrogen fixation, the added bacteria are intolerant to antimony, thus failing to guarantee a sustained and efficient nitrogen-fixing effect. Therefore, metal-tolerant nitrogen-fixing bacteria are key to ensuring the efficient nitrogen-fixing effect of the bacterial gel.

[0096] 4. The nitrogen fixation efficiency of D4, D6, and D9 was all below 12 nmol C2H2 / (g·h), accounting for approximately 6% of the examples. A common characteristic of the three comparative examples is that none possessed photoelectric quantum dots, meaning that the microorganisms within them could not utilize light to drive stronger oxidation. The results indicate that, under nutrient-poor conditions, while metal-tolerant nitrogen-fixing bacteria have the potential for nitrogen fixation, they are almost unable to complete the process due to a lack of energy sources. Therefore, increasing the number of photoelectric quantum dots is crucial to ensuring that the microorganisms in the product obtain energy.

[0097] 5. D5 exhibits a certain nitrogen-fixing effect, with a nitrogenase activity of 120.47 nmolC2H2 / (g·h), significantly higher than that of D4, D6, and D9. Experimental results indicate that nitrogen-fixing bacteria can utilize photoelectrons for nitrogen fixation; however, the non-conductive backbone reduces the bacteria's opportunities to access photogenerated electrons. Therefore, it is necessary to increase the conductive backbone to expand the space for nitrogen-fixing bacteria to utilize photogenerated electrons.

[0098] 6. Compared to C1-C3, the nitrogenase activity of D7 is only 73.56 nmolC2H2 / (g·h). Similar to its metal oxidation ability, the lack of carboxylation modification during the preparation of photogenerating quantum dots prevents them from undergoing amidation with 2-aminopyrrole and stably binding to the conductive framework, thus affecting the electron-hole pair separation effect. Therefore, carboxylation modification of photogenerating quantum dots is an important step in improving nitrogen fixation efficiency.

[0099] 7. The metal oxidation effect of C1~C3 is higher than that of D8, which means that metal-tolerant nitrogen-fixing bacteria can use photogenerated electrons for nitrogen fixation, and their nitrogen fixation effect is higher than that of known autotrophic nitrogen-fixing bacteria.

[0100] III. Stability Testing:

[0101] The stability of samples C1~C3, D1, D4, and D5 was tested by incubation in actual mining area soil, and the stability was measured by the degradation rate of the samples. First, 5 g of sample was freeze-dried to remove moisture, and the mass at this point was recorded as M0. The sample was then placed in a mesh bag, and the bag was tightly sealed. Next, it was buried in a pot of soil at a depth of 0-5 cm. The potted soil was then incubated for 30 days in a constant temperature incubator (purchased from Beijing Zhongxing Weiye Instrument Co., Ltd., model RGX-800) under conditions of 12 h light / 12 h darkness per day. The light intensity of the incubator was set to level 3, and the soil moisture content was maintained at 30%. After 30 days, the mesh bag was removed, the sample was cleaned, freeze-dried, weighed, and the result was recorded as M1. The degradation rate (D, %) was calculated using the following formula:

[0102] ;

[0103] Table 2 Degradation rate of different samples (average of three results)

[0104] Based on Table 2, the following conclusions can be drawn:

[0105] C1~C3, D1, and D5 exhibit similar stability, losing less than 20% of their mass after 30 days of incubation in soil. In contrast, D4, lacking the sponge-like dual-network structure, lost 32.48% of its mass, a loss of 60%. The experimental results demonstrate that forming a sponge-like dual-network structure by topologically bonding the conductive framework and the sponge-supporting network can improve product stability and benefit long-term product use.

[0106] IV. Potted plant experiment verification:

[0107] The effects of C1-C3 and D1-D9 on simulated mining area soil were verified through pot experiments. The simulated mining area soil was prepared by mixing 10-24 mesh quartz sand and 800 mesh kaolin in a 1:3 mass ratio, and aged with potassium antimony tartrate solution. The final result showed that the simulated mining area soil contained 1000 mg / kg of antimony and almost no nitrogen. Specifically, 10 g of the sample and 500 g of simulated mining area soil were added to each pot, mixed evenly, and the soil layer was kept to a thickness of about 3 cm. Then, four germinated Arabidopsis thaliana seeds were transplanted into each pot and cultured in a constant temperature incubator at 25℃ for 60 days (the constant temperature incubator was purchased from Beijing Zhongxing Weiye Instrument Co., Ltd., model RGX-800). During the period, there was 12 h of light / 12 h of darkness. The light intensity in the constant temperature incubator was set to level 3, the humidity was controlled at 30%, and the soil moisture content was maintained at about 30%. Fresh weight and plant height were measured immediately after the culture was completed.

[0108] Table 3. Average fresh weight and average plant height of Arabidopsis thaliana (average of three results)

[0109] The data for each pot is represented by the average of 4 seeds. There are three pots for each treatment. The average of the three results is the average of the results of the three pots.

[0110] Based on Table 3, the following conclusions can be drawn:

[0111] In simulated mining soils using C1-C3, the growth of Arabidopsis thaliana was similar. Among them, the average fresh weight of Arabidopsis was 6.88-7.33 g, and the average plant height was 13.69-15.24 cm, showing the best growth among all groups, exceeding that of known antimony-oxidizing autotrophic nitrogen-fixing bacteria. Experimental results indicate that, within the parameter range required by this invention, the obtained light-driven antimony oxidation-nitrogen fixation synergistic sponge-like mycelium can generate photogenerated electrons and holes through the metal oxidation zone under light, improving the oxidation dynamics of microorganisms. The photogenerated electrons can provide energy for metal-tolerant nitrogen-fixing bacteria, maintaining nitrogen fixation, while the generated photogenerated holes can oxidize metals, reducing their toxicity in the soil, demonstrating promising application prospects.

[0112] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A sponge-like bacterial gel with synergistic light-driven antimony oxidation and nitrogen fixation, characterized in that, It consists of a metal oxidation zone and antimony-tolerant nitrogen-fixing bacteria, wherein the antimony-tolerant nitrogen-fixing bacteria are uniformly dispersed in the metal oxidation zone; The metal oxide region has a sponge-like double network structure, which is a porous structure formed by the topological bonding of a conductive skeleton and a sponge-supporting network. The conductive framework is formed by the polymerization of pyrrole containing amine groups and is tightly connected to the photoelectric quantum dots through an amidation reaction; The sponge-supporting network is formed by cross-linking sodium alginate with polyvalent metal ions.

2. The sponge-like mycelium with photo-driven antimony oxidation and nitrogen fixation synergistic effect according to claim 1, characterized in that, The conductive framework is formed by polymerization of at least one of 2-aminopyrrole and 3-aminopyrrole; the photoelectric quantum dots are obtained by electrolyzing an acid solution using a high-purity conductive graphite electrode plate, followed by dialyzing and filtering the electrolyte, and drying the product to obtain photoelectric quantum dots with carboxyl groups on the surface.

3. The sponge-like mycelium with photo-driven antimony oxidation and nitrogen fixation synergistic effect according to claim 1, characterized in that, The antimony-tolerant nitrogen-fixing bacteria retain nitrogen-fixing activity in a system containing 1-10 mM Sb(III), and the antimony-tolerant nitrogen-fixing bacteria include: Venetian Azotobacter (Venelandia diffusa). Azotobacter vinelandii D50645 strain, Bacillus polymyxa ( Paenibacillus polymyxa At least one of the D16876 strains.

4. A method for preparing a sponge-like mycelium with synergistic photo-driven antimony oxidation and nitrogen fixation, characterized in that, Pyrrole containing amino groups was dissolved in deionized water with photoelectrogenerating quantum dots. A catalyst was added, and the reaction was heated to complete the amidation reaction. After the solution was cooled, a polymerization initiator was added to induce pyrrole polymerization. The product was washed and dried to obtain a conductive framework, which was then sterilized. Then, sterilized sodium alginate was added to a solution of nitrogen-fixing bacteria tolerant to antimony and stirred. Subsequently, a sterilized conductive framework was added to obtain a uniform gel solution. The gel solution was dropped into a sterilized multivalent metal solution and reacted, followed by washing to obtain a sponge-like bacterial gel with photo-driven antimony oxidation and nitrogen fixation synergy.

5. The preparation method according to claim 4, characterized in that, Specifically, the steps include the following: Step a: Electrolyze the acid solution at constant voltage using a high-purity conductive graphite electrode plate, then filter the electrolyte using a dialysis bag, and vacuum dry the product collected in the dialysis bag to obtain photoelectric quantum dots with carboxyl groups on the surface; Step b: Dissolve at least one of 2-aminopyrrole and 3-aminopyrrole with the photoelectric quantum dots obtained in step a in deionized water, add a catalyst, heat in an oil bath to complete the amidation reaction, cool the solution and add a polymerization initiator to induce pyrrole polymerization, wash the product with deionized water and vacuum dry to obtain a conductive framework and sterilize it. Step c: Add sterilized sodium alginate to the solution of antimony-tolerant nitrogen-fixing bacteria and stir evenly. Then add sterilized conductive framework to obtain a uniform gel solution. Drop the gel solution evenly into a sterilized and pre-cooled multivalent metal solution. After the reaction, wash with deionized water to obtain a sponge-like bacterial gel with photo-driven antimony oxidation and nitrogen fixation synergy.

6. The preparation method according to claim 5, characterized in that, The acid solution used in step a is at least one of acetic acid, citric acid, oxalic acid, and malic acid, with a concentration controlled at 0.01~0.1 M and an electrolysis voltage controlled at 5~15 V / cm. During dialysis, a 30~60 kDa dialysis bag is first used for dialysis for 60~72 h to collect the filtrate. Then, the collected filtrate is dialyzed through a 500~2000 Da dialysis bag for 3~5 days to collect the product. In step b, the mass ratio of at least one of 2-aminopyrrole and 3-aminopyrrole to the photoelectric quantum dots is controlled at 10~100:1, the total mass of the two is 3~5 g, and the amount of deionized water is 90~110 mL; the catalyst is 4-N,N-dimethylpyridine or 1-hydroxybenzotriazole, the amount of catalyst is 0.1~0.3 g, the oil bath temperature is controlled at 100~120℃, the oil bath reaction time is 3~5 h, the polymerization initiator includes persulfate, the mass is 5~10 g, and the polymerization reaction time is 1~2 h.

7. The preparation method according to claim 5, characterized in that, Step c: Nitrogen-fixing bacteria solution OD 600 The concentration of sodium alginate is controlled at 0.1-0.5 g / 100 mL, the stirring time is controlled at 18-24 h, the amount of conductive framework added is 3-5 g / 100 mL, the polyvalent metal solution is a 3-5% mass concentration calcium chloride or ferric chloride solution, the droplet diameter is controlled at 1-3 mm, 500-1000 mL of polyvalent metal solution is added dropwise to every 100 mL of gel solution, and the reaction time is 5-8 h after the addition is completed.

8. The application of the spongy mycelium with photo-driven antimony oxidation and nitrogen fixation synergistic as described in any one of claims 1-3, or the spongy mycelium with photo-driven antimony oxidation and nitrogen fixation synergistic prepared by the method described in any one of claims 4-7, characterized in that, It is used to simultaneously achieve Sb(III) oxidation and biological nitrogen fixation under light conditions.

9. The application according to claim 8, characterized in that, Enhance Sb(III) oxidation and biological nitrogen fixation remediation in oligotrophic soils of mining areas.

10. The application according to claim 9, characterized in that, Lighting conditions include natural light or artificial light.

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