Method for repairing soil heavy metal pollution by coupling soybean urease with biochar cementing agent

Abstract

The invention discloses a method for restoring soil heavy metal pollution by coupling soybean urease with a charcoal cementing agent, and relates to the technical field of soil heavy metal pollution restoration, and the method comprises the following steps: dynamic microbiome regulation and control: introducing photosensitive protein into soil functional bacteria; the acyl homoserine lactone analogue is synthesized and applied to soil, and is competitively combined with a microbial quorum sensing receptor to block an inhibitor to secrete a signal channel; according to the method, microbial urease secretion is regulated and controlled through optogenetics, an inhibitor signal is blocked through quorum sensing interference, and a microbiome is precisely regulated and controlled; the pH-responsive nanocapsule releases an inhibitor in a targeted manner, and the magnetic-responsive nanochain dynamically captures interfering bacteria, so that the limitation of passive adsorption is broken through; the gene editing plant releases coumarin to inhibit fungal metabolism, and the engineering mycorrhizal fungi physically shields an inhibitor to construct cross-biosphere cooperative defense; the urease activity and the heavy metal removal rate are remarkably improved, meanwhile, the soil microbial diversity is maintained, and the soil specificity interference problem is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of soil heavy metal pollution remediation, and specifically to a method for remediating soil heavy metal pollution by coupling soybean urease with a biochar binder. Background Art

[0002] Soil heavy metal pollution remediation plays a crucial role in the field of environmental science and engineering, and is essential for maintaining soil ecological functions, ensuring the safety of agricultural products, and promoting the development of sustainable agriculture. In recent years, with the deepening of the understanding of the soil ecosystem, the impact of the soil microbiome on the soil remediation process has become increasingly prominent.

[0003] In the practice of soil heavy metal pollution remediation, the interference of the soil microbiome on the remediation system exhibits significant non-linear characteristics. On the one hand, some native microorganisms in the soil can compete with soil urease for urea, a key substrate, through metabolite competition mechanisms, resulting in a reduction in the amount of substrate available for urease, and thus reducing the catalytic efficiency of urease in the soil nitrogen cycle and pollutant transformation processes. On the other hand, certain fungi can secrete urease inhibitors, which directly inhibit the catalytic activity of urease by binding to the urease active site or changing the urease molecular conformation. It should be noted that this interference shows a high degree of specificity due to differences in soil type, geographical region, climate conditions, and pollution degree, and it is difficult to predict and regulate with a unified model or method.

[0004] Traditional soil heavy metal pollution remediation technologies mainly focus on the physical fixation, chemical leaching, and biological enrichment of heavy metals. However, these technologies mostly focus on the removal or stabilization of heavy metals themselves, seriously neglecting the complex ecological interaction relationships between the soil microbiome and the remediation agents. In addition, due to the high complexity of the soil environment, including variable physical and chemical properties, rich microbial communities, and complex organic-inorganic interactions, it is difficult to accurately reproduce the interference of the soil microbiome on the remediation system in a laboratory simulation environment. This leads to the inability of traditional remediation technologies to precisely regulate the interference of the soil microbiome in the face of actual soil remediation scenarios, making it difficult to effectively improve the remediation efficiency and fully ensure the stability and health of the soil ecosystem.

[0005] In view of this, a method for remediating soil heavy metal pollution by coupling soybean urease with a biochar binder is provided to overcome the above problems. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for remediating soil heavy metal pollution by coupling soybean urease with a biochar binder to solve the problems raised in the above background art.

[0007] To solve the above technical problems, the method for remediating soil heavy metal pollution by coupling soybean urease with a biochar binder provided by the present invention includes the following steps:

[0008] Dynamic microbiome regulation: introducing photosensitive proteins into soil functional bacteria; synthesizing acyl-homoserine lactone analogs and applying them to the soil to competitively bind to microbial quorum sensing receptors and block the inhibitor secretion signaling pathway;

[0009] Nanoscale intelligent response repair: preparing chitosan-

[0010] silica hybrid nanoparticles, encapsulating soybean urease and catechol with them as carriers to form pH-responsive nanocapsules, enabling them to release inhibitors only in acidic soil and neutralize the urease inhibitors secreted by fungi; connecting magnetic Fe3O4 nanoparticles and biochar through DNA strands to form a three-dimensional network structure under an external magnetic field to dynamically capture and enrich interfering bacteria;

[0011] Microbe-plant symbiosis regulation: using the CRISPR / Cas9 technology to edit the organic acid secretion pathway of plants so that they release coumarin when detecting interfering bacteria, inhibiting the synthesis of fungal urease inhibitors; inoculating genetically engineered arbuscular mycorrhizal fungi to express urease protective proteins on the surface of their hyphae to physically shield the interaction between inhibitors and urease.

[0012] Furthermore, the soil functional bacteria are Bacillus subtilis, and the photosensitive protein system is the CRY2 / CIB1 system.

[0013] Furthermore, the pH-responsive nanocapsules release inhibitors only in acidic soil with a pH < 6.0 by regulating the degree of deacetylation and crosslinking density of chitosan.

[0014] Furthermore, in the microbe-plant symbiosis regulation, the plant is ryegrass.

[0015] Furthermore, in the microbe-

[0016] plant symbiosis regulation, the synthesis of fungal urease inhibitors is inhibited by releasing coumarin.

[0017] Furthermore, in the dynamic microbiome regulation, the acyl-homoserine lactone analogs are synthesized by remote activation with light.

[0018] Furthermore, in the dynamic microbiome regulation, it also includes synthesizing acyl-homoserine lactone analogs by inhibiting the expression of their urease secretion genes.

[0019] Furthermore, the wavelength of the light source is 450 - 495 nanometers.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] Precise regulation of the microbiome: In the present invention, a dynamic microbiome regulation system is constructed. By using optogenetics regulation, the CRY2 / CI B1 system is introduced into Bacillus subtilis, and a light source with a wavelength of 450 - 495 nanometers is used to remotely activate or inhibit the expression of its urease secretion gene. At the same time, the quorum sensing interference strategy is utilized, and an artificially synthesized acyl-homoserine lactone (AHL) analogue competitively binds to the quorum sensing receptor of microorganisms, blocking the inhibitor secretion signaling pathway. This changes the existing practice of only killing microorganisms with inhibitors, achieves precise regulation of the microbiome, and solves the problem that traditional methods cannot respond in real time to soil-specific disturbances.

[0022] Intelligent response repair: The design of nano-scale intelligent response repair agents breaks through the limitation of traditional repair agents relying on passive adsorption. The pH-responsive nano-capsules use chitosan-

[0023] silica hybrid nanoparticles as carriers, encapsulating urease and catechol, and releasing inhibitors only in acidic soil (pH < 6.0) to neutralize the urease inhibitors secreted by fungi; the magnetic response nano-chains connect magnetic Fe3O4 nanoparticles and biochar through DNA strands, forming a three-dimensional network structure under an external magnetic field to dynamically capture and enrich interfering bacteria. This design realizes active defense through the intelligent response of nano-materials, breaking through the dual technical barriers of material synthesis and biocompatibility.

[0024] Constructing an ecological synergy network: In the aspect of the microbial-

[0025] plant symbiotic regulation network, the present invention uses the CRISPR / Cas9 technology to edit the organic acid secretion pathway of ryegrass, enabling it to release coumarin when detecting interfering bacteria, inhibiting the synthesis of fungal urease inhibitors, and inoculating genetically engineered arbuscular mycorrhizal fungi (AMF), whose hyphal surface expresses urease protective proteins to physically shield the interaction between inhibitors and urease. This breaks through the limitation of the existing technology that only focuses on the microbiome itself. By constructing a cross-biological kingdom synergy mechanism, it realizes the systematic regulation of the plant-

[0026] microbe interaction network.

[0027] Balancing repair efficiency and ecological protection: Compared with traditional methods, which rely on broad-spectrum antibiotics or static inhibitors and are prone to causing microbial community imbalance and secondary pollution. The present invention effectively improves urease activity, reduces soil heavy metal content, while maintaining the diversity and stability of the soil microbial community through precise regulation and ecological synergy, achieving a win-win situation of "improving repair efficiency" and "protecting the ecosystem". Brief Description of the Drawings

[0028] Figure 1 This is the schematic diagram of the method for repairing soil heavy metal pollution with the soybean urease-coupled biochar binder of the present invention. Detailed implementation manners

[0029] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0030] Please refer to Figure 1 :

[0031] I. Embodiment 1

[0032] (1) Material preparation

[0033] Soil functional bacteria: Select Bacillus subtilis as the recipient bacterium for introducing photosensitive protein.

[0034] Photosensitive protein system: Obtain the CRY2 / CIB1 system for introducing into Bacillus subtilis.

[0035] Quorum sensing interference substance: Synthesize acyl-homoserine lactone (AHL) analogs.

[0036] Nanomaterials: Prepare chitosan-silica hybrid nanoparticles and magnetic Fe3O4 nanoparticles, and obtain DNA strands for connecting magnetic particles and biochar.

[0037] Plants and fungi: Select ryegrass as the gene editing object, prepare arbuscular mycorrhizal fungi (AMF), and perform protein engineering modification on it.

[0038] (2) Specific steps

[0039] Dynamic microbiome regulation system

[0040] Optogenetic regulation: Through molecular biology techniques, introduce the CRY2 / CIB1 system into Bacillus subtilis, arrange a light source with a wavelength of 450 - 495 nanometers in the test area, and remotely activate or inhibit the expression of the urease secretion gene of Bacillus subtilis according to the metabolic conditions of soil microorganisms.

[0041] Quorum sensing interference: Apply the synthesized AHL analogs to the soil to competitively bind to the quorum sensing receptors of microorganisms and block the inhibitor secretion signaling pathway.

[0042] Nanoscale intelligent response repair agent

[0043] pH-responsive nanocapsules: Using chitosan-

[0044] Silica hybrid nanoparticles are used as carriers to encapsulate soybean urease and catechol, enabling the release of inhibitors in acidic soil (pH < 6.0) to neutralize the urease inhibitors secreted by fungi.

[0045] Magnetoresponsive nanochains: Magnetic Fe3O4 nanoparticles are linked to biochar through DNA strands to form a three-dimensional network structure under an external magnetic field, dynamically capturing and enriching interfering bacteria.

[0046] Microbe-plant symbiotic regulation network

[0047] Engineered plant root exudates: The organic acid secretion pathway of ryegrass is edited using CRISPR / Cas9 technology to release coumarin when interfering bacteria are detected, inhibiting the synthesis of fungal urease inhibitors.

[0048] Mycorrhizal fungi synergy: Inoculate genetically engineered AMF, and urease protection proteins are expressed on the surface of its hyphae to physically shield the interaction between inhibitors and urease.

[0049] (III) Effect evaluation

[0050] In-situ monitoring: Set up multiple monitoring points, regularly collect soil samples, and detect indicators such as urease activity and heavy metal content.

[0051] Data analysis: Evaluate the repair effect by comparing the data before and after the experiment. The results show that Example 1 can effectively improve urease activity, reduce the heavy metal content in the soil, and at the same time maintain the diversity and stability of the soil microbial community, achieving a win-win situation of "improved repair efficiency" and "ecosystem protection".

[0052] II. Example 2

[0053] (I) Material preparation

[0054] It is basically the same as Example 1, only removing the synthesis and use of quorum sensing interfering substances.

[0055] (II) Specific steps

[0056] Dynamic microbiome regulation system: Only perform optogenetic regulation. The CRY2 / CIB1 system is introduced into Bacillus subtilis, and the expression of its urease secretion gene is activated or inhibited by specific wavelength light illumination.

[0057] Nanoscale intelligent responsive repair agent: The same as Example 1.

[0058] Microbe-plant symbiotic regulation network: The same as Example 1.

[0059] (III) Effect evaluation

[0060] In-situ monitoring: Set the same monitoring points as in Example 1, collect soil samples, and detect indicators such as urease activity and heavy metal content.

[0061] Data analysis: The experimental results show that due to the lack of quorum sensing interference strategy, some microorganisms can still secrete inhibitors, resulting in a lower increase in urease activity than in Example 1 and a decrease in the soil heavy metal remediation efficiency.

[0062] III. Example 3

[0063] (I) Material preparation

[0064] Basically the same as in Example 1, only removing the optogenetic regulation part.

[0065] (II) Specific steps

[0066] Dynamic microbiome regulation system: Only perform quorum sensing interference, apply AHL analogs to the soil to block the inhibitor secretion signaling pathway.

[0067] Nanoscale intelligent response repair agent: The same as in Example 1.

[0068] Microorganism-plant symbiotic regulation network: The same as in Example 1.

[0069] (III) Effect evaluation

[0070] In-situ monitoring: Set the same monitoring points as in Example 1, collect soil samples, and detect indicators such as urease activity and heavy metal content.

[0071] Data analysis: The experimental results show that due to the inability to precisely regulate the urease secretion of soil functional bacteria, the soil remediation effect is affected to a certain extent, and both the urease activity and the heavy metal remediation efficiency are lower than those in Example 1.

[0072] IV. Example 4

[0073] (I) Material preparation

[0074] Basically the same as in Example 1, only using ordinary ryegrass without gene editing.

[0075] (II) Specific steps

[0076] Dynamic microbiome regulation system: The same as in Example 1.

[0077] Nanoscale intelligent response repair agent: The same as in Example 1.

[0078] Microorganism-

[0079] Plant symbiotic regulation network: Only inoculate genetically engineered AMF and use the urease-protecting protein on the surface of its hyphae to shield the interaction between the inhibitor and urease.

[0080] (III) Effect evaluation

[0081] In-situ monitoring: Set the same monitoring points as in Example 1, collect soil samples, and detect indicators such as urease activity and heavy metal content.

[0082] Data analysis: Since ryegrass cannot release coumarin when detecting interfering bacteria, the synthesis of fungal urease inhibitor is not effectively inhibited, resulting in a soil remediation effect inferior to that of Example 1.

[0083] V. Example 5

[0084] (I) Material preparation

[0085] Basically the same as in Example 1, only using ordinary AMF without protein engineering modification.

[0086] (II) Specific steps

[0087] Dynamic microbiome regulation system: The same as in Example 1.

[0088] Nanoscale intelligent response repair agent: The same as in Example 1.

[0089] Microorganism-

[0090] Plant symbiosis regulation network: Only by editing the organic acid secretion pathway of ryegrass, making it release coumarin when detecting interfering bacteria, and inhibiting the synthesis of fungal urease inhibitor.

[0091] (III) Effect evaluation

[0092] In-situ monitoring: Set the same monitoring points as in Example 1, collect soil samples, and detect indicators such as urease activity and heavy metal content.

[0093] Data analysis: Since ordinary AMF cannot effectively shield the interaction between the inhibitor and urease, the soil remediation effect decreases compared with Example 1.

[0094] VI. Comparative Example 1

[0095] (I) Material preparation

[0096] Prepare broad-spectrum antibiotics.

[0097] (II) Specific steps

[0098] Use broad-spectrum antibiotics to kill soil microorganisms, trying to reduce the influence of interfering bacteria on the repair system, and do not adopt the innovative technologies of Example 1 in other repair steps.

[0099] (III) Effect evaluation

[0100] In-situ monitoring: Set the same monitoring points as in Example 1, collect soil samples, and detect indicators such as urease activity, heavy metal content, and microbial community structure.

[0101] Data analysis: The experimental results show that while broad-spectrum antibiotics kill interfering bacteria, they also disrupt the community structure of beneficial microorganisms in the soil, leading to the imbalance of the soil ecosystem. Although the urease activity increases in the short term, in the long run, the soil remediation effect is not good, and there is a risk of secondary pollution.

[0102] VII. Comparative Example 2

[0103] (I) Material preparation

[0104] Prepare static inhibitors.

[0105] (II) Specific steps

[0106] Apply the static inhibitor to the soil to inhibit the secretion of urease inhibitors by microorganisms, and do not adopt the innovative technology of Example 1 in other remediation steps.

[0107] (III) Effect evaluation

[0108] In-situ monitoring: Set the same monitoring points as in Example 1, collect soil samples, and detect indicators such as urease activity and heavy metal content.

[0109] Data analysis: Although the static inhibitor can inhibit the secretion of urease inhibitors to a certain extent, due to its inability to dynamically regulate according to the changes in the soil environment and its insufficient ability to cope with soil-specific interferences, the soil remediation efficiency is lower than that of Example 1.

[0110] VIII. Comparative Example 3

[0111] (I) Material preparation

[0112] Prepare traditional biochar adsorbents.

[0113] (II) Specific steps

[0114] Only use traditional biochar adsorbents to fix soil heavy metals, without considering the interference of the soil microbiome on the remediation system.

[0115] (III) Effect evaluation

[0116] In-situ monitoring: Set the same monitoring points as in Example 1, collect soil samples, and detect indicators such as urease activity and heavy metal content.

[0117] Data analysis: Although traditional biochar adsorbents can fix some heavy metals, due to ignoring the ecological interaction between the microbiome and the remediation agent, the soil urease activity is greatly affected, and the remediation effect is not ideal.

[0118] IX. Comparative Example 4

[0119] (I) Material Preparation

[0120] Prepare a repair agent with only a single function, such as a reagent that can only neutralize urease inhibitors.

[0121] (II) Specific Steps

[0122] Use a reagent that can only neutralize urease inhibitors for soil remediation, and do not adopt the multi - technology synergy scheme of Example 1.

[0123] (III) Effect Evaluation

[0124] In - situ monitoring: Set the same monitoring points as in Example 1, collect soil samples, and detect indicators such as urease activity and heavy metal content.

[0125] Data analysis: Since this repair agent can only solve a single problem and cannot cope with the complex interference of the soil microbiome on the repair system, the soil remediation effect is far inferior to that of Example 1.

[0126] X. Comparative Example 5

[0127] (I) Material Preparation

[0128] Prepare conventional soil remediation materials, without involving nanomaterials and gene editing technologies.

[0129] (II) Specific Steps

[0130] Adopt conventional soil remediation materials and methods for remediation, and do not adopt the innovative technologies of Example 1.

[0131] (III) Effect Evaluation

[0132] In - situ monitoring: Set the same monitoring points as in Example 1, collect soil samples, and detect indicators such as urease activity and heavy metal content.

[0133] Data analysis: Conventional soil remediation methods cannot achieve precise regulation and ecological synergy of the soil microbiome, the soil remediation efficiency is low, and the protection effect on the soil ecosystem is limited.

[0134] Summary:

[0135] By comparing Examples 2 to 5 and Comparative Examples 1 to 5 with Example 1, it can be clearly seen that the multi-dimensional collaborative repair system constructed in Example 1 shows significant advantages in dealing with the non-linear interference of the soil microbiome on the repair system. The absence of the quorum sensing interference strategy and optogenetic regulation in Examples 2 and 3 respectively leads to insufficient regulation of the microbiome and a decrease in the repair efficiency, indicating the importance of the cooperation between the two technical means in the dynamic microbiome regulation system. The absence of plant gene editing and mycorrhizal fungal protein engineering modification in Examples 4 and 5 respectively weakens the function of the microbial-plant symbiotic regulation network, further demonstrating the key role of multi-technology collaboration in enhancing the repair effect.

[0136] The traditional repair methods used in Comparative Examples 1 to 5 cannot compare with Example 1 in terms of repair efficiency and ecological protection because they fail to fully consider the complex interactions between the soil microbiome and the repair system. Although broad-spectrum antibiotics and static inhibitors can inhibit interfering bacteria to a certain extent, they will damage the soil ecosystem and are difficult to cope with soil-specific interference. Traditional biochar adsorbents and single-functional repair agents cannot solve the problem of microbiome interference on the repair system, and the repair effect is limited.

[0137] Practical application potential

[0138] The repair method of Example 1 not only achieved good results under laboratory conditions but also has great potential in practical applications. Through precise regulation and ecological collaboration, this method can effectively improve the repair efficiency, reduce the repair cost, and at the same time reduce the negative impact on the soil ecosystem, meeting the requirements of sustainable development. For example, when applying this method to farmland severely polluted by heavy metals, it can repair the soil while protecting the beneficial microorganisms in the soil, maintaining the ecological balance of the soil, and providing a good soil environment for the growth of crops.

[0139] Future research directions

[0140] Although the repair method of Example 1 has achieved remarkable results, there are still some problems that need further research. For example, the application of optogenetic technology in soil remediation still faces the problem of limited light penetration depth in the soil, and more efficient light transmission technologies or new photosensitive proteins need to be developed. The long-term stability and environmental safety of nanomaterials in the soil also need to be further evaluated. In addition, how to optimize the plant-microbe interaction network to improve the stability and durability of the repair effect is also an important direction for future research.

[0141] Through a series of examples and comparative examples, this study demonstrated that the innovative method of using soybean urease-coupled biochar binder to repair heavy metal-contaminated soil has significant advantages in dealing with the non-linear interference of the soil microbiome on the repair system. This method achieves a win-win situation between repair efficiency and ecological protection, providing new ideas and technical means for the repair of heavy metal-contaminated soil. In the future, with the continuous development and improvement of related technologies, this method is expected to play a greater role in practical applications and make important contributions to improving soil environmental quality.

Claims

1. A method for repairing soil heavy metal pollution by coupling soybean urease with biochar binder, characterized in that: The following steps are involved: Dynamic microbiome regulation: introducing photosensitive proteins into soil functional bacteria; synthesizing acyl homoserine lactone analogs and applying them into the soil to competitively bind to microbial quorum sensing receptors and block inhibitor secretion signaling pathways; Nanoscale smart response repair: preparation of chitosan- Silica hybrid nanoparticles are used as carriers to encapsulate soybean urease and catechol to form pH-responsive nanocapsules, which release inhibitors only in acidic soils and neutralize the urease inhibitors secreted by fungi; magnetic Fe3O4 nanoparticles are connected to biochar through DNA chains to form a three-dimensional network structure under an external magnetic field, dynamically capturing and enriching interfering bacteria; microorganism- Plant symbiotic regulation: Use CRISPR / Cas9 technology to edit the organic acid secretion pathway of plants, so that they release coumarins when interfering bacteria are detected, inhibiting the synthesis of fungal urease inhibitors; inoculate genetically engineered arbuscular mycorrhizal fungi to express urease protective proteins on the surface of their hyphae, physically shielding the interaction between inhibitors and urease.

2. The method for repairing soil heavy metal pollution by coupling soybean urease with a biochar binder according to claim 1, characterized in that: The soil functional bacteria is Bacillus subtilis, and the photosensitive protein system is the CRY2 / CIB1 system.

3. The method for repairing soil heavy metal pollution by coupling soybean urease with a biochar binder according to claim 1, characterized in that: The pH-responsive nanocapsules release inhibitors only in acidic soils with a pH value less than 6.0 by regulating the deacetylation degree and cross-linking density of chitosan.

4. The method for repairing soil heavy metal pollution by coupling soybean urease with a biochar binder according to claim 1, characterized in that: In the microbe-plant symbiotic regulation, the plant is ryegrass.

5. The method for repairing soil heavy metal pollution by coupling soybean urease with a biochar binder according to claim 1, characterized in that: In microbial- In plant symbiotic regulation, it inhibits the synthesis of fungal urease inhibitors by releasing coumarins.

6. The method for repairing soil heavy metal pollution by coupling soybean urease with a biochar binder according to claim 1, characterized in that: Synthesis of acyl homoserine lactone analogs via remote activation by light for dynamic microbiome regulation.

7. The method for repairing soil heavy metal pollution by coupling soybean urease with a biochar binder according to claim 6, characterized in that: Dynamic microbiome regulation also includes the synthesis of acyl homoserine lactone analogs by inhibiting the expression of its urease secretion gene.

8. The method for repairing soil heavy metal pollution by coupling soybean urease with a biochar binder according to claim 6, characterized in that: The wavelength of the light source is 450-495 nanometers.

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