Biological microcapsule for repairing heavy metal pollution as well as preparation method and application of biological microcapsule

Abstract

The invention discloses a biological microcapsule for repairing heavy metal pollution as well as a preparation method and application of the biological microcapsule. The biological microcapsule consists of a core material and a wall material, the core material is prepared from bacillus subtilis, aspergillus niger, azospirillum brasilense and starch; the wall material comprises modified polyethylene glycol and sodium alginate; the modified polyethylene glycol is prepared by reacting polyethylene glycol, 2-[(tert-butyl) amino] acetyl chloride hydrochloride and 2-chloroethyl sodium sulfonate. The biological microcapsule for repairing heavy metal pollution can further enrich soil heavy metal through high adsorption capacity and adsorption capacity of the wall material, remarkably reduces the degree of heavy metal pollution of soil through excellent reduction and adsorption capacity of the complex microbial inoculant, and can be widely applied to the field of heavy metal repairing of soil.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a biological microcapsule for repairing heavy metal pollution, and a preparation method and application thereof. Background Art

[0002] Since heavy metals in the soil cannot be biodegraded, their accumulation in plants will seriously affect the physiological and molecular activities of plants, directly or indirectly slowing down plant growth and causing crop yield reductions. Accumulation in the food chain will also cause irreversible harm to the human body.

[0003] Therefore, it is necessary to treat heavy metal ions in the soil to reduce environmental and health hazards. Methods for treating heavy metals in soil include physical methods such as soil replacement, isolation, and heat treatment, as well as chemical methods such as leaching, solidification, and reduction. However, physical or chemical methods are not only costly to treat soil but also often pose the risk of further contamination. Compared to physical or chemical treatment methods, biological methods such as phytoremediation and microbial remediation are more suitable for low-cost and environmentally friendly treatment needs.

[0004] Among them, microbial remediation of heavy metal pollution often requires embedding microorganisms in carriers or wall materials to maintain their biological activity. Microcapsules are tiny capsules with micron size composed of a core material and a film agent that encapsulates the core material. In order to suppress the volatilization of active ingredients and improve transportability, microcapsules that encapsulate spices, medicines, pesticides, etc. in capsules with polymer compounds as film agents are used in industry. As representative methods for manufacturing microcapsules, spray drying as a physical method, coacervation as a physicochemical method, interfacial polymerization or in situ polymerization as chemical methods are known.

[0005] The existing biological microcapsules cannot meet the needs of soil microbial remediation. On the one hand, the embedded microbial agents have low effect and efficiency in heavy metal remediation, long remediation cycles, and cannot adapt to environments with high heavy metal concentrations. On the other hand, the microcapsules have insufficient adsorption capacity and treatment range for heavy metals, and cannot comprehensively treat heavy metal pollution. Large-scale spraying will lead to performance waste and further pollution.

[0006] Therefore, there is an urgent need for a biological microcapsule that can adapt to high-concentration heavy metal pollution and can repair heavy metal pollution in liquid soil on a large scale and with high efficiency. Summary of the Invention

[0007] Purpose of the invention: In view of the defects of the prior art, the purpose of the present invention is to provide a biological microcapsule that can adapt to high-concentration heavy metal pollution and can repair heavy metal pollution on a large scale and with high efficiency, as well as its preparation method and application.

[0008] Technical solution: On one hand, the present invention provides a biological microcapsule for repairing heavy metal pollution, wherein the biological microcapsule is composed of a core material and a wall material; The core material comprises Bacillus subtilis, Aspergillus niger, Azospirillum brasiliensis and starch; The wall material includes modified polyethylene glycol and sodium alginate; The modified polyethylene glycol is prepared by reacting polyethylene glycol, 2-[(tert-butyl)amino]acetyl chloride hydrochloride and sodium 2-chloroethylsulfonate.

[0009] The Bacillus subtilis in the core material of the present invention can secrete extracellular polymers, which contain a large number of negatively charged functional groups, such as carboxyl, hydroxyl, carbonyl, etc. These functional groups can combine with heavy metal ions through ion exchange, complexation, covalent adsorption, etc., thereby fixing heavy metals in the environment; Aspergillus niger can, on the one hand, chelate and adsorb heavy metal ions based on chitin and glucan in the cell wall, and on the other hand, it can secrete oxalic acid, which can further dissolve heavy metal precipitates and accelerate the adsorption and reduction of heavy metals; Azospirillum brasiliensis can, on the one hand, provide a nitrogen source through nitrogen fixation, and on the other hand, promote the formation of biofilms and enhance the overall stress resistance of the bacterial community; the composite bacterial agent can achieve long-term and efficient remediation of heavy metals in soil through the triple synergistic effects of adsorption, reduction and protection.

[0010] Furthermore, the modified polyethylene glycol is prepared by the following steps: (1) Add polyethylene glycol, 2-[(tert-butyl)amino]acetyl chloride hydrochloride and an organic solvent into a reactor, mix in an ice bath for 20-40 minutes, then add triethylamine, heat to 50-60°C, react under nitrogen protection for 8-12 hours, filter, wash and dry to obtain amino polyethylene glycol; (2) Add amino polyethylene glycol, sodium 2-chloroethylsulfonate and an organic solvent into a reactor, stir evenly and then heat to 40-50°C. Keep the temperature to react for 14-18 hours, filter, wash and dry to obtain the modified polyethylene glycol.

[0011] In the present invention, polyethylene glycol is modified to enable successful grafting of tertiary amine structures and sulfonate structures. On the one hand, the number of pores on the surface of the microcapsule can be increased through the multi-branched tertiary amine structure, thereby improving the enrichment effect and adsorption capacity of the wall material for soil heavy metals; on the other hand, the sulfonate structure can not only react with sodium alginate to form a stable wall material structure, but also further improve the pore-forming effect, dispersion effect and adsorption effect, thereby significantly improving the enrichment capacity of the microcapsule wall material for soil heavy metals.

[0012] Furthermore, the molecular weight of the polyethylene glycol is 200-4000; the organic solvent is selected from one of tetrahydrofuran and dimethyl sulfoxide; In step (1), the mass ratio of polyethylene glycol, 2-[(tert-butyl)amino]acetyl chloride hydrochloride and triethylamine is (12-15):(2-3):(2-3); In the step (2), the mass ratio of the amino polyethylene glycol to sodium 2-chloroethylsulfonate is (5-8): (1-2).

[0013] Furthermore, the starch is selected from at least one of resistant starch and cross-linked starch.

[0014] The present invention further defines starch as resistant starch or cross-linked starch, which can not only provide nutrition for the survival and growth of microorganisms in the core material, but also form a cross-linked network or increase resistant components to improve the activity of the composite bacterial agent in a high concentration heavy metal environment, thereby improving the stability of the repair process.

[0015] Another aspect of the present invention provides a method for preparing any of the above-mentioned biological microcapsules for repairing heavy metal pollution, comprising the following steps: (1) Bacillus subtilis, Aspergillus niger and Azospirillum brasiliensis were mixed in proportion and resuspended in sterile saline to form a composite bacterial suspension; (2) Add starch to the composite bacterial suspension, stir and mix thoroughly to obtain a composite core material; (3) The composite core material, modified polyethylene glycol and sodium alginate are mixed in proportion, stirred evenly and then spray-dried to obtain the biological microcapsules for repairing heavy metal pollution.

[0016] In the present invention, modified polyethylene glycol and sodium alginate are used as wall materials, which can not only improve the adsorption effect and adsorption capacity of microcapsules for heavy metal ions in the soil, achieve the enrichment effect of heavy metals in the soil, and thus expand the scope of soil remediation, but also significantly improve the protection and isolation effect of the microcapsules for the core material, so that microorganisms can remain active in an environment with high heavy metal concentrations, thereby achieving a high remediation area effect, reducing the investment in biological microcapsules, lowering costs and improving efficiency.

[0017] Furthermore, the effective viable bacteria count in the composite bacterial suspension in step (1) is not less than 3×10 9 CFU / g; The effective viable bacterial count ratio of the Bacillus subtilis, Aspergillus niger and Azospirillum brasiliensis is (1-1.5): (0.8-1.2): (0.5-0.8).

[0018] Furthermore, in step (2), the mass ratio of the composite bacterial suspension to starch is (5-8): (1-2).

[0019] Furthermore, in step (3), the mass ratio of the composite core material, modified polyethylene glycol and sodium alginate is (1.5-2): (3-5): (3-5).

[0020] Furthermore, the spray drying conditions in step (3) are: inlet air temperature 120-140°C, fan frequency 40-50Hz, peristaltic speed 5-10rpm, and outlet air temperature 50-60°C.

[0021] Finally, the present invention also provides the use of any of the above-mentioned biological microcapsules for repairing heavy metal pollution in the field of soil heavy metal pollution repair.

[0022] Beneficial effects: (1) The biological microcapsules for repairing heavy metal pollution provided by the present invention are prepared by using a composite bacterial agent of Bacillus subtilis, Aspergillus niger and Azospirillum brasiliensis as the core repair material, compounding it with starch as the core material, and using modified polyethylene glycol and sodium alginate as the wall material. The biological microcapsules can further enrich the soil heavy metals through the high adsorption capacity and adsorption capacity of the wall material, and significantly reduce the degree of heavy metal pollution in the soil through the excellent reduction and adsorption capacity of the composite bacterial agent. The microcapsules can be widely used in the field of heavy metal remediation of soil.

[0023] (2) The biological microcapsules for repairing heavy metal pollution provided by the present invention use Bacillus subtilis, Aspergillus niger and Azospirillum brasiliensis as composite bacterial agents, and achieve efficient repair of heavy metal pollution through the synergistic effect among the three.

[0024] (3) In the biological microcapsules for repairing heavy metal pollution provided by the present invention, Bacillus subtilis can secrete extracellular polymers, which contain a large number of negatively charged functional groups, such as carboxyl, hydroxyl, carbonyl, etc. These functional groups can combine with heavy metal ions through ion exchange, complexation, covalent adsorption, etc., thereby fixing heavy metals in the environment; Aspergillus niger can, on the one hand, chelate and adsorb heavy metal ions based on chitin and glucan in the cell wall, and on the other hand, can secrete oxalic acid, which can further dissolve heavy metal precipitates and accelerate the adsorption and reduction of heavy metals; Azospirillum brasiliensis can, on the one hand, provide a nitrogen source through nitrogen fixation, and on the other hand, promote the formation of biofilm and enhance the overall stress resistance of the bacterial community; the composite bacterial agent can achieve long-term and efficient repair of soil heavy metals through the triple synergistic effects of adsorption, reduction and protection.

[0025] (4) The biological microcapsules for repairing heavy metal pollution provided by the present invention use modified polyethylene glycol and sodium alginate as wall materials, which can not only improve the adsorption effect and adsorption capacity of microcapsules for heavy metal ions in the soil, achieve the enrichment effect of heavy metals in the soil, and thus expand the scope of soil remediation, but also significantly improve the protection and isolation effect of microcapsules on the core material, so that microorganisms can remain active in an environment with high heavy metal concentrations, thereby achieving a high remediation area effect, reducing the investment in biological microcapsules, reducing costs and improving efficiency.

[0026] (5) In the biological microcapsules for repairing heavy metal pollution provided by the present invention, tertiary amine structure and sulfonate structure are successfully grafted to polyethylene glycol after modification. On the one hand, the number of pores on the surface of the microcapsules can be increased by the multi-branched tertiary amine structure, thereby improving the enrichment effect and adsorption capacity of the wall material for heavy metals in the soil; on the other hand, the sulfonate structure can not only react with sodium alginate to form a stable wall material structure, but also further improve the pore-forming effect, dispersion effect and adsorption effect, significantly improving the enrichment capacity of the microcapsule wall material for heavy metals in the soil. DETAILED DESCRIPTION

[0027] The present invention will be described below in conjunction with specific embodiments. It should be noted that the following examples are illustrative of the present invention and are intended only to illustrate the present invention and are not intended to limit the present invention. Other combinations and various modifications within the scope of the present invention may be made without departing from the spirit or scope of the present invention.

[0028] The Bacillus subtilis MES 810 strain in the present invention was deposited by the applicant in the China General Microbiology Center (CGMCC) of the China Culture Collection Administration on August 10, 2017, with the deposit number CGMCC 14514. It should be noted that the Bacillus subtilis strain of the present invention has been patented by our company with application number 201710862006.3; Aspergillus niger strain was purchased from the General Microbiology Center of China Culture Collection Administration of Microorganisms (CGMCC) on July 14, 2008, with the accession number CGMCC 3.11571. Azospirillum brasilense strain was purchased from the General Microbiology Center of China General Microbiology Culture Collection (CGMCC) on June 27, 2005, with the accession number CGMCC 1.5808. The molecular weight of polyethylene glycol is 2000; the CAS number of 2-[(tert-butyl)amino]acetyl chloride hydrochloride is 915725-52-9; and the remaining reagents and equipment are conventional reagents and equipment in this technical field.

[0029] Preparation of modified polyethylene glycol-1 (1) In a reactor, add 6 g of polyethylene glycol, 1.5 g of 2-[(tert-butyl)amino]acetyl chloride hydrochloride and 50 mL of tetrahydrofuran, mix in an ice bath for 30 minutes, then add 1.5 g of triethylamine, heat to 50°C, react under nitrogen protection for 12 hours, filter, wash and dry to obtain amino polyethylene glycol; (2) In a reactor, 4 g of amino polyethylene glycol, 1 g of sodium 2-chloroethylsulfonate and 50 mL of tetrahydrofuran were added, stirred evenly and then heated to 40°C. After keeping the temperature for 18 hours, the mixture was filtered, washed and dried to obtain the modified polyethylene glycol-1.

[0030] Preparation of modified polyethylene glycol-2 The preparation is basically the same as that of modified polyethylene glycol-1, except that step (2) is not performed and 2-[(tert-butyl)amino]acetyl chloride hydrochloride in step (1) is replaced by an equal amount of acryloyl chloride.

[0031] Example 1 The bio-microcapsules for repairing heavy metal pollution were prepared by the following steps: (1) After mixing Bacillus subtilis, Aspergillus niger and Azospirillum brasiliensis, resuspend the bacteria in sterile saline to form a composite bacterial suspension; (2) Add 1.5 parts of cross-linked starch to 6 parts of the composite bacterial suspension by weight, and stir thoroughly to obtain a composite core material; (3) Mixing 2 parts of the composite core material, 4 parts of modified polyethylene glycol-1 and 4 parts of sodium alginate by weight, stirring evenly and spray drying to obtain the biological microcapsules for repairing heavy metal pollution; The effective viable bacteria count in the composite bacterial suspension in step (1) is 4×10 9 CFU / g; among them, the effective viable bacterial count ratio of Bacillus subtilis, Aspergillus niger and Azospirillum brasiliensis was 1.5:1:0.5; The spray drying conditions in step (3) are as follows: an inlet air temperature of 130°C, a fan frequency of 45 Hz, a creep speed of 10 rpm, and an outlet air temperature of 55°C.

[0032] Example 2 The method is basically the same as that of the embodiment, except that the cross-linked starch in step (2) is replaced by an equal amount of resistant starch; the composite core material in step (3) is replaced by 1.5 parts, the modified polyethylene glycol-1 is replaced by 5 parts, and the sodium alginate is replaced by 5 parts.

[0033] Example 3 The method is basically the same as the embodiment, except that in step (3), the composite core material is changed to 2 parts, the modified polyethylene glycol-1 is changed to 3 parts, and the sodium alginate is changed to 3 parts.

[0034] Comparative Example 1 The same as Example 1, except that the effective viable bacteria count of the composite bacterial suspension in step (1) is changed to 4×10 9 CFU / g of Bacillus subtilis suspension.

[0035] Comparative Example 2 The process is basically the same as Example 1, except that the cross-linked starch in step (2) is replaced by an equal amount of wheat starch.

[0036] Comparative Example 3 The process is basically the same as Example 1, except that the modified polyethylene glycol-1 in step (3) is replaced by an equal amount of polyethylene glycol.

[0037] Comparative Example 4 The process is basically the same as Example 1, except that the modified polyethylene glycol-1 in step (3) is replaced by an equal amount of polyethylene glycol-2.

[0038] Performance Testing Adsorption capacity and adsorption amount test: 0.1 g of each product of Examples 1-3 and Comparative Examples 1-4 were weighed and placed in a 500 ml wide-mouth conical flask. Cd was added to each sample at a concentration of 10 mg / L. 2+ Place the conical flask in a double-layer constant temperature shaker at (25±5)℃ and shake at 150r / min for 12h. Then take out the solution and filter it through a 0.45μm microporous membrane. Use a flame atomic absorption spectrometer to determine the Cd content in the filtrate. 2+ , and calculate the effect of different products on Cd in solution 2+ The maximum adsorption capacity and adsorption rate.

[0039] Repair ability test: Take 10g of each of the products of Examples 1-3 and Comparative Examples 1-4, add them to 5000g of soil heavily contaminated with heavy metals, mix them evenly and simulate the environment of the contaminated soil area. After 14 days, record the Cd content in the soil. 2+ Removal rate.

[0040] The test results are shown in the following table: According to the comparison of the test results of Examples 1-3 with those of Comparative Example 1, the biological microcapsules for repairing heavy metal pollution provided by the present invention have a better heavy metal repair ability than the microbial agents in the prior art by rationally matching the bacterial species of the composite bacterial agent and based on the synergistic repair effect of Bacillus subtilis, Aspergillus niger and Azospirillum brasiliensis, and can efficiently and stably repair soils contaminated with severe heavy metals.

[0041] According to the comparison of the test results of Examples 1-3 and Comparative Example 2, the addition of cross-linked starch or resistant starch to the core material of the biological microcapsule for repairing heavy metal pollution provided by the present invention can not only provide nutrition for the survival and growth of microorganisms in the core material, but also form a cross-linked network or increase the resistance components to improve the activity of the composite bacterial agent in a high concentration heavy metal environment, thereby improving the stability of the repair process.

[0042] According to the comparison of the test results of Examples 1-3 and Comparative Examples 3-4, the biological microcapsules for repairing heavy metal pollution provided by the present invention can significantly improve the enrichment capacity of the biological microcapsules for heavy metals in the soil by modifying the amino and sulfonic acid groups of polyethylene glycol and compounding it with sodium alginate to form the wall material, thereby effectively increasing the repair area, reducing material input and reducing costs.

[0043] The above embodiments are only for illustrating the technical concept and features of the present invention. Its purpose is to enable people familiar with this technology to understand the content of the present invention and implement it. It cannot be used to limit the scope of protection of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A biological microcapsule for repairing heavy metal pollution, characterized in that: The biological microcapsule is composed of a core material and a wall material; The core material comprises Bacillus subtilis, Aspergillus niger, Azospirillum brasiliensis and starch; The wall material includes modified polyethylene glycol and sodium alginate; The modified polyethylene glycol is prepared by reacting polyethylene glycol, 2-[(tert-butyl)amino]acetyl chloride hydrochloride and sodium 2-chloroethylsulfonate.

2. The biological microcapsule for repairing heavy metal pollution according to claim 1, characterized in that: The modified polyethylene glycol is prepared by the following steps: (1) Add polyethylene glycol, 2-[(tert-butyl)amino]acetyl chloride hydrochloride and an organic solvent into a reactor, mix in an ice bath for 20-40 minutes, then add triethylamine, heat to 50-60°C, react under nitrogen protection for 8-12 hours, filter, wash and dry to obtain amino polyethylene glycol; (2) Add amino polyethylene glycol, sodium 2-chloroethylsulfonate and an organic solvent into a reactor, stir evenly and then heat to 40-50°C. Keep the temperature to react for 14-18 hours, filter, wash and dry to obtain the modified polyethylene glycol.

3. The biological microcapsule for repairing heavy metal pollution according to claim 2, characterized in that: The molecular weight of the polyethylene glycol is 200-4000; the organic solvent is selected from tetrahydrofuran or dimethyl sulfoxide; In step (1), the mass ratio of polyethylene glycol, 2-[(tert-butyl)amino]acetyl chloride hydrochloride and triethylamine is (12-15):(2-3):(2-3); In the step (2), the mass ratio of the amino polyethylene glycol to sodium 2-chloroethylsulfonate is (5-8): (1-2).

4. The biological microcapsule for repairing heavy metal pollution according to claim 1, characterized in that: The starch is selected from at least one of resistant starch and cross-linked starch.

5. The method for preparing the biological microcapsule for repairing heavy metal pollution according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Bacillus subtilis, Aspergillus niger and Azospirillum brasiliensis were mixed in proportion and resuspended in sterile saline to form a composite bacterial suspension; (2) Add starch to the composite bacterial suspension, stir and mix thoroughly to obtain a composite core material; (3) The composite core material, modified polyethylene glycol and sodium alginate are mixed in proportion, stirred evenly and then spray-dried to obtain the biological microcapsules for repairing heavy metal pollution.

6. The method for preparing biological microcapsules for repairing heavy metal pollution according to claim 5, characterized in that: The effective viable bacteria count in the composite bacterial suspension in step (1) is not less than 3×10 9 CFU / g; The effective viable bacterial count ratio of the Bacillus subtilis, Aspergillus niger and Azospirillum brasiliensis is (1-1.5): (0.8-1.2): (0.5-0.8).

7. The method for preparing biological microcapsules for repairing heavy metal pollution according to claim 5, characterized in that: The mass ratio of the composite bacterial suspension to starch in step (2) is (5-8): (1-2).

8. The method for preparing biological microcapsules for repairing heavy metal pollution according to claim 5, characterized in that: In the step (3), the mass ratio of the composite core material, modified polyethylene glycol and sodium alginate is (1.5-2): (3-5): (3-5).

9. The method for preparing biological microcapsules for repairing heavy metal pollution according to claim 5, characterized in that: The spray drying conditions in step (3) are: inlet air temperature 120-140°C, fan frequency 40-50 Hz, peristaltic speed 5-10 rpm, and outlet air temperature 50-60°C.

10. Use of the biological microcapsule for repairing heavy metal pollution according to any one of claims 1 to 4 in the field of soil heavy metal pollution remediation.

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