Biological microcapsule for repairing heavy metal pollution and preparation method and application thereof

Abstract

The application discloses a kind of biological microcapsules for repairing heavy metal pollution and its preparation method and application, the biological microcapsules are composed of core material and wall material;The core material includes bacillus subtilis, aspergillus niger, brazilian nitrogen-fixing spirillum and starch;The wall material includes modified polyethylene glycol and sodium alginate;The modified polyethylene glycol is prepared by polyethylene glycol, 2-[(tert-butyl) amino] acetyl chloride hydrochloride and 2-chloroethyl sodium sulfonate reaction.The biological microcapsules for repairing heavy metal pollution of the application can further enrich soil heavy metals by the high adsorption capacity and adsorption capacity of wall material, and significantly reduce the degree of soil heavy metal pollution by the excellent reduction and adsorption capacity of composite microbial agent, and can be widely applied in the field of soil heavy metal repair.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a bio-microcapsule for remediating heavy metal pollution, its preparation method, and its application. Background Technology

[0002] Because heavy metals in the soil cannot be biodegraded, their accumulation in plants can seriously affect plant physiology and molecular activities, directly or indirectly slowing down plant growth and causing crop yield reduction. Their accumulation in the food chain can also cause irreversible damage to the human body.

[0003] Therefore, it is necessary to treat heavy metal ions in soil to reduce harm to the environment and health. Methods for treating heavy metals in soil include physical methods such as soil replacement, isolation, and thermal treatment, as well as chemical methods such as leaching, solidification, and reduction. However, physical or chemical methods not only incur high costs but also often pose the possibility of further pollution. Compared to physical or chemical methods, biological methods such as phytoremediation and microbial remediation are more suitable for addressing the need for low-cost and environmentally friendly treatment.

[0004] Microbial remediation of heavy metal pollution often requires encapsulating microorganisms within a carrier or wall material to maintain their biological activity. Microcapsules are tiny capsules with a micrometer-sized core material and an encapsulating membrane. Industrially, microcapsules containing fragrances, pharmaceuticals, pesticides, etc., are used to inhibit the volatilization of active ingredients and improve transportability. Representative manufacturing methods for microcapsules include spray drying (physical method), condensation (physicochemical method), and interfacial polymerization or in-situ polymerization (chemical method).

[0005] Existing bio-microcapsules cannot meet the needs of soil microbial remediation. On the one hand, the encapsulated microbial agents have low efficacy and efficiency in heavy metal remediation, long remediation cycles, and cannot adapt to environments with high heavy metal concentrations. On the other hand, microcapsules have insufficient adsorption capacity and treatment range for heavy metals, and cannot comprehensively treat heavy metal pollution. Furthermore, large-scale application can lead to wasted performance and further pollution.

[0006] Therefore, there is an urgent need for a type of bio-microcapsule that can adapt to high concentrations of heavy metal pollution and can remediate 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 shortcomings of the prior art, the purpose of this invention is to provide a biological microcapsule that can adapt to high concentrations of heavy metal pollution and can remediate heavy metal pollution on a large scale and with high efficiency, as well as its preparation method and application.

[0008] Technical solution:

[0009] In one aspect, this invention provides a bio-microcapsule for remediating heavy metal pollution, the bio-microcapsule being composed of a core material and a wall material;

[0010] The core material includes Bacillus subtilis, Aspergillus niger, Azotobacter brasiliensis, and starch;

[0011] The wall material includes modified polyethylene glycol and sodium alginate;

[0012] The modified polyethylene glycol was prepared by reacting polyethylene glycol, 2-[(tert-butyl)amino]acetyl chloride hydrochloride and sodium 2-chloroethylsulfonate.

[0013] The Bacillus subtilis in the core material of this invention can secrete extracellular polymers containing a large number of negatively charged functional groups, such as carboxyl, hydroxyl, and carbonyl groups. These functional groups can bind to heavy metal ions through ion exchange, complexation, and covalent adsorption, thereby fixing heavy metals in the environment. Aspergillus niger can chelate and adsorb heavy metal ions based on chitin and dextran in its cell wall, and can also secrete oxalic acid, which can further dissolve heavy metal precipitates and accelerate the adsorption and reduction of heavy metals. Azotobacter brasiliensis can provide a nitrogen source through nitrogen fixation and promote the formation of biofilms, enhancing the overall stress resistance of the bacterial community. The compound bacterial agent can achieve long-term and efficient remediation of heavy metals in soil through the triple synergistic effect of adsorption, reduction, and protection.

[0014] Furthermore, the modified polyethylene glycol is prepared through the following steps:

[0015] (1) In a reactor, polyethylene glycol, 2-[(tert-butyl)amino]acetyl chloride hydrochloride and organic solvent are added. After mixing in an ice bath for 20-40 minutes, triethylamine is added. The temperature is raised to 50-60℃ and reacted under nitrogen protection for 8-12 hours. After filtration, washing and drying, aminated polyethylene glycol is obtained.

[0016] (2) In a reactor, add aminated polyethylene glycol, sodium 2-chloroethyl sulfonate and organic solvent, stir evenly and heat to 40-50°C, keep the reaction at the temperature for 14-18 hours, filter, wash and dry to obtain the modified polyethylene glycol.

[0017] In this invention, by modifying polyethylene glycol to successfully graft tertiary amine and sulfonate structures, on the one hand, the number of pores on the surface of microcapsules can be increased through the multi-branched tertiary amine structure, thereby improving the enrichment effect and adsorption capacity of the wall material for heavy metals in soil; on the other hand, the sulfonate structure can not only cross-link with sodium alginate to form a stable wall material structure, but also further improve the pore formation, dispersion and adsorption effects, significantly improving the enrichment capacity of microcapsule wall material for heavy metals in soil.

[0018] Furthermore, the molecular weight of the polyethylene glycol is 200-4000; the organic solvent is selected from tetrahydrofuran or dimethyl sulfoxide.

[0019] 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);

[0020] In step (2), the mass ratio of aminated polyethylene glycol to sodium 2-chloroethylsulfonate is (5-8):(1-2).

[0021] Furthermore, the starch is selected from at least one of resistant starch or cross-linked starch.

[0022] By further defining the starch in this invention as resistant starch or cross-linked starch, it can not only provide nutrients 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 compound microbial agent in a high-concentration heavy metal environment, thereby improving the stability of the repair process.

[0023] Another aspect of the present invention provides a method for preparing bio-microcapsules for remediating heavy metal pollution according to any one of the above-mentioned methods, comprising the following steps:

[0024] (1) Bacillus subtilis, Aspergillus niger and Azotobacter brasiliensis were mixed in proportion and then the bacterial cells were resuspended in sterile physiological saline to form a compound bacterial suspension.

[0025] (2) Add starch to the composite bacterial suspension, stir and mix thoroughly to obtain the composite core material;

[0026] (3) The composite core material, modified polyethylene glycol and sodium alginate are mixed in proportion, stirred evenly and then spray-dried to obtain the bio-microcapsule for remediating heavy metal pollution.

[0027] In this invention, using modified polyethylene glycol and sodium alginate as wall materials not only enhances the adsorption capacity and absorption capacity of microcapsules for heavy metal ions in soil, achieving the enrichment effect of heavy metals in soil and thus expanding the scope of soil remediation, but also significantly improves the protective and isolating effect of microcapsules on the core material, allowing microorganisms to maintain activity in environments with high heavy metal concentrations, thereby achieving a high remediation area, reducing the investment in biological microcapsules, lowering costs, and improving efficiency.

[0028] Furthermore, the number of viable bacteria in the compound bacterial suspension in step (1) is not less than 3 × 10⁻⁶. 9 CFU / g;

[0029] The effective viable count ratio of Bacillus subtilis, Aspergillus niger and Azotobacter brasiliensis is (1-1.5):(0.8-1.2):(0.5-0.8).

[0030] Further, in step (2), the mass ratio of the compound bacterial suspension to starch is (5-8):(1-2).

[0031] 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).

[0032] Furthermore, the conditions for spray drying in step (3) are: inlet air temperature 120-140℃, fan frequency 40-50Hz, creeping speed 5-10rpm, and outlet air temperature 50-60℃.

[0033] Finally, the present invention also provides the application of any of the above-mentioned bio-microcapsules for remediating heavy metal pollution in the field of soil heavy metal pollution remediation.

[0034] Beneficial effects:

[0035] (1) The bio-microcapsule for remediating heavy metal pollution provided by the present invention uses a compound microbial agent of Bacillus subtilis, Aspergillus niger and Azotobacter brasiliensis as the core remediation material and starch as the core material. Modified polyethylene glycol and sodium alginate are used as wall materials to make the bio-microcapsule. The high adsorption capacity and adsorption capacity of the wall material can further enrich the soil heavy metals. The excellent reduction and adsorption capacity of the compound microbial agent can significantly reduce the degree of soil heavy metal pollution. It can be widely used in the field of soil heavy metal remediation.

[0036] (2) The biological microcapsules for remediating heavy metal pollution provided by the present invention use Bacillus subtilis, Aspergillus niger and Azotobacter brasiliensis as compound bacterial agents, and achieve efficient remediation of heavy metal pollution through the synergistic effect among the three.

[0037] (3) In the bio-microcapsules for remediating heavy metal pollution provided by the present invention, Bacillus subtilis can secrete extracellular polymers containing a large number of negatively charged functional groups, such as carboxyl, hydroxyl, and carbonyl groups. These functional groups can bind to heavy metal ions through ion exchange, complexation, covalent adsorption, etc., thereby fixing heavy metals in the environment. Aspergillus niger can chelate and adsorb heavy metal ions based on chitin and dextran in the cell wall, and can also secrete oxalic acid, which can further dissolve heavy metal precipitates and accelerate the adsorption and reduction of heavy metals. Azotobacter brasiliensis can provide nitrogen sources through nitrogen fixation and promote the formation of biofilms, thereby enhancing the overall stress resistance of the bacterial community. The compound bacterial agent can achieve long-term and efficient remediation of soil heavy metals through the triple synergistic effect of adsorption, reduction and protection.

[0038] (4) The bio-microcapsules for remediating 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 capacity of microcapsules for heavy metal ions in soil and achieve the enrichment effect of heavy metals in soil, thereby expanding the soil remediation range, but also significantly improve the protective and isolation effect of microcapsules on core materials, so that microorganisms can maintain activity in the environment of high heavy metal concentration, thereby achieving the effect of high remediation area, reducing the investment of bio-microcapsules, reducing costs and improving efficiency.

[0039] (5) In the bio-microcapsules for remediating heavy metal pollution provided by the present invention, after modifying polyethylene glycol, tertiary amine structure and sulfonate structure are successfully grafted. 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 heavy metals in the soil. On the other hand, the sulfonate structure can not only cross-link with sodium alginate to form a stable wall material structure, but also further improve the pore formation effect, dispersion effect and adsorption effect, significantly improving the enrichment capacity of the microcapsule wall material for heavy metals in the soil. Detailed Implementation

[0040] The present invention will be described below with reference to specific embodiments. It should be noted that the following embodiments are examples of the present invention and are used only to illustrate the invention, not to limit it. Other combinations and various modifications within the scope of the present invention can be made without departing from its spirit or scope.

[0041] The Bacillus subtilis MES 810 strain in this invention was deposited by the applicant at the China General Microbiological Culture Collection Center (CGMCC) on August 10, 2017, with accession number CGMCC 14514.

[0042] It should be noted that the Bacillus subtilis strain of the present invention has been patented by our company, application number 201710862006.3;

[0043] The Aspergillus niger strain was purchased from the China General Microbiological Culture Collection Center (CGMCC), with a deposit date of July 14, 2008, and accession number CGMCC 3.11571;

[0044] The strain of Azospirillum brasilense was purchased from the China General Microbiological Culture Collection Center (CGMCC), with a deposit date of June 27, 2005, and accession number CGMCC 1.5808.

[0045] The polyethylene glycol has a molecular weight of 2000; the CAS number of 2-[(tert-butyl)amino]acetyl chloride hydrochloride is 915725-52-9; the remaining reagents and equipment are conventional reagents and equipment in this technical field.

[0046] Preparation of modified polyethylene glycol-1

[0047] (1) In a reactor, add 6g of polyethylene glycol, 1.5g of 2-[(tert-butyl)amino]acetyl chloride hydrochloride and 50mL of tetrahydrofuran. After mixing in an ice bath for 30 minutes, add 1.5g of triethylamine. Heat to 50°C and react under nitrogen protection for 12 hours. Filter, wash and dry to obtain aminated polyethylene glycol.

[0048] (2) In the reactor, add 4g of aminated polyethylene glycol, 1g of sodium 2-chloroethylsulfonate and 50mL of tetrahydrofuran, stir evenly and heat to 40°C, keep the reaction at the temperature for 18 hours, filter, wash and dry to obtain the modified polyethylene glycol-1.

[0049] Preparation of modified polyethylene glycol-2

[0050] The preparation is basically the same as that of modified polyethylene glycol-1, except that step (2) is omitted and in step (1) 2-[(tert-butyl)amino]acetyl chloride hydrochloride is replaced with an equal amount of acryloyl chloride.

[0051] Example 1

[0052] Biocapsules for remediating heavy metal pollution were prepared using the following steps:

[0053] (1) After mixing Bacillus subtilis, Aspergillus niger and Azotobacter brasiliensis, the bacterial cells were resuspended in sterile physiological saline to form a compound bacterial suspension;

[0054] (2) Add 1.5 parts of cross-linked starch to 6 parts of the composite bacterial suspension by weight, and mix thoroughly to obtain the composite core material;

[0055] (3) By weight, 2 parts of composite core material, 4 parts of modified polyethylene glycol-1 and 4 parts of sodium alginate are mixed, stirred evenly and then spray-dried to obtain the bio-microcapsules for remediating heavy metal pollution.

[0056] The effective viable bacteria count in the compound bacterial suspension in step (1) is 4 × 10⁻⁶. 9 CFU / g; among which, the effective viable count ratio of Bacillus subtilis, Aspergillus niger and Azotobacter brasiliensis was 1.5:1:0.5;

[0057] The conditions for spray drying in step (3) are: inlet air temperature 130℃, fan frequency 45Hz, peristalsis speed 10rpm, and outlet air temperature 55℃.

[0058] Example 2

[0059] The process is basically the same as the previous example, except that in step (2), the cross-linked starch is replaced with an equal amount of resistant starch; in step (3), the composite core material is replaced with 1.5 parts, the modified polyethylene glycol-1 is replaced with 5 parts, and the sodium alginate is replaced with 5 parts.

[0060] Example 3

[0061] The basic method is the same as the example, 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.

[0062] Comparative Example 1

[0063] Basically the same as in Example 1, except that in step (1), the compound bacterial suspension is changed to have an effective viable count of 4 × 10⁻⁶. 9 CFU / g Bacillus subtilis suspension.

[0064] Comparative Example 2

[0065] The process is basically the same as in Example 1, except that in step (2), the cross-linked starch is replaced with an equal amount of wheat starch.

[0066] Comparative Example 3

[0067] The process is basically the same as in Example 1, except that in step (3), modified polyethylene glycol-1 is replaced with an equal amount of polyethylene glycol.

[0068] Comparative Example 4

[0069] The process is basically the same as in Example 1, except that in step (3), modified polyethylene glycol-1 is replaced with an equal amount of polyethylene glycol-2.

[0070] Performance testing

[0071] Adsorption capacity and adsorption amount detection: Weigh 0.1g of each product from Examples 1-3 and Comparative Examples 1-4 and place them in 500ml wide-mouth conical flasks. Add 10mg / L Cd to each sample. 2+ Solution. The conical flask was placed in a double-layered constant-temperature shaker at (25±5)℃ and shaken at 150 r / min for 12 h. After passing through a 0.45 μm microporous membrane, the Cd content in the filtrate was determined using flame atomic absorption spectrometry. 2+ And calculate the effect of different products on Cd in the solution. 2+ The maximum adsorption capacity and adsorption rate.

[0072] Remediation capacity testing: 10g of each of the products from Examples 1-3 and Comparative Examples 1-4 were added to 5000g of heavily contaminated soil containing heavy metals. After thorough mixing, the soil environment was simulated. The Cd concentration in the soil was recorded after 14 days.2+ Removal rate.

[0073] The test results are shown in the table below:

[0074]

[0075] According to the comparison of the test results of Examples 1-3 and Comparative Example 1, the bio-microcapsules for remediating heavy metal pollution provided by the present invention, through the reasonable combination of bacterial species in the compound bacterial agent, based on the synergistic remediation effect of Bacillus subtilis, Aspergillus niger and Azotobacter brasiliensis, have a better heavy metal remediation ability than the microbial agents in the prior art, and can efficiently and stably remediate soils with severe heavy metal pollution.

[0076] 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 bio-microcapsule core material for remediating heavy metal pollution provided by the present invention can not only provide nutrients 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 compound bacterial agent in a high-concentration heavy metal environment, thereby improving the stability of the remediation process.

[0077] The comparison of the test results of Examples 1-3 and Comparative Examples 3-4 shows that the bio-microcapsules for remediating heavy metal pollution provided by the present invention, by modifying polyethylene glycol with amino and sulfonic acid groups and then combining it with sodium alginate to form a wall material, can significantly improve the bio-microcapsule's ability to enrich heavy metals in the soil, thereby effectively increasing the remediation area, reducing material input, and lowering costs.

[0078] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A bio-microcapsule for remediating heavy metal pollution, characterized in that, The bio-microcapsule is composed of a core material and a wall material; The core material includes Bacillus subtilis, Aspergillus niger, Azotobacter brasiliensis, and starch; The wall material includes modified polyethylene glycol and sodium alginate; The modified polyethylene glycol was prepared by reacting polyethylene glycol, 2-[(tert-butyl)amino]acetyl chloride hydrochloride and sodium 2-chloroethylsulfonate. The modified polyethylene glycol is prepared by the following steps: (1) In a reactor, polyethylene glycol, 2-[(tert-butyl)amino]acetyl chloride hydrochloride and organic solvent are added. After mixing in an ice bath for 20-40 minutes, triethylamine is added. The temperature is raised to 50-60℃ and reacted under nitrogen protection for 8-12 hours. After filtration, washing and drying, aminated polyethylene glycol is obtained. (2) In a reactor, add aminated polyethylene glycol, sodium 2-chloroethyl sulfonate and organic solvent, stir evenly and heat to 40-50°C, keep the reaction at the temperature for 14-18 hours, filter, wash and dry to obtain the modified polyethylene glycol; The starch is selected from at least one of resistant starch or cross-linked starch; The polyethylene glycol has a molecular weight of 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 step (2), the mass ratio of aminated polyethylene glycol to sodium 2-chloroethylsulfonate is (5-8):(1-2).

2. The method for preparing the bio-microcapsules for remediating heavy metal pollution as described in claim 1, characterized in that, Includes the following steps: (1) Bacillus subtilis, Aspergillus niger and Azotobacter brasiliensis were mixed in proportion and then the bacterial cells were resuspended in sterile physiological saline to form a compound bacterial suspension. (2) Add starch to the composite bacterial suspension, stir and mix thoroughly to obtain the 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 bio-microcapsule for remediating heavy metal pollution.

3. The method for preparing bio-microcapsules for remediating heavy metal pollution according to claim 2, characterized in that, In step (1), the number of viable bacteria in the compound bacterial suspension is not less than 3 × 10⁻⁶. 9 CFU / g; The effective viable count ratio of Bacillus subtilis, Aspergillus niger and Azotobacter brasiliensis is (1-1.5):(0.8-1.2):(0.5-0.8).

4. The method for preparing bio-microcapsules for remediating heavy metal pollution according to claim 2, characterized in that, In step (2), the mass ratio of the compound bacterial suspension to starch is (5-8):(1-2).

5. The method for preparing bio-microcapsules for remediating heavy metal pollution according to claim 2, characterized in that, 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).

6. The method for preparing bio-microcapsules for remediating heavy metal pollution according to claim 2, characterized in that, The conditions for spray drying in step (3) are: inlet air temperature 120-140℃, fan frequency 40-50Hz, creeping speed 5-10rpm, and outlet air temperature 50-60℃.

7. The application of the bio-microcapsules for remediating heavy metal pollution as described in claim 1 in the field of soil heavy metal pollution remediation.

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