A preparation method of a living bacteria composite biomaterial for repairing Cr(VI) contaminated water body

Abstract

The application belongs to the technical field of contaminated water body remediation, and discloses a preparation method of a living bacteria composite biomaterial for remediation of Cr(VI) contaminated water bodies, wherein modified melamine sponge is used as a composite material support for fixing living bacteria, and in-situ modification is carried out by adding polypyrrole; an electroactive bacteria with Cr(VI) reduction capacity is used as a model strain, and a hybrid strain obtained by in-situ assembly of amino-modified carbon dots and polydopamine is used as a core-embedded bacteria agent of the modified sponge; the core-embedded bacteria agent is heterojunction grafted onto the modified melamine sponge by using a mixed solution of sodium lactate, polyethylene glycol and bis[tris(hydroxymethyl)aminopropane] as an outer layer embedding agent, and a living bacteria composite material is prepared. The application uses modified melamine sponge as a fixing support of living bacteria material, modifies the electroactive strain, and carries out in-situ assembly of the modified sponge, so that the whole preparation process is simple and has the performances of green and environmental protection.

Description

Technical Field

[0001] This invention relates to the field of Cr(VI) polluted water remediation technology, specifically to a method for preparing a live bacteria composite biomaterial for remediating Cr(VI) polluted water. Background Technology

[0002] In the natural environment, chromium (Cr) exists primarily in the forms of Cr(VI) and Cr(III). Cr(VI) is highly toxic and readily soluble, while Cr(III) is less toxic and easily forms insoluble precipitates. Therefore, converting the highly toxic and readily soluble Cr(VI) into the insoluble and less toxic Cr(III) for further separation is a widely accepted and effective method for chromium removal from water bodies or sediments. Currently, electrolysis, chemical methods, ion exchange, and microbial reduction are considered effective methods for chromium removal from water bodies. Compared to electrolysis and ion exchange, these methods are costly; while adding chemical agents can efficiently reduce and remove Cr(VI), excessive consumption of these agents often leads to secondary pollution. Therefore, considering the low cost and considerable treatment effect, the use of microbial reduction of Cr(VI) is gaining increasing attention.

[0003] Microorganisms are characterized by rapid reproduction, high reaction rates, and strong environmental tolerance. Utilizing microorganisms to remediate Cr(VI) contaminated water is an effective, green, and inexpensive remediation technology that minimizes environmental disturbance during remediation and shows promise for treating Cr(VI) contaminated water. Shewanella (… Shewanella Microorganisms are typical dissimilar Cr(VI) reduction model microorganisms, widely distributed in soil and water. To ensure complete reduction of the electron acceptor Cr(VI), traditional microbial Cr(VI) reduction mostly requires the microbial oxidation and decomposition of lactic acid to provide biosource electrons. However, adding large amounts of lactic acid to maintain microbial Cr(VI) reduction increases treatment costs. Therefore, how to expand the electron flux of Cr(VI) in the microbial reduction process and simultaneously improve the electron transfer rate is the main bottleneck of microbial Cr(VI) reduction at present. Furthermore, due to the small size and easy dispersion of individual microbial cells, their resistance in highly polluted environmental media is limited. If pollutant transformation and removal are achieved simply by inoculating functional active bacteria, the treatment efficiency is low. In addition, frequent addition of microbial agents is required to improve the purification effect, which will greatly increase the treatment cost. Summary of the Invention

[0004] To promote the application of microorganisms in chromium removal from water bodies, and addressing the problems existing in the current technology, this invention provides a method for preparing a live bacterial composite biomaterial for remediating Cr(VI) polluted water bodies. The method is optimized and improved in the following three aspects: (1) At the microscale, the intracellular and extracellular interface layers of individual functional microorganisms can be modified to enhance the electron transfer efficiency between the non-biological / biological interface layers and strengthen the resistance and tolerance of individual microbial cells. (2) At the large scale, large-scale biofilms can be formed through the aggregation and reproduction of individual microorganisms to improve biological conversion efficiency. (3) To promote the recycling and reuse of functional active bacteria, novel composite live biomaterials can be prepared using microbial grafting / solidification technology. The advantages are: effective functional bacteria can be immobilized, promoting the directional reproduction of biofilms and maintaining the biological activity of functional bacteria; at the same time, the active bacteria can be recycled and reactivated through composite solidification materials, thereby reducing treatment costs.

[0005] The technical solution adopted in this invention is as follows: A method for preparing a live bacteria composite biomaterial for remediating Cr(VI) polluted water, comprising the following specific steps:

[0006] (1) Preparation of live bacteria composite material scaffold: melamine sponge was used as the main scaffold of live bacteria composite material, polypyrrole was added for in-situ surface modification, and the modified sponge was obtained after vacuum drying;

[0007] (2) Live bacteria modification: Electroactive bacteria with the ability to reduce Cr(VI) were selected as model strains and pre-cultured. After centrifugation, the expanded cells were obtained. Under anaerobic conditions, the expanded cells were first resuspended in bis[tris(hydroxymethyl)aminopropane] buffer and then blown with nitrogen. Then, surface modification and hybrid assembly were carried out in a mixture of polydopamine and amino-modified carbon dots. Afterward, the cells were washed with bis[tris(hydroxymethyl)aminopropane] buffer and centrifuged to obtain hybrid strain cells.

[0008] (3) Immobilization of hybrid strains / modified sponges: A mixture of sodium lactate, bis[tris(hydroxymethyl)aminopropane] and polyethylene glycol was prepared as an outer layer embedding agent; under an anaerobic nitrogen atmosphere, the hybrid strains obtained in step (2) were used as the core embedding agent, and the outer layer embedding agent and the modified sponge obtained in step (1) were grafted to achieve multidimensional heterogeneous grafting to obtain a solidified live bacteria composite material.

[0009] In step (1), the process of in-situ surface modification of melamine sponge includes the following steps: First, the melamine sponge is rinsed and dried with disinfectant alcohol and deionized water; then, the obtained melamine sponge is fully immersed in the modified polypyrrole solution to allow it to fully absorb the polypyrrole; then, ferric chloride solution is added to the above system and stirred thoroughly in an ice-water bath; after the above system has reacted, it is taken out and washed with deionized water until the rinsing solution is colorless.

[0010] The polypyrrole solution uses deionized water as a solvent and contains 0.37% to 1.48% polypyrrole monomer by mass.

[0011] In step (1), vacuum drying involves placing the cleaned modified melamine sponge in a vacuum drying oven. The drying conditions are set at 50 ºC for 12 h.

[0012] Electroactive bacteria capable of reducing Cr(VI) are Shewanella ( Shewanella ) ,like Shewanella oneidensis MR-1 Shewanella xiamenensis BC01 and Shewanella putrefaciens CN32, etc.

[0013] In step (2), amino-modified carbon dots are added to the culture dishes used for pre-culture.

[0014] In step (2), the concentration of the polydopamine solution used for in-situ hybrid assembly is 4 g / L.

[0015] The surface modification and hybrid assembly process was carried out under a shaker, with the culture parameters set at 200 rpm, 30°C, and 2 h under aerobic conditions; the centrifugation parameters for separating the hybrid strains were set at 5000 rpm and 3 min.

[0016] In the outer embedding agent, the mass ratio of bis[tris(hydroxymethyl)aminopropane], sodium lactate, and polyethylene glycol is 198:157~314:2100~5600.

[0017] This invention utilizes modified melamine sponge as a scaffold for grafting and immobilizing live bacteria, through the electroactive bacteria with Cr(VI) reducing ability (… Shewanella Hybrid strains were obtained by modifying (a type of microorganism) and used as the core embedding agent for live bacteria composite materials. In-situ assembly of the hybrid strains and modified sponges was achieved by adding an outer embedding agent and a cross-linking immobilizer. The entire preparation process is simple, and the strains can be repeatedly recycled and reused in water chromium removal applications, exhibiting good performance. This provides a new approach to enhancing the application of microorganisms in chromium removal.

[0018] Compared with existing technologies, this invention has the following advantages: 1. This invention achieves in-situ grafting, assembly, and fixation of large-scale biofilms on composite materials by heterojunction grafting and fixing electroactive Shewanella bacteria with Cr(VI) reduction capabilities onto melamine sponges, thus avoiding the defects of traditional microbial chromium removal technologies that require the addition of excessive microbial agents and are difficult to recycle; 2. At the microscale of the live bacteria composite material, the addition of polypyrrole for in-situ polymerization and assembly modification greatly enhances the active contact sites and electron conduction capabilities between the live bacteria and the melamine sponge framework, thereby effectively improving the catalytic efficiency for Cr(VI) reduction; 3. The added amino-modified carbon dots not only improve the robustness and metabolic activity of the microorganisms, but also exhibit certain reducing properties, providing more reducing power for chromium removal; 4. The composite material does not require excessive addition of sodium lactate as a carbon source to maintain microbial growth during use. It can utilize visible light catalysis to maximize the use of light energy. Photocatalysis generates photoelectrons and amino-modified carbon dots with reducing properties to provide more reducing power for the removal of Cr(VI). 5. It is easy to recycle. It can be directly separated from the liquid. After separation, the living material can be directly added to the next reaction system after removing surface impurities with deionized water. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0020] Figure 1 A schematic diagram of the microstructure of a live bacteria composite biomaterial used to remediate Cr(VI) contaminated water.

[0021] In the figure, 2 is a melamine sponge modified with polypyrrole; 3 is a living heterozygous Shewanella cell that can reduce Cr(VI);

[0022] Figure 2 Flowchart of the preparation process of live bacteria composite biomaterials for remediating Cr(VI) contaminated water;

[0023] In the figure, 1. Melamine sponge scaffold; 1-1. Scaffold size cutting; 1-2. Rinsing, drying and storage; 2. Polypyrrole-modified melamine sponge; 2-1. Preparation of polypyrrole solution; 2-2. Soaking and thorough mixing; 2-3. Oxidation reaction of FeCl3 solution; 2-4. Separation, washing and drying; 3. Live heterozygous Shewanella cells capable of reducing Cr(VI); 3-1. Activation and aerobic expansion of the strain; 3-2. Centrifugation, removal of supernatant and resuspension; 3-3. External hybridization with polydopamine and amino-modified carbon dots; 3-4. Aerobic culture; 3-5. Centrifugation, washing and collection of hybrid cells; 4. Preparation of outer layer embedding agent; 5. Immobilized live bacteria composite material; 5-1. Resuspension of live hybrid bacteria; 5-2. Grafting of hybrid bacterial solution onto modified melamine sponge; 5-3. Aerobic culture, cleaning, separation and preservation of composite materials. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0025] This invention provides a live bacteria composite biomaterial for remediating Cr(VI) contaminated water, such as... Figure 1 As shown, it includes a polypyrrole-modified melamine sponge and a live heterozygous Shewanella cell that can reduce Cr(VI).

[0026] The specific preparation process of the above-mentioned live bacteria composite biomaterial for remediating Cr(VI) polluted water is as follows: Figure 2 As shown, taking some specific embodiments as examples, the steps include:

[0027] (1) Preparation of modified melamine sponge with immobilized live bacteria

[0028] First, melamine sponge was cut into small cylindrical strips with dimensions of Φ10 mm × H40 mm. These strips were rinsed twice with deionized water and 70 wt% ethanol, and then vacuum-dried at 100 ℃ for later use. In a 200 mL beaker, 0.15–0.60 mL of polypyrrole monomer standard was sequentially dissolved in 40 mL of deionized water and ultrasonically dispersed to obtain a mixed solution containing 0.37%–1.48% polypyrrole monomer by mass. The pre-cut melamine sponge cylindrical strips were then immersed in the polypyrrole solutions of different concentrations, and repeatedly squeezed with PTFE tweezers to ensure full absorption of the polypyrrole solution. After the sponge had fully absorbed the polypyrrole solution, 40 mL of a 0.18 mol / L ferric chloride solution was added to the system. The mixture was then magnetically stirred for 4 h in an ice-water bath. After stirring is complete, the melamine sponge loaded with polypyrrole is removed and washed repeatedly with deionized water at least 5 times until the rinsing solution is colorless. The washed modified melamine sponge is then placed in a vacuum drying oven. The drying conditions are set at 50 ºC for 12 hours.

[0029] (2) Modification and hybridization of wild-type Shewanella cells capable of reducing Cr(VI)

[0030] Wild-type electroactive materials with Cr(VI) reducing ability were selected. Shewanella The genus is the type strain (e.g. Shewanella oneidensis MR-1 Shewanella xiamenensis BC01 and Shewanella putrefaciensCN32 and other strains can be used as supply strains. Take 200 μL of wild-type strain from the cryopreservation tube and incubate aerobically in 20 mL of modified LB medium (with 0.102–0.204 mL of 10.0 g / L amino-modified carbon dot stock solution added to the LB medium, resulting in an amino-modified carbon dot concentration of 50–100 mg / L) for a period of time. The aerobic culture conditions are: shaker culture, 200 rpm, 16 h, 30 °C. Centrifuge the resulting culture at 5000 rpm for 5 min, remove the supernatant, and resuspend the obtained bacterial cells in 1.0 mL of 10 mmol / L (pH 7.0–7.2) bis[tris(hydroxymethyl)aminopropane] buffer. Resuspension is performed under anaerobic nitrogen aeration for 30 min. After resuspension, 1 mL of 4.0 g / L polydopamine solution and 0.3–0.6 mL of 10.0 g / L amino-modified carbon dot stock solution were anaerobically injected into the resuspension system under anaerobic nitrogen aeration. The resulting hybrid bacterial culture was then continuously cultured aerobically for a period of time. The aerobic culture conditions were set as follows: shaker culture, shaker speed 200 rpm, culture time 2 h, culture temperature 30 ℃. After the culture was completed, the bacterial cells were centrifuged at 5000 rpm for 3 min, and the supernatant was removed to obtain hybrid bacterial cells. The separated hybrid cells were washed twice with 10.0 mL of 10 mmol / L (pH=7.0–7.2) bis[tris(hydroxymethyl)aminopropane] buffer, and then centrifuged again at 5000 rpm for 3 min to obtain hybrid bacterial cells for later use.

[0031] (3) Preparation of outer layer embedding agent

[0032] Add 0.198 g of bis[tris(hydroxymethyl)aminopropane], 0.157~0.314 g of sodium lactate, and 2.1~5.6 g of polyethylene glycol powder particles to 70 mL of sterile water and dissolve them. After ultrasonic vibration for 5 min, an outer embedding agent is prepared to fix the living hybrid Cr(VI) reducible Shewanella cells prepared in (2) onto the modified melamine sponge scaffold prepared in (1).

[0033] (4) Grafting and fixation of heterozygous strains on modified melamine sponge

[0034] The grafting of the modified melamine sponges and hybrid strains prepared in (1) and (2) above was achieved by adding an outer embedding agent. The living hybrid Cr(VI)-reducible Shewanella cells prepared in (2) were resuspended in the outer embedding agent obtained in (3) above, maintaining the OD600 of the mixture at approximately 0.8–1.2. This resuspension system was then transferred to a 100 mL sterilized conical flask. Six or more modified melamine sponges obtained in (1) were transferred into this conical flask culture system. The modified melamine sponges were squeezed using sterilized polytetrafluoroethylene tweezers to ensure they fully absorbed the resuspended bacterial solution. After the modified melamine sponges had fully absorbed the bacterial solution, the conical flask was transferred to a shaker for aerobic culture. The shaker culture conditions were set as follows: 150 rpm, aerobic culture for 5 h, and culture temperature of 30 °C. After the culture is completed, the melamine sponge grafted with the bacterial solution is separated from the conical flask, rinsed three times with a 10 mmol / L bis[tris(hydroxymethyl)aminopropane] solution, and then stored in a 50 mL sterile centrifuge tube for subsequent use in the removal of Cr(VI) from wastewater.

[0035] Some specific embodiments of the present invention are as follows: according to the preparation method described above, in step (1), 0.15 mL, 0.45 mL and 0.60 mL of polypyrrole monomer standard are added sequentially for modifying melamine sponge; in step (2), select... Shewanella oneidensis MR-1 is the model strain for Cr(VI) reduction. 0.2 mL of 10.0 g / L amino-modified carbon dot stock solution was added to modified LB medium. After resuspension, 0.4 mL of 10.0 g / L amino-modified carbon dot stock solution was anaerobically injected. In step (3), 0.30 g of sodium lactate and 4.3 g of polyethylene glycol powder particles were added for the preparation of the outer layer embedding agent. Finally, the corresponding live bacterial composite biomaterials were prepared according to step (4). Except for the specific dosage (concentration and mention) of the reagents or strains mentioned above, unless otherwise specified, the dosage of other reagents is the same as that of other reagents described in steps (1) to (4). Finally, the prepared live bacterial composite biomaterials were sequentially numbered as Material 1, Material 2, and Material 3.

[0036] Table 1. Catalytic performance and recyclability evaluation of live bacteria composite biomaterials for chromium removal from wastewater.

[0037]

[0038] Eighty mL of Cr(VI)-contaminated wastewater with an initial concentration of 50 mg / L was placed in a 100 mL anaerobic serum bottle. The three composite materials were added to the reaction system. The reaction temperature was 25–30 °C in the dark, and the system was sealed after deoxygenation with nitrogen for 20 min before the reaction. Five random sampling points were set during the reaction, and 2 mL liquid samples were taken from the system to monitor the chromium removal efficiency in the wastewater. After the reaction was complete (the residual Cr(VI) concentration in the wastewater was <0.01 mg / L), the composite materials were removed, rinsed 2–3 times with distilled water, and then added back to the same reaction system for recycling experiments to evaluate the recyclability and catalytic efficiency of the materials. As shown in Table 1, all three materials exhibited good catalytic performance after four cycles, with catalytic efficiencies reaching 100%. The composite material modified with a larger dose of polypyrrole monomer showed superior catalytic performance, and the reaction time was shorter, ranging from 18 to 36 h.

[0039] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for preparing a live bacteria composite biomaterial for remediating Cr(VI) contaminated water, characterized in that... The specific steps include the following: (1) Preparation of live bacteria composite material scaffold: melamine sponge was used as the main scaffold of live bacteria composite material, polypyrrole was added for in-situ surface modification, and the modified sponge was obtained after vacuum drying; (2) Live bacteria modification: Electroactive bacteria with the ability to reduce Cr(VI) were selected as model strains and pre-cultured. After centrifugation, the expanded cells were obtained. Under anaerobic conditions, the expanded cells were first resuspended in bis[tris(hydroxymethyl)aminopropane] buffer and then blown with nitrogen. Then, surface modification and hybrid assembly were carried out in a mixture of polydopamine and amino-modified carbon dots. Afterward, the cells were washed with bis[tris(hydroxymethyl)aminopropane] buffer and centrifuged to obtain hybrid strain cells. (3) Immobilization of hybrid strains / modified sponges: A mixture of sodium lactate, bis[tris(hydroxymethyl)aminopropane] and polyethylene glycol was prepared as an outer layer embedding agent; under an anaerobic nitrogen atmosphere, the hybrid strains obtained in step (2) were used as the core embedding agent, and the outer layer embedding agent and the modified sponge obtained in step (1) were grafted to achieve multidimensional heterogeneous grafting to obtain a solidified live bacteria composite material.

2. The method for preparing the live bacteria composite biomaterial for remediating Cr(VI) polluted water according to claim 1, characterized in that: In step (1), the process of in-situ surface modification of melamine sponge includes the following steps: First, the melamine sponge was rinsed and dried with rubbing alcohol and deionized water. Then, the melamine sponge was fully immersed in the modified and loaded polypyrrole solution to allow it to fully absorb the polypyrrole. Next, ferric chloride solution was added to the above system and stirred thoroughly in an ice-water bath. After the reaction was completed, the sponge was removed and washed with deionized water until the rinsing solution was colorless.

3. The method for preparing the live bacteria composite biomaterial for remediating Cr(VI) polluted water according to claim 2, characterized in that: The polypyrrole solution uses deionized water as a solvent and contains 0.37% to 1.48% polypyrrole monomer by mass.

4. The method for preparing the live bacteria composite biomaterial for remediating Cr(VI) polluted water according to claim 2, characterized in that: In step (1), vacuum drying involves placing the cleaned modified melamine sponge in a vacuum drying oven.

5. The method for preparing the live bacteria composite biomaterial for remediating Cr(VI) polluted water according to claim 4, characterized in that: The drying conditions were set at 50 ºC for 12 hours.

6. The method for preparing the live bacteria composite biomaterial for remediating Cr(VI) polluted water according to claim 1, characterized in that: Electroactive bacteria capable of reducing Cr(VI) are Shewanella ( Shewanella ).

7. The method for preparing the live bacteria composite biomaterial for remediating Cr(VI) polluted water according to claim 1, characterized in that: In step (2), amino-modified carbon dots are added to the culture dishes used for pre-culture.

8. The method for preparing the live bacteria composite biomaterial for remediating Cr(VI) polluted water according to claim 1, characterized in that: In step (2), the concentration of the polydopamine solution used for in-situ hybrid assembly is 4 g / L.

9. The method for preparing the live bacteria composite biomaterial for remediating Cr(VI) polluted water according to claim 1, characterized in that: The surface modification and hybrid assembly process was carried out under a shaker, with the culture parameters set at 200 rpm, 30 ℃, and 2 h under aerobic conditions; the centrifugation parameters for separating the hybrid strains were set at 5000 rpm and 3 min.

10. The method for preparing the live bacteria composite biomaterial for remediating Cr(VI) polluted water according to claim 1, characterized in that: In the outer embedding agent, the mass ratio of bis[tris(hydroxymethyl)aminopropane], sodium lactate, and polyethylene glycol is 198:157~314:2100~5600.

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