WO2024060516A1 - 一种用于修复Cr(VI)污染水体的活菌复合生物材料的制备方法 - Google Patents

一种用于修复Cr(VI)污染水体的活菌复合生物材料的制备方法 Download PDF

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WO2024060516A1
WO2024060516A1 PCT/CN2023/079364 CN2023079364W WO2024060516A1 WO 2024060516 A1 WO2024060516 A1 WO 2024060516A1 CN 2023079364 W CN2023079364 W CN 2023079364W WO 2024060516 A1 WO2024060516 A1 WO 2024060516A1
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modified
repairing
sponge
contaminated water
composite biological
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陈铮
李建
王�锋
曾艳琼
黄鹏
赵聪媛
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温州医科大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

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  • the present invention relates to the technical field of remediation of Cr(VI)-contaminated water bodies, and specifically relates to a preparation method of live bacteria composite biological materials for remediation of Cr(VI)-polluted water bodies.
  • Chromium (Cr) is one of the five heavy metal pollutants strictly controlled by the country. Especially in industries such as electroplating and tanning, the high concentration of chromium in wastewater is one of the most typical pollutants that are difficult to control and seriously pollutes the environment. The surrounding ecological environment such as regional water bodies and sediments. Cr mainly exists in the natural environment in the form of Cr(VI) and Cr(III). Among them, Cr(VI) is highly toxic and easily soluble, while Cr(III) is low in toxicity and prone to form insoluble precipitates. Therefore, converting highly toxic and easily soluble Cr(VI) into insoluble and low-toxic Cr(III) for re-separation is currently a generally recognized and effective way to achieve chromium removal from water or sediment.
  • electrolysis, chemical methods, ion exchange methods and microbial reduction methods are considered to be effective methods for removing chromium from water bodies.
  • the treatment cost is high; although adding chemicals can effectively reduce and remove Cr(VI), excessive consumption of chemicals often easily causes secondary pollution. Therefore, considering the low economic treatment cost and considerable treatment effect, the use of microorganisms to reduce Cr(VI) has gradually attracted attention.
  • Microorganisms have the characteristics of fast reproduction, high reaction rate and strong environmental tolerance.
  • the use of microorganisms to remediate Cr(VI)-contaminated water bodies is an effective, green, cheap and feasible remediation technology. It can minimize the disturbance to the environment during remediation and has certain application prospects in the treatment of Cr(VI)-contaminated water bodies. .
  • Shewanella (
  • the present invention provides a preparation method of live bacteria composite biological materials for repairing Cr(VI) contaminated water bodies, which mainly works in the following three aspects. Optimization and improvement: (1). On a micro scale, by modifying the intracellular and extracellular interface layers of individual cells of functional microorganisms, the electron transfer efficiency between the abiotic/biological interface interface layers can be improved and the individual microorganisms can be strengthened. Cell resistance tolerance. (2). On a large scale, individual microorganisms can be aggregated and multiplied into large-scale biofilms to improve biological transformation efficiency. (3).
  • new composite living biological materials can be prepared through microbial grafting/curing technology. Its advantages are: it can immobilize effective functional bacteria and promote biological The directional propagation of the membrane maintains the biological activity of functional bacteria; at the same time, the recovery, activation and reuse of active bacteria can be achieved through composite curing materials, thereby reducing treatment costs.
  • the technical solution adopted by the present invention is as follows: a preparation method of live bacteria composite biological materials used to repair Cr(VI) contaminated water bodies, including the following specific steps:
  • Modification of live bacteria Select electroactive bacteria with the ability to reduce Cr(VI) as the model strain, conduct pre-culture, and then centrifuge to obtain expanded cells; in an anaerobic environment, first culture the expanded cells First resuspend in bis[tris(hydroxymethyl)aminopropane] buffer and then blow with nitrogen; then perform surface modification and hybrid assembly in a mixed system of polydopamine and amino-modified carbon dots; and then use bis[tris(hydroxymethyl)aminopropane] base) aminopropane] buffer, and centrifuged to obtain hybrid strain cells;
  • Hybrid strain/modified sponge immobilization Configure a mixture of sodium lactate + bis[tris(hydroxymethyl)aminopropane] + polyethylene glycol as the outer embedding agent; under anaerobic nitrogen atmosphere, The hybrid strain obtained in step (2) is used as the core-embedded bacterial agent, and multi-dimensional heterojunction grafting is achieved through the outer layer of embedding agent and the modified sponge obtained in step (1) to obtain a solidified viable bacterial composite material.
  • step (1) the process of in-situ surface modification of the melamine sponge includes the following steps: first, rinse and dry the melamine sponge with sterilized alcohol and deionized water; then, fully soak the obtained melamine sponge in the modified loaded polypyrrole. solution to fully absorb the polypyrrole; then, add ferric chloride solution to the above system and stir thoroughly in an ice water bath; after the reaction of the above system is completed, take it out and wash it with deionized water until the rinse solution is colorless until.
  • the polypyrrole solution uses deionized water as a solvent, and contains a polypyrrole monomer mass fraction of 0.37% to 1.48%.
  • step (1) vacuum drying involves placing the cleaned modified melamine sponge in a vacuum drying box.
  • the drying conditions were set to 50 oC and the drying time was 12 h.
  • the electroactive bacteria with the ability to reduce Cr(VI) are Shewanella , such as Shewanella oneidensis MR-1, Shewanella xiamenensis BC01 and Shewanella putrefaciens CN32, etc.
  • step (2) amino-modified carbon dots are added to the petri dish used for pre-culture.
  • step (2) the concentration of polydopamine solution used for in-situ hybrid assembly is 4 g/L.
  • the surface modification and hybrid assembly process was carried out under a shaker, the culture parameters were set to 200 rpm, the temperature was 30°C, the time was set to 2 h, and aerobic conditions were used; the centrifugal parameters for the isolation of hybrid strains were set is 5000 rpm and 3 min.
  • the mass ratio of bis[tris(hydroxymethyl)aminopropane], sodium lactate, and polyethylene glycol is 198:157 ⁇ 314:2100 ⁇ 5600.
  • the present invention utilizes modified melamine sponge as a fixed scaffold for grafting and immobilizing live bacteria, by treating electroactive bacteria with Cr(VI) reducing ability (
  • the entire preparation process is simple, can be recycled and reused in the application process of chromium removal from water, and has good performance, providing a new idea for strengthening the application of microorganisms in chromium removal.
  • the present invention achieves large-scale biological control by grafting and fixing electroactive Shewanella heterojunctions with the ability to reduce Cr(VI) on melamine sponges.
  • the in-situ grafting, assembly and fixation of the membrane on the composite material avoids the shortcomings of traditional microbial chromium removal technology that requires the addition of excessive microbial agents and the difficulty of recycling the agents;
  • On the microscale of viable bacteria composite materials By adding polypyrrole in-situ polymerization assembly modification, the active contact sites and electronic conductivity between the living bacteria and the melamine sponge skeleton were greatly improved, thereby effectively improving the reduction catalytic efficiency of Cr(VI); 3.
  • the amino-modified carbon dots not only improve the robustness and metabolic activity of microorganisms, but also the amino-modified carbon dots show certain reducing properties and can provide more reducing power for reductive chromium removal; 4.
  • Composite materials in There is no need to add excessive sodium lactate as a carbon source during use to maintain the growth of microorganisms. It can be used for the catalysis of visible light, maximizing the use of light energy, and generating photoelectrons and amino-modified carbon dots with reducing properties through photocatalysis to remove Cr. (VI) Provide more reducing power; 5. It is easy to recycle and can be directly clamped and separated from the liquid. The separated living materials can be directly added to the next reaction system after using deionized water to remove surface impurities. .
  • Figure 1 is a schematic diagram of the microstructure of a living bacteria composite biomaterial used to remediate Cr(VI) contaminated water;
  • 2 is a polypyrrole-modified melamine sponge
  • 3 is a living hybrid Shewanella cell that can reduce Cr(VI);
  • Figure 2 is a flow chart of the preparation process of live bacteria composite biomaterials used to remediate Cr(VI) contaminated water
  • Oxidation reaction of solution 2-4. Separation, cleaning and drying, 3. Living hybrid Shewanella cells that can reduce Cr(VI), 3-1. Activation and aerobic expansion of bacterial strains, 3- 2. Centrifuge, remove the supernatant and resuspend, 3-3. Add polydopamine and amino-modified carbon dots for external hybridization, 3-4. Aerobic culture, 3-5. Centrifuge, clean and Collection, 4. Configuration of the outer layer of embedding agent, 5. Immobilized viable bacteria composite material, 5-1. Resuspension of living hybrid bacteria, 5-2. Grafting of hybrid bacterial solution on modified melamine sponge, 5-3. Aerobic culture, cleaning, separation and preservation of composite materials.
  • the present invention provides a living bacteria composite biological material for repairing Cr(VI)-polluted water bodies. As shown in Figure 1, it includes a polypyrrole-modified melamine sponge and a living hybrid Shewanella that can reduce Cr(VI). Bacterial cells, living hybrid Shewanella cells that can reduce Cr(VI).
  • the melamine sponge was cut into small cylindrical strips with a specification of ⁇ 10 mm ⁇ H40 mm, rinsed twice with deionized water and 70 wt% ethanol, and vacuum dried at 100°C for later use.
  • 0.15 ⁇ 0.60 mL of polypyrrole monomer standard was dissolved in 40 mL of deionized water, and dispersed by ultrasonic vibration to obtain a mixed solution containing polypyrrole monomer with a mass fraction of 0.37% ⁇ 1.48%.
  • the wild-type electroactive Shewanella with Cr(VI) reduction ability was selected as the model strain (such as Shewanella oneidensis MR-1, Shewanella xiamenensis BC01 and Shewanella putrefaciens CN32). 200 mL of wild-type strains were taken from the frozen tube of bacterial storage and aerobically cultured for a period of time in 20 mL of modified LB medium (0.102 ⁇ 0.204 mL of 10.0 g/L amino-modified carbon dot stock solution was added to the LB medium, so that the concentration of amino-modified carbon dots in the modified medium was 50 ⁇ 100 mg/L).
  • the aerobic culture conditions were set as follows: shaking culture, shaking speed of 200 rpm, culture for 16 h, and culture temperature of 30 °C.
  • the resuspension conditions were nitrogen anaerobic aeration blowing for 30 min.
  • the grafting of the modified melamine sponge obtained in (1) and (2) above with the hybrid strain is assembled by adding an outer layer embedding agent.
  • the living hybrid Cr(VI)-reducing Shewanella cells obtained in (2) are resuspended in the outer layer embedding agent obtained in (3) above, so that the OD 600 in the mixed system is roughly maintained between 0.8 and 1.2, and the resuspended system is transferred to a 100 mL sterilized conical flask.
  • More than 6 modified melamine sponges obtained in (1) are transferred to the conical flask culture system, and the modified melamine sponges are squeezed with sterilized polytetrafluoroethylene tweezers to allow the sponges to fully absorb the resuspended bacterial solution.
  • the conical flask is transferred to a shaker for aerobic culture.
  • the shaker culture conditions are set as follows: rotation speed 150 rpm, aerobic culture 5 h, and culture temperature 30 °C.
  • 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 stored in a 50 mL sterile centrifuge tube for subsequent use in removing Cr(VI) from wastewater.
  • step (1) 0.15 mL, 0.45 mL and 0.60 mL of polypyrrole monomer standard are respectively added for modified melamine sponge; step (2) Shewanella oneidensis MR-1 was selected as the model strain for Cr(VI) reduction, and 0.2 mL of amino-modified carbon dot stock solution with a concentration of 10.0 g/L was added to the modified LB medium; after resuspension, anaerobically injected with a concentration of 0.4 mL It is a 10.0 g/L stock solution of amino-modified carbon dots; in step (3), add 0.30 g sodium lactate and 4.3 g polyethylene glycol powder particles for the preparation of the outer embedding agent; finally prepare according to step (4) to obtain Corresponding live bacteria composite biomaterials.
  • the dosage of other reagents is the same as the dosage of other reagents described in steps (1) to (4).
  • the prepared living bacteria composite biological materials were numbered as material 1, material 2 and material 3 in sequence.
  • a 100 mL anaerobic serum bottle contains 80 mL of Cr(VI)-contaminated wastewater with an initial concentration of 50 mg/L.
  • the above three composite materials were added to the reaction system.
  • the reaction temperature is 25 ⁇ 30 °C, in a dark environment, deoxidize with nitrogen for 20 minutes before the reaction and then seal.
  • Five sampling time points were randomly set during the reaction process, and a 2 mL liquid sample was extracted from the system to monitor the chromium removal efficiency in wastewater.
  • the concentration of residual Cr(VI) in the waste liquid is ⁇ 0.01 mg/L
  • take out the composite material rinse it with distilled water 2 to 3 times, and follow the same steps above to clean the composite material again.

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Abstract

一种用于修复Cr（VI）污染水体的活菌复合生物材料的制备方法，将改性三聚氰胺海绵作为所述用于固定活菌所用的复合材料支架，通过添加聚吡咯进行原位修饰改性；以具有Cr（VI）还原能力的电活性菌作为模式菌株，通过氨基修饰碳点与聚多巴胺的原位组装得到杂合菌株作为所述改性海绵的内核包埋菌剂；通过乳酸钠、聚乙二醇与双[三（羟甲基）氨基丙烷]的混合液作为外层包埋剂将所述的内核包埋菌剂异质结接枝到所述的改性三聚氰胺海绵上，制得活菌复合材料。利用改性三聚氰胺海绵作为活菌材料的固定支架，通过电活性菌株的修饰，并将其与改性海绵进行原位组装，整个制备过程简单，具有绿色、环保等性能。

Description

一种用于修复Cr(VI)污染水体的活菌复合生物材料的制备方法 技术领域
本发明涉及Cr(VI)污染水体的修复技术领域，具体涉及一种用于修复Cr(VI)污染水体的活菌复合生物材料的制备方法。
 背景技术
铬(Cr)是国家严格控制的五种重金属污染物之一，尤其是在电镀和制革等产业中，废水中的高浓度铬是最典型难被控制的污染物代表之一，严重污染了区域的水体及底泥等周边生态环境。Cr在自然环境中主要以Cr(VI)和Cr(III)形式存在。其中，Cr(VI)毒性高且易溶解，而Cr(III)毒性低并易形成难溶沉淀。因此，将高毒易溶的Cr(VI)转化为难溶低毒的Cr(III)从而进行再分离是现阶段普遍认可的实现水体或者底泥除铬的一种有效途径。现阶段，采用电解法、化学法、离子交换法及微生物还原法被认为是水体除铬的有效方法。相比较电解法和离子交换法，处理成本高；虽然添加化学药剂能高效还原去除Cr(VI)，但过量消耗药剂往往容易造成二次污染。因此，考虑到低廉的经济处理成本和可观的处理效果，利用微生物还原Cr(VI)逐渐备受关注。
微生物具有繁殖能力快、反应速率高及环境耐受性强等特征。利用微生物来修复含Cr(VI)污染的水体是一种有效、绿色、廉价可行的修复技术，可最大限度地降低修复时对环境的扰动，对治理Cr(VI)污染的水体有一定应用前景。希瓦氏菌(
Shewanella
)是典型的异化Cr(VI)还原模式微生物，在土壤及水体广泛分布。为确保电子受体Cr(VI)彻底还原，传统的微生物还原Cr(VI)多数需要通过微生物氧化分解乳酸来提供生物源电子。但是，若通过添加大量乳酸来维持微生物还原Cr(VI)反而又增大了处理成本。因此，如何扩充Cr(VI)在微生物还原过程中的电子通量并同时提升电子传递速率是现阶段微生物还原除铬的主要瓶颈。再有，由于微生物个体细胞小并且易分散，在高污染的环境介质中的抵抗能力有限。倘若通过单纯地依靠接种功能活性菌来实现对污染物的转化去除，其处理效率较低。另外，为了提升净化效果还需频繁地投加菌剂，这将极大程度地增加处理成本。
 技术问题
为推动微生物在水体除铬的应用，针对现有技术所存在的问题，本发明提供一种用于修复Cr(VI)污染水体的活菌复合生物材料的制备方法，其主要在以下三方面作了优化与改进：(1). 在微小尺度上，可通过对功能微生物的个体细胞的胞内和胞外界面层进行修饰，提升非生物/生物接口界面层间的电子传递效能并强化微生物个体细胞的抵抗耐受性。(2). 在大尺度上，可通过个体微生物的聚集性繁殖成大尺度的生物膜，以提升生物的转化效率。(3). 为促进功能活性菌的回收再利用，可通过微生物接枝/固化技术手段，制备出新型的复合活体生物材料，其优点表现在：能将有效功能菌实现固定化，促进了生物膜的定向繁殖，从而保持了功能菌的生物活性；同时又可通过复合固化材料实现对活性菌的回收及活化再利用，从而降低处理费用。
 技术解决方案
本发明所采取的技术方案如下：一种用于修复Cr(VI)污染水体的活菌复合生物材料的制备方法，包括以下具体步骤：
（1）活菌复合材料支架的制备：以三聚氰胺海绵作为活菌复合材料的主要支架，添加聚吡咯进行原位表面修饰，真空干燥后得到改性海绵；
（2）活菌修饰：选择对Cr(VI)具有还原能力的电活性细菌为模式菌株，进行预培养，之后离心获得扩培后的细胞；在厌氧环境下，先将扩培得到的细胞先重悬浮于双[三(羟甲基)氨基丙烷]缓冲液后氮吹；再依次在聚多巴胺及氨基修饰碳点混合体系中进行表面修饰及杂合组装；随后用双[三(羟甲基)氨基丙烷]缓冲液冲洗，离心分离得到杂合菌株细胞；
（3）杂合菌株/改性海绵固定化：配置乳酸钠+双[三(羟甲基)氨基丙烷]+聚乙二醇的混合液作为外层包埋剂；在厌氧氮气氛围下，将步骤（2）所得的杂合菌株作为内核包埋菌剂，通过外层包埋剂与步骤（1）所得的改性海绵实现多维化异质结接枝得到固化活菌复合材料。
步骤（1）中，对三聚氰胺海绵进行原位表面修饰的过程包括以下步骤：首先，将三聚氰胺海绵经过消毒酒精和去离子水漂洗干燥；之后，将所得三聚氰胺海绵充分浸泡在的修饰负载的聚吡咯溶液中，使其充分吸收聚吡咯；之后，往以上体系加入氯化铁溶液，并且在冰水浴中充分搅拌；待以上体系反应结束后，取出，用去离子水清洗，直至漂洗液为无色为止。
所述的聚吡咯溶液是以去离子水作为溶剂，含聚吡咯单体质量分数为0.37%~1.48%。
步骤（1）中，真空干燥为将清洗后的改性三聚氰胺海绵置于真空干燥箱中。所述的干燥条件设定为50 ºC，干燥时间为12 h。
对Cr(VI)具有还原能力的电活性细菌为希瓦氏菌( Shewanella) ，如 Shewanella oneidensis MR-1、 Shewanella xiamenensis BC01与 Shewanella putrefaciens CN32等。
步骤（2）中，预培养所采取的培养皿中添加有氨基修饰碳点。
步骤（2）中，用于原位杂化组装的聚多巴胺溶液浓度为4 g/L。
所述表面修饰及杂合组装过程在摇床下进行，培养参数设定为200 rpm，温度为30 ℃，时间设定为2 h，有氧条件；所述对杂合菌株分离的离心参数设定为5000 rpm与3 min。
所述的外层包埋剂中，双[三(羟甲基)氨基丙烷]、乳酸钠、聚乙二醇的质量比为198：157~314：2100~5600。
本发明利用改性三聚氰胺海绵作为接枝固定活菌的固定支架，通过对具有Cr(VI)还原能力的电活性菌(
Shewanella
属)进行修饰得到杂合菌株并作为活菌复合材料的内核包埋菌剂，通过添加外层包埋剂与交联固定剂实现杂合菌株与改性海绵的原位组装。整个制备过程简单，在用于水体除铬的应用过程中可实现重复回收再利用，并且性能良好，为强化微生物在除铬的治理应用提供了新的思路。
 有益效果
与现有技术相比，本发明具有以下优点：1. 本发明通过将具有还原Cr(VI)能力的电活性希瓦氏菌异质结接枝固定在三聚氰胺海绵上，实现了大尺度的生物膜在复合材料上的原位接枝、组装与固定，规避了传统微生物法除铬技术中需通过添加过量微生物菌剂并且菌剂难以再回收的缺陷；2. 在活菌复合材料的微尺度上，通过添加聚吡咯原位聚合组装修饰，大大提升了活菌与三聚氰胺海绵骨架之间的活性接触位点及电子传导能力，从而有效提升了对Cr(VI)的还原催化效率；3. 添加的氨基修饰碳点的碳点不仅提升了微生物的鲁棒性及代谢活力，并且氨基修饰碳点的碳点表现出一定的还原特性可为还原除铬提供更多还原力；4. 复合材料在使用过程中无需过量地投加乳酸钠作为碳源以维持微生物的生长，可借用于可见光的催化作用，最大限度地利用光能，通过光催化产生光电子与具有还原特性的氨基修饰碳点为去除Cr(VI)提供更多的还原力；5. 回收方便，可直接从液体中夹取分离，分离后的活体材料直接用去离子水去除表面的杂质后又可继续投加到下一个反应体系中。
 附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，根据这些附图获得其他的附图仍属于本发明的范畴。
图1为用于修复Cr(VI)污染水体的活菌复合生物材料的微观结构示意图；
图中，2为聚吡咯修饰的三聚氰胺海绵；3为活体杂合型可还原Cr(VI)的希瓦氏菌细胞；
图2为用于修复Cr(VI)污染水体的活菌复合生物材料的制备过程流程图；
图中，1. 三聚氰胺海绵支架、1-1. 支架尺寸切割、1-2. 漂洗、干燥与保存、2. 聚吡咯修饰的三聚氰胺海绵、2-1. 聚吡咯溶液的配置、2-2. 浸泡与充分混合、2-3. FeCl
3
溶液的氧化反应、2-4. 分离、清洗与干燥、3. 活体杂合型可还原Cr(VI)的希瓦氏菌细胞、3-1. 菌种的活化与有氧扩培、3-2. 离心、去上清液与重悬浮、3-3. 加聚多巴胺与氨基修饰的碳点进行外杂合、3-4. 有氧培养、3-5. 杂合细胞的离心、清洗与收集、4. 外层包埋剂的配置、5. 固定化活菌复合材料、5-1. 活体杂合菌的重悬浮、5-2. 杂合菌液在改性三聚氰胺海绵的接枝、5-3. 复合材料的有氧培养、清洗、分离与保存。
 本发明的最佳实施方式
>
为使本发明的目的、技术方案和优点更加清楚，下面将结合附图对本发明作进一步地详细描述。
本发明提供一种用于修复Cr(VI)污染水体的活菌复合生物材料，如图1所示，其包括聚吡咯修饰的三聚氰胺海绵和活体杂合型可还原Cr(VI)的希瓦氏菌细胞，活体杂合型可还原Cr(VI)的希瓦氏菌细胞。
上述的用于修复Cr(VI)污染水体的活菌复合生物材料的具体制备过程如图2所示，以一些具体实施例为例，包括以下步骤：
（1）固定活菌的改性三聚氰胺海绵的制备
首先，将三聚氰胺海绵切割成规格为Φ10 mm ´ H40 mm的小圆柱条，用去离子水和70 wt%的乙醇漂洗两次后并于100 ℃真空干燥后以备用。在200 mL的烧杯中，将0.15 ~ 0.60 mL的聚吡咯单体标准品依次溶解在40 mL的去离子水中，超声震荡分散得到含聚吡咯单体质量分数为0.37%~1.48%的混合溶液。将已切割成型的三聚氰胺海绵圆柱条浸泡以上不同浓度的聚吡咯溶液，用聚四氟镊子反复挤压使海绵条充分吸收聚吡咯溶液。待海绵充分吸收聚吡咯溶液后，往上述体系中添加40 mL含0.18 mol/L的三氯化铁溶液，将以上混合体系在冰水浴环境下磁力充分搅拌4 h。待搅拌结束后，把被聚吡咯负载得到的三聚氰胺海绵取出，用去离子水反复清洗5次以上，直至漂洗液为无色，将清洗后的改性三聚氰胺海绵置于真空干燥箱中。所述的干燥条件设定为50 ºC，干燥时间为12 h。
（2）可还原Cr(VI)的野生型希瓦氏菌细胞的修饰及杂合
选择具有Cr(VI)还原能力的野生型电活性 Shewanella属为模式菌株(如 Shewanella oneidensis MR-1、 Shewanella xiamenensis BC01与 Shewanella putrefaciens CN32等可作为供用菌种)。从菌保藏冷冻管种吸取200 mL野生型菌种，先在20 mL的改性LB培养基(在LB培养基中添加0.102~0.204 mL浓度为10.0 g/L的氨基修饰碳点储备液，使得改性培养基中氨基修饰碳点浓度为50~100 mg/L)中有氧连续培养一段时间。有氧培养条件设定为：摇床培养，摇床转速200 rpm，培养16 h，培养温度30 ℃。将上述扩培得到的菌液在5000 rpm下离心5 min，去除上清液，将所获得的菌体细胞在1.0 mL的10 mmol/L(pH=7.0~7.2)的双[三(羟甲基)氨基丙烷]缓冲液中进行重悬浮。重悬浮条件采用氮气厌氧曝气吹30 min。重悬浮结束后，在厌氧氮气曝气的环境下继续往重悬浮体系中厌氧注射1 mL浓度为4.0 g/L的聚多巴胺溶液与0.3~0.6 mL的浓度为10.0 g/L的氨基修饰碳点储备液。将以上获得的杂合菌液继续在有氧连续培养一段时间。所述的有氧培养条件设定为：摇床培养，摇床转速200 rpm，培养2 h，培养温度30 ℃。待培育结束后，将菌体在5000 rpm下离心3 min后，去除上清液后得到杂合菌株细胞，用10.0 mL的10 mmol/L(pH=7.0~7.2)的双[三(羟甲基)氨基丙烷]缓冲液重复冲洗分离后的杂合细胞2次，再二次离心分离5000 rpm下离心3 min，得到杂合菌株细胞以备用。
（3）外层包埋剂的制备
在70 mL无菌水，分别添加0.198 g双[三(羟甲基)氨基丙烷]、0.157~0.314 g乳酸钠、2.1~5.6 g聚乙二醇粉末颗粒溶解于其中，超声震荡5 min后制得用于将(2)制得的活体杂合型可还原Cr(VI)的希瓦氏菌细胞固定于(1)制得的改性三聚氰胺海绵支架上的外层包埋剂。
（4）杂合菌株在改性三聚氰胺海绵上的接枝固定
上述(1)和(2)制得改性三聚氰胺海绵与杂合菌株的接枝是通过添加外层包埋剂组装。将(2)制得的活体杂合型可还原Cr(VI)的希瓦氏菌细胞重悬浮在以上(3)得到的外层包埋剂中，使得混合体系中的OD 600大致维持在0.8~1.2 之间，将该重悬浮体系转入100 mL灭菌过的锥形瓶。将6个以上(1)得到的改性三聚氰胺海绵转入到该锥形瓶培养体系中，使用灭菌过的聚四氟镊子挤压改性三聚氰胺海绵，使海绵充分吸收重悬浮的菌液。待改性三聚氰胺海绵充分吸收菌液后，将锥形瓶转入到摇床有氧培养。摇床培养条件设定为：转速150 rpm，有氧培养5 h，培养温度30 ℃。待培养结束后，将被菌液接枝后的三聚氰胺海绵从锥形瓶中分离取出，用浓度为10 mmol/L双[三(羟甲基)氨基丙烷]溶液冲洗3次后，保存在50 mL无菌离心管中，以用于后续废水除Cr(VI)中使用。
本发明的一些具体实施例为根据以上所述的准备方法，在步骤（1）中依次分别添加0.15 mL、0.45 mL与0.60 mL聚吡咯单体标准品用于改性三聚氰胺海绵；步骤（2）中选择 Shewanella oneidensis MR-1为Cr(VI)还原的模式菌株，改性LB培养基中添加0.2 mL浓度为10.0 g/L的氨基修饰碳点储备液；重悬浮后，厌氧注射0.4 mL浓度为10.0 g/L的氨基修饰碳点储备液；步骤（3）中添加0.30 g乳酸钠与4.3 g聚乙二醇粉末颗粒用于外层包埋剂的制备；最后根据步骤（4）制备分别得到对应的活菌复合生物材料。除了以上提及到具体特殊使用试剂或者菌种的对应具体用量（浓度与提及），如果无特殊说明，其他试剂的用量都与步骤（1）~（4）所述的其他试剂用量相同。最后，将制备得到的活菌复合生物材料依次编号为材料1、材料2与材料3。
表1 活菌复合生物材料用于废水除铬的催化性能及其可回收利用性能评价
在100 mL厌氧血清瓶中装有体积80 mL且含有初始浓度为50 mg/L的Cr(VI)污染废水，分别将以上3种复合材料添加到反应体系中。反应温度为25~30 ℃，黑暗环境下，反应前用氮气脱氧20 min后进行密封。反应过程中随机设置5个取样时间点，从体系中抽取2 mL的液体样品用于监测废水中的除铬效率。待反应完全结束后（废液中所残余Cr(VI)浓度<0.01 mg/L），将复合材料给取出来，用蒸馏水冲洗2~3次后，按照以上相同步骤再次将清洗后的复合材料又依次投加到相同的反应体系中进行循环可回收实验，评价材料的可回收性能及其催化效率。如表1所示，3种材料经循环4次后仍表现出良好的催化性能，催化效率均达到100%；用更大剂量的聚吡咯单体修饰得到的复合材料的催化性能更加优越，催化反应的时间越短，反应时间维持在18~36 h。
 以上所揭露的仅为本发明较佳实施例而已，当然不能以此来限定本发明之权利范围，因此依本发明权利要求所作的等同变化，仍属本发明所涵盖的范围。

Claims (9)

  1. 一种用于修复Cr(VI)污染水体的活菌复合生物材料的制备方法，其特征在于包括以下具体步骤：
    （1）活菌复合材料支架的制备：以三聚氰胺海绵作为活菌复合材料的主要支架，添加聚吡咯进行原位表面修饰，真空干燥后得到改性海绵；
    （2）活菌修饰：选择对Cr(VI)具有还原能力的电活性细菌为模式菌株，进行预培养，之后离心获得扩培后的细胞；在厌氧环境下，先将扩培得到的细胞先重悬浮于双[三(羟甲基)氨基丙烷]缓冲液后氮吹；再依次在聚多巴胺及氨基修饰碳点混合体系中进行表面修饰及杂合组装；随后用双[三(羟甲基)氨基丙烷]缓冲液冲洗，离心分离得到杂合菌株细胞；
    （3）杂合菌株/改性海绵固定化：配置乳酸钠+双[三(羟甲基)氨基丙烷]+聚乙二醇的混合液作为外层包埋剂；在厌氧氮气氛围下，将步骤（2）所得的杂合菌株作为内核包埋菌剂，通过外层包埋剂与步骤（1）所得的改性海绵实现多维化异质结接枝得到固化活菌复合材料。
  2. 根据权利要求1所述的用于修复Cr(VI)污染水体的活菌复合生物材料的制备方法，其特征在于：步骤（1）中，对三聚氰胺海绵进行原位表面修饰的过程包括以下步骤：首先，将三聚氰胺海绵经过消毒酒精和去离子水漂洗干燥；之后，将所得三聚氰胺海绵充分浸泡在的修饰负载的聚吡咯溶液中，使其充分吸收聚吡咯；之后，往以上体系加入氯化铁溶液，并且在冰水浴中充分搅拌；待以上体系反应结束后，取出，用去离子水清洗，直至漂洗液为无色为止。
  3. 根据权利要求2所述的用于修复Cr(VI)污染水体的活菌复合生物材料的制备方法，其特征在于：所述的聚吡咯溶液是以去离子水作为溶剂，含聚吡咯单体质量分数为0.37%~1.48%。
  4. 根据权利要求2所述的用于修复Cr(VI)污染水体的活菌复合生物材料的制备方法，其特征在于：步骤（1）中，真空干燥为将清洗后的改性三聚氰胺海绵置于真空干燥箱中。所述的干燥条件设定为50 ºC，干燥时间为12 h。
  5. 根据权利要求1所述的用于修复Cr(VI)污染水体的活菌复合生物材料的制备方法，其特征在于：对Cr(VI)具有还原能力的电活性细菌为希瓦氏菌( Shewanella)。
  6. 根据权利要求1所述的用于修复Cr(VI)污染水体的活菌复合生物材料的制备方法，其特征在于：步骤（2）中，预培养所采取的培养皿中添加有氨基修饰碳点。
  7. 根据权利要求1所述的用于修复Cr(VI)污染水体的活菌复合生物材料的制备方法，其特征在于：步骤（2）中，用于原位杂化组装的聚多巴胺溶液浓度为4 g/L。
  8. 根据权利要求1所述的用于修复Cr(VI)污染水体的活菌复合生物材料的制备方法，其特征在于：所述表面修饰及杂合组装过程在摇床下进行，培养参数设定为200 rpm，温度为30 ℃，时间设定为2 h，有氧条件；所述对杂合菌株分离的离心参数设定为5000 rpm与3 min。
  9. 根据权利要求1所述的用于修复Cr(VI)污染水体的活菌复合生物材料的制备方法，其特征在于：所述的外层包埋剂中，双[三(羟甲基)氨基丙烷]、乳酸钠、聚乙二醇的质量比为198：157~314：2100~5600。
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