Method for accelerating microbial reduction of selenite by immobilized riboflavin

Abstract

The invention belongs to the field of environmental biotechnology and nano material application, and relates to a method for accelerating microorganisms to reduce selenite based on immobilized riboflavin. Aiming at the problems of poor stability, difficulty in recovery, non-repeated use and the like of dissolved riboflavin serving as an electron shuttle, riboflavin is grafted on the surface of a multi-walled carbon nanotube through a covalent binding method, and immobilized riboflavin capable of being recycled is constructed. The material can accelerate extracellular electron transfer of shewanella MR-1, and toxic sodium selenite is efficiently reduced into low-toxicity or non-toxic elemental selenium. Results show that the immobilized riboflavin not only maintains high reduction activity of riboflavin, but also has excellent cyclic utilization performance, still maintains high reduction rate after being repeated for multiple times, and effectively reduces operation cost and resource loss. According to the invention, an efficient, economical and sustainable application scene is provided for a microorganism-electron shuttle combined remediation technology, and a new thought is also provided for application of a functional nano material in an environmental biotechnology.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of environmental biotechnology and nanomaterial application, and specifically relates to a method for accelerating microbial reduction of selenite based on multi-walled carbon nanotube immobilized riboflavin. TECHNICAL BACKGROUND

[0002] Selenium is an important metalloid element, widely used in electronics, glass, metallurgy, chemical industry, agriculture and food industry. With the increasing industrial activities, the discharge of selenium-containing wastewater has become increasingly prominent. Selenium in wastewater mainly exists in the form of selenate and selenite, among which selenite is the focus of selenium pollution control due to its high water solubility, strong mobility and high biological toxicity. Excessive discharge of selenium not only poses a serious threat to the environment and ecological system, but also may have an impact on human health through the food chain, causing damage to the nervous system and even increasing the risk of cancer. Therefore, developing efficient, low-cost and environmentally friendly treatment technologies for selenium-containing wastewater has become an important research direction in the field of environmental protection.

[0003] Currently, the main treatment technologies for selenium-containing wastewater include adsorption, coagulation sedimentation, membrane separation, chemical reduction and electrochemical reduction. Although the above technologies are widely used, they still have many shortcomings: adsorption and coagulation sedimentation methods are prone to secondary pollution, membrane separation treatment costs are high, and chemical reduction and electrochemical reduction methods require additional catalysts or external electric fields, resulting in complex systems and high energy consumption. In contrast, the microbial method uses the metabolic action of microorganisms to reduce high-toxicity oxidized selenium to low-toxicity and insoluble elemental selenium, which has the advantages of being green, environmentally friendly and low-cost. However, the reduction process of the microbial method is relatively slow and mostly occurs intracellularly. Due to the high toxicity of selenite, it can easily inhibit microbial activity and even cause cell death, severely affecting the reduction efficiency. Therefore, improving the electron transfer efficiency in the microbial treatment process is the key to improving the reduction rate and system stability.

[0004] To enhance the extracellular electron transfer efficiency, researchers have proposed introducing electron shuttles in biological systems. Electron shuttles are a class of substances with redox activity that can accelerate the transfer of extracellular electrons, reduce the biological toxicity of selenite, and improve the reduction rate. Common electron shuttles include riboflavin, anthraquinone 2, 6-disulfonic acid sodium and humic acid, but such substances are expensive and have a soluble structure, which can flow out with the effluent in continuous operation, requiring continuous addition, significantly increasing the operating cost. In addition, low concentrations of electron shuttles have limited effect, while high concentrations may cause secondary pollution. Therefore, developing a fixed and recyclable electron shuttle system is the key to improving the efficiency and economy of the microbial method.

[0005] In this context, the present application proposes to use multi-walled carbon nanotubes as carrier materials, and to fix riboflavin on the surface thereof by covalent bonding to construct a stable, efficient and recyclable immobilized electron shuttle system. Multi-walled carbon nanotubes have high specific surface area, good electrical conductivity and chemical stability, which can significantly improve the efficiency of microbial extracellular electron transfer, and at the same time have good mechanical strength and repeated use performance. This strategy not only solves the problem of difficult recovery of traditional dissolved electron shuttles, but also significantly improves the reduction capacity of microbial system to selenite, providing a new solution for the green and efficient treatment of selenium-containing wastewater. SUMMARY

[0006] The primary purpose of the present application is to overcome the shortcomings of high cost, easy loss and non-recyclability of existing electron shuttles, and to propose a method for accelerating microbial reduction of selenite based on multi-walled carbon nanotube immobilized riboflavin. Compared with direct addition of soluble riboflavin, the immobilized riboflavin prepared by the present application has stable structure and can be reused, and is less likely to cause secondary pollution, and is more suitable for engineering promotion and long-term operation.

[0007] To achieve the above-mentioned purpose, the present application provides a method for accelerating microbial reduction of selenite by immobilized riboflavin, which specifically comprises the following steps:

[0008] (1) Bacterial culture: Shewanella MR-1 is inoculated into LB liquid medium, and cultured to the logarithmic growth phase, and the bacterial cells are collected to obtain a bacterial suspension;

[0009] (2) Preparation of immobilized riboflavin: riboflavin and acidified multi-walled carbon nanotubes are subjected to oil bath reaction, and a protective agent and a catalyst are added to make the reaction more complete, and after the reaction, suction filtration and drying are performed to obtain immobilized riboflavin powder;

[0010] (3) Construction of reaction system: the bacterial suspension obtained in step (1) is inoculated into a serum bottle containing a basic salt culture solution, and an electron donor, a selenite solution and the immobilized riboflavin powder obtained in step (2) are added, and N2 is blown to create anaerobic conditions, and then the serum bottle is sealed and cultured.

[0011] (4) Reaction and sampling: the serum bottle in step (3) is incubated at a constant temperature with shaking, and samples are taken at regular intervals, and after centrifugation and filtration separation, the concentration of selenite is detected.

[0012] Preferably, the buffer solution for collecting bacterial cells in step (1) is physiological saline, the pH of the LB liquid medium used in step (1) is 6.0-8.0, and the pH of the basic salt culture solution in step (3) is 6.0-8.0.

[0013] Preferably, the acidification conditions of the multi-walled carbon nanotubes in step (2) are mixed acid acidification, and the volume ratio of sulfuric acid to nitric acid is 3:1-1:3.

[0014] Preferably, the concentration of the mixed acid in step (2) is 1 g: 0.5-1 L, and the oil bath condition is 60-100 o C, and the time is 2-20 h.

[0015] Preferably, in step (2), the riboflavin is grafted to the acidified multi-walled carbon nanotubes through an amidation reaction, a hydroxyl protecting agent triphenylchloromethane is added, the concentration of the triphenylchloromethane is 1-5 M, the catalyst is a 4-dimethylaminopyridine and acetonitrile solution, the concentration of the 4-dimethylaminopyridine is 0.5-2 M, and the concentration of the acetonitrile is 10-30%.

[0016] Preferably, in step (2), the reaction temperature is 40-100 o C, the oil bath time is 2-20 h, and the vacuum drying temperature is 60-100 o C, and the time is 8-36 h.

[0017] Preferably, in step (3), the final concentration OD 600 of the bacterial suspension is between 0.5 and 1.5, and the electron donor is sodium lactate, and the concentration of the sodium lactate is 10-40 mM.

[0018] Preferably, in step (3), the dosage of the immobilized riboflavin in the reaction system is 0.5-2 g / L, and the initial concentration of the sodium selenite is 0.2-1 mM.

[0019] Preferably, in step (4), the oscillation condition is 30 o C, the oscillation frequency is 150-200 rmp, the sampling interval is 2-12 h, and after centrifugation, the sample is filtered through a 0.22 µm filter membrane.

[0020] Preferably, the centrifugation uses a high-speed refrigerated centrifuge, the speed is 8000 x g, the temperature is 4 o C, and the time is 10 min.

[0021] Compared with the prior art, the method has the following advantages:

[0022] (1) high efficiency: the immobilized riboflavin significantly improves the reduction rate of Shewanella MR-1 to selenite; (2) stability: the material structure is stable, and has good reusability; (3) recyclability: the material is not easy to lose with the effluent, and is easy to separate and recycle, thereby effectively reducing the operation cost; and (4) environmental protection: the material does not need to be frequently added, thereby avoiding the risk of secondary pollution. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 A flow chart for preparing the immobilized riboflavin;

[0024] Figure 2is an FTIR graph of the multi-walled carbon nanotube, riboflavin, acidified multi-walled carbon nanotube and immobilized riboflavin of the present application;

[0025] Figure 3 is an SEM graph of the multi-walled carbon nanotube, acidified multi-walled carbon nanotube and immobilized riboflavin of the present application;

[0026] Figure 4 is a cyclic voltammetry pulse curve of Shewanella MR-1 and Shewanella MR-1+ immobilized riboflavin obtained according to Example 1;

[0027] Figure 5 is an effect graph of the accelerated reduction of selenite by immobilized riboflavin obtained according to Example 1;

[0028] Figure 6 is a reduction effect graph of selenite by immobilized riboflavin used for 5 times according to Example 2. DETAILED DESCRIPTION

[0029] The present application will be further described in detail below with reference to the accompanying drawings and by describing the embodiments, so as to help the skilled in the art to have a more complete, accurate and in-depth understanding of the inventive concept and technical solutions of the present application.

[0030] Example 1:

[0031] The method described in the present example mainly comprises the following steps:

[0032] (1) Cultivation and enrichment of Shewanella MR-1: Shewanella MR-1 strain is purchased from China Microbial Strain Query Network, platform number: Bio-097658. Shewanella MR-1 is inoculated into LB solid culture medium (5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, 15 g / L agar powder), and cultured in a constant temperature incubator at 30 o C for 24 h. The colonies are transferred to LB liquid culture medium (components are the same as above, without agar) for enrichment culture. The initial pH of the culture is 7.0±0.1, and the temperature is 30 o C, and the shaking speed is 180 rmp. When the bacterial solution reaches the logarithmic growth phase (OD 600 ≈1.5), the culture solution is collected and centrifuged (8000×g, 10 min, 4 oC) Collect bacterial cells, wash three times with physiological saline, and resuspend in basal salt culture medium for later use. Basal salt culture medium: 0.46 g / L NaCl, 0.2067 g / L NH4Cl, 0.014 g / L MgCl2, 0.05 mM PBS, and 1 mL / L trace elements (5 mM ZnSO4·6H2O, 5 mM MnCl2·4H2O, 8 mM CoCl2·4H2O, 1 mM CuCl2·2H2O, 1 mM NiCl2·6H2O, 1.5 mM NaMoO4·2H2O).

[0033] (2) Acidification of multi-walled carbon nanotubes: multi-walled carbon nanotubes are reacted with mixed acid (V 硫酸 V 硝酸 The mixture (ratio 1:3) was sonicated in a brown round-bottom flask for 30 min (power 240 W), and then heated to 80°C. o The reaction was carried out under magnetic stirring in an oil bath for 4 hours. The mixture was then cooled to room temperature (25°C). o C) Filter the solution through a 0.45 µm PVDF membrane and wash with deionized water until neutral (pH = 7.0 ± 0.1). Dry in a vacuum dryer at 80 °C. o Acidified multi-walled carbon nanotube powder was obtained by vacuum drying at -0.1 MPa for 24 h.

[0034] (3) Preparation of riboflavin immobilized in multi-walled carbon nanotubes: The acidified multi-walled carbon nanotube powder obtained in step (2) and riboflavin were dissolved in 20% acetonitrile solution at a molar ratio of 2:1. 1 M 4-dimethylaminopyridine was added as a catalyst and 2 M triphenylchloromethane was added as a hydroxyl protectant. The mixture was ultrasonically treated for 30 min (power 240 W) to make the solution uniform. Then, it was heated to 60 °C. o Under C-oil bath conditions, the suspension was stirred magnetically for 16 h, and then cooled to room temperature (25°C). o After C), it is filtered through a 0.45 µm PVDF membrane, washed with pure water until neutral (pH=7.0±0.1), and then dried in a vacuum dryer at 80°C. o Vacuum drying at -0.1 MPa for 24 h yielded multi-walled carbon nanotube-immobilized riboflavin powder. The preparation flowchart is shown below. Figure 1 As shown, its characterization is as follows Figures 2-4 As shown.

[0035] (4) Microbial anaerobic reaction system construction: the bacterial suspension obtained in step (1) was added to a serum bottle containing a basic salt medium. The basic salt medium composition included: 0.46 g / L NaCl, 0.2067 g / L NH4Cl, 0.014 g / L MgCl2, 0.05 mM PBS, and 1 mL / L trace elements (5 mM ZnSO4·6H2O, 5 mM MnCl2·4H2O, 8 mM CoCl2·4H2O, 1 mM CuCl2·2H2O, 1 mM NiCl2·6H2O, 1.5 mM NaMoO4·2H2O). The concentration of the strain in the system was OD600≈1.5, 20 mM sodium lactate was added as an electron donor, 0.5 mM sodium selenite was added as a target pollutant, and 1 g / L of the multi-walled carbon nanotube immobilized riboflavin obtained in step (3) was added. N2 was introduced for 10 min, the dissolved oxygen concentration was controlled to be lower than 0.2 mg / L, and an anaerobic environment was created. 600

[0036] (5) Reaction and detection: the serum bottle in step (4) was placed in a 30 o C constant temperature shaker and cultured (180 rmp) under shaking. The reaction solution was collected periodically (0-72 h), centrifuged (8000xg, 4 o C, 10 min), filtered with a 0.22 µm filter membrane, and the separated solution was detected for selenite content. The detection results are shown in Figure 5

[0037] (6) Results: under the conditions of a selenite concentration of 0.5 mM and a Shewanella MR-1 concentration of OD600≈1.5, the addition of immobilized riboflavin can achieve 100% removal of selenite within 72 h.

[0038] Example 2

[0039] The method described in this example mainly includes the following steps:

[0040] (1) Cultivation and enrichment of Shewanella MR-1: Shewanella MR-1 strain was purchased from China Microbial Strain Query Network, platform number: Bio-097658. Shewanella MR-1 was inoculated into LB solid culture medium (5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, 15 g / L agar powder), and cultured in a constant temperature incubator at 30 o C for 24 h. The colonies were transferred to LB liquid culture medium (components as above, without agar) for enrichment culture. The initial pH of the culture was 7.0±0.1, the temperature was 30 o C, and the shaking speed was 180 rmp. The culture was incubated until the bacterial solution reached the logarithmic growth phase (OD 600 ​​≈1.5), collect the culture solution and centrifuge (8000 x g, 10 min, 4 o C) Collect the bacteria, wash with physiological saline for three times, and resuspend in the base salt culture solution for standby. Base salt culture solution: 0.46 g / L NaCl, 0.2067 g / L NH4Cl, 0.014 g / L MgCl2, 0.05 mM PBS, and 1 mL / L trace elements (5 mM ZnSO4·6H2O, 5 mM MnCl2·4H2O, 8 mM CoCl2·4H2O, 1 mM CuCl2·2H2O, 1 mM NiCl2·6H2O, 1.5 mM NaMoO4·2H2O).

[0041] (2) Collection of immobilized riboflavin: After the end of the shake flask, the solution added with immobilized riboflavin in Example 1 is collected, and the precipitate is collected after centrifugal filtration. Finally, vacuum drying is performed in a vacuum drying machine at 80 o C for 24 h to obtain a powder.

[0042] (3) Construction of a microbial anaerobic reaction system: The bacterial suspension in step (1) is added to 50 mL of base salt medium, and the final concentration of the strain in the system is OD600≈1.5. 20 mM sodium lactate, 0.5 mM selenite solution, and 1 g / L of immobilized riboflavin collected in step (2) are added to the system. The system is placed in a serum bottle for anaerobic treatment, and the anaerobic bottle is placed in a 30 o C, 180 rpm shaker for culture;

[0043] (4) Reaction and detection: The serum bottle in step (3) is placed in a 30 o C constant temperature shaker for shaking culture (180 rmp), and the reaction solution is collected periodically (0~72 h), centrifuged (8000 x g, 4 o C, 10 min), filtered with a 0.22 µm filter, and the separated solution is detected for selenite content. The selenite content detection results of 5 cycles are shown in Figure 6 .

[0044] (5) Results: Under the conditions of a selenite concentration of 0.5 mM and a Shewanella MR-1 concentration of OD600≈1.5, the removal rate of selenite by immobilized riboflavin can reach 100% within 72 h after 5 cycles of use.

Claims

1. A method for accelerating the microbial reduction of selenite using immobilized riboflavin, characterized in that, It includes the following steps. (1) Bacterial culture: Shewanella MR-1 was inoculated into LB liquid medium and cultured to the logarithmic growth phase. The bacterial cells were collected to obtain a bacterial suspension. (2) Preparation of immobilized riboflavin: Riboflavin was reacted with acidified multi-walled carbon nanotubes in an oil bath. Protective agents and catalysts were added to make the reaction more complete. After the reaction, the mixture was filtered and dried to obtain immobilized riboflavin powder. (3) Construction of reaction system: The bacterial suspension obtained in step (1) was inoculated into a serum bottle containing basic salt culture medium, and electron donors, selenite solution and immobilized riboflavin powder obtained in step (2) were added. After N2 purging to create anaerobic conditions, the system was sealed and cultured. (4) Reaction and sampling: The serum bottle from step (3) was kept at a constant temperature and shaken. Samples were taken periodically, and the concentration of selenite was detected after centrifugation and filtration.

2. The method according to claim 1, characterized in that, The buffer solution for collecting bacterial cells in step (1) is physiological saline, the pH of the LB liquid medium used in step (1) is 6.0~8.0, and the pH of the basal salt culture medium in step (3) is 6.0~8.

0.

3. The method according to claim 1, characterized in that, The acidification conditions for multi-walled carbon nanotubes in step (2) are mixed acid acidification, with a volume ratio of sulfuric acid to nitric acid of 3:1 to 1:

3.

4. The method according to claim 3, characterized in that, In step (2), the mixed acid concentration of the multi-walled carbon nanotubes is 1 g: 0.5~1 L, and the oil bath conditions are 60~100. o C, the time is 2~20 hours.

5. The method according to claim 1, characterized in that, In step (2), riboflavin and acidified multi-walled carbon nanotubes are grafted through an amidation reaction. The hydroxyl protectant triphenylchloromethane is added at a concentration of 1-5 M, and the catalyst is a solution of 4-dimethylaminopyridine and acetonitrile, with the concentration of 4-dimethylaminopyridine being 0.5-2 M and the concentration of acetonitrile being 10-30%.

6. The method according to claim 1, characterized in that, The reaction temperature in step (2) is 40~100℃. o C, oil bath time is 2~20 h, vacuum drying temperature is 60~100℃ o C, the time is 8~36 hours.

7. The method according to claim 1, characterized in that, The final concentration OD of the bacterial suspension described in step (3) 600 The concentration is between 0.5 and 1.5, with sodium lactate as the electron donor and a concentration of 10 to 40 mM.

8. The method according to claim 1, characterized in that, In step (3), the amount of immobilized riboflavin added to the reaction system is 0.5~2 g / L, and the initial concentration of selenite is 0.2~1 mM.

9. The method according to claim 1, characterized in that, The oscillation condition described in step (4) is 30. o C, with an oscillation frequency of 150~200 rmp, sampling interval of 2~12 h, and filtered through a 0.22 µm filter membrane after centrifugation.

10. The method according to claim 9, characterized in that, The centrifugation was performed using a high-speed refrigerated centrifuge at a speed of 8000 × g and a temperature of 4 °C. o C, duration is 10 minutes.

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