CN114906941A - Shewanella-nano material hybrid cell construction method and application thereof - Google Patents
Shewanella-nano material hybrid cell construction method and application thereof Download PDFInfo
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- 238000006731 degradation reaction Methods 0.000 claims abstract description 24
- 230000015556 catabolic process Effects 0.000 claims abstract description 22
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims abstract description 9
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- VCJMYUPGQJHHFU-UHFFFAOYSA-N iron(3+);trinitrate Chemical compound [Fe+3].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O VCJMYUPGQJHHFU-UHFFFAOYSA-N 0.000 claims description 8
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- 235000019345 sodium thiosulphate Nutrition 0.000 claims description 6
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- 239000002054 inoculum Substances 0.000 claims description 4
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- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical compound [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 claims description 4
- NPFOYSMITVOQOS-UHFFFAOYSA-K iron(III) citrate Chemical compound [Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NPFOYSMITVOQOS-UHFFFAOYSA-K 0.000 claims description 4
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- 230000010355 oscillation Effects 0.000 claims description 2
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
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- 241001538194 Shewanella oneidensis MR-1 Species 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
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- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 1
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 1
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- 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
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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
- 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
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- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a construction method and application of Shewanella-nano material hybrid cells. The invention transfers high-activity Shewanella to an anaerobic reaction system, and adds an iron source and a sulfur source to generate the Shewanella-nano material hybrid cell. And transferring the heterozygous cells to an anaerobic degradation reaction solution, and adding methyl violet to obtain a heterozygous cell degradation methyl violet reaction system. The Shewanella-nano material hybrid cell constructed by the invention can continuously output electrons in the metabolic process based on cells, so that the nano material on the surface of the Shewanella-nano material is continuously repaired and regenerated, and high-efficiency catalytic activity can be kept, thereby realizing the recycling of the Shewanella-nano material; the advantages of microbes and the nano material are complemented, the advantages of biological reducing power and strong catalytic performance of the nano material are combined, the loss of the nano material is reduced, and efficient and lasting degradation and decoloration of organic pollutants are realized.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a construction method and application of a Shewanella-nanometer material heterozygous cell.
Background
With the development of dye and printing and dyeing industry, dye wastewater has become one of the most important water pollution sources at present. The waste water has the characteristics of deep color, high Biochemical Oxygen Demand (BOD) and Chemical Oxygen Demand (COD) values, complex and variable composition, large discharge amount, wide distribution range, difficult degradation and the like. If the waste water is directly discharged, serious harm is brought to the ecological environment. Therefore, the search for efficient and economical dye degradation methods becomes a hotspot in the environmental field.
At present, a lot of researches are carried out on the treatment method of printing and dyeing wastewater at home and abroad, and the common methods mainly comprise a coagulating sedimentation method, a chemical oxidation method, an adsorption method, a membrane separation method, a biological method and the like. The chemical flocculation method has the advantages of low investment and high decolorization efficiency, but can generate a large amount of sludge; the chemical oxidation method has high decolorization efficiency but has limited removal: the electrochemical method has the advantages of simple operation, high removal rate, good decolorization, large energy consumption, high cost and side reaction; the adsorption method has reliable operation, good treatment effect and high cost; the membrane separation method has high separation efficiency, but high cost and easy blockage. In the newly emerging field of nano materials, nano ferrous sulfide is widely applied to chromium-containing wastewater treatment due to high catalytic efficiency and strong reducibility. However, the nano ferrous sulfide mainly has the problems of large consumption and high cost of nano materials.
Compared with the traditional method, the biodegradation decoloring method has the characteristics of low cost, environmental friendliness, wide applicable dye range and the like, but has the problem of low degradation efficiency. Based on the method, the novel Shewanella-nano material hybrid cell is constructed, and efficient dye-polluted water body restoration is realized by utilizing the advantage complementation of the microorganism and the nano material.
Disclosure of Invention
Aiming at the defects of the prior art, the Shewanella-nano material hybrid cell is constructed, the advantages of microorganisms and nano materials are combined, the nano materials with high-efficiency catalytic performance are regenerated in situ in real time by utilizing the biological reducibility of the microorganisms, the high-efficiency multi-cycle treatment of organic dyes is realized, and the treatment efficiency of the nano materials of unit mass is improved.
In order to achieve the purpose, the invention adopts the following technical means:
the invention provides a construction method of Shewanella-nano material hybrid cells, which comprises the following steps:
(1) inoculating Shewanella to an LB culture medium, and performing shake culture to obtain a bacterial liquid;
(2) centrifuging the bacterial liquid obtained in the step (1), adding the precipitated bacterial sludge into an anaerobic reaction buffer solution, and adding a water-soluble ferric salt solution and a water-soluble sulfur source salt solution; and (4) performing shake culture to obtain the Shewanella-nano material hybrid cell.
Further, the Shewanella bacteria in step (1) is Shewanella oneidensis MR-1.
The ratio of the Shewanella to the LB culture medium in the step (1) is that the volume ratio of the inoculum size to the LB culture medium is 0.05-0.8: 100.
The conditions of the shaking culture in the step (1) are as follows: the temperature is 20-37 ℃, the oscillation rotation speed is 50-300 rpm, and the time is 10-20 h.
The OD600 value of the bacterial liquid in the step (1) is 0.5-4.
The rotating speed of the centrifugation in the step (2) is 2000-8000 rpm, and the time of the centrifugation is 2-10 min.
The anaerobic reaction buffer solution in the step (2) is prepared by mixing an LB liquid culture medium and an M9 culture medium according to the volume ratio of 1: 4-99, heating and boiling, introducing nitrogen, autoclaving and adding sterile sodium lactate to the final concentration of 0.1-50 mM.
And (3) in the step (2), the final concentration OD600 value of the Shewanella-nanometer material hybrid cell is 0.05-5.
The water-soluble ferric salt in the step (2) is one of ferric chloride, ferric nitrate, ferric sulfate or ferric citrate; the water-soluble sulfur source salt is one of sodium thiosulfate, sodium sulfite, low sodium sulfate or sodium sulfate.
And (3) in the step (2), the final concentrations of the water-soluble ferric salt solution and the water-soluble sulfur source salt solution are both more than 50 mu M.
The conditions of the shaking culture in the step (2) are as follows: the temperature is 4-37 ℃, the rotating speed is 50-300 rpm, and the time is more than 5 h.
Furthermore, the invention also provides the Shewanella-nano material hybrid cell constructed by the method.
Further, the invention also provides application of the Shewanella-nanometer material hybrid cell in dye degradation. The application is degrading organic dye methyl violet.
Compared with the prior art, the invention has the beneficial effects that:
the Shewanella is activated and cultured to obtain high-activity bacteria; transferring the bacteria to an anaerobic reaction system, and adding an iron source and a sulfur source to generate the Shewanella-nano material hybrid cell. And transferring the hybrid cell to an anaerobic degradation reaction solution, and adding methyl violet to obtain a hybrid cell degradation methyl violet reaction system. The invention fully utilizes the reducing power of microorganisms to continuously regenerate the nano material with high-efficiency degradation performance outside cells, and the constructed Shewanella-nano material heterozygous system can continuously output electrons based on cells in the metabolic process, so that the nano material on the surface of the Shewanella-nano material is continuously repaired and regenerated, and high-efficiency catalytic activity can be kept, thereby realizing the reutilization of the Shewanella-nano material; the invention utilizes the advantage complementation of the microorganism and the nano material, combines the advantages of biological reducing power and strong catalytic performance of the nano material, reduces the loss of the nano material, realizes the high-efficiency and durable degradation and decoloration of organic pollutants, and realizes the aim of restoring the dye polluted water body.
Drawings
FIG. 1 is a SEM representation of cells; in the figure, a is a Shewanella SEM electron microscope picture, b is a Shewanella-nano material hybrid cell SEM electron microscope picture;
FIG. 2 is an EDS element characterization diagram;
FIG. 3 is a graph showing the effect of methyl violet degradation;
FIG. 4 is a graph showing the effect of periodic methyl violet degradation of Shewanella-nanomaterial hybrid cells prepared in example 1.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be illustrative of the present invention and are not intended to limit the scope of the present invention. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. The basic reagents and materials described in the examples of the following examples are all available from reagent companies. The experimental procedures, for which specific conditions are not specified, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
Reagent:
LB culture medium: 10 g/L tryptone, 5g/L yeast extract, 5g/L, pH =7 sodium chloride.
M9 medium: na (Na) 2 HPO 4 ·12H 2 O 17.8 g/L、KH 2 PO 4 3 g/L、NaCl 0.5 g/L、NH 4 Cl 10.5 g/L, 10mM sodium lactate, 0.1mM CaCl 2 And 1mM MgSO 4 。
Example 1
(1) Shewanella (Shewanella oneidensis MR-1, purchased from ATCC American type culture Collection, Strain number ATCC 700550) was inoculated into LB medium for culture, and the volume ratio of Shewanella inoculum to LB medium was 0.4: 100, respectively; culturing in a shaking table at 30 ℃ for 12h at a shaking revolution of 200rpm to obtain a bacterial liquid with a substantially smooth surface and no obvious particles;
(2) fully mixing an LB liquid culture medium and an M9 culture medium according to the volume ratio of 20:80, heating and boiling, filling nitrogen, then autoclaving, and then adding sterile sodium lactate to the final concentration of 18 mM to obtain an anaerobic reaction buffer solution; centrifuging the Shewanella bacteria liquid cultured in the step (1) at the rotation speed of 5000rpm for 5min, adding the sediment bacteria mud into 300mL of prepared anaerobic reaction buffer solution, and controlling the final concentration to be OD 600 = 0.1; adding an iron chloride solution and a sodium thiosulfate solution with initial concentrations of 0.1M into the anaerobic reaction buffer solution after inoculation of the bacterial sludge, wherein the final concentrations of the iron chloride and the sodium thiosulfate are 100 mu M; culturing in a shaking table at 30 ℃ for 13h at the shaking revolution of 200rpm to obtain the Shewanella-nano material hybrid cell.
FIG. 1 is a SEM representation of cells; in the figure, a is a Shewanella SEM electron microscope picture, and b is a Shewanella-nano material hybrid cell SEM electron microscope picture. As can be seen in FIG. 1, Shewanella is oval, and the constructed Shewanella-nanomaterial hybrid cell has obvious nanoparticles outside the Shewanella. The color of the hybrid cells gradually darkened by visual observation during the culture period, and the concentration of ferrous iron is gradually increased.
Taking out Shewanella-nanometer material heterozygous cells stored in an anaerobic workstation, centrifuging at 8000rpm for 5min, discarding supernatant, and centrifuging and washing for 3 times by using anoxic water; and washing the precipitate by using 75%, 100% ethanol and acetone respectively, placing the finally centrifuged precipitate in an anaerobic workstation for drying, grinding the black solid into powder by using an agate mortar after the precipitate is dried, and taking a proper amount of powder sample for EDS elemental analysis and characterization. FIG. 2 is an EDS element characterization diagram; as shown in fig. 2, the presence of elemental iron and elemental sulfur can be confirmed, demonstrating that the extracellular nanoparticles are iron sulfides.
Example 2
In this example, wild-type shewanella, carbon-source-free shewanella and carbon-source-free hybrid cells were used as comparative examples, and the removal efficiency of methyl violet by the hybrid cells constructed in example 1 and the comparative examples was verified. The breeding and construction method of the comparative example is basically the same as that of example 1, and the differences are as follows: the wild Shewanella culture environment is not added with ferric chloride and sodium thiosulfate; LB and sodium lactate are not contained in the anaerobic reaction buffer of the carbon-source-free hybrid cells: the carbon-source-free Shewanella does not contain LB and sodium lactate in an anaerobic reaction buffer solution, and ferric chloride and sodium thiosulfate are not added.
Taking the Shewanella-nanomaterial hybrid cell obtained in example 1 out of the anaerobic workstation, and centrifuging at 6000rpm for 8 min; washed with fresh M9, resuspended in 60mL of simulated methyl Violet wastewater (7.5 mg/L final methyl Violet dye added to the anaerobic reaction buffer prepared in example 1), and maintained at OD 600 =0.1, shake culture at 30 ℃. Setting the sampling time to be 0h, 0.25h, 1h, 2h, 14h, 26h and 34h respectively, sampling 1mL each time, centrifuging the obtained sample at 12000rpm for 5min, measuring the concentration of the pollutant methyl violet in the supernatant at 580nm by using a spectrophotometer, and drawing a degradation curve. By the same methodAnd (4) determining the removal efficiency of the methyl violet of the comparative example, and drawing a degradation curve.
FIG. 3 is a graph showing the effect of methyl violet degradation; as can be seen in FIG. 3, the removal efficiency of the constructed Shewanella-nanomaterial hybrid cell on methyl violet is 4.3 times that of wild Shewanella and 3 times that of a carbon-source-free hybrid cell. The Shewanella-nano material hybrid cell has a good methyl violet degradation effect.
Example 3
Resuspending the Shewanella-nanomaterial hybrid cell constructed in the example 1 into simulated methyl violet wastewater with initial concentration of 7.5mg/L under an anaerobic condition, sampling every 2h, centrifuging a sample at 12000rpm for 5min, and measuring the methyl violet concentration at 580nm by using a spectrophotometer; and after the methyl violet of the reaction system is degraded, injecting the methyl violet wastewater solution again to ensure that the concentration of the methyl violet of the reaction system is 7.5mg/L until the methyl violet dye is not degraded any more, and drawing a degradation curve.
FIG. 4 is a graph showing the effect of multi-cycle methyl violet degradation of Shewanella-nanomaterial hybrid cells prepared in example 1; as can be seen from FIG. 4, the Shewanella-nanomaterial hybrid cell prepared can realize periodic reductive degradation of ferrous sulfide (FeS), the FeS processing capacity per unit mass is improved by 3.6 times, and the Shewanella-nanomaterial hybrid cell has good cell tolerance. Has good prospect of multicycle reduction degradation of organic dye.
Example 4
(1) Shewanella (purchased from ATCC American type culture Collection, and the strain number ATCC 700550) is inoculated into an LB culture medium for culture, and the volume ratio of the Shewanella inoculum to the LB culture medium is 0.8:100, respectively; placing the mixture in a shaking table at the temperature of 20 ℃ and shaking at the rotation speed of 50rpm for culturing for 20 hours to obtain a bacterial liquid, wherein the OD600 value of the bacterial liquid is 0.5;
(2) mixing LB liquid culture medium and M9 culture medium at a volume ratio of 1: 99, heating to boil, introducing nitrogen gas, autoclaving, and adding sterile sodium lactate to a final concentration of 20mM to obtain anaerobic reaction buffer solution; centrifuging overnight-cultured Shewanella bacteria liquid at 2000rpm for 10min, adding the precipitate bacteria mud into 300mL anaerobic reaction buffer solution, and controlling the final concentration at OD 600 = 0.05; mixing ferric nitrate solution and sulfurous acidAdding the sodium acid solution into the anaerobic reaction buffer solution inoculated with the bacterial sludge, wherein the final concentration of ferric nitrate and sodium sulfite is 100 mu M; placing in a shaking table at 4 ℃ and shaking at the speed of 50rpm for shaking culture for 20h to obtain the Shewanella-nano material hybrid cell.
Centrifuging the obtained Shewanella-nano material hybrid cell at 3000rpm for 10min, washing with M9 culture medium, adding into anaerobic reaction buffer solution containing organic dye methyl violet to obtain organic dye degradation system, and culturing in a shaking table until the final concentration OD600 of bacterial liquid is 0.05.
Example 5
(1) Inoculating Shewanella to an LB culture medium for culture, wherein the volume ratio of the Shewanella inoculation amount to the LB culture medium is 0.8:100, respectively; placing the mixture in a shaking table at the temperature of 10 ℃, and culturing for 15h at the shaking revolution of 200rpm to obtain a bacterial liquid, wherein the OD600 value of the bacterial liquid is 2;
(2) mixing an LB liquid culture medium and an M9 culture medium according to a volume ratio of 1:50, heating and boiling, introducing nitrogen, autoclaving, and adding sterile sodium lactate to a final concentration of 50mM to obtain an anaerobic reaction buffer solution; centrifuging overnight-cultured Shewanella bacteria liquid at 8000rpm for 2min, adding the precipitate bacteria mud into 300mL anaerobic reaction buffer solution, and controlling the final concentration at OD 600 = 4; adding a ferric sulfate solution and a low sodium sulfate solution into the anaerobic reaction buffer solution inoculated with the bacterial sludge, wherein the final concentration of ferric sulfate and low sodium sulfate is 50 mu M; culturing in a shaking table at 37 ℃ for 5h at the shaking revolution of 300rpm to obtain the Shewanella-nano material hybrid cell.
Centrifuging the obtained Shewanella-nano material hybrid cell at 7000rpm for 5min, washing with M9 culture medium, adding into anaerobic reaction buffer solution containing organic dye methyl violet to obtain organic dye degradation system, and culturing in a shaking table until the final concentration OD600 of bacterial liquid is 5.
Example 6
(1) Shewanella is inoculated into an LB culture medium for culture, and the volume ratio of the Shewanella inoculation amount to the LB culture medium is 0.2: 100, respectively; placing the mixture in a shaking table at the temperature of 37 ℃, and culturing for 10 hours at the shaking revolution of 300rpm to obtain a bacterial liquid, wherein the OD600 value of the bacterial liquid concentration is 4;
(2) mixing LB liquid culture medium and M9 culture medium at a volume ratio of 1: 20, heating to boil, introducing nitrogen gas, autoclaving, and adding sterile sodium lactate to a final concentration of 0.1mM to obtain anaerobic reaction buffer solution; centrifuging the overnight cultured Shewanella bacteria liquid at 6000rpm for 5min, adding the precipitate bacteria mud into 300mL anaerobic reaction buffer solution, and controlling the final concentration to be OD600= 2; adding a ferric citrate solution and a sodium sulfate solution into the anaerobic reaction buffer solution inoculated with the bacterial sludge, wherein the final concentration of ferric citrate and sodium sulfate is 80 mu M; placing the mixed cells in a shaking table at 37 ℃ and shaking at the shaking revolution speed of 300rpm for shaking and culturing for 8h to obtain the Shewanella-nano material hybrid cells.
The above examples are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any obvious improvements, substitutions or modifications can be made by those skilled in the art without departing from the spirit of the present invention.
Claims (10)
1. A method for constructing Shewanella-nanomaterial hybrid cells, comprising the steps of:
(1) inoculating Shewanella to an LB culture medium, and performing shake culture to obtain a bacterial liquid;
(2) centrifuging the bacterial liquid obtained in the step (1), adding the precipitated bacterial sludge into an anaerobic reaction buffer solution, and adding a water-soluble ferric salt solution and a water-soluble sulfur source salt solution; and (4) performing shake culture to obtain the Shewanella-nano material hybrid cell.
2. The construction method according to claim 1, wherein the Shewanella is inoculated into the LB medium in the step (1) in a ratio of an inoculum size to the LB medium volume of 0.05-0.8: 100.
3. The method of claim 1, wherein the conditions of shake culture in step (1) are as follows: the temperature is 20-37 ℃, the oscillation revolution is 50-300 rpm, and the time is 10-20 h; the OD600 value of the bacterial liquid in the step (1) is 0.5-4.
4. The construction method according to claim 1, wherein the rotation speed of the centrifugation in the step (2) is 2000-8000 rpm, and the time of the centrifugation is 2-10 min; the OD600 value of the final concentration of the Shewanella-nano material hybrid cell in the step (2) is 0.05-5.
5. The construction method according to claim 1, wherein the anaerobic reaction buffer solution in step (2) is prepared by mixing LB liquid medium and M9 medium at a volume ratio of 1: 4-99, boiling, introducing nitrogen gas, autoclaving, and adding sterile sodium lactate to a final concentration of 0.1-50 mM.
6. The construction method according to claim 1, wherein the water-soluble ferric salt in step (2) is one of ferric chloride, ferric nitrate, ferric sulfate or ferric citrate; the water-soluble sulfur source salt is one of sodium thiosulfate, sodium sulfite, low sodium sulfate or sodium sulfate.
7. The method according to claim 1, wherein the final concentrations of the water-soluble ferric salt solution and the water-soluble sulfur source salt solution in step (2) are both greater than 50 μ M.
8. The method of claim 1, wherein the conditions of shake culture in step (2) are as follows: the temperature is 4-37 ℃, the rotating speed is 50-300 rpm, and the time is more than 5 h.
9. The Shewanella-nanomaterial hybrid cell constructed according to the construction method of claim 1.
10. Use of the Shewanella-nanomaterial hybrid cell of claim 9 in dye degradation.
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