CN113960141B - Preparation method of immobilized microbial membrane for measuring BOD of seawater - Google Patents
Preparation method of immobilized microbial membrane for measuring BOD of seawater Download PDFInfo
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
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- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- DEXFNLNNUZKHNO-UHFFFAOYSA-N 6-[3-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]piperidin-1-yl]-3-oxopropyl]-3H-1,3-benzoxazol-2-one Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1CCN(CC1)C(CCC1=CC2=C(NC(O2)=O)C=C1)=O DEXFNLNNUZKHNO-UHFFFAOYSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
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- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- 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
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- 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
- C12N1/36—Adaptation or attenuation of cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/18—Water
- G01N33/1806—Biological oxygen demand [BOD] or chemical oxygen demand [COD]
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Abstract
The invention provides a preparation method of an immobilized microbial membrane for measuring seawater BOD, which comprises the following steps: s1, domesticating microorganisms suitable for high-salinity seawater; s2, generating a layer of electron mediator which promotes microorganism metabolism on the surface of the electrode; and S3, polymerizing the conductive solution doped with microorganisms on the surface of the electronic medium layer of the electrode by a cyclic voltammetry to form a microbial film with a double-layer structure on the surface of the electrode. The microbial film has the advantages of high measurement accuracy, good stability and long service life, and can realize the rapid and accurate measurement of the BOD of the high-salt seawater.
Description
Technical Field
The invention relates to the technical field of sewage treatment, in particular to a preparation method of an immobilized microbial membrane for measuring seawater BOD.
Background
Microbial membranes are a central part of rapid BOD measurement, and generally use single species or mixed species of microorganisms as biosensing elements. When BOD substances are added into water or degradation metabolism occurs, the change or conversion of the internal and external source respiration modes of microorganisms is caused, the intensity of the coupling output current is changed, the current value output by the sensor and the BOD concentration are in a linear relation under certain conditions, and the BOD concentration of a sample can be measured according to the linear relation between the current value and the BOD concentration.
The existing microorganism fixing method comprises an adsorption method, a crosslinking method and an embedding method, but the adsorption method has the problems that microorganisms are easy to fall off and the service life is influenced; the embedding method is not suitable for the reaction of macromolecular substances, and the embedding material is easy to introduce larger mass transfer resistance when in use; the survival rate of the immobilized microbial film by the crosslinking method is low, the preparation is complex, and the dosage of the crosslinking agent and the microbial biomass are mutually restricted.
The existing calcium alginate embedding method and polyvinyl alcohol embedding method have the defects of easy swelling, weak acid-base adaptability, no osmotic pressure resistance, easy absorption of colored substances, more flocs and the like to different degrees, and influence the detection stability and sensitivity. Therefore, a microbial membrane fixing method with strong stability and high sensitivity and capable of detecting BOD of high-salinity seawater needs to be developed.
Disclosure of Invention
In view of the above, the invention provides a microbial membrane with high sensitivity, strong stability, suitability for high salinity seawater, good repeatability and long service life and a preparation method thereof.
The technical scheme of the invention is realized as follows: the invention provides a preparation method of an immobilized microbial membrane for measuring seawater BOD, which comprises the following steps:
s1, domesticating microorganisms suitable for high-salinity seawater;
s2, generating a layer of electron mediator which promotes microorganism metabolism on the surface of the electrode;
s3, polymerizing the conductive solution doped with domesticated microorganisms on the surface of the electronic medium layer of the electrode by a cyclic voltammetry to form a microbial film with a double-layer structure on the surface of the electrode.
Based on the above technical solution, preferably, the operation steps of step S1 are as follows:
s11, taking microorganisms separated from seawater sludge, and carrying out domestication and 2-3 times of plate separation to obtain pure bacterial strains;
s12, the pure strain in the step S11 is subjected to shaking culture, is inoculated to a culture medium after being washed by a sterile buffer solution, and is prepared into bacterial suspension of 8X 109CFU/mL for standby when the concentration of the bacterial body reaches 10X 109 CFU/mL.
Based on the above technical solution, preferably, the operation steps of step S2 are as follows:
s21, sequentially carrying out ultrasonic treatment on the electrode in an organic solvent and deionized water for 5min, drying by nitrogen, immersing in 0.1-0.3mol/L sulfuric acid, and scanning by adopting an electrochemical cyclic voltammetry to obtain a working electrode;
and S22, doping the methylene blue on the surface of the working electrode in the step S21 by electrochemical cyclic voltammetry to obtain the electrode covered with the methylene blue film.
On the basis of the technical scheme, preferably, the organic solvent is one or a combination of more of ethanol, acetone and cyclohexane.
Based on the above technical solution, preferably, the operation steps of step S3 are as follows:
s31, dissolving a conductive substance in the bacterial suspension in the step S12 to prepare a mixed solution;
s32, immersing the electrode covered with the methylene blue film in the step S22 into the mixed solution in the step S31, and scanning by adopting electrochemical cyclic voltammetry to obtain the microbial film electrode covered with the double-layer structure.
On the basis of the technical scheme, preferably, the conductive substance is one or a combination of more of polyacetylene, polythiophene, polyaniline, polyphenylene, polystyrene and polydiacetylene, and the concentration is 0.01-0.03mol/L.
On the basis of the above technical scheme, preferably, the microorganism in the step S1 is one of bacillus licheniformis, candida, hansenula, klebsiella, escherichia coli, clostridium butyricum and pseudomonas aeruginosa.
On the basis of the above technical solution, preferably, the electrode in the step S2 is a disc electrode, and the diameter of the disc electrode is 1-5mm.
On the basis of the technical scheme, preferably, the disc electrode is one of a gold disc electrode, a silver disc electrode and a titanium disc electrode.
Compared with the prior art, the preparation method of the immobilized microbial film for measuring the BOD of the seawater has the following beneficial effects:
(1) The microbial film of the invention is used for measuring the BOD of seawater, and has the advantages of short measurement time and real-time monitoring on site; the classical 5 day method takes 5 days in the whole measurement process and cannot be detected in real time on site, and the sensor method of the microbiological method can finish measurement within 10 minutes.
(2) The microbial film of the invention has high measurement accuracy, and can reduce the error of the general BOD rapid detection method from +/-20% to +/-5%.
(3) The microbial film of the invention also has the advantages of good stability and long service life, and the stable service life exceeds 3 months, and the service life of the far-reaching calcium alginate gel is 1 month.
(4) The strain used in the invention is separated and domesticated from the seawater sludge, so that the rapid and accurate measurement of the BOD of the high-salt seawater can be realized.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a standard graph of an immobilized microbial membrane for measuring BOD of seawater according to the present invention;
FIG. 2 is a graph showing the results of a microbial film stability test of an immobilized microbial film for measuring BOD of seawater according to the present invention.
Detailed Description
The following description of the embodiments of the present invention will clearly and fully describe the technical aspects of the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, are intended to fall within the scope of the present invention.
Example 1
The preparation process of immobilized microbial film for measuring BOD of sea water includes the following steps:
s1, preparing strain suspension; the strain is selected from bacillus licheniformis separated from seawater sludge, and the selected bacillus licheniformis is domesticated and subjected to plate separation for 2 times to obtain a pure strain. Shake culturing pure strain in culture medium solution, PBS solution and 0.8% sodium chloride salt water at 30deg.C and 140rpm for 24 hr, centrifuging at 25deg.C and 7000r/min for 20min, washing with equal volume of sterile buffer solution for 2 times to obtain Bacillus licheniformis suspension, inoculating Bacillus licheniformis to nutrient agar plate, culturing for 6 hr, and placing in refrigerator at 4deg.C. Before use, a loop of strain is taken from the agar plane and inoculated into 200mL of sterile culture medium again for culturing for 12h to ensure that the concentration of the strain reaches 10 multiplied by 10 9 CFU/mL, diluted with sterile physiological saline to obtain 8×10 9 The CFU/mL bacterial suspension is ready for use.
S2, preparing a working electrode, namely sequentially carrying out ultrasonic treatment on a gold plate electrode with the diameter of 1mm in ethanol and deionized water for 5min respectively, drying by nitrogen, then putting the gold plate electrode into a 0.1mol/L dilute sulfuric acid solution, and scanning for a plurality of times by adopting an electrochemical cyclic voltammetry within the range of-0.2-1.2V until the superposition of scanning curves is stable, and taking the gold plate electrode as the working electrode for standby.
S3, preparing a microbial membrane electrode; the methylene blue is doped on the surface of a working electrode by an electrochemical cyclic voltammetry to obtain a new methylene blue film NMB/Au; then preparing a conductive material containing 0.01mol/L and 8×10 9 A mixed solution of a CFU/mL concentration bacillus licheniformis suspension; and (3) immersing NMB/Au into the mixed solution, and obtaining the microbial membrane electrode with a double-layer structure by adopting an electrochemical cyclic voltammetry.
The conductive substances are polyacetylene, polythiophene and polystyrene, and polyacetylene: polythiophene: the mass ratio of the polyphenylene ethylene is 1:1:1.
Example 2
The preparation process of immobilized microbial film for measuring BOD of sea water includes the following steps:
s1, preparing strain suspension; the strain is selected from Hansenula polymorpha separated from seawater sludge, and the Hansenula polymorpha after selection is domesticated and subjected to plate separation for 3 times to obtain a pure strain. Shake culturing pure strain in culture medium solution, PBS solution and 0.8% sodium chloride salt water at 30deg.C and 140rpm for 24 hr, centrifuging cultured Hansenula polymorpha at 25deg.C and 7000r/min for 20min, washing with equal volume of sterile buffer solution for 2 times to obtain Hansenula polymorpha suspension, inoculating Hansenula polymorpha to nutrient agar plate, culturing for 6 hr, and placing in refrigerator at 4deg.C. Before use, a loop of strain is taken from the agar plane and inoculated into 200mL of sterile culture medium again for culturing for 12h to ensure that the concentration of the strain reaches 10 multiplied by 10 9 CFU/mL, diluted with sterile physiological saline to obtain 8×10 9 The CFU/mL bacterial suspension is ready for use.
S2, preparing a working electrode, namely sequentially carrying out ultrasonic treatment on a silver plate electrode with the diameter of 5mm in acetone and deionized water for 5min respectively, drying by nitrogen, then putting the silver plate electrode into a 0.3mol/L dilute sulfuric acid solution, and scanning for a plurality of times by adopting an electrochemical cyclic voltammetry within the range of-0.2-1.2V until the superposition of scanning curves is stable, and taking the silver plate electrode as the working electrode for standby.
S3, preparing a microbial membrane electrode; the methylene blue is doped on the surface of a working electrode by an electrochemical cyclic voltammetry to obtain a new methylene blue film NMB/Ag; then preparing a conductive material containing 0.03mol/L and 8×10 10 The mixed solution of the microbial bacteria solution with the concentration of CFU/mL; and immersing NMB/Ag into the mixed solution, and obtaining the microbial membrane electrode with a double-layer structure by adopting an electrochemical cyclic voltammetry.
The conductive substances are polyaniline and polyphenylene; polyaniline: the volume ratio of the polyphenylene is 1:1.
Example 3
The preparation process of immobilized microbial film for measuring BOD of sea water includes the following steps:
s1, preparing strain suspension; the strain is selected from colibacillus separated from seawater sludge, and the selected colibacillus is domesticated and subjected to plate separation for 2 times to obtain a pure strain. Shake culturing pure strain in culture medium solution, PBS solution and 0.8% sodium chloride salt water at 30deg.C and 140rpm for 24 hr, centrifuging at 25deg.C and 7000r/min for 20min, washing with equal volume of sterile buffer solution for 2 times to obtain Escherichia coli suspension, inoculating Escherichia coli into nutrient agar plate, culturing for 6 hr, and placing in refrigerator at 4deg.C. Before use, a loop of strain is taken from the agar plane and inoculated into 200mL of sterile culture medium again for culturing for 12h to ensure that the concentration of the strain reaches 10 multiplied by 10 9 CFU/mL, diluted with sterile physiological saline to obtain 8×10 9 The CFU/mL bacterial suspension is ready for use.
S2, preparing a working electrode, namely sequentially carrying out ultrasonic treatment on a titanium disk electrode with the diameter of 2mm in cyclohexane and deionized water for 5min respectively, drying by nitrogen, then putting the titanium disk electrode into a 0.1mol/L dilute sulfuric acid solution, and scanning for a plurality of times by adopting an electrochemical cyclic voltammetry within the range of-0.2-1.2V until the superposition of scanning curves is stable, and taking the titanium disk electrode as the working electrode for standby.
S3, preparing a microbial membrane electrode; the methylene blue is doped on the surface of a working electrode by an electrochemical cyclic voltammetry to obtain a new methylene blue film NMB/Ti; then preparing a conductive material containing 0.01mol/L and 8×10 9 The mixed solution of the microbial bacteria solution with the concentration of CFU/mL; and immersing NMB/Ti into the mixed solution, and obtaining the microbial membrane electrode with a double-layer structure by adopting an electrochemical cyclic voltammetry.
The conductive substances are polystyrene and polydiacetylene, and the polystyrene: the volume ratio of the polydiacetylene is 1:1.
Example 4
The preparation process of immobilized microbial film for measuring BOD of sea water includes the following steps:
s1, preparing strain suspension; the strain is selected from candida separated from seawater sludge, and the pure strain is obtained by domestication and 2-3 times of plate separation of the selected candida. Pure strain was grown in medium solution, PBS solution and 0.8% at 30℃and 140rpmAnd (3) carrying out shake culture on sodium chloride salt water for 24 hours, centrifuging the cultured candida at 25 ℃ and 7000r/min for 20 minutes, obtaining candida suspension after washing for 2 times by using an equal volume of sterile buffer solution, inoculating the candida to a nutrient agar plate, culturing for 6 hours, and placing in a refrigerator at 4 ℃. Before use, a loop of strain is taken from the agar plane and inoculated into 200mL of sterile culture medium again for culturing for 12h to ensure that the concentration of the strain reaches 10 multiplied by 10 9 CFU/mL, diluted with sterile physiological saline to obtain 8×10 9 The CFU/mL bacterial suspension is ready for use.
S2, preparing a working electrode, namely sequentially carrying out ultrasonic treatment on a gold plate electrode with the diameter of 3mm in acetone and deionized water for 5min respectively, drying by nitrogen, then putting the gold plate electrode into a 0.3mol/L dilute sulfuric acid solution, and scanning for a plurality of times by adopting an electrochemical cyclic voltammetry within the range of-0.2-1.2V until the superposition of scanning curves is stable, and taking the gold plate electrode as the working electrode for standby.
S3, preparing a microbial membrane electrode; the methylene blue is doped on the surface of a working electrode by an electrochemical cyclic voltammetry to obtain a new methylene blue film NMB/Au; then preparing a conductive material containing 0.01mol/L and 8×10 9 The mixed solution of the microbial bacteria solution with the concentration of CFU/mL; and (3) immersing NMB/Au into the mixed solution, and obtaining the microbial membrane electrode with a double-layer structure by adopting an electrochemical cyclic voltammetry.
The conductive liquid is polyacetylene, polythiophene and polyaniline, and polyacetylene: polythiophene: the volume ratio of polyaniline is 1:1:1.
Example 5
The preparation process of immobilized microbial film for measuring BOD of sea water includes the following steps:
s1, preparing strain suspension; the strain is selected from klebsiella isolated from seawater sludge, and pure strain is obtained by domestication and 2 times of plate separation of the selected klebsiella. Shake culturing pure strain in culture medium solution, PBS solution and 0.8% sodium chloride salt water at 30deg.C and 140rpm for 24 hr, centrifuging at 25deg.C and 7000r/min for 20min, washing with equal volume of sterile buffer solution for 2 times to obtain suspension, and inoculating the strain to nutrient agarThe plates were incubated for 6h and then placed in a refrigerator at 4 ℃. Before use, a loop of strain is taken from the agar plane and inoculated into 200mL of sterile culture medium again for culturing for 12h to ensure that the concentration of the strain reaches 10 multiplied by 10 9 CFU/mL, diluted with sterile physiological saline to obtain 8×10 9 The CFU/mL bacterial suspension is ready for use.
S2, preparing a working electrode, namely sequentially carrying out ultrasonic treatment on a titanium disk electrode with the diameter of 4mm in cyclohexane and deionized water for 5min respectively, drying by nitrogen, then putting the titanium disk electrode into a 0.1mol/L dilute sulfuric acid solution, and scanning for a plurality of times by adopting an electrochemical cyclic voltammetry within the range of-0.2-1.2V until the superposition of scanning curves is stable, and taking the titanium disk electrode as the working electrode for standby.
S3, preparing a microbial membrane electrode; the methylene blue is doped on the surface of a working electrode by an electrochemical cyclic voltammetry to obtain a new methylene blue film NMB/Ti; then preparing a conductive material containing 0.01mol/L and 8×10 9 The mixed solution of the microbial bacteria solution with the concentration of CFU/mL; and immersing NMB/Ti into the mixed solution, and obtaining the microbial membrane electrode with a double-layer structure by adopting an electrochemical cyclic voltammetry.
The conductive substances are polystyrene and polydiacetylene, and the polystyrene: the volume ratio of the polydiacetylene is 1:1.
Example 6
The preparation process of immobilized microbial film for measuring BOD of sea water includes the following steps:
s1, preparing strain suspension; the strain is selected from clostridium butyricum separated from seawater sludge, and the selected strain is domesticated and subjected to plate separation for 3 times to obtain a pure strain. Shake culturing pure strain in culture medium solution, PBS solution and 0.8% sodium chloride salt water at 30deg.C and 140rpm for 24 hr, centrifuging at 25deg.C and 7000r/min for 20min to obtain mixed strain, washing with equal volume of sterile buffer solution for 2 times to obtain clostridium butyricum suspension, inoculating clostridium butyricum into nutrient agar plate, culturing for 6 hr, and placing in refrigerator at 4deg.C. Before use, a loop of strain is taken from the agar plane and inoculated into 200mL of sterile culture medium again for culturing for 12h to ensure that the concentration of the strain reaches 10 multiplied by 10 9 CFU/mL, diluted with sterile physiological saline to obtain 8×10 9 The CFU/mL bacterial suspension is ready for use.
S2, preparing a working electrode, namely sequentially carrying out ultrasonic treatment on a silver plate electrode with the diameter of 5mm in acetone and deionized water for 5min respectively, drying by nitrogen, then putting the silver plate electrode into a 0.3mol/L dilute sulfuric acid solution, and scanning for a plurality of times by adopting an electrochemical cyclic voltammetry within the range of-0.2-1.2V until the superposition of scanning curves is stable, and taking the silver plate electrode as the working electrode for standby.
S3, preparing a microbial membrane electrode; the methylene blue is doped on the surface of a working electrode by an electrochemical cyclic voltammetry to obtain a new methylene blue film NMB/Ag; then preparing a conductive material containing 0.03mol/L and 8×10 10 The mixed solution of the microbial bacteria solution with the concentration of CFU/mL; and immersing NMB/Ag into the mixed solution, and obtaining the microbial membrane electrode with a double-layer structure by adopting an electrochemical cyclic voltammetry.
The conductive substances are polyaniline and polyphenylene; polyaniline: the volume ratio of the polyphenylene is 1:1.
Example 7
The preparation process of immobilized microbial film for measuring BOD of sea water includes the following steps:
s1, preparing strain suspension; the strain is selected from Pseudomonas aeruginosa separated from seawater sludge, and the selected Pseudomonas aeruginosa is domesticated and subjected to plate separation for 2 times to obtain a pure strain. Shake culturing pure strain in culture medium solution, PBS solution and 0.8% sodium chloride salt water at 30deg.C and 140rpm for 24 hr, centrifuging cultured Pseudomonas aeruginosa at 25deg.C and 7000r/min for 20min, washing with equal volume of sterile buffer solution for 2 times to obtain Pseudomonas aeruginosa suspension, inoculating Pseudomonas aeruginosa into nutrient agar plate, culturing for 6 hr, and placing in refrigerator at 4deg.C. Before use, a loop of strain is taken from the agar plane and inoculated into 200mL of sterile culture medium again for culturing for 12h to ensure that the concentration of the strain reaches 10 multiplied by 10 9 CFU/mL, diluted with sterile physiological saline to obtain 8×10 9 The CFU/mL bacterial suspension is ready for use.
S2, preparing a working electrode, namely sequentially carrying out ultrasonic treatment on a gold plate electrode with the diameter of 3mm in ethanol and deionized water for 5min respectively, drying by nitrogen, then putting the gold plate electrode into a 0.1mol/L dilute sulfuric acid solution, and scanning for a plurality of times by adopting an electrochemical cyclic voltammetry within the range of-0.2-1.2V until the superposition of scanning curves is stable, and taking the gold plate electrode as the working electrode for standby.
S3, preparing a microbial membrane electrode; the methylene blue is doped on the surface of a working electrode by an electrochemical cyclic voltammetry to obtain a new methylene blue film NMB/Au; then preparing a conductive material containing 0.01mol/L and 8×10 9 The mixed solution of the pseudomonas aeruginosa suspension with the concentration of CFU/mL; and (3) immersing NMB/Au into the mixed solution, and obtaining the microbial membrane electrode with a double-layer structure by adopting an electrochemical cyclic voltammetry.
The conductive substances are polyacetylene and polythiophene, polyacetylene: the volume ratio of polythiophene was 1:1.
Comparative example 1
Comparative example 1, doped with the absence of methylene blue, comprises the following specific steps:
s1, preparing strain suspension; the strain is selected from bacillus licheniformis separated from seawater sludge, and the selected bacillus licheniformis is domesticated and subjected to plate separation for 2 times to obtain a pure strain. Shaking culture of pure strain in culture medium solution, PBS solution and NaCl with volume fraction of 0.8% at 30deg.C and 140rpm for 24 hr, centrifuging at 25deg.C and 7000r/min for 20min, washing with equal volume of sterile buffer solution for 2 times to obtain Bacillus licheniformis suspension, inoculating Bacillus licheniformis to nutrient agar plate, culturing for 6 hr, and placing in refrigerator at 4deg.C. Before use, a loop of strain is taken from the agar plane and inoculated into 200mL of sterile culture medium again for culturing for 12h to ensure that the concentration of the strain reaches 10 multiplied by 10 9 CFU/mL, diluted with sterile physiological saline to obtain 8×10 9 The CFU/mL bacterial suspension is ready for use.
S2, preparing a working electrode, namely sequentially carrying out ultrasonic treatment on the gold plate electrode in ethanol and deionized water for 5min respectively, drying by nitrogen, then putting the gold plate electrode in a 0.1mol/L dilute sulfuric acid solution, and scanning for a plurality of times by adopting an electrochemical cyclic voltammetry within the range of-0.2-1.2V until the superposition of scanning curves is stable, and taking the gold plate electrode as the working electrode for standby.
S3, preparing a microbial membrane electrode; then preparing a conductive material containing 0.01mol/L and 8×10 9 CFU/mL concentration bacillus licheniformisThe mixed solution of the bacterial suspension; and immersing the working electrode into the mixed solution, and obtaining the microbial membrane electrode covered with microorganisms by adopting an electrochemical cyclic voltammetry. The conductive substances are polyacetylene, polythiophene and polystyrene, and polyacetylene: polythiophene: the mass ratio of the polyphenylene ethylene is 1:1:1.
Comparative example 2
The existing calcium alginate gel embedding method is taken as a comparative example 2, and the specific steps are as follows:
and (3) dripping sodium alginate solution and bacterial liquid on the surface of a working electrode, immersing in calcium chloride solution for solidification, and covering a polyamide membrane on the surface of cell gel to prevent the embedded cells from falling off. The prepared sensor is used for BOD rapid detection. The electrode and the bacterial liquid are the same as in the first embodiment.
Determination of BOD in seawater Using microbial films prepared in examples and comparative examples as sensors 5 The content is as follows. Drawing standard curves of the biomembrane modified electrodes prepared in the above examples and comparative examples, and determining BOD in seawater according to the standard curves 5 Concentration.
FIG. 1 is a standard curve of example 1, and FIG. 1 shows that in this example 1, BOD is used 5 The concentration of (2) is the abscissa, the current delta mA is the ordinate, and a linear relation can be obtained by drawing a standard curve of the microbial membrane modified electrode: y= 0.0521x-0.027, r 2 ＝0.9983。
The bacterial suspensions of examples 1 to 7 and comparative examples 1 to 2 were diluted to 10X 10 in order 8 CFU/mL、10×10 7 CFU/mL、10×10 6 CFU/mL、10×10 5 CFU/mL、10×10 4 CFU/mL; preparing microbial films on the surfaces of the electrodes by using the obtained bacterial suspensions with different concentrations, measuring the cyclic voltammetry curves of the microbial films in sulfuric acid solution with the concentration of 0.1mol/L after rinsing by distilled water, wherein the scanning voltage is-0.2-1.2V, and the scanning rate is 50mV/s; and drawing a standard curve of the microbial membrane modified electrode by taking the logarithm of the bacterial concentration as an abscissa and the peak current of the obtained cyclic voltammogram at 0.2V as an ordinate. The microbial films of the present invention were verified for sensitivity and accuracy by taking samples of the same concentration and measuring the colony count using the microbial films prepared in example 1 and comparative examples 1-2, and each example or comparative example was repeatedly examined 5 times to obtain a flatAnd (5) an average value.
TABLE 1 colony count measurement results
Table 1 shows that the concentration of the bacterial suspension measured in example 1 of the present invention was substantially the same as that measured by the plate count method, and the relative standard deviation was lower than that of the plate colony count method. The invention has better accuracy, and the counting result is superior to the traditional plate colony technology method and calcium alginate gel embedding method. The accuracy and the service life of the microbial film without being doped with methylene blue are lower than those of the microbial film with being doped with methylene blue, which shows that the methylene blue and the microorganisms have synergistic effect, and the detection accuracy and the service life can be increased. The accuracy of example 2 is the lowest and the relative standard deviation is the greatest. The service life of the microbial films in examples 1-7 reaches 98-110d and is far longer than that of the microbial films in comparative examples 1-2, which shows that the biological film has the advantages of high accuracy and long service life. The microorganisms of examples 1 to 7 have strong adsorptivity on the electrode surface and do not fall off; the gel of example 2 swells and the microorganisms are easily dispersed and have weak adsorptivity, thereby affecting the detection result and the service life.
To verify the stability of the immobilization of the biofilm of the invention, the biological membranes were immobilized with a specific BOD 5 Seawater with concentration as detection object, BOD of 0-105d is tested 5 The concentration and the result are shown in FIG. 2. FIG. 2 shows the BOD of seawater detected in 105d using the biofilms of example 1 and comparative example 2 5 The current at the initial detection of seawater (at 0 d) was 10mA, and the concentration was consistent with that of example and comparative example 2. The current of example 1 was always between 9.5-10.4mA at 0-105d, the maximum and minimum values differing by 0.9mA; the current of comparative example 2 was between 8.0 and 11.8mA at 0-35d, the maximum and minimum values were 3.8mA, and there was a continuous decrease after 35d, with no sign of recovery, and the current was 4.9mA at 105 d. The microbial film has the advantages of high measurement accuracy and good stability, can reduce the error from +/-20% to within +/-5%, and also shows that the comparative example 2 detects after the service life is longerThe result is in a continuous decline trend and cannot be recovered, and needs to be replaced in time to detect.
The microbial film of the invention is used for measuring the BOD of seawater, and has the advantages of short measurement time and real-time monitoring on site; the classical 5-day method takes 5 days in the whole measurement process and cannot be detected in real time on site, and the sensor method of the microbiological method can finish measurement within 10 minutes.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (6)
1. The preparation method of the immobilized microbial membrane for measuring seawater BOD is characterized by comprising the following steps of: the method comprises the following steps:
s1, domesticating microorganisms suitable for high-salinity seawater, wherein the operation steps are as follows:
s11, taking microorganisms separated from seawater sludge, and carrying out domestication and 2-3 times of plate separation to obtain pure bacterial strains;
s12, the pure strain in the step S11 is subjected to shaking culture, is washed by a sterile buffer solution and is inoculated in a culture medium, and when the concentration of thalli reaches 10 multiplied by 10 9 CFU/mL was prepared into 8X 10 9 The CFU/mL bacterial suspension is reserved;
s2, generating a layer of electron mediator for promoting microorganism metabolism on the surface of the electrode, wherein the operation steps are as follows:
s21, sequentially carrying out ultrasonic treatment on the electrode in an organic solvent and deionized water for 5min, drying by nitrogen, immersing in 0.1-0.3mol/L sulfuric acid, and scanning by adopting an electrochemical cyclic voltammetry to obtain a working electrode;
s22, doping methylene blue on the surface of the working electrode in the step S21 by electrochemical cyclic voltammetry to obtain an electrode covered with a methylene blue film;
s3, polymerizing a conductive solution doped with domesticated microorganisms on the surface of an electronic medium layer of the electrode by a cyclic voltammetry to form a microbial film with a double-layer structure on the surface of the electrode, wherein the operation steps are as follows:
s31, dissolving a conductive substance in the bacterial suspension in the step S12 to prepare a mixed solution;
s32, immersing the electrode covered with the methylene blue film in the step S22 into the mixed solution in the step S31, and scanning by adopting electrochemical cyclic voltammetry to obtain the microbial film electrode covered with the double-layer structure.
2. The method for preparing the immobilized microbial membrane for measuring seawater BOD as claimed in claim 1, wherein the method comprises the following steps: the organic solvent is one or more of ethanol, acetone and cyclohexane.
3. The method for preparing the immobilized microbial membrane for measuring seawater BOD as claimed in claim 2, wherein: the conductive substance is one or more of polyacetylene, polythiophene, polyaniline, polyphenylene ethylene and polydiacetylene, and the concentration is 0.01-0.03mol/L.
4. The method for preparing the immobilized microbial membrane for measuring seawater BOD as claimed in claim 1, wherein the method comprises the following steps: the microorganism in the step S1 is one of bacillus licheniformis, candida, hansenula, klebsiella, escherichia coli, clostridium butyricum and pseudomonas aeruginosa.
5. The method for preparing the immobilized microbial membrane for measuring seawater BOD as claimed in claim 1, wherein the method comprises the following steps: the electrode in the step S2 is a disc electrode, and the diameter of the disc electrode is 1-5mm.
6. The method for preparing the immobilized microbial membrane for measuring BOD of seawater according to claim 5, wherein the method comprises the following steps: the electrode is one of a gold plate electrode, a silver plate electrode and a titanium plate electrode.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101105470A (en) * | 2006-07-10 | 2008-01-16 | 北京化工大学 | Biosensor enzyme sensitive film and its preparation method |
| CN102320687A (en) * | 2011-06-18 | 2012-01-18 | 山东大学 | The preparation method of a kind of polyaniline-mikrobe combined electrode |
| CN103512938A (en) * | 2012-06-15 | 2014-01-15 | 中国科学院理化技术研究所 | Electrochemical sensor and device for immediately monitoring and detecting water body biotoxicity |
| CN104007154A (en) * | 2013-02-22 | 2014-08-27 | 中国科学院理化技术研究所 | Redox hydrosol, preparation method and application of integrated microbial electrode for rapid detection of water body biotoxicity |
| CN106981668A (en) * | 2017-05-04 | 2017-07-25 | 天津天狮学院 | A kind of microorganism electrode and its preparation method and application |
| CN108535343A (en) * | 2018-04-01 | 2018-09-14 | 桂林理工大学 | The preparation method and applications of methylene blue-gold composite nano particle modified electrode |
| CN111398382A (en) * | 2020-04-21 | 2020-07-10 | 普洱学院 | Polymethylene blue modified glassy carbon electrode and preparation method and application thereof |
-
2021
- 2021-09-26 CN CN202111131062.2A patent/CN113960141B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101105470A (en) * | 2006-07-10 | 2008-01-16 | 北京化工大学 | Biosensor enzyme sensitive film and its preparation method |
| CN102320687A (en) * | 2011-06-18 | 2012-01-18 | 山东大学 | The preparation method of a kind of polyaniline-mikrobe combined electrode |
| CN103512938A (en) * | 2012-06-15 | 2014-01-15 | 中国科学院理化技术研究所 | Electrochemical sensor and device for immediately monitoring and detecting water body biotoxicity |
| CN104007154A (en) * | 2013-02-22 | 2014-08-27 | 中国科学院理化技术研究所 | Redox hydrosol, preparation method and application of integrated microbial electrode for rapid detection of water body biotoxicity |
| CN106981668A (en) * | 2017-05-04 | 2017-07-25 | 天津天狮学院 | A kind of microorganism electrode and its preparation method and application |
| CN108535343A (en) * | 2018-04-01 | 2018-09-14 | 桂林理工大学 | The preparation method and applications of methylene blue-gold composite nano particle modified electrode |
| CN111398382A (en) * | 2020-04-21 | 2020-07-10 | 普洱学院 | Polymethylene blue modified glassy carbon electrode and preparation method and application thereof |
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