A method for preparing a microbial electrode for a water body toxicity detection device

Abstract

The present application relates to a kind of preparation method of microbial electrode for water toxicity detection equipment, it is related to electrode preparation technical field.The present application provides a kind of preparation method of microbial electrode for water toxicity detection equipment, first, the well-cultured microorganism is deposited to the bottom of test tube using the way of centrifugation, then the upper layer solution is poured off.Microorganism is dispersed again with physiological saline.Subsequently, the solution containing microorganism is dropped on the surface of electrode, after the solution is dried, the extracellular polymer remaining in the solution can be used as adhesive, realize the fixation of microorganism on the surface of electrode.Microorganism is film stable on the surface of electrode, and can be used for flow analysis detection of water toxicity.In addition, the extracellular polymer in fixed biofilm is less than in situ growth biofilm, so the response to toxicity is more sensitive than in situ growth biofilm.Compared with gel method, the present application is more simple to operate.

Description

Technical Field

[0001] This invention relates to the field of electrode preparation technology, and more specifically to a method for preparing a microbial electrode for a water toxicity detection device. Background Technology

[0002] Electrochemical methods for detecting water toxicity using microorganisms as toxicity-responsive test subjects have received increasing attention. In our previous work, we developed an instrument for detecting the total toxicity of water using suspended microorganisms as toxicity test subjects (Development of an online instrument for detecting total toxicity of water. Analytical Chemistry, 2017, 45(9):1415-1419). Suspended microorganisms are sensitive to toxicity, but their low-temperature (4℃) storage time is short (2 weeks), thus increasing the instrument's maintenance frequency. Although lyophilization technology can extend the storage time of suspended microorganisms, using lyophilized powder will significantly increase the instrument's operating cost. To reduce the instrument's maintenance frequency, we modified the instrument by using microbial electrodes to replace the original suspended microorganisms for toxicity detection. The fixed microorganisms can be preserved at low temperature (4℃) for at least 2 months, significantly reducing the instrument's maintenance frequency.

[0003] There are generally two methods for immobilizing microorganisms on electrode surfaces in existing technologies. One method is in-situ growth, where the electrode is placed in a microbial culture medium, prompting the microorganisms to form a biofilm on the electrode surface. However, due to the large amount of extracellular polymers present in the biofilm, this type of in-situ cultured biofilm is not sensitive to toxicity. The other commonly used method for immobilizing microorganisms is to use gel materials, such as chitosan, agarose, polyvinyl alcohol, and calcium alginate, utilizing the steric hindrance of the gel material's pores to immobilize the microorganisms on the electrode surface. Microorganisms immobilized by gel fixation are sensitive to toxicity. However, gel fixation of microorganisms requires culturing and washing the microorganisms, then mixing and dispersing the washed microorganisms with a sol, and finally solidifying them through cooling or chemical reaction. Therefore, the preparation process is cumbersome. Summary of the Invention

[0004] The present invention aims to solve the technical problems in the prior art and provides a method for preparing a microbial electrode for a water toxicity detection device.

[0005] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0006] This invention provides a method for preparing a microbial electrode for a water toxicity detection device, comprising the following steps:

[0007] (1) Prepare a mixed solution of peptone, yeast extract powder, sodium chloride and water. After sterilization, the mixed solution is sealed and stored for later use.

[0008] (2) Inoculate the bacterial strain into the mixed solution obtained in step (1) and culture it to obtain a microbial solution;

[0009] (3) Prepare an aqueous solution of sodium chloride. Centrifuge the microbial solution obtained in step (2), remove the supernatant, and then add an aqueous solution of sodium chloride to redisperse the microorganisms to obtain a redispersed microbial solution.

[0010] (4) Spread the microbial solution obtained in step (3) onto the electrode surface. After the electrode surface dries, a microbial electrode for water toxicity detection equipment is obtained.

[0011] In the above technical solution, in step (1), in the mixed solution of peptone, yeast extract, sodium chloride and water, the mass ratio of peptone to water is 4-10:1000, the mass ratio of yeast extract to water is 4-10:1000, and the mass ratio of sodium chloride to water is 7-12:1000.

[0012] In the above technical solution, in step (2), the culture temperature of the microorganism is 20-42℃ and the time is 12-24h.

[0013] In the above technical solution, in step (3), the sodium chloride aqueous solution contains 0.7% to 1.2% sodium chloride by mass.

[0014] In the above technical solution, in step (3), the centrifugation speed is 5000 rpm and the centrifugation time is 3 to 8 min.

[0015] In the above technical solution, in step (3), the volume ratio of the sodium chloride aqueous solution used to redisperse the microorganisms to the volume of the microbial solution before centrifugation is 1 to 2:10.

[0016] In the above technical solution, in step (4), the drying temperature is 30℃~60℃ and the drying time is 15min~60min.

[0017] In the above technical solution, in step (4), the electrode is ITO conductive glass or FTO conductive glass.

[0018] The beneficial effects of this invention are:

[0019] This invention provides a method for preparing a microbial electrode for a water toxicity detection device. In this method, cultured microorganisms are first deposited at the bottom of a test tube by centrifugation, and then the supernatant solution is discarded. The microorganisms are redispersed with physiological saline. Subsequently, the solution containing the microorganisms is dropped onto the electrode surface. After the solution dries, the extracellular polymers remaining in the solution act as a binder, immobilizing the microorganisms on the electrode surface. The microbial film formed on the electrode surface is stable and can be used for flow analysis detection of water toxicity. In the formation of in-situ cultured microbial films, microorganisms need to secrete and adsorb large amounts of extracellular polymers to ensure they do not detach from the microbial film during proliferation and growth. Compared to microorganisms in in-situ cultured microbial films, suspended microorganisms adsorb very little extracellular polymer. Therefore, the microbial film provided by this invention has a lower extracellular polymer content than in-situ grown biofilms and is more sensitive to toxicity. For example, 1 mg / L Hg 2+ The activity inhibition rate of the microbial membrane prepared by this invention was 23%, while 2 mg / L Hg 2+ The inhibition rate of in-situ cultured microbial membrane activity was only 13.99% (Toxicity detection in water containing heavy metal ions with a self-powered microbial fuel cell-based biosensor. Talanta, 2017, 168, 210-216). Compared with the gelation method, this method is simpler to operate. Attached Figure Description

[0020] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0021] Figure 1 This is a schematic diagram of the microbial electrode prepared in Example 1 used for toxicity detection.

[0022] Figure 2 This is a schematic diagram of the microbial electrode prepared in Example 2 used for toxicity detection.

[0023] Figure 3 This is a schematic diagram of the microbial electrode prepared in Example 3 used for toxicity detection.

[0024] Figure 4 This is a schematic diagram of the microbial electrode prepared in Example 4 used for toxicity detection. Detailed Implementation

[0025] Example 1

[0026] A method for preparing a microbial electrode for a water toxicity detection device includes the following steps:

[0027] (1) Add 1.6g peptone, 4g yeast extract and 4g sodium chloride to 400mL of deionized water. Place the solution in an autoclave and maintain the temperature at 121℃ for 15min to obtain a sterilized solution;

[0028] (2) Add 100 μL of tap water to the sterilized solution obtained in step (1). Then place the solution in a shaker and incubate at 30°C and 200 rpm for 16 h. This yields a microbial solution.

[0029] (3) Dissolve 1g of sodium chloride in 100mL of deionized water to obtain a sodium chloride solution. Pour 10mL of the microbial solution obtained in step (2) into a centrifuge tube and centrifuge at 5000rpm for 5min. After pouring the supernatant out of the centrifuge tube, add 2mL of sodium chloride solution to the centrifuge tube and shake to disperse the microorganisms at the bottom of the centrifuge tube. A microbial solution with low extracellular polymer content is obtained;

[0030] (4) According to 80 μL / cm 2 The amount of microbial solution with low extracellular polymeric content obtained in step (3) is drop-coated onto the ITO electrode. After drying at 50°C for 15 minutes, a microbial electrode for water toxicity detection equipment is obtained. When this electrode is used for toxicity detection, 5 mg / L Hg... 2+ The inhibition rate was 76%, see [reference needed]. Figure 1 .

[0031] Example 2

[0032] A method for preparing a microbial electrode for a water toxicity detection device includes the following steps:

[0033] (1) Add 4.0g peptone, 1.6g yeast extract powder and 4.0g sodium chloride to 400mL of deionized water. Place the solution in an autoclave and maintain the temperature at 121℃ for 15min to obtain a sterilized solution;

[0034] (2) Add E. coli to the sterilized solution obtained in step (1). Then place the solution in a shaker and incubate at 37°C and 220 rpm for 12 hours. This yields the microbial solution.

[0035] (3) Dissolve 0.8g of sodium chloride in 100mL of deionized water to obtain a sodium chloride solution. Pour 10mL of the microbial solution obtained in step (2) into a centrifuge tube and centrifuge at 5000rpm for 3min. After pouring the supernatant out of the centrifuge tube, add 1mL of sodium chloride solution to the centrifuge tube and shake to disperse the microorganisms at the bottom of the centrifuge tube. A microbial solution with low extracellular polymer content is obtained;

[0036] (4) According to 60 μL / cm2 The amount of microbial solution with low extracellular polymeric content obtained in step (3) is drop-coated onto the FTO electrode. After drying at 40°C for 60 minutes, a microbial electrode for water toxicity detection equipment is obtained. When this electrode is used for toxicity detection, 20 mg / L Hg... 2+ The inhibition rate was 76%, see [reference needed]. Figure 2 .

[0037] Example 3

[0038] A method for preparing a microbial electrode for a water toxicity detection device includes the following steps:

[0039] (1) Add 0.8g peptone, 2g yeast extract powder and 1.4g sodium chloride to 200mL of deionized water. Place the solution in an autoclave and maintain the temperature at 121℃ for 15min to obtain a sterilized solution;

[0040] (2) Add BODseed to the sterilized solution obtained in step (1). Then place the solution in a shaker and incubate at 20°C and 200 rpm for 24 hours. This yields a microbial solution.

[0041] (3) Dissolve 1.2g of sodium chloride in 100mL of deionized water to obtain a sodium chloride solution. Pour 10mL of the microbial solution obtained in step (2) into a centrifuge tube and centrifuge at 5000rpm for 8min. After pouring the supernatant out of the centrifuge tube, add 2mL of sodium chloride solution to the centrifuge tube and shake to disperse the microorganisms at the bottom of the centrifuge tube. A microbial solution with low extracellular polymeric content is obtained.

[0042] (4) According to 40 μL / cm 2 The amount of microbial solution with low extracellular polymeric content obtained in step (3) is drop-coated onto the ITO electrode. After drying at 30°C for 60 min, a microbial electrode for water toxicity detection equipment is obtained. When this electrode is used for toxicity detection, 5 mg / L Hg... 2+ The inhibition rate was 98%, see [reference needed]. Figure 3 .

[0043] Example 4

[0044] A method for preparing a microbial electrode for a water toxicity detection device includes the following steps:

[0045] (1) Add 1.6g peptone, 3g yeast extract powder and 4.8g sodium chloride to 400mL of deionized water. Place the solution in an autoclave and maintain the temperature at 121℃ for 15min to obtain a sterilized solution;

[0046] (2) Add 100 μL of tap water to the sterilized solution obtained in step (1). Then place the solution in a shaker and incubate at 40℃ and 200 rpm for 16 h. This yields a microbial solution.

[0047] (3) Dissolve 1.0 g of sodium chloride in 100 mL of deionized water to obtain a sodium chloride solution. Pour 10 mL of the microbial solution obtained in step 12) into a centrifuge tube and centrifuge at 5000 rpm for 5 min. After pouring the supernatant out of the centrifuge tube, add 1 mL of sodium chloride solution to the centrifuge tube and shake to disperse the microorganisms at the bottom of the centrifuge tube. A microbial solution with low extracellular polymer content is obtained;

[0048] (4) According to 80 μL / cm 2 The amount of microbial solution with low extracellular polymeric content obtained in step (3) is drop-coated onto the ITO electrode. After drying at 60°C for 15 minutes, a microbial electrode for water toxicity detection equipment is obtained. When this electrode is used for toxicity detection, 5 mg / L Hg... 2+ The inhibition rate was 94%, see Figure 4 .

[0049] Example 5

[0050] A method for preparing a microbial electrode for a water toxicity detection device includes the following steps:

[0051] (1) Add 2.0g peptone, 2.0g yeast extract and 4.0g sodium chloride to 400mL of deionized water. Place the solution in an autoclave and maintain the temperature at 121℃ for 15min to obtain a sterilized solution;

[0052] (2) Add 100 μL of tap water to the sterilized solution obtained in step (1). Then place the solution in a shaker and incubate at 42℃ and 200 rpm for 18 h. This yields a microbial solution.

[0053] (3) Dissolve 1.2g of sodium chloride in 100mL of deionized water to obtain a sodium chloride solution. Pour 10mL of the microbial solution obtained in step (2) into a centrifuge tube and centrifuge at 5000rpm for 6min. After pouring the supernatant out of the centrifuge tube, add 1mL of sodium chloride solution to the centrifuge tube and shake to disperse the microorganisms at the bottom of the centrifuge tube. A microbial solution with low extracellular polymeric content is obtained.

[0054] (4) According to 40 μL / cm 2 The amount of microbial solution with low extracellular polymeric content obtained in step (3) is drop-coated onto the ITO electrode. After drying at 60°C for 15 minutes, a microbial electrode for water toxicity detection equipment is obtained. When this electrode is used for toxicity detection, 5 mg / L Hg... 2+The inhibition rate was 96%.

[0055] Example 6

[0056] A method for preparing a microbial electrode for a water toxicity detection device includes the following steps:

[0057] (1) Add 0.6g peptone, 1.0g yeast extract and 1g sodium chloride to 100mL of deionized water. Place the solution in an autoclave and maintain the temperature at 121℃ for 15min to obtain a sterilized solution;

[0058] (2) Add 100 μL of tap water to the sterilized solution obtained in step (1). Then place the solution in a shaker and incubate at 37°C and 180 rpm for 18 h. This yields a microbial solution.

[0059] (3) Dissolve 1.2g of sodium chloride in 100mL of deionized water to obtain a sodium chloride solution. Pour 10mL of the microbial solution obtained in step (2) into a centrifuge tube and centrifuge at 5000rpm for 7min. After pouring the supernatant out of the centrifuge tube, add 1mL of sodium chloride solution to the centrifuge tube and shake to disperse the microorganisms at the bottom of the centrifuge tube. A microbial solution with low extracellular polymeric content is obtained.

[0060] (4) According to 40 μL / cm 2 The amount of microbial solution with low extracellular polymeric content obtained in step (3) is drop-coated onto the ITO electrode. After drying at 50°C for 15 minutes, a microbial electrode for water toxicity detection equipment is obtained. When this electrode is used for toxicity detection, 5 mg / L Hg... 2+ The inhibition rate was 97%.

[0061] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a microbial electrode for a water body toxicity detection device, characterized by, The method comprises the following steps: (1) preparing a mixed solution of proteose peptone, yeast extract powder, sodium chloride and water, sterilizing the mixed solution and sealing it for later use; (2) inoculating a strain into the mixed solution obtained in step (1) and culturing to obtain a microbial solution; (3) preparing an aqueous sodium chloride solution, centrifuging the microbial solution obtained in step (2) to remove the upper solution, adding the aqueous sodium chloride solution to re-disperse the microorganisms, and obtaining a microbial solution with low extracellular polymer content; (4) spreading the microbial solution obtained in step (3) on the surface of an electrode, and obtaining a microbial electrode for a water toxicity detection device after the electrode surface is dried.

2. The production method according to claim 1, characterized by, In step (1), the mass ratio of proteose peptone to water in the mixed solution of proteose peptone, yeast extract powder, sodium chloride and water is 4-10:1000, the mass ratio of yeast extract powder to water is 4-10:1000, and the mass ratio of sodium chloride to water is 7-12:1000.

3. The preparation method according to claim 1, characterized in that, In step (2), the culture temperature of the microorganisms is 20-42°C, and the culture time is 12-24 hours.

4. The method of claim 1, wherein, In step (3), the mass percentage of sodium chloride in the aqueous sodium chloride solution is 0.7%-1.2%.

5. The preparation method according to claim 1, characterized in that, In step (3), the centrifugation speed is 5000 rpm, and the centrifugation time is 3-8 minutes.

6. The method of claim 1, wherein, In step (3), the volume ratio of the aqueous sodium chloride solution used for re-dispersing the microorganisms to the volume of the microbial solution before centrifugation is 1-2:

10.

7. The preparation method according to claim 1, characterized in that, In step (4), the drying temperature is 30-60°C, and the drying time is 15-60 minutes.

8. The method of claim 1, wherein, In step (4), the electrode is ITO conductive glass or FTO conductive glass.

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