Method and device for detecting toxicity of water body through photoelectric double signals

Abstract

The invention belongs to the technical field of water body detection, and particularly relates to a method and a device for detecting water body toxicity through photoelectric double signals. The method comprises the following steps: adding a microbial solution into a to-be-detected sample to obtain a first solution; adding a tungsten trioxide suspension and a first culture medium into the first solution to obtain a second solution, and obtaining a first signal through an electric signal detection device, and / or adding a second culture medium into the first solution to obtain a third solution, and obtaining a second signal through an optical signal detection device, wherein the microorganisms are microorganisms with anaerobic electrogenesis and aerobic luminescence metabolic pathways at the same time. According to the method, the toxicity of the water body can be effectively and stably detected in anaerobic and oxygen-enriched environments at the same time, microorganisms are kept in a suspended state during testing, a natural biological membrane does not need to be formed on the surface of an electrode, and the microorganisms can be in direct contact with toxic substances, so that the defects of a traditional single-signal biosensor in the aspects of broad spectrum and reliability of water quality early warning are overcome.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of water body detection, and particularly relates to a method and device for detecting water toxicity through photoelectric dual signals. BACKGROUND

[0002] Water pollution has become a major environmental challenge that needs to be solved globally, and its complexity is due to the diversity of pollutant sources, the concealment of migration and transformation processes, and the accumulation of ecological risks. Under this background, water quality early warning technology is of great significance to protect water environment safety, prevent pollution incidents and maintain ecosystem health. Biological detection has attracted widespread attention due to its intuitive results. Among them, electroactive biosensors have the advantages of rapid response, simple maintenance, low running cost, and can identify unknown pollutants or the toxicity risk of multiple pollutants.

[0003] However, in order to avoid the competition of dissolved oxygen in water samples for electrons leading to low electrical signals, electroactive biosensors generally need to be operated in low-oxygen environments, which limits their application in different water environments. Secondly, it takes several days for electroactive biosensors to naturally form a biological membrane on the electrode surface, and the three-dimensional multi-layer structure of the electroactive biological membrane and the extracellular polymers such as polysaccharides and proteins inside affect the transmission of toxic substances, and the biological capacitance characteristics also affect the transmission of electrical signals, resulting in signal lag. In addition, the morphological structure and community composition of different naturally formed biological membranes are different, and the reproducibility and standardization are difficult.

[0004] Therefore, there is an urgent need for a standardized method that can detect water toxicity in both anaerobic and aerobic environments effectively and stably. SUMMARY

[0005] The present application aims to at least partially solve one of the technical problems in the related art.

[0006] To this end, an embodiment of the first aspect of the present application proposes a method for detecting water toxicity through photoelectric dual signals, comprising: adding a microbial solution to a sample to be tested to obtain a first solution; adding tungsten trioxide suspension and a first culture medium to the first solution to obtain a second solution, and obtaining a first signal through an electrical signal detection device, and / or adding a second culture medium to the first solution to obtain a third solution, and obtaining a second signal through a light signal detection device, wherein the microorganism is a microorganism that has both anaerobic electricity production and aerobic luminescence metabolic pathways. This method can effectively and stably detect water toxicity in both anaerobic and aerobic environments, and the microorganism remains in a suspended state during testing, without the need to slowly form a natural biological membrane on the electrode surface, and the microorganism can directly contact with the toxic substances, enhancing the mass transfer process, and making up for the shortcomings of traditional single signal biosensors in terms of broad spectrum and reliability in water quality early warning.

[0007] In some embodiments, the microorganism is Shewanella wisseniensis MS32, having the accession number of ATCC 51908 or CIP 105547 or DSM 12036.

[0008] In some embodiments, the OD of the microorganism solution after being diluted by 50 times is 0.1-0.4, optionally, the OD of the microorganism solution after being diluted by 50 times is 0.2. 600 600 In some embodiments, the OD of the microorganism solution after being diluted by 50 times is 0.1-0.4, optionally, the OD of the microorganism solution after being diluted by 50 times is 0.2.

[0009] In some embodiments, the concentration of the tungsten trioxide suspension is 10-40 g / L, preferably, the concentration of the tungsten trioxide suspension is 25 g / L.

[0010] In some embodiments, the method further comprises: adding the tungsten trioxide suspension and the first medium to the microorganism solution only, as a non-contamination control group; and / or adding the second medium to the microorganism solution only, as a non-contamination control group.

[0011] In some embodiments, wherein the first medium is a deoxygenated medium, based on the anaerobic electricity-generating metabolic pathway of the microorganism, the obtaining the first signal by the electrical signal detection device comprises: taking 0.2 mL of the second solution in a 96-well quartz glass enzyme-labeled plate every 10 minutes, imaging the color of the tungsten trioxide in the second solution with a scanner, and quantitatively characterizing the color as the first signal; and / or wherein the second medium is an oxygen-exposed medium, based on the aerobic luminescence metabolic pathway of the microorganism, the obtaining the second signal by the light signal detection device comprises detecting the light signal of the microorganism by a photomultiplier tube as the second signal.

[0012] In some embodiments, the method further comprises: comparing the first signal of the second solution and the non-contamination control group, determining that the water body has toxicity based on the first signal of the second solution being lower than the first signal of the non-contamination control group, and / or comparing the second signal of the third solution and the non-contamination control group, determining that the water body has toxicity based on the second signal of the second solution being lower than the second signal of the non-contamination control group.

[0013] In some embodiments, the detecting the toxicity of the water body comprises detecting metals and metal oxides, non-metal inorganic substances, and organic pollutants in the water body, optionally, the organic pollutants comprise phenols, ethers, alcohols, aldehydes, preferably, detecting copper ions and / or formaldehyde in the water body.

[0014] ​The embodiment of the second aspect of the present application provides a detection device for detecting water toxicity, comprising: a solution mixing chamber for mixing a microbial solution and a sample solution to be detected; and an electrical signal detection device for adding tungsten trioxide suspension, a first culture medium and detecting the color change of tungsten trioxide caused by the electricity generation of the microorganism; and / or a light signal detection device for adding a second culture medium and detecting the change of light signal caused by the luminescence of the microorganism, wherein the microorganism is a microorganism having both anaerobic electricity generation and aerobic luminescence metabolic pathways.

[0015] In some embodiments, the first culture medium is a deoxygenated culture medium, the second culture medium is an oxygenated culture medium, the microorganism is Shewanella woonjiensis MS32, the preservation number of which is ATCC 51908 or CIP 105547 or DSM12036, the electrical signal detection device comprises a scanner, and the light signal detection device comprises a photomultiplier tube.

[0016] The advantages and technical effects brought by the independent claims according to the embodiments of the present application are as follows:

[0017] (1) The embodiments of the present application first provide Shewanella woonjiensis MS32 as a toxicity sensitive element, which has both anaerobic electricity generation and aerobic luminescence metabolic pathways, and can realize water quality monitoring in different dissolved oxygen water environments.

[0018] (2) The method of the embodiments of the present application keeps the microorganism in a suspended state when applied, and does not need to form a natural biofilm slowly on the electrode surface, so that the microorganism can directly contact with the toxic substance, thereby enhancing the mass transfer process. The electrochromic material (such as tungsten trioxide) is introduced to construct an electrical-optical signal conversion module, so that the output electrical signal change of the suspended state electroactive microorganism can be quickly and timely mapped through the color change of tungsten trioxide.

[0019] (3) The method of the embodiments of the present application unifies the toxicity response of anaerobic electricity generation and aerobic luminescence pathways into light signals, so that the test results are more comparable.

[0020] In summary, the microbial light-electricity dual-channel water quality toxicity sensor proposed in the embodiments of the present application makes up for the shortcomings of traditional single signal biological sensors in terms of broad spectrum and reliability of water quality early warning. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is the XRD analysis result of tungsten trioxide used in the method of the embodiments of the present application.

[0022] Figure 2The results of detecting water pollution by the method for detecting water body toxicity through photoelectric double signal in the embodiment of the application are shown. DETAILED DESCRIPTION

[0023] Embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings. The embodiments described below by reference to the drawings are exemplary and are intended to explain the present application, and cannot be understood as a limitation of the present application.

[0024] The present application is made based on the findings and recognitions of the inventors on the following facts and problems:

[0025] Water environmental pollution has become a major environmental challenge that needs to be solved globally, and its complexity is due to the diversity of pollutant sources, the concealment of migration and transformation process, and the accumulation of ecological risk. In this context, water quality early warning technology is of great significance to protect water environmental safety, prevent pollution incidents, and maintain the health of the ecological system.

[0026] Traditional water quality monitoring methods rely on single-dimensional analysis of physical and chemical indicators, which can reflect the content of pollutants, but it is difficult to directly link to biological effects and ecological risks, and indirect assessment is needed according to established standards, which is difficult to quickly respond to comprehensive risks in complex pollution scenarios. In contrast, electroactive biosensors have the advantages of rapid response, simple maintenance, and low operating cost, and can identify the toxicity risk of unknown pollutants or the combined action of multiple pollutants. Electroactive microorganisms can produce electrons through intracellular metabolism and transfer to the electrode through extracellular electron transfer, and then output an electrical signal. Since the pollutants in the water sample will inhibit the intracellular metabolic activity and extracellular electron transfer process of electroactive microorganisms, the output electrical signal will decrease. Then the electrochemical workstation is used to quantitatively analyze the inhibition rate of the electrical signal, so as to evaluate the acute toxicity of the water body.

[0027] However, most electroactive biosensors use electroactive biofilms naturally grown on the electrode surface as sensitive elements. In order to avoid the competition of dissolved oxygen in the water sample for electrons leading to low electrical signals, electroactive biosensors generally need to be operated in a low-oxygen environment, which limits their application in different water environments. Secondly, the three-dimensional multilayer structure of the electroactive biofilm and the extracellular polymers such as polysaccharides and proteins inside it affect the transmission of toxic substances, and its bioelectricity capacity characteristics also affect the transmission of electrical signals, which has signal lag. The morphological structure and community composition of different naturally formed biofilms are different, and it is difficult to reproduce and standardize.

[0028] To this end, embodiments of the first aspect of the application propose a method for detecting toxicity of a water body by photoelectric dual signal, comprising: adding a microbial solution to a sample to be tested to obtain a first solution; adding tungsten trioxide suspension and a first culture medium to the first solution to obtain a second solution, and obtaining a first signal by an electrical signal detection device, and / or adding a second culture medium to the first solution to obtain a third solution, and obtaining a second signal by a light signal detection device, wherein the microorganism is a microorganism having both anaerobic electricity generation and aerobic luminescence metabolic pathways. The method can effectively and stably detect the toxicity of the water body in anaerobic and oxygen-rich environments at the same time. The microorganism remains in a suspended state during testing, without the need for slow formation of a natural biofilm on the electrode surface. The microorganism can directly contact the toxic substance, enhancing the mass transfer process and making up for the lack of broad-spectrum and reliability of traditional single-signal biosensors in water quality early warning.

[0029] In some embodiments, the microorganism is Shewanella wissu MS32, which has a preservation number of ATCC 51908 or CIP 105547 or DSM 12036.

[0030] In some embodiments, the OD 600 of the microbial solution after being diluted 50 times is 0.1-0.4. Optionally, the OD 600 of the microbial solution after being diluted 50 times is 0.2. It should be noted that the concentration of the microorganism in the microbial solution is defined in this way.

[0031] In some embodiments, the concentration of the tungsten trioxide suspension is 10-40 g / L, and preferably, the concentration of the tungsten trioxide suspension is 25 g / L.

[0032] In some embodiments, the method further comprises: only adding the tungsten trioxide suspension and the first culture medium to the microbial solution as a non-pollution control group; and / or only adding the second culture medium to the microbial solution as a non-pollution control group.

[0033] In some embodiments, wherein the first culture medium is a deoxygenated culture medium, based on the anaerobic electricity generation metabolic pathway of the microorganism, the obtaining of the first signal by the electrical signal detection device comprises: taking 0.2 mL of the second solution in a 96-well quartz glass enzyme plate every 10 minutes, imaging the color of the tungsten trioxide in the second solution with a scanner, and quantitatively characterizing the color as the first signal; and / or wherein the second culture medium is an oxygen-exposed culture medium, based on the aerobic luminescence metabolic pathway of the microorganism, the obtaining of the second signal by the light signal detection device comprises detecting the light signal of the microorganism by a photomultiplier tube as the second signal.

[0034] In some embodiments, the method further comprises comparing the first signal of the second solution and the first signal of the non-polluted control group, determining that the water body is toxic based on the first signal of the second solution being lower than the first signal of the non-polluted control group, and / or comparing the second signal of the third solution and the second signal of the non-polluted control group, determining that the water body is toxic based on the second signal of the second solution being lower than the second signal of the non-polluted control group.

[0035] In some embodiments, the detecting water body toxicity comprises detecting metals and metal oxides, non-metal inorganic substances and organic pollutants in the water body, optionally, the organic pollutants comprise phenols, ethers, alcohols, aldehydes, preferably, detecting copper ions and / or formaldehyde in the water body. In some embodiments, solutions with gradient concentrations of water body pollutants can also be detected, and the corresponding obtained concentration-light / electric signal curves can be used as standard curves. Based on the standard curves, the concentration of water body pollutants in the sample to be tested can be quantified when the light / electric signal of the sample to be tested is obtained.

[0036] Embodiments of the second aspect of the present application propose a detection device for detecting water body toxicity, comprising: a solution mixing chamber for mixing a microbial solution and a sample solution to be tested; and an electric signal detection device for adding tungsten trioxide suspension, a first culture medium and detecting the color change of tungsten trioxide caused by the electricity generation of the microorganism; and / or a light signal detection device for adding a second culture medium and detecting the light signal change caused by the luminescence of the microorganism, wherein the microorganism is a microorganism with both anaerobic electricity generation and aerobic luminescence metabolic pathways.

[0037] In some embodiments, the first culture medium is a deoxygenated culture medium, the second culture medium is an oxygen-exposed culture medium, the microorganism is Shewanella wissuensis MS32 with accession number ATCC 51908 or CIP 105547 or DSM12036, the electric signal detection device comprises a scanner, and the light signal detection device comprises a photomultiplier tube.

[0038] In some specific embodiments, the microorganism is a microorganism with both anaerobic electricity generation and aerobic luminescence metabolic pathways.

[0039] When the microbial solution and the deoxygenated medium are added to the sample to be tested, the microorganism produces an electrical signal, i.e., a first signal, through an anaerobic electricity-producing metabolic pathway. For the electrical signal, those skilled in the art can use various electrical signal detection devices for detection. In the present embodiment, tungsten trioxide suspension is also added at the same time as the medium, and the solution, at a concentration obtained through experiments, can obtain the electrons produced by the microorganism and further convert them into a light signal. The signal can be quantitatively detected by a scanner. In some specific embodiments, the microorganism is a microorganism having both anaerobic electricity-producing and aerobic light-emitting metabolic pathways. When the microbial solution and the oxygen-rich / exposed oxygen medium are added to the sample to be tested, the microorganism produces a light signal, i.e., a second signal, through an aerobic light-emitting metabolic pathway. For the light signal, those skilled in the art can use various light signal detection devices for detection. In the present embodiment, the light signal is quantitatively detected by a photomultiplier tube.

[0040] The schemes of the present application will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. If no specific technique or condition is specified in the examples, the technique or condition described in the literature in the art or according to the product manual is used. If no manufacturer of the reagent or instrument is specified, it is a conventional product that can be obtained commercially.

[0041] Unless otherwise specified, the quantitative analysis test in the following examples is set up with three repeated experiments, and the average value is taken.

[0042] Example

[0043] In this embodiment, copper ions and formaldehyde are detected in anaerobic and oxygen-rich water bodies by the method of photoelectric dual-signal detection of water toxicity.

[0044] (1) Bacterial culture: The Shewanella wuana MS32 is enriched and cultured using a commercially available 2216E medium (item number: LA0341; purchased from: Solarbio), and reaches the logarithmic growth phase after about 20-24 hours. It is collected by centrifugation at 6000-8000 rpm and washed 3 times with 2.5 times phosphate buffer (20.02 g / L NaCl; 1.00 g / L KCl; 1.22 g / L KH2PO4; 8.95 g / L Na2HPO4·12H2O, adjust pH to 7.4±0.2) to remove the residual medium in the bacterial solution. Finally, it is resuspended in 2.5 times phosphate buffer. The concentrated bacterial solution is diluted 50 times, and the OD600 is measured to be 0.2.

[0045] (2) Preparation of tungsten trioxide: A solution containing 41.25 g / L Na2WO4·2H2O and 14.5 g / L NaCl was prepared, and 3 mol / L HCl was slowly added to adjust the pH value to 2.0 ± 0.1. 45 mL of the solution was taken into a 100 mL stainless steel autoclave, and heated in an oven at 180°C for 16 hours. After the autoclave was cooled, all the solution was collected, filtered with a 0.45 μm microporous filter membrane, and the white substance on the surface of the filter membrane was collected, washed with ultrapure water, and filtered again, repeated for 3 times. Finally, the collected white substance was dried at 60°C, and carefully ground with a mortar for XRD analysis. Finally, a 25 g / L tungsten trioxide suspension was prepared.

[0046] (3) Water quality early warning in anaerobic electricity production mode: 1 mL of the concentrated bacterial solution prepared in step (1), 1 mL of the 25 g / L tungsten trioxide suspension, and 8 mL of nitrogen-deaerated medium (its composition is 5 g / L tryptone, 1 g / L yeast extract powder, 30.15 g / L NaCl, 6.16 g / L MgSO4·7H2O, 5.08 g / L MgCl2·6H2O, 1.49 g / L KCl, and the pH value is adjusted to 7.4 ± 0.2 with 1 mol / L NaOH) were added into a serum bottle. Then different concentrations of toxic substances (1 mg / L, 2 mg / L, 3 mg / L, 5 mg / L, 7 mg / L of copper ions (by adding reagent CuCl2) and 0.0025%, 0.005%, 0.0010%, 0.015%, 0.020% of formaldehyde) were added as the contaminated water sample to be tested. Every 10 minutes, 0.2 mL of water sample was taken into a 96-well quartz glass enzyme-labeled plate, and the color of tungsten trioxide in the water sample was imaged with a scanner, and the color change was quantitatively characterized by image analysis software Fiji imageJ.

[0047] (4) Water quality early warning in aerobic luminescence mode: 1 mL of the concentrated bacterial solution and 8 mL of fully aerated medium (its composition is the same as (3)) were added into a quartz glass bottle, and different concentrations of toxic substances (1 mg / L, 2 mg / L, 3 mg / L, 5 mg / L, 7 mg / L of copper ions (by adding reagent CuCl2) and 0.0025%, 0.005%, 0.0010%, 0.015%, 0.020% of formaldehyde) were added as the contaminated water sample to be tested. The light signal produced by the microorganism at different times was monitored in a dark environment by using a photomultiplier tube.

[0048] Figure 1 is the XRD analysis result of the tungsten trioxide used in the method in the embodiment of the present application. The result shows that the synthesized tungsten trioxide is of hexagonal crystal structure, and has strong electrochromic ability.

[0049] Figure 2The result of detecting water pollution by the method for detecting water body toxicity by photoelectric double signal in the embodiment of the application.

[0050] According to Figure 2 The water body toxicity detection method and device provided by the embodiment of the application can respond to various toxic substances (such as heavy metals and organic pollutants), the concentration of the toxic substance has a significant linear correlation with the signal inhibition rate, and the rapid early warning of water body toxicity can be realized.

[0051] In addition, the terms "first", "second", "third", etc. are used only for descriptive purposes and should not be construed as indicating or implying relative importance or an indicated number of technical features. Therefore, the features defined as "first", "second", etc. can explicitly or implicitly include at least one of the features. In the description of the application, the meaning of "a plurality of" is at least two, for example, two, three, etc., unless otherwise explicitly and specifically limited.

[0052] In the present application, the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, the skilled person in the art can combine and combine the different embodiments or examples described in the specification and the features of the different embodiments or examples without contradiction.

[0053] Although the embodiments of the present application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. A method for detecting toxicity of a water body by photoelectric dual signal, characterized in that, comprising: adding a microorganism solution to a sample to be tested to obtain a first solution; adding tungsten trioxide suspension and a first medium to the first solution to obtain a second solution, and obtaining a first signal by an electrical signal detection device, and / or adding a second medium to the first solution to obtain a third solution, and obtaining a second signal by a light signal detection device, wherein the microorganism is a microorganism having both anaerobic electricity generation and aerobic luminescence metabolic pathways.

2. The method of claim 1, wherein, The microorganism is Shewanella wuismanii MS32, which has the accession number ATCC 51908 or CIP 105547 or DSM 12036.

3. The method of claim 1, wherein, OD of the microbial solution after 50-fold dilution 600 is 0.1-0.4, optionally, OD of the microbial solution after 50-fold dilution 600 is 0.

2.

4. The method of claim 1, wherein, The concentration of the tungsten trioxide suspension is 10-40 g / L, preferably, the concentration of the tungsten trioxide suspension is 25 g / L.

5. The method of claim 1, wherein, Further comprising: adding only the tungsten trioxide suspension and the first medium to the microorganism solution as a non-contaminated control group; and / or adding only the second medium to the microorganism solution as a non-contaminated control group.

6. The method of claim 1, wherein the first medium is a deoxygenated medium, and based on the anaerobic electricity generation metabolic pathway of the microorganism, the obtaining a first signal by an electrical signal detection device comprises taking 0.2 mL of the second solution in a 96-well quartz glass enzyme-coated plate every 10 minutes, imaging the color of the tungsten trioxide in the second solution with a scanner, and quantitatively characterizing the color as the first signal; and / or the second medium is an oxygen-exposed medium, and based on the aerobic luminescence metabolic pathway of the microorganism, the obtaining a second signal by a light signal detection device comprises detecting the light signal of the microorganism by a photomultiplier tube as the second signal.

7. The method of claim 5, wherein, Further comprising: comparing the first signal of the second solution and the non-contaminated control group, and based on the first signal of the second solution being lower than the first signal of the non-contaminated control group, determining that the water body has toxicity, and / or comparing the second signal of the third solution and the non-contaminated control group, and based on the second signal of the second solution being lower than the second signal of the non-contaminated control group, determining that the water body has toxicity.

8. The method of claim 1, wherein, The detecting water body toxicity comprises detecting metal and metal oxide, non-metal inorganic substance and organic pollutant in the water body, optionally, the organic pollutant comprises phenol, ether, alcohol, aldehyde, preferably, detecting copper ion and / or formaldehyde in the water body.

9. A detection device for detecting toxicity of a water body, characterized in that, comprising: a solution mixing chamber for mixing a microorganism solution and a sample solution to be tested; and an electrical signal detection device for adding tungsten trioxide suspension, a first medium, and detecting the color change of tungsten trioxide caused by electricity generation of the microorganism; and / or a light signal detection device for adding a second medium and detecting the light signal change caused by luminescence of the microorganism, wherein the microorganism is a microorganism having both anaerobic electricity generation and aerobic luminescence metabolic pathways.

10. The device of claim 9, wherein said first culture medium is a deoxygenated culture medium, said second culture medium is an oxygenated culture medium, said microorganism is Shewanella wooledensis MS32, having the accession number ATCC 51908 or CIP 105547 or DSM 12036, said electrical signal detection device comprises a scanner, said optical signal detection device comprises a photomultiplier.

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