CN114965617A - Method for identifying false positive of electrochemically active microorganisms for detecting toxic pollutants caused by electron acceptor - Google Patents
Method for identifying false positive of electrochemically active microorganisms for detecting toxic pollutants caused by electron acceptor Download PDFInfo
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Abstract
The invention relates to a method for identifying false positive of an electrochemically active microorganism detection toxic pollutant caused by an electron acceptor. The method can realize the identification of false positive problem of water quality biotoxicity detection alarm caused by electron acceptor by designing a two-step detection method and utilizing the electrochemical active microorganisms with bioelectrochemical system output electric signal inhibition rate and bidirectional electron transfer capability to generate reverse current. The two-step detection process established by the invention has the advantages of rapidness, high efficiency, convenient operation, high sensitivity and the like of a bioelectrochemical system in water quality detection, and simultaneously eliminates the problem of false positive alarm caused by an electron acceptor in detection. In addition, the electrochemical system in the two-step detection process runs relatively independently, so that the detection cost is saved and the detection efficiency is improved while the accuracy of the detection result is ensured.
Description
Technical Field
The invention relates to the field of water quality detection, in particular to a water quality biotoxicity false positive detection method by using electrochemical active microorganisms.
Background
The water quality detection plays a crucial role in the whole water environment protection, water pollution control and water environment health maintenance. For drinking water, harmful bacteria, heavy metal pesticides, etc. in the water can cause various diseases; for industrial water, mineral impurities, acid and alkali ions in water affect product quality or damage containers and pipelines, so that water quality detection is a big concern for people and is not a little worth. Although the traditional water quality detection methods such as a chemical pyrolysis method, an atomic fluorescence detection method, a liquid chromatography method and the like have the advantages of high detection precision, good stability, strong repeatability and the like, the problems of complex operation steps, dangerous reaction conditions, long detection time, high technical requirements, easy secondary pollution and the like exist at the same time. The water quality biotoxicity detection technology based on the biosensor is more and more emphasized because of its fast analysis speed, wide detection range and capability of analyzing the comprehensive toxicity of various pollutants at the same time, and has become one of the basic measures for ensuring the safety of water supply and ecological environment.
The water quality biotoxicity detection is a detection method developed based on biological toxicology, aquatic organisms live in a water environment for a long time, and the health state of the aquatic organisms can reflect the pollution degree of the water environment. Under certain conditions, the change of the physiological behavior of the aquatic organisms can be used as an important index for evaluating the water environment safety. Such as fish, fleas, algae, and microorganisms, are often selected as test organisms and water toxicity is assessed by changes in their movement, respiratory activity, and physiological metabolism in water samples. The microorganism is a water quality biotoxicity detection receptor which is researched most internationally at present due to the advantages of multiple types, easy culture, small individual, rapid propagation and the like. The luminous bacteria contain luminescent elements such as fluorescein, luciferase, ATP and the like, and can generate fluorescence as an alarm signal through intracellular biochemical reaction under the aerobic condition. The toxic substances reduce the fluorescence intensity by inhibiting the activity of enzyme or inhibiting the intracellular metabolic process related to the luminescence reaction, thereby characterizing the water toxicity by observing the fluorescence intensity of the luminescent bacteria. The luminous bacteria method has the advantages of high speed, high sensitivity, simplicity, convenience, low price and the like, but the luminous intensity is easily interfered by the color and the turbidity of pollutants when the luminous bacteria method is actually applied, so that the problem of false positive alarm is caused.
In recent years, with the attention of more and more researchers on electrochemically active microorganisms, the application range thereof relates to a plurality of fields such as sewage treatment detection, microbial electrogenesis and MFC sensors. The water quality detection technology based on the electrochemical active microorganism can not only avoid false positive caused by luminous bacteria, but also has the advantages of the water quality toxicity biological detection technology of the luminous bacteria. In the early days, a lot of researches found that part of the electrochemically active microorganisms can utilize insoluble solids such as electrodes as electron acceptors to transfer electrons generated by respiratory chain metabolism to the outside of cells through membranes, which is called an electricity generation process. Under certain conditions, the electrical signal output by the electrochemically active microorganisms is directly related to the metabolic activity of the microorganisms. The principle of detecting the biotoxicity of water quality by the electrochemical active microorganisms is based on that toxic and harmful substances weaken normal metabolism which damages microorganisms, so that electric signals are reduced to reflect the concentration of the toxic and harmful substances.
With the actual application and research of the electrochemical active microorganisms in detecting the biotoxicity of the water quality, electron acceptors such as nitrate, nitrite and fumaric acid existing in the water body can interfere the electric signals of the electrochemical active microorganisms, so that the electric signals are reduced, and false alarm is generated. The method and technology of identifying the problem of false positive alarm caused by electron acceptor in detecting water quality biotoxicity by using electrochemically active microbes is to be further researched.
Disclosure of Invention
The invention relates to a method for identifying false positive of electrochemically active microorganisms caused by electron acceptors to detect toxic pollutants, which has the following principle: the output current of the electrochemically active microorganisms reduced by toxic pollutants in the water body is based on the toxicological effect, and the output current reduced by the electron acceptor without the toxicological effect in the water body is because of electron competition with the electrode, so that the false positive problem caused by the electron acceptor can be identified according to different mechanisms of reducing the output current of the electrochemically active microorganisms by the toxic pollutants and the electron acceptor; the method is characterized in that: and constructing a secondary alarm process, judging whether to start a primary alarm according to the influence of the water on the output current of the electrochemically active microorganisms, judging whether to start a secondary alarm according to the influence of the water on the metabolic activity of the electrochemically active microorganisms, analyzing the biotoxicity of the water quality by integrating the primary and secondary alarm results, identifying the false positive of the primary alarm by using the secondary alarm result, and judging the false positive of the primary alarm caused by the existence of an electron acceptor in the water according to the influence of the water sample on the reverse current of a bioelectrochemical system with reverse extracellular electron transfer capacity if the primary alarm is the false positive.
The method provided by the invention comprises the following specific steps:
(1) constructing a pure-culture three-electrode bioelectrochemical system A by taking electrochemically active microorganisms in logarithmic growth phase as seed sources;
(2) injecting distilled water into the system A, and recording a stable current parameter i output by the system A 1 ；
(3) Injecting the sterilized water to be tested into the system A, and recording a stable current parameter i output by the system 2 ；
(4) Setting a first-level alarm coefficient as P 1 The current suppression ratio is calculated with reference to equation (1):
when P is present 1 If the alarm is more than or equal to 30 percent, starting a first-level alarm, and if the alarm is more than or equal to 30 percent>P 1 If the alarm is more than or equal to 0, the first-level alarm is not started;
(5) replacing the electrolyte in the electrochemical system A causing the first-level alarm with the normal electrolyte, injecting distilled water into the belt system A, and recording a stable current parameter i output by the system A 3 ；
(6) Setting a first-level alarm coefficient as P 2 The current suppression rate is calculated with reference to equation (2):
when P is present 2 If the alarm is more than or equal to 30 percent, starting a secondary alarm, and if the alarm is more than or equal to 30 percent>P 2 If the alarm is more than or equal to 0, the secondary alarm is not started;
(7) analyzing the biotoxicity of the water quality by integrating the first-level and second-level alarm results, and if the first-level alarm result is the first-level alarm result, analyzing the biotoxicity of the water qualityThe alarm coefficient is 30%>P 1 Not less than 0, normal water body and no biotoxicity; if first class alarm coefficient P 1 More than or equal to 30 percent and a secondary alarm coefficient of 30 percent>P 1 If the alarm is more than or equal to 0, the first-level alarm is false positive; first order alarm coefficient P 1 More than or equal to 30 percent, and a secondary alarm coefficient P 2 More than or equal to 30 percent, the water body has toxic pollutants.
(8) Constructing a pure-culture three-electrode bioelectrochemical system B by taking an electrochemically active microorganism with bidirectional electron transfer capacity in logarithmic growth phase as a seed source;
(9) recording the baseline current i when System B is operating steadily 4 ；
(10) Injecting the sterilized false positive water body causing the first-level alarm into an electrochemical system B, and recording a stable current parameter i input by the system B 5 ；
(11) And (3) calculating the signal-to-noise ratio value by taking the signal-to-noise ratio coefficient as S and referring to the formula (3):
if S is more than or equal to 3, the water body contains an electron acceptor.
The invention has the following advantages: compared with the prior art, the method has the advantages of the bioelectrochemical system in water quality detection, namely the advantages of rapidness, high efficiency, convenient operation, high sensitivity and the like, and simultaneously eliminates the problem of false positive alarm caused by an electron acceptor possibly encountered by the bioelectrochemical system in the detection; in addition, the electrochemical system in the two-step detection process runs relatively independently, so that the detection cost is saved and the detection efficiency is improved while the accuracy of the detection result is ensured.
Drawings
FIG. 1 is a flow chart of the method of the present invention for identifying false positive of an electrochemically active microorganism for toxic contaminants by electron acceptor detection;
FIG. 2 is a graph showing the variation of input current of a water sample containing an electron acceptor to which the S.loihica PV-4 bioelectrochemical system according to the embodiment of the present invention is added;
Detailed Description
Example 1
And constructing a single-chamber three-electrode bioelectrochemical system consisting of a working electrode, a counter electrode and a reference electrode. The working electrode was a square carbon cloth (HCP330, Shanghai Hesen electric Co., Ltd., China) with a side length of 2 cm. The counter electrode and the reference electrode were respectively a square platinum sheet electrode (Pt210, tianjin aida hengshan limited, china) and a standard Ag/AgCl electrode (R0303, tianjin aida hengshan limited, china; 0.205V vs. standard hydrogen electrode) with a side length of 1cm, the carbon cloth was soaked overnight in an acetone solution before use, rinsed sufficiently with ultrapure water to remove the acetone solution, and finally dried and subjected to high-temperature ammoniation treatment. The effective volume of the electrolytic cell is 50mL, the electrolytic cell is sealed by a tetrafluoro cover, the cover is provided with 5 holes, three holes are respectively inserted into a working electrode, a counter electrode and a reference electrode, and the other two holes are a water inlet and a water outlet. All the parts except the reference electrode are sterilized at high temperature and high pressure, the reference electrode is soaked in 75% alcohol overnight, and finally the electrochemical system assembly is completed in a clean bench (SW-CJ-1F, Sujingtai).
The strain Shewanella loihica PV-4 frozen at-80 ℃ is completely thawed and inoculated into LB liquid culture medium for shaking table overnight culture. The next day, 5ml of fresh bacterial liquid was added to 300ml of LB liquid medium for reactivation, and the culture was terminated when the bacterial liquid) OD 600. apprxeq.1. And adding 15mL of the resuspended bacterial liquid and 25mL of the culture medium of the LDM into a bioelectrochemical system by using a disposable sterile syringe, wherein a microorganism growth carbon source in the DM culture medium is 10mM sodium lactate, and a microorganism growth nitrogen source in the DM culture medium is 0.5g/L of yeast extract. The electrochemical system was placed in a 22 ℃ incubator (HPS-500, Tokyo electronic technology development Co., Ltd., Harbin) and the input current of the bioelectrochemical system was recorded and monitored.
And after the system is started, adjusting the potential to be-0.5V, and when the current signal is basically stable, monitoring the baseline current value input by the system to be 0 uA. And opening a water inlet and a water outlet of the electrolytic cell, and respectively adding water samples containing fumaric acid, dimethyl sulfoxide (DMSO), nitrate and trimethylamine oxide (TMAO) into the bioelectrochemical system. As can be seen from fig. 2, when the baseline current inputted by the system is stabilized, and then a water sample containing an electron acceptor is added to the system, a reverse current is generated, wherein the water sample containing fumaric acid causes the electrochemical system to generate the average value of the reverse current peak value to be 98.7uA (97uA 104uA 95uA), the water sample containing DMSO causes the electrochemical system to generate the average value of the reverse current peak value to be 110.3uA (96uA 99uA 136uA), the water sample containing nitrate causes the electrochemical system to generate the average value of the reverse current peak value to be 55.7uA (55uA 60uA 52uA), the water sample containing TMAO causes the electrochemical system to generate the average value of the reverse current peak value to be 117.3uA (125uA 131uA 96uA), and the water sample containing the electron acceptor generates the reverse current so as to obtain the signal-to-noise ratio coefficient S which is far greater than 3, and as the electron acceptor is consumed, the input current to the electrochemical system returns to the baseline current of 0 uA.
The experimental result shows that the bioelectrochemical system constructed by taking S.loihica PV-4 as a seed source can identify a plurality of electron acceptors in a water sample and reduce the electron acceptors to generate reverse current, and can be used for judging the false positive problem of primary alarm caused by the electron acceptors in the water body.
Claims (6)
1. A method for identifying false positive of electrochemically active microorganisms caused by electron acceptors in toxic contaminant detection, which comprises the following steps: the output current of the electrochemically active microorganisms reduced by toxic pollutants in the water body is based on the toxicological effect, and the output current reduced by the electron acceptor without the toxicological effect in the water body is because of electron competition with the electrode, so that the false positive problem caused by the electron acceptor can be identified according to different mechanisms of reducing the output current of the electrochemically active microorganisms by the toxic pollutants and the electron acceptor; the method is characterized in that: and constructing a secondary alarm process, judging whether to start a primary alarm according to the influence of the water on the output current of the electrochemically active microorganisms, judging whether to start a secondary alarm according to the influence of the water on the metabolic activity of the electrochemically active microorganisms, analyzing the biotoxicity of the water quality by integrating the primary and secondary alarm results, identifying the false positive of the primary alarm by using the secondary alarm result, and judging the false positive of the primary alarm caused by the existence of an electron acceptor in the water according to the influence of the water sample on the reverse current of a bioelectrochemical system with reverse extracellular electron transfer capacity if the primary alarm is the false positive.
2. The electron acceptor of claim 1 comprising one or more of nitrate, nitrite, fumaric acid, trimethylamine oxide, and dimethyl sulfoxide.
3. The primary alarm detection process of claim 1 comprising:
1) detecting the influence of distilled water on electrochemically active microorganisms, and recording the output current i 1 ；
2) Detecting the influence of water on electrochemically active microorganisms, and recording the output current i 2 ；
3) Setting a first-level alarm coefficient as P 1 Calculating a current suppression rate with reference to formula (1);
4) when P is present 1 If the alarm is more than or equal to 30 percent, starting a first-level alarm, and if the alarm is more than or equal to 30 percent>P 1 If the alarm is more than or equal to 0, the first-level alarm is not started.
4. The secondary alarm detection process of claim 1 comprising:
1) judging whether the first-level alarm is started or not, and if so, performing a second-level alarm detection process;
2) detecting the influence of distilled water on electrochemically active microorganisms, and recording the output current i 3 ；
3) Setting the secondary alarm coefficient as P 2 Calculating a current suppression rate with reference to formula (2);
4) when P is present 2 If the alarm is more than or equal to 30 percent, starting a secondary alarm, and if the alarm is more than or equal to 30 percent>P 1 And if the alarm is more than or equal to 0, the secondary alarm is not started.
5. The method for analyzing the biotoxicity of the water quality by integrating the primary and secondary alarm results as claimed in claim 1, and identifying the false positive of the primary alarm by using the secondary alarm result, which is characterized in that:
1) the first-level alarm coefficient is 30%>P 1 Not less than 0, normal water body and no biotoxicity;
2) first order alarm coefficient P 1 More than or equal to 30 percent and a secondary alarm coefficient of 30 percent>P 2 If the alarm is more than or equal to 0, the first-level alarm is false positive;
3) first order alarm coefficient P 1 Not less than 30 percent, and a secondary alarm coefficient P 2 More than or equal to 30 percent, the water body has toxic pollutants.
6. The method for determining false positive of primary alarm caused by electron acceptor in water body according to the influence of water sample on reverse current of bioelectrochemical system with reverse extracellular electron transfer capability as claimed in claim 1, is characterized in that:
1) constructing a bioelectrochemical system with reverse extracellular electron transfer;
2) recording the baseline current i during steady operation of the system 4 ；
3) Detecting the influence of false positive water body of first-level alarm on the bioelectrochemical system, and recording the input current i 5 ；
4) Setting the signal-to-noise ratio as S, and calculating the signal-to-noise ratio by referring to a formula (3);
5) if S is more than or equal to 3, the water body contains an electron acceptor.
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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2005156204A (en) * | 2003-11-21 | 2005-06-16 | Fuji Electric Systems Co Ltd | Monitoring method for toxic substance |
| US20090215110A1 (en) * | 2005-05-26 | 2009-08-27 | Colin Gibson | Water monitoring systems |
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