CN114965617B - Method for detecting false positive of toxic pollutant by identifying electrochemical active microorganism caused by electron acceptor - Google Patents
Method for detecting false positive of toxic pollutant by identifying electrochemical active microorganism caused by electron acceptor Download PDFInfo
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Classifications
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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
-
- 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
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A20/00—Water conservation; Efficient water supply; Efficient water use
- Y02A20/20—Controlling water pollution; Waste water treatment
Abstract
The invention relates to a method for detecting false positives of toxic pollutants by identifying electrochemically active microorganisms caused by an electron acceptor. The method is characterized in that a two-step detection method is designed, and electrochemical active microorganisms with the output electric signal inhibition rate and the bidirectional electron transfer capacity of a bioelectrochemical system are utilized to generate reverse current, so that the false positive problem of water biotoxicity detection alarm caused by the recognition of an electron acceptor can be realized. The two-step detection flow established by the invention has the advantages of high speed, high efficiency, convenient operation, high sensitivity and the like in water quality detection of the bioelectrochemical system, and simultaneously eliminates the false positive alarm problem caused by the electron acceptor possibly encountered in the detection. In addition, the operation of the electrochemical system in the two-step detection process is relatively independent, so that the accuracy of a detection result is ensured, the detection cost is saved, and the detection efficiency is improved.
Description
Technical Field
The invention relates to the field of water quality detection, in particular to detection of water quality biotoxicity false positive by electrochemical active microorganisms.
Background
The water quality detection plays a vital role in the aspects of whole water environment protection, water pollution control and water environment health maintenance. For drinking water, harmful bacteria, heavy metal pesticides and the like in the water can cause various diseases; for industrial water, mineral impurities, acid and alkali ions and the like in the water can affect the quality of products or damage containers and pipelines, so that water quality detection is relevant to civil affairs and cannot be changed. The traditional water quality detection method such as a chemical pyrolysis method, an atomic fluorescence detection method, a liquid chromatography method and the like has the advantages of high detection precision, good stability, strong repeatability and the like, but also has the problems of complex operation steps, dangerous reaction conditions, long detection time, high technical requirements, easiness in secondary pollution and the like. The water quality biotoxicity detection technology based on the biosensor has been paid more attention to because of its rapid analysis speed, wide detection range and capability of simultaneously analyzing the comprehensive toxicity of various pollutants, 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 the 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 behaviors of the aquatic organisms can be used as an important index for evaluating whether the water environment is safe or not. Such as fish, fleas, algae, microorganisms, etc., are commonly selected as test organisms, and water toxicity is assessed by their movement in a water sample, respiratory activity, and changes in physiological metabolism. Microorganisms are the most studied water quality biotoxicity detection receptor internationally at present because of the advantages of multiple types, easy culture, small individuals, rapid propagation and the like. The toxicity test of the luminous bacteria is most widely studied, and the luminous bacteria contain luminous elements such as fluorescein, luciferase, ATP and the like, and can generate fluorescence as an alarm signal through biochemical reaction in cells under the aerobic condition. Toxic substances reduce the fluorescence intensity by inhibiting the activity of enzymes or inhibiting the metabolic processes related to luminous reaction in cells, so that the water toxicity is characterized by observing the fluorescence intensity of luminous bacteria. The luminous bacteria method has the advantages of rapidness, high sensitivity, simplicity, convenience, low cost and the like, but the luminous intensity is extremely easy to be interfered by pollutant color and turbidity when the method is actually applied, and the false positive alarm problem is generated.
In recent years, as electrochemically active microorganisms are attracting more and more attention from researchers, the application range thereof relates to various fields such as sewage treatment detection, microbial electricity generation and MFC sensors. The electrochemical active microorganism is a special environment microorganism with extracellular electronic transmission capability and can realize bidirectional conversion of chemical energy and electric energy, and the water quality detection technology based on the electrochemical active microorganism can not only avoid false positive caused by luminescent bacteria, but also take the advantages of the luminescent bacteria water quality toxicity biological detection technology into consideration. Early studies have found that some electrochemically active microorganisms can use insoluble solids, such as electrodes, as electron acceptors to transport electrons produced by respiratory chain metabolism across the membrane to the outside of the cell, a process known as electrogenesis. Under certain conditions, the electrical signal output by an electrochemically active microorganism is directly related to the metabolic activity of the microorganism. The principle of detecting water biotoxicity by electrochemically active microorganisms is based on that toxic and harmful substances can weaken normal metabolism of damaging microorganisms, so that electric signals are reduced to reflect the concentration of the toxic and harmful substances, and the principle has been proved to be capable of detecting various pollutants such as heavy metals, organic pesticides, antibiotics and the like in a water sample, and has wide application prospect.
With the deep practical application and research of electrochemically active microorganisms in detecting water biotoxicity, electron acceptors existing in water bodies, such as nitrate, nitrite, fumaric acid and the like, interfere with the electric signals of the electrochemically active microorganisms, so that the electric signals are reduced, and false alarms appear. Methods and techniques for identifying false positive alarm problems caused by electron acceptors that may occur when electrochemically active microorganisms are used to detect water biotoxicity are further under investigation.
Disclosure of Invention
The invention relates to a method for detecting false positive of toxic pollutants by electrochemically active microorganisms caused by recognition of electron acceptors, which comprises the following steps: the output current of the electrochemical active microorganisms is reduced by toxic pollutants in the water body based on toxicological effects, and the output current is reduced by the electron acceptors without toxicological effects in the water body due to competition with the electrodes, so that false positive problems caused by the electron acceptors can be identified according to different mechanisms of the toxic pollutants and the electron acceptors for reducing the output current of the electrochemical active microorganisms; the method is characterized in that: constructing a secondary alarm flow, judging whether to start primary alarm according to the influence of the water body on the output current of the electrochemical active microorganisms, judging whether to start secondary alarm according to the influence of the water body on the metabolic activity of the electrochemical active microorganisms, analyzing the biotoxicity of water quality by synthesizing the primary alarm result and the secondary alarm result, identifying the false positive of the primary alarm by utilizing the secondary alarm result, and judging that the false positive of the primary alarm is caused by the existence of an electron acceptor in the water body according to the influence of the water sample on the reverse current of the bioelectrochemical system with reverse extracellular electronic transmission capability if the primary alarm is 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 a logarithmic growth phase as seed sources;
(2) Adding distilled water into the system A by injection, and recording stable current parameter i output by the system A 1 ；
(3) Injecting the sterilized water body to be detected into the system A, and recording the stable current parameter i output by the system 2 ；
(4) Let the primary alarm coefficient be P 1 The current suppression ratio is calculated with reference to formula (1):
when P 1 If the alarm is more than or equal to 30 percent, the first-stage alarm is started, and when the alarm is more than or equal to 30 percent>P 1 If the alarm is more than or equal to 0, the primary alarm is not started;
(5) Replacing the electrolyte in the electrochemical system A which causes primary alarm with normal electrolyte, injecting distilled water into the belt system A, and recording the stable current parameter i output by the system A 3 ；
(6) Let the primary alarm coefficient be P 2 The current suppression ratio is calculated with reference to formula (2):
when P 2 If the temperature is more than or equal to 30 percent, a secondary alarm is started, and when the temperature 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 water quality biotoxicity by combining the primary and secondary alarm results, if the primary alarm coefficient is 30%>P 1 Not less than 0, normal water body and no biological toxicity; if the first-level alarm coefficient P 1 More than or equal to 30 percent, and the secondary alarm coefficient is 30 percent>P 1 If the value is more than or equal to 0, the first-level alarm is false positive; first-level alarm coefficient P 1 More than or equal to 30 percent, and the secondary alarm coefficient P 2 And if the water content is more than or equal to 30 percent, toxic pollutants exist in the water body.
(8) Constructing a pure-culture three-electrode bioelectrochemical system B by taking electrochemically active microorganisms in logarithmic growth phase with bidirectional electron transfer capability as seed sources;
(9) Recording the baseline current of system B in steady operationi 4 ；
(10) After the false positive water body which causes the primary alarm is sterilized, the false positive water body is injected into the electrochemical system B, and the stable current parameter i input by the system B is recorded 5 ；
(11) Let the signal-to-noise ratio coefficient be S, calculate the signal-to-noise ratio value with reference to equation (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 method has the advantages of high speed, high efficiency, convenient operation, high sensitivity and the like, and simultaneously eliminates the false positive alarm problem caused by the electron acceptor possibly encountered by the bioelectrochemical system in detection; in addition, the operation of the electrochemical system in the two-step detection process is relatively independent, so that the accuracy of a detection result is ensured, the detection cost is saved, and the detection efficiency is improved.
Drawings
FIG. 1 is a flow chart of a method of the invention for identifying false positives of toxic contaminants by electrochemically active microorganisms caused by electron acceptors;
FIG. 2 is a graph showing the change in input current of an S.loihica PV-4 bioelectrochemical system incorporating an electron acceptor-containing water sample in accordance with an embodiment of the present invention;
Detailed Description
Example 1
A single-chamber three-electrode bioelectrochemical system consisting of a working electrode, a counter electrode and a reference electrode is constructed. The working electrode was a square carbon cloth (HCP 330, shanghai forest electric company, china) with a side length of 2 cm. The counter electrode and the reference electrode were respectively square platinum sheet electrodes (Pt 210, tianjin Aida Heng Cheng, inc., china) and standard Ag/AgCl electrodes (R0303, tianjin Aida Heng Cheng, inc., china; 0.205V vs. standard hydrogen electrode) with a side length of 1cm, respectively, and the carbon cloth was immersed overnight in an acetone solution before use, rinsed with ultrapure water to remove the acetone solution, and finally baked and aminated at a high temperature. 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 accessories except the reference electrode were sterilized at high temperature and high pressure, the reference electrode was immersed in 75% alcohol overnight, and finally the electrochemical system assembly was completed in a clean bench (SW-CJ-1F, threzaine).
The strain Shewanella loihica PV-4 frozen at-80 ℃ is completely melted and inoculated into LB liquid culture medium for shaking overnight culture. The next day, 5ml of fresh bacterial liquid is added into 300ml of LB liquid culture medium for reactivation, and the culture is stopped when the bacterial liquid) OD600 is approximately equal to 1. 15mL of the resuspended bacterial solution and 25mL of LDM medium were added to the bioelectrochemical system using a disposable sterile syringe, wherein the microbial growth carbon source in DM medium was 10mM sodium lactate and the microbial growth nitrogen source was 0.5g/L yeast extract. The electrochemical system was placed in a constant temperature incubator (HPS-500, haerbin, tonka electronic technology development Co., ltd.) at 22℃and the input current to the bioelectrochemical system was recorded and monitored.
After the system is started, the potential is regulated to be-0.5V, and after the current signal is basically stable, the baseline current value input by the monitoring system is 0uA. 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 radical and trimethylamine oxide (TMAO) into a bioelectrochemical system. As can be seen from fig. 2, when the baseline current input by the system is stable, reverse current is generated by adding water samples containing electron acceptors to the system, wherein the average value of the reverse current peaks generated by the electrochemical system by the water samples containing fumaric acid is 98.7uA (97 uA 104uA 95uA), the average value of the reverse current peaks generated by the electrochemical system by DMSO is 110.3uA (96 uA 99uA 136 uA), the average value of the reverse current peaks generated by the electrochemical system by nitrate water samples is 55.7uA (55 uA 60uA 52uA), the average value of the reverse current peaks generated by the electrochemical system by TMAO is 117.3uA (125 uA 131uA 96 uA), and the reverse current is generated by the water samples containing electron acceptors to obtain a signal to noise ratio coefficient S which is far greater than 3, and the input current of the electrochemical system is restored to the baseline current of 0uA with consumption of the electron acceptors.
Experimental results show that the bioelectrochemical system constructed by taking the S.loihica PV-4 as a seed source can identify various 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 existence of the electron acceptors in the water body.
Claims (2)
1. A method for identifying false positives of toxic pollutants detected by electrochemically active microorganisms caused by an electron acceptor, which is characterized by comprising the following steps: constructing a secondary alarm flow, judging whether to start primary alarm according to the influence of a water body on output current of electrochemical active microorganisms, judging whether to start secondary alarm according to the influence of the water body on metabolic activity of the electrochemical active microorganisms, analyzing water biotoxicity by synthesizing primary and secondary alarm results, identifying false positive of the primary alarm by utilizing the secondary alarm results, and judging that the existence of an electron acceptor in the water body leads to the false positive of the primary alarm according to the influence of a water sample on reverse current of a bioelectrochemical system with reverse extracellular electron transfer capability if the primary alarm is false positive, wherein the method specifically comprises the following steps:
(1) Setting a first-level alarm detection flow:
constructing a bioelectrochemical system by taking electrochemically active microorganisms as seed sources, detecting the influence of distilled water on the electrochemically active microorganisms, and recording output current i 1 Detecting the influence of a water body on electrochemical active microorganisms, and recording output current i 2 Let the primary alarm coefficient be P 1 Calculating the current suppression ratio with reference to formula (1), when P 1 If the alarm is more than or equal to 30%, starting a first-level alarm, and when the alarm is more than 30% P 1 If the alarm is more than or equal to 0, the primary alarm is not started;
(2) Setting a secondary alarm detection flow:
judging whether the primary alarm is started or not, if so, performing a secondary alarm detection flow, and replacing the normal electrolyte to cause the primary alarmElectrolyte in police bioelectrochemical system, detecting influence of distilled water on electrochemically active microorganisms, and recording output current i 3 Let the secondary alarm coefficient be P 2 Calculating the current suppression ratio with reference to formula (2), when P 2 If the temperature is more than or equal to 30%, a secondary alarm is started, and when the temperature is more than 30% P 2 If the alarm is more than or equal to 0, the secondary alarm is not started;
(3) Analyzing the biotoxicity of water quality by synthesizing the primary and secondary alarm results:
the primary alarm coefficient is more than 30 percent and is more than P 1 Not less than 0, normal water body and no biological toxicity; first-level alarm coefficient P 1 More than or equal to 30 percent, and the secondary alarm coefficient is more than 30 percent and is more than P 2 If the value is more than or equal to 0, the first-level alarm is false positive; first-level alarm coefficient P 1 More than or equal to 30 percent, and the secondary alarm coefficient P 2 More than or equal to 30 percent, toxic pollutants exist in the water body;
(4) Judging false positive of primary alarm caused by the existence of an electron acceptor in the water body:
constructing a bioelectrochemical system capable of generating reverse current according to the influence of a water sample on reverse current of electrochemically active microorganisms with reverse extracellular electron transfer capability, and recording a baseline current i when the system is stably operated 4 Detecting the influence of a false positive water body with primary alarm on a bioelectrochemical system, and recording an input current i 5 Setting the signal-to-noise ratio coefficient as S, and calculating the signal-to-noise ratio value by referring to the formula (3);
if S is more than or equal to 3, the water body contains an electron acceptor.
2. A method of identifying false positives for detecting toxic contaminants by an electrochemically active microorganism resulting from an electron acceptor according to claim 1 wherein said electron acceptor comprises one or more of nitrate, nitrite, fumaric acid, trimethylamine oxide and dimethyl sulfoxide.
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