阅读说明：本技术 一种基于微生物燃料电池的毒性评价方法 (Toxicity evaluation method based on microbial fuel cell ) 是由 于茵 周岳溪 邢飞 席宏波 于 2020-04-02 设计创作，主要内容包括：本发明公开了一种基于微生物燃料电池的毒性评价方法,包括微生物燃料电池系统及电信号检测装置。微生物燃料电池系统由阴阳极基质、双室型微生物燃料电池系统和蠕动泵组成。阴阳极基质通过蠕动泵流经微生物燃料电池,电池的阴阳极室连接到电信号检测器,信号输出端连接到电脑上。当基质中出现不同浓度毒性物质时,输出电信号相应减小,计算单位时间的产电量即平均电流抑制率,与未投加毒性物质进行对比,判断废水的生物抑制程度。本发明灵敏、结果直观,检测范围宽,计算方法准确,误报率低,较好的预测微生物受抑制的程度。(The invention discloses a toxicity evaluation method based on a microbial fuel cell, which comprises a microbial fuel cell system and an electric signal detection device. The microbial fuel cell system consists of a cathode substrate, an anode substrate, a double-chamber microbial fuel cell system and a peristaltic pump. The cathode and anode substrates flow through the microbial fuel cell through the peristaltic pump, the cathode and anode chambers of the cell are connected to an electric signal detector, and the signal output end is connected to a computer. When toxic substances with different concentrations appear in the matrix, the output electric signal is correspondingly reduced, the electricity generation amount in unit time, namely the average current inhibition rate, is calculated, and compared with the situation that no toxic substance is added, the biological inhibition degree of the wastewater is judged. The method has the advantages of sensitivity, visual result, wide detection range, accurate calculation method, low false alarm rate and better prediction of the degree of the inhibition of the microorganisms.)

1. A toxicity evaluation method based on a microbial fuel cell is characterized by comprising the following steps:

(1) adding nutrient substances into a container with stable microbial performance, and adding a sample to be detected into the container to form a mixed solution serving as an anode matrix, wherein the mixed solution is defined as an experimental group; taking another container with stable microbial performance, adding nutrient substances as an anode matrix, and defining the container as a blank group;

(2) connecting the blank group of anode matrixes obtained in the step (1) into a pipeline, and conveying the blank group of anode matrixes into an anode chamber of a double-chamber fuel cell through a peristaltic pump;

(3) connecting the cathode substrate to the cathode chamber of the dual-chamber fuel cell by a peristaltic pump;

(4) respectively connecting the cathode and the anode of the double-chamber fuel cell to an electric signal detection device, wherein the electric signal detection device is connected to a computer and records an electric signal;

(5) recording the current value of the electric signal when the electric signal is stable, and recording the current value as I_nor；

(6) Replacing the blank group of the anode substrate in the step (2) with the experimental group of the anode substrate, connecting the blank group of the anode substrate into a pipeline, conveying the blank group of the anode substrate into the anode chamber of the double-chamber fuel cell through a peristaltic pump, repeating the steps (3) to (4), monitoring the current value of the electrical signal when the electrical signal is stable to obtain the average current in unit time, and recording the average current as the average current

(7) Calculating the average current inhibition rate, wherein the average current inhibition rate is calculated according to the following formula:

wherein I_norIs the blank set stabilization current value and,is the average current per unit time of the experimental group after the sample to be measured is added;

(8) and evaluating the inhibition condition of the sample to be tested of the experimental group on the microorganisms in the anode matrix through the current inhibition rate, thereby evaluating the toxicity of the sample to be tested.

2. The microbial fuel cell-based toxicity evaluation method according to claim 1, wherein the nutrient substances in step (1) are: glucose, acetate or mixtures thereof.

3. The microbial fuel cell-based toxicity evaluation method according to claim 1, wherein the sample to be tested in step (1) contains a toxic substance, and the toxic substance is 2, 4-dichlorophenol, acrylonitrile, or a mixture thereof.

4. The microbial fuel cell-based toxicity evaluation method of claim 1, wherein the cathode substrate in step (3) is an aqueous solution of potassium ferricyanide.

5. The microbial fuel cell-based toxicity evaluation method according to claim 1, wherein step (8) further comprises the steps of:

presetting three ACIR values, namely ACIR₁、ACIR₂、ACIR₃；

When the ACIR is less than or equal to ACIR1, showing that no microorganism inhibiting substance exists in the sample to be detected;

when the ACIR is not less than 2 and not more than 1, the existence of microbe inhibiting substances in the sample to be detected is shown, but the inhibition is not strong;

when the ACIR is not less than 2 and not more than 3, the existence of microorganism inhibiting substances in the sample to be detected is shown, and the inhibition is strong;

when the ACIR is more than or equal to ACIR3, the existence of substances inhibiting microorganisms in the sample to be detected is shown, and the inhibition is very strong.

6. The microbial fuel cell-based toxicity evaluation method of claim 5, wherein ACIR 1-5%, ACIR 2-30%, and ACIR 3-50%.

7. The microbial fuel cell-based toxicity evaluation method according to claim 1, wherein the microorganism in step (1) is an electrogenic anaerobic microorganism selected from one or a combination of two or more of α -proteus, β -proteus, clostridium, shewanella, and geobacillus.

8. The method for evaluating toxicity based on a microbial fuel cell according to claim 1, wherein in the step (2), the flow rate of the peristaltic pump is 1 to 10 ml/min.

9. The microbial fuel cell-based toxicity evaluation method of claim 1, wherein in step (6), the electrical signal is stabilized to continuously run for three cycles, and the maximum value of the electrical signal varies by less than 10% during the three cycles.

Technical Field

The invention relates to the field of water treatment, in particular to a monitoring and method for evaluating the inhibition of microorganisms and the influence of toxic substances on the inhibition of the microorganisms based on a microbial fuel cell.

Background

In recent years, water pollution events are commonly reported, and monitoring water quality in time is an important link for preventing and treating water pollution. The conventional water quality monitoring is realized by a physical chemical biological method which is widely applied but cannot reflect the toxic degree of toxic substances to human beings and other organisms. The water environment monitoring organisms mainly comprise microorganisms, phytoplankton, zooplankton, higher aquatic plants, fish and the like. Biosensors are increasingly used as a tool for implementing supplementary water quality assessment, can be used for evaluating the biotoxicity effect of a water body, can reflect the cumulative effect of toxic substances existing in organisms, and has the advantages of sensitivity and diversity.

The Microbial Fuel Cell (MFC) is a novel biosensor, can be used for measuring the inhibition of wastewater on anaerobic microorganisms, has high sensitivity and high detection speed, can realize on-line monitoring, and has good application prospect. The electrogenesis microorganism is very sensitive to environmental change, when the influent contains toxic substances, the life activity of the electrogenesis microorganism is weakened, and the generated voltage or current changes, so that the rapid and effective indication effect can be achieved, and the rapid and economic evaluation on the water body biotoxicity can be realized. The toxic substances such as phenols, aldehydes, halogenated hydrocarbons, cyanides, heavy metals and the like contained in the wastewater can inhibit microorganisms, reduce biological activity, damage cell structures, inhibit metabolic processes and the like, influence the biological treatment effect and the running stability of a sewage treatment plant, and cause food chain shortage, acute and chronic poisoning events and even ecological imbalance for a water ecosystem. Therefore, the scientific and effective evaluation and control of the biological inhibition of water are urgent and important.

At present, the characterization indexes of the microorganisms under inhibition mainly comprise voltage inhibition rate, current difference, current, voltage, coulombic capacity and the like, when the external resistance is constant, the results of inhibition conditions are indicated to be the same by adopting voltage or current according to ohm's law, but the electric signals of the method usually have larger time variation of the results of calculating the inhibition rate by instantaneous values, the true degree of the inhibition effect cannot be effectively reflected, and the action degree of toxic substances cannot be effectively distinguished when the final voltage is the same. Therefore, it is very important to provide an efficient and effective evaluation method.

Disclosure of Invention

The invention provides a toxicity evaluation method based on a microbial fuel cell. The method is sensitive, has a wide detection range for different toxic substances, and is a good toxicity evaluation index.

In a first aspect, the present invention provides a toxicity evaluation method based on a microbial fuel cell, comprising the steps of:

(1) adding nutrient substances into an anode matrix container with stable microbial culture, and adding a sample to be detected into the anode matrix container to form a mixed solution. The nutrient substances are glucose or acetate and the like. The sample to be tested is a sample containing toxic substances such as 2, 4-dichlorophen, acrylonitrile and the like. And defining the mixed liquid containing the sample to be tested in the anode substrate container as an anode substrate as a test group.

And adding nutrient substances into another anode substrate container with stable microbial culture, and mixing to form a mixed solution. And taking the mixed solution without the sample substance to be detected as an anode matrix, and defining the mixed solution as a blank group.

(2) And (3) connecting the mixed solution of the anode substrate in the blank group prepared in the step (1) into a pipeline, and connecting the mixed solution into the anode chamber of the double-chamber fuel cell through a peristaltic pump.

(3) The cathode substrate was connected to the two-chamber fuel cell cathode chamber by a peristaltic pump.

(4) The cathode and the anode of the microbial fuel cell are respectively connected to an electric signal detection device through one or more inert metals such as carbon, graphite, silver, platinum and the like, and the electric signal detection device is connected to a computer and records electric signals.

(5) And obtaining the current or the voltage of the electric signal through the electric signal when the electric signal is stable.

Because the calculation results of the voltage and the current are the same according to the ohm's law under the condition of constant external resistance, the current calculation is adopted in the research. Therefore, in the step (5), after the electric signal is stabilized, the current value of the electric signal is recorded and is marked as I_nor；

(6) Replacing the blank group of the anode substrate in the step (2) with the experimental group of the anode substrate, connecting the blank group of the anode substrate into a pipeline, conveying the blank group of the anode substrate into the anode chamber of the double-chamber fuel cell through a peristaltic pump, repeating the steps (3) to (4), monitoring the current value of the electrical signal when the electrical signal is stable to obtain the average current in unit time, and recording the average current as the average current

(7) Calculating the average current inhibition rate, wherein the average current inhibition rate is calculated according to the following formula:

wherein I_norIs the blank set stabilization current value and,is the average current per unit time of the experimental group after the sample to be measured is added;

(8) and evaluating the inhibition condition of the sample to be tested of the experimental group on the microorganisms in the anode matrix through the current inhibition rate, thereby evaluating the toxicity of the sample to be tested.

Further, the present invention presets three ACIR values, ACIR1, ACIR2, and ACIR3, and compares them to:

when ACIR is less than or equal to ACIR1, no microbe-inhibiting substance is present in the water;

when the ACIR2 is less than or equal to the ACIR1, the existence of microbe inhibiting substances in water is indicated, but the inhibition is not strong, so that attention needs to be paid;

when the ACIR2 is less than or equal to the ACIR3, the existence of microbe inhibiting substances in water is shown, the inhibition is strong, and attention needs to be paid and appropriate measures need to be taken;

the ACIR is more than or equal to ACIR3, which shows that the water has microorganism-inhibiting substances with strong inhibition and needs emergency measures.

In the step (1), the microorganism is an electrogenic anaerobic microorganism, and is specifically selected from one or more of α -Proteus, β -Proteus, Clostridium, Shewanella and Geobacillus, the Shewanella is MR-1 or DSP-10, and the Geobacillus is thioredocillus.

In the above step (1), in a preferred embodiment, the microorganism is a combination of S.terreus and Shewanella MR-1.

In the step (1), the stable microbial performance refers to a stable microbial community obtained by adding the microbes into a container and further culturing the microbes by adding nutrient substances. The microbiological performance stability is expressed in that the electric signal of the microbiological fuel cell is stable, namely the maximum current change amplitude of the microbiological fuel cell is less than 10% in three periods, so that the microbiological performance stability in the container is determined. In the present application, a stable microbial community is also used in the solution of the present application for I_norThe measurement of (1).

The amount of the nutrients added in the above step (1) is 1-5 g/L based on the volume of the liquid in the container, and in a preferred embodiment, the amount of the nutrients added is 1-2 g/L.

In the step (5) or (6), the signal stabilization means that the maximum current of the microbial fuel cell of 3 cycles changes by less than 10%. And the duration of the maximum current is the same over 3 cycles.

In this application, the period is a period in which a nutrient is added to a container containing a microorganism until the microorganism completes digestion and absorption of the nutrient. The one cycle is a period from the fluctuation of the generated electric signal to the electric signal being again stable after the addition of the nutrient substance in the container with the microbial community. The period is 10-30 hours; preferably, the period is 20 to 30 hours.

In the present application, in step (6), the current value sampling time for calculating the average current suppression rate refers to the time from the time when the current value of the electric signal changes by less than 10% after the sample to be measured is added, and I is calculated_norThe sampling time of the electric signal current value is the same as the sampling time of the current value for calculating the average current inhibition rate by adding the sample to be measured.

In the present application, in a preferred embodiment, the sample to be tested and the addition of the nutrient substance are simultaneous.

Further preferably, in step (1), the microbial fuel cell is cultured at a constant temperature.

Further preferably, in step (7), the performance curves of the microbial fuel cells are similar in the toxicity test of the same batch.

In a second aspect, the invention provides a microbiological fuel cell based toxicity evaluation method, specifically:

in the step (1), nitrogen is required to be blown for more than 40 minutes before the sample to be measured is added to the anode substrate.

In the step (2), the flow rate of the peristaltic pump is 1-10 ml/min, and the anode chamber microorganisms are taken from the bacterial liquid with the culture period of more than 1 year.

In step (3), the electric signal detection device is set to a constant external resistance mode.

In the step (5), the trend of the electric signal to be stable is the calculation end point of the toxicity test, and the change amplitude of the electric signal is less than 10 percent;

in the above steps (2) and (3), the dual chamber fuel cell is a conventional dual chamber fuel cell in the art. A diaphragm is arranged between the cathode chamber and the anode chamber, and the diaphragm is a proton exchange membrane.

The ACIR1, the ACIR2, and the ACIR3 were 5%, 30%, and 50%, respectively.

In the embodiment of the invention, the anode substrate blank group is glucose or acetate, trace elements, metal elements and the like. The cathode matrix is potassium ferricyanide. The mixing of the nutrient substrate with the toxic substance does not affect the concentration of the nutrient in the substrate. The cathode and anode materials of the microbial fuel cell are respectively inert nonmetal or metal, such as one or more of inert metals such as carbon, graphite, silver, platinum and the like.

The toxicity evaluation method based on the microbial fuel cell provided by the invention has the following effects: (1) the linear range is wide, and the method is suitable for measuring the condition of large wastewater concentration change range; (2) the result is visual, the linear relation is better, and the error is small; (3) can react the impact of the microorganism on toxic substances, and has reliable result; (4) the applicability is stronger, does benefit to actual popularization and use.

Drawings

FIG. 1 is a schematic view of the structure of the present invention.

FIG. 2 shows the result of the biotoxicity test of 2, 4-dichlorophen.

FIG. 3 shows the result of the biotoxicity test of acrylonitrile.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples.

The invention provides a toxicity evaluation method based on a microbial fuel cell, which comprises the following steps as shown in figure 1:

(1) adding sodium acetate into a container with stable microbial performance, wherein the adding amount of the sodium acetate is 1 g/L, taking the sodium acetate as a blank group, adding sodium acetate into another two containers with stable microbial performance, wherein the adding amount of glucose is 1 g/L, further adding a sample to be tested containing 2, 4-dichlorophenol and acrylonitrile toxic substances into the two containers respectively to form a mixed solution as an anode substrate, defining the mixed solution as a test group, blowing nitrogen for more than 40 minutes for the anode substrate before the toxic substances are added, connecting the mixed solution obtained in the step (1) into a pipeline, connecting the mixed solution into an anode chamber of a double-chamber fuel cell through a peristaltic pump, connecting a cathode substrate into a cathode chamber of the double-chamber fuel cell through the peristaltic pump, wherein the flow rate of the peristaltic pump is 1-10 ml/min, microorganisms in the anode chamber are taken from a bacterial solution with the culture period of more than 1 year, and culturing the microbial fuel cell at a constant temperature.

The cathode and the anode of the microbial fuel cell are respectively connected to an electric signal detection device through a metal sheet, the electric signal detection device is connected to a computer and records an electric signal, and the electric signal detection device is set to be in a constant external resistance mode.

When the output electric signal of the microbial fuel cell is stable, the microbial fuel cell can be used for a toxicity test, and the signal stability means that the maximum current error of the microbial fuel cell in 3 periods is less than 10%, and the duration time of the maximum current is the same in 3 periods. In the examples of the present application, the above-mentioned one cycle is 30 hours. Toxicity test of the same batch, microorganismsThe performance curves of the fuel cells are similar. The change amplitude of the electric signal is less than 10%, the electric signal is considered to tend to be stable, and the calculation end point of the toxicity test is reached. And calculating the average current inhibition rate through the average current of the action time, namely the electricity generation amount per unit time, and representing the inhibited condition of the microorganisms. Calculating the action time of the average current inhibition rate means that the change amplitude of the electric signal is less than 10 percent after toxic substances are added, I_norThe measuring time of the electric signal is the same as the action time of the toxic substance. The formula is as follows:

wherein I_norIs a stable electrical signal without toxic substances,is the average current per unit time after addition of the toxic substance;

three ACIR values, ACIR, are preset in the system₁、ACIR₂And ACIR₃，ACIR₁＝5％、ACIR₂＝30％，ACIR₃50% and compared to ACIR:

when ACIR is less than or equal to 5 percent, showing that no microorganism inhibiting substances exist in the water;

when the ACIR is more than or equal to 30 percent and less than or equal to 5 percent, the water has microbe inhibiting substances, but the inhibition is not strong, and attention needs to be paid;

when the ACIR is more than or equal to 30% and less than or equal to 50%, the water has microorganism inhibiting substances, the inhibition is strong, attention needs to be paid, and appropriate measures need to be taken;

when the ACIR is more than or equal to 50 percent, the existence of microorganism inhibiting substances in water is shown, the inhibition is very strong, and emergency measures need to be taken.

FIG. 2 shows the real-time results of the biotoxicity assay of 2, 4-dichlorophenol, the detected concentration of 2, 4-dichlorophenol is 0.4-1000 mg/L and the lowest detected concentration is 0.4 mg/L₅₀It was 36.18 mg/L.

FIG. 3 shows the results of real-time detection of the biotoxicity of acrylonitrile, the detected concentration of acrylonitrile being 0.5-7 by the average current suppression ratio000 mg/L, with the lowest detectable concentration of 0.5 mg/L₅₀Greater than 1000 mg/L.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and simplifications made in the spirit of the present invention are intended to be included in the scope of the present invention.