CN113358722B - Method for realizing rapid detection of water toxicity based on suspended electrochemical active microorganisms - Google Patents

Abstract

The invention relates to a method for realizing rapid detection of water toxicity by utilizing suspended electrochemically active microorganisms, mainly aiming at the problem of low sensitivity existing in the existing electrochemically active microorganisms detection technology for water quality. By constructing a microbial electrochemical water toxicity sensor with suspended electrochemical microorganisms as the core, the method does not need to incubate biofilms, and only uses the bacterial suspension of electrochemically active microorganisms to directly transduce water toxicity information into electrical signals. Rapid detection of water toxicity to meet the needs of emergency detection and mobile detection of water toxicity.

Description

A method for rapid detection of water toxicity based on suspended electrochemically active microorganisms

technical field

The invention relates to the field of biological monitoring, in particular to a method for realizing rapid detection of water toxicity by utilizing suspended electrochemically active microorganisms.

Background technique

Early warning of acute water pollution is of great significance for ensuring ecological security and people's health. The existing water toxicity testing methods mainly include physical and chemical analysis methods and biological methods. The physicochemical method is to estimate the toxicity of water quality through physical or chemical analysis. It can quantitatively analyze a certain pollutant, and has high measurement accuracy and sensitivity for a single substance, but it cannot directly reflect the biological toxicity of various pollutants, and the operation is complicated and the test time is long, so it is not suitable for on-site rapid detection and continuous online. Therefore, traditional physical and chemical methods cannot make timely responses to sudden water pollution.

The biological method is based on the principle of adapting organisms to the environment. When toxic pollutants enter the water body, the toxic pollutants will inhibit or promote the movement, growth, and respiration of normal living organisms in the water body. Changes in activity, metabolism, etc. to assess water quality. Commonly used indicator organisms such as fish, algae, luminescent bacteria, etc., make up for the deficiency of physical and chemical analysis methods to a certain extent. Fish is the earliest indicator organism to be used. By examining the effect of the tested sample on the activity and behavior of fish, the toxicity of the sample can be determined. The fish toxicity test is simple to operate and has high reliability, but it has shortcomings such as long detection period and relatively high monitoring lower limit. Compared with fish, algae are easier to cultivate, have a shorter cycle, and are more suitable as indicator materials. Therefore, this assay method has been developed rapidly, but it has the disadvantage of poor repeatability due to the influence of the quality of benchmark algae cultivation. Due to their unique physiological characteristics, luminescent bacteria are used as indicators for the determination of environmental toxicants in environmental monitoring. Its relative luminosity based on luminescent bacteria is significantly negatively correlated with the total concentration of toxic components in water samples, so the relative luminosity of water samples can be measured by a bioluminescence photometer to express its acute toxicity level. However, the complex signal transduction process required in the detection process of the luminescent bacteria method leads to its high cost, and the luminescent bacteria are more sensitive to Cl- and have stricter pH requirements. The experimental conditions are relatively harsh, and false alarms are prone to occur. In general, the development of traditional biological toxicity testing methods has been relatively mature, which makes up for the shortcomings of physical and chemical analysis methods to a certain extent, but there are shortcomings such as long cycle, complex operation, poor repeatability, false alarms, etc., which cannot meet the requirements of acute toxicity monitoring of water quality. need.

Electrochemically active microorganisms (EABs) are environmental microorganisms with a unique extracellular electron transfer function, which can convert the chemical energy of organic matter into electrical energy while maintaining growth and metabolism. EAB research originated in 1921, when American scientists first discovered that yeast and E. coli can generate electrical signals under specific conditions, and envisaged their application in space activities to improve the reliability of manned space missions. After this study, EAB's unique energy conversion method has attracted more and more researchers' attention. Korean researchers Kim et al. first introduced EAB into the field of biomonitoring in 2007 and demonstrated the feasibility of EAB to detect water toxicity. The basic principle of EAB detection of water toxicity is that the electrical signal generated by microorganisms can directly reflect its metabolic activity. When EAB comes into contact with toxic pollutants in water, the pollutants will affect the metabolic activity of electricity-producing microorganisms, thereby promoting the current signal. or inhibit. Therefore, compared with the existing biological methods, EAB can realize online monitoring of water toxicity by utilizing the output current signal of EAB without additional signal transduction devices.

Based on the research of Kim et al., domestic and foreign scholars have further expanded the detection range of EAB detection technology for water toxicity, and successfully realized the detection of Cr(VI), Cd(II), Cu(II), Ni(II), nitrofen The detection of common toxic pollutants such as acephate and neomycin sulfate and new toxic pollutants such as neomycin sulfate has shown a good application prospect. Low sensitivity is the main factor limiting the application of EAB technology to detect water toxicity. Researchers at home and abroad have optimized a series of process parameters of EAB water quality detection technology, including electrode material, external resistance, control mode, sensor configuration, electronic mediator concentration and substrate concentration, etc. The above optimization has improved EAB detection to a certain extent. Sensitivity and accuracy of water toxicity techniques. However, despite a large number of optimization studies, the detection limits of the existing EAB detection technology for water toxicity detection of major pollutants still cannot meet the increasingly stringent environmental standards.

In general, the EAB detection technologies for water toxicity reported by the existing research all use the biofilm formed by EAB as the detection element. Although the biofilm has the ability of self-renewal, self-repair and self-maintenance, it is more conducive to long-term online monitoring of water quality. This is also the main reason for the poor sensitivity and low accuracy of EAB detection of water toxicity. This is because the biofilm contains a large amount of EPS composed of polysaccharides and proteins, which makes the biofilm structure complex and dense, which is not conducive to the diffusion of toxic pollutants into the biofilm or even into the cells. Moreover, EPS can adsorb and complex toxic pollutants, reducing the adverse effects of toxic pollutants on microorganisms. In addition, a large number of studies have proved that the biofilm formed by microorganisms has heterogeneity, and the metabolic activity presents a gradient change, that is, the bacterial activity on the surface is high, and the bacterial growth in the central area is slow or even non-growing, resulting in a decrease in the sensitivity of the biofilm to pollutants. . In addition, since the biofilm is a three-dimensional structure, its thickness and density will change significantly with the incubation time, so the repeatability and accuracy of the detection results using the biofilm as the detection element are poor. Due to the lack of biofilm barriers, suspended bacteria will be directly exposed to the full dose of pollutants when in contact with toxic pollutants, so suspended bacteria are more sensitive to external adverse environments than biofilm bacteria, suggesting that suspended bacteria may be more suitable as Sensing element to detect water toxicity. A large number of previous studies have shown that the suspended state of mixed bacteria EAB can hardly complete the extracellular electron transfer, and cannot output the current signal to realize the detection of water quality toxicity. The applicant's recent research found that the suspended state of several model EAB strains (eg, Shewanella oneidensis MR-1 and Shewanella loihica PV-4) has the ability to transfer electrons in different phases, and can output electrical signals without incubating biofilms. Therefore, the construction of water quality toxicity detection technology with these microorganisms as the core is expected to solve the application bottleneck faced by the current EAB water quality toxicity detection technology.

SUMMARY OF THE INVENTION

The invention establishes a method for realizing rapid detection of water toxicity based on suspended electrochemically active microorganisms. The method constructs a microbial electrochemical water toxicity sensor with suspended electrochemical microorganisms as the core, does not need to incubate biofilms, and only uses electricity The bacterial suspension of chemically active microorganisms can directly transduce the information of water toxicity into electrical signals, realize the rapid detection of water toxicity, and meet the needs of emergency detection and mobile detection of water toxicity.

The principle of this method is as follows: electrochemically active microorganisms can convert chemical energy in organic matter into electrical energy, but most of them need to form a biofilm to output electrical signals. The applicant previously screened out the electrochemical activity with heterogeneous electron transfer ability Microorganisms, without the need to form a biofilm, can also transfer electrons generated by the respiratory chain to the outside of the cell and use electrodes as electron acceptors to form an output current. Therefore, the electrochemically active microorganisms that can output current in a suspended state are The core is to build a bioelectrochemical system. When the solution does not contain toxic pollutants, electrochemically active microorganisms can form a stable output current through the heterogeneous electron transfer process in a suspended state, and when the solution contains toxic pollutants, the electrochemically active microorganisms can form a stable output current. The activity of chemically active microorganisms will be gradually inhibited, and the output current signal will also show a downward trend. Therefore, the rapid detection of water toxicity can be achieved by quantifying the degree of inhibition of the output current of the suspended electrochemically active microorganisms by the water body to be tested.

The specific steps of this method are as follows:

1) Construct a three-electrode electrochemical system. The effective volume of the electrochemical system is 50 mL and the working volume is 40 mL. The working electrode, counter electrode and reference electrode are 2cm×2cm carbon cloth, 1cm×1cm platinum sheet electrode and Ag/AgCl respectively. Electrode (0.205V vs. standard hydrogen electrode);

2) Using Luria-Bertani medium, the electrochemically active microorganism Shewanella oneidensis MR-1 with heterogeneous electron transfer in suspension was activated overnight, and then 20 mL of the bacterial suspension with an OD600 of between 1 and 2 was added to the electro- chemical system;

3) Add 20mL of water sample to be tested into the electrochemical system, then use high-purity nitrogen to deoxygenate the electrochemical system, the aeration time is 15-30min, the aeration flow rate is 300-100mL/min, the electrolyte is The liter contains 1 g NaHCO ₃ , 0.13 g KCl, 0.027 g CaCl _{2.2H 2} O, 0.2 g MgCl ₂ .6H ₂ O, 5.85 g NaCl and 7.2 g HEPES;

4) Place the electrochemical system in a temperature-controlled environment, with a temperature control range of 22±1°C, use an electrochemical workstation to apply a constant potential of +0.5V to the electrochemical system, and set the system static time to 30min before recording the current;

5) After the resting time is over, continue to record the current of the electrochemical system for 30min, and the recording interval is 1s;

6) Calculate the water toxicity coefficient according to formula (1), wherein i ₀ is the current of the electrochemical system at the beginning of detection, and i _t is the current of the electrochemical system at the 30th minute;

T _W =(1-i _t /i ₀ )×100 (1)

7) When Tw≥10, it can be judged that the water quality is toxic; when 10>Tw≥0, it can be judged that the water quality is non-toxic.

The beneficial effects that the present invention can achieve: compared with the prior art, the present invention can complete the water toxicity detection only by using suspended electrochemically active microorganisms without pre-incubating biofilms (generally, the incubation time is 3-7 days), which can satisfy At the same time, the method established by the present invention has high water quality toxicity detection sensitivity, can more effectively early warning of water pollution, and has wide application prospects.

Further, when the present invention uses suspended electrochemically active microorganisms to detect water toxicity, the method has high sensitivity to both heavy metal pollution and organic pollution. It has higher sensitivity and can be used for early warning of heavy metal pollution and organic pollution in water, especially for early warning of organic pollution.

Due to the selection of electrochemically active microorganisms with heterogeneous electron transfer ability in suspension, a biofilm-free water quality detection system can be constructed.

Description of drawings

Fig. 1 is the schematic diagram that the present invention realizes rapid detection of water toxicity based on suspended electrochemically active microorganisms

2 is the detection of three different heavy metals based on suspended electrochemically active microorganisms in Example 1 of the present invention and the linear fitting (ab: detection of different concentrations of Cd ²⁺ ; cd: detection of different concentrations of Hg ²⁺ ; ef: detection of different concentrations of Ni ²⁺ )

3 shows the detection of three different organic pollutants and linear fitting based on suspended electrochemically active microorganisms in Example 1 of the present invention (a-b: detection of doxycycline hydrochloride at different concentrations; c-d: detection of phenol at different concentrations; e-f: detection of different concentrations concentration of perchlorophenol)

Detailed ways

Example 1

Construct an electrochemical system consisting of a working electrode, a counter electrode, and a reference electrode. The working electrode, counter electrode and reference electrode were respectively selected from 2cm×2cm carbon cloth (WOS1009, Taiwan Carbon Energy Corporation, China), 1cm×1cm platinum sheet (Pt210, Tianjin Aida Hengsheng Technology Co., Ltd., China) and standard Ag/AgCl electrode (R0303, Tianjin Aida Hengsheng Technology Co., Ltd., China; 222.4 mV vs. SHE). The carbon cloth undergoes the following pretreatment process before use. First, it is soaked in a mixture of acetone and ethanol (volume 1:1) overnight to remove surface residues, then rinsed with deionized water for several times, dried, and finally subjected to high-temperature ammonia treatment. All accessories except the reference electrode are sterilized by high temperature and high pressure, and the reference electrode is soaked in 75% alcohol overnight. After the sterilization of each accessory is completed, the assembly is completed in a clean workbench (SW-CJ-1F, Sujing Antai).

Shewanella oneidensis MR-1 strain was stored at -80°C. Before use, the strains were inoculated in Luria-Bertani medium and placed in a 22°C incubator overnight for activation. After two overnight activations, the OD600 of the Shewanellaoneidensis MR-1 bacterial suspension was approximately 1.5. 20mL bacterial suspension and 20mL sterile electrolyte were added to the electrochemical system, and each liter of electrolyte included 1g NaHCO ₃ , 0.13g KCl, 0.027g CaCl _{2.2H 2} O, 0.2g MgCl ₂ .6H ₂ O, 5.85g NaCl and 7.2g HEPES. After adding the liquid, nitrogen was continuously aerated for 20 min to remove the dissolved oxygen in the solution. The electrochemical system was placed in a 22°C constant temperature incubator (HPS-500, Harbin Donglian Electronic Technology Development Co., Ltd.), and a multi-channel potentiostat (CHI1030C, Shanghai Chenhua Instrument Co., Ltd., China) was used to apply +0.5V Potential (vs. reference electrode), the rest time was set to 30 min, and the output current of the electrochemical system was recorded after 30 min.

It can be seen from Figure 2 (Figures 2a, 2c, 2e) that when the output current of the electrochemical system is collected, the output current of the electrochemical system is basically stable. Specifically, the output current of the electrochemical system did not fluctuate by more than 3% within 20 min, which indicated that the suspended Shewanella oneidensis MR-1 could output current stably under normal water quality conditions. At 20 min, toxic pollutants were added to the electrochemical system to simulate an acute toxic shock. As can be seen from the figure, when 0.1 mg/L Cd ²⁺ was added to the electrochemical system, the output current of Shewanella oneidensis MR-1 decreased significantly, indicating that the use of suspended Shewanella oneidensis MR-1 can effectively achieve water quality Toxicity testing.

_Tw was calculated by Equation 1 to evaluate the degree of inhibition of the output current of Shewanella oneidensis MR-1 by toxic pollutants. The specific experimental results are:

When the microorganisms were exposed to 0.1mg/L Cd ²⁺ for 30min, the current inhibition rate reached 12.3%; when the Cd ²⁺ concentration increased to 0.2mg/L, the current inhibition rate was 20.4%; when the Cd ²⁺ concentration increased to 0.5mg /L, the current inhibition rate reached 30.6% (Fig. 2a). Using Prism GraphPad to fit the relationship between _Tw and Cd ²⁺ concentration (Fig. 2b), it was found that the linear relationship was good, R ² = 0.97, and the sensitivity of suspended electrochemically active microorganism Shewanella oneidensis MR-1 to detect Cd ²⁺ was 36.7% (mg/L) ^-1 .

When the microorganisms were exposed to 0.1mg/L Hg ²⁺ for 30min, the current inhibition rate reached 15.7%; when the Hg ²⁺ concentration increased to 0.2mg/L, the current inhibition rate was 23.1%; when the Hg ²⁺ concentration increased to 0.5mg /L, the current inhibition rate reached 31.9% (Fig. 2c), and the sensitivity of Hg ²⁺ detection using suspended electrochemically active microorganism Shewanella oneidensis MR-1 was calculated to be 38.7% (mg/L) ^-1 (Fig. 2d) .

When the microorganisms were exposed to 0.1mg/L Ni ²⁺ for 30min, the current inhibition rate reached 11.5%; when the Ni ²⁺ concentration increased to 0.2mg/L, the current inhibition rate was 18.8%; when the Ni ²⁺ concentration increased to 0.5mg /L, the current inhibition rate reached 29.3% (Fig. 2e), and the sensitivity of using suspended electrochemically active microorganism Shewanella oneidensis MR-1 to detect Ni ²⁺ was calculated to be 35.1% (mg/L) ^-1 (Fig. 2f) .

When the microorganism was exposed to 0.1 mg/L doxycycline hydrochloride for 30 minutes, the current inhibition rate reached 12.6%; when the concentration of doxycycline hydrochloride increased to 0.2 mg/L, the current inhibition rate was 21.4%; When the concentration of doxycycline increased to 0.5 mg/L, the current inhibition rate reached 30.2% (Fig. 3a). The sensitivity of using the suspended electrochemically active microorganism Shewanellaoneidensis MR-1 to detect doxycycline hydrochloride was calculated to be 39.8% (mg/L). ) ^-1 (Fig. 3b).

When the microorganisms were exposed to 0.1mg/L phenol for 30min, the current inhibition rate reached 13.8%; when the phenol concentration increased to 0.2mg/L, the current inhibition rate was 20.4%; when the phenol concentration increased to 0.5mg/L, the current inhibition rate It reached 32.1% (Fig. 3c), and the calculated sensitivity for the detection of phenol using the suspended electrochemically active microorganism Shewanella oneidensis MR-1 was 44.9% (mg/L) ^-1 (Fig. 3d).

When the microorganisms were exposed to 0.1mg/L perchlorophenol for 30min, the current inhibition rate reached 14.5%; when the perchlorophenol concentration increased to 0.2mg/L, the current inhibition rate was 19.6%; when the perchlorophenol concentration increased to 0.5mg /L, the current inhibition rate reached 31.9% (Fig. 3e), and the calculated sensitivity of using suspended electrochemically active microorganism Shewanella oneidensis MR-1 to detect perchlorophenol was 41.9% (mg/L) ^-1 (Fig. 3f).

It can be seen from the above experimental results that the experimental results fully demonstrate that this technique can effectively evaluate water quality toxicity, and the method has high sensitivity to heavy metal pollution and organic pollution. Furthermore, the present invention has significantly better sensitivity results for the detection of organic pollutants. Compared with the detection sensitivity of heavy metal pollutants, the sensitivity is 35.1%-38.7%, and the detection sensitivity of organic pollutants is as high as 39.8%-44.9%.

When using Shewanella loihica PV-4 and Pseudomonas aeruginosa as electrochemically active microorganisms for experiments, a similar trend of results was obtained with Shewanella oneidensis MR-1, which may be because the above three belong to the heterogeneous electron transfer ability in the suspended state Therefore, they can be used as electrochemically active microorganisms to construct a water quality detection system without biofilm.

Therefore, this technology can be applied to the early warning of heavy metal pollution and organic pollution in water, especially for the early warning of organic pollution.

Claims (8)

1. a method for realizing rapid detection of water toxicity based on suspended electrochemically active microorganisms, is characterized in that: the method is by constructing a microbial electrochemical water toxicity sensor with suspended electrochemical microorganisms as the core, does not incubate biofilm, only utilizes The bacterial suspension of electrochemically active microorganisms can directly transduce water quality toxicity information into electrical signals, realize rapid detection of water quality toxicity, and meet the needs of emergency detection and mobile detection of water quality toxicity; the electrochemically active microorganisms specifically include Shewanella oneidensis One or more of MR-1, Shewanella loihica PV-4 or Pseudomonasaeruginosa; the specific steps of the method include: (1) Construct a three-electrode electrochemical system, whose working electrode, counter electrode and reference electrode are carbon cloth, platinum sheet electrode and Ag/AgCl electrode respectively, or use a wire mesh containing working electrode, counter electrode and reference electrode at the same time printed electrodes; (2) overnight activation treatment with electrochemically active microorganisms capable of heterogeneous electron transfer in a suspended state, and then the bacterial suspension with an OD600 between 1 and 2 is added to the electrochemical system; (3) adding the water sample to be tested into the electrochemical system, and deoxidizing the electrochemical system; (4) Place the electrochemical system in a temperature-controlled environment, apply a constant potential of about +0.5V to the electrochemical system using an electrochemical workstation, and set the static time of the system before recording the current; (5) After the static time is over, continue to record the current of the electrochemical system; (6) Calculate the water quality toxicity coefficient according to the recorded results, and judge the toxicity of water quality; Wherein, step (6) calculates the water toxicity coefficient according to formula (1), wherein i ₀ is the current of the electrochemical system at the beginning of detection, and i _t is the current of the electrochemical system at the 30th minute: T _W =(1-i ^t /i ₀ )×100 (1) When Tw≥10, it can be judged that the water quality is toxic, and when 10>Tw≥0, it can be judged that the water quality is non-toxic. 2. method as claimed in claim 1 is characterized in that, the carbon cloth specification of step (1) is 2cm * 2cm, the platinum sheet specification is 1cm * 1cm, and described three-electrode electrochemical system effective volume is 50mL and working volume is 40 mL, or the three-electrode electrochemical system is a small system with an effective volume ≤ 10 mL or a micro system with an effective volume ≤ 1 mL; Luria-Bertani medium is used for the overnight activation treatment in step (2). 3. method as claimed in claim 1, is characterized in that, the described deoxygenation treatment of step (3) utilizes high-purity nitrogen to carry out aeration, and aeration time is 15-30min, and aeration flow rate is 300-100mL/min; Step (3) The electrochemical system contains an electrolyte, and each liter of the electrolyte contains 1 g of NaHCO ₃ , 0.13 g of KCl, 0.027 g of CaCl ₂ .2H ₂ O, 0.2 g of MgCl ₂ .6H ₂ O, 5.85 g of NaCl and 7.2 g of NaCl gHEPES. 4. method as claimed in claim 1 is characterized in that, the temperature control range during step (4) temperature control is 22 ± 1 ℃, and the system static time before recording current is set to 30min; Step (5) continues recording electrochemical system The current was 30 min, and the recording interval was 1 s. 5. The microbial electrochemical water toxicity sensor constructed by the method of any one of claims 1-4. 6. The application of the microbial electrochemical water toxicity sensor according to claim 5 in the early warning of heavy metal pollution in water. 7. The application of the microbial electrochemical water toxicity sensor according to claim 5 in the early warning of organic pollution in water bodies. 8 . The application according to claim 7 , wherein the organic matter pollution detection sensitivity of the microbial electrochemical water toxicity sensor is higher than the heavy metal pollution detection sensitivity. 9 .

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