Microbial electrode and microbial sensor for nitrification toxicity detection

Abstract

The invention discloses a microbial electrode and a microbial sensor for nitrification toxicity detection. The microbial electrode provided by the invention comprises a substrate electrode and an artificial microbial film, the artificial microbial film is loaded with nitrifying bacteria and coats the surface of the substrate electrode, and the substrate electrode is modified by a redox medium. The redox medium is utilized to modify the substrate electrode, so that on one hand, nitrifying bacteria taking non-electroactive microorganisms as dominant flora have the capability of carrying out electron transfer with the electrode, and the activity change of the nitrifying bacteria can be expressed by an electric signal; and on the other hand, on the premise that the activity of the redox medium is not affected, the redox medium can be fixed on the surface of the substrate electrode, so that the loss of the redox medium can be effectively avoided, the use cost is effectively saved, secondary pollution to the environment is avoided, and the electron transfer rate between the mediator and the electrode can be increased.

Description

Technical Field

[0001] The invention relates to the technical field of wastewater toxicity detection, and in particular to a microbial electrode and a microbial sensor for nitrification toxicity detection. Background Art

[0002] Industrial wastewater refers to wastewater discharged during industrial production, which has the characteristics of a wide variety, large discharge volume, high pollution load and complex composition. Industrial wastewater contains a large number of organic pollutants, heavy metals, chemical substances and other pollution sources, some of which are highly toxic. Direct discharge into the natural environment will have a serious impact on human health and the ecosystem.

[0003] Biological treatment technology is the core process for removing organic pollutants from industrial wastewater. Common forms include activated sludge method, anoxic-aerobic (A / O) method, fluidized bed reactor and membrane bioreactor. These technologies mainly rely on activated sludge formed by microorganisms to remove pollutants from water, and the activity state of activated sludge is affected by toxic substances in wastewater. When the concentration of toxic substances in wastewater is high, the activity of activated sludge will decrease, the flocs will be loose, and the sedimentation performance will be reduced, resulting in poor pollutant removal effect and sludge loss, thereby affecting the effluent quality and the subsequent operation of the wastewater treatment plant.

[0004] In biological treatment systems, organic pollutants and toxic substances contained in industrial wastewater can have a serious impact on microbial activity. Biological treatment systems rely on microorganisms to degrade organic matter in wastewater, including nitrifying bacteria and other microorganisms that oxidize ammonia nitrogen. However, toxic substances in industrial wastewater can inhibit the metabolic activity of microorganisms, resulting in a decrease in microbial activity and affecting the wastewater treatment effect. In particular, the toxic effect of toxic substances on nitrifying bacteria is particularly prominent, because the oxidation of ammonia nitrogen in wastewater by nitrifying bacteria is a key step in the biological treatment system. Therefore, the evaluation and monitoring of toxic substances in industrial wastewater, especially the evaluation and monitoring of nitrification toxicity, has important scientific significance and practical value.

[0005] At present, the assessment, monitoring and early warning technologies for nitrification toxicity in industrial wastewater mainly include traditional chemical analysis methods, biological monitoring methods, and biosensor-based technologies developed in recent years.

[0006] Traditional chemical analysis methods assess the nitrification toxicity level by detecting the concentration of specific toxic substances in wastewater, and often use chromatography, mass spectrometry, and electrochemical methods to quantitatively analyze toxic substances in wastewater. However, these methods are usually time-consuming and costly, and cannot fully reflect the comprehensive situation of nitrification toxic substances in wastewater.

[0007] In order to reflect the comprehensiveness of wastewater toxicity, researchers further adopted biological monitoring methods to evaluate the toxicity level of wastewater by observing indicators such as the growth status and metabolic activity of nitrifying bacteria in wastewater. Commonly used biological monitoring methods include observing and evaluating the consumption of dissolved oxygen and ammonia nitrogen, production of nitrite, dehydrogenase activity, ATP concentration, mass transfer rate, cell integrity, etc. of nitrifying bacteria to indicate the degree of toxicity of wastewater. However, these biological monitoring methods have problems such as cumbersome operation and poor reproducibility, and usually require several hours of culture and detection time, which is inefficient.

[0008] In recent years, with the development of microbial electrochemical technology, researchers have constructed microbial sensor devices by combining microorganisms with carrier electrodes to accurately evaluate indicators such as microbial growth status and metabolic activity, thereby quickly and accurately monitoring toxic substances in wastewater. Although there are many electrodes in the field of electrochemistry that can specifically detect the concentration of certain substances, such as oxygen electrodes, ammonia nitrogen electrodes, and nitrite electrodes, these electrodes are combined with nitrifying bacteria to characterize the biological activity of nitrifying bacteria by detecting the rate at which the nitrifying bacteria consume oxygen and ammonia nitrogen or produce nitrite and nitrate in the ammonia oxidation reaction, thereby evaluating the nitrification toxicity in wastewater. Although this biosensor method simplifies the operation process and shortens the detection time, the principle of its toxicity evaluation is the same as that of the traditional biological monitoring method, except that the substance concentration detection method in the traditional biological monitoring method is replaced by an electrochemical electrode.

[0009] However, most of the microorganisms in biosensors are electroactive microorganisms that are enriched by activated sludge or cultured after screening specific strains. They exchange electrons through a variety of extracellular electron transfer mechanisms (EET). Most nitrifying bacteria are non-electroactive bacteria, usually do not have nanowire structures, and the redox mediators they secrete cannot directly exchange electrons with electrodes.

[0010] Considering that electroactive microorganisms can secrete redox mediators as intermediaries to exchange electrons with carrier electrodes, adding suitable soluble redox mediators has become an effective way to make nitrification toxicity sensors with nitrifying sludge as the test microorganisms. Although the redox mediators added to the culture medium can realize the electron exchange between the nitrifying sludge and the electrode, the added redox mediators exist in a free state in the culture medium and will be discharged after each detection, which not only increases the cost of use, but also pollutes the environment. Summary of the invention

[0011] The purpose of the present application is to solve the problem in the prior art of high cost and environmental pollution caused by adding soluble redox media when making nitrification toxicity sensors using nitrifying sludge as test microorganisms, and to provide a microbial electrode and microbial sensor for nitrification toxicity detection.

[0012] In order to achieve the above invention purpose, the technical solution adopted in this application is:

[0013] In a first aspect, the present application provides a microbial electrode for nitrification toxicity detection, wherein the microbial electrode comprises a substrate electrode and an artificial microbial membrane;

[0014] The artificial microbial membrane is coated on the surface of the substrate electrode, and the substrate electrode is modified by a redox medium;

[0015] The artificial microbial membrane is loaded with nitrifying bacteria.

[0016] In an optional embodiment, the redox mediator is a redox mediator that can enter and exit cells;

[0017] Optionally, the redox mediator comprises at least one of phenolic and quinone compounds, organic redox mediators and artificially synthesized redox mediators;

[0018] Optionally, the phenolic and quinone compounds include p-benzoquinone and / or coenzyme Q; the organic redox mediator includes ferrocene or its derivatives; and the artificially synthesized redox mediator includes at least one of methyl viologen, methylene blue and neutral red.

[0019] In an optional embodiment, the method of modifying the substrate electrode using the redox mediator includes at least one of a cyclic voltammetric electropolymerization method, an electrochemical deposition method, an adsorption method, a chemical bonding method, and a drop coating method;

[0020] And / or, when the base electrode is modified by the redox medium, the concentration of the redox medium in the modification solution is 0.1-100 mmol / L.

[0021] Among them, the cyclic voltammetry electropolymerization method may include the following operations:

[0022] The redox medium is placed in a suitable electrolyte, with the substrate electrode as the working electrode, the graphite electrode or the platinum sheet electrode or the titanium mesh electrode as the counter electrode, and the reference electrode such as the silver-silver chloride electrode or the mercury oxide electrode as the reference electrode. The potential range obtained according to the obvious redox peak shape of the redox medium is the scanning potential range, the scanning speed is 5 to 100 mv / s, and the scanning speed is 25 to 100 circles, so that the redox medium in the electrolyte is polymerized on the electrode surface.

[0023] The electrochemical deposition method may include the following operations:

[0024] The redox medium is placed in a suitable electrolyte, with the base electrode as the working electrode, the graphite electrode or platinum sheet electrode or titanium mesh electrode as the counter electrode, and the reference electrode such as silver-silver chloride electrode or mercury oxide electrode as the reference electrode, and a constant current, constant voltage or cyclic scanning potential is applied to the working electrode to deposit the redox medium in the electrolyte on the electrode surface.

[0025] The adsorption method may include the following operations:

[0026] The surface of the substrate electrode is immersed in a solution containing a redox medium, and the redox medium is adsorbed on the electrode surface by utilizing the chemical adsorption or intermolecular interaction between the substrate electrode surface and the redox medium, thereby preparing a chemically modified electrode with specific properties, and the redox medium is loaded on the electrode surface by adsorption.

[0027] Chemical bonding can include the following operations:

[0028] The chemical bonding method requires that the base electrode be pre-treated by oxidation-reduction and other methods to introduce a large number of oxygen-containing groups, and then use the covalent connection between the oxygen-containing groups and the compound groups in the redox medium to form a stable covalent bond, so that the redox medium is tightly adsorbed on the electrode surface and fixed on the electrode surface by chemical bonds.

[0029] The drop coating method may include the following operations:

[0030] The redox medium is dissolved in a suitable solvent to prepare a uniformly dispersed solution, an appropriate amount of the redox medium solution is pipetted and drop-coated on the treated bare electrode surface, and the solvent is evaporated under an infrared lamp or natural conditions so that the redox medium is uniformly covered on the electrode surface.

[0031] In an optional embodiment, the raw materials of the artificial microbial membrane include embedding material, nitrifying bacteria product, conductive material and cross-linking agent;

[0032] In parts by weight, the ratio of the embedding material, the nitrifying bacteria product, the conductive material and the cross-linking agent is (1-5): (1-10): (1-10): 1.

[0033] In an optional embodiment, the embedding material includes at least one of sodium alginate, polyacrylamide, polyvinyl alcohol and cellulose;

[0034] and / or, the conductive material comprises at least one of carbon nanotube powder, activated carbon powder, graphene powder and graphite powder;

[0035] And / or, the cross-linking agent includes at least one of calcium ion salt, ammonium persulfate, N,N,N',N'-tetramethylethylenediamine, sodium borate and copper sulfate. Specifically, different embedding materials can use different cross-linking agents. The cross-linking agent of sodium alginate material can be calcium ion salt; the cross-linking agent of polyacrylamide material can be ammonium persulfate and N,N,N',N'-tetramethylethylenediamine; the cross-linking agent of polyvinyl alcohol material can be sodium borate; the cross-linking agent of cellulose material can be copper sulfate or calcium salt.

[0036] For example, the nitrifying bacteria product, the embedding material and the conductive material can be mixed and then coated on the surface of the base electrode, and then the electrode is immersed in a cross-linking agent and kept at 20 to 25°C for a suitable time to obtain the artificial microbial membrane. The prepared artificial microbial membrane can be rinsed with distilled water, then placed in a refrigerator at -20°C for a certain period of time, and then stored at 0°C, and thawed and activated using an electrolyte solution containing a matrix when used.

[0037] The nitrifying bacteria product may be any product containing nitrifying bacteria, for example, it may be bacterial powder of nitrifying bacteria, or other products loaded with nitrifying bacteria. In an optional embodiment, the nitrifying bacteria product is nitrification sludge, and the preparation process of the nitrification sludge includes:

[0038] The aerobic sludge is acclimated and cultured using an acclimation medium. During the acclimation and culture process, the sludge concentration is controlled to be 1-5 g / L. The ammonia nitrogen removal rate is not less than 90% after acclimation for 4 hours. The sludge is then co-cultured with the redox medium for 0.5-12 hours, then washed and fixed to the original volume, and centrifuged at 3000-7000 revolutions for 3-10 minutes.

[0039] Wherein, the acclimation medium can be a commonly used nitrifying bacteria acclimation medium in the field. Exemplarily, the acclimation medium can contain only an inorganic carbon source without an organic carbon source, and only contains ammonium chloride that can be used by nitrifying bacteria (such as ammonia oxidizing bacteria) as an energy source.

[0040] In a specific embodiment, the acclimation medium includes water, stock solution A, stock solution B, stock solution C, FeSO4·7H2O and NH4Cl. The ammonia nitrogen concentration of the water can be 50-100 mg / L; the stock solution A can be K2HPO 4[and KH2PO4 aqueous solution, the concentration of K2HPO4 is 36.9g / L, the concentration of KH2PO4 is 5.17g / L; the stock solution B can be an aqueous solution of MgSO4 and CaCl2, the concentration of MgSO4 is 1.05g / L, and the concentration of CaCl2 is 0.53g / L; the stock solution C can be ZnSO4·7H2O, MnCl2·H2O, CoCl2·6H2O, CuSO4·5H2O, Ni The aqueous solution of Cl2·6H2O and Na2MoO4·2H2O, the concentration of ZnSO4·7H2O is 0.50g / L, the concentration of MnCl2·H2O is 0.50g / L, the concentration of CoCl2·6H2O is 0.40g / L, the concentration of CuSO4·5H2O is 0.40g / L, the concentration of NiCl2·6H2O is 0.20g / L, and the concentration of Na2MoO4·2H2O is 0.05g / L.

[0041] In an optional embodiment, relative to 1L of water, the amount of the stock solution A can be 0.2-0.4mL, the amount of the stock solution B can be 1-2mL, the amount of the stock solution C can be 0.5-1mL, the amount of FeSO4·7H2O can be 0.04-0.1g, and the amount of NH4Cl can be 0.44-1g.

[0042] In an optional embodiment, the substrate electrode includes at least one of a glassy carbon electrode, a gold electrode, a platinum electrode and a boron-doped diamond thin film electrode.

[0043] In a second aspect, the present application provides a microbial sensor for nitrification toxicity detection, wherein the biosensor comprises the above-mentioned microbial electrode.

[0044] In an optional embodiment, the microbial sensor includes the microbial electrode, an electrochemical workstation, an aeration pump, a reactor body, a reference electrode, a counter electrode, an injection needle and an electrolyte solution; wherein,

[0045] The top of the reactor body is provided with a working electrode hole, a reference electrode hole, a counter electrode hole, an injection hole and an air inlet hole; the microbial electrode, the reference electrode and the counter electrode extend into the reactor body through the working electrode hole, the reference electrode hole and the counter electrode hole respectively; the injection needle extends into the reactor body through the injection hole; the aeration pump is connected to the air inlet hole;

[0046] The electrochemical workstation is electrically connected to the microbial electrode, the reference electrode and the counter electrode respectively.

[0047] Exemplarily, the reference electrode may be a silver-silver chloride reference electrode, and the counter electrode may be a platinum counter electrode. The effective volume of the reactor body may be 100 mL, the working volume may be 90 mL, and the material thereof may be an organic or inorganic material other than a metal material.

[0048] The function of the electrolyte solution is to provide a stable pH environment for the nitration reaction, and the pH value is generally controlled to be 7-8. In a specific embodiment, the electrolyte solution may include 8.95 g / L of Na2HPO4·12H2O, 3.00 g / L of NaH2PO4, and 0.22 g / L of NH4Cl.

[0049] The technical solution of this application has at least the following beneficial effects:

[0050] (1) The microbial electrode provided by the present application includes a substrate electrode and an artificial microbial membrane, wherein the artificial microbial membrane is loaded with nitrifying bacteria and coated on the surface of the substrate electrode, and the substrate electrode is modified with a redox medium. Using a redox medium to modify the substrate electrode can, on the one hand, enable non-electroactive microbial nitrifying bacteria to have the ability to transfer electrons with the electrode, thereby allowing the activity changes of the nitrifying bacteria to be expressed by electrical signals; on the other hand, it can be fixed on the surface of the substrate electrode without affecting the activity of the redox medium, which can not only effectively avoid the loss of the redox medium, thereby effectively saving the use cost and avoiding secondary pollution to the environment, but also accelerate the electron transfer rate between the mediator and the electrode.

[0051] The microbial sensor, which realizes indirect electron exchange between nitrifying bacteria and electrodes by fixing redox mediators on the substrate electrode, can detect nitrification toxicity in wastewater in a very short time, and because the microorganisms are in a living state, this sensor can achieve long-term stable operation.

[0052] (2) The raw materials of the artificial microbial membrane of the microbial electrode provided in the present application include an embedding material, a nitrifying bacteria product, a conductive material and a cross-linking agent. Among them, nitrifying bacteria are used as the test microorganisms, so that the nitrification toxicity results obtained by the test are more representative; embedding with the embedding material can significantly reduce the formation time of the artificial microbial membrane. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0054] Figure 1 The structural diagram of the microbial sensor provided in Example 1 of the present application is schematically shown.

[0055] Description of reference numerals:

[0056] 1. Electrochemical workstation; 2. Aeration pump; 3. Reactor body; 4. Counter electrode; 5. Reference electrode; 6. Microbial electrode; 7. Injection needle. DETAILED DESCRIPTION

[0057] In order to further explain the technical means and results taken by the present invention to achieve the predetermined invention purpose, the specific implementation methods, technical solutions and features of the present invention application are described in detail below with preferred embodiments. The specific features, structures, or characteristics of the multiple embodiments in the following description can be combined in any suitable form.

[0058] Example 1

[0059] This embodiment provides a microbial sensor for nitrification toxicity detection. Figure 1 The structural diagram of the microbial sensor of this embodiment is schematically shown.

[0060] like Figure 1 As shown, the microbial sensor of this embodiment includes a microbial electrode 6, an electrochemical workstation 1, an aeration pump 2, a reactor body 3, a reference electrode 5, a counter electrode 4, an injection needle 7 and an electrolyte solution located in the reactor body. The top of the reactor body 3 is provided with a working electrode hole, a reference electrode hole, a counter electrode hole, an injection hole and an air inlet hole; the microbial electrode 6, the reference electrode 5 and the counter electrode 4 extend into the reactor body 3 through the working electrode hole, the reference electrode hole and the counter electrode hole respectively; the injection needle 7 extends into the reactor body 3 through the injection hole; the aeration pump 2 is connected to the air inlet hole. The electrochemical workstation 1 is electrically connected to the microbial electrode 6, the reference electrode 5 and the counter electrode 4 respectively.

[0061] The microbial electrode 6 is composed of a base electrode and an artificial microbial membrane, and the artificial microbial membrane is coated on the surface of the base electrode. The base electrode is a glassy carbon electrode modified by electrochemical polymerization of neutral red. The artificial microbial membrane is made of an embedding material (sodium alginate), wet bacteria of nitrified sludge, a conductive material (carbon nanotubes) and a cross-linking agent (calcium chloride).

[0062] The reference electrode 5 is a silver-silver chloride reference electrode, and the counter electrode 4 is a platinum sheet counter electrode. The effective volume of the reactor body 3 is 100 mL, and the working volume is 90 mL. The electrolyte solution is 8.95 g / L Na2HPO4·12H2O, 3.00 g / L NaH2PO4, and 0.22 g / L NH4Cl.

[0063] Example 2

[0064] This example introduces the domestication method of nitrifying sludge.

[0065] Nitrifying sludge is domesticated from aerobic sludge from municipal sewage treatment plants. The domestication medium consists of 1L water, 0.2mL stock solution A, 1.2mL stock solution B, 0.6mL stock solution C, 0.041g FeSO4·7H2O, and 0.44g NH4Cl. The influent ammonia nitrogen concentration is controlled to be 50mg / L (a higher ammonia nitrogen load is used to improve the enrichment efficiency of nitrifying bacteria).

[0066] Stock solution A: K2HPO4 (36.9 g / L) and K2HPO4 (5.17 g / L).

[0067] Stock solution B: MgSO4 (1.05 g / L) and CaCl2 (0.53 g / L).

[0068] Stock solution C: ZnSO4·7H2O (0.50g / L), MnCl2·H2O (0.50g / L), CoCl2·6H2O (0.40g / L), CuSO4·5H2O (0.40g / L), NiCl2·6H2O (0.20g / L), Na2MoO4·2H2O (0.05g / L).

[0069] The concentration of domesticated nitrifying sludge is controlled at about 3g / L. When the ammonia nitrogen removal rate reaches 90% within 4 hours, the nitrifying sludge domestication is considered complete.

[0070] Example 3

[0071] This embodiment introduces the method for making and using the microbial sensor described in Embodiment 1.

[0072] (1) The glassy carbon electrode was electrochemically modified using a polymerization solution containing 8.954 g / L Na2HPO4·12H2O, 3.00 g / L NaH2PO4, 8.4 g / L NaNO3, and 0.1 mM neutral red. The modification was performed using an electrochemical workstation and cyclic voltammetry was performed, scanning 25 cycles at 100 mV / s in the range of -1 V to 0.8 V.

[0073] (2) 10 mL of the nitrification sludge prepared in Example 2 was taken and co-cultured with 0.1 mL of a 0.1 mM neutral red solution for 1 hour, then washed with tap water several times to remove the neutral red in the culture medium, and the volume was fixed to the original volume. The washed nitrification sludge was centrifuged at 5000 rpm for 5 minutes to obtain nitrification sludge wet cells.

[0074] (3) Prepare a 5% sodium alginate solution, mix 1 mL of the sodium alginate solution, 1 g of wet nitrified sludge bacteria, and 0.1 g of carbon nanotubes to obtain a mixed sol; apply the mixed sol dropwise on the surface of the glassy carbon electrode modified in step (1), and place the electrode in a 5% calcium chloride solution for cross-linking; then wash it three times with phosphate buffer and freeze it in a -20°C refrigerator for 1 hour to obtain a microbial electrode.

[0075] (4) Add 70 mL of electrolyte solution (8.95 g / L Na2HPO4·12H2O, 3.00 g / L NaH2PO4, 0.22 g / L NH4Cl) to the reactor body, and add 10 mL of the test solution. The aeration pump is used to aerate the air at a rate of 30 mL / min. The working electrode, counter electrode and reference electrode are placed in the corresponding electrode holes. The corresponding electrochemical workstation wires are connected. The cyclic voltammetry in the electrochemical workstation is started. The voltage range is set to the potential range of the corresponding redox medium, the scan rate is set to 100 mv / s, and the number of scans is set to 200. In this process, the electrochemical workstation records the cyclic voltammetry changes of the nitrification toxicity sensor, with the number of scans as the horizontal axis and the peak current of the oxidation peak corresponding to the redox medium of each circle as the vertical axis. The descending part of the obtained curve after reaching the peak is linearly fitted to obtain the corresponding slope.

[0076] Data analysis is achieved by converting the toxicity of unknown toxic wastewater into 3,5-dichlorophenol (3,5-DCP) equivalent toxicity using 3,5-dichlorophenol as an intermediate medium: using different concentrations of 3,5-dichlorophenol and the slope of the fitting straight line obtained from the change in the peak current of 3,5-dichlorophenol at each concentration, a standard curve representing the correlation between the concentration and the slope of 3,5-dichlorophenol is established. When the wastewater to be tested enters the detector, the corresponding slope of the wastewater is first obtained by fitting according to the above-mentioned cyclic voltammetry method, and then based on the above-mentioned standard curve, the corresponding slope of the wastewater is used to reversely infer the corresponding 3,5-dichlorophenol equivalent concentration to achieve quantitative evaluation.

[0077] For each concentration of 3,5-dichlorophenol, the data of the peak current change of the oxidation peak taken by the sensor cyclic voltammetry are fitted with the best straight line using the least squares method, and an array describing the straight line, such as the slope a and the intercept b, is obtained and returned. Table 1 shows the slope and intercept of the fitted straight line corresponding to 6 concentrations of 3,5-dichlorophenol.

[0078] Table 1 Slopes and intercepts of the fitted lines corresponding to 6 concentrations of 3,5-dichlorophenol

[0079] serial number concentration Slope a Intercept b 1 0.71 -1.34E-09 1.49E-05 2 1.41 -2.74E-09 2.33E-05 3 4.11 -6.59E-09 1.61E-05 4 6.67 -1.66E-08 1.61E-05 5 9.09 -3.31E-08 1.44E-05 6 12.50 -5.25E-08 2.69E-05

[0080] The obtained 3,5-DCP corresponding slope and the corresponding 3,5-dichlorophenol concentration were fitted with Dose Response to obtain the slope-3,5-dichlorophenol concentration model, which is the aforementioned standard curve, and embedded in the data analysis software. The Dose Response fitting standard curve is:

[0081]

[0082] Where x is the equivalent concentration of 3,5-DCP, y is the slope of the best straight line, and the parameter values ​​(standard deviation) are as follows: A1 = 0.49348 (0.56655), A2 = 61.72372 (1.91343), LOG x0 =8.82528(0.15626), P=0.20467(0.01314), correlation is 0.99945.

[0083] In actual testing, the corresponding slope of the wastewater is obtained by fitting according to the cyclic voltammetry method, and then the corresponding slope of the wastewater is substituted into the formula (1) as the y value to reversely infer the corresponding 3,5-dichlorophenol equivalent concentration to achieve quantitative evaluation.

[0084] Test Case

[0085] The following three methods were used to test the nitrification toxicity of two wastewaters with different toxicity levels (pharmaceutical wastewater A and chemical wastewater B).

[0086] Method 1: The method described in Example 3.

[0087] Method 2: Neutral red solution (not polymerized on the electrode surface) is directly added to the suspension of nitrification sludge prepared in Example 2 (sodium alginate is not used to embed on the electrode surface), and then the electrode is inserted into the mixed solution; under constant aeration and sufficient substrate conditions, the wastewater sample is added to the mixed solution, and 350 cyclic voltammetry scans are performed between the electrode potential of -0.8v and 0.8v.

[0088] Method 3: Traditional nitrification inhibition test Water quality--Toxicity test for assessing the inhibition of nitrification of activated sludge microorganisms (ISO 9509:2006). The main principle is to expose the nitrifying sludge to the toxic sample and fully contact it. After 4 hours, the amount of ammonia nitrogen converted into nitrate and nitrite is determined, and compared with the blank control without toxic samples, the percentage reduction of nitrate and nitrite production is calculated, that is, the nitrification inhibition rate; at the same time, this method is used to determine the nitrification inhibition rate of 3,5-dichlorophenol at different concentrations, and a concentration-effect curve is drawn; the nitrification inhibition rate of the wastewater sample is converted into the equivalent concentration of 3,5-dichlorophenol.

[0089] The sample addition ratios under the above three methods were kept consistent; the substrate volume and neutral red content used in methods 1 and 2 were kept consistent; and the nitrification sludge addition concentrations in methods 2 and 3 were kept consistent. The test results are shown in Table 2.

[0090] Table 2 Nitrification toxicity test results of pharmaceutical wastewater A and chemical wastewater B

[0091]

[0092] The test results in Table 2 show that the technical solution of the present application has significant accuracy, sensitivity and timeliness in the detection of toxic wastewater, as follows:

[0093] (1) Method 3 (ISO 9509:2006) is the most classic and currently the only international standard method for wastewater nitrification toxicity testing. The equivalent concentrations of 3,5-dichlorophenol in the two wastewater samples measured by Method 1 are very close to the results of Method 3, with a difference of <7%, indicating that the microbial sensor of the present application has high accuracy in wastewater toxicity testing.

[0094] (2) The initial current value of Method 1 in the blank control test is much greater than that of Method 2, indicating that the use of sodium alginate to embed microorganisms and the use of neutral red polymer modified electrodes in Method 1 played an important role in the transfer of electrons from nitrifying bacteria to the electrode. Although the toxicity of the two wastewaters was significantly different, Method 1 achieved ideal results in the toxicity tests of both wastewaters, while Method 2 only showed a toxic response to Sample A and the slope was 3 orders of magnitude lower than that of Method 1, indicating that the use of sodium alginate to embed microorganisms and the use of neutral red polymer modified electrodes in Method 1 played an important role in the sensitivity of the sensor. The above results show that the microbial sensor of the present application has significant sensitivity in wastewater toxicity testing.

[0095] (3) In terms of test time, Method 1 and Method 2 complete the test within 1 hour, but Method 3 requires more than 4 hours. This indicates that the microbial sensor of the present application has high timeliness in wastewater toxicity testing. Since the test time is greatly shortened and continuous automatic online monitoring can be achieved, it provides favorable technical support for the monitoring and early warning of nitrification toxicity of actual wastewater and has broad market prospects.

[0096] The above is only a preferred specific implementation of the present invention; however, the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solution and its improved conception within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A microbial electrode for nitrification toxicity detection, characterized in that: The microbial electrode comprises a substrate electrode and an artificial microbial membrane; The artificial microbial membrane is coated on the surface of the substrate electrode, and the substrate electrode is modified by a redox medium; The artificial microbial membrane is loaded with nitrifying bacteria.

2. The microbial electrode according to claim 1, characterized in that: The redox mediator is a redox mediator that can enter and exit cells; Optionally, the redox mediator comprises at least one of phenolic and quinone compounds, organic redox mediators and artificially synthesized redox mediators; Optionally, the phenolic and quinone compounds include p-benzoquinone and / or coenzyme Q; the organic redox mediator includes ferrocene or its derivatives; and the artificially synthesized redox mediator includes at least one of methyl viologen, methylene blue and neutral red.

3. The microbial electrode according to claim 1, characterized in that: The method of modifying the substrate electrode using the redox medium comprises at least one of cyclic voltammetric electropolymerization, electrochemical deposition, adsorption, chemical bonding and drop coating; And / or, when the base electrode is modified by the redox medium, the concentration of the redox medium in the modification solution is 0.1-100 mmol / L.

4. The microbial electrode according to claim 1, characterized in that: The raw materials of the artificial microbial membrane include embedding material, nitrifying bacteria product, conductive material and cross-linking agent; In parts by weight, the ratio of the embedding material, the nitrifying bacteria product, the conductive material and the cross-linking agent is (1-5): (1-10): (1-10):

1.

5. The microbial electrode according to claim 4, characterized in that: The embedding material comprises at least one of sodium alginate, polyacrylamide, polyvinyl alcohol and cellulose; and / or, the conductive material comprises at least one of carbon nanotube powder, activated carbon powder, graphene powder and graphite powder; And / or, the cross-linking agent includes at least one of calcium ion salt, ammonium persulfate, N,N,N',N'-tetramethylethylenediamine, sodium borate and copper sulfate.

6. The microbial electrode according to claim 4 or 5, characterized in that: The nitrifying bacteria product includes nitrifying sludge, and the preparation process of the nitrifying sludge includes: The aerobic sludge is acclimated and cultured using an acclimation medium, the sludge concentration is controlled to be 1-5 g / L during the acclimation and culture process, and the ammonia nitrogen removal rate is not less than 90% after acclimation for 4 hours, and then co-cultured with the redox medium for 0.5-12 hours, and then washed and fixed to the original volume, and centrifuged at 3000-7000 rpm for 3-10 minutes; Optionally, the acclimation medium comprises water, stock solution A, stock solution B, stock solution C, FeSO4·7H2O and NH4Cl; wherein, The ammonia nitrogen concentration of water is 50-100 mg / L; And / or, the stock solution A is an aqueous solution of K2HPO4 and KH2PO4, the concentration of K2HPO4 is 36.9 g / L, and the concentration of KH2PO4 is 5.17 g / L; And / or, the stock solution B is an aqueous solution of MgSO4 and CaCl2, the concentration of MgSO4 is 1.05 g / L, and the concentration of CaCl2 is 0.53 g / L; And / or, the stock solution C is an aqueous solution of ZnSO4·7H2O, MnCl2·H2O, CoCl2·6H2O, CuSO4·5H2O, NiCl2·6H2O and Na2MoO4·2H2O, the concentration of ZnSO4·7H2O is 0.50 g / L, the concentration of MnCl2·H2O is 0.50 g / L, the concentration of CoCl2·6H2O is 0.40 g / L, the concentration of CuSO4·5H2O is 0.40 g / L, the concentration of NiCl2·6H2O is 0.20 g / L, and the concentration of Na2MoO4·2H2O is 0.05 g / L.

7. The microbial electrode according to claim 6, characterized in that: Relative to 1L of water, the amount of the stock solution A is 0.2-0.4mL, the amount of the stock solution B is 1-2mL, the amount of the stock solution C is 0.5-1mL, the amount of FeSO4·7H2O is 0.04-0.1g, and the amount of NH4Cl is 0.44-1g.

8. The microbial electrode according to claim 1, characterized in that: The substrate electrode includes at least one of a glassy carbon electrode, a gold electrode, a platinum electrode and a boron-doped diamond film electrode.

9. A microbial sensor for nitrification toxicity detection, characterized in that: The microbial sensor comprises the microbial electrode according to any one of claims 1 to 8.

10. The microbial sensor according to claim 9, characterized in that: The microbial sensor comprises the microbial electrode, an electrochemical workstation, an aeration pump, a reactor body, a reference electrode, a counter electrode, an injection needle and an electrolyte solution; wherein, The top of the reactor body is provided with a working electrode hole, a reference electrode hole, a counter electrode hole, an injection hole and an air inlet hole; the microbial electrode, the reference electrode and the counter electrode extend into the reactor body through the working electrode hole, the reference electrode hole and the counter electrode hole respectively; the injection needle extends into the reactor body through the injection hole; the aeration pump is connected to the air inlet hole; The electrochemical workstation is electrically connected to the microbial electrode, the reference electrode and the counter electrode respectively.

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