Spectroscopic characterization method, system and application for dissimilatory iron-reducing bacteria electron transfer

Abstract

The application discloses a spectrum characterization method and system for dissimilatory iron-reducing bacteria electron transfer and application, relates to the field of microbial electrochemistry, and solves the technical problems that the traditional characterization method of extracellular electron transfer efficiency of DIRB is complicated to operate, high in cost, low in sensitivity, and unable to realize real-time monitoring on the extracellular electron transfer process of DIRB. The method comprises the following steps: S1, synthesizing a fluorescent specific probe; S2, culturing and detecting dissimilatory iron-reducing bacteria; S3, detecting fluorescence spectrum; and S4, modeling and analyzing a quantitative relationship. According to the position and intensity change of the fluorescence spectrum characteristic peak, the quantitative relationship between the fluorescence quenching degree and the extracellular electron transfer efficiency is established, the electron transfer efficiency of the dissimilatory iron-reducing bacteria is judged, and thus the qualitative and quantitative characterization of the extracellular electron transfer efficiency is realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial electrochemistry, and relates to spectroscopic analysis technology, in particular to a spectroscopic characterization method, system and application for dissimilatory iron-reducing bacteria electron transfer. BACKGROUND

[0002] Dissimilatory iron-reducing bacteria (DIRB) is a kind of microorganism that can transmit electrons to external electron acceptors such as ferric ions by extracellular electron transfer (EET). DIRB has shown great application value in biogeochemical cycles, environmental pollution remediation, microbial fuel cells and other fields. The extracellular electron transfer efficiency of DIRB is one of the key factors affecting its application performance, and it is of great significance to develop a rapid, simple and sensitive EET efficiency characterization method.

[0003] Currently, the commonly used EET efficiency characterization methods include traditional electrochemical methods, spectroscopic methods and isotope labeling methods. The current characterization methods for the extracellular electron transfer efficiency of DIRB still have the following problems:

[0004] Firstly, the traditional electrochemical method (such as cyclic voltammetry) requires complex electrode modification operation and expensive instrument equipment, and the operation process is extremely cumbersome and the cost is also relatively high.

[0005] Secondly, the existing spectroscopic method (such as ultraviolet-visible absorption spectrum) has relatively low sensitivity and cannot achieve high-precision quantitative analysis.

[0006] Thirdly, there is a lack of a simple, rapid and highly sensitive characterization method to monitor the extracellular electron transfer process of DIRB in real time.

[0007] In view of the above problems, the present application proposes a new spectroscopic characterization method based on the fluorescence characteristics difference of the synthesized anthraquinone riboflavin fluorescence specific probe redox state, which realizes the qualitative and quantitative analysis of the extracellular electron transfer of DIRB by using the fluorescence quenching principle. SUMMARY

[0008] The present application aims to solve at least one of the technical problems existing in the prior art; for this purpose, the present application proposes a spectroscopic characterization method, system and application for dissimilatory iron-reducing bacteria electron transfer to solve the technical problems proposed in the above background technology. The present application realizes the qualitative and quantitative analysis of the extracellular electron transfer of DIRB by using the fluorescence quenching principle, and solves the problems of traditional DIRB extracellular electron transfer efficiency characterization methods, such as complicated operation, high cost, low sensitivity and inability to monitor the extracellular electron transfer process of DIRB in real time.

[0009] To achieve the above object, the first aspect of the present application provides a spectral characterization method for dissimilatory iron-reducing bacteria electron transfer, comprising:

[0010] Preparation of fluorescent specific probes made of materials with significant fluorescence property difference in oxidation state and reduction state;

[0011] Add the fluorescent specific probes to deionized water to form fluorescent probe solutions with different concentrations of fluorescent specific probes, and determine the concentration of the fluorescent specific probes and the fluorescence intensity standard curve;

[0012] Preparation of dissimilatory iron-reducing bacteria suspensions with different concentrations;

[0013] Determination of the concentration of the dissimilatory iron-reducing bacteria in the bacterial suspension and the change curve of the fluorescence intensity;

[0014] Construction of a number of co-culture samples and corresponding fluorescence spectrum detection to obtain co-culture fluorescence spectrum detection information;

[0015] According to the co-culture fluorescence spectrum detection information, a quantitative relationship model of fluorescence quenching degree and extracellular electron transfer efficiency is constructed for judging and / or characterizing the efficiency of dissimilatory iron-reducing bacteria electron transfer.

[0016] Another aspect of the present application provides a spectral characterization system for dissimilatory iron-reducing bacteria electron transfer, comprising:

[0017] Comprising:

[0018] The probe analysis module is used to determine the concentration of the fluorescent specific probes and the fluorescence intensity standard curve;

[0019] The bacterial suspension analysis module is used to determine the concentration of the dissimilatory iron-reducing bacteria in the bacterial suspension and the change curve of the fluorescence intensity;

[0020] The co-culture fluorescence spectrum detection information acquisition module is used to acquire co-culture fluorescence spectrum detection information;

[0021] And the extracellular electron transfer efficiency characterization module is used to construct a quantitative relationship model of fluorescence quenching degree and extracellular electron transfer efficiency for judging and / or characterizing the efficiency of dissimilatory iron-reducing bacteria electron transfer.

[0022] Another aspect of the present application provides an application of the above-mentioned spectral characterization system for dissimilatory iron-reducing bacteria electron transfer in the field of microbial electrochemistry, environmental science or energy in electron transfer efficiency spectral characterization.

[0023] Compared with the prior art, the present application has the following advantages:

[0024] The application can realize high-sensitivity detection by the fluorescence quenching principle through the difference between the fluorescence characteristics of the synthetic fluorescence specific probe in the oxidation state and the reduction state, can monitor the extracellular electron transfer process of DIRB in real time, has the advantages of high sensitivity, does not need a complex sample pretreatment process, is simple to operate, is suitable for laboratory and on-site detection, and only needs a conventional fluorescence spectrophotometer to complete the detection, is low in cost, can be applied to different types of dissimilatory iron-reducing bacteria and electron transfer efficiency characterization under different environmental conditions, can be widely applied to the research and application in the fields of microbial electrochemistry, environmental science and energy, and can be used for metal ion detection in water environment and soil environment, and has the advantage of wide application range. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0026] Fig. 1 It is a method flowchart of the present application.

[0027] Fig. 2 It is a synthetic structure formula of the anthraquinone disulfonic acid and riboflavin fluorescence specific probe in the present application. DETAILED DESCRIPTION

[0028] The technical solutions of the present application will be described in detail below in combination with the embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0029] Please refer to Figs. 1-2 The first aspect of the present application provides the embodiment 1

[0030] The spectral characterization method for electron transfer of dissimilatory iron-reducing bacteria comprises the following steps:

[0031] S1, synthesis of fluorescent specific probe: a material exhibiting significant fluorescence characteristics difference between oxidation state and reduction state is prepared, which will exhibit significantly different fluorescence characteristics in oxidation state and reduction state. The anthraquinone-sulfonic acid group is used to enhance water solubility and complexing ability with metal ions, realize electron acceptor specific recognition, and provide redox sensitive fluorescence signal by using riboflavin. The anthraquinone-riboflavin fluorescent specific probe, i.e. fluorescent specific probe, is synthesized and prepared. Specifically, anthraquinone disulfonic acid sodium AQDS is used as the parent structure, and riboflavin is introduced as the fluorescence signal unit. The sulfonic acid group (-SO3H) of anthraquinone disulfonic acid and the amino group (-NH2) of riboflavin are condensed under N2 protection to form a sulfonamide bond by removing water molecules: R-SO2-NH-R ， The anthraquinone disulfonic acid sodium and riboflavin are combined by amide bond chemical covalent coupling to obtain the fluorescent specific probe, and the structural formula is shown in FIG. 1. Fig. 2

[0032] In this embodiment, after obtaining the fluorescent specific probe, the structure of the fluorescent specific probe is verified. The vibration peak changes of the sulfonic acid group (1200 cm -1 ) of anthraquinone disulfonic acid sodium AQDS and the amino group (1650 cm -1 ) of riboflavin in the fluorescent specific probe are detected by FTIR analysis to confirm the formation of chemical bond. The fluorescence intensity decay rate is measured under different pH and temperature conditions to determine the stability of the fluorescent specific probe, wherein the pH range is pH 5-9, and the temperature range is 20-40°C. The interference degree of common anions such as SO4 2- , NO3 - , and reducing substances such as sulfide is tested to verify the specificity.

[0033] S2, cultivation and detection of dissimilatory iron-reducing bacteria: Geobacter sulfurreducens PCA is inoculated into LB medium containing 10 mM ferric citrate and 10 mg / L fulvic acid, and cultured under anaerobic conditions at 30°C to logarithmic growth phase. 10 mL of cultured bacterial solution is centrifuged at 5000 rpm for 10 min to collect the bacterial cells, which are washed twice with 0.1 M Tris-HCl buffer solution with pH=8 and resuspended to 5 mL to obtain bacterial suspension. The bacterial suspension is placed in a fluorescence spectrophotometer, the excitation wavelength is set to 350 nm, the emission wavelength is set to 450 nm, the fluorescence spectrum is recorded, and the concentration change curve of dissimilatory iron-reducing bacteria in the bacterial suspension and the fluorescence intensity is established.

[0034] ​S3, fluorescence spectrum detection: the fluorescence specific probe is added to deionized water, the excitation wavelength is set to 446 nm, the emission wavelength is set to 525 nm, the change of fluorescence intensity of the composite probe is detected, the quantitative relationship between fluorescence intensity and its oxidation state concentration is determined, and the concentration and fluorescence intensity standard curve of the fluorescence specific probe is drawn;

[0035] The fluorescence specific probe is co-cultured with dissimilatory iron-reducing bacteria, extracellular electron transfer is utilized to make the material change from the oxidation state to the reduction state, fluorescence quenching occurs, different concentrations of ferric ions are set, the fluorescence spectrum instrument is used for real-time monitoring of the change of fluorescence intensity, and co-culturing fluorescence spectrum detection information is acquired.

[0036] S4, quantitative relationship modeling and analysis: according to the co-culturing fluorescence spectrum detection information, the fluorescence intensity of the co-culturing sample before and after the dissimilatory iron-reducing bacteria are added is recorded, analyzed and calculated, the concentration and fluorescence intensity curve of the dissimilatory iron-reducing bacteria is drawn, the quantitative relationship between the fluorescence quenching degree and the extracellular electron transfer efficiency is determined according to the position and intensity change of the fluorescence spectrum characteristic peak, the quantitative relationship model of the fluorescence quenching degree and the extracellular electron transfer efficiency is formed, the electron transfer efficiency of Geobacter sulfurreducens PCA is judged, and qualitative and quantitative characterization of the extracellular electron transfer efficiency is realized.

[0037] Based on the above-mentioned spectrum characterization method for dissimilatory iron-reducing bacteria electron transfer,

[0038] The application can also construct a plurality of co-culturing samples and corresponding fluorescence spectrum detection, acquire co-culturing fluorescence spectrum detection information, and the construction method of the plurality of co-culturing samples comprises co-culturing a bacterial suspension and a fluorescence specific probe and adding an electron acceptor, and different co-culturing conditions are set;

[0039] According to the co-culturing fluorescence spectrum detection information, the quantitative relationship model of the fluorescence quenching degree and the extracellular electron transfer efficiency is constructed, and the efficiency of dissimilatory iron-reducing bacteria electron transfer is judged and / or characterized.

[0040] The first aspect of the application also provides an embodiment 2

[0041] The embodiment is different from embodiment 1 in that:

[0042] S2, culturing and detecting of dissimilatory iron-reducing bacteria: Shewanella oneidensis MR-1 is inoculated into LB medium containing 10 mM hydrous ferric oxide and 10 mg / L humic acid, and cultured under anaerobic conditions at 30°C to the logarithmic growth phase, 10 mL of the cultured bacterial solution is centrifuged at 5000 rpm for 10 min to collect the bacterial cells, the bacterial cells are washed twice with 0.1 M Tris-HCl buffer solution with pH=7 and then resuspended to 5 mL, the bacterial suspension is obtained, and the bacterial suspension is placed in a fluorescence spectrophotometer, the excitation wavelength is set to 350 nm, the emission wavelength is set to 450 nm, the fluorescence spectrum is recorded, and the change curve of the bacterial concentration and the fluorescence intensity is established.

[0043] S4, quantitative relationship modeling and analysis: according to the co-culture fluorescence spectrum detection information, the fluorescence intensity before and after the addition of dissimilatory iron-reducing bacteria in the co-culture sample is recorded, analyzed and calculated, the concentration of the dissimilatory iron-reducing bacteria and the fluorescence intensity curve of the fluorescence specific probe are drawn, the quantitative relationship between the fluorescence quenching degree and the extracellular electron transfer efficiency is determined according to the position and intensity change of the fluorescence spectrum characteristic peak, the quantitative relationship model of the fluorescence quenching degree and the extracellular electron transfer efficiency is formed, the electron transfer efficiency of Shewanella oneidensis MR-1 is determined, and thus the qualitative and quantitative characterization of the extracellular electron transfer efficiency is realized.

[0044] Another aspect of the present application provides example 3

[0045] The spectrum characterization system for electron transfer of dissimilatory iron-reducing bacteria comprises a probe analysis module for determining the concentration and fluorescence intensity standard curve of the fluorescence specific probe;

[0046] A bacterial suspension analysis module is configured to determine the concentration and fluorescence intensity change curve of the dissimilatory iron-reducing bacteria in the bacterial suspension.

[0047] A co-culture fluorescence spectrum detection information acquisition module is configured to acquire co-culture fluorescence spectrum detection information.

[0048] An extracellular electron transfer efficiency characterization module is configured to construct a quantitative relationship model of the fluorescence quenching degree and the extracellular electron transfer efficiency, and to determine and / or characterize the efficiency of electron transfer of dissimilatory iron-reducing bacteria.

[0049] Another aspect of the present application provides an application of the spectrum characterization system for electron transfer of dissimilatory iron-reducing bacteria in the field of microbial electrochemistry, environmental science or energy in spectrum characterization of electron transfer efficiency.

[0050] The above examples are only used to illustrate the technical method of the present application but not limit the present application. Although the present application is described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical method of the present application can be modified or equivalently replaced without departing from the spirit and scope of the technical method of the present application.

Claims

1. A spectroscopic characterization method for electron transfer in dissimilatory iron-reducing bacteria, characterized in that, include, A fluorescent specific probe is prepared, which is made of a material that has significant differences in fluorescence characteristics in the oxidized and reduced states; the fluorescent specific probe is added to deionized water to form fluorescent probe solutions with different concentrations of fluorescent specific probe, and a standard curve of fluorescent specific probe concentration versus fluorescence intensity is determined. Prepare bacterial suspensions of different concentrations of dissimilatory iron reducing bacteria; determine the variation curves of the concentration of dissimilatory iron reducing bacteria and fluorescence intensity in the bacterial suspensions; construct several co-culture samples and corresponding fluorescence spectra detection to obtain co-culture fluorescence spectra detection information; based on the co-culture fluorescence spectra detection information, construct a quantitative relationship model between fluorescence quenching degree and extracellular electron transfer efficiency to determine and / or characterize the electron transfer efficiency of dissimilatory iron reducing bacteria. The method for constructing several co-culture samples includes co-culturing bacterial suspension with fluorescent specific probes and adding electron acceptors, and setting different co-culture conditions; the co-culture conditions include the concentration of dissimilatory iron-reducing bacteria in the bacterial suspension, the culture time, and the concentration of electron acceptors; the method for preparing fluorescent specific probes includes using anthraquinone-sulfonic acid groups to enhance water solubility and complexation ability with metal ions to achieve electron acceptor specific recognition, using riboflavin to provide redox-sensitive fluorescent signals, and synthesizing anthraquinone-riboflavin fluorescent specific probes; The method for preparing the fluorescent specific probe includes: using sodium anthraquinone disulfonate as the parent structure, introducing riboflavin as the fluorescent signal unit, and under nitrogen protection, condensing the sulfonic acid group of anthraquinone disulfonate with the amino group of riboflavin to remove water molecules to form a sulfonamide bond, and then binding sodium anthraquinone disulfonate to riboflavin through chemical covalent coupling of the amide bond. The method for constructing a quantitative relationship model between fluorescence quenching degree and extracellular electron transfer efficiency based on co-culture fluorescence spectral detection information includes: recording, analyzing and calculating the fluorescence intensity of the co-culture sample before and after the addition of dissimilar iron-reducing bacteria, plotting the concentration of dissimilar iron-reducing bacteria and the fluorescence intensity curve of the fluorescence-specific probe, determining the quantitative relationship between fluorescence quenching degree and extracellular electron transfer efficiency based on the position and intensity changes of the characteristic peaks in the fluorescence spectrum, and forming a quantitative relationship model between fluorescence quenching degree and extracellular electron transfer efficiency.

2. The spectroscopic characterization method for electron transfer in dissimilar iron-reducing bacteria according to claim 1, characterized in that, The method for preparing fluorescent specific probes further includes: structural verification of the fluorescent specific probes; detecting the vibrational peak changes of the sulfonic acid group and riboflavin amino group of sodium anthraquinone disulfonate in the complex by FTIR analysis to confirm the formation of chemical bonds; determining the fluorescence intensity decay rate under different pH and temperature conditions to determine the stability of the composite probe; and testing the degree of interference from anions and reducing substances to verify specificity.

3. The spectroscopic characterization method for electron transfer in dissimilar iron-reducing bacteria according to claim 1, characterized in that, Preparation method of bacterial suspension of dissimilatory iron reducing bacteria: dissimilatory iron reducing bacteria are inoculated into a culture medium containing electron acceptors and cultured under anaerobic conditions to the logarithmic growth phase to obtain bacterial suspension; the bacterial suspension is taken, the bacterial cells are collected by centrifugation, washed with buffer solution and resuspended to obtain bacterial suspension.

4. The spectroscopic characterization method for electron transfer in dissimilar iron-reducing bacteria according to claim 1, characterized in that, The method for fluorescence spectroscopy detection includes: placing the co-cultured sample in a fluorescence spectrophotometer, setting the excitation wavelength to 300nm-500nm and the emission wavelength to 400nm-600nm for detection.

5. The spectroscopic characterization method for electron transfer in dissimilar iron-reducing bacteria according to claim 3, characterized in that, The electron acceptor is hydrated iron oxide.

6. A spectroscopic characterization system for electron transfer in dissimilar iron-reducing bacteria based on the method of claim 1, characterized in that, include: The probe analysis module is used to determine the standard curve of the concentration and fluorescence intensity of fluorescent specific probes. The bacterial suspension analysis module determines the change curve of the concentration of dissimilatory iron-reducing bacteria and the fluorescence intensity in the bacterial suspension; The co-culture fluorescence spectroscopy detection information acquisition module is used to acquire co-culture fluorescence spectroscopy detection information; It also includes an extracellular electron transfer efficiency characterization module, used to construct a quantitative relationship model between fluorescence quenching degree and extracellular electron transfer efficiency, for judging and / or characterizing the electron transfer efficiency of dissimilatory iron-reducing bacteria.

7. The application of the spectroscopic characterization system for electron transfer in dissimilar iron-reducing bacteria as described in claim 6 in the fields of microbial electrochemistry, environmental science, or energy for the spectroscopic characterization of electron transfer efficiency.

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