CN113607793A - Method for constructing biological membrane catalytic electrode with high activity - Google Patents
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
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- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
- Y02E60/30—Hydrogen technology
- Y02E60/50—Fuel cells
Abstract
The invention discloses a method for constructing a biomembrane catalytic electrode with high activity, belonging to the field of bioelectrochemistry. The invention constructs a bioelectrochemical tank, inoculates electrochemically active bacteria to a culture medium in the bioelectrochemical tank, operates the bioelectrochemical tank, keeps the anode potential constant to domesticate the electrochemical activity of the bacteria directionally, maintains low-level oxygen in the culture medium to promote the formation of a biofilm, applies hydraulic shearing to make the bacteria which are not firmly adhered on the surface of an electrode fall off, and continuously changes the culture medium to eliminate thallus cells which grow in suspension. The invention can quickly construct a biomembrane catalytic electrode with high activity at low cost to form a thicker and stable biomembrane, and has application value in the aspect of sensing chemicals or pollutants.
Description
Technical Field
The invention relates to the field of bioelectrochemistry, in particular to a method for constructing a biomembrane catalytic electrode with high activity.
Background
With the increasing national demand for pollution control and pollution prevention, microbial electrochemical technology has shown outstanding value as a typical solution of green chemistry. The core of microbial electrochemical technology lies in the interfacial electrochemical activity between microbial cells or proteins and solid electrodes. Since the first discovery by Potter of british biology in 1911 that microorganisms can generate electric current, various enzymes and bacteria are immobilized on the surface of an electrode in order to construct an electrochemical interface with excellent performance, but the electrochemical interface is hindered by enzyme inactivation and insulation of bacterial outer membrane/cell wall. Part of dissimilatory metal reducing bacteria in the electrochemically active microorganisms exist in a conductive channel crossing the outer membrane and are subjected to respiratory metabolism, so that molecular conditions are provided for constructing a high-efficiency bioelectrochemical system. By virtue of its conductive pathway, this part of the microorganisms has the ability to utilize a variety of natural or unnatural electron acceptors (e.g., azo dyes and high-valence metal/metalloid ions) for reductive respiration, which is an excellent choice for the application of bioelectrochemical catalysis techniques.
Establishing an effective electrically conductive connection with the electrodes is a prerequisite for the use of these novel electrochemically active microorganisms for increasing the level of bioelectrochemical technology. However, in the case of the classical facultative aerobic electrochemical microorganism, shewanella onadawsei, the adhesion capability to the electrode is limited, it is difficult to form a thick and stable biofilm, or the formed biofilm has only weak electrochemical activity. Therefore, the technicians in the field carry out gene modification on electrochemically active bacteria such as Shewanella onadatumi, and express more extracellular polysaccharide to improve the adhesion effect of the bacteria, and express more cytochrome c to improve the power generation capacity. However, these modifications have limited improvements in the electrochemical activity of the bacteria, but greatly increase the technical complexity and economic technical costs of their application. Therefore, it is very important to develop a simple, convenient, easy-to-use and reliable electrochemical catalysis technical scheme for electrochemically active bacteria to greatly improve the bioelectrochemical activity of the bacteria.
Disclosure of Invention
The invention aims to provide a simple and low-cost method for constructing a stable biomembrane catalytic electrode with high activity.
The method for constructing the biomembrane catalytic electrode with high activity comprises the following steps:
the method comprises the steps of establishing a bioelectrochemical tank, inoculating electrochemically active bacteria to a culture medium in the bioelectrochemical tank, operating the bioelectrochemical tank under an anode potential to enable the bacteria to be acclimated electrochemically on the surface of an electrode, maintaining low-level oxygen in the culture medium to enable the bacteria to perform aerobic respiration to a certain degree and simultaneously promote the bacteria to adhere and grow on the surface of the electrode, applying hydraulic shearing to enable the bacteria with weak adhesion on the surface of the electrode to fall off, and continuously replacing the culture medium to eliminate thallus cells with suspension growth and enable nutrient substances to be mainly supplied to the bacteria on the surface of the electrode for growth and utilization.
Preferably, the low level of oxygen in the maintenance medium is a maintenance medium dissolved oxygen level of 0.1mg/L to 8 mg/L.
Preferably, the application of hydraulic shear to detach the weakly adhered bacteria from the culture medium is stirring the culture medium to make it rotate. It is further preferred that a stirrer is added to the medium at a rotation speed of 80 to 400 rpm.
Preferably, the continuous medium replacement is performed every 24 hours.
Preferably, the bacteria are aerobic and facultative aerobic electrochemically active bacteria, such as Shewanella oneidensis MR-1.
Preferably, the bioelectrochemical cell is a single-chamber three-electrode bioelectrochemical cell, the three electrodes comprise a graphite sheet working electrode, a platinum wire counter electrode and a silver-silver chloride reference electrode, wherein the graphite sheet working electrode is connected into an electrochemical loop through a titanium wire, 1/2 volume of culture medium is added into the electrochemical cell to be used as electrolyte and inoculated with electrochemically active bacterial liquid, the potential of the working electrode needs to be kept constant between-0.2V and +0.6V vs. SHE, oxygen in the overhead space of the electrochemical cell is used for growth by bacteria, the cell and the electrolyte are replaced by a fresh and sterile cell and culture medium every 24 hours, the surface of the electrode is cleaned by sterile water, the replaced fresh culture medium is added with a yeast extract with the concentration of 1g/L by using a sodium lactate mineral salt culture medium, constant potential control is temporarily interrupted before the process of replacing the cell and the culture medium, running cyclic voltammetry scanning, wherein the potential range is from-0.5V to +0.4V vs. SHE, and standard hydrogen electrode SHE; the scan rate was 5 mV/s.
Preferably, the electrochemical workstation of the bioelectrochemical cell is CHI1040C, providing a potential of the working electrode of +0.4vvs.
The invention utilizes the physiological characteristics of aerobic and facultative aerobic electrochemical active microorganisms to design a special culture scheme to optimize the formation and strength of a biological membrane on the surface of an electrode material, so that the conductive cytochrome c is fully expressed, and the biological membrane electrode with high-level electrochemical activity and resistance to hydraulic shearing damage is obtained. The specific principle is that aerobic and facultative aerobic electrochemical active microorganisms can rapidly grow and synthesize cytochrome c when carrying out aerobic respiration in a rich culture medium, and meanwhile, the oxidative extracellular environment is favorable for curing the cytochrome c; on the other hand, the agglomeration and film forming ability of the composite material are regulated and controlled by the oxygen level, and a biological film can be well formed when a certain dissolved oxygen level is maintained for a long time. By utilizing the principle, the invention provides rich nutrient medium and continuous low-level oxygen in the bioelectrochemical pool for aerobic respiration, growth and adhesion on the surface of the electrode; continuously replacing the culture medium to eliminate the thallus cells growing in suspension, removing the competition of the thallus cells on nutrients and oxygen, promoting the growth and division of the thallus cells on the surface of the electrode, and improving the expression level of cytochrome c; applying an oxidative potential to enable bacteria on the surface of the electrode to perform limited aerobic respiration and electrode respiration simultaneously, so that the gradually growing bacteria are subjected to electrochemical acclimation to form a more effective extracellular electron transfer channel; the applied hydraulic shear makes the weak adhered bacteria fall off, the strength of the biological membrane is more stable, and the electrochemical interaction between the thallus and the electrode is stronger.
The invention can quickly construct a biomembrane catalytic electrode with high activity at low cost to form a thicker and stable biomembrane, and has application value in the aspect of sensing chemicals or pollutants.
Drawings
FIG. 1 is a schematic diagram of a single-chamber three-electrode electrochemical cell device for biofilm culture
FIG. 2 is a schematic diagram of the working electrode structure of FIG. 1
FIG. 3 is a current monitoring graph in the process of culturing Shewanella onadatumi MR-1 biofilm
FIG. 4 is a fluorescence microscopic field view of a biofilm of Shewanella onadatumi MR-1
FIG. 5 shows cyclic voltammetric signals during the culture of Shewanella knatz MR-1 biofilm
FIG. 6 shows the cyclic voltammetry signals of amaranth on the catalytic electrode of Shewanella onantha MR-1 biomembrane
FIG. 7 is a cyclic voltammetric signal of dichromate ions on a catalytic electrode of Shewanella onadawa bacteria MR-1 biofilm
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1
One-chamber three-electrode bioelectrochemical cell
As shown in FIGS. 1 and 2, before the single-chamber three-electrode bioelectrochemical cell of the experimental group is assembled, the glass bottle 1, the culture medium 2, the stirrer 3, the rubber stopper 4 and the working electrode 5 need to be sterilized at high temperature and high pressure together. Wherein the rubber stopper 4 is used as a sealing material and an electrode fixing material, the mouth of the glass bottle is sealed, and holes are drilled in advance before use so that the reference electrode 6 and the counter electrode 7 pass through the holes. The working electrodes 5 are two pieces of conductive electrode material 52 connected by wires 51. Before sterilization, the wire 51 of the working electrode 5 is assembled through the rubber stopper 4 in advance so that the conductive electrode material is located in the glass bottle 1. When assembled, the reference electrode 6 and the counter electrode 7 sterilized with 75% alcohol and ultraviolet rays for a short time are inserted into the glass bottle 1 through the rubber stopper 4 in a sterile work station. A250 mL glass blue-covered bottle is selected as the glass bottle 1, a 125mL LB culture medium is selected as the culture medium 2, a butyl rubber plug is selected as the rubber plug 4, a titanium wire is selected as the metal wire 51, a graphite sheet is selected as the conductive electrode material 52, a silver-silver chloride reference electrode is selected as the reference electrode 6, and a platinum wire counter electrode is selected as the counter electrode 7.
The conventional biomembrane culture scheme is taken as a control group, and the single-chamber three-electrode bioelectrochemical cell constructed by the conventional biomembrane culture scheme is the same as the single-chamber three-electrode bioelectrochemical cell of the experimental group, except that oxygen is not provided, stirring is not carried out, and oxidizing potential is still applied to enable bacteria to carry out electrode respiration. Specifically, the medium 2 in the glass bottle 1 was filled with nitrogen gas to remove all dissolved and headspace oxygen while constructing a single-chamber three-electrode bioelectrochemical cell, and the glass bottle 1 was closed with rubber stoppers 4 fixed to the respective electrodes under the condition that oxygen was not re-introduced. To avoid microbial contamination, the nitrogen source was passed through a sterile 0.22 μm filter head before entering the vial 1 and the entire system was allowed to proceed in a sterile clean bench until deoxygenation and sealing was complete, with other operations and other experimental conditions being the same as those in the experimental group.
Secondly, culturing the high-activity biological membrane
The Shewanella kandaensis MR-1 strain stored at-80 ℃ was inoculated into 100mL LB in a 250mL Erlenmeyer flask and activated overnight in a shaker at 30 ℃. Inoculating 1mL of bacterial liquid cultured overnight in advance into the culture medium 2; the electrochemical cell was connected to a multi-channel electrochemical workstation CHI1040C, running a chronoamperometric measurement, setting the potential of the working electrode to +0.2vvs. silver-silver chloride reference electrode (i.e. +0.4V vs. she). The current was monitored over time during the incubation and periodically interrupted to perform cyclic voltammetric scans as shown in figures 3 and 5.
For the experimental group, medium 2 was slowly stirred using stirrer 3, maintaining the stirring speed at 120rpm and the dissolved oxygen level at 0.1 to 8mg/L, and the experiment was performed at room temperature (28-30 ℃). After the above system was run for 24 hours ("LB cultivation" stage in fig. 3a and 3 b), the electrochemical cell was changed, wherein the new medium was sodium lactate mineral salt medium plus 1g/L yeast extract (specific formulation of sodium lactate mineral salt medium see literature Scientific Reports,2014,4:3735), electrochemical cultivation was continued with unchanged conditions, and the medium was changed every 24 hours for 4 consecutive times.
For the control group, the LB culture period was extended to 48 hours due to weak bacterial growth, and then replaced with a sodium lactate mineral salt medium supplemented with 1g/L yeast extract.
Thirdly, detecting the physiological activity and the electrochemical activity of the biological membrane
Within 5 days, the current produced by the experimental group of biofilms gradually increased. Followed byThe current generated by the biomembrane gradually becomes stable after the culture time is continuously prolonged, and finally reaches 13.25 muA/cm2A graphite electrode. After the experimental group of biofilms were stained with LIVE/DEAD back Bacterial Viability Kit, typical aggregation of the cells in the biofilms was observed under a fluorescence microscope (fig. 4a), and the cells in the biofilms were proved to have good physiological activity by the general yellow-green fluorescence. The best phase of biofilm current production in the control group was that during the initial LB culture, the highest current was only 13.6% of the experimental group. The red fluorescence which is commonly existed in the control group biomembrane is observed under a fluorescence microscope (figure 4b), which proves that a great amount of thallus cells with poor physiological activity and even death exist in the biomembrane.
Cyclic voltammetry tests run during the culture of experimental group biofilms (fig. 5a) found that the peak signals representing oxidation and reduction of cytochrome c increased rapidly over the first three days, indicating biofilm formation and rapid synthesis of cytochrome c. The redox peak signal is weakened but tends to be stable when the culture is continued, which indicates that the electrochemical activity of the biological membrane reaches maturity. The peak cyclic voltammetry signals of the control biofilms reached the strongest level on the sixth day (fig. 5b), and then were also attenuated and stabilized. From the magnitude of the peak current, the electrochemical activity of the control group of biological membranes is obviously weaker than that of the experimental group of biological membranes.
Example 2 detection of Activity of catalytic Amaranthus mangostanus and dichromate with biofilm catalytic electrode
The qualitative and quantitative analysis of amaranth (representing azo dye) and potassium dichromate (representing high-valent metal ion) was performed using the cultured biofilm catalysis electrodes of the experimental group and the control group of test example 1. Experiments were performed using a three electrode cell that can be sealed from oxygen. The working electrode is a cultured biomembrane catalytic electrode, the counter electrode is a platinum wire, and the reference electrode is a silver-silver chloride electrode. The electrolyte was 60mL of phosphate buffer (20mM NaH) containing supporting electrolyte2PO4，80mM Na2HPO45g/LNaCl, pH 7.4), previously deoxygenated. The parts here were assembled into a three-electrode cell for testing in an anaerobic glove box and the subsequent testing was completed in an anaerobic glove box.During testing, firstly, before amaranth and potassium dichromate are added, cyclic voltammetry scanning is carried out to obtain a basic electrochemical signal of the biomembrane catalytic electrode. Amaranth and potassium dichromate were then added to the electrolyte to the indicated concentrations, and cyclic voltammetric scanning was run again. The procedures and parameters of cyclic voltammetric scanning were: pre-polarization (+0.4V vs. she,20 s); scanning (+0.4V → -0.5V → +0.4V, rate 5mV/s, two cycles were run, with data from the second cycle taken as a standard).
In cyclic voltammetry (fig. 6) in which dichromate ions are subjected to reduction-oxidation conversion, a pair of newly generated redox peaks are observed in a low potential region and a high potential region, respectively, and serve as a marker signal of amaranth. The magnitude of the redox peak current can be used as the basis for judging the amaranth concentration, wherein the response of the experimental group of biological membranes to amaranth (figure 6a) is obviously stronger than that of the control group of biological membranes (figure 6 b).
In cyclic voltammetry (FIG. 7) in which the dichromate ion is subjected to reduction-oxidation conversion, no newly generated redox peak is observed. But the cyclic voltammetric signal representing the concentration of cytochrome c gradually decreased with increasing chromium concentration, reflecting the competition of dichromate ions for cytochrome c catalytic sites at the electrode surface, and could be used as a sensor for trace chromium contamination. The experimental group showed a clear response to dichromate (fig. 7a), while the control group showed no clear response due to too low activity (fig. 7 b).
It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the principles of the invention, and it is intended that such changes and modifications also fall within the scope of the appended claims.
Claims (9)
1. A method of constructing a biofilm catalytic electrode having high activity, comprising the steps of:
the method comprises the steps of establishing a bioelectrochemical tank, inoculating electrochemically active bacteria to a culture medium in the bioelectrochemical tank, operating the bioelectrochemical tank under an anode potential to enable the bacteria to be acclimated electrochemically on the surface of an electrode, maintaining low-level oxygen in the culture medium to enable the bacteria to perform aerobic respiration to a certain degree and simultaneously promote the bacteria to adhere and grow on the surface of the electrode, applying hydraulic shearing to enable the bacteria with weak adhesion on the surface of the electrode to fall off, and continuously replacing the culture medium to eliminate thallus cells with suspension growth and enable nutrient substances to be mainly supplied to the bacteria on the surface of the electrode for growth and utilization.
2. The method of claim 1, wherein the low level of oxygen in the maintenance medium is a maintenance medium dissolved oxygen level of 0.1mg/L to 8 mg/L.
3. The method of claim 1, wherein the step of applying hydraulic shear to dislodge the loosely adhered bacteria from the culture medium comprises agitating the culture medium and rotating the culture medium.
4. The method according to claim 3, wherein the stirrer is added to the culture medium at a rotation speed of 80-400 rpm.
5. The method of claim 1, wherein the continuous medium change is performed every 24 hours.
6. The method of claim 1, wherein said bacteria are aerobic and facultative aerobic electrochemically active bacteria.
7. The method according to claim 6, wherein the bacterium is Shewanella oneidensis (Shewanella oneidensis) MR-1.
8. The method of claim 1, wherein the bioelectrochemical cell is a single-chamber three-electrode bioelectrochemical cell comprising a graphite sheet working electrode, a platinum wire counter electrode and a silver-silver chloride reference electrode, wherein the graphite sheet working electrode is connected to an electrochemical circuit by a titanium wire, 1/2 volume of medium is added to the electrochemical cell as an electrolyte and inoculated with an electrochemically active microbial liquid, the potential of the working electrode is kept constant between-0.2V and +0.6V vs. SHE (standard hydrogen electrode, the same applies hereinafter), oxygen in the headspace of the electrochemical cell is used for growth by bacteria, the cell and the electrolyte are replaced with fresh and sterile cells and medium every 24 hours, the electrode surface is washed with sterile water, the replaced fresh medium uses a sodium lactate mineral salt medium plus a yeast extract with a concentration of 1g/L, before the process of replacing the electrolytic cell and the culture medium, the constant potential control is temporarily interrupted, the cyclic voltammetry scanning is operated, and the potential range is from-0.5V to +0.4V vs. SHE; the scan rate was 5 mV/s.
9. The method of claim 8, wherein the electrochemical workstation of the bioelectrochemical cell is CHI1040C, and the potential of the working electrode is +0.4V vs. SHE.
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