Microbial reduction graphene modified electrode, preparation method and application thereof

Abstract

The application discloses a kind of microbial reduction graphene modified electrode and its preparation method and application, the preparation method includes: the basic electrode material is mixed, and initial concentration <1.0mg / mL of graphene oxide suspension and sulfur-reducing bacillus are added, culture, and microbial reduction graphene modified electrode is obtained.The microbial reduction graphene modified electrode prepared by the application has the advantages of good conductivity, high specific capacitance, and high strain adhesion amount, is a new type of electrode material with excellent performance, and can be widely used to construct a bioelectrochemical system, while significantly improving the electrochemical performance and acid production performance of the bioelectrochemical system, has high use value and good application prospect.In addition, the preparation method also has the advantages of simple process, easy operation, high preparation efficiency, etc., is convenient for large-scale preparation, and is beneficial to industrial application.

Description

Technical Field

[0001] This invention belongs to the field of bioelectrochemical electrode technology, and relates to a microbial reduced graphene modified electrode, its preparation method and application. Background Technology

[0002] Bioelectrochemical systems (BES) are generally two-chamber H-type reactors. Structurally, electrode materials are the most critical factor affecting the performance of a BES system. Electrode materials need to possess characteristics such as high conductivity, high specific surface area, low internal impedance, strong mechanical strength, good biocompatibility, and chemical stability. Metallic electrodes have smooth surfaces and low biocompatibility, which is unfavorable for bio-attachment and growth, and they are easily eroded and corroded. Therefore, the most commonly used basic electrode materials in BES are primarily non-metallic carbon materials, such as carbon felt, graphite rods, carbon cloth, and carbon paper.

[0003] Graphene is a novel nanomaterial of carbon, consisting of a 2D honeycomb structure formed by the close packing of single-layer six-membered carbon rings. It possesses optical, electrical, and catalytic properties and is widely used in electrochemistry. The main methods for modifying electrodes with graphene are physicochemical and bioreduction methods. Physicochemically prepared graphene-modified BES electrodes can significantly optimize electrode electrochemical activity, accelerate electron transfer rates, and reduce electrode impedance. However, the physicochemical preparation method is demanding, requiring high temperatures and specific reactors, and may introduce toxic substances. Therefore, the use of green and efficient bioreduced graphene-modified electrodes is essential.

[0004] Bioreduced graphene oxide-modified electrodes are rarely reported. Among the existing reports, some researchers have used anaerobic activated sludge as inoculated microorganisms to reduce graphene oxide and prepare bioreduced graphene oxide-modified MFC anodes, achieving a maximum power density of 1905±80 mW / m³. 2 Its coulombic efficiency reached 54%, improving the energy output and electron transfer efficiency of MFC. Yong et al. (2014) assembled 3D porous graphene composite biomembrane electrodes using the extracellular electron transfer capability of Shewanella, achieving a maximum power density of 843±31 mW / m. 2 However, the above methods still have the following drawbacks or shortcomings: the reduction of GO by microorganisms is incomplete and uneven, making it difficult to stably load graphene onto the surface of the base electrode and to form a loose, porous graphene structure layer. This results in poor electrode conductivity, difficulty in increasing microbial adhesion and bioactivity, and difficulty in forming a structurally stable biofilm, ultimately leading to poor electrochemical performance of the electrode. Furthermore, there are no reports on the preparation method of electrodes modified with graphene oxide using *Geobacterium thioreduction* and its application in BES systems.

[0005] Therefore, obtaining a microbially reduced graphene-modified electrode with good conductivity and high specific capacity is of great significance for constructing bioelectrochemical systems with excellent electrochemical performance and promoting the widespread use of bioelectrochemical systems. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a microbial reduced graphene modified electrode with good conductivity, high specific capacity and high strain attachment, as well as its preparation method and application.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] A method for preparing a microbially reduced graphene-modified electrode includes the following steps:

[0009] S1. Mix the basic electrode material and culture medium to obtain a mixture;

[0010] S2. Add the graphene oxide suspension and sulfur-reducing Gastrobacterium to the mixture obtained in step S1 and culture it to obtain a microbial reduced graphene modified electrode; the initial concentration of graphene oxide in the culture system is <1.0 mg / mL.

[0011] In a further improvement to the above preparation method, in step S2, the initial concentration of graphene oxide in the culture system is 0.2 mg / mL to 0.8 mg / mL; and the initial volume concentration of thioreactive bacteria in the culture system is 5%.

[0012] In a further improvement to the above preparation method, in step S2, the addition of the graphene oxide suspension and the sulfur-reducing Gastrobacterium tumefaciens can be carried out in any of the following ways:

[0013] Method 1: First, add sulfur-reducing Gastrobacterium to the mixture obtained in step S1, and after an interval of 1 to 3 days, add graphene oxide suspension.

[0014] Method 2: Add the graphene oxide suspension and sulfur-reducing Gastrobacterium to the mixture obtained in step S1 simultaneously;

[0015] Method 3: First, add the graphene oxide suspension to the mixture obtained in step S1, and after an interval of 1 to 3 days, add sulfur-reducing bacteria.

[0016] In a further improvement to the above preparation method, in step S2, the sulfur-reducing Geobacterium is added once or three times.

[0017] The above preparation method is further improved in that the concentration of graphene oxide in the graphene oxide suspension is 2 mg / mL; the graphene oxide suspension is prepared by the following method: mixing graphene oxide with a sterile salt solution and sonicating for 1 h to 3 h to obtain the graphene oxide suspension; the sterile salt solution contains NH4Cl, Na2HPO4 and KCl; the sulfur-reducing bacillus is G. sulfurreducens PCA; the sulfur-reducing bacillus is a strain in the exponential growth phase; the OD of the sulfur-reducing bacillus... 600 The value ranges from 0.2 to 0.8.

[0018] The above preparation method is further improved in step S1, wherein the basic electrode material is at least one of carbon felt, graphite rod, carbon cloth, and carbon paper; the basic electrode material is also loaded with titanium wire; the basic electrode material further includes the following pretreatment before use: the basic electrode material is sequentially immersed in acetone, acid solution, and alkaline solution; the immersion time of the basic electrode material in acetone, acid solution, and alkaline solution is 24h, 12h, and 12h respectively; the acid solution is hydrochloric acid solution; the concentration of the acid solution is 1M; the alkaline solution is sodium hydroxide solution; the concentration of the alkaline solution is 1M; the culture medium is modified DMSZ culture medium; the modified DMSZ culture medium contains: 1.5g / L NH4Cl, 0.6g / L Na2HPO4, 0.1g / L KCl, 0.48g / L Na2S·9H2O, 10mL / L trace element solution, 10mL / L Wolfe vitamin solution, 1 mL / L selenite-tungstate solution, 20 mM sodium acetate, 50 mM sodium fumarate.

[0019] In a further improvement to the above preparation method, in step S2, the culture is carried out under light-proof and static conditions; nitrogen and carbon dioxide are introduced into the mixed solution during the culture process; the volume ratio of nitrogen to carbon dioxide is 4:1; the temperature is controlled at 25℃ to 35℃ during the culture process; and the culture time is 7 to 15 days.

[0020] As a general technical concept, the present invention also provides a microbial reduced graphene modified electrode, which is prepared by the above-described preparation method.

[0021] The above-mentioned microbial reduced graphene modified electrode is further improved in that the microbial reduced graphene modified electrode includes a basic electrode material, the surface of which is modified with microbial reduced graphene, and the interior and surface of the microbial reduced graphene are attached with thioreducing bacteria to form a multi-layer sandwich structure of strain-microbial reduced graphene-strain; the microbial reduced graphene has a porous two-dimensional sheet structure.

[0022] As a general technical concept, the present invention also provides an application of the above-mentioned microbial reduced graphene modified electrode in the construction of a bioelectrochemical system.

[0023] In a further improvement to the above application, the bioelectrochemical system includes a microbial fuel cell and a microbial electrolytic cell; in the microbial fuel cell, the microbial reduced graphene-modified electrode is used as the anode; in the microbial electrolytic cell, the microbial reduced graphene-modified electrode is used as the cathode.

[0024] Compared with the prior art, the advantages of the present invention are as follows:

[0025] (1) To address the shortcomings of existing methods for preparing microbially reduced graphene-modified electrodes, such as slow GO reduction rate, incomplete reduction and poor uniformity, low microbial adhesion, and difficulty in forming biofilms on the electrode surface or poor structural stability of the formed biofilms, this invention creatively proposes a method for preparing microbially reduced graphene-modified electrodes. First, a mixture of basic electrode materials and culture medium is prepared. Then, a suspension of graphene oxide and *Germella sulfolithotrigen* are added for cultivation. During cultivation, the strong reducing action of *Germella sulfolithotrigen* rapidly and completely reduces graphene oxide to graphene. Furthermore, the bioreduction force and aggregation characteristics allow graphene to stably adhere to the surface of the basic electrode material. As graphene continuously adheres, a wavy and wrinkled porous structure is formed on the surface of the basic electrode material. On the one hand, the porous graphene nanosheets formed during cultivation can serve as a carrier material for *Geobacterium thioreductoides* and provide more habitats for its growth. This not only further increases the amount of bacteria attached to the surface of the base electrode material but also improves the reduction rate and degree of graphene oxide. This results in a functional structural layer composed of a large number of bacteria and graphene on the surface of the base electrode material. More importantly, modifying the surface of the base electrode material with this functional structural layer significantly improves the electrode's conductivity and specific capacity, while also significantly enhancing the bioactivity and attachment amount on the electrode surface. This facilitates the formation of a highly bioactive and stable biofilm, ultimately significantly improving the electrode's electrochemical and acid-producing performance. The microbial reduced graphene-modified electrode prepared in this invention possesses advantages such as good conductivity, high specific capacity, and high bacterial attachment amount. It is a novel electrode material with excellent performance and can be widely used in constructing bioelectrochemical systems. It can significantly improve the electrochemical and acid-producing performance of bioelectrochemical systems, demonstrating high practical value and promising application prospects. In addition, the preparation method of the present invention has the advantages of simple process, easy operation and high preparation efficiency, which facilitates large-scale preparation and industrial application.

[0026] (2) In the preparation method of the present invention, the initial concentration of graphene oxide in the culture system is optimized to be <1.0 mg / mL. In particular, when the initial concentration of graphene oxide in the culture system is 0.2 mg / mL to 0.8 mg / mL, it is more conducive to preparing microbial reduced graphene modified electrodes with better conductivity and higher specific capacity. However, when the initial concentration of graphene oxide in the culture system is too high or too low, it is not conducive to improving the conductivity and specific capacity of the electrode. This is because if the GO concentration is too low, the amount of bioreduced graphene oxide will be less, and less graphene oxide will be formed on the electrode surface. This will make it difficult for the biofilm on the electrode surface to form or the formation rate will be slower when the electrode is used, which will obviously affect the electrochemical performance of the electrode. Excessive GO will inhibit the growth of thioreducing bacteria, resulting in a decrease in the degree of GO reduction or a decrease in graphene adhesion. At the same time, in the experiment, transmission electron microscopy (TEM) also showed that the cell membrane of thioreducing bacteria was damaged under high GO addition (>0.8 mg / mL). In this invention, graphene is a porous carbon material with a certain electron storage capacity due to its porous structure. Therefore, the more graphene anchored on the electrode, the better the electrode's electrical performance. Simultaneously, *Geobacterium thioreductoides*, an electrogenic bacterium, forms an electrochemically active biofilm on the carbon felt surface. Its extracellular cytochromes have electron storage capabilities, i.e., a capacitor-like effect. Therefore, the faster the *Geobacterium thioreductoides* grows on the electrode and the greater its attachment amount, the more beneficial it is to forming a high-performance electrode material.

[0027] (3) In the preparation method of the present invention, the addition method of graphene oxide suspension and sulfur-reducing bacteria is also optimized. In this method, sulfur-reducing bacteria are added first, and then graphene oxide suspension is added. On the one hand, this can promote the reduction degree and reduction rate of graphene oxide by sulfur-reducing bacteria, so that more strains and graphene can be attached to the electrode surface, thereby forming an electrode with higher conductivity and higher specific capacitance. On the other hand, under the combined action of sulfur-reducing bacteria and graphene, the structural stability of the electrode can be significantly improved, and it is more conducive to the formation of a highly active and stable biofilm on the electrode surface.

[0028] (4) In the preparation method of the present invention, the number of times of adding sulfur-reducing Globulus is 1 or 3. By adding sulfur-reducing Globulus multiple times, not only can the dispersibility of graphene on the electrode surface be improved, but also the amount of strain attached to the electrode can be increased. More importantly, a multi-layer sandwich structure of "strain-graphene-strain" can be formed, which can not only make microbial reduced graphene (MRGO) more stably attached to the electrode surface, but also significantly improve the specific capacity and conductivity of the electrode.

[0029] (5) This invention also provides an application of microbially reduced graphene modified electrode in the construction of a bioelectrochemical system. Specifically, the microbially reduced graphene modified electrode is used as the anode of a microbial fuel cell (MFC), which can not only reduce the consumption of organic acids and enhance glucose metabolism, but also improve the transfer efficiency of output electrons. At the same time, it can increase the acid production through carbon chain elongation reaction (especially significantly increase the production of butyric acid). Alternatively, the microbially reduced graphene modified electrode is used as the cathode of a microbial fuel cell (MEC), which can not only significantly increase the richness and diversity of the biological community in the cathode chamber of the MEC, but also accelerate the microbial electron transfer rate and significantly increase the production of volatile fatty acids (VFAs) (especially significantly increase the production of high-value butyric acid and hexanoic acid), with good application prospects. Attached Figure Description

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0031] Figure 1 The images show SEM images of the microbial reduced graphene modified electrode (A2) prepared in Example 2 of this invention and the PCA / carbon felt prepared in Example 1.

[0032] Figure 2 This is the output voltage curve of the microbial fuel cell prepared by PCA / carbon felt and microbial reduced graphene modified electrode (A2) in Example 3 of the present invention.

[0033] Figure 3 This is a graph showing the change in VFAs production over time over 10 days after the microbial fuel cell prepared from PCA / carbon felt and microbially reduced graphene modified electrode (A2) in Example 3 of the present invention has been operating stably.

[0034] Figure 4 This is a graph showing the change in VFAs production over time over 22 days after the microbial electrolysis cell, which was prepared from PCA / carbon felt and microbially reduced graphene modified electrode (A2) in Example 4 of the present invention, was put into stable operation. Detailed Implementation

[0035] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Example 1

[0037] A method for preparing a microbially reduced graphene-modified electrode, specifically investigating the effect of different concentrations of graphene oxide on electrode performance, includes the following steps:

[0038] Step 1: Disperse graphene oxide in a sterile salt solution (containing 1.5 g / L NH4Cl, 0.6 g / L Na2HPO4 and 0.1 g / L KCl), seal, and sonicate for 3 hours to form a graphene oxide suspension with a concentration of 2 mg / mL.

[0039] Step 2: Prepare carbon felt (30mm x 30mm) loaded with titanium wire (0.4mm in length). Soak the carbon felt loaded with titanium wire in acetone for 24 hours, rinse with ultrapure water, soak in 1M HCl solution for 12 hours, rinse with ultrapure water, soak in 1M NaOH solution for 12 hours, and rinse with ultrapure water to complete the pretreatment of carbon felt.

[0040] The pretreated carbon felt was added to 20 mL of modified DMSZ culture medium to mix the titanium wire-loaded carbon felt with the modified DMSZ culture medium evenly, resulting in a mixture. The modified DMSZ culture medium contained: 1.5 g / L NH4Cl, 0.6 g / L Na2HPO4, 0.1 g / L KCl, 0.48 g / L Na2S·9H2O, 10 mL / L trace element solution, 10 mL / L Wolfe vitamin solution, 1 mL / L selenite-tungstate solution, 20 mM sodium acetate, and 50 mM sodium fumarate.

[0041] Step 3: The mixture is autoclaved to ensure sterility and then divided into 6 groups.

[0042] Step 4: Add an appropriate amount of 2 mg / mL graphene oxide suspension and G. sulfurreducens PCA strain (commercially available). The inoculated strain is in the exponential growth phase with an OD of [missing value]. 600 Add 0.8 mg / mL to the sterilized mixture to make the final concentrations of graphene oxide in the mixture 0, 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1.0 mg / mL, respectively, and make the final volume concentration of Thiobacillus thioreductoids in the mixture 5% (v / v), thus obtaining mixtures with different graphene oxide concentrations.

[0043] Step 5: Place the mixture of different graphene oxide concentrations in an incubator at 30°C and incubate for 9 days under static and light-proof conditions. During the incubation process, nitrogen (flow rate 1.6 L / min) and carbon dioxide (flow rate 0.4 L / min) are introduced into the system, with a volume ratio of nitrogen to carbon dioxide of 4:1. During the incubation process, Geobacterium thioreduction rapidly and completely reduces graphene oxide to graphene and attaches it to the surface of the carbon felt, thus obtaining the microbial graphene-modified electrode, which is the Geobacterium thioreduction graphene-modified electrode.

[0044] In this embodiment, the prepared microbial reduced graphene modified electrode includes a basic electrode material, on which microbial reduced graphene is modified. Thiobacillus thioretinibacterium is attached to the inside and surface of the microbial reduced graphene, forming a functional structural layer composed of a large number of strains and graphene. It is a multi-layer sandwich structure composed of strain-microbial reduced graphene-strain, wherein the microbial reduced graphene is a porous two-dimensional sheet structure.

[0045] In this embodiment, when the final concentrations in the modified DMSZ culture medium were 0, 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1.0 mg / mL, the prepared microbial reduced graphene modified electrodes were respectively denoted as PCA / carbon felt, 0.2-MRGO, 0.4-MRGO, 0.6-MRGO, 0.8-MRGO, and 1.0-MRGO.

[0046] The electrochemical performance of the prepared microbially reduced graphene modified electrodes was tested, specifically by cyclic voltammetry (CV) analysis and electrochemical impedance spectroscopy (EIS) analysis. The results are shown in Table 1.

[0047] The cyclic voltammetry (CV) test results show that the capacitance is 0.8-MRGO (90.75F / m). 2 >0.6-MRGO(88.88F / m 2 >0.4-MRGO(84.03F / m 2 >0.2-MRGO(43.87F / m 2 >1.0-MRGO(9.43F / m 2 PCA / carbon felt (4.92F / m) 2 > Blank group (2.42F / m) 2 This shows that both MRGO and PCA strains can promote the storage of electrode charge.

[0048] As shown in Table 1, electrochemical impedance spectroscopy (EIS) analysis reveals that the electrochemical polarization impedance (Ri) is... ct) and solution resistance (R S The results show that the R of the MRGO-modified electrode group S and R ct All values ​​were lower than those in the PCA / carbon felt group and the blank group, with the 0.6-MRGO group showing the lowest R value. S and R ct .

[0049] Table 1 Electrochemical polarization impedance (R0) of graphene-modified electrodes with different microbial reduction. ct ) and solution resistance (R S )

[0050] Group Name <![CDATA[R S (Oh)]]> <![CDATA[R ct (Oh)]]> Group Name <![CDATA[R S (Oh)]]> <![CDATA[R ct (Oh)]]> Blank group 27.28 105.01 0.6-MRGO 9.58 29.23 PCA / Carbon Felt 15.41 70.65 0.8-MRGO 13.54 75.67 0.2-MRGO 13.14 67.71 1.0-MRGO 11.23 30.57 0.4-MRGO 10.19 50.38

[0051] Therefore, in the preparation method of the present invention, when the initial concentration of graphene oxide in the culture system is <1.0 mg / mL, and especially when the initial concentration of graphene oxide in the culture system is 0.2 mg / mL to 0.8 mg / mL, it is more conducive to preparing microbial reduced graphene modified electrodes with better conductivity and higher specific capacitance.

[0052] Example 2

[0053] A method for preparing a microbially reduced graphene-modified electrode, specifically investigating the effects of different access sequences and access times on electrode performance, includes the following steps:

[0054] Step 1: Disperse graphene oxide in a sterile salt solution (containing 1.5 g / L NH4Cl, 0.6 g / L Na2HPO4 and 0.1 g / L KCl), seal, and sonicate for 3 hours to form a graphene oxide suspension with a concentration of 2 mg / mL.

[0055] Step 2: Prepare carbon felt (30mm x 30mm) loaded with titanium wire (0.4mm in length) in a carbon felt preparation device. Soak the carbon felt loaded with titanium wire in acetone for 24 hours, rinse with ultrapure water, soak in 1M HCl solution for 12 hours, rinse with ultrapure water, soak in 1M NaOH solution for 12 hours, and rinse with ultrapure water to complete the pretreatment of carbon felt.

[0056] The pretreated carbon felt was added to 20 mL of modified DMSZ culture medium to mix the titanium wire-loaded carbon felt with the modified DMSZ culture medium evenly, resulting in a mixture. The modified DMSZ culture medium contained: 1.5 g / L NH4Cl, 0.6 g / L Na2HPO4, 0.1 g / L KCl, 0.48 g / L Na2S·9H2O, 10 mL / L trace element solution, 10 mL / L Wolfe vitamin solution, 1 mL / L selenite-tungstate solution, 20 mM sodium acetate, and 50 mM sodium fumarate.

[0057] Step 3: The mixture is autoclaved to ensure sterility and divided into 6 groups, numbered 1 to 6.

[0058] Step 4: In mixtures 1, 3, and 5, *Germolomia sulfadiazine* was added once; in mixtures 2, 4, and 6, it was added three times. In mixtures 1 and 2, *Germolomia sulfadiazine* was added first, followed by the graphene oxide suspension. In mixtures 3 and 4, *Germolomia sulfadiazine* and the graphene oxide suspension were added simultaneously. In mixtures 5 and 6, the graphene oxide suspension was added first, followed by *Germolomia sulfadiazine*. The time interval between each inoculation of the strain or GO in each group was 3 days.

[0059] Add an appropriate amount of 2 mg / mL graphene oxide suspension and G. sulfurreducens PCA strain (commercially available) to the mixtures 1 through 6. The inoculated strain is in the exponential growth phase with an OD of [missing value]. 600 After setting the concentration of graphene oxide to 0.8, the final concentration of graphene oxide in the mixture was set to 0.6 mg / mL, and the final volume concentration of thioreactive bacteria in the mixture was set to 5% (v / v), resulting in mixtures with different inoculation sequences and inoculation times.

[0060] Step 5: Place the mixtures with different inoculation sequences and inoculation times in an incubator at a temperature of 25℃~35℃ and incubate for 9 days under static and light-proof conditions. During the incubation process, nitrogen (flow rate of 1.6L / min) and carbon dioxide (flow rate of 0.4L / min) are introduced into the system, with a volume ratio of nitrogen to carbon dioxide of 4:1. During the incubation process, Geobacterium thioreduction rapidly and completely reduces graphene oxide to graphene and attaches it to the surface of the carbon felt, thus obtaining the microbial reduced graphene modified electrode, which is the Geobacterium thioreduction reduced graphene modified electrode.

[0061] In this embodiment, the microbial reduced graphene modified electrode prepared in the mixture of solutions No. 2, No. 4, and No. 6 includes a basic electrode material, on which microbial reduced graphene is modified. Thiobacillus thioreductoids are attached to the inside and surface of the microbial reduced graphene, forming a multi-layer sandwich structure composed of strain-microbial reduced graphene-strain, wherein the microbial reduced graphene is a porous two-dimensional sheet structure.

[0062] In this embodiment, the microbial reduced graphene modified electrodes prepared in the mixture of solutions 1 to 6 are denoted as A1-A6 respectively.

[0063] The electrochemical performance of the prepared microbially reduced graphene modified electrodes was tested, specifically by cyclic voltammetry (CV) analysis and electrochemical impedance spectroscopy (EIS) analysis. The results are shown in Table 2.

[0064] The CV results show that A2(138.07F / m) 2 )≈A6(139.47F / m 2 )>A4(124.75F / m 2 )>A1(122.81F / m 2 A3 (108.95F / m) 2 A5 (93.74F / m) 2 Therefore, it can be seen that the specific capacitance of strains inoculated three times is greater than that of strains inoculated once; in addition, the microbial reduced graphene modified electrodes prepared in the mixtures of PCA strains first (No. 1 and No. 2) showed greater capacitance than those prepared in other inoculation sequences.

[0065] As shown in Table 2, the resistance R of the microbially reduced graphene-modified electrodes (A1 and A2) prepared by first inoculating the PCA strain is... S and R ct Both have lower resistance than the other two connection sequences.

[0066] Table 2. R values ​​of microbial-reduced graphene-modified electrodes prepared in mixtures with different access sequences and access times. S and R ct

[0067] Group Name <![CDATA[R S (Oh)]]> <![CDATA[R ct (Oh)]]> Group Name <![CDATA[R S (Oh)]]> <![CDATA[R ct (Oh)]]> A1 11.76 48.87 A4 12.02 62.42 A2 9.37 20.27 A5 12.73 67.35 A3 11.96 56.30 A6 11.59 51.92

[0068] Therefore, in this invention, adding *Germolomum sulfadiazine* first, followed by a suspension of graphene oxide, can, on the one hand, promote and enhance the reduction degree and rate of graphene oxide by *Germolomum sulfadiazine*, thereby enabling more bacteria and graphene to adhere to the electrode surface. This results in an electrode with higher conductivity and specific capacitance. On the other hand, the combined effect of *Germolomum sulfadiazine* and graphene significantly improves the structural stability of the electrode and facilitates the formation of a highly active and stable biofilm on the electrode surface. Furthermore, by adding *Germolomum sulfadiazine* multiple times, this invention not only improves the dispersibility of graphene on the electrode surface but also increases the amount of bacteria attached. More importantly, it forms a multi-layered sandwich structure of "strain-graphene-strain," which not only allows microbial reduced graphene (MRGO) to adhere more stably to the electrode surface but also significantly improves the electrode's specific capacitance and conductivity.

[0069] In addition, the microbial reduced graphene modified electrode (A2) prepared in Example 2 of this invention and the PCA / carbon felt prepared in Example 1 were characterized by SEM, and the results are as follows: Figure 1 As shown.

[0070] Figure 1 The images show SEM images of the microbial reduced graphene modified electrode (A2) prepared in Example 2 of this invention and the PCA / carbon felt prepared in Example 1. Figure 1 In the diagram, a represents PCA / carbon felt, and b represents a microbially reduced graphene-modified electrode (A2). Figure 1 It is known that G. sulfurreducens PCA strain immobilizes MRGO on the carbon felt electrode during the GO reduction process, and a large number of PCA strains are embedded or anchored on the graphene sheet, forming a multi-layer sandwich structure of "PCA strain-MRGO-PCA strain". The MRGO-modified electrode is significantly loose and porous, providing more habitats for microbial growth.

[0071] Example 3

[0072] An application of a microbially reduced graphene-modified electrode in the construction of a bioelectrochemical system specifically involves using the microbially reduced graphene-modified electrode (A2) prepared in Example 2 as the anode to construct a microbial fuel cell (MFC), and using this microbial fuel cell (MFC) for acid production, including the following steps:

[0073] Step 1: The microbial reduced graphene modified electrode (A2) is placed in the anode chamber containing the electrolyte of the anode chamber as the working electrode, and the stone rod electrode is placed in the cathode chamber containing the electrolyte of the cathode chamber as the counter electrode. The two electrodes are connected by copper wire, and the anode chamber and cathode chamber are separated by a proton exchange membrane. A gas collection bag is connected outside the anode chamber to prepare a microbial fuel cell.

[0074] In this embodiment, the electrolyte solution in the anode chamber contains: 6000 mg COD / L glucose solution, 0.31 g / L NH4Cl, 2.0 g / L NaHCO3, 0.2 g / L MgCl2·H2O, 0.05 g / L yeast extract, 3.4 g / L K2HPO4·3H2O, and 4.4 g / L K2HPO4; the electrolyte solution in the cathode chamber contains: 10 g / L K3[Fe(CN)6], 3.4 g / L K2HPO4·3H2O, and 4.4 g / L K2HPO4; 10% acclimated residual sludge is introduced into the anode chamber as inoculum microorganisms. The residual sludge used is taken from the secondary sedimentation tank sludge of a sewage treatment plant in Changsha, Hunan, China.

[0075] Step 2: Aerate the anolyte solution with N2 for 20-40 minutes, seal the anolyte, maintain a constant temperature of 25℃-35℃, start the MFC and record the voltage across the external resistor.

[0076] Step 3: When the voltage is detected to be below 10mV and the K3[Fe(CN)6] solution turns green, the solutions in the anode and cathode chambers are replaced again, which means the MFC water replacement is completed. This is one cycle. After three consecutive stable cycles, the MFC can be considered to have started successfully. After the start-up period, the acclimation sludge is no longer inoculated and the MFC power generation / acid generation experimental operation period begins, still using 6000mg COD / L glucose solution as the anode electrolyte.

[0077] Control group: The microbial reduced graphene modified electrode (A2) prepared in Example 2 was replaced with the PCA / carbon felt prepared in Example 1, and all other conditions were the same.

[0078] Experimental testing and analysis of MFC:

[0079] (1) Electrochemical performance

[0080] 1) MFC voltage output performance

[0081] A microbial fuel cell (MFC) was fabricated using the aforementioned PCA / carbon felt and microbially reduced graphene modified electrode (A2) as the anode, and then MFC power generation tests were conducted. Figure 2 As shown.

[0082] Figure 2 This is the output voltage curve of the microbial fuel cell prepared by PCA / carbon felt and microbial reduced graphene modified electrode (A2) in Example 3 of the present invention. Figure 2 In the diagram, F0 represents PCA / carbon felt, and F represents a microbially reduced graphene-modified electrode (A2). Figure 2 It can be seen that the output voltage of the microbial fuel cell prepared by the microbial reduced graphene modified electrode (A2) rises to 400mV in a short time and remains at a high level throughout the cycle; while the output voltage of the microbial fuel cell prepared by PCA / carbon felt only reaches a maximum of 100mV and remains low throughout the cycle.

[0083] (2) Analysis of the production of volatile fatty acids (VFAs) in MFC

[0084] A microbial fuel cell was fabricated using the aforementioned PCA / carbon felt and microbially reduced graphene-modified electrode (A2) as the anode. Then, during 10 days of operation, the glucose solution (6000 mg COD / L) in the anode chamber was subjected to anaerobic fermentation to produce acid. The results were as follows: Figure 3 As shown.

[0085] Figure 3 This is a graph showing the change in VFAs production over time over 10 days after the microbial fuel cell prepared from PCA / carbon felt and microbially reduced graphene modified electrode (A2) in Example 3 of the present invention has been operating stably. Figure 3 In the diagram, a represents PCA / carbon felt, and b represents a microbially reduced graphene-modified electrode (A2). Figure 3 It can be seen that at the end of the experiment, the acetic acid content of the microbial fuel cell prepared by the MFC anode made of PCA / carbon felt and microbially reduced graphene modified electrode (A2) was 545.81±20.98 mg COD / L and 321.276±25.52 mg COD / L, respectively, and the butyric acid content was 2184.36±62.82 mg COD / L and 450.99±33.03 mg COD / L, respectively.

[0086] As can be seen from the above data, when the microbial reduced graphene modified electrode of the present invention is used as the anode of the MFC, the constructed microbial fuel cell has a greater power output and a greater VFA production.

[0087] Example 4

[0088] An application of a microbially reduced graphene-modified electrode in the construction of a bioelectrochemical system specifically involves using the microbially reduced graphene-modified electrode (A2) prepared in Example 2 as the anode to construct a microbial electrolytic cell (MEC), and using this microbial electrolytic cell (MEC) for acid production, including the following steps:

[0089] Step 1: The microbial reduced graphene modified electrode (A2) is placed in the cathode chamber containing the cathode electrolyte as the working electrode, and the stone rod electrode is placed in the anode chamber containing the anode electrolyte as the counter electrode. The two electrodes are connected by a copper wire and connected to a DC power supply (IT3602 type DC power supply). The anode chamber and cathode chamber are separated by a proton exchange membrane. A gas collection bag is connected outside the MEC cathode chamber to prepare the microbial electrolysis cell.

[0090] In this embodiment, the cathode chamber electrolyte solution contains: 10000 mg COD / L glucose solution, 0.31 g / L NH4Cl, 2.0 g / L NaHCO3, 0.2 g / L MgCl2·H2O, 0.05 g / L yeast extract, 3.4 g / L K2HPO4·3H2O, and 4.4 g / L K2HPO4; the anode chamber electrolyte solution contains: 10 g / L K3[Fe(CN)6], 3.4 g / L K2HPO4·3H2O, and 4.4 g / L K2HPO4; 10% acclimated residual sludge is introduced into the cathode chamber as inoculated microorganisms. The residual sludge used is taken from the secondary sedimentation tank sludge of a sewage treatment plant in Changsha, Hunan, China.

[0091] Step 2: Aerate the anolyte solution with N2 for 20-40 minutes, seal the anolyte, maintain a constant temperature of 25℃-35℃, start the MEC and record the voltage across the external resistor.

[0092] Step 3: When the voltage is detected to be below 10mV and the K3[Fe(CN)6] solution turns green, the solutions in the anode and cathode chambers are replaced again, which means the MEC water replacement is completed. This is one cycle. After three consecutive stable cycles, the MEC can be considered to have started up successfully. After the start-up period, the acclimation sludge is no longer inoculated and the MEC enters the experimental operation period of electricity / acid production. The 6000mg COD / L glucose solution is still used as the anode electrolyte.

[0093] Control group: The microbial reduced graphene modified electrode (A2) prepared in Example 2 was replaced with the PCA / carbon felt prepared in Example 1, and all other conditions were the same.

[0094] Experimental testing and analysis of MEC:

[0095] (1) Electrochemical performance

[0096] The MEC is started under conditions of 0.5–0.9V, and the voltage across the external resistor is recorded. The status of VFAs in the reactor is monitored. When the hexanoic acid production reaches its maximum, the solution is extracted from the bottom of each chamber, and then new solution is added. This completes the water exchange of the MEC and constitutes one operating cycle. After three cycles of continuous and stable production of hexanoic acid, the MEC is considered to have started successfully. After the start-up period, acclimation sludge is no longer inoculated, and the MEC enters the experimental operation period for acid production, still using 10000 mg COD / L glucose solution as the cathode electrolyte.

[0097] 1) Cyclic voltammetry test

[0098] Cyclic voltammetry (CV) tests were performed on the microbial electrolytic cell prepared in Example 4 using an electrochemical workstation (CHI760E, Shanghai Chenhua). The specific test conditions were as follows: At the end of the MEC experiment, using the CHI760E electrochemical workstation manufactured by Shanghai Chenhua Co., Ltd., with the PCA / carbon felt and microbially reduced graphene-modified electrode (A2) as the working electrode, and the stone rod anode serving as both the auxiliary and reference electrode, the test was conducted in a sealed three-electrode system with phosphate buffer (0.2720 g / L KH2PO4, 0.2840 g / L Na2HPO4, pH 7.0) as the electrolyte. N2 aeration was performed for 20–30 min, with continuous N2 flow during the test. The scan voltage was -1.2 V to 0.2 V, and the scan rate was 0.01 V / s. Microbially reduced graphene-modified electrode (A2) (55.68 F / m) 2 PCA / carbon felt (9.97F / m) 2 As can be seen, the microbial reduced graphene modified electrode of the present invention can significantly increase the charge storage capacity of the electrode.

[0099] (2) Analysis of volatile fatty acid production in MEC

[0100] A microbial electrolytic cell was prepared using the above-mentioned PCA / carbon felt and microbially reduced graphene modified electrode (A2) as the cathode. Then, the glucose solution (10000 mg COD / L) in the cathode chamber was subjected to anaerobic fermentation to produce acid over 22 days. The results are as follows: Figure 4 As shown.

[0101] Figure 4 This is a graph showing the change in VFAs production over time over 22 days after the microbial electrolysis cell, which was prepared from PCA / carbon felt and microbially reduced graphene modified electrode (A2) in Example 4 of the present invention, was put into stable operation. Figure 3 In the diagram, a represents PCA / carbon felt, and b represents a microbially reduced graphene-modified electrode (A2). From... Figure 4 As can be seen, on the 6th day of operation, the butyric acid yield of the microbial electrolyzer constructed with the microbially reduced graphene-modified electrode (A2) exceeded 60% of the total VFAs, while the butyric acid yield of the microbial electrolyzer prepared with PCA / carbon felt did not exceed 60% until the 20th day of operation. On the 20th day of operation, the yields of hexanoic acid in the microbial electrolyzers prepared with PCA / carbon felt and microbially reduced graphene-modified electrode (A2) were 1241.90±82.88 mg COD / L and 84.44±33.26 mg COD / L, respectively, and the yields of butyric acid were 3081.06±142.00 mg COD / L and 751.62±116.46 mg COD / L, respectively. It is evident that the microbial electrolyzer prepared with the microbially reduced graphene-modified electrode significantly improved the VFAs yield.

[0102] As can be seen from the above results, compared with conventional bioelectrodes, the microbial reduced graphene modified electrode prepared in this invention has the advantages of good conductivity and high specific capacity. It is a novel electrode material with excellent performance and can be widely used to construct bioelectrochemical systems. At the same time, it can significantly improve the electrochemical performance and acid production performance of bioelectrochemical systems. It has high application value and good application prospects.

[0103] The above embodiments are merely preferred embodiments of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a microbially reduced graphene-modified electrode, characterized in that, Includes the following steps: S1. Mix the basic electrode material and culture medium to obtain a mixture; S2. Add the graphene oxide suspension and sulfur-reducing Gastrobacterium to the mixture obtained in step S1 and culture it to obtain a microbial reduced graphene modified electrode; the initial concentration of graphene oxide in the culture system is <1.0 mg / mL.

2. The preparation method according to claim 1, characterized in that, In step S2, the initial concentration of graphene oxide in the culture system is 0.2 mg / mL to 0.8 mg / mL; the initial volume concentration of thioreducing Geobacterium in the culture system is 5%.

3. The preparation method according to claim 1, characterized in that, In step S2, the graphene oxide suspension and sulfur-reducing Gastrobacterium are added in any of the following ways: Method 1: First, add sulfur-reducing Gastrobacterium to the mixture obtained in step S1, and after an interval of 1 to 3 days, add graphene oxide suspension; Method 2: Add the graphene oxide suspension and sulfur-reducing Gastrobacterium to the mixture obtained in step S1 simultaneously; Method 3: First, add the graphene oxide suspension to the mixture obtained in step S1, and after an interval of 1 to 3 days, add sulfur-reducing bacteria.

4. The preparation method according to claim 3, characterized in that, In step S2, the sulfur-reducing bacteria are added once or three times.

5. The preparation method according to claim 4, characterized in that, The graphene oxide suspension contains 2 mg / mL of graphene oxide. The graphene oxide suspension is prepared by mixing graphene oxide with a sterile salt solution and sonicating for 1-3 hours to obtain the graphene oxide suspension. The sterile salt solution contains NH4Cl, Na2HPO4, and KCl. The sulfur-reducing bacillus is *G. sulfurreducens* PCA. The sulfur-reducing bacillus is a strain in the exponential growth phase. The OD of the sulfur-reducing bacillus... 600 The value ranges from 0.2 to 0.

8.

6. The preparation method according to any one of claims 1 to 5, characterized in that, In step S1, the base electrode material is at least one of carbon felt, graphite rod, carbon cloth, and carbon paper; titanium wire is also loaded on the base electrode material; the base electrode material also includes the following pretreatment before use: the base electrode material is sequentially immersed in acetone, acid solution, and alkaline solution; the immersion time of the base electrode material in acetone, acid solution, and alkaline solution is 24h, 12h, and 12h respectively; the acid solution is hydrochloric acid solution; the concentration of the acid solution is 1M; the alkaline solution is sodium hydroxide solution; the concentration of the alkaline solution is 1M; the culture medium is modified DMSZ culture medium; the modified DMSZ culture medium contains: 1.5g / L NH4Cl, 0.6g / L Na2HPO4, 0.1g / L KCl, 0.48g / L Na2S·9H2O, 10mL / L trace element solution, 10mL / L Wolfe vitamin solution, 1mL / L selenite-tungstate solution, 20mM sodium acetate, and 50mM sodium fumarate; In step S2, the culture is carried out under light-proof and static conditions; nitrogen and carbon dioxide are introduced into the mixture during the culture process; the volume ratio of nitrogen to carbon dioxide is 4:1; the temperature is controlled at 25℃ to 35℃ during the culture process; and the culture time is 7 to 15 days.

7. A microbially reduced graphene-modified electrode, characterized in that, The microbial reduced graphene modified electrode is prepared by the preparation method according to any one of claims 1 to 6.

8. The microbial reduced graphene modified electrode according to claim 7, characterized in that, The microbial reduced graphene modified electrode includes a base electrode material, the surface of which is modified with microbial reduced graphene. The microbial reduced graphene has sulfur-reducing bacteria attached to its interior and surface, forming a multi-layer sandwich structure of strain-microbial reduced graphene-strain. The microbial reduced graphene has a porous two-dimensional sheet structure.

9. The application of a microbially reduced graphene modified electrode as described in claim 7 or 8 in the construction of a bioelectrochemical system.

10. The application according to claim 9, characterized in that, The bioelectrochemical system includes a microbial fuel cell and a microbial electrolytic cell; in the microbial fuel cell, the microbial reduced graphene modified electrode is used as the anode; in the microbial electrolytic cell, the microbial reduced graphene modified electrode is used as the cathode.

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