A porous bioelectrode and its preparation method and application

Abstract

This invention provides a porous bioelectrode, its preparation method, and its applications. The method for preparing the porous bioelectrode utilizes ink-to-ink printing technology to achieve precise design and controllable fabrication of the bioelectrode's pore size, porosity, and distribution, constructing a three-dimensional bioelectrode system with a hierarchical pore structure. This balances efficient microbial attachment, stable biofilm growth, and smooth reactant transport. By constructing a multi-scale structure with macroscopic and micron-level pores coexisting, this invention effectively reduces the resistance to mass transfer and electron transfer. Furthermore, through controllable surface roughness and micron-level pit design, it enhances the mechanical and interfacial bonding between microorganisms and the electrode, promoting the enrichment and stable attachment of electrogenic microorganisms. Simultaneously, this invention introduces a template sacrifice method to achieve highly repeatable control of the micron-level pore structure, significantly improving the consistency of electrode structure and performance.

Description

Technical Field

[0001] This invention relates to the field of bioelectrochemical material preparation technology, and in particular to a porous bioelectrode, its preparation method, and its application. Background Technology

[0002] With socio-economic development, the large-scale consumption of traditional fossil fuels has led to problems such as the greenhouse effect and environmental pollution. Simultaneously, the discharge of industrial and domestic wastewater has exacerbated water pollution. While traditional wastewater treatment technologies (such as activated sludge processes and anaerobic digestion) can effectively remove organic pollutants, they generally suffer from drawbacks such as high energy consumption, large sludge production, and resource waste due to the inability to recover energy. Therefore, developing new technologies that combine environmental remediation and energy recovery has become a research hotspot.

[0003] Bioelectrochemical systems (BES) are an interdisciplinary technology combining microbiology, electrochemistry, and engineering. This technology utilizes electroactive microorganisms (such as Geobacter and Shewanella) to catalyze the oxidation of organic substrates on the anode surface, releasing electrons. These electrons are then transferred to the cathode via an external circuit, while protons migrate to the cathode through diffusion or ion exchange membranes, ultimately achieving the conversion of chemical energy into electrical energy or the synthesis of specific products. BES has broad application prospects in fields such as microbial fuel cells (MFC), microbial electrolyzers (MEC), biosensing, and ecological restoration.

[0004] In a bioelectrode system (BES), the microbial electrode (anode or cathode) is a key component that determines the overall performance of the system. Its main function is to provide attachment sites for microorganisms and enable efficient extracellular electron transfer. Currently, traditional microbial electrodes mostly use carbon-based materials, such as carbon cloth, carbon paper, carbon felt, graphite rods, and activated carbon particles. Although these materials possess certain biocompatibility and conductivity, their microstructure largely depends on the natural deposition of raw materials, resulting in random and uncontrollable pore distribution. This leads to the following main technical problems in practical applications:

[0005] First, uncontrollable pore structure leads to limited mass transfer and low biofilm utilization. Random pore structures easily cause local blockage, making it difficult for substrates and protons to penetrate deep into the electrode, resulting in "dead zones" or local acidification inside the electrode, increasing the internal resistance of the system. At the same time, microorganisms have difficulty effectively colonizing deep within the electrode, limiting the effective biomass loading and electron production rate.

[0006] Second, the consistency and stability of electrode performance are poor. Traditional porous materials exhibit significant structural differences between different batches, leading to marked fluctuations in the electrochemical performance of the electrodes. During long-term operation, due to biofilm shedding or scaling in the pores, the electrodes are prone to passivation, resulting in a substantial decrease in output power.

[0007] Third, they are difficult to meet the high precision requirements of biosensing. When used as biosensors, traditional electrodes suffer from poor reproducibility and low signal-to-noise ratio due to their random structural design, making it difficult to obtain stable response signals in micro-scale detection.

[0008] To address these issues, existing research has attempted to improve electrode performance through nanomaterial modification (such as carbon nanotubes and graphene composites). However, while surface modification can increase specific surface area, it cannot fundamentally solve the problems of disordered pore structure, high mass transfer resistance, and limited geometric configuration of the matrix material.

[0009] Given the current shortcomings in the preparation of microbial electrodes, it is necessary to improve them. Summary of the Invention

[0010] This invention addresses the problems of low microbial attachment efficiency, uneven biofilm distribution, high resistance to interfacial mass transfer and electron transfer, poor repeatability of electrode performance, and poor long-term operational stability caused by insufficient material performance and random, uncontrollable pore structure in existing bioelectrodes of bioelectrodes. It proposes a porous bioelectrode, its preparation method, and its applications. This invention utilizes ink-to-ink printing technology to achieve precise design and controllable fabrication of the bioelectrode's pore size, porosity, and distribution, constructing a three-dimensional bioelectrode system with a hierarchical pore structure, thus balancing efficient microbial attachment, stable biofilm growth, and smooth reactant transport. By constructing a multi-scale structure with macroscopic and micron-level pores coexisting, this invention effectively reduces the resistance to mass transfer and electron transfer. Furthermore, through controllable surface roughness and micron-level pit design, it enhances the mechanical and interfacial bonding between microorganisms and the electrode, promoting the enrichment and stable attachment of electrogenic microorganisms. Simultaneously, this invention introduces a template sacrifice method to achieve highly repeatable control of the micron-level pore structure, significantly improving the consistency of electrode structure and performance. Based on the additive manufacturing process driven by digital modeling, the prepared bioelectrodes have advantages such as high structural consistency, stable performance, rapid iteration and strong potential for large-scale preparation, and are suitable for related engineering applications of microbial electrochemical systems.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] In a first aspect, the present invention provides a method for preparing a porous bioelectrode, comprising the following steps:

[0013] The substrate material is dissolved in a first solvent to obtain a precursor solution;

[0014] After adding conductive filler and gelatin pore-forming agent to the precursor solution and mixing, the mixture is degassed to obtain the ink material.

[0015] The ink material is placed in the syringe of the ink direct writing 3D printing device, the printing parameters are set, and a three-dimensional electrode is printed.

[0016] The three-dimensional electrode is then cured and shaped.

[0017] The solidified and shaped three-dimensional electrode is then dried.

[0018] The dried three-dimensional electrode was immersed in a second solvent to obtain a porous three-dimensional electrode.

[0019] The porous three-dimensional electrode is then allowed to dry naturally, thus creating a porous bioelectrode.

[0020] Preferably, the substrate material includes polyvinyl alcohol, sodium alginate, or chitosan;

[0021] The conductive filler includes at least one of carbon black, carbon nanotubes, or graphene.

[0022] Preferably, the gelatin has a particle size of 30~224μm;

[0023] The ink material contains 20-60% gelatin by mass.

[0024] The first solvent includes at least one of water, ethanol, and glycerol;

[0025] The viscosity of the precursor solution is 10. 3 ~10 7 mPa·s;

[0026] The mass ratio of the substrate material to the conductive filler is (53~63):(2~10).

[0027] Preferably, the printing parameters are: printing speed of 1~60 mm / s, needle diameter of 0.1~2 mm, extrusion pressure of 0.02~0.6 MPa, linear filling spacing of 0.5~5.0 mm, and rotation between adjacent layers of 0~360°.

[0028] Preferably, if the substrate material is sodium alginate, the curing and shaping includes: placing the three-dimensional electrode in a calcium chloride aqueous solution for cross-linking curing;

[0029] If the substrate material is polyvinyl alcohol, the curing and shaping includes: freezing the three-dimensional electrode, thawing it, repeating the freeze-thaw cycle, and performing cross-linking curing.

[0030] If the substrate is chitosan, the fixation and shaping includes: placing the three-dimensional electrode in an alkaline coagulation bath for curing.

[0031] Preferably, the three-dimensional electrode is placed in a calcium chloride aqueous solution for cross-linking and curing, wherein the curing temperature is 1~30℃, the curing time is not less than 30s, and the mass concentration of the calcium chloride aqueous solution is 0.1~20%.

[0032] Preferably, the freezing temperature is -40 to -30°C, the freezing time is 1 to 24 hours, the thawing temperature is 20 to 25°C, the thawing time is 1 to 24 hours, and the number of repeated freezing-thawing cycles is 2 to 10.

[0033] The alkaline coagulation bath is a 1.0-3.0 M sodium hydroxide aqueous solution or potassium hydroxide aqueous solution, and the curing time is 30-120 min.

[0034] Preferably, the fixed three-dimensional electrode is immersed in a second solvent for treatment, wherein the immersion treatment temperature is 35~90 ℃ and the immersion time is not less than 30s, and the second solvent includes at least one of ethanol, acetone, aqueous ethanol solution, and aqueous acetone solution.

[0035] In the step of drying the solidified and shaped three-dimensional electrode, the drying temperature is above the sol-gel transition temperature of the gelatin, and the drying time is not less than 0.5 hours.

[0036] Secondly, the present invention also provides a porous bioelectrode, which is prepared using the aforementioned preparation method.

[0037] Thirdly, the present invention also provides a porous bioelectrode prepared by the preparation method described above, or the application of the porous bioelectrode described above in the preparation of microbial fuel cells, microbial electrolyzers, biosensors, enrichment of Geobacterium, removal of COD from water, and detection of nitrobenzene.

[0038] The porous bioelectrode, its preparation method, and its application of the present invention have the following advantages compared to the prior art:

[0039] The method for preparing porous bioelectrodes of this invention utilizes ink-to-ink printing technology to achieve precise design and controllable fabrication of the pore size, porosity, and distribution of the bioelectrode, constructing a three-dimensional bioelectrode system with a hierarchical pore structure. This approach balances efficient microbial attachment, stable biofilm growth, and unimpeded reactant transport. By constructing a multi-scale structure with both macroscopic and micron-level pores, this invention effectively reduces the resistance to mass transfer and electron transfer. Furthermore, through controllable surface roughness and micron-level pit design, it enhances the mechanical and interfacial bonding between microorganisms and the electrode, promoting the enrichment and stable attachment of electrogenic microorganisms. Simultaneously, this invention introduces a template sacrifice method to achieve highly repeatable control of the micron-level pore structure, significantly improving the consistency of electrode structure and performance. Based on a digital modeling-driven additive manufacturing process, the prepared bioelectrode exhibits advantages such as high structural consistency, stable performance, rapid iteration capability, and strong potential for large-scale fabrication, making it suitable for related engineering applications in microbial electrochemical systems. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0041] Figure 1 Optical microscope images of porous bioelectrodes prepared with pore-forming agents of different particle sizes in Example 1;

[0042] Figure 2 The porosity variation results of porous bioelectrodes prepared with different mass fractions of gelatin in Example 2;

[0043] Figure 3 Images of porous bioelectrodes prepared in Example 3 with different linear filling spacings of 1.0 mm, 1.5 mm, 2.0 mm and 2.5 mm;

[0044] Figure 4 The operation of bioelectrodes prepared after drying but not after drying;

[0045] Figure 5 Comparison of peak current magnitudes after operation of porous bioelectrodes prepared with four different linear filling spacings in Example 3;

[0046] Figure 6 Cyclic voltammetry testing of porous bioelectrodes after biofilm formation and comparison of COD removal rates;

[0047] Figure 7Nyquist plot and equivalent circuit biomembrane characterization for electrochemical impedance spectroscopy of porous bioelectrodes after biofilm attachment;

[0048] Figure 8 Comparison of SEM images of the biofilm surface morphology on the electrochemical surface of the porous bioelectrode after biofilm attachment;

[0049] Figure 9 The composition of the microbial community on the biofilm of the porous bioelectrode electrochemical surface after biofilm attachment;

[0050] Figure 10 The total EDS spectrum of the biofilm on the electrochemical surface of the porous bioelectrode after biofilm attachment.

[0051] Figure 11 EDS elemental spectrum of the biofilm on the electrochemical surface of the porous bioelectrode after biofilm attachment;

[0052] Figure 12 The current-time curve of the porous bioelectrode after nitrobenzene stimulation is shown. Detailed Implementation

[0053] The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0054] It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of embodiments. Furthermore, in the description of this application, the term "comprising" means "including but not limited to". Various embodiments of the present invention may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single digits within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Additionally, whenever a numerical range is indicated herein, it means including any referenced number (fraction or integer) within the indicated range.

[0055] This application provides a method for preparing a porous bioelectrode, comprising the following steps:

[0056] S1. Dissolve the substrate material in the first solvent to obtain the precursor solution;

[0057] S2. After adding conductive filler and gelatin pore-forming agent to the precursor solution and mixing, the mixture is degassed to obtain the ink material.

[0058] S3. Place the ink material into the syringe of the ink direct writing 3D printing equipment, set the printing parameters, and print a three-dimensional electrode.

[0059] S4. Solidify and shape the three-dimensional electrode;

[0060] S5. Dry the solidified and shaped three-dimensional electrode;

[0061] S6. Immerse the dried three-dimensional electrode in the second solvent to obtain a porous three-dimensional electrode.

[0062] S7. Then let the porous three-dimensional electrode dry naturally to obtain a porous bioelectrode.

[0063] The method for preparing porous bioelectrodes of the present invention selects commercially available gelatin particles as a pore-forming agent raw material, serving as a sacrificial template (pore-forming agent) for preparing porous bioelectrodes; a precursor solution with a certain viscosity is prepared by dissolving the substrate material in deionized water; conductive filler is added and uniformly dispersed in the precursor solution by mechanical stirring or ultrasonic dispersion to form a conductive network; gelatin pore-forming agent particles are added to the above mixture and stirred until uniformly mixed to obtain a solid-liquid mixed ink material; the ink material is transferred to a vacuum stirring and degassing machine for stirring and degassing under vacuum conditions to eliminate air bubbles generated inside the ink material and during stirring, ensuring that the ink material is dense and its rheological properties meet printing requirements; the degassed ink is transferred to a DIW printing syringe, and corresponding printing parameters are set according to the rheological characteristics of the ink and the preset micron-level pore design requirements, and the printing path is set using slicing software. According to the process parameters, a three-dimensional electrode is printed; the three-dimensional electrode is cured and shaped to maintain the stability of the electrode structure; the cured and shaped three-dimensional electrode is dried until the electrode shrinks to a size with no significant change; the dried three-dimensional electrode is then immersed in a second solvent to obtain a porous three-dimensional electrode; the porous three-dimensional electrode is naturally dried to obtain a porous bioelectrode; specifically, the porous three-dimensional electrode in the wet state is placed in a sterile and ventilated environment for natural drying. Unlike the existing technology where DIW hydrogel electrodes are usually used directly in a swollen wet state after primary curing, this invention introduces a natural drying dehydration and densification step: as the water gradually evaporates, the electrode matrix undergoes controlled volume shrinkage. The internal stress generated by the physical shrinkage process causes the conductive fillers (such as carbon black and carbon nanotubes) dispersed in the matrix to approach and accumulate in space, thereby reconstructing a more compact and continuous electronic conductive network. The dry electrode transforms from a wet state to a dry, rigid structure, reducing contact resistance, increasing overall conductivity, and enhancing the electrode's mechanical strength and structural rigidity. This enables it to withstand stronger hydraulic shearing, solving the technical problems of poor mechanical properties and easy deformation during long-term operation of traditional hydrogel electrodes.

[0064] In some embodiments, the substrate material includes polyvinyl alcohol, sodium alginate, or chitosan.

[0065] The conductive filler includes at least one of carbon black, carbon nanotubes, or graphene.

[0066] In some embodiments, the gelatin particle size is 30–224 μm; specifically, the gelatin particles are pre-dried in a drying oven. Subsequently, the dried gelatin particles are pulverized in a mechanical grinder (such as a ball mill or high-speed pulverizer). After grinding, the resulting powder is graded and screened using a standard sieve. By selecting sieves with different mesh sizes, specific particle size ranges (30–40 μm, 50–60 μm, 100–110 μm, 150–160 μm, and 200–224 μm) are chosen. The selected dried gelatin particles with the target particle size are collected and sealed for storage as sacrificial templates (pore-forming agents) for preparing graded porous bioelectrodes. Pore-forming agents of different particle sizes are used to control the micron-scale pore size of the electrode.

[0067] In some embodiments, the mass fraction of gelatin in the ink material is 20-60%, and the porosity of the electrode shows a significant upward trend with the increase of the mass fraction of the pore-forming agent.

[0068] In some embodiments, the first solvent includes at least one of water, ethanol, and glycerol;

[0069] In some embodiments, the viscosity of the precursor solution is 10. 3 ~10 7 mPa·s;

[0070] In some embodiments, the mass ratio of the substrate material to the conductive filler is (53~63):(2~10).

[0071] In some embodiments, the printing parameters are: printing speed of 1~60 mm / s, needle diameter of 0.1~2 mm, extrusion pressure of 0.02~0.6 MPa, linear filling spacing of 0.5~3.0 mm, and rotation between adjacent layers of 0~360°.

[0072] Specifically, linear fill spacing refers to the center-to-center distance between two adjacent ink filaments (printing lines) deposited parallel to a predetermined printing path within the same layer of 3D printing. Simply put, it's the center-to-center distance between two printing lines in the same layer. This parameter directly determines the width of the gap between the printing lines in the same layer, and this gap forms the final millimeter-scale macroscopic through-hole of the electrode, the core channel for substrate, proton transport, and deep colonization of microorganisms. Specifically, interlayer rotation ranges from 0 to 360°. For example, a 90° rotation between adjacent layers refers to the printing path direction of the subsequent layer rotating 90° relative to the printing path direction of the previous layer during the layer-by-layer printing process of the electrode's three-dimensional structure (usually orthogonal rotation). Rotation (such as horizontal to vertical, vertical to horizontal) is a key printing parameter design for achieving the macroscopic porosity, structural mechanical stability, and mass transfer uniformity of the electrodes. Specifically, when printing the first layer, the print head deposits ink along a preset path in a horizontal direction (such as left and right), forming parallel horizontal ink lines. The spacing between the ink lines forms the macroscopic channels of the first layer. When printing the second layer, the print head rotates 90° to become vertical (such as up and down), and the new vertical ink lines cover the intersection of the horizontal ink lines of the first layer, forming an orthogonal grid structure between the layers. When printing the third layer, it rotates 90° again to return to the horizontal direction, and so on, to complete the layer-by-layer stacking of the entire three-dimensional electrode.

[0073] This invention employs a dual-level porosity control strategy combining macroscopic and microscopic approaches. By setting the filling line spacing to be greater than the diameter of the extruded filaments, regularly arranged macroscopic interconnected channels are constructed between adjacent deposited filaments. The design size of these macroscopic channels is typically on the order of hundreds of micrometers to millimeters, aiming to form a fluid transport network connected with the micrometer-sized pores created by the pore-forming agent in subsequent steps. After setting the parameters, the printer is started, and ink is deposited layer by layer to construct a three-dimensional electrode.

[0074] In some embodiments, if the substrate material is sodium alginate, the curing and shaping includes: placing the three-dimensional electrode in a calcium chloride aqueous solution for cross-linking curing; wherein the curing temperature is 1~30℃, the curing time is not less than 30s, and the mass concentration of the calcium chloride aqueous solution is 0.1~20%.

[0075] In some embodiments, if the substrate material is polyvinyl alcohol, the curing and shaping includes: freezing the three-dimensional electrode, thawing it, repeating the freeze-thaw cycle, and performing cross-linking curing; the freezing temperature is -40~-30℃, the freezing time is 1~24h, the thawing temperature is 20~25℃, the thawing time is 1~24h, and the number of freeze-thaw cycles is 2~10 times.

[0076] In some embodiments, if the substrate is chitosan, the fixing and shaping includes: placing the three-dimensional electrode in an alkaline coagulation bath for curing, so that the chitosan molecular chains undergo a deprotonation reaction and form a physically cross-linked three-dimensional network structure through hydrogen bonding and hydrophobic interactions; the alkaline coagulation bath is a sodium hydroxide aqueous solution or potassium hydroxide aqueous solution with a concentration of 1.0~3.0 M, and the curing time is 30~120 min.

[0077] Primary Curing and Shaping: After printing, an appropriate post-curing treatment method is selected based on the characteristics of the substrate material to maintain the stability of the electrode structure. For example, if the substrate material is sodium alginate (SA), the printed three-dimensional electrode is immersed in a calcium chloride aqueous solution with a mass concentration of 0.1~20% for ionic cross-linking curing; if the substrate material is polyvinyl alcohol (PVA), the printed three-dimensional electrode is placed in a low-temperature environment (-40~-30℃) for freezing, and then thawed at room temperature (20~25℃), undergoing multiple freeze-thaw cycles to achieve physical cross-linking curing.

[0078] In some embodiments, the fixed three-dimensional electrode is immersed in a second solvent for treatment, wherein the immersion treatment temperature is 35~90°C and the immersion time is not less than 30s, and the second solvent includes at least one of ethanol, acetone, aqueous ethanol solution, and aqueous acetone solution. Template removal and micropore pre-forming based on the second solvent: The electrode device after primary curing is transferred to a second solvent for the substrate material for immersion treatment, and the temperature of the solvent system needs to be maintained above the sol-gel transition temperature of gelatin (e.g., 35°C~90°C). By selecting the type of solvent, the following are achieved: 1. Suppression of substrate swelling and collapse: Since ethanol or acetone are poor solvents for substrate materials (sodium alginate or PVA), the polymer chains of the substrate are in a contracted state and cannot undergo violent swelling or dissolution, avoiding defects such as collapse, deformation, or closure of micron-level pore structures caused by excessive water absorption and swelling of the substrate; 2. Efficient template removal: Under high temperature environment, the gelatin pore-forming agent particles dispersed in the electrode skeleton undergo phase change liquefaction and diffuse into the external solvent using the concentration gradient, thereby preserving micron-level pores in situ while maintaining the rigidity and integrity of the electrode skeleton. The solvent is then replaced and washed multiple times until no gelatin residue remains in the washing solution, thereby obtaining a hierarchical porous precursor with a non-collapsed structure and well-preserved pore morphology, which is then prepared for natural drying and densification.

[0079] In some embodiments, in the step of drying the solidified and shaped three-dimensional electrode, the drying temperature is above the sol-gel transition temperature of the gelatin (e.g., 35°C to 90°C), and the drying time is not less than 0.5 hours.

[0080] In some embodiments, the porous three-dimensional electrode is left to air dry naturally in a sterile, ventilated, room-temperature environment at room temperature (20~30°C) for 12~48 hours.

[0081] Based on the same inventive concept, the present invention also provides a porous bioelectrode, which is prepared by the above-described preparation method.

[0082] Based on the same inventive concept, the present invention also provides a porous bioelectrode prepared by the above-mentioned preparation method or the application of the above-mentioned porous bioelectrode in the preparation of microbial fuel cells, microbial electrolyzers, biosensors, enrichment of Geobacterium, removal of COD from water, and detection of nitrobenzene.

[0083] Specifically, the application of porous bioelectrodes in enriched Geobacterium includes:

[0084] The porous bioelectrode of the present invention is placed in an electrochemical reactor, and an electrolyte, which is a culture medium solution containing Geobacterium, is added to the electrochemical reactor. A potential is applied to the porous bioelectrode, and through an electrochemical reaction, Geobacterium is enriched on the surface of the porous bioelectrode and a biofilm (i.e., biofilm formation) is formed. Geobacterium is a typical electroactive microorganism that can efficiently oxidize organic matter and transfer electrons to the electrode. The surface of the present invention is enriched with a highly electroactive group of Geobacterium, which is beneficial for electricity generation and COD degradation.

[0085] Specifically, a three-electrode system is adopted, with the porous bioelectrode of the present invention as the working electrode, a platinum sheet as the counter electrode, and an Ag / AgCl reference electrode. The working electrode potential is kept constant at +0.3~0.4 V (vs. Ag / AgCl) by a potentiostat, that is, Geobacterium is enriched on the surface of the porous bioelectrode and a biofilm is formed.

[0086] The culture medium solution comprises: PBS as the buffer, and per liter contains the following components: sodium acetate: 1.2~1.4 g, ammonium chloride (NH4Cl): 0.31~0.35 g, disodium hydrogen phosphate (Na2HPO4): 4.58~4.87 g, sodium dihydrogen phosphate (NaH2PO4): 1.88~2.0 g, sodium chloride (NaCl): 0.13~0.18 g;

[0087] In addition to Geobacter, the culture medium solution contains other bacteria, such as Allomonas, Petrimonas, hydrogen-eating bacteria, Chlorella, Polymorphosomal, Flavobacterium, Desulfovibrio, Clostridium, proteinophilic bacteria, Actinomycetes, Propionibacterium, Acinetobacter, Rhodococcus, Pseudomonas, Sedibacterium, anaerobic acid-eating bacteria, Bacillus lysine, Oscillatoria, Christensen, Scherescher, Acetobacter, Corynebacterium, Brucella, etc. Even if the electrolyte contains other bacteria, the porous bioelectrode of this invention can still enrich Geobacter in large quantities.

[0088] Specifically, the application of porous bioelectrodes in the detection of nitrobenzene includes:

[0089] The porous bioelectrode with attached membrane is placed in an electrochemical reactor, and an electrolyte solution (a culture medium solution containing nitrobenzene, as described above) is added to the reactor. A potential is applied to the porous bioelectrode with attached membrane, and the current-time curve is recorded to detect the concentration of nitrobenzene. Specifically, a three-electrode system is used, with the porous bioelectrode with attached membrane as the working electrode, a platinum sheet as the counter electrode, and an Ag / AgCl reference electrode. The working electrode potential is maintained constant at +0.3~0.4 V (vs. Ag / AgCl) using a potentiostat, and the current-time curve is recorded. The following further illustrates the porous bioelectrode, its preparation method, and its application with specific embodiments. This section further illustrates the content of the invention with specific embodiments, but should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0090] Example 1

[0091] This embodiment provides a method for preparing a porous bioelectrode, including the following steps:

[0092] S1. Dissolve the substrate material in a first solvent to obtain a precursor solution; the first solvent is water;

[0093] S2. After adding conductive filler and gelatin pore-forming agent to the precursor solution and mixing, the mixture is degassed to obtain the ink material.

[0094] S3. Place the ink material into the syringe of the ink direct writing 3D printing equipment, set the printing parameters, and print a three-dimensional electrode.

[0095] S4. Solidify and shape the three-dimensional electrode;

[0096] S5. Dry the solidified and shaped three-dimensional electrode;

[0097] S6. Immerse the dried three-dimensional electrode in the second solvent to obtain a porous three-dimensional electrode.

[0098] S7. Allow the porous three-dimensional electrode to dry naturally to obtain a porous bioelectrode.

[0099] The substrate material is sodium alginate;

[0100] Conductive fillers include carbon black and carbon nanotubes;

[0101] The gelatin content in the ink material is 40% by mass;

[0102] The viscosity of the precursor solution is 10. 5 mPa·s;

[0103] The mass ratio of the substrate material, carbon black, and carbon nanotubes is 53:5:2.

[0104] The printing parameters are as follows: printing speed is 30 mm / s, needle diameter is 0.74 mm, extrusion pressure is 0.3 MPa, linear fill spacing is 2.5 mm, rotation between adjacent layers is 90°, and the number of printing layers is 12.

[0105] The curing and shaping process includes: placing the three-dimensional electrode in a calcium chloride aqueous solution for cross-linking and curing; wherein the curing temperature is 4℃, the time is 2h, and the mass concentration of the calcium chloride aqueous solution is 5%;

[0106] In the step of drying the solidified and shaped three-dimensional electrode, the drying temperature is 50℃ and the time is 2h.

[0107] The dried three-dimensional electrode was immersed in a second solvent for 1 hour at a temperature of 40°C. The second solvent was ethanol.

[0108] The porous three-dimensional electrode was naturally dried at room temperature (25℃) for 48 hours to obtain a porous bioelectrode.

[0109] The D50 particle sizes of the gelatin were 35 μm, 55 μm, 105 μm, 155 μm and 212 μm, respectively.

[0110] In Example 1, the pore size of the electrode at the micron scale was controlled by using pore-forming agents with different particle sizes; the porous bioelectrodes prepared with pore-forming agents of different particle sizes showed significant differences in pore size. Figure 1 Optical microscope images of porous bioelectrodes prepared with pore-forming agents of different particle sizes in Example 1. Figure 1 i) corresponds to 35 μm, ii) corresponds to 55 μm, iii) corresponds to 105 μm, iv) corresponds to 155 μm, and v) corresponds to 212 μm.

[0111] from Figure 1 As can be seen, with the increase of gelatin pore-forming agent particle size, the micropore size inside the 3D printed electrode increases accordingly, but the specific surface area decreases. By optimizing the pore-forming agent particle size, a balance can be achieved between eliminating internal mass transfer limitations and providing sufficient bio-attachment sites, thereby maximizing biocolonization.

[0112] Example 2

[0113] This embodiment provides a method for preparing a porous bioelectrode, including the following steps:

[0114] S1. Dissolve the substrate material in a first solvent to obtain a precursor solution; the first solvent is water;

[0115] S2. After adding conductive filler and gelatin pore-forming agent to the precursor solution and mixing, the mixture is degassed to obtain the ink material.

[0116] S3. Place the ink material into the syringe of the ink direct writing 3D printing equipment, set the printing parameters, and print a three-dimensional electrode.

[0117] S4. Solidify and shape the three-dimensional electrode;

[0118] S5. Dry the solidified and shaped three-dimensional electrode;

[0119] S6. Immerse the dried three-dimensional electrode in the second solvent to obtain a porous three-dimensional electrode.

[0120] S7. Allow the porous three-dimensional electrode to dry naturally to obtain a porous bioelectrode.

[0121] The substrate material is sodium alginate;

[0122] Conductive fillers include carbon black and carbon nanotubes;

[0123] The ink material contains 20-60% gelatin by mass (20%, 30%, 40%, 50% and 60% respectively); the gelatin has a D50 particle size of 55 μm.

[0124] The viscosity of the precursor solution is 10. 5 mPa·s;

[0125] The mass ratio of the substrate material, carbon black, and carbon nanotubes is 53:5:2.

[0126] The printing parameters are as follows: printing speed is 30 mm / s, needle diameter is 0.74 mm, extrusion pressure is 0.3 MPa, linear fill spacing is 2.5 mm, rotation between adjacent layers is 90°, and the number of printing layers is 12.

[0127] The curing and shaping process includes: placing the three-dimensional electrode in a calcium chloride aqueous solution for cross-linking and curing; wherein the curing temperature is 4℃, the time is 2h, and the mass concentration of the calcium chloride aqueous solution is 5%;

[0128] In the step of drying the solidified and shaped three-dimensional electrode, the drying temperature is 50℃ and the time is 2h.

[0129] The dried three-dimensional electrode was immersed in a second solvent for 1 hour at a temperature of 40°C. The second solvent was ethanol.

[0130] The porous three-dimensional electrode was naturally dried at room temperature (25℃) for 48 hours to obtain a porous bioelectrode.

[0131] Figure 2The porosity changes of porous bioelectrodes prepared with different mass fractions of gelatin in Example 2 are shown.

[0132] from Figure 2 As can be seen, the porosity of the electrode shows a significant upward trend with the increase of the proportion of pore-forming agent. When the mass fraction of gelatin is 30%, the porosity is about 5.15%, and when the mass fraction of gelatin increases to 60%, the porosity increases to about 15.69%.

[0133] Example 3

[0134] This embodiment provides a method for preparing a porous bioelectrode, including the following steps:

[0135] S1. Dissolve the substrate material in water to obtain a precursor solution;

[0136] S2. After adding conductive filler and gelatin pore-forming agent to the precursor solution and mixing, the mixture is degassed to obtain the ink material.

[0137] S3. Place the ink material into the syringe of the ink direct writing 3D printing equipment, set the printing parameters, and print a three-dimensional electrode.

[0138] S4. Solidify and shape the three-dimensional electrode;

[0139] S5. Dry the solidified and shaped three-dimensional electrode;

[0140] S6. Immerse the dried three-dimensional electrode in the second solvent to obtain a porous three-dimensional electrode.

[0141] S7. Allow the porous three-dimensional electrode to dry naturally to obtain a porous bioelectrode.

[0142] The substrate material is sodium alginate;

[0143] Conductive fillers include carbon black and carbon nanotubes;

[0144] The ink material contains 40% gelatin by mass; the D50 particle size of the gelatin is 55 μm.

[0145] The viscosity of the precursor solution is 10. 5 mPa·s;

[0146] The mass ratio of the substrate material, carbon black, and carbon nanotubes is 53:5:2.

[0147] The printing parameters are as follows: printing speed is 30 mm / s, needle diameter is 0.74 mm, extrusion pressure is 0.3 MPa, linear filling spacing is 1~2.5 mm (1.0 mm, 1.5 mm, 2.0 mm and 2.5 mm respectively), adjacent layers are rotated 90°, and the number of printing layers is 12.

[0148] The curing and shaping process includes: placing the three-dimensional electrode in a calcium chloride aqueous solution for cross-linking and curing; wherein the curing temperature is 4℃, the time is 2h, and the mass concentration of the calcium chloride aqueous solution is 5%;

[0149] In the step of drying the solidified and shaped three-dimensional electrode, the drying temperature is 50℃ and the time is 2h.

[0150] The dried three-dimensional electrode was immersed in a second solvent for 1 hour at a temperature of 40°C. The second solvent was ethanol.

[0151] The porous three-dimensional electrode was naturally dried at room temperature (25℃) for 48 hours to obtain a porous bioelectrode.

[0152] Figure 3 The images show porous bioelectrodes prepared in Example 3 with different linear filling spacings of 1.0 mm, 1.5 mm, 2.0 mm and 2.5 mm.

[0153] from Figure 3 Significant millimeter-scale pore size variations can be observed; meanwhile, the magnified inset in the upper left corner shows that the surface of the electrode-printed lines still maintains a dense microporous structure.

[0154] Furthermore, Figure 4 a) is a case where a porous three-dimensional electrode obtained according to the method in Example 3 (linear filling spacing of 2.5 mm) (i.e. without the drying step in step S7) breaks during operation; Figure 4 (b) shows that the porous bioelectrode finally prepared according to the method in Example 3 (with a linear filling spacing of 2.5 mm) (after the drying step in step S7) still maintains the integrity of the main structure of the electrode after more than two months of continuous operation, and no macroscopic deformation or collapse was observed, which confirms the structural reliability of the 3D printed electrode under long-term operating conditions.

[0155] Furthermore, in Example 3, four porous bioelectrodes with different linear packing spacings (1.0 mm, 1.5 mm, 2.0 mm, and 2.5 mm) were prepared, with a conventional commercial carbon felt (using SGL Group commercial carbon felt, model KFD 2.5 EA) as the control group. All test electrodes maintained a consistent projected area of ​​approximately 15 mm × 15 mm. A two-chamber microbial electrochemical reactor was constructed for testing, employing a three-electrode system. The anode chamber was equipped with the working electrode (the porous bioelectrode prepared in Example 3 or a conventional commercial carbon felt and an Ag / AgCl reference electrode), and the cathode chamber was equipped with a platinum sheet counter electrode. The two chambers were separated by a Nafion 212 proton exchange membrane. The electrolyte in the anode chamber consisted of an 80% (v / v) nutrient culture medium solution and a 20% (v / v) Geobacterium enrichment mixture.

[0156] The nutrient culture medium solution is prepared as follows: using 50 mM PBS (pH 7.2) as the buffer solution, each liter contains the following components:

[0157] Sodium acetate: 1.2 g, ammonium chloride (NH4Cl): 0.31 g, disodium hydrogen phosphate (Na2HPO4): 4.58 g, sodium dihydrogen phosphate (NaH2PO4): 1.88 g, sodium chloride (NaCl): 0.13 g;

[0158] The preparation method of the Geobacter enrichment mixture is as follows: Inoculate the mixed bacterial solution containing Geobacter into the nutrient culture medium solution (the composition of which is shown above), and carry out enrichment culture under constant temperature of 30℃, anaerobic, static and light-protected conditions. After the bacterial cells grow stably, take the bacterial solution and transfer it to fresh anaerobic culture medium at an inoculation amount of 20% (v / v). Repeat the subculturing 5 times to obtain a bacterial solution with stable growth and uniform activity, which is the Geobacter enrichment mixture.

[0159] Cathode chamber electrolyte: 50 mM PBS (pH 7.2) buffer solution was used.

[0160] The potential of the working electrode (i.e., the porous bioelectrode prepared with different printing line spacings in Example 3 or a traditional commercial carbon felt) was kept constant at +0.3 V (vs. Ag / AgCl) using a potentiostat, and the current change curve over time was recorded. The test results are as follows: Figure 5 As shown, Figure 5 The 1mm~2.5mm in the figure represents the linear filling spacing in Example 3, and CC represents conventional commercial carbon felt.

[0161] Figure 5 (a) shows a comparison of the time required for different electrodes to reach the first peak and the peak size. The test results show that the time to reach the first peak decreases as the printed line spacing increases, such as... Figure 5 (b) The current-time results show that electrodes with larger line spacing achieve higher peak currents during long-term operation, and the peak currents achieved by all printed electrode groups exceed those of commercial carbon felt. The relatively faster start-up time and higher peak current indicate that the large-pore structure has superior mass transfer characteristics, better mitigating substrate diffusion resistance compared to smaller-pore structures, and promoting rapid and high-density enrichment of electroactive bacteria within the deep structure of the 3D-printed stereo electrode.

[0162] according to Figure 5 Similarly, after 35 days of electrochemical reaction, a biofilm (i.e., biofilm formation) was formed on the electrode surface. The porous bioelectrode with the biofilm and a traditional commercial carbon felt were both analyzed by cyclic voltammetry (voltage range -0.5~0.4 V, scan rate 0.1 mV / s, test system same as...). Figure 5 (three-electrode system), results are as follows Figure 6 As shown;

[0163] Figure 6 a) In this context, 3D Printed CB / SA-1.0 ~ 2.5mm represents the linear infill spacing in Example 3, and Carbon Fiber Felt represents conventional commercial carbon felt. b) In this context, the horizontal axis CC represents conventional commercial carbon felt, and 1~2.5 represents the linear infill spacing in Example 3.

[0164] like Figure 6 As shown, with the increase of linear packing spacing, the peak current of the oxidation peak of the electrode increases significantly, indicating that the macroporous structure enhances the biocatalytic reaction rate. The larger line spacing electrode has a higher COD removal rate, while the COD removal rate of traditional commercial carbon felt is about 30% / day, while the COD removal rate of the porous bioelectrode of this invention is between 43% and 57% / day.

[0165] according to Figure 5 Similarly, after 35 days of electrochemical reaction, a biofilm (i.e., biofilm formation) is formed on the electrode surface. Using porous bioelectrodes with biofilms formed by four different linear packing spacings (1.0 mm, 1.5 mm, 2.0 mm, and 2.5 mm) as described in Example 3, and with conventional commercially available carbon felt with biofilm as a control group, a two-chamber microbial electrochemical reactor was constructed. A three-electrode system was used to test AC impedance spectroscopy. The anode chamber was equipped with either the porous bioelectrode with biofilm or the conventional commercially available carbon felt with biofilm. Figure 5 The reference electrode was Ag / AgCl, and the cathode chamber was equipped with a platinum plate counter electrode. The two chambers were separated by a Nafion 112 proton exchange membrane. Both the cathode and cathode chamber electrolytes were 50 mM PBS (pH 7.2) buffer. Test results are as follows: Figure 7 As shown.

[0166] In AC impedance spectroscopy analysis, all porous bioanodes exhibited similar Nyquist plots, including high-frequency semicircles and low-frequency curves. Figure 7This paper presents an equivalent circuit model of the electrochemical reaction process based on a physical model and its impedance fitting results. With the linear filler spacing increasing from 1 mm to 2.5 mm, both the series resistance Rs (9.49 Ω ~ 18.02 Ω) and the charge transfer resistance Rct (0.43 Ω ~ 12.25 Ω) significantly increase. This may be due to the elongation of the electron / ion transport path caused by the sparse mesh structure, or the change in effective contact area caused by the reduction of contact points between the electrodes and electrode clamps. Furthermore, this work compares the changes in electrochemical impedance of the electrodes before and after biofilm inoculation. The fitting parameters show that both Rs and Rct decrease, confirming that the formation of the microbial biofilm on the electrode surface effectively establishes electron transport channels and accelerates the interfacial redox reaction kinetics.

[0167] Furthermore, according to Figure 5 The method described in the article involves the formation of a biofilm (i.e., biofilm attachment) on the electrode surface after 35 days of electrochemical reaction. The morphology of the biofilm on the anode (i.e., the working electrode) after biofilm attachment was studied using scanning electron microscopy (SEM). Figure 8 As shown, the SEM image of the porous bioelectrode prepared in Example 3 with a linear filling spacing of 1.0 mm shows a dense biofilm, with the anode surface almost completely covered by the biofilm, while the surface of conventional commercial carbon felt only has some sparse microbial films attached.

[0168] The composition of the microbial community on the porous bioelectrode prepared in Example 3 with a linear packing spacing of 1.0 mm and on a conventional commercial carbon felt was analyzed using 16S rRNA amplicon sequencing technology. The results are as follows: Figure 9 As shown, the porous bioelectrode community structure obtained by the present invention is highly similar, with Geobacter, the main electrogenic bacterium, dominating, and its relative abundance is as high as 70% or more, indicating that the printed electrode has good biocompatibility and that a highly efficient electrogenic community has been successfully domesticated on the surface of the printed electrode.

[0169] Figures 10-11 The EDS elemental spectrum of the porous bioelectrode prepared in Example 3 with a linear filling spacing of 1.0 mm after biofilm formation is shown. The electrode is mainly composed of carbon (C, accounting for 76.48%), nitrogen (N, accounting for 10.82%) and oxygen (O, accounting for 9.89%), and contains small amounts of phosphorus (P), sulfur (S) and sodium (Na), which further indicates that the electrode surface has been covered by a microbial biofilm rich in protein and nucleic acid.

[0170] To verify the application performance of the porous bioelectrode of this invention in the field of biosensing, a porous bioelectrode that had been successfully domesticated and implanted into a biosensor was constructed. A series of nitrobenzene solutions with concentration gradients (5 ppm, 10 ppm, 20 ppm, 40 ppm, 80 ppm) were selected for toxicity shock tests. Specifically, the tests were conducted using... Figure 5 The method described above involves an electrochemical reaction lasting 35 days, after which a biofilm (i.e., biofilm formation) is formed on the electrode surface. A three-electrode system is used, with the anode chamber containing a working electrode (either a porous bioelectrode with biofilm formation after different linear filling spacings as described in Example 3, or a traditional commercial carbon felt with biofilm formation) and an Ag / AgCl reference electrode, and the cathode chamber containing a platinum sheet electrode. The two chambers are separated by a Nafion 212 proton exchange membrane. The electrolyte in the anode chamber is a nutrient culture medium solution containing different concentrations (5 ppm, 10 ppm, 20 ppm, 40 ppm, 80 ppm) of nitrobenzene (composition as shown above). The electrolyte in the cathode chamber is a buffer solution of 50 mM PBS (pH 7.2).

[0171] The working electrode potential was maintained at a constant +0.3 V (vs. Ag / AgCl) using a potentiostat, and the current-time curve was recorded. The results are as follows: Figure 12 As shown. Figure 12 Medium indicates that the nutrient medium solution does not contain nitrobenzene. Figure 12 In this context, 1 to 2.5 represent the linear filling spacing in Example 3, and CC represents conventional commercial carbon felt.

[0172] The current suppression ratio is used as a key indicator for evaluating the sensitivity and detection range of a sensor. For example... Figure 12As shown, the test results indicate that in the low-concentration detection range (sensitivity test): when the nitrobenzene concentration is 5 ppm, the bioelectrode with a line spacing of 1.0 mm in this embodiment exhibits the best response characteristics, with a current suppression ratio of 10.65%, significantly higher than the 8.14% of the comparative example (traditional electrode). This demonstrates that under low-concentration toxic stimulation, the effective specific surface area and superior electron transfer efficiency of the electrode of this invention result in higher detection sensitivity. In the high-concentration detection range: as the nitrobenzene concentration increases to 80 ppm, the current suppression ratio of the comparative electrode reaches as high as 73.37%. This indicates that the bioactivity of the comparative electrode is severely suppressed, the signal response is close to saturation, reaching its detection limit, and it cannot further distinguish higher concentrations of pollutants. In contrast, the bioelectrode with a line spacing of 2.0 mm in this embodiment has a current suppression ratio of only 38.33% at this high concentration. This lower suppression ratio means that the electrode still retains a large number of active sites and signal response margin. This demonstrates that the electrode described in this invention effectively improves mass transfer flux by regulating the macroscopic pore structure, significantly broadening the linear detection range of the sensor and enabling it to detect higher concentrations of nitrobenzene.

[0173] It is understood that the technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0174] The above are merely preferred embodiments of this application, and only specifically describe the technical principles of this application. These descriptions are only for explaining the principles of this application and should not be construed as limiting the scope of protection of this application in any way. Based on this explanation, any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application, as well as other specific embodiments of this application that can be conceived by those skilled in the art without creative effort, should be included within the scope of protection of this application.

Claims

1. A method for preparing a porous bioelectrode, characterized in that, Includes the following steps: The substrate material is dissolved in a first solvent to obtain a precursor solution; After adding conductive filler and gelatin pore-forming agent to the precursor solution and mixing, the mixture is degassed to obtain the ink material. The ink material is placed in the syringe of the ink direct writing 3D printing device, the printing parameters are set, and a three-dimensional electrode is printed. The three-dimensional electrode is then cured and shaped. The solidified and shaped three-dimensional electrode is then dried. The dried three-dimensional electrode was immersed in a second solvent to obtain a porous three-dimensional electrode. The porous three-dimensional electrode is then allowed to dry naturally, thus creating a porous bioelectrode.

2. The method for preparing a porous bioelectrode as described in claim 1, characterized in that, The substrate material includes polyvinyl alcohol, sodium alginate, or chitosan; The conductive filler includes at least one of carbon black, carbon nanotubes, or graphene.

3. The method for preparing a porous bioelectrode as described in claim 1, characterized in that, The gelatin has a particle size of 30~224μm; The ink material contains 20-60% gelatin by mass. The first solvent includes at least one of water, ethanol, and glycerol; The viscosity of the precursor solution is 10. 3 ~10 7 mPa·s; The mass ratio of the substrate material to the conductive filler is (53~63):(2~10).

4. The method for preparing a porous bioelectrode as described in claim 1, characterized in that, The printing parameters are as follows: printing speed of 1~60 mm / s, needle diameter of 0.1~2 mm, extrusion pressure of 0.02~0.6 MPa, linear filling spacing of 0.5~5.0 mm, and rotation between adjacent layers of 0~360°.

5. The method for preparing a porous bioelectrode as described in claim 2, characterized in that, If the substrate material is sodium alginate, the curing and shaping includes: placing the three-dimensional electrode in a calcium chloride aqueous solution for cross-linking and curing; If the substrate material is polyvinyl alcohol, the curing and shaping includes: freezing the three-dimensional electrode, thawing it, repeating the freeze-thaw cycle, and performing cross-linking curing. If the substrate is chitosan, the fixation and shaping includes: placing the three-dimensional electrode in an alkaline coagulation bath for curing.

6. The method for preparing a porous bioelectrode as described in claim 5, characterized in that, The three-dimensional electrode is placed in a calcium chloride aqueous solution for cross-linking and curing, wherein the curing temperature is 1~30℃ and the curing time is not less than 30s, and the mass concentration of the calcium chloride aqueous solution is 0.1~20%.

7. The method for preparing a porous bioelectrode as described in claim 5, characterized in that, The freezing temperature is -40~-30℃, the freezing time is 1~24h, the thawing temperature is 20~25℃, the thawing time is 1~24h, and the number of repeated freezing-thawing cycles is 2~10. The alkaline coagulation bath is a 1.0-3.0 M sodium hydroxide aqueous solution or potassium hydroxide aqueous solution, and the curing time is 30-120 min.

8. The method for preparing a porous bioelectrode as described in claim 1, characterized in that, The fixed three-dimensional electrode is immersed in a second solvent for treatment, wherein the immersion treatment temperature is 35~90℃ and the immersion time is not less than 30s, and the second solvent includes at least one of ethanol, acetone, aqueous ethanol solution, and aqueous acetone solution. In the step of drying the solidified and shaped three-dimensional electrode, the drying temperature is above the sol-gel transition temperature of the gelatin, and the drying time is not less than 0.5 hours.

9. A porous bioelectrode, characterized in that, It is prepared by any one of the preparation methods described in claims 1 to 8.

10. The application of a porous bioelectrode prepared by any one of the preparation methods described in claims 1 to 8 or the porous bioelectrode described in claim 9 in the preparation of microbial fuel cells, microbial electrolyzers, biosensors, enrichment of Geobacterium, removal of COD from water, and detection of nitrobenzene.

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