Three-dimensional bio-printing electrogenesis anode and preparation method and application thereof

Abstract

The invention belongs to the technical field of microbial fuel cells, and discloses a three-dimensional bio-printing electrogenesis anode and a preparation method and application thereof. High-concentration carbon nanotubes are used as a conductive material, polyethylene glycol diacrylate and dithiothreitol are mixed to prepare bio-ink, and a bioactive material is constructed through 3D printing. And a thiol-ene click reaction between acrylate and thiol is utilized to form primary crosslinking. And after the 3D biological printing is finished, soaking in an ammonium persulfate solution to accelerate C-S cross-linking and form C-C secondary cross-linking, thereby finally obtaining the PDCB biological anode with a double-cross-linked network and high CNTs concentration. When the bioactive material is used as an anode, the maximum current density of the MFCs is 509.3 A / m < 3 >. Besides, for the bioactive material with any bacterial density, the maximum current density of the MFCs is basically kept unchanged in three complete operation cycles, which indicates that the click cross-linking curing has a promoting effect on the stability of the bioactive material.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fuel cells, in particular to a method for preparing a battery anode. Background Art

[0002] With the rapid development of the economy, the continued use of traditional fossil fuels has brought a series of challenges to mankind, such as energy shortages and environmental degradation. How to obtain energy without producing toxic waste has always been a hot research topic. Microbial fuel cells (MFCs) have broad application prospects in the fields of energy recovery and environmental remediation because they can use organic waste as fuel while producing clean by-products (H2O and CO2). However, the low output performance limits the practical application of MFCs. The anode of MFCs, as the site for biocatalyst attachment and electron generation, is crucial to the output performance of MFCs, so people are committed to developing anodes with good performance.

[0003] The key factors in MFC performance are the formation of a biofilm on the anode and the extracellular electron transfer (EET) between the biofilm and the anode interface. The formation of the anode biofilm is directly related to the number of bacteria it carries. Therefore, 3D bioprinting technology based on organisms (cells, bacteria, etc.) has attracted widespread attention due to its unique advantages. It can prepare bioactive materials with a three-dimensional porous structure by stacking and bonding layer by layer. This type of printed material can not only accommodate more organisms, but also accelerate the transfer of substances and electrons with the help of the three-dimensional spatial structure, ensuring the long-term activity of the organism. Therefore, 3D bioprinted anodes are gradually developing in the field of MFC.

[0004] Cross-linking and curing is a key step in the printing process, which has an important impact on the mechanical stability of the bioprinted structure and its subsequent long-term application in MFCs. Traditional cross-linking is generally a physical forming method such as heat-induced cross-linking (such as gelatin thermal gelation) or freeze-drying. For example, patent CN113308148A discloses a preparation method for using carbon nanotubes as conductive fillers to form printable hydrogel inks, and uses freeze-thaw cycles to increase the crystallinity of the polyvinyl alcohol physical cross-linking network. However, as a 3D printed anode loaded with active organisms, this method cannot be applied because it destroys the activity of microorganisms. At the same time, fuel cell application scenarios usually require the electrodes to be immersed in electrolyte solutions for a long time, which makes it difficult for physical cross-linking strategies based on hydrogen bonds or ionic interactions to meet long-term stability requirements.

[0005] Therefore, it is crucial to propose a 3D bioprinted anode with good conductivity and fast, stable and universal cross-linking curing method for use in microbial fuel cells to improve the anode output performance. Summary of the Invention

[0006] In response to the above technical problems, the present invention proposes a three-dimensional bioprinted electricity-generating anode and its preparation method and application.

[0007] To achieve the above object, the technical solution of the present invention is implemented as follows:

[0008] The thiol groups of dithiothreitol (DTT) and the double bonds of polyethylene glycol diacrylate (PEGDA) undergo a thiol-ene click reaction to form a CS covalent network. Immersion in an ammonium sulfate (APS) solution accelerates the thiol-ene click reaction and induces free radical polymerization to form C—C bonds, ultimately forming a secondary cross-linked interpenetrating network. The resulting three-dimensional anode is used as a microbial fuel cell anode, achieving high output performance.

[0009] A method for preparing a three-dimensional bioprinted electrogenic anode, comprising the following steps:

[0010] S1. Dissolve a certain amount of polyethylene glycol diacrylate (PEGDA) in water at 50°C and heat and stir to prepare a polyethylene glycol diacrylate aqueous solution. Then, add bacteria, dithiothreitol (DTT), and carbon nanotubes (CNTs) and stir until evenly mixed to obtain PDCB bio-ink, referred to as bio-ink.

[0011] S2. Loading the bio-ink obtained in step S1 into a 3D printer for printing to obtain a printed structure;

[0012] S3. The printed structure obtained in step S2 is completely immersed in an ammonium persulfate (APS) solution and further solidified to obtain a conductive bioscaffold (PDCB), namely a three-dimensional bioprinted electrogenic anode (also known as a PDCB three-dimensional bioanode).

[0013] The concentration of the polyethylene glycol diacrylate aqueous solution in the above step S1 is 20 wt % to 60 wt %.

[0014] In the above step S1, 2-6 mg of dithiothreitol and 140-180 mg of conductive material are added to each mL of the polyethylene glycol diacrylate aqueous solution.

[0015] Furthermore, in step S1 above, the bacteria were grown by 600 The bacterial suspension with a value of 1-2 is centrifuged and the upper liquid is removed to obtain the bacterial suspension; the volume of the bacterial suspension required for each mL of polyethylene glycol diacrylate aqueous solution is 15-135 mL.

[0016] The preparation method of the bacterial suspension is as follows: adding the bacterial culture preservation solution to the culture medium, culturing at a constant temperature, washing by centrifugation, and adding water to obtain the bacterial suspension.

[0017] In the above step S1, the bacteria is at least one of Shewanella oneidensis MR-1 and Escherichia coli K-12.

[0018] The printing parameters in step S2 above are set to an extrusion speed of 2.5 mm. 3 / s, the printing speed is 3.0mm / s, and the printing temperature is 20-35℃.

[0019] The concentration of the ammonium persulfate solution in step S3 is 3 wt%-8 wt%, and the soaking time is ≥10 min.

[0020] The 3D bioprinted electrogenic anode fabricated using the aforementioned method was crosslinked via a thiol-ene click reaction (CS) between acrylates (derived from PEGDA) and thiols (derived from DTT). High concentrations of carbon nanotubes served as conductive elements, successfully overcoming the limitations of photocuring processes imposed by high concentrations of dark conductive fillers, resulting in a highly conductive and stable bioanode.

[0021] Application of the above-mentioned 3D bioprinted electricity-generating anode in microbial fuel cells.

[0022] The beneficial effects produced by the present invention are:

[0023] 1. The present invention utilizes a direct ink writing technology platform and click crosslinking curing, and innovatively adopts a thiol-ene click chemistry crosslinking strategy to break through the optical limitations of traditional photocuring technology to construct a highly stable bioanode.

[0024] 2. In the PDCB three-dimensional bioanode prepared by the present invention, high-concentration conductive carbon nanotubes are used as the printing support material to achieve a synergistic improvement in conductivity and long-term stability.

[0025] 3. The maximum power density of the PDCB three-dimensional bioanode prepared by the present invention can reach 508.0W / m 3 Moreover, within three complete operating cycles (over 800 hours), the maximum current density of MFCs remained basically unchanged, confirming the promoting effect of click crosslinking on the stability of bioactive materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1This is a schematic diagram of the preparation process and cross-linking mechanism of the PDCB three-dimensional bioprinted electrogenic anode based on click cross-linking curing in Example 1 of the present invention.

[0028] Figure 2 This is the PDCB three-dimensional bioprinted electrogenic anode prepared in Example 1 of the present invention and SEM magnification images at different magnifications.

[0029] Figure 3 CLSM image of a natural bacterial suspension (a); CLSM image of a bacterial suspension observed after breaking up an incompletely solidified PDCB-printed scaffold (b) to demonstrate that the printing process has no effect on bacterial activity; CLSM image of a block hydrogel containing only bacteria, PEGDA, DTT, and APS but no CNTs (c).

[0030] Figure 4 The printing conditions of PDCB inks with different CNTs concentrations in the examples of the present invention, comparative examples 3 and 4 are shown, including the extrusion state and the deposition state.

[0031] Figure 5 The sol-gel fractions obtained by immersing the PDCB three-dimensional biological anode prepared in Example 1 and Comparative Example 5 in the electrolyte for different times.

[0032] Figure 6 is the sol-gel fraction of the electrodes prepared in Example 1 and Comparative Example 1, wherein PEGDA-light represents the photocrosslinked and cured hydrogel scaffold of Comparative Example 1, and PEGDA-DTT represents the click-cured hydrogel scaffold of Example 1 by adding DTT.

[0033] Figure 7 1 and 2 are the swelling equilibrium curves of the electrodes prepared in Example 1 and Comparative Example 2, wherein SA-CNTs is an ionically cross-linked hydrogel scaffold (Comparative Example 2) and PEGDA-CNTs is a click-cured hydrogel scaffold (Example 1).

[0034] Figure 8 Impedance curve (a) and It curve (b) measured by the bioelectrode prepared in Example 1 of the present invention.

[0035] Figure 9 Current density curve (a) and power density curve (b) measured for dual-chamber MFCs loaded with the bioanode prepared in Example 1 of the present invention.

[0036] Figure 10 Protein content of the bioanode with different bacterial loadings (a) and current density curves of the dual-compartment MFCs constructed with bioelectrodes with different bacterial loadings (b). DETAILED DESCRIPTION

[0037] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

[0038] The polyethylene glycol diacrylate (PEGDA), dithiothreitol (DTT), and ammonium persulfate (APS) used in the present invention were purchased from MacLean Biochemical Technology Co., Ltd.; carboxylated multi-walled carbon nanotubes (CNTs) were purchased from Shenzhen Suiheng Graphene Technology; the LIVE / DEAD BacLight Viability Kit was purchased from Thermo Fisher Scientific Inc.; Luria-Bertani broth was purchased from Beijing Aoboxing Biotechnology Co., Ltd.; the water used (resistance greater than 18 MΩcm at 25°C) was purchased from the company. -1 ) was prepared by a Millipore Milli-Q water purification system (Billerica, MA, USA) and sterilized by high temperature.

[0039] The bacteria Shewanella oneidensis MR-1 used in the present invention was purchased from ATCC American Type Culture Collection with the strain number ATCC700550; and Escherichia coli K-12 was purchased from ATCC American Type Culture Collection with the strain number ATCC10798.

[0040] Example 1

[0041] A method for preparing a three-dimensional bioprinted electrogenic anode in this embodiment, the preparation process is as follows Figure 1 As shown, the steps are as follows:

[0042] (1) Preparation of bacterial suspension

[0043] 0.1 mL of the electrogenic bacterium Shewanella oneidensis MR-1 was inoculated into 15 mL of Luria-Bertani broth and cultured in a constant temperature incubator (parameters: 150 rpm, 30°C) for 24 h. After centrifugation, the cells were washed three times with water (centrifugation conditions: 5000 rpm, 5 min), and water was added to adjust the OD 600 =1.5 bacterial suspension.

[0044] (2) Preparation of bio-ink

[0045] After centrifugation of 75 mL of the bacterial suspension obtained in step (1), the upper liquid was removed, and the bacteria settled at the bottom were added to a 20 wt% PEGDA aqueous solution (1 mL). 180 mg of CNTs and 3 mg of DTT were added in sequence, and the mixture was stirred evenly to obtain PDCB bio-ink.

[0046] (3) PDCB three-dimensional bioanode

[0047] The PDCB bio-ink obtained in step (2) was placed in the bio-3D printing platform (SunP BioMaker 1) and extruded through a 20G flat-tip needle (diameter 0.30 mm) at a speed of 2.5 mm. 3 The printing process was carried out at room temperature (20-35°C) at a speed of 3.0 mm / s. The final printed structure measured 1 cm × 1 cm × 1 cm. After printing, the printed structure was completely immersed in a 4 wt% APS solution for 10 minutes to promote further cross-linking and curing of the PDCB bioscaffold, resulting in a PDCB 3D bioprinted electrogenic anode, named PDC-B3.

[0048] The three-dimensional bioprinted anode body prepared in this embodiment based on click cross-linking curing is constructed with a customized hierarchical network structure through a three-dimensional bioprinting platform. A high concentration of carbon nanotubes (CNTs) is used as the conductive element, and polyethylene glycol diacrylate (PEGDA) and dithiothreitol (DTT) are mixed. An innovative thiol-ene click chemistry cross-linking strategy is adopted to break through the optical limitations of traditional photocuring technology to construct a highly conductive and stable bioanode.

[0049] Figure 2 This is a physical picture of the PDCB three-dimensional bioprinted electrogenic anode prepared in this example ( Figure 2 a) and SEM images at different magnifications. Due to the presence of high concentration of CNTs, the scaffold appears dark black. Low magnification SEM images ( Figure 2 b) shows that the diameter of the macro-grid pores of PDCB is 0.8 mm. In addition, the high-magnification SEM image ( Figure 2 c) shows a dense pore structure, which is due to the fact that the bioscaffold is composed of a high concentration of polymers and carbon nanotubes. However, it still shows microscopic pores of 3 to 5 μm, which facilitate the input of substances and the discharge of metabolic waste. The porous anode with a macroporous structure allows the substrate to penetrate into the interior of the anode, but the microporous anode can promote the diffusion of endogenous electron shuttles (such as riboflavin) to improve the efficiency and kinetics of extracellular electron transfer. In addition, a higher magnification SEM shows ( Figure 2 d), Rod-shaped bacteria are distributed in the hydrogel network, and tubular CNTs are wrapped around the bacteria, promoting the transfer of electrons generated by the bacteria and improving the conductive properties of the bioscaffold.

[0050] Figure 3 CLSM images of a natural bacterial suspension (a); CLSM images of a bacterial suspension observed after breaking up an incompletely cured PDCB-printed scaffold (b), demonstrating that the printing process had no effect on bacterial activity; and CLSM images of a bulk hydrogel containing only bacteria, PEGDA, DTT, and APS, without CNTs (c). These two steps demonstrate that the printing process and printed materials have negligible effects on bacterial activity.

[0051] Example 2

[0052] A method for preparing a three-dimensional bioprinted electrogenic anode in this embodiment comprises the following steps:

[0053] (1) Preparation of bacterial suspension

[0054] 0.1 mL of the electrogenic bacterium Shewanella oneidensis MR-1 was inoculated into 15 mL of Luria-Bertani broth and cultured in a constant temperature incubator (parameters: 150 rpm, 30°C) for 24 h. After centrifugation, the cells were washed three times with water (centrifugation conditions: 5000 rpm, 5 min), and water was added to adjust the OD 600 =1.5 bacterial suspension.

[0055] (2) Preparation of bio-ink

[0056] After centrifugation of 15 mL of the bacterial suspension obtained in step (1), the upper liquid was removed, and the bacteria settled at the bottom were added to a 20 wt% PEGDA aqueous solution (1 mL). 180 mg of CNTs and 3 mg of DTT were added in sequence, and the mixture was stirred evenly to obtain PDCB bio-ink.

[0057] (3) PDCB three-dimensional bioanode

[0058] The PDCB bio-ink obtained in step (2) was placed in the bio-3D printing platform (SunP BioMaker 1) and extruded through a 20G flat-tip needle (diameter 0.30 mm) at a speed of 2.5 mm. 3 The printing process was carried out at room temperature (20-35°C) at a speed of 3.0 mm / s. The final printed structure measured 1 cm × 1 cm × 1 cm. After printing, the printed structure was completely immersed in a 4 wt% APS solution for 10 minutes to promote further cross-linking and curing of the PDCB bioscaffold, resulting in a PDCB 3D bioprinted electrogenic anode, named PDC-B1.

[0059] Example 3

[0060] A method for preparing a three-dimensional bioprinted electrogenic anode in this embodiment comprises the following steps:

[0061] (1) Preparation of bacterial suspension

[0062] 0.1 mL of the electrogenic bacterium Shewanella oneidensis MR-1 was inoculated into 15 mL of Luria-Bertani broth and cultured in a constant temperature incubator (parameters: 150 rpm, 30°C) for 24 h. After centrifugation, the cells were washed three times with water (centrifugation conditions: 5000 rpm, 5 min), and water was added to adjust the OD 600 =1.5 bacterial suspension.

[0063] (2) Preparation of bio-ink

[0064] After centrifugation of the 45 mL bacterial suspension obtained in step (1), the upper liquid was removed, and the bacteria settled at the bottom were added to a 20 wt% PEGDA aqueous solution (1 mL). 180 mg of CNTs and 3 mg of DTT were added in sequence, and the mixture was stirred evenly to obtain PDCB bio-ink.

[0065] (3) PDCB three-dimensional bioanode

[0066] The PDCB bio-ink obtained in step (2) was placed in the bio-3D printing platform (SunP BioMaker 1) and extruded through a 20G flat-tip needle (diameter 0.30 mm) at a speed of 2.5 mm. 3 The printing process was carried out at room temperature (20-35°C) at a speed of 3.0 mm / s. The final printed structure measured 1 cm × 1 cm × 1 cm. After printing, the printed structure was completely immersed in a 4 wt% APS solution for 10 minutes to promote further cross-linking and curing of the PDCB bioscaffold, resulting in a PDCB 3D bioprinted electrogenic anode, named PDC-B2.

[0067] Example 4

[0068] A method for preparing a three-dimensional bioprinted electrogenic anode in this embodiment comprises the following steps:

[0069] (1) Preparation of bacterial suspension

[0070] 0.1 mL of the electrogenic bacterium Shewanella oneidensis MR-1 was inoculated into 15 mL of Luria-Bertani broth and cultured in a constant temperature incubator (parameters: 150 rpm, 30°C) for 24 h. After centrifugation, the cells were washed three times with water (centrifugation conditions: 5000 rpm, 5 min), and water was added to adjust the OD 600 =1.5 bacterial suspension.

[0071] (2) Preparation of bio-ink

[0072] After centrifugation of 105 mL of the bacterial suspension obtained in step (1), the upper liquid was removed, and the bacteria settled at the bottom were added to a 20 wt% PEGDA aqueous solution (1 mL). 180 mg of CNTs and 3 mg of DTT were added in sequence, and the mixture was stirred evenly to obtain PDCB bio-ink.

[0073] (3) PDCB three-dimensional bioanode

[0074] The PDCB bio-ink obtained in step (2) was placed in the bio-3D printing platform (SunP BioMaker 1) and extruded through a 20G flat-tip needle (diameter 0.30 mm) at a speed of 2.5 mm. 3 The printing process was carried out at room temperature (20-35°C) at a speed of 3.0 mm / s. The final printed structure measured 1 cm × 1 cm × 1 cm. After printing, the printed structure was completely immersed in a 4 wt% APS solution for 10 minutes to promote further cross-linking and curing of the PDCB bioscaffold, resulting in a PDCB 3D bioprinted electrogenic anode, named PDC-B4.

[0075] Example 5

[0076] A method for preparing a three-dimensional bioprinted electrogenic anode in this embodiment comprises the following steps:

[0077] (1) Preparation of bacterial suspension

[0078] 0.1 mL of the electrogenic bacterium Shewanella oneidensis MR-1 was inoculated into 15 mL of Luria-Bertani broth and cultured in a constant temperature incubator (parameters: 150 rpm, 30°C) for 24 h. After centrifugation, the cells were washed three times with water (centrifugation conditions: 5000 rpm, 5 min), and water was added to adjust the OD 600 =1.5 bacterial suspension.

[0079] (2) Preparation of bio-ink

[0080] After centrifugation of 135 mL of the bacterial suspension obtained in step (1), the upper liquid was removed, and the bacteria settled at the bottom were added to a 20 wt% PEGDA aqueous solution (1 mL). 180 mg of CNTs and 3 mg of DTT were added in sequence, and the mixture was stirred evenly to obtain PDCB bio-ink.

[0081] (3) PDCB three-dimensional bioanode

[0082] The PDCB bio-ink obtained in step (2) was placed in the bio-3D printing platform (SunP BioMaker 1) and extruded through a 20G flat-tip needle (diameter 0.30 mm) at a speed of 2.5 mm. 3 The printing process was carried out at room temperature (20-35°C) at a speed of 3.0 mm / s. The final printed structure measured 1 cm × 1 cm × 1 cm. After printing, the printed structure was completely immersed in a 4 wt% APS solution for 10 minutes to promote further cross-linking and curing of the PDCB bioscaffold, resulting in a PDCB 3D bioprinted electrogenic anode, named PDC-B5.

[0083] Example 6

[0084] A method for preparing a three-dimensional bioprinted electrogenic anode in this embodiment comprises the following steps:

[0085] (1) Preparation of bacterial suspension

[0086] 0.1 mL of the electrogenic bacterium Shewanella oneidensis MR-1 was inoculated into 15 mL of Luria-Bertani broth and cultured in a constant temperature incubator (parameters: 150 rpm, 30°C) for 24 h. After centrifugation, the cells were washed three times with water (centrifugation conditions: 5000 rpm, 5 min), and water was added to adjust the OD 600 =1 bacterial suspension.

[0087] (2) Preparation of bio-ink

[0088] After centrifugation of 75 mL of the bacterial suspension obtained in step (1), the upper liquid was removed, and the bacteria settled at the bottom were added to a 60 wt% PEGDA aqueous solution (1 mL). 140 mg of CNTs and 6 mg of DTT were added in sequence, and the mixture was stirred evenly to obtain PDCB bio-ink.

[0089] (3) PDCB three-dimensional bioanode

[0090] The PDCB bio-ink obtained in step (2) was placed in the bio-3D printing platform (SunP BioMaker 1) and extruded through a 20G flat-tip needle (diameter 0.30 mm) at a speed of 2.5 mm. 3 The printing process was carried out at room temperature, with a printing speed of 3.0 mm / s. The final printed structure measured 1 cm × 1 cm × 1 cm. After printing, the printed structure was completely immersed in a 3 wt% APS solution for 10 minutes to promote further cross-linking and curing of the PDCB bioscaffold, producing a 3D bioprinted PDCB electrogenic anode.

[0091] Example 7

[0092] A method for preparing a three-dimensional bioprinted electrogenic anode in this embodiment comprises the following steps:

[0093] (1) Preparation of bacterial suspension

[0094] 0.1 mL of the electrogenic bacterium Shewanella oneidensis MR-1 was inoculated into 15 mL of Luria-Bertani broth and cultured in a constant temperature incubator (parameters: 150 rpm, 30°C) for 24 h. After centrifugation, the cells were washed three times with water (centrifugation conditions: 5000 rpm, 5 min), and water was added to adjust the OD 600 =2 bacterial suspension.

[0095] (2) Preparation of bio-ink

[0096] After centrifugation of 75 mL of the bacterial suspension obtained in step (1), the upper liquid was removed, and the bacteria settled at the bottom were added to a 40 wt% PEGDA aqueous solution (1 mL). 160 mg of CNTs and 4 mg of DTT were added in sequence, and the mixture was stirred evenly to obtain PDCB bio-ink.

[0097] (3) PDCB three-dimensional bioanode

[0098] The PDCB bio-ink obtained in step (2) was placed in the bio-3D printing platform (SunP BioMaker 1) and extruded through a 20G flat-tip needle (diameter 0.30 mm) at a speed of 2.5 mm. 3 The printing process was carried out at room temperature, with a printing speed of 3.0 mm / s. The final printed structure measured 1 cm × 1 cm × 1 cm. After printing, the printed structure was completely immersed in an 8 wt% APS solution for 10 minutes to promote further cross-linking and curing of the PDCB bioscaffold, producing a 3D bioprinted PDCB electrogenic anode.

[0099] Example 8

[0100] A method for preparing a three-dimensional bioprinted electrogenic anode in this embodiment comprises the following steps:

[0101] (1) Preparation of bacterial suspension

[0102] 0.1 mL of electrogenic bacteria Escherichia coli K-12 was inoculated into 15 mL of Luria-Bertani broth and cultured in a constant temperature incubator (parameters: 150 rpm, 30°C) for 24 h. After centrifugation, the cells were washed three times with water (centrifugation conditions: 5000 rpm, 5 min), and water was added to adjust the OD 600 =1.5 bacterial suspension.

[0103] (2) Preparation of bio-ink

[0104] After centrifugation of 75 mL of the bacterial suspension obtained in step (1), the upper liquid was removed, and the bacteria settled at the bottom were added to a 20 wt% PEGDA aqueous solution (1 mL). 180 mg of CNTs and 2 mg of DTT were added in sequence, and the mixture was stirred evenly to obtain PDCB bio-ink.

[0105] (3) PDCB three-dimensional bioanode

[0106] The PDCB bio-ink obtained in step (2) was placed in the bio-3D printing platform (SunP BioMaker 1) and extruded through a 20G flat-tip needle (diameter 0.30 mm) at a speed of 2.5 mm. 3 The printing process was carried out at room temperature, with a printing speed of 3.0 mm / s. The final printed structure measured 1 cm × 1 cm × 1 cm. After printing, the printed structure was completely immersed in a 4 wt% APS solution for 10 minutes to promote further cross-linking and curing of the PDCB bioscaffold, producing a 3D bioprinted PDCB electrogenic anode.

[0107] Comparative Example 1

[0108] The preparation method of the PEGDA-LAP-CNTs-Bacteria-Light (PLCB-Light) three-dimensional bioprinted electrogenic anode based on photocrosslinking curing in this comparison is different from that in Example 1 in that photocrosslinking curing is used. The specific steps are as follows:

[0109] (1) Preparation of bacterial suspension

[0110] 0.1 mL of the electrogenic bacterium Shewanella oneidensis MR-1 was inoculated into 15 mL of Luria-Bertani broth and cultured in a constant temperature incubator (parameters: 150 rpm, 30°C) for 24 h. After centrifugation, the cells were washed three times with water (centrifugation conditions: 5000 rpm, 5 min), and water was added to adjust the OD 600 =1.5 bacterial suspension.

[0111] (2) Preparation of bio-ink

[0112] After centrifugation, 75 mL of the bacterial suspension obtained in step (1) was added with a 20 wt% PEGDA aqueous solution (1 mL), and then 180 mg of CNTs and 3 mg of photoinitiator LAP (phenyl-2,4,6-trimethylbenzoyl lithium phosphite) were added in sequence. After stirring evenly, PDCB bio-ink was obtained.

[0113] (3) PDCB three-dimensional bioanode

[0114] The PDCB bio-ink obtained in step (2) was placed in the bio-3D printing platform (SunP BioMaker 1) and extruded through a 20G flat-tip needle (diameter 0.30 mm) at a speed of 2.5 mm. 3 The printing process was carried out at room temperature, with a printing speed of 3.0 mm / s. The final printed structure had a size of 1 cm × 1 cm × 1 cm. After printing, the printed structure was irradiated with a 405 nm UV lamp for 30 minutes to achieve photocrosslinking, thus producing the PLCB-Light 3D bioprinted electrogenic anode.

[0115] Comparative Example 2

[0116] The preparation method of the SA-CNTs-Bacteria (SCB) three-dimensional bioprinting electrogenic anode based on ionic crosslinking curing in this comparative example differs from that in Example 1 in terms of the bio-ink composition and crosslinking method. The specific steps are as follows:

[0117] (1) Preparation of bacterial suspension

[0118] 0.1 mL of the electrogenic bacterium Shewanella oneidensis MR-1 was inoculated into 15 mL of Luria-Bertani broth and cultured in a constant temperature incubator (parameters: 150 rpm, 30°C) for 24 h. After centrifugation, the cells were washed three times with water (centrifugation conditions: 5000 rpm, 5 min), and water was added to adjust the OD 600 =1.5 bacterial suspension.

[0119] (2) Preparation of bio-ink

[0120] After centrifugation, 75 mL of the bacterial suspension obtained in step (1) was added with 2 wt % sodium alginate (SA) aqueous solution (1 mL), and 180 mg of CNTs were added. The mixture was stirred evenly to obtain bio-ink.

[0121] (3) Three-dimensional bioanode

[0122] The bio-ink obtained in step (2) was placed in the bio-3D printing platform (SunP BioMaker 1) and extruded through a 20G flat-tip needle (diameter 0.30 mm) at a speed of 2.5 mm. 3 The printing process was carried out at room temperature, with a printing speed of 3.0 mm / s. The final printed structure had a size of 1 cm × 1 cm × 1 cm. After printing, the printed structure was completely immersed in a 4% CaCl2 solution for 10 minutes to achieve ionic crosslinking, thus producing the SCB 3D bioprinted electrogenic anode.

[0123] Comparative Example 3

[0124] The preparation method of a three-dimensional bioprinting electrogenic anode in this comparative example differs from that in Example 1 in that the amount of CNTs added is less than 140 mg. The steps are as follows:

[0125] (1) Preparation of bacterial suspension

[0126] 0.1 mL of the electrogenic bacterium Shewanella oneidensis MR-1 was inoculated into 15 mL of Luria-Bertani broth and cultured in a constant temperature incubator (parameters: 150 rpm, 30°C) for 24 h. After centrifugation, the cells were washed three times with water (centrifugation conditions: 5000 rpm, 5 min), and water was added to adjust the OD 600 =1.5 bacterial suspension.

[0127] (2) Preparation of bio-ink

[0128] After centrifugation, 75 mL of the bacterial suspension obtained in step (1) was added with 20 wt% PEGDA aqueous solution (1 mL), and 120 mg of CNTs and 3 mg of DTT were added in sequence. The mixture was stirred evenly to obtain PDCB bio-ink.

[0129] (3) PDCB three-dimensional bioanode

[0130] The PDCB bio-ink obtained in step (2) was placed in the bio-3D printing platform (SunP BioMaker 1) and extruded through a 20G flat-tip needle (diameter 0.30 mm) at a speed of 2.5 mm. 3 The printing process was carried out at room temperature, with a printing speed of 3.0 mm / s. The final printed structure measured 1 cm × 1 cm × 1 cm. After printing, the printed structure was completely immersed in a 4 wt% APS solution for 10 minutes to promote further cross-linking and curing of the PDCB bioscaffold, thus producing the PDCB 3D bioprinted electrogenic anode.

[0131] Comparative Example 4

[0132] The preparation method of a three-dimensional bioprinting electrogenic anode in this comparative example differs from that in Example 1 in that the amount of CNTs added is higher than 180 mg. The steps are as follows:

[0133] (1) Preparation of bacterial suspension

[0134] 0.1 mL of the electrogenic bacterium Shewanella oneidensis MR-1 was inoculated into 15 mL of Luria-Bertani broth and cultured in a constant temperature incubator (parameters: 150 rpm, 30°C) for 24 h. After centrifugation, the cells were washed three times with water (centrifugation conditions: 5000 rpm, 5 min), and water was added to adjust the OD600 =1.5 bacterial suspension.

[0135] (2) Preparation of bio-ink

[0136] 75 mL of the bacterial suspension obtained in step (1) was centrifuged and then added with 20 wt% PEGDA aqueous solution (1 mL), followed by 200 mg of CNTs and 3 mg of DTT, and stirred to obtain PDCB bio-ink.

[0137] (3) PDCB three-dimensional bioanode

[0138] The PDCB bio-ink obtained in step (2) was placed in the bio-3D printing platform (SunP BioMaker 1) and extruded through a 20G flat-tip needle (diameter 0.30 mm) at a speed of 2.5 mm. 3 The printing process was carried out at room temperature, with a printing speed of 3.0 mm / s. The final printed structure measured 1 cm × 1 cm × 1 cm. After printing, the printed structure was completely immersed in a 4 wt% APS solution for 10 minutes to promote further cross-linking and curing of the PDCB bioscaffold, thus producing the PDCB 3D bioprinted electrogenic anode.

[0139] Figure 4 The printing conditions of PDCB inks with different CNTs concentrations (Example 1, Comparative Example 3 and Comparative Example 4) include extrusion state and deposition state. The actual printing process shows that the CNTs concentration is in the optimal printing stage when it is 140-180 mg / mL. At this concentration stage, the ink has good shear thinning properties. The high shear rate destroys the interaction between the macromolecular chains in the PDCB ink, causing the viscosity to drop, and the ink is extruded in a uniform filamentous form. When the shear disappears, it quickly returns to its original viscosity and quickly self-heals into a solid gel. However, when the CNTs concentration is lower than this range (taking 120 mg / mL as an example), the viscosity of the ink material is too low, causing the extrusion state to be droplet-like, and the material support is insufficient to be deposited. When the CNTs concentration is higher than this range (taking 200 mg / mL as an example), the ink cannot be extruded due to excessive viscosity.

[0140] Comparative Example 5

[0141] The method for preparing a three-dimensional bioprinting electrogenic anode in this comparative example differs from that in Example 1 in that the immersion time in the APS solution is less than 10 minutes, and the steps are as follows:

[0142] (1) Preparation of bacterial suspension

[0143] 0.1 mL of the electrogenic bacterium Shewanella oneidensis MR-1 was inoculated into 15 mL of Luria-Bertani broth and cultured in a constant temperature incubator (parameters: 150 rpm, 30°C) for 24 h. After centrifugation, the cells were washed three times with water (centrifugation conditions: 5000 rpm, 5 min), and water was added to adjust the OD 600 =1.5 bacterial suspension.

[0144] (2) Preparation of bio-ink

[0145] After centrifugation, 75 mL of the bacterial suspension obtained in step (1) was added with 20 wt% PEGDA aqueous solution (1 mL), and 180 mg of CNTs and 3 mg of DTT were added in sequence. The mixture was stirred evenly to obtain PDCB bio-ink.

[0146] (3) PDCB three-dimensional bioanode

[0147] The PDCB bio-ink obtained in step (2) was placed in the bio-3D printing platform (SunP BioMaker 1) and extruded through a 20G flat-tip needle (diameter 0.30 mm) at a speed of 2.5 mm. 3 The printing process was carried out at room temperature, with a printing speed of 3.0 mm / s. The final printed structure measured 1 cm × 1 cm × 1 cm. After printing, the printed structure was completely immersed in a 4 wt% APS solution for 8 minutes to promote further cross-linking and curing of the PDCB bioscaffold, thus producing the PDCB 3D bioprinted electrogenic anode.

[0148] Figure 5 The 3D-printed modules were immersed in APS for 8 minutes (Comparative Example 5) and 10 minutes (Example 1), and the sol-gel fractions obtained by soaking them in the electrolyte for different times showed that after soaking in APS for 8 minutes, the sol-gel fraction decreased significantly with time in the electrolyte, eventually leading to collapse and affecting its application in MFC. Therefore, the soaking time affects the curing degree of the bioanode. When the time is less than 10 minutes, the curing degree is poor, and long-term soaking in the electrolyte will cause collapse. When the soaking time is less than 10 minutes, the sulfate ions generated by APS can split into sulfate radicals, which accelerate the reaction of acrylates and thiols, greatly accelerating the gelation process. At the same time, sulfate radicals can also trigger the free radical polymerization of PEGDA (-CC-), and the construction of a hydrogel double-crosslinked network is achieved under the joint crosslinking action of -CS- and -CC-.

[0149] Implementation effect examples

[0150] (1) Sol-gel fraction test

[0151] After vacuum drying, the solidified sample was weighed (M1). The soluble portion was extracted in a dichloromethane solution at a ratio of 1 mL of dichloromethane per 10 mg of sample. After 24 hours of extraction, the dichloromethane was removed and the sample was vacuum dried at room temperature for 24 hours. The final sample mass (M2) was calculated and divided by the initial mass to obtain the sol-gel fraction (%).

[0152] Figure 6 It is the sol-gel fraction of the samples prepared in Example 1 and Comparative Example 1, wherein PEGDA-light represents the hydrogel scaffold cured by photocrosslinking (the three-dimensional bioprinting electrogenic anode prepared in Comparative Example 1), and PEGDA-DTT represents the hydrogel scaffold cured by adding DTT (the three-dimensional bioprinting electrogenic anode prepared in Example 1). The sol-gel fraction can directly reflect the degree of gelation of the hydrogel. The higher the score, the more perfect the three-dimensional network structure formed in the system, the more monomers or polymer chains undergo crosslinking reactions, and a gel phase is formed, and the higher the degree of curing of the hydrogel. It can be found that due to the dark black color of the scaffold, it absorbs light, resulting in the inability of PEGDA to absorb light and cannot be photocured and crosslinked. Therefore, Figure 5 It can be clearly observed that the sol-gel fractions of PEGDA-light are all below 20%, which is much lower than that of the scaffolds cured by click cross-linking.

[0153] (2) Swelling rate test

[0154] The swelling rate of a hydrogel is positively correlated with its water absorption capacity. Samples were placed in a 37°C drying oven and dried to constant weight, then weighed (W1). The samples were then placed in 30°C water for a period of constant swelling and absorption. After removal, the sample surface moisture was removed with filter paper and weighed again (W2). The swelling rate (%) was calculated using the following formula. The sample was allowed to swell for a total of 14 hours. The swelling rate, calculated as (W2 - W1) / W1 × 100%, was plotted against the swelling time to create a swelling equilibrium curve. Three measurements were taken for each sample, and the average was taken.

[0155] Figure 7 Shown are the swelling equilibrium curves of different samples, where SA-CNTs is an ionically cross-linked hydrogel scaffold (the three-dimensional bioprinting electricity-generating anode prepared in Comparative Example 2), and PEGDA-CNTs is a click-cured hydrogel scaffold (the three-dimensional bioprinting electricity-generating anode prepared in Example 1). The hydrogel swelling rate represents the ability and degree of the hydrogel to absorb water and expand in volume. When the hydrogel absorbs water and swells, its internal structure will change, thereby affecting the mechanical properties. The hydrogel network structure with low cross-linking density is relatively loose, with more space for water molecules to enter, and the swelling rate is usually higher; the hydrogel network structure with high cross-linking density is tight, and it is relatively difficult for water molecules to enter, so the swelling rate is lower. Figure 7The swelling equilibrium curves for the different samples show that the swelling rate of all samples gradually increases over time, eventually reaching a plateau. Specifically, after 2 hours, the swelling rate of PEGDA-CNTs reaches over 200%, while that of SA-CNTs is only around 100%. This indicates that the swelling capacity of the sodium alginate hydrogel is much higher than that of the PEGDA hydrogel, indicating that ionic crosslinking is weaker than chemical crosslinking, which would be detrimental to subsequent immersion of the bioanode.

[0156] (3) Electrochemical impedance spectroscopy

[0157] Inject 20 mL of electrolyte into a three-electrode electrolytic cell. Use the prepared PDCB bioscaffold as the working electrode, a platinum wire electrode as the counter electrode, and a calomel electrode as the reference electrode. Obtain a Nyquist plot using an electrochemical workstation.

[0158] Electrolyte formula: 0.6g / L K3[Fe(CN)6], 0.8g / L K4[Fe(CN)6] and 7.5g / L KCl.

[0159] (4) Current response over time (It) curve

[0160] A titanium wire-fixed 3D bioprinted electrogenic anode served as the working electrode, a platinum wire electrode served as the counter electrode, and a calomel electrode served as the reference electrode. The electrolyte for the three-electrode system consisted of 20 mL of M9 buffer solution (3.0 g / L KH2PO4, 15.0 g / L Na2HPO4, 5.0 g / L NaCl, and 0.1 g / L MgSO4) containing 5% LB broth and 18 mM sodium lactate. After assembly, the cell was aerated with nitrogen to remove dissolved oxygen and then sealed. The cell was connected to an electrochemical workstation and the time-dependent current response (It) curve was measured at constant potential.

[0161] Figure 8 The impedance curve (a) and It curve (b) measured for the three-dimensional bioprinted electrogenic anode prepared in Example 1. EIS was used to investigate the electron transfer capacity of the bioanode in Example 1. The impedance was 32.8Ω. The carbon nanotubes participated in the construction of the conductive network, providing more electron transfer paths, making the bioanode have good conductivity. The It curve showed 529.0A / m 3 From the maximum current density, it can be seen that the bioelectrode prepared in this embodiment has good electrochemical performance.

[0162] Application Examples

[0163] The PDCB three-dimensional bioprinted electricity-generating anode prepared in Example 1 of the present invention was applied to the anode chamber of a typical H-type dual-chamber microbial fuel cell. The specific steps are as follows:

[0164] A PDCB bioelectrode (or PDCB 3D bioprinted electrogenic anode) was used as the anode, and 100 mL of anolyte was injected into the anodic chamber. Simultaneously, a piece of carbon paper (2 cm × 4 cm) was selected as the cathode, and 100 mL of catholyte was injected into the cathodic chamber. An external 1000Ω resistor was connected. When the cell reached a steady state, a resistance box (9900-50Ω) was connected. The output voltage (U) was collected by adjusting the resistance value (R) to plot power density and polarization curves.

[0165] Anolyte formula: potassium dihydrogen phosphate 3 g / L, disodium hydrogen phosphate dodecahydrate 15 g / L, sodium chloride 5 g / L, magnesium sulfate 0.12 g / L, sodium lactate 2 g / L, Luria-Bertani broth 1 g / L.

[0166] Catholyte formula: sodium dihydrogen phosphate 8.5g / L, sodium bicarbonate 10g / L, potassium ferrocyanide 16.45g / L.

[0167] Figure 9 The current density curve (a) and power density curve (b) of the dual-chamber MFCs are shown. The power density reaches 495.6A / m in the dual-chamber MFCs. 3 The maximum output current density of MFCs remained basically unchanged within three complete operating cycles (more than 800 hours), further confirming the promoting effect of click cross-linking on the stability of bioactive materials. The power density curve of the dual-chamber MFCs loaded with PDCB bioelectrodes was tested by adjusting the external circuit load, showing 508.0W / m 3 Maximum power density. It can be seen that the invention's three-dimensional bioprinted anode based on click cross-linking curing can achieve high performance output when used in MFC.

[0168] Low bacterial loading capacity and low extracellular electron transfer (EET) efficiency are two major bottlenecks that limit the performance of bioelectrochemical systems to practical applications. Increasing the bacterial loading capacity of the bioanode can facilitate practical applications. A bacterial suspension with OD600=1.5 was first cultured, and 15mL, 45mL, 75mL, 105mL and 135mL of bacterial suspension (Examples 1-5) were centrifuged and added to the bio-ink to prepare five bioscaffolds with different bacterial loadings, PDC-B1, PDC-B2, PDC-B3, PDC-B4 and PDC-B5. By analyzing the protein content of the five bioscaffolds, it was found that the protein content was 3.8mg, 11.6mg, 19.0mg, 26.5mg and 33.6mg, respectively. The protein content basically conforms to 1:3:5:7:9, which is consistent with the amount of bacterial suspension used ( Figure 10a). Then, H-type dual-chamber MFCs were constructed as described above to study the long-term power generation performance of different electrodes. All anodes could operate for more than 600 hours, demonstrating the long-term stability of each bioscaffold ( Figure 10 b) and the chemical materials used will not be toxic to bacteria during the biofilm formation process.

[0169] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a three-dimensional bioprinted electrogenic anode, characterized in that: Here are the steps: S1. Add bacteria to a polyethylene glycol diacrylate aqueous solution, then add dithiothreitol and carbon nanotubes, and stir to obtain a bio-ink. S2. Printing the bio-ink obtained in step S1 to obtain a printed structure; S3. Immerse the printed structure obtained in step S2 in an ammonium persulfate solution and solidify it to obtain a conductive bioscaffold, namely a 3D bioprinted electrogenic anode.

2. The method for preparing a three-dimensional bioprinting electrogenic anode according to claim 1, characterized in that: The concentration of the polyethylene glycol diacrylate aqueous solution in step S1 is 20 wt%-60 wt%.

3. The method for preparing a three-dimensional bioprinting electrogenic anode according to claim 2, characterized in that: In step S1, 2-6 mg of dithiothreitol and 140-180 mg of conductive material are added to each mL of polyethylene glycol diacrylate aqueous solution.

4. The method for preparing a three-dimensional bioprinting electrogenic anode according to claim 3, characterized in that: In step S1 Bacteria were isolated by OD 600 The bacterial suspension with a value of 1-2 is centrifuged and the upper liquid is removed to obtain the bacterial suspension; the volume of the bacterial suspension required for each mL of polyethylene glycol diacrylate aqueous solution is 15-135 mL.

5. The method for preparing a three-dimensional bioprinting electrogenic anode according to claim 4, characterized in that: The preparation method of the bacterial suspension comprises the following steps: adding a bacterial seed preservation solution into a culture medium, culturing at a constant temperature, washing by centrifugation, and adding water to obtain a bacterial suspension.

6. The method for preparing a three-dimensional bioprinting electrogenic anode according to any one of claims 1 to 5, characterized in that: In step S1, the bacteria are Shewanella oneidensis MR-1 and Escherichia coli At least one of K-12.

7. The method for preparing a three-dimensional bioprinting electrogenic anode according to claim 6, characterized in that: The printing parameters in step S2 are set to an extrusion speed of 2.5 mm. 3 / s, printing speed 3.0 mm / s, printing temperature 20-35℃.

8. The method for preparing a three-dimensional bioprinting electrogenic anode according to claim 7, characterized in that: The concentration of the ammonium persulfate solution in step S3 is 3 wt%-8 wt%, and the soaking time is ≥10 min.

9. A three-dimensional bioprinted electrogenic anode prepared using the preparation method described in any one of claims 1-5 or 7 or 8.

10. Use of the three-dimensional bioprinted electricity-generating anode according to claim 9 in a microbial fuel cell.

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