A method of regulating the structure of electroactive biofilm in a microbial fuel cell

Abstract

The present application relates to the technical field of fuel cell, and aims to provide a method for regulating and controlling the structure of electroactive biofilm of microbial fuel cell. The method is to add amino acid to the domesticated microbial fuel cell to regulate and control the inner and outer structure of the electroactive biofilm, so that the electroactive layer inside the electroactive biofilm is densified, and the growth of the aerobic layer outside the electroactive biofilm is limited and thinned. The addition amount of the amino acid is controlled, so that the concentration of the amino acid in the nutrient solution is 50-500 mg / L, and the nutrient solution refers to the mixture of electrolyte, fuel and amino acid. The present application can regulate and control the structure of the electroactive biofilm, and the process is energy-saving, environment-friendly and simple to operate, and the potential environmental pollution and the risk of secondary pollution are reduced. The biofilm is denser and functionalized, the electron transfer path is optimized, and the electron transfer resistance is reduced. Organic matter can be more effectively converted into electric energy, and the energy conversion efficiency is improved.

Description

Technical Field

[0001] The invention belongs to the field of fuel cells and relates to a method for regulating the electroactive biofilm structure of a microbial fuel cell. Background Art

[0002] Microbial fuel cells (MCUs) are a green energy technology that utilizes an electroactive biofilm on the anode surface to convert chemical energy from organic matter into electricity. Electrogenic microorganisms within the MCUs transfer extracellular electrons through direct or indirect pathways. The structural characteristics of the MCUs, such as thickness, porosity, and microbial diversity, directly influence the efficiency of extracellular electron transfer and the power generation performance of MCUs.

[0003] Currently, strategies for improving the performance of electroactive biofilms at the anode include genetically engineering electrogenic bacteria, optimizing external resistance and shear stress, and adding heavy metal ions. For example, adjusting the external voltage from 0.7 V to 0.9 V can increase current output by 61% and promote the formation of a uniform, highly active biofilm. Regulating external resistance and shear stress can form a dense, Geobacter-rich electroactive biofilm, enabling a compact MFC with a power density as high as 4300 mW·m -2 However, the above methods still have many limitations, such as high cost and complex operation of genetic modification, possible environmental toxicity caused by the addition of heavy metals, and difficulty in adapting to complex application scenarios by relying on physical parameter regulation.

[0004] Therefore, a low-cost, environmentally friendly bioregulatory strategy is urgently needed to optimize the electroactive biofilm construction process. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide a method for regulating the electroactive biofilm structure of a microbial fuel cell.

[0006] To solve the technical problem, the solution of the present invention is:

[0007] Provided is a method for regulating the structure of an electroactive biofilm in a microbial fuel cell. The method comprises adding amino acids to an already domesticated microbial fuel cell to regulate the inner and outer layer structures of the electroactive biofilm, thereby densifying the electroactive layer inside the electroactive biofilm and restricting the growth and thinning of the outer aerobic layer. The amount of amino acid added is controlled so that its concentration in a nutrient solution is 50 to 500 mg / L. The nutrient solution is a mixture of electrolytes, fuel, and amino acids.

[0008] As a preferred embodiment of the present invention, the amino acid is any one of aspartic acid, glutamic acid or histidine.

[0009] As a preferred embodiment of the present invention, during the control process, the microbial fuel cell and an external resistor of 1000Ω form a loop and are placed in a constant temperature incubator at 30°C for cultivation.

[0010] As a preferred embodiment of the present invention, during the control process, the voltage data output by the microbial fuel cell is recorded at intervals. If 3 to 5 stable output voltage cycles are recorded, the control is considered to be completed.

[0011] As a preferred embodiment of the present invention, during the regulation process, all the nutrient solutions of the microbial fuel cell are replaced every 24 hours.

[0012] As a preferred embodiment of the present invention, the microbial fuel cell uses a graphite sheet as an anode, a nickel foam air cathode as a cathode, a phosphate buffer solution with a pH of 7 as an electrolyte, and an appropriate amount of fuel is added to the electrolyte.

[0013] As a preferred embodiment of the present invention, the fuel used in the microbial fuel cell is any one of carbohydrates, organic acids, alcohols or nitrogen-containing organic matter.

[0014] As a preferred embodiment of the present invention, the fuel used in the microbial fuel cell is glucose, and the added amount is controlled so that its concentration in the electrolyte is 1 g / L.

[0015] Description of the invention principle:

[0016] Microbial fuel cell systems typically use nutrients such as carbohydrates, organic acids, alcohols, or nitrogen-containing organic compounds as fuels to provide the energy needed for microbial survival. Therefore, glucose, acetic acid, ethanol, urea, and amino acids can all serve as primary carbon sources for biofuel cells. However, prior art uses of amino acids as fuels have primarily focused on their nutritional value, specifically to sustain microbial growth.

[0017] In the research on constructing high-performance anodic electroactive biofilms, existing strategies include genetic manipulation, optimization of external resistance and shear force, and the addition of substances such as heavy metals. However, genetic engineering modification (such as overexpression of conductive protein genes) is costly and complex to operate, difficult to apply on a large scale, and prone to biological contamination. In addition, although adding heavy metal ions to the system can increase the electron transfer rate, there is a risk of environmental toxicity and it is easy to cause secondary pollution. The regulation of physical parameters (such as external voltage and shear force) is highly dependent on equipment and is difficult to adapt to complex and changeable actual operating conditions. In addition, existing strategies mostly focus on optimizing biofilm conductivity, ignoring the importance of coordinated regulation of microbial metabolic activity and biofilm structure.

[0018] Microorganisms can adapt to changing nutritional conditions by dynamically adjusting their morphology, gene expression, and protein synthesis. Amino acids, as core components of microbial metabolism, play a key role in cell proliferation, energy metabolism, and interspecies signaling. Therefore, the addition of amino acids to the culture medium has been proposed to significantly accelerate microbial growth and optimize its function by redistributing proteomic resources, thereby regulating the structure of electroactive biofilms. This approach can improve the electron transfer efficiency and power generation performance of microbial fuel cells, thereby reducing reliance on heavy metals or synthetic mediators and minimizing the risk of secondary contamination.

[0019] Amino acids play a key role in microbial metabolism, not only as components of proteins and active residues in many enzymes, but also participating in a variety of metabolic pathways. The present application significantly increases the growth rate of microorganisms by supplementing amino acids, which is particularly important in the formation of electroactive biofilms that require high metabolic activity. By adjusting the added concentration of amino acids, the structure of the electroactive biofilm can be significantly optimized, especially in terms of the density and activity of the biofilm. Microbial fuel cells with added amino acids exhibit higher maximum power density and coulombic efficiency than microbial fuel cells cultured only with glucose. This shows that amino acids not only promote the growth of electroactive bacteria, but also optimize the structure of the biofilm, thereby improving the electron transfer efficiency and overall battery performance.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The present invention uses amino acids as additives to achieve regulation of the electroactive biofilm structure. This process is energy-saving, environmentally friendly, and easy to operate, reducing the risk of potential environmental pollution and secondary pollution.

[0022] 2. The present invention significantly improves the maximum power density and Coulombic efficiency of microbial fuel cells by adding only a small amount of amino acids. Compared to traditional microbial fuel cells cultivated using only conventional fuels, the present invention can more effectively convert organic matter into electricity, improving energy conversion efficiency.

[0023] 3. The present invention promotes the growth of electroactive bacteria by adding amino acids, accelerating the formation and maturation of electroactive biofilms. This makes the biofilm denser and more functional, optimizes the electron transfer pathway, and reduces electron transfer resistance.

[0024] 4. This invention differs from existing methods for optimizing electroactive biofilm construction by using environmentally friendly amino acids as additives to optimize the inner and outer layers of the electroactive biofilm, creating a high-performance electroactive biofilm that improves the startup speed and power generation performance of the MFC. This simultaneous optimization strategy of building both the inner and outer layers is expected to improve the overall performance of MFCs using complex organic matter as fuel, providing important innovative insights for the further development and application of electroactive biofilm research. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the structural diagram of the electroactive biofilm in Comparative Example 1.

[0026] Figure 2 This is a diagram of the electroactive biofilm structure in Example 1.

[0027] Figure 3 This is the activity diagram of the electroactive biofilm in Comparative Example 1.

[0028] Figure 4 This is the activity diagram of the electroactive biofilm in Example 1.

[0029] Figure 5 2 are the electrochemical impedance curves of the microbial fuel cells in Example 2 and Comparative Example 2.

[0030] Figure 6 Impedance fitting results of the microbial fuel cells in Example 2 and Comparative Example 2.

[0031] Figure 7 2 are the power density curves of the microbial fuel cells in Example 2 and Comparative Example 2.

[0032] Figure 8 This is the construction process of the electroactive biofilm on the graphite sheet in Comparative Example 3 and Example 3.

[0033] Figure 9 This is the complete in situ structure of the electroactive microorganism in Example 4.

[0034] Figure 10 This is the in-situ complete structure of the electroactive microorganism in Comparative Example 4.

[0035] Figure 11 This is the in-situ complete structure of the electroactive microorganism in Comparative Example 5.

[0036] Figure 12 This is the electroactive biofilm structure in Example 5.

[0037] Figure 13 This is the electroactive biofilm structure in Comparative Example 5.

[0038] Figure 14This is the electroactive biofilm structure in Comparative Example 6.

[0039] exist Figure 1 and Figure 3 In the figure, A0 refers to the electroactive biofilm of group A0 after acclimation without adding glutamate in Comparative Example 1; Figure 2 and Figure 4 In the above, A2 refers to the electroactive biofilm of group A2 after adding glutamate for acclimatization in Example 1. Figure 8 In the figure, AD are the electroactive biofilms at the initial moment, 12h, 24h and 72h in the acclimation process using only glucose in comparative example 3, and EH are the electroactive biofilms at the initial moment, 12h, 24h and 72h in the acclimation process using histidine in example 3. Figure 9 In the figure, R1 is the electroactive biofilm of group R1 after adding aspartic acid and acclimating with a 1000Ω external resistor in Example 4. Figure 10 In the figure, R2 is the electroactive biofilm of group R2 in comparative example 4 after adding aspartic acid and acclimating with a 500Ω external resistor. Figure 11 In the figure, R3 is the electroactive biofilm of group R3 in comparative example 5 after adding aspartic acid and acclimating with a 2000Ω external resistor. Figure 12 In the example 5, D1 is the electroactive biofilm of group D1 after acclimation by replacing all nutrient solutions every 24 hours. Figure 13 In the figure, D2 is the electroactive biofilm of group D2 after acclimation by pumping nutrient solution in a continuous flow manner in Example 5. DETAILED DESCRIPTION

[0040] The present invention will be described in detail below in conjunction with specific embodiments and accompanying drawings, but the present invention is not limited to the following embodiments. Without departing from the content and scope of the present invention, all variations and implementations should be included in the technical scope of the present invention.

[0041] In each of the Examples and Comparative Examples, the concentrations of amino acids and glucose refer to their respective added (or acclimated) concentrations in a nutrient solution, which is a mixture of electrolytes, fuel, and amino acids.

[0042] Example 1

[0043] Addition of glutamate modulates the electroactive biofilm structure of microbial fuel cells:

[0044] (1) As Example 1, a microbial fuel cell system was prepared by adding glutamate (Group A2, 50 mg / L) to a 1 g / L glucose-acclimated system. A graphite sheet was used as the anode of the microbial fuel cell, a nickel foam air cathode was used as the cathode, and a phosphate buffer solution with a pH of 7 was used as the electrolyte. The microbial fuel cell was connected to a circuit, an external resistor of 1000 Ω was connected, and then placed in a constant temperature incubator at 30°C for incubation. The system voltage data was recorded every 5 minutes, and the output voltage data was recorded during this period. After recording 3 to 5 stable output voltage cycles, a microbial fuel cell with a mature electroactive biofilm was obtained, and the inner and outer layer structures and activity of the electroactive biofilm were observed.

[0045] (2) As comparative example 1, except that glutamic acid was not added (Group A0, 0 mg / L), other operations remained unchanged.

[0046] (3) If Figure 1 、 2 As shown in Figure 2, the in situ complete structure of electroactive microorganisms was photographed using OCT technology. The A2 group with added glutamate showed a more suitable double-layer structure, with a loose aerobic layer on the outer layer and a dense electroactive layer on the inner layer. Figure 3 、 4 As shown, the activity of electroactive microorganisms was characterized by CLSM technology, and the activity of group A2 with added glutamate was higher than that of group A0.

[0047] Example 2

[0048] Addition of aspartate modulates the electroactive biofilm structure of microbial fuel cells:

[0049] (1) As Example 2, aspartic acid (GA20 group, 200 mg / L) was added to a microbial fuel cell system acclimated with 1 g / L glucose. A graphite sheet was used as the anode of the microbial fuel cell, a nickel foam air cathode was used as the cathode, and a phosphate buffer solution with a pH of 7 was used as the electrolyte. The microbial fuel cell was connected to a circuit, an external resistor of 1000 Ω was connected, and then placed in a constant temperature incubator at 30°C for incubation. The system voltage data was recorded every 5 minutes, and the output voltage data was recorded during this period. After recording 3 to 5 stable output voltage cycles, a microbial fuel cell with a mature electroactive biofilm was obtained.

[0050] (2) As comparative example 2, except that aspartic acid was not added (GA0 group, 0 mg / L), other operations remained unchanged.

[0051] (3) After the electroactive biofilm matured, the microbial fuel cell was subjected to electrochemical impedance spectroscopy (EIS) and power density testing using an electrochemical workstation, and the coulombic efficiency was calculated. The electrochemical reaction system was a three-electrode system, with saturated KCl-filled Ag / AgCl as the reference electrode. The EIS was operated in a frequency range of 100 kHz to 0.01 Hz with an amplitude of 10 mV. The power density test was performed using polarization tests at different resistances from 10,000 Ω to 50 Ω, with each resistance setting for 20 minutes. The COD concentration in the solution was measured using a spectrophotometric screening method. Before measuring the COD value, the solution was sampled and filtered to remove bacteria.

[0052] (4) If Figure 5 The electrochemical impedance curves and Figure 6 As shown in the impedance fitting results, compared with the GA0 group without aspartic acid, the electron transfer impedance and diffusion impedance of the electroactive biofilm in the GA20 group with aspartic acid added were greatly reduced.

[0053] (5) If Figure 7 As shown in the power density curve, the maximum power density increased to 910 mW·m after adding aspartic acid. -2 The coulombic efficiency results showed that the addition of aspartic acid increased the coulombic efficiency from 28.9% to 38.93%.

[0054] Example 3

[0055] Adding histidine to regulate the electroactive biofilm structure of microbial fuel cells:

[0056] (1) As Example 3, a microbial fuel cell system was prepared by adding histidine (GA20 group, 500 mg / L) to 1 g / L glucose. A graphite sheet was used as the anode of the microbial fuel cell, a nickel foam air cathode was used as the cathode, and a phosphate buffer solution with a pH of 7 was used as the electrolyte. The microbial fuel cell was connected to a circuit, an external resistor of 1000 Ω was connected, and then the cell was placed in a constant temperature incubator at 30°C for incubation.

[0057] (2) As comparative example 3, except that histidine was not added (GA0 group, 0 mg / L), other operations remained unchanged.

[0058] (3) If Figure 8 As shown in the figure, OCT technology was used to record the construction process of the electroactive biofilm on the graphene sheet every 12 hours. The results show that the addition of histidine can promote the densification of the electroactive layer within the biofilm, thereby maintaining the high activity of the inner layer and reducing internal impedance, while also limiting the growth of the outer aerobic layer and reducing diffusion impedance.

[0059] Example 4

[0060] Regulation of electroactive biofilm structure of microbial fuel cells under different external resistance values:

[0061] (1) As Example 4, aspartic acid (R1 group, 100 mg / L) was added to a microbial fuel cell system acclimated with 1 g / L glucose. A graphite sheet was used as the anode of the microbial fuel cell, a nickel foam air cathode was used as the cathode, and a phosphate buffer solution with pH = 7 was used as the electrolyte. The microbial fuel cell was connected to the circuit, an external resistor of 1000 Ω (R1, 1000 Ω) was connected, and then placed in a constant temperature incubator at 30°C for incubation. The system voltage data was recorded every 5 minutes, and the output voltage data was recorded during this period. After recording 3 to 5 stable output voltage cycles, a microbial fuel cell with a mature electroactive biofilm was obtained, and the inner and outer layer structures of the electroactive biofilm were observed;

[0062] (2) As comparative example 4, except for adjusting the resistance value of the external resistor (R2 group, 500Ω), other operations remain unchanged.

[0063] (3) As comparative example 5, except for adjusting the resistance value of the external resistor (R3 group, 2000Ω), other operations remain unchanged.

[0064] (4) Using OCT technology to photograph the complete in situ structure of electroactive microorganisms under different external resistor values, Figure 9 、 Figure 10 、 Figure 11 Corresponding to Example 4, Comparative Example 4, and Comparative Example 5 respectively. As can be seen from the figure, the R1 group with the addition of aspartic acid showed a suitable double-layer structure, with a loose aerobic layer as the outer layer and a dense electroactive layer as the inner layer. The R2 group showed a thicker inner electroactive membrane, increased the dead zone of the biofilm, and the bacterial activity decreased by 31%. The R3 group showed a looser and thicker outer layer and a thinner electroactive layer. The reason for the above differences is that the current density passing through the fuel cell is greater at a relatively small external resistance value. Although it can promote the growth of the dense electroactive layer in the inner layer, it will form a dead zone of the biofilm; the current density passing through the fuel cell is smaller at a relatively large external resistance value, which is relatively unfavorable for the formation of a dense electroactive layer. Therefore, a relatively moderate external resistance of 1000Ω was selected in this embodiment.

[0065] Example 5

[0066] Regulating the electroactive biofilm structure of microbial fuel cells under different addition methods:

[0067] (1) Aspartic acid (100 mg / L) was added to a microbial fuel cell system acclimated with 1 g / L glucose. A graphite sheet was used as the anode of the microbial fuel cell, a nickel foam air cathode was used as the cathode, and a pH = 7 phosphate buffer was used as the electrolyte. The microbial fuel cell was connected to a circuit with an external resistor of 1000 Ω. As Example 5, the entire nutrient solution (i.e., a mixture of electrolyte, fuel, and amino acids) was replaced every 24 hours in Group D1. As Comparative Example 5, the nutrient solution was pumped into Group D2 using a continuous flow method to ensure that the total amount of nutrients in the two groups was consistent daily.

[0068] Then, the cells were cultured in a constant temperature incubator at 30°C, and the system voltage data was recorded every 5 minutes. During this period, the output voltage data was recorded. After 3 to 5 stable output voltage cycles were recorded, a microbial fuel cell with a mature electroactive biofilm was obtained, and the inner and outer layer structures of the electroactive biofilm were observed.

[0069] (2) The in situ complete structures of the electroactive microorganisms in Example 5 and Comparative Example 5 were photographed using OCT technology, as shown in FIG. Figure 12 、 Figure 13 As shown in the figure, group D1, cultured with regular replacement of the entire nutrient solution, exhibited a suitable bilayer structure, with a loose aerobic outer layer and a dense electroactive inner layer. Group D2, cultured with continuous nutrient solution pumping, exhibited a thicker biofilm structure with increased bacterial dead zones. This difference is due to the fact that the regular flow of nutrient solution during solution replacement disturbs and impacts the biofilm structure attached to the anode and cathode plates of the fuel cell, causing some of the bacterial dead zones to fall off, reducing the electron transfer and diffusion impedance of the electroactive microbial film.

[0070] Comparative Example 6

[0071] Electroactive biofilm structure of microbial fuel cells using amino acids alone as fuel:

[0072] (1) As comparative example 6, aspartic acid was used alone as the fuel, and the microbial fuel cell system was acclimated at an addition concentration of 1 g / L. A graphite sheet was used as the anode of the microbial fuel cell, a nickel foam air cathode was used as the cathode, and a phosphate buffer solution with a pH of 7 was used as the electrolyte. The microbial fuel cell was connected to the circuit, an external resistor of 1000 Ω (R1, 1000 Ω) was connected, and then placed in a constant temperature incubator at 30°C for incubation. The system voltage data was recorded every 5 minutes, and the output voltage data was recorded during this period. After recording 3 to 5 stable output voltage cycles, a microbial fuel cell with a mature electroactive biofilm was obtained, and the inner and outer layer structures (E1) of the electroactive biofilm were observed.

[0073] (2) If Figure 14As shown in the figure, the in situ complete structure of electroactive microorganisms was photographed using OCT technology. The electroactive biofilm acclimated with pure aspartic acid was thicker overall and had internal void structures due to the production of more gas. Figure 9 、 Figure 12 Compared with the membrane structures of Examples 4 and 5 in which aspartic acid is used as an additive, this membrane structure is obviously not conducive to the electron exchange between the electroactive bacteria and the electrode.

[0074] (3) The same experiment verified that when glutamate and histidine were used alone as fuels, compared with when they were used separately as additives, the electroactive biofilm was also thicker overall after acclimation and had an internal void structure.

[0075] In summary, the present invention proposes to use a small amount of amino acid addition to promote the densification of the electroactive layer in the biofilm, maintain the high activity of the inner layer, reduce the internal impedance, and at the same time limit the growth of the outer aerobic layer, reduce the diffusion impedance, and improve the efficiency of the electrochemical reaction. The adjustment means include the selection of different amino acids, adjustment of the external resistor value, adjustment of the amino acid concentration and the addition method, and the optimization of the electroactive biofilm structure to improve the electrochemical performance of the MFC. The final biofilm has a clear double-layer structure, the outer layer is a loose aerobic layer, and the inner layer is a dense and highly active electroactive layer. Based on the electrochemical impedance spectroscopy, power density test and coulomb efficiency test, it was confirmed that the appropriate addition of amino acids can reduce the electron transfer impedance and diffusion impedance of the electroactive microbial membrane, enhance the power density of the microbial fuel cell, and improve the coulomb efficiency of the microbial fuel cell.

[0076] The present invention is not limited to the above-described preferred embodiment. Based on the teachings of this invention, anyone can derive various other methods for regulating the electroactive biofilm structure of a microbial fuel cell. All equivalent changes and modifications made within the scope of the present invention are intended to fall within the scope of this invention.

Claims

1. A method for regulating the electroactive biofilm structure of a microbial fuel cell, characterized in that: By adding amino acids to the domesticated microbial fuel cell, the inner and outer layer structures of the electroactive biofilm are regulated, resulting in a densified electroactive layer inside the electroactive biofilm and a restricted and thinned outer aerobic layer. The amount of amino acid added is controlled to achieve a concentration of 50 to 500 mg / L in a nutrient solution, wherein the nutrient solution is a mixture of electrolytes, fuel, and amino acids. The amino acid is any one of aspartic acid, glutamic acid, or histidine. The microbial fuel cell uses a graphite sheet as an anode, a nickel foam air cathode as a cathode, and a phosphate buffer solution with a pH of 7 as an electrolyte. A proper amount of fuel is added to the electrolyte; the fuel is any one of carbohydrates, organic acids, alcohols or nitrogen-containing organic matter.

2. The method according to claim 1, characterized in that During the regulation process, the microbial fuel cell and an external resistor of 1000Ω formed a loop and were placed in a constant temperature incubator at 30°C for cultivation.

3. The method according to claim 1, characterized in that During the control process, the voltage data output by the microbial fuel cell is recorded at intervals. If 3 to 5 stable output voltage cycles are recorded, the control is considered to be completed.

4. The method according to claim 1, wherein During the regulation process, all the nutrient solution of the microbial fuel cell was replaced every 24 hours.

5. The method according to claim 1, wherein The fuel used in the microbial fuel cell is glucose, and the added amount is controlled so that its concentration in the electrolyte is 1 g / L.

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