A method for detecting direct and indirect electron transfer capabilities of biochar

Abstract

The present invention discloses a method for detecting the direct and indirect electron transfer capabilities of biochar. The method comprises the following steps: grinding and sieving the biochar, and then adding ultrapure water to prepare a biochar solution; activating a Shewanella stock solution, inoculating it into a sterilized LB culture medium, placing it in a shaker for cultivation, and repeatedly transferring it multiple times before adding ultrapure water to prepare a Shewanella solution; using an anaerobic state, using a three-electrode system connected to an electrochemical workstation, adding ultrapure water to a glassy carbon cup electrode, setting the voltage, and after the current stabilizes, injecting the Shewanella solution into the glassy carbon cup electrode of a control group, and injecting the Shewanella solution and the biochar solution into the glassy carbon cup electrode of an experimental group. In a current-time curve, the difference in peak current between the experimental group and the control group represents the direct electron transfer capability of the biochar, and the difference in stable current represents the indirect electron transfer capability of the biochar. The method of the present invention is simple and easy to implement, and directly demonstrates the direct and indirect electron transfer capabilities of the biochar.

Description

Technical Field

[0001] The invention relates to a method for detecting direct and indirect electron transfer capabilities of biochar, and belongs to the technical field of biochar. Background Art

[0002] In recent years, electrochemically active bacteria have shown promising applications in environmental pollution control and sustainable green energy. Shewanella is the most representative of these active bacteria. They can transfer electrons generated by organic matter metabolism to electron acceptors through extracellular electron transfer, playing a vital role in energy production, environmental remediation, and geochemical recycling systems. However, the extracellular electron transfer process in Shewanella is typically slow, limiting the application of bioelectrochemical systems and attracting extensive research attention.

[0003] It is currently known that biochar can act as an electron mediator to promote electron transfer during the degradation and transformation of pollutants by Shewanella. Because biochar can directly transfer electrons released by microorganisms through the carbon matrix, it acts as an "electron shuttle" or "redox mediator" in the reduction system, enhancing the microorganism's ability to transfer extracellular electrons, thereby effectively increasing the reduction rate of target pollutants. Therefore, the direct and indirect electron transfer capacity of biochar is considered to be key to promoting the extracellular electron transfer process of Shewanella. However, there is currently no accurate experimental method to quantitatively characterize and determine the direct and indirect electron transfer capacity of biochar. Summary of the Invention

[0004] In response to the problems existing in the prior art, the present invention provides a simple, direct and low-cost detection method for quantitatively characterizing the direct and indirect electron transfer capacity of biochar, thereby specifically improving the extracellular electron transfer rate of Shewanella.

[0005] The technical solutions of the present invention are as follows:

[0006] A method for detecting the direct and indirect electron transfer capabilities of biochar, comprising the following steps:

[0007] (1) Preparation of biochar solution: The biochar obtained after pyrolysis was cooled to room temperature, ground and sieved, and then added with ultrapure water to prepare a biochar solution;

[0008] (2) Cultivation of Shewanella solution: After activating the Shewanella stock solution, inoculate it into sterilized LB medium and culture it in a shaker. After repeated transfers, add ultrapure water to prepare a Shewanella solution.

[0009] (3) Under anaerobic conditions, a three-electrode system (Ag / AgCl electrode, platinum wire and glassy carbon cup electrode as reference electrode, counter electrode and working electrode, respectively) was used and connected to an electrochemical workstation. Ultrapure water was added to the glassy carbon cup electrode and the voltage was set. When the current stabilized, the Shewanella solution was slowly injected into the glassy carbon cup electrode of the control group, and the Shewanella solution and biochar solution were slowly injected into the glassy carbon cup electrode of the experimental group to obtain the current i-time t curve. In the it curve, the difference in peak current between the experimental group and the control group is the direct electron transfer capacity of the biochar, and the difference in stable current is the indirect electron transfer capacity of the biochar.

[0010] In step (1), the biochar is pyrolyzed at a temperature of 200 to 800° C. and sieved through a mesh size of 100 to 300 meshes.

[0011] The concentration of the biochar solution in step (1) is 0.5 to 3 g / L.

[0012] In step (2), the shaking incubation temperature is 25-35° C., the time is 12-24 hours, the speed is 100-200 rpm, and the number of repeated transfers is 3-5 times.

[0013] The concentration of the Shewanella solution in step (2) is 0.3×10 7 ~2.4×10 7 CFU / mL.

[0014] Step (3) Add 20-40 mL of ultrapure water to the glassy carbon cup electrode and set the voltage to 0.2-0.5 V.

[0015] In step (3), the amount of Shewanella solution added to the glassy carbon electrodes in the control group and the experimental group was the same, and both were 1 to 5 mL. The amount of biochar solution added to the experimental group was 5 to 15 mL; the solution injection flow rate was 20 to 25 mL / min.

[0016] The advantages of the present invention are:

[0017] 1. The biochar used in the present invention is not restricted, and the experimental process is green and environmentally friendly.

[0018] 2. The process of the present invention is simple and easy to operate, and can directly demonstrate the direct and indirect electron transfer capabilities of biochar. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the it curve diagram of Example 1;

[0020] Figure 2 It is the it curve diagram of Example 2;

[0021] Figure 3 It is the it curve diagram of Example 3. DETAILED DESCRIPTION

[0022] The present invention will be further described below with reference to specific embodiments, but the protection scope of the present invention is not limited to the contents described above.

[0023] The Shewanella stock solution and LB culture medium used in the present invention are conventional commercial products.

[0024] Example 1

[0025] A method for detecting the direct and indirect electron transfer capabilities of biochar, comprising the following steps:

[0026] (1) Preparation of biochar solution: Biomass straw was heated to 500°C under nitrogen atmosphere for 3 hours to obtain biochar after pyrolysis. The biochar was cooled to room temperature, ground through a 100-mesh sieve, and then added with ultrapure water to prepare a 0.5 g / L biochar solution.

[0027] (2) Cultivation of Shewanella solution: After activating the Shewanella stock solution, inoculate it into sterilized LB medium and culture it in a shaker at 25°C at 100 rpm. After 12 h of culture, transfer it three times and add ultrapure water to make it 0.3×10 7 CFU / mL of Shewanella solution;

[0028] (3) Under anaerobic conditions, a three-electrode system (Ag / AgCl electrode, platinum wire and glassy carbon cup electrode as reference electrode, counter electrode and working electrode, respectively) was used and connected to an electrochemical workstation. 20 mL of ultrapure water was added to the glassy carbon cup electrode and the voltage was set to 0.2 V. When the current stabilized, 1 mL of Shewanella solution was slowly injected into the glassy carbon cup electrode of the control group, and 1 mL of Shewanella solution and 5 mL of biochar solution were slowly injected into the glassy carbon cup electrode of the experimental group at an injection rate of 20 mL / min. The it curve was obtained. The difference in peak current between the experimental group and the control group was the direct electron transfer capacity of the biochar, and the difference in stable current was the indirect electron transfer capacity of the biochar.

[0029] Figure 1 It is the it curve of Example 1. It can be seen that the peak current of the control group is 2.53μA, the stable current is 0.27μA, the peak current of the experimental group is 2.78μA, and the stable current is 0.59μA. Therefore, the direct electron transfer capacity of the biochar in this example is 0.25μA, and the indirect electron transfer capacity is 0.32μA.

[0030] Example 2

[0031] A method for detecting the direct and indirect electron transfer capabilities of biochar, comprising the following steps:

[0032] (1) Preparation of biochar solution: Biomass straw was heated to 800°C under nitrogen atmosphere for 3 hours. The biochar obtained after pyrolysis was cooled to room temperature, ground through a 150-mesh sieve, and then ultrapure water was added to prepare a 1 g / L biochar solution.

[0033] (2) Cultivation of Shewanella solution: After activating the Shewanella stock solution, inoculate it into sterilized LB medium and culture it in a shaker at 30°C at 150 rpm. After 16 h of culture, transfer it four times and add ultrapure water to make it 0.4×10 7 CFU / mL of Shewanella solution;

[0034] (3) Under anaerobic conditions, a three-electrode system (Ag / AgCl electrode, platinum wire and glassy carbon cup electrode as reference electrode, counter electrode and working electrode, respectively) was used and connected to an electrochemical workstation. 25 mL of ultrapure water was added to the glassy carbon cup electrode and the voltage was set to 0.3 V. When the current stabilized, 2 mL of Shewanella solution was slowly injected into the glassy carbon cup electrode of the control group, and 2 mL of Shewanella solution and 6 mL of biochar solution were slowly injected into the glassy carbon cup electrode of the experimental group at an injection rate of 23 mL / min. The it curve was obtained. The difference in peak current between the experimental group and the control group was the direct electron transfer capacity of the biochar, and the difference in stable current was the indirect electron transfer capacity of the biochar.

[0035] Figure 2 From the IT curve of Example 2, it can be seen that the peak current of the control group is 2.53 μA, and the stable current is 0.27 μA, while the peak current of the experimental group is 4.73 μA, and the stable current is 0.31 μA. Therefore, the direct electron transfer capacity of the biochar in this example is 2.2 μA, and the indirect electron transfer capacity is 0.04 μA.

[0036] Example 3

[0037] A method for detecting the direct and indirect electron transfer capabilities of biochar, comprising the following steps:

[0038] (1) Preparation of biochar solution: Biomass straw was heated to 320°C under nitrogen atmosphere for 3 hours. The biochar obtained after pyrolysis was cooled to room temperature, ground through a 300-mesh sieve, and then added with ultrapure water to prepare a 3 g / L biochar solution.

[0039] (2) Cultivation of Shewanella solution: After activating the Shewanella stock solution, inoculate it into sterilized LB medium and culture it in a shaker at 35°C at a speed of 100 rpm. After 24 hours of culture, after five repeated transfers, add ultrapure water to prepare 2.4×10 7 CFU / mL of Shewanella solution;

[0040] (3) Under anaerobic conditions, a three-electrode system (Ag / AgCl electrode, platinum wire and glassy carbon cup electrode as reference electrode, counter electrode and working electrode, respectively) was used and connected to an electrochemical workstation. 40 mL of ultrapure water was added to the glassy carbon cup electrode and the voltage was set to 0.5 V. When the current stabilized, 5 mL of Shewanella solution was slowly injected into the glassy carbon cup electrode of the control group, and 5 mL of Shewanella solution and 20 mL of biochar solution were slowly injected into the glassy carbon cup electrode of the experimental group at an injection rate of 25 mL / min. The it curve was obtained. The difference in peak current between the experimental group and the control group was the direct electron transfer capacity of the biochar, and the difference in stable current was the indirect electron transfer capacity of the biochar.

[0041] Figure 3 From the IT curve of Example 3, it can be seen that the peak current of the control group is 0.91 μA and the stable current is 0.05 μA, while the peak current of the experimental group is 1.08 μA and the stable current is 0.14 μA. Therefore, the direct electron transfer capacity of the biochar in this example is 0.17 μA, and the indirect electron transfer capacity is 0.09 μA.

[0042] The above examples also illustrate that biochars obtained at different pyrolysis temperatures have different electron transfer capabilities, providing a selection basis for those skilled in the art to subsequently use biochar to improve the extracellular electron transfer capacity of microorganisms.

Claims

1. A method for detecting the direct and indirect electron transfer capacity of biochar, characterized in that: The specific steps are as follows: (1) Grind and sieve the biochar, then add ultrapure water to prepare a biochar solution; The mesh size of biochar screening is 100~300 mesh; The concentration of biochar solution is 0.5~3g / L; (2) After activating the Shewanella stock solution, inoculate it into sterilized LB medium and culture it in a shaker. After repeated transfers, add ultrapure water to prepare a Shewanella solution. (3) Under anaerobic conditions, a three-electrode system was used, with the Ag / AgCl electrode, platinum wire, and glassy carbon cup electrode as the reference electrode, counter electrode, and working electrode, respectively. The electrodes were connected to an electrochemical workstation, ultrapure water was added to the glassy carbon cup electrode, and the voltage was set. When the current stabilized, the Shewanella solution was injected into the glassy carbon cup electrode of the control group, and the Shewanella solution and biochar solution were injected into the glassy carbon cup electrode of the experimental group to obtain a current-time curve. In the current-time curve, the difference in peak current between the experimental group and the control group was the direct electron transfer capacity of the biochar, and the difference in stable current was the indirect electron transfer capacity of the biochar.

2. The method for detecting the direct and indirect electron transfer capacity of biochar according to claim 1, characterized in that: In step (2), the shaking incubation temperature is 25-35°C, the time is 12-24 hours, the speed is 100-200 rpm, and the number of repeated transfers is 3-5 times.

3. The method for detecting the direct and indirect electron transfer capacity of biochar according to claim 1, characterized in that: Step (2) The concentration of Shewanella solution is 0.3×10 7 ~2.4×10 7 CFU / mL.

4. The method for detecting the direct and indirect electron transfer capacity of biochar according to claim 1, characterized in that: Step (3) Add 20-40 mL of ultrapure water to the glassy carbon cup electrode and set the voltage to 0.2-0.5 V.

5. The method for detecting the direct and indirect electron transfer capabilities of biochar according to claim 1, characterized in that: In step (3), the amount of Shewanella solution added to the glassy carbon electrodes in the control group and the experimental group was the same, and both were 1-5 mL. The amount of biochar solution added to the experimental group was 5-15 mL; the solution injection flow rate was 20-25 mL / min.

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