Method for rapidly screening and enriching electroactive microorganisms
By using the periodic step potential control mode to screen and enrich electric active microorganisms in the H-type fuel cell reactor, the problems of long periods and excessive screening of screening and enrichment processes in the prior art are solved, and the rapid and efficient enrichment of electroactive microorganisms is achieved.
Patent Information
- Application Number
- CN202510199968.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-24
- Publication Date
- 2025-06-13
AI Technical Summary
The prior art has problems of long periods and over-screening when screening and enriching electroactive microorganisms, especially in the range of specific electrode potentials, microorganisms that cannot effectively transmit electrons are difficult to enrich.
The three-electrode system H-type fuel cell reactor is used to screen and enrich electroactive microorganisms through the periodic step electrode potential control mode, and the capacitance effect of the electroactive biofilm is stimulated by alternating high and low electric potentials to promote the growth and electron transfer of electroactive microorganisms.
The rapid and efficient enrichment of electroactive microorganisms is achieved, the growth and metabolism process of target microorganisms is accelerated, the enrichment speed and efficiency are improved, and the problem of over-screening is avoided.
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Figure CN120142418A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the fields of environmental microbial electrochemistry and green environmental bioengineering, and particularly relates to a method for rapidly screening and enriching electroactive microorganisms. Background Art
[0002] Electroactive microorganisms are a class of facultative or obligate anaerobic microorganisms that can transfer electrons extracellularly. They are the key components of microbial electrochemical technologies such as microbial fuel cells, microbial electrochemical sensors, and microbial electrosynthesis, acting as living "catalysts" in these electrochemical devices and having a decisive impact on the efficiency and operating conditions of the system.
[0003] The traditional processes for screening and enriching electroactive microorganisms mainly adopt two modes: the microbial fuel cell control mode without artificial intervention, and the fixed electrode potential mode. In the microbial fuel cell control mode, environmental microorganisms are added to the anodic end of a bipolar chamber separated by an ion exchange membrane or a monopolar chamber electrochemical cell without an ion exchange membrane, and sufficient organic and inorganic nutrients are provided for them, enabling electroactive microorganisms to gradually attach to the anode surface to form a specific biofilm. These microorganisms transfer electrons extracellularly through metabolic activities, and the electrons are finally guided through an external circuit to reach the cathode. The cathode reaction is mostly the reduction of oxygen (from air) or protons, and sometimes also includes the reduction of some consumable oxidants (such as potassium ferricyanide, in the bipolar chamber mode). The electrode potential of the anode fluctuates freely within a certain range due to the metabolic action of the microorganisms, but is limited by the cathode potential and must be more negative than the cathode. In the fixed electrode potential mode, an electrochemical workstation is used to control the anode potential of the electrochemical cell to screen and enrich electroactive microorganisms that can grow preferentially at a specific fixed electrode potential.
[0004] Industrial application of microbial electrochemical technology often requires enriching a relatively diverse electroactive microbial community for subsequent targeted screening and enrichment. However, in the microbial fuel cell mode, the diffusion of oxygen from the cathode to the anode due to the use of an economical aeration method to introduce air into the cathode will disrupt the anaerobic environment of the anode, leading to inefficient growth of electroactive microorganisms. In the fixed electrode potential mode, the pre-selected potential will screen out potential electroactive microorganisms that can survive at higher potentials or those that conduct electricity through mediators, so there is a problem of over-screening. Specifically, in the fixed electrode potential mode, the preset potential may be lower than the apparent potential of the active sites of key extracellular electron transfer enzymes or protein groups in environmental microbial cells. In this case, microorganisms cannot transfer electrons to the electrode, so there is a possibility that electroactive microorganisms cannot be obtained in the system. To sum up, when applying conventional screening and enrichment methods, the main problems faced are: one is that the process cycle is relatively long, and the other is that it is difficult to enrich electroactive microorganisms that cannot effectively transfer electrons within a specific controlled electrode potential range. Summary of the Invention
[0005] The purpose of the present invention is to provide a method for rapidly screening and enriching electroactive microorganisms, which can screen and enrich electroactive microorganisms from environmental microorganisms, accelerate the growth and metabolic process of target microorganisms, and thus achieve rapid and efficient enrichment of electroactive microorganisms.
[0006] To achieve the above purpose, the present invention provides the following technical solutions:
[0007] A method for rapidly screening and enriching electroactive microorganisms, comprising the following steps:
[0008] Step 1: Use a three-electrode system H-type fuel cell reactor. Among them, carbon fiber or carbon felt is selected as the anode, an Ag / AgCl electrode is selected as the reference electrode, and a stainless steel mesh is used as the cathode; the anode chamber and the cathode chamber are separated by an ion exchange membrane.
[0009] Step 2: Add power generation medium to the anode chamber and the cathode chamber and purge with nitrogen for more than 30 minutes. The power generation medium in the cathode chamber does not contain Na 2 Ac, FeCl 2 and Na 2Add inoculum to the anode chamber of S, then connect the electrochemical workstation, set the electrode potential to the periodic step mode, start enriching electroactive microorganisms, and record the magnitude of the current in the electrochemical reactor over time. A positive current indicates that electroactive microorganisms have adhered to the anode; a continuously negative current indicates that there are insufficient electroactive microorganisms in the inoculum or there is over-selection in the electrode potential manipulation, resulting in the inability to enrich electroactive microorganisms; a current that trends from positive to zero or becomes negative indicates insufficient carbon source, and the culture medium needs to be replaced; perform multiple cycles of enrichment culture continuously according to the above process.
[0010] The periodic step mode is to instantaneously change the working potential between high and low potentials within a set time interval, where the high and low potentials respectively correspond to the potentials that can cause electron accumulation and release.
[0011] Furthermore, in step one, the working electrode is pretreated: soak the working electrode in acetone and absolute ethanol for 5 minutes in sequence, and then air-dry it.
[0012] Furthermore, in step one, the ion exchange membrane is pretreated: soak the ion exchange membrane in a 5% NaCl solution for more than half a day, and wash it with deionized water before use.
[0013] Furthermore, in step one, the anode chamber and cathode chamber of the H-type fuel cell reactor are made of high borosilicate glass, with two openings for connecting the reference electrode or the inlet / outlet pipes provided on the opposite side of the flange and sealed with an opening screw cap and a silicone rubber ring. The entire reactor is wrapped with aluminum foil to avoid light and prevent the growth of photosynthetic microorganisms.
[0014] Furthermore, in step two, the formula of the electricity-generating culture medium contains organic matter, nitrogen, sulfur, and trace metal elements required for the growth and reproduction of microorganisms, and has buffering capacity to maintain a neutral pH for the growth and reproduction of microorganisms.
[0015] Furthermore, in step two, the formula of the electricity-generating culture medium includes KH 2 PO 4 2.27 g / L, K 2 HPO 4 ·6H 2 O 7.44 g / L, NH 4 Cl 0.037 g / L, MgCl 2 ·6H 2 O 0.025 g / L, Na 2 Ac 2.05 g / L, trace element stock solution 1 mL / L, FeCl solution with a concentration of 20 mmol / L 2 1 mL / L, and Na solution with a concentration of 77 mmol / L 2 S 1 mL / L.
[0016] Furthermore, the formulation of the trace element stock solution includes 5.00 g / L of EDTA-2Na, 0.50 g / L of ZnCl 2 0.50 g / L of CoCl 2 ·6H 2 O 0.80 g / L of MnCl 2 ·4H 2 O 5.90 g / L of CuSO 4 ·5H 2 O 0.10 g / L of H 3 BO 3 0.10 g / L of NiCl 2 ·6H 2 O 0.20 g / L of Na 2 SeO 3 0.01 g / L of Na 2 MoO 4 ·2H 2 O 0.20 g / L of Na and 2 WO 4 ·2H 2 O 0.10 g / L.
[0017] Furthermore, in the periodic step mode, the high and low electric potentials are selected between -0.4 V and 0.4 V relative to the Ag / AgCl reference electrode, and the duration controlled by each level of electric potential can be arbitrarily selected from several minutes to several hours.
[0018] Furthermore, the duration controlled by each level of electric potential is maintained for an equal length of time, and the value range of this duration is 2 minutes ≤ t ≤ 2 hours.
[0019] Compared with the prior art, the present invention has the following beneficial effects:
[0020] The present invention applies an H-type fuel cell reactor with good sealing performance to avoid air entering the anode, and purges it with nitrogen for more than 30 minutes to create an anaerobic environment at the anode, which is beneficial to the growth and metabolism of electroactive microorganisms. Under the periodic step electrode potential control mode, electroactive microorganisms are screened and enriched from environmental microorganisms. The alternation of high and low electric potentials will stimulate the capacitive effect of the electroactive biofilm, which is beneficial to the synthesis of outer membrane cytochromes, pili, certain redox compounds and biopolymers by electroactive microorganisms, further facilitating the electron transfer between electroactive microorganisms and electrodes, accelerating the growth and metabolism of target microorganisms, and achieving rapid and efficient enrichment.
[0021] Further, in the periodic step mode of the present invention, the high and low electric potentials are selected between -0.4 V and 0.4 V relative to the Ag / AgCl reference electrode. According to the electric potential of the redox center of the biofilm determined by cyclic voltammetry scanning, a potential close to or higher than the redox center of the biofilm is selected. Reasonable electric potential is used to enrich electroactive organisms, and the electric field force is used to actively guide the directional migration of electroactive organisms, while stimulating their metabolic activity, thereby improving the enrichment speed and enabling electroactive organisms to aggregate in a shorter time. The selected high electric potential in the step mode should not be too high. If it is higher than 0.4 V, it may cause microbial oxidative stress, reduce biofilm activity or cause microbial death. The selected low electric potential in the step mode should not be too low either. If it is lower than -0.4 V, 2 NaAc cannot be oxidized, which may cause microorganisms to be unable to transfer electrons to the electrode and ultimately unable to enrich electroactive microorganisms. Description of the Drawings
[0022] Figure 1 Schematic diagram of the current density of potentiostatic (initial low potential) enrichment culture in the soil system;
[0023] Figure 2 Schematic diagram of the current density of potentiostatic (initial high potential) enrichment culture in the soil system;
[0024] Figure 3 Schematic diagram of the current density of enrichment culture under periodic step potential in the soil system;
[0025] Figure 4(a) shows the bacterial community structure of anode microorganisms in the soil system;
[0026] Figure 4(b) shows the archaeal community structure of anode microorganisms in the soil system;
[0027] Figure 5 Schematic diagram of the current density of potentiostatic (initial high potential, 0.2 V relative to the reference) enrichment culture in the activated sludge system;
[0028] Figure 6 Schematic diagram of the current density of enrichment culture under periodic step potential in the activated sludge system;
[0029] Figure 7(a) shows the bacterial community structure of anode microorganisms in the activated sludge system;
[0030] Figure 7(b) shows the archaeal community structure of anode microorganisms in the activated sludge system. Detailed Embodiments
[0031] To make the above objects, features, and advantages of the present invention more obvious and understandable, the present invention will be described in detail below with reference to the drawings and specific embodiments.
[0032] In the following embodiments, the formula of the electricity-producing culture medium includes KH 2PO 4 2.27 g / L, K 2 HPO 4 ·6H 2 O 7.44 g / L, NH 4 Cl 0.04 g / L, MgCl 2 ·6H 2 O 0.025 g / L, Na 2 Ac 2.05 g / L, trace element stock solution 1 mL / L, FeCl solution with a concentration of 20 mmol / L 2 1 mL / L and Na solution with a concentration of 77 mmol / L 2 S 1 mL / L. The formula of the trace element stock solution includes 5.00 g / L of EDTA-2Na, ZnCl 2 0.50 g / L, CoCl 2 ·6H 2 O 0.80 g / L, MnCl 2 ·4H 2 O 5.90 g / L, CuSO 4 ·5H 2 O 0.10 g / L, H 3 BO 3 0.10 g / L, NiCl 2 ·6H 2 O 0.20 g / L, Na 2 SeO 3 0.01 g / L, Na 2 MoO 4 ·2H 2 O 0.20 g / L and Na 2 WO 4 ·2H 2 O 0.10 g / L.
[0033] The formula of the electricity-producing culture medium is not limited to the listed components, but needs to contain organic matter, nitrogen, sulfur, and trace metal elements required for the growth and reproduction of microorganisms, and has buffering capacity to maintain a neutral pH for the growth and reproduction of microorganisms.
[0034] Example 1: Enrichment of electroactive microorganisms in soil
[0035] Obtaining the inoculum: Select the topsoil in an area with normal vegetation cover. First, remove the surface plant sediments, and then dig out the moist soil with a depth of 5 - 10 cm. Add the electricity-producing culture medium to the dug soil to form a mud-water mixture, filter out the coarse mineral particles in the soil with an 80-mesh sieve, and then use a filter membrane with a pore size of 20 μm for suction filtration. The resulting liquid is the inoculum.
[0036] Construct an H-type fuel cell reactor using a three-electrode system. The anode uses a carbon brush made of carbon fiber filaments with a diameter of 3 cm, which is wound with titanium wire on the outside and connected to the working electrode clip of the electrochemical workstation. The cathode uses a stainless steel mesh with dimensions of 12 cm × 9 cm and is connected to the counter electrode clip of the electrochemical workstation. The reference electrode is selected as Ag / AgCl and is connected to the reference electrode clip of the electrochemical workstation.
[0037] Add 280 mL of electricity-producing medium and 1 mL of inoculum to the anode chamber, and add an electricity-producing medium without Na 2 Ac, FeCl 2 and Na 2 S to the cathode chamber, and wrap it with aluminum foil to avoid light and prevent the growth of photosynthetic microorganisms. Connect the electrochemical workstation, set the electrode potential to the periodic step mode, and the electrode potential jumps back and forth between -0.15 V and 0.2 V (relative to the Ag / AgCl reference), and each electrode potential is maintained for 2 min, that is, a sawtooth wave period containing a low potential and a high potential is 4 min, and this cycle continues continuously to start the enrichment culture of electroactive microorganisms. Use the electrochemical workstation to record the current magnitude every 60 s. When the current is greater than 0 A, it indicates successful startup. After entering the period with a positive current, when the average current starts to decrease or drops nearly to 0 A, replace the solution in the anode chamber and continuously run for three cycles for enrichment culture.
[0038] Comparative Example 1: The difference from Example 1 is that the electrode potential is set to the low potential constant potential mode.
[0039] Comparative Example 2: The difference from Example 1 is that the electrode potential is set to the high potential constant potential mode.
[0040] After the startup of Example 1 and Comparative Examples 1 and 2 is successful, record the current density situation during the three cycles of enrichment culture. As Figure 1 shown, in the constant potential mode, if the enrichment culture starts from the low potential initially, the system cannot generate a current for oxidizing organic matter in the positive direction for a long time, that is, electroactive microorganisms cannot be enriched. Only when the electrode potential is adjusted to -0.1 V (relative to the Ag / AgCl reference) can a current be generated. Constant potential enrichment culture requires gradually adjusting the potential according to the electricity production situation until electroactive microorganisms can transfer electrons to the electrode, that is, the system generates a positive current. After the system undergoes further high potential control, a relatively large positive current can be generated at potentials of 0.1 V and 0.2 V. As Figure 2 shown, in the constant potential mode, if the enrichment culture starts from the high potential (0.2 V relative to the Ag / AgCl reference) initially, the system shows a trend consistent with the conventional constant potential enrichment culture, that is, as electroactive microorganisms are enriched, the highest current density in each cycle of the system gradually increases. As Figure 3As shown, the gray block represents the measured values of the periodic step current density fluctuating up and down, and the black line represents the moving average. The highest current density in the second enrichment culture cycle of the system can reach a relatively high level. It can be seen that within several enrichment culture cycles, the highest current density in the periodic step mode far exceeds that in the potentiostatic control mode.
[0041] By comparing Figure 1 , Figure 2 and Figure 3 it can be seen that the highest current densities in the low-potential potentiostatic mode and the high-potential potentiostatic mode are approximately 2 mA / m 2 and 0.5 mA / m 2 respectively, while the highest current density in the periodic step mode is approximately 15 mA / m 2 . Since the current will increase or decrease sharply when the potential changes instantaneously in the periodic step mode (as shown by the gray block in Figure 3 ), the stable value reached after a certain time (as shown by the black line in Figure 3 ) is selected as the highest current density. The current density in the periodic step mode far exceeds that in the potentiostatic mode, indicating that the biofilm activity is higher and the biomass is more in the periodic step mode during enrichment culture. At the same time, the time for the first positive current generation in Example 1 is shorter than that in Comparative Examples 1 and 2, indicating that the periodic step mode can efficiently and rapidly enrich electroactive microorganisms.
[0042] After three cycles of enrichment culture, the reactor was opened in the glove box, and the biofilm on the anode was collected for sequencing. As shown in Figures 4(a) and 4(b), the sequencing results of the anode microorganisms show that the abundances of electroactive bacterial communities in the potentiostatic mode are 42.11% (initial high potential of 0.2 V) and 51.19% (initial low potential) respectively, and the abundances of electroactive archaeal communities are 0.05% (initial high potential of 0.2 V) and 0 (initial low potential). The abundances of electroactive bacterial communities in the step mode are 58.88%, and the abundances of electroactive archaeal communities are 26.23%. The abundances of electroactive bacteria and archaea in the periodic step mode are both greater than those in the two potentiostatic modes.
[0043] If the anodic potential in the low-potential potentiostatic mode is not continuously increased, the first reactor is likely to be discarded without electroactive microorganisms, indicating that there is indeed an over-screening problem in the conventional potentiostatic screening and enrichment mode. For the same inoculum, enrichment culture is initially carried out at a high potential of 0.2 V. The current becomes positive in the first cycle, indicating that electroactive microorganisms can be enriched and cultured in the potentiostatic mode, but a suitable constant electrode potential is required. It is not difficult to see that it is generally difficult to find a suitable electrode potential due to random factors, which is an inherent drawback of the potentiostatic control mode. The microbial community compositions enriched by the two different potentiostatic modes are different. The dominant species of electroactive bacteria enriched by the initial high potential of 0.2 V are Fluviicola_sp. and unclassified_f_PHOS-HE36, while the dominant species of electroactive bacteria enriched by gradually increasing the potential after the initial low potential are Dysgonomonas_hofstadii, unclassified_g_Dysgonomonas, and unclassified_p_Firmicutes. Among the archaeal communities enriched by the three modes, the only electroactive archaea proven so far is unclassified_g_Methanobrevibacter. In addition, the overall culture time of the cyclic step mode is the shortest, indicating that the cyclic step mode indeed has the effect of quickly screening and enriching electroactive microorganisms and has advantages over the potentiostatic mode.
[0044] Example 2: Enrichment of electroactive microorganisms in activated sludge
[0045] Obtaining the inoculum: Activated sludge is taken from the outlet of the activated sludge tank of the sewage treatment plant. Within 2 hours after sampling, it is left to stand and the lower deposited activated sludge is extracted. After being concentrated by centrifugation at 8000 rpm for 5 minutes using a centrifuge, it is moderately dispersed using a culture medium and resuspended for inoculation.
[0046] Construct an H-type fuel cell reactor and adopt a three-electrode system. The anode is a carbon felt (with a thickness of 2 mm), which is connected to the outside of the reactor through a titanium wire and connected to the working electrode clip of the electrochemical workstation; the cathode is a stainless steel mesh (12 cm × 9 cm), which is connected to the counter electrode clip of the electrochemical workstation; Ag / AgCl is used as the reference electrode and is connected to the reference electrode clip.
[0047] Add 280 mL of electricity-generating culture medium and 1 mL of inoculum to the anode chamber, and add no Na 2 Ac, FeCl 2 and Na 2The electricity-producing medium of S is wrapped with aluminum foil to avoid light and prevent the growth of photosynthetic microorganisms. Connect an electrochemical workstation, set the electrode potential to a periodic step mode, and the electrode potential jumps back and forth between -0.3 V and 0.2 V (relative to the Ag / AgCl reference electrode), with each electrode potential maintained for 2 min, that is, a staircase wave cycle including a low potential and a high potential is 4 min, and this cycle continues. Start the enrichment culture of electroactive microorganisms, and use the electrochemical workstation to record the current magnitude every 60 s. When the current is greater than 0 A, it indicates successful startup. After entering the cycle with a positive current, when the current starts to decrease or drops close to 0 A, replace the solution in the anodic chamber and continuously run for three cycles for enrichment culture.
[0048] Comparative Example 3: The difference from Example 2 is that the electrode potential is set to a high potential constant potential mode.
[0049] After successful startup of Example 2 and Comparative Example 3, carry out enrichment culture for three cycles and record the current density situation. As Figure 5 shown, in the constant potential mode, when starting the enrichment culture from the initial high potential (0.2 V relative to the Ag / AgCl reference electrode), the system shows a trend consistent with the conventional constant potential enrichment culture, that is, with the enrichment of electroactive microorganisms, the highest current density in each cycle of the system gradually increases or reaches stability. Figure 6 The gray blocks in the figure are the measured values of the periodic step current density fluctuating up and down, and the black line is the moving average value. With the enrichment culture of electroactive microorganisms, the highest current density in each cycle of the system also gradually increases or reaches stability.
[0050] By comparing Figure 5 and Figure 6 it can be seen that the maximum current densities in the two modes are not much different, both about 1500 mA / m 2 , because in the periodic step mode, when the potential changes instantaneously, it will cause the current to increase or decrease sharply (as shown by the gray blocks in Figure 6 ), so the stable value reached after a certain time (as shown by the black line in Figure 6 ) is selected as the highest current density. And the time difference between the first occurrence of positive current in Example 2 and Comparative Example 3 is not much different.
[0051] After three enrichment cycles, the reactor was opened in a glove box, and the biofilm on the anode was collected for sequencing. As shown in Figures 7(a) and 7(b), the abundance of electroactive bacteria in the periodic step mode was 57.73%, and the abundance of electroactive archaea was 96.68%. The abundance of electroactive bacteria in the potentiostatic mode was 52.01%, and the abundance of electroactive archaea was 91%. The dominant electroactive bacteria in both modes were unclassified_o_Sphingobacteriales and unclassified_g_Petrimonas. The electroactive bacterial species Geobacter_anodireducens also existed in the periodic step mode, indicating that more electroactive bacteria could be enriched simultaneously in the periodic step mode than in the potentiostatic mode. The dominant electroactive archaea in both modes were unclassified_g_Methanobacterium and unclassified_g_Methanobrevibacter. It can be seen that although the start-up time and maximum current density of the two modes were not much different, the time to reach a steady state in the periodic step mode was shortened by nearly 5 days compared with the potentiostatic mode. Considering that the general doubling time of electroactive bacteria was 5 - 8 hours, the periodic step mode had an obvious advantage over the potentiostatic mode. In addition, the abundance of electroactive microbial bacteria and archaea enriched in the periodic step mode was greater than that in the potentiostatic mode, and the electroactive microbial community enriched was more diverse, indicating that the periodic step mode could efficiently enrich electroactive microorganisms and had an advantage over the potentiostatic mode.
[0052] Example 3: The difference from Example 2 was that the electrode potential was set to the periodic step mode, and the anode potential of the system was controlled to jump between -0.4V and 0.4V (both relative to the Ag / AgCl reference electrode), and each potential was maintained for 2 hours.
[0053] Example 4: The difference from Example 2 was that the electrode potential was set to the periodic step mode, and the anode potential of the system was controlled to jump between -0.2V and 0.3V (both relative to the Ag / AgCl reference electrode), and each potential was maintained for 30 minutes.
[0054] Example 5: The difference from Example 2 was that the electrode potential was set to the periodic step mode, and the anode potential of the system was controlled to jump between -0.1V and 0.25V (both relative to the Ag / AgCl reference electrode), and each potential was maintained for 10 minutes.
[0055] Example 6: The difference from Example 2 was that the electrode potential was set to the periodic step mode, and the anode potential of the system was controlled to jump between -0.35V and 0.1V (both relative to the Ag / AgCl reference electrode), and each potential was maintained for 45 minutes.
Claims
1. A method for rapid screening and enrichment of electroactive microorganisms, characterized in that: The following steps are involved: Step 1: A three-electrode system H-type fuel cell reactor is used, wherein carbon fiber or carbon felt is selected as the anode, Ag / AgCl electrode is selected as the reference electrode, and a stainless steel mesh is used as the cathode; the anode chamber and the cathode chamber are separated by an ion exchange membrane; Step 2: Add electrogenic culture medium to the anode chamber and cathode chamber and purge with nitrogen for more than 30 minutes, wherein the electrogenic culture medium in the cathode chamber does not contain Na2Ac, FeCl2 and Na2S, and add inoculum to the anode chamber, then connect the electrochemical workstation, set the electrode potential to the periodic step mode, start enriching and culturing electroactive microorganisms, and record the current size of the electrochemical reactor over time. If the current is positive, it indicates that electroactive microorganisms have attached to the anode; if the current is continuously negative, it indicates that there is a lack of electroactive microorganisms in the inoculum or the electrode potential manipulation is over-screened and cannot enrich electroactive microorganisms; If the current changes from positive to zero or turns negative, it indicates that the carbon source is insufficient and the culture medium needs to be replaced. Multiple cycles of enrichment culture are carried out continuously according to the above process. The periodic step mode is to instantly change the working potential between high and low potentials within a set time interval, and the high and low potentials correspond to the potentials that can cause electron accumulation and release respectively.
2. A method for rapid screening and enrichment of electroactive microorganisms according to claim 1, characterized in that: The working electrode described in step 1 is pretreated by soaking the working electrode in acetone and anhydrous ethanol for 5 minutes respectively and then drying it.
3. A method for rapid screening and enrichment of electroactive microorganisms according to claim 1, characterized in that: In step 1, the ion exchange membrane is pretreated by soaking the ion exchange membrane in a 5% NaCl solution for more than half a day and washing it with deionized water before use.
4. A method for rapid screening and enrichment of electroactive microorganisms according to claim 1, characterized in that: The anode chamber and cathode chamber of the H-type fuel cell reactor described in step 1 are made of high borosilicate glass. Two openings for connecting a reference electrode or an air / liquid inlet pipe are provided on the opposite side of the flange and are sealed by an open screw cover and a silicone ring. The entire reactor is wrapped with aluminum foil to avoid light and prevent the growth of photosynthetic microorganisms.
5. A method for rapid screening and enrichment of electroactive microorganisms according to claim 1, characterized in that: The formula of the electrogenic medium in step 2 contains organic matter, nitrogen, sulfur and trace metal elements required for the growth and reproduction of microorganisms, and has buffering capacity and can maintain a neutral pH to facilitate the growth and reproduction of microorganisms.
6. A method for rapid screening and enrichment of electroactive microorganisms according to claim 5, characterized in that: The formula of the electrogenic medium described in step 2 includes KH2PO4 2.27g / L, K2HPO4·6H2O 7.44g / L, NH4Cl 0.037g / L, MgCl2·6H2O 0.025g / L, Na2Ac 2.05g / L, trace element stock solution 1mL / L, 20mmol / L FeCl2 solution 1mL / L and 77mmol / L Na2S solution 1mL / L.
7. A method for rapid screening and enrichment of electroactive microorganisms according to claim 6, characterized in that: The formula of the trace element stock solution includes 5.00 g / L of EDTA-2Na, 0.50 g / L of ZnCl2, 0.80 g / L of CoCl2·6H2O, 5.90 g / L of MnCl2·4H2O, 0.10 g / L of CuSO4·5H2O, 0.10 g / L of H3BO3, 0.20 g / L of NiCl2·6H2O, 0.01 g / L of Na2SeO3, 0.20 g / L of Na2MoO4·2H2O and 0.10 g / L of Na2WO4·2H2O.
8. A method for rapid screening and enrichment of electroactive microorganisms according to claim 1, characterized in that: In the periodic step mode, the high and low potentials are selected between -0.4 V and 0.4 V relative to the Ag / AgCl reference electrode, and the duration of each level of potential control is optionally selected from several minutes to several hours.
9. A method for rapid screening and enrichment of electroactive microorganisms according to claim 8, characterized in that: The duration of each level of potential control is maintained for a constant time, and the value range of the duration is 2 minutes ≤ t ≤ 2 hours.