Method and device for rapid enrichment culture of salt-tolerant electroactive HNAD target bacteria

Abstract

The invention discloses a method and a device for rapid enrichment culture of salt-tolerant electroactive HNAD target bacteria, and aims to solve the problems of low enrichment efficiency, long screening period and difficulty in pure bacteria separation of salt-tolerant HNAD florae in the prior art. The method is used for rapidly enriching and separating the salt-tolerant electroactive HNAD strain, so that the denitrification performance and the application potential of the strain in a high-salt environment are improved.

Description

Technical Field

[0001] This invention relates to the fields of environmental engineering and bioelectrochemical technology, specifically to a method and apparatus for the rapid enrichment and culture of salt-tolerant electroactive HNAD target bacteria. Background Technology

[0002] In recent years, industrial wastewater and aquaculture wastewater often exhibit high salinity and high nitrogen loads. Traditional biological nitrogen removal processes are frequently constrained by reduced microbial activity and decreased nitrogen removal efficiency under high salinity conditions. Heterotrophic nitrification-aerobic denitrification (HNAD) processes have attracted widespread attention due to their simplified reaction pathway and the ability to simultaneously remove ammonia and nitrate nitrogen. However, in high-salt environments, the low enrichment efficiency of HNAD bacterial communities and the difficulty in isolating pure strains limit their engineering applications.

[0003] Bioelectrochemical systems (BESs) are used for the enrichment and screening of denitrifying bacteria because they can provide external electron donors / acceptors to promote specific metabolic pathways. Among them, single-chamber microbial electrolyzers (SCMECs) have the advantages of low resistance and high electron transfer efficiency compared to two-chamber microbial electrolyzers (TCMECs), making them suitable for the enrichment of electroactive bacteria. However, most existing technologies focus on electroactive denitrifying bacteria under ambient temperature and low salinity conditions, and there is a lack of technical methods for the rapid and effective enrichment of salt-tolerant electroactive HNAD bacteria under high salinity environments.

[0004] CN116332324B discloses a single-chamber, diaphragm-free electrochemically enhanced aerobic microbial wastewater electrolysis device, which has the advantage of reducing internal resistance and improving treatment efficiency. However, it is mainly aimed at the removal of organic matter, rather than the enrichment and separation of high-salt-tolerant electroactive denitrifying bacteria.

[0005] CN110697877B discloses a bioelectrolysis method for denitrification and methanogenesis in wastewater with a low C / N ratio. It constructs a bioanode through potential control to achieve a coupled reaction between organic carbon and ammonia nitrogen. Although related to electrochemical biological processes, it does not address the enrichment and screening of microbial communities under high-salt environments.

[0006] CN211339214U: A microbial electrolysis cell-membrane bioreactor composite device for landfill leachate treatment. Although the device is also an electrochemical biological system, its structure is complex. It combines membrane treatment but does not enrich or denitrify high-salt-tolerant bacteria.

[0007] CN101710626A discloses a single-chamber MFC structure and its application in wastewater treatment, emphasizing its advantages of simple structure and low energy consumption, making it suitable for power generation and wastewater treatment. However, this technology is mainly used for degrading organic pollutants in wastewater and energy recovery, and does not involve the enrichment and denitrification process of salt-tolerant electroactive bacteria in high-salt environments. Summary of the Invention

[0008] In view of the above-mentioned shortcomings of the existing technology, the present invention aims to provide a method and apparatus for rapid enrichment and culture of salt-tolerant electroactive HNAD target bacteria, so as to solve the problems of low enrichment efficiency, long screening cycle and difficulty in pure bacterial isolation of salt-tolerant HNAD bacterial groups in the existing technology.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for rapid enrichment culture of salt-tolerant electroactive HNAD target bacteria, the specific steps of which are as follows: Step 1: Rapid enrichment is performed using a single-chamber microbial electrolysis device. Activated sludge containing HNAD bacteria is added to the reaction chamber of the device, along with electrolyte; the concentration of NaCl in the electrolyte is at least 3 wt% (by mass percentage). Step 2: Start the electrochemical workstation using the potential control method. The cathode potential is -0.5V to 1.0V, the initial C / N ratio is set to 4, and aeration is performed. The aeration intensity is 15mL / min to 25mL / min, and each cycle is 6 days. After each cycle, the electrolyte in the reaction chamber is replaced. Step 3: After the denitrification rate stabilizes, adjust the C / N value to 2, keep other parameters unchanged, and continue running for multiple cycles; after the denitrification rate stabilizes again, adjust the C / N value to 0, keep other parameters unchanged, and continue running for multiple cycles until the denitrification rate stabilizes again, then the target bacterial community has been enriched. Step 4: Remove the electrode biofilm from the reaction chamber and prepare it into a bacterial suspension. Perform serial dilutions on the bacterial suspension, and spread the suspensions obtained from each serial dilution on LB medium for culture and purification to obtain the initial screening strain. Step 5: Inoculate the initial screening strain obtained in Step 4 into LB medium for expansion culture. When the absorbance of the bacterial solution at a wavelength of 600 nm reaches 0.5, transfer the bacterial solution into a centrifuge tube, add mineral salt medium, WO3 nanoparticles and sterile paraffin oil, and continue to culture at a constant temperature of 25±5℃ and record the color development. Step 6: When the bacterial culture from Step 5 turns blue, inoculate the bacterial culture into LB medium for further expansion. When the absorbance of the bacterial culture at a wavelength of 600 nm reaches 0.6, perform low-speed freeze centrifugation to obtain bacterial cells. Subsequently, inoculate the bacterial cells into sterilized nitrification medium, denitrification medium, and simultaneous nitrification-denitrification medium, respectively, and culture them at a constant temperature of 25℃ and 200 r / min to obtain the salt-tolerant electroactive HNAD target bacteria.

[0010] Preferably, in step 1, the electrolyte comprises the following components: anhydrous sodium acetate 2-3 g / L, NH4Cl 0.5-1.0 g / L, NaCl 10-15 g / L, K2HPO4·3H2O 1-2 g / L, KH2PO4 0.2-0.5 g / L, trace element solution 1-2 ml / L, vitamin solution 1-2 ml / L; and water as the solvent.

[0011] Preferably, the trace element solution comprises the following components: 1-2 g / L nitroglycerin, 2-4 g / L magnesium sulfate, 0.1-1.0 g / L manganese sulfate, 1.0-2.0 g / L sodium chloride, 0.1-0.5 g / L ferrous sulfate heptahydrate, 0.01-0.05 g / L copper sulfate pentahydrate, 0.01-0.05 g / L potassium aluminum sulfate dodecahydrate, 0.1-0.5 g / L cobalt chloride hexahydrate, 0.1-0.5 g / L calcium chloride dihydrate, 0.1-0.5 g / L zinc chloride, 0.01-0.05 g / L boric acid, 0.02-0.05 g / L sodium molybdate, 0.02-0.05 g / L nickel chloride hexahydrate, and 0.02-0.05 g / L sodium tungstate dihydrate; the solvent is water.

[0012] Preferably, the vitamin solution comprises the following components: biotin 0.1-0.5 g / L, vitamin B6 1-2 g / L, vitamin B12 0.1-0.5 g / L, folic acid 0.1-0.5 g / L, pantothenic acid 0.5-1.0 g / L, nicotinic acid 0.5-1.0 g / L, lipoic acid 0.5-1.0 g / L, P-aminobenzoic acid 0.5-1.0 g / L, thiamine 0.5-1.0 g / L, and riboflavin 0.5-1.0 g / L; the solvent is water.

[0013] Preferably, in step 4, the step of preparing the electrode biofilm into a bacterial suspension is as follows: The removed electrode biofilm is placed under sterile conditions and gently rinsed with sterile physiological saline (0.85 wt% NaCl solution) or sterile phosphate buffered saline (PBS, pH 7.2-7.4) to remove loose impurities from the biofilm surface without damaging it. Subsequently, the electrode biofilm is cut into small pieces (approximately 0.5 cm × 0.5 cm), placed in sterile centrifuge tubes, and 5-10 times the electrode volume of sterile buffer is added. The biofilm is then fully detached using ultrasonic dispersion (80-120 W power, 3-5 s sonication, 2 s interval, repeated 5-10 times) or vortexing (2-3 min) to form a homogeneous bacterial suspension. The resulting suspension is allowed to stand for 1-2 min to allow large particles to settle; the supernatant is the bacterial suspension, which can be used for subsequent serial dilution and plate coating operations. Then, the bacterial suspension was serially diluted and plated. The LB medium consisted of 30 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract, and 20 g / L agar. The bacterial suspension was diluted using a 1 mL bacterial suspension + 9 mL diluent ratio, resulting in a total dilution of 10 times, denoted as 10⁻⁶. -1 Then, take 1 mL of this diluted solution and add 10 mL of diluent to further dilute it, recording the result as 10 mL. -2 Following this logic, we can obtain 10. -3 10 -4 10 -5 10 -6 The suspension resulting from gradient dilution.

[0014] Preferably, in step 5, the amount of mineral salt culture medium added is 1-3 ml / L; the amount of WO3 nanoparticles added is 1%-5% based on the total mass of LB culture medium, preferably 3%; and the amount of sterile paraffin oil added is 0.5-1.5 ml / L.

[0015] Preferably, in step 6, the nitrification medium comprises the following components: (NH4)2SO4 0.5-1.0 g / L, C6H5Na3O7·2H2O 12-15 g / L, NaH2PO4 0.2-0.5 g / L, K2HPO4·3H2O 0.5-1.0 g / L, MgSO4·7H2O 0.05-0.1 g / L, MnSO4·H2O 0.01-0.05 g / L, FeSO4·7H2O 0.01-0.05 g / L, NaCl 30-50 g / L, and pH 7.0-7.5; the solvent is water.

[0016] Preferably, in step 6, the denitrification medium comprises the following components: (NH4)2SO4 0.5-1.0 g / L, C6H5Na3O7·2H2O 12-15 g / L, NaH2PO4 0.2-0.5 g / L, K2HPO4·3H2O 0.5-1.0 g / L, MgSO4·7H2O 0.05-0.1 g / L, MnSO4·H2O 0.01-0.05 g / L, FeSO4·7H2O 0.01-0.05 g / L, NaCl 30-50 g / L, NaNO3 (or NaNO2) 0.5-1.0 g / L (or NaNO2 0.4-0.5 g / L), and pH 7.0-7.5; the solvent is water.

[0017] Preferably, in step 6, the components of the simultaneous nitrification-denitrification medium are as follows: CH3COONa 3-5 g / L, Na2HPO4 4-5 g / L, KH2PO4 1-2 g / L, MgSO4·7H2O 0.1-0.5 g / L, trace element solution 1-5 mL, NH4Cl 0.1-0.5 g / L, KNO3 0.1-0.5 g / L, NaCl 30-50 g / L, pH 7.0-7.5; the solvent is water.

[0018] Secondly, the present invention provides a single-chamber microbial electrolysis device for implementing the rapid enrichment process in the above-described method. The device includes a reaction chamber with a closed cavity structure. A grid plate is horizontally arranged within the reaction chamber, with its edges fixedly connected to the inner wall of the chamber. The grid plate divides the cavity within the reaction chamber into a cathode region and an anode region. Liquids in the cathode region and the anode region can flow between each other through the grid plate, but solid substances in the two regions cannot move through it. Activated carbon particles are filled in the anode region. One end of an anode conductive carbon brush passes through the side wall of the reaction chamber and is inserted into the activated carbon particles; the other end is connected to an electrochemical workstation via a wire. A reference electrode and a working electrode are provided in the cathode region. One end of the reference electrode passes through the side wall of the reaction chamber and extends into the liquid in the cathode region, while the other end of the reference electrode is connected to the circuit of the electrochemical workstation. A cathode conductive carbon brush is used as the working electrode. One end of the cathode conductive carbon brush passes through the side wall of the reaction chamber and extends into the liquid in the cathode region, while the other end of the cathode conductive carbon brush is connected to the circuit of the electrochemical workstation. The cathode conductive carbon brush, anode conductive carbon brush, and reference electrode are all connected to the electrochemical workstation via wires. At least one aeration head is also provided in the cathode region. The aeration head is connected to an aeration device via an aeration pipe, and the aeration device can aerate the liquid in the cathode region through the aeration head. A water outlet is provided in the anode region to discharge the liquid in the anode region. An overflow outlet is provided in the cathode region to allow the liquid in the cathode region to flow out when it rises to a certain height.

[0019] Preferably, the activated carbon particles have a particle size of 2mm-6mm.

[0020] Preferably, the reference electrode is an Ag / AgCl electrode.

[0021] Preferably, it also includes a gas flow meter for detecting the gas flow rate in the aeration pipe.

[0022] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention employs a single-chamber microbial electrolysis cell structure, eliminating the resistive interference caused by the ion exchange membrane in a dual-chamber system and significantly reducing charge transfer impedance. Simultaneously, the activated carbon particles filled in the anode region possess a high specific surface area, providing ample attachment sites for electroactive microorganisms and accelerating the electron transfer process. Furthermore, combined with external constant potential regulation, it enhances extracellular electron transfer in electroactive microorganisms, avoiding the competitive consumption of electron donors in traditional biological denitrification, and selectively inhibits the growth of non-target bacterial communities under high-salt conditions, selectively enriching the target bacterial community. Finally, the acclimatization method of gradient reduction of the C / N ratio and a fixed water exchange cycle significantly shortens the time required for the target bacterial community to reach stable denitrification efficiency, resulting in a significantly faster acclimatization speed compared to traditional enrichment methods.

[0023] 2. In the process of pure bacterial isolation, this invention utilizes the colorimetric reaction of WO3 nanoparticles to achieve rapid initial screening. After the bacterial culture reaches a suitable state, a culture medium containing WO3 nanoparticles is added, allowing for the identification of electroactive strains through color changes within a short time, significantly improving screening efficiency compared to traditional electrochemical detection methods. Then, the initially screened strains are inoculated onto various specialized culture media, and their nitrification, denitrification, and simultaneous nitrification-denitrification capabilities are verified by detecting nitrogen metabolism indicators, avoiding the limitations of traditional streak plating methods that rely solely on colony morphology. Simultaneously, the colorimetric positive strains are repeatedly streaked until the colony morphology is uniform. Combined with functional verification and purity testing, this ensures the final acquisition of pure cultured salt-tolerant electroactive HNAD target strains, making the screening process both highly efficient and accurate.

[0024] 3. The target strain obtained by the method described in this invention can still maintain high nitrification and denitrification activities in a high-salt environment, breaking through the bottleneck of efficiency decline of conventional HNAD bacteria under salt stress. The screened strains adapt to high-salt conditions through their own metabolic regulation, including synthesizing osmotic regulators to maintain intracellular osmotic pressure balance, optimizing cell membrane structure to improve stability and reduce salt ion damage, and regulating the conformation of the active site of key denitrifying enzymes to ensure stable enzyme activity. In addition, during the enrichment process, the C / N ratio is gradually reduced to simulate the carbon-scarce environment of actual high-salt wastewater, further enhancing the adaptability of the bacterial community to high-salt conditions, so that the target strains can still stably perform denitrification function in complex high-salt scenarios.

[0025] 4. This invention provides feasible microbial resources and an electrochemical process basis for the treatment of high-salt and nitrogen-polluted wastewater, with promising engineering application prospects. In practical treatment, the method described in this invention can be coupled with existing wastewater treatment units to form an integrated system, meeting the compliant treatment requirements of different high-salt and nitrogen-polluted wastewater. Simultaneously, the method described in this invention features low aeration intensity during operation, reducing aeration energy consumption, and the electrode materials used are cost-effective and have a long service life, significantly reducing electrode investment costs. The high stability of the target strains also reduces sludge production and the maintenance costs of periodically adding microbial strains. Furthermore, the method described in this invention can simultaneously achieve energy recovery during denitrification, reducing the use of chemical agents, lowering carbon emissions, and balancing treatment effectiveness, economic cost, and environmental benefits, thus possessing significant engineering promotion value. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the structure of the single-chamber microbial electrolysis device described in this invention.

[0027] Figure 2 This shows the changes in ammonia nitrogen removal rate of the target strain screened by the method described in this invention under different C / N ratios.

[0028] Figure 3The changes in nitrite, nitrate, and OD600 over time for the target strains screened by the method described in this invention under different C / N ratios.

[0029] Figure 4 The changes in nitrate and nitrite over time are shown when the target strain screened by the method described in this invention is placed on the working electrode in a three-electrode system and a voltage of -0.5V is applied using a potentiostat.

[0030] In the diagram: 1. Reaction chamber; 2. Grating plate; 3. Cathode area; 4. Anode area; 5. Anode conductive carbon brush; 6. Electrochemical workstation; 7. Reference electrode; 8. Working electrode; 9. Aeration head; 10. Aeration pipe; 11. Aeration device; 12. Water outlet; 13. Overflow outlet; 14. Gas flow meter. Detailed Implementation

[0031] The technical solutions of the present invention will be clearly and completely described in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the present invention are within the scope of protection of the present invention.

[0032] Unless otherwise specified in the specific circumstances, the numerical ranges listed herein include upper and lower limits, as well as all integers and fractions within that range, but are not limited to the specific values ​​listed when the range is defined.

[0033] I. A method for rapid enrichment and culture of salt-tolerant, electroactive HNAD target bacteria Step 1: Rapid enrichment is performed using a single-chamber microbial electrolysis device. Activated sludge containing HNAD bacteria is added to the reaction chamber of the device, along with electrolyte; the concentration of NaCl in the electrolyte is at least 3 wt% (by mass percentage). Step 2: Start the electrochemical workstation using the potential control method, with the cathode potential ranging from -0.5V to 1.0V and the initial C / N ratio set to 4, and perform aeration treatment; the aeration intensity is 15mL / min-25mL / min, with each cycle lasting 6 days, and the electrolyte in the reaction chamber is replaced after each cycle; Step 3: After the denitrification rate stabilizes, adjust the C / N value to 2, keep other parameters unchanged, and continue running for multiple cycles; after the denitrification rate stabilizes again, adjust the C / N value to 0, keep other parameters unchanged, and continue running for multiple cycles until the denitrification rate stabilizes again, then the target bacterial community has been enriched. Step 4: Remove the electrode biofilm from the reaction chamber and prepare it into a bacterial suspension. Perform serial dilutions on the bacterial suspension, and spread the suspensions obtained from each serial dilution on LB medium for culture and purification to obtain the initial screening strain. Step 5: Inoculate the initial screening strain obtained in Step 4 into LB medium for expansion culture. When the absorbance of the bacterial solution at a wavelength of 600 nm reaches 0.5, transfer the bacterial solution into a centrifuge tube, add mineral salt medium, WO3 nanoparticles and sterile paraffin oil, and continue to culture at a constant temperature of 25±5℃ and record the color development. Step 6: When the bacterial culture from Step 5 turns blue, inoculate the bacterial culture into LB medium for further expansion. When the absorbance of the bacterial culture at a wavelength of 600 nm reaches 0.6, perform low-speed freeze centrifugation to obtain bacterial cells. Subsequently, inoculate the bacterial cells into sterilized nitrification medium, denitrification medium, and simultaneous nitrification-denitrification medium, respectively, and culture them at a constant temperature of 25℃ and 200 r / min to obtain the salt-tolerant electroactive HNAD target bacteria.

[0034] Existing technologies face several limitations in the enrichment and denitrification of salt-tolerant electroactive bacterial communities under high-salt conditions. First, the enrichment efficiency of salt-tolerant electroactive bacterial communities under high-salt conditions is extremely low. While halophilic bacteria exhibit some activity in high-salt MFCs, there is still a lack of mature and effective methods for the rapid enrichment of HNAD bacteria. This is mainly because HNAD bacteria grow slowly in high-salt environments, have low electrode adhesion efficiency, and exhibit weak electroactivity, making it difficult to achieve large-scale enrichment in a short period. Second, existing technologies generally lack purification and screening mechanisms specifically for HNAD bacteria. Most studies focus on the electrochemical process itself, paying little attention to how to efficiently isolate and screen target electroactive strains from complex bacterial communities, and lacking simple and effective colorimetric or screening mechanisms, making the acquisition of target strains extremely difficult. Furthermore, although heterotrophic denitrification is highly efficient, it relies on large amounts of chemical carbon sources, which not only increases operating costs but also introduces potential secondary pollution risks. Furthermore, there is currently a lack of specific enrichment methods for salt-tolerant electroactive HNAD bacteria. Although single-chamber MEC denitrification research and electrochemical denitrification and methanogenesis technologies exist, a complete enrichment culture and screening protocol suitable for salt-tolerant electroactive heterotrophic nitrifying-aerobic denitrifying bacteria under high-salt conditions has not yet been established. At the same time, the screening and purification of high-salt-tolerant bacteria still rely on traditional methods, lacking rapid identification techniques such as colorimetric screening, resulting in a cumbersome and inefficient screening process.

[0035] Addressing the numerous challenges of existing technologies in treating high-salt, high-nitrogen wastewater, this invention ingeniously combines a single-chamber microbial electrolysis cell with electrochemical control methods to achieve rapid enrichment and efficient separation of salt-tolerant, electroactive HNAD strains. Through precise potential control and C / N gradient adjustment, this invention achieves a dual improvement in enrichment efficiency and salt-tolerant denitrification capacity: potential control can regulate the metabolic pathways of microorganisms as needed, promoting the growth and reproduction of salt-tolerant, electroactive HNAD bacteria; while C / N gradient adjustment provides a suitable nutrient environment for the microorganisms, optimizing the carbon-nitrogen ratio to meet their nutrient needs at different growth stages, further enhancing their activity and salt tolerance; simultaneously, in a high-salt environment, by rationally adjusting the potential and C / N gradient, salt-tolerant, electroactive HNAD bacteria can be rapidly enriched, significantly improving denitrification efficiency. Furthermore, this invention introduces WO3 nanocolorization technology. When WO3 nanomaterials interact with electroactive strains, a unique colorimetric reaction occurs. This reaction enables researchers to quickly and accurately identify target strains. Compared with traditional screening methods, this WO3 nanocolorization-based screening mechanism has higher sensitivity and accuracy, greatly shortens the screening cycle, and improves screening efficiency, providing strong technical support for the rapid acquisition of salt-tolerant electroactive HNAD strains.

[0036] The method and device described in this invention have achieved unexpected technical results in the enrichment and separation of salt-tolerant electroactive HNAD bacteria. Regarding the enrichment efficiency of salt-tolerant electroactive HNAD bacteria, this invention increases the number of enriched bacteria several times compared to traditional methods within the same timeframe. This significant improvement enables the faster establishment of highly efficient denitrifying microbial communities in high-salt environments, laying a solid foundation for subsequent denitrification processes. In actual high-salt wastewater treatment simulation experiments, the concentration of salt-tolerant electroactive HNAD bacteria in the wastewater treated by the device of this invention reached several times that of traditional methods, greatly improving the enrichment efficiency of the bacteria and enhancing the system's denitrification capacity. In terms of pure bacterial isolation, the WO3 nanocolorimetric screening mechanism introduced in this invention plays a crucial role. Previous pure bacterial isolation processes were often time-consuming and laborious, requiring multiple tedious cultivation and detection steps. However, the screening mechanism of this invention can quickly and accurately identify target electroactive strains, shortening the pure bacterial isolation time from several weeks in traditional methods to several days. Using WO3 nanocolorization technology, researchers can directly observe the colorimetric reaction produced by the interaction between electroactive bacterial strains and nanomaterials, thereby rapidly screening HNAD strains with high electroactivity and salt tolerance. This innovative mechanism not only improves screening efficiency but also significantly reduces screening costs, making the pure bacterial isolation process more efficient and convenient. From a practical application perspective, this invention provides a highly promising new approach for the treatment of high-salt, high-nitrogen polluted wastewater. High-salt, high-nitrogen polluted wastewater poses a significant threat to the ecological environment, and the limitations of traditional treatment technologies have made its treatment a major challenge in the environmental protection field. The method and apparatus described in this invention can effectively enrich and separate salt-tolerant, electroactive HNAD bacterial communities, achieving efficient denitrification treatment of these wastewaters and reducing environmental pollution. Furthermore, this invention can also optimize process parameters to achieve purification treatment of these wastewaters based on their high salt and high nitrogen characteristics.

[0037] In some embodiments of the present invention, in step 1, the electrolyte comprises the following components: anhydrous sodium acetate 2-3 g / L, NH4Cl 0.5-1.0 g / L, NaCl 10-15 g / L, K2HPO4·3H2O 1-2 g / L, KH2PO4 0.2-0.5 g / L, trace element solution 1-2 ml / L, and vitamin solution 1-2 ml / L; the solvent is water. More preferably, the electrolyte comprises the following components: anhydrous sodium acetate 2.5641 g / L, NH4Cl 0.7641 g / L, NaCl 10 g / L, K2HPO4·3H2O 1.9171 g / L, KH2PO4 0.2177 g / L, trace element solution 1 ml / L, and vitamin solution 1 ml / L; the remainder is water.

[0038] In some embodiments of the present invention, the components of the trace element solution are as follows: 1-2 g / L of nitroglycerin, 2-4 g / L of magnesium sulfate, 0.1-1.0 g / L of manganese sulfate, 1.0-2.0 g / L of sodium chloride, 0.1-0.5 g / L of ferrous sulfate heptahydrate, 0.01-0.05 g / L of copper sulfate pentahydrate, 0.01-0.05 g / L of potassium aluminum sulfate dodecahydrate, 0.1-0.5 g / L of cobalt chloride hexahydrate, 0.1-0.5 g / L of calcium chloride dihydrate, 0.1-0.5 g / L of zinc chloride, 0.01-0.05 g / L of boric acid, 0.02-0.05 g / L of sodium molybdate, 0.02-0.05 g / L of nickel chloride hexahydrate, and 0.02-0.05 g / L of sodium tungstate dihydrate; the solvent is water. More preferably, the trace element solution comprises the following components: 1.5 g / L nitroglycerin, 3.0 g / L magnesium sulfate, 0.5 g / L manganese sulfate, 1.0 g / L sodium chloride, 0.1 g / L ferrous sulfate heptahydrate, 0.01 g / L copper sulfate pentahydrate, 0.01 g / L potassium aluminum sulfate dodecahydrate, 0.1 g / L cobalt chloride hexahydrate, 0.1 g / L calcium chloride dihydrate, 0.13 g / L zinc chloride, 0.01 g / L boric acid, 0.025 g / L sodium molybdate, 0.024 g / L nickel chloride hexahydrate, and 0.025 g / L sodium tungstate dihydrate; the solvent is water.

[0039] In some embodiments of the present invention, the vitamin solution comprises the following components: biotin 0.1-0.5 g / L, vitamin B6 1-2 g / L, vitamin B12 0.1-0.5 g / L, folic acid 0.1-0.5 g / L, pantothenic acid 0.5-1.0 g / L, nicotinic acid 0.5-1.0 g / L, lipoic acid 0.5-1.0 g / L, P-aminobenzoic acid 0.5-1.0 g / L, thiamine 0.5-1.0 g / L, and riboflavin 0.5-1.0 g / L; the solvent is water. More preferably, the vitamin solution comprises the following components: biotin 0.2 g / L, vitamin B6 1.00 g / L, vitamin B12 0.1 g / L, folic acid 0.2 g / L, pantothenic acid 0.5 g / L, nicotinic acid 0.5 g / L, lipoic acid 0.5 g / L, P-aminobenzoic acid 0.5 g / L, thiamine 0.5 g / L, and riboflavin 0.5 g / L; the solvent is water.

[0040] In some embodiments of the present invention, step 4, the step of preparing the electrode biofilm into a bacterial suspension, is as follows: The removed electrode biofilm is placed under sterile conditions and gently rinsed with sterile physiological saline (0.85 wt% NaCl solution) or sterile phosphate buffer (PBS, pH 7.2-7.4) to remove loose impurities from the biofilm surface without damaging it. Subsequently, the electrode biofilm is cut into small pieces (approximately 0.5 cm × 0.5 cm), placed in sterile centrifuge tubes, and 5-10 times the electrode volume of sterile buffer is added. The biofilm is then fully detached using ultrasonic dispersion (80-120 W power, 3-5 s sonication, 2 s interval, repeated 5-10 times) or vortexing (2-3 min) to form a uniform bacterial suspension. The resulting suspension is allowed to stand for 1-2 min to allow large particles to settle; the supernatant is the bacterial suspension, which can be used for subsequent serial dilution and plate coating operations. Then, the bacterial suspension was serially diluted and plated. The LB medium consisted of 30 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract, and 20 g / L agar. The bacterial suspension was diluted using a 1 mL bacterial suspension + 9 mL diluent ratio, resulting in a total dilution of 10 times, denoted as 10⁻⁶. -1 Then, take 1 mL of this diluted solution and add 10 mL of diluent to further dilute it, recording the result as 10 mL. -2 Following this logic, we can obtain 10. -3 10 -4 10 -5 10 -6 The suspension resulting from gradient dilution.

[0041] In some embodiments of the present invention, in step 5, the amount of mineral salt culture medium added is 1-3 ml / L; the amount of WO3 nanoparticles added is 1%-5% based on the total mass of LB culture medium, preferably 3%; and the amount of sterile paraffin oil added is 0.5-1.5 ml / L. The present invention constructs a solid-phase electron acceptor system by introducing WO3 nanoparticles into the mineral salt culture medium. WO3 nanoparticles can simulate the electrode environment and act as semiconductor electron acceptors to accept electrons released from the extracellular environment of electroactive bacteria, thereby promoting the preferential growth and enrichment of strains with extracellular electron transfer capabilities. The mechanism mainly includes: (1) Electron acceptor function: Electroactive microorganisms release electrons when metabolizing organic substrates. WO3 particles are reduced to low-valence oxides (WO3→WO2) while accepting these electrons, thereby providing a continuous electron outflow channel, which can simulate the cathode environment and promote the growth of strains with extracellular electron transfer (EET) capabilities. (2) Selective enrichment effect: During the screening of target bacteria, non-electroactive bacteria lack effective electron transport pathways. These bacteria are metabolically restricted and grow slowly in WO3-containing systems, which inhibits their growth and prevents them from forming enriched communities. On the contrary, electroactive bacteria with outer membrane polyhemoglobin or conductive nanowire structures can efficiently utilize WO3 particles as electron acceptors through the EET pathway, thus being selectively enriched in the community. (3) Adsorption on particle surface forms a local electroactive microenvironment, resulting in surface adsorption and microenvironment effects: The surface of WO3 nanoparticles has abundant hydroxyl groups and oxygen vacancies, which can adsorb extracellular polysaccharides, extracellular electron transport proteins, and flavin mediators, forming a locally enriched electron transport interface, further improving the colonization and electron exchange efficiency of electroactive bacteria. (4) Stability and reusability: WO3 particles can provide a stable environment for the enrichment of target bacteria and soluble electron acceptors (such as Fe) 3+ NO3 - Unlike other materials, WO3 particles are insoluble solid phases, making the system stable and not easily consumed. This helps maintain a constant supply of electron acceptors during the screening process, facilitating long-term enrichment culture.

[0042] II. A single-chamber microbial electrolysis device as described in claim 1 This invention employs a single-chamber microbial electrolysis device, such as... Figure 1As shown, the device includes a reaction chamber 1, which is a closed structure with an internal cavity. A grid plate 2 is horizontally arranged within the reaction chamber, with its edges fixedly connected to the inner wall of the chamber. The grid plate divides the cavity into a cathode region 3 and an anode region 4. Liquids in the cathode and anode regions can flow between each other through the grid plate, but solid substances in the two regions cannot move through it. Activated carbon particles are filled in the anode region 4. One end of an anode conductive carbon brush 5 passes through the side wall of the reaction chamber and is inserted into the activated carbon particles; its other end is connected to an electrochemical workstation 6 via a wire. A reference electrode 7 and a working electrode 8 are provided in the cathode region. One end of the reference electrode passes through the side wall of the reaction chamber and extends into the liquid in the cathode region, while the other end of the reference electrode is connected to the circuit of the electrochemical workstation. A cathode conductive carbon brush is used as the working electrode. One end of the cathode conductive carbon brush passes through the side wall of the reaction chamber and extends into the liquid in the cathode region, while the other end of the cathode conductive carbon brush is connected to the circuit of the electrochemical workstation. The cathode conductive carbon brush, anode conductive carbon brush, and reference electrode are respectively connected to the electrochemical workstation via wires. At least one aeration head 9 is also provided in the cathode region. The aeration head 9 is connected to the aeration device 11 through an aeration pipe 10, and the aeration device can aerate the liquid in the cathode region through the aeration head. A water outlet 12 is provided in the anode region to discharge the liquid in the anode region. An overflow outlet 13 is provided in the cathode region to allow the liquid in the cathode region to flow out when it rises to a certain height.

[0043] In terms of structural design, this invention adopts a simplified single-chamber electrochemical structure (SCMEC). This structure has significant advantages. It eliminates the complex membrane design of traditional two-chamber structures, resulting in a significant reduction in internal resistance and enabling electrons to be transferred more quickly and efficiently within the system. At the same time, the improved electron conduction efficiency provides more favorable conditions for the metabolic activities of microorganisms, promotes the enrichment of electroactive bacteria, and makes the entire system operate more efficiently. In some embodiments of the present invention, the activated carbon particles have a particle size of 2 mm-6 mm. Before use, the activated carbon particles are sequentially soaked in solutions of 1 mol / L HCl, 1 mol / L NaOH, and 90% ethanol, with each soaking time being 12 h.

[0044] In some embodiments of the present invention, the reference electrode is an Ag / AgCl electrode.

[0045] In some embodiments of the present invention, a gas flow meter 14 is also included for detecting the gas flow rate in the aeration pipe.

[0046] III. Examples Example 1 The device used in this embodiment is as follows: Figure 1As shown, and the device used in this embodiment is merely an example, the device described in this invention is not limited to practical applications. Figure 1 The materials and structure are shown. The device is made of a cylindrical acrylic bottle with an inner diameter of 8 cm and a height of 18 cm, with an effective working volume of 500 ml. The lower part of the reactor is the anode area, and the upper part is the cathode area. The anode area is filled with activated carbon particles with a diameter of 2-6 mm (the activated carbon is soaked in 1 mol / L HCl, 1 mol / L NaOH, and 90% ethanol solutions sequentially for 12 h before use), with a filling height of 4 cm. There is a protruding bottle opening on the right side of the anode area of ​​the reactor. A conductive carbon brush is inserted into it and covered with activated carbon particles, sealed with a rubber stopper with a 1 mm central opening. A titanium rod connected to the conductive carbon brush protrudes from the central opening of the rubber stopper, and any remaining gaps are sealed with quick-drying adhesive to prevent leakage. A conductive carbon brush is inserted at the upper end of the cathode area of ​​the reactor, i.e., at the top of the bottle opening, and sealed with a rubber sheet with a 1mm central hole. A reference electrode (silver chloride (Ag / AgCl) electrode) and an aeration head are then inserted through this point. The conductive carbon brushes and reference electrode of the anode and cathode are then connected to an electrochemical workstation (CHI1040C) using wires (the cathode is the working electrode, and the anode is the counter electrode). An overflow port is located at the upper left of the reactor body, and a water outlet is located at the lower left for easy water replacement.

[0047] The salinity was set at 3% in the experiment, adjusted with NaCl. A C / N ratio gradient acclimation and enrichment process was then carried out at this salinity level. The reactor was started using a potential-controlled method. The reactor was operated in single-chamber MEC mode, with the cathode potential set to -0.5V (vs. Ag / AgCl), and the initial C / N ratio set to 4. 20 mL of treated activated sludge was inoculated into the reactor, and the electrolyte solution shown in Table 1 was added until the water level reached the overflow outlet, followed by aeration. The aeration rate was set at 20 mL / min, and the dissolved oxygen concentration was maintained at 2-3 mg / L daily. A water exchange cycle was performed every 6 days, with fresh water added to the reactor at the end of each cycle. After the denitrification efficiency stabilized, the C / N ratio was reduced to 2, while other parameters remained unchanged. The cycle and operation were the same as described above. After the denitrification efficiency stabilized at this stage, the C / N ratio was reduced to 0, while other parameters remained unchanged, and the process continued for several cycles until the denitrification efficiency stabilized, at which point the target bacterial community was considered enriched. The electrolyte solution configuration used in the device is shown in the table below.

[0048] Table 1 Electrolyte Formulation Table 2. Formulas for Trace Element Solutions Table 3 Vitamin Solution Formula 1. Initial screening of salt-tolerant electroactive HNAD target strains After domestication and enrichment, a certain amount of electrode biofilm with excellent electro-denitrification performance was used to prepare a bacterial suspension. Then, 1 mL of the bacterial suspension was serially diluted (10⁻⁶). -3 -10 -6 Then, each gradient suspension was evenly spread onto sterile (LB) solid plates (containing 30g L). -1 NaCl, 10g L -1 Tryptone, 5g / L -1 Yeast extract powder, 20 g / L -1 (Use technical agar powder), and after single colonies grow, pick them out and perform multiple streaking purifications on sterile LB plates until the colony morphology remains consistent. To further obtain highly efficient salt-tolerant electroactive HNAD target strains, the initially screened pure bacteria were inoculated into LB medium for expansion. When the OD600 of the bacterial solution reached 0.5, it was transferred to centrifuge tubes, and a certain volume of mineral salt medium, 5% WO3 nanoparticles, and sterile paraffin oil were added. The culture was continued for 2 hours in a constant temperature incubator, and the color development (blue) was recorded. The electroactive candidate strains screened using WO3 nanoparticles were expanded again. When the OD600 of the bacterial solution reached 0.6, the bacterial solution was centrifuged using a low-speed refrigerated centrifuge to obtain bacterial cells. The bacterial cells were inoculated into sterilized nitrification medium, denitrification medium, and simultaneous nitrification-denitrification medium at pH 7.5 (specific components are shown in Tables 4-6 below). The culture was carried out at 25 ℃ and 200 r min⁻¹. The nitrification and denitrification activities of the strains were detected every 1 hour. The pure bacteria with highly efficient nitrification, denitrification, and simultaneous nitrification-denitrification performance were the salt-tolerant electroactive HNAD target strains required for this project.

[0049] Table 4. Composition of Nitrification Medium Table 5 Composition of Denitrification Medium Table 6. Composition of SND Culture Medium 2. Performance of the target bacteria obtained after screening The selected single bacterial strains were placed in 50ml centrifuge tubes and divided into two groups, with nitrification and denitrification culture media added to one group respectively. When the OD600 reached approximately 0.8, 1ml of the bacterial culture was pipetted into a new 50ml centrifuge tube to examine the nitrification and denitrification capabilities. The compositions of the nitrification and denitrification culture media are shown in Tables 7 and 8.

[0050] Table 7 Nitrification Culture Medium Table 8 Denitrification Culture Medium The nitrogen content is fixed, and the C / N ratio is calculated accordingly to change the amount of COD added (for example, if the nitrate concentration in Table 8 is 200 mg / L, then the C / N ratio is 0, and the amount of sodium acetate added is 0; if the C / N ratio is 2, the amount of sodium acetate added is converted to COD equivalent of 400 mg / L, only the COD content is changed, and other components remain unchanged).

[0051] Nitrification capacity assessment: 50ml centrifuge tubes were filled with nitrification medium, 1ml of bacterial solution was added, and the sample was tested.

[0052] Denitrification capacity assessment: 50ml centrifuge tubes were filled with nitrification medium, 1ml of bacterial solution was added, and the sample was tested.

[0053] Electrotrophic denitrification capacity assessment: The strain was transferred to the working electrode in the three-electrode system, a voltage of -0.5V was applied using a potentiostat, 80ml of denitrification medium was added, and the sample was measured.

[0054] The changes in ammonia nitrogen removal rate of the screened strains under different C / N ratios are as follows: Figure 2 As shown. From Figure 2 It can be seen that under simulated high-salt environment (the electrolyte in the screening process contains a high concentration of NaCl, and salt-tolerant bacteria are acclimatized in a targeted manner), ammonia nitrogen is efficiently removed under different C / N ratios. Especially in the suitable range of C / N=4~6, the ammonia nitrogen removal rate is fast, the final residue is low, and the bacterial cell (OD600) grows vigorously. This indicates that after the screening process of "high-salt acclimatization + potential regulation + gradient C / N acclimatization" of this invention, the target strain can maintain stable and efficient nitrification activity under high salt stress, breaking through the bottleneck of the decline in activity of conventional denitrifying strains in high-salt environments, and verifying the effectiveness of the screening method in achieving the dual goals of salt tolerance and efficient denitrification.

[0055] The changes in nitrite, nitrate, and OD600 of the screened strains over time under different C / N ratios are shown in the following figures. Figure 3 As shown. From Figure 3 As can be seen, the target strains obtained by the screening method of this invention can achieve efficient nitrate removal at different C / N ratios, with no significant accumulation of nitrite, and the denitrification process is complete and smooth. Simultaneously, at suitable C / N ratios (e.g., C / N=4, 6), the bacterial cell growth (OD600) is vigorous, and the biomass is sufficient. This proves that the screening method of this invention can efficiently obtain target strains that possess both complete denitrification function (no intermediate product accumulation) and vigorous metabolic growth capacity, demonstrating the beneficial effects of the screening process on the integrity of the strain's denitrification function and biomass stability.

[0056] The changes in nitrate and nitrite over time after the selected strains were placed on the working electrode of a three-electrode system and a -0.5V voltage was applied using a potentiostat are shown below. Figure 4 As shown. From Figure 4 As can be seen, under the condition of applying a voltage of -0.5V to the three-electrode system, the nitrate concentration decreases rapidly over time, nitrite accumulates briefly and then degrades rapidly, and total nitrogen decreases significantly and is eventually almost completely removed. This proves that the screening method of the present invention can efficiently obtain target strains with excellent electroactive denitrification function. These strains can not only rapidly convert nitrate under electrochemical drive, but also have no intermediate product (nitrite) accumulation during the denitrification process, and the total nitrogen is completely removed. This demonstrates the beneficial effect of the screening process in the targeted enrichment of electroactive HNAD bacteria and ensuring the integrity and efficiency of their denitrification function.

[0057] 3. Summary The above experiments demonstrate that the target bacterial strains screened using the method described in this invention exhibit outstanding salt tolerance and stable denitrification in high-salt environments. This is because existing denitrifying bacterial strains often experience a sharp drop in denitrification efficiency under high-salt conditions (e.g., NaCl ≥ 3%) due to osmotic pressure damage and enzyme activity inhibition. However, the target bacterial strains of this invention, after undergoing "high-salt electrolyte acclimatization + potential regulation," can still maintain highly efficient removal of ammonia nitrogen and nitrate in simulated high-salt systems (e.g., nitrification / denitrification culture media containing high concentrations of NaCl). This is evidenced by the rapid degradation of ammonia nitrogen and near-complete removal of nitrate when C / N = 4~6. This proves that the target bacterial strains, through high-salt directional acclimatization using the described screening method, have acquired metabolic regulatory abilities to adapt to salt stress (e.g., synthesizing osmotic substances and optimizing cell membrane structure), overcoming the bottleneck of conventional bacterial strains' inactivation under high salt conditions.

[0058] Meanwhile, the target bacterial strains screened by the method described in this invention exhibit excellent electroactivity and strong adaptability to low-carbon / carbon-free scenarios. Existing denitrifying bacteria of the same type mostly rely on organic carbon sources for electron donation, limiting nitrogen removal in low-carbon or carbon-free wastewater (such as high-salt industrial wastewater). The target bacteria of this invention, under a three-electrode system (-0.5V voltage), can directly utilize electrochemical electron donors to achieve complete metabolism of nitrate → nitrite → nitrogen (with no nitrite accumulation and near-complete removal of total nitrogen), demonstrating the high efficiency and process integrity of electroactive denitrification. This provides unique advantages in the treatment of low-carbon, high-salt wastewater, thanks to the targeted enrichment strategy of "potential regulation + electroactive function verification" in the screening method.

[0059] Moreover, the target bacterial strains screened by the method described in this invention have a wide C / N ratio adaptation range and strong resistance to environmental fluctuations. Existing similar bacterial strains have a narrow adaptability to C / N ratios (e.g., efficient nitrogen removal only at C / N=5~8), and are prone to metabolic imbalance when the carbon source is too low or too high. The target bacteria of this invention can achieve vigorous growth (high OD600) and efficient nitrogen removal (rapid degradation of ammonia nitrogen and nitrate) in the C / N range of 2, 4, 6, and 8, and still have functional elasticity even at the extreme C / N ratio of C / N=0. This is a direct result of the screening method of "gradient C / N acclimatization", enabling the strains to adapt to the complex scenario of large fluctuations in the C / N ratio in actual wastewater.

[0060] The target bacterial strains screened by the method described in this invention have complete denitrification function and no risk of intermediate product accumulation. Some existing denitrifying bacteria are prone to nitrite accumulation during denitrification (intermediate product residue rate ≥10%), increasing the risk of secondary contamination. The target bacteria of this invention do not exhibit nitrite retention during nitrification, denitrification, and electroactive denitrification (as shown in the denitrification diagram, nitrite accumulates briefly and then rapidly degrades, eventually approaching zero), ensuring thorough removal of total nitrogen. This is due to the dual guarantee of multi-medium culture function verification (nitrification, denitrification, simultaneous nitrification-denitrification) + colorimetric initial screening in the screening method, ensuring the integrity of the strain's metabolic pathway.

[0061] In summary, the screening method of this invention, through a strategy of high-salt directional acclimatization and potential regulation, utilizes a single-chamber microbial electrolysis cell, high-salt electrolyte, and constant potential regulation to achieve precise enrichment of salt-tolerant electroactive HNAD bacteria, solving the technical problem that conventional methods cannot simultaneously achieve both salt tolerance and electroactivity. The process of gradient-reduced C / N acclimatization followed by nitrification / denitrification / electroactivity multi-medium verification systematically enhances the environmental adaptability of target strains to carbon and nitrogen fluctuations, while ensuring the integrity and absence of their denitrification metabolic pathways, overcoming the limitations of traditional screening methods such as single function and poor environmental adaptability. Furthermore, the method of WO3 nanoparticle colorimetric primary screening followed by functional secondary screening can rapidly identify electroactive strains and accurately verify their denitrification function, making it more efficient and accurate than the traditional streak plating method and avoiding the functional mismatch problem caused by screening based solely on colony morphology.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the technical solutions. Those skilled in the art should understand that any modifications or equivalent substitutions to the technical solutions of the present invention without departing from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A method for rapid enrichment culture of salt-tolerant, electroactive HNAD target bacteria, characterized in that, The specific steps are as follows: Step 1: Rapid enrichment is achieved using a single-chamber microbial electrolysis device. Activated sludge containing HNAD bacteria is added to the reaction chamber of the device, along with electrolyte. The concentration of NaCl in the electrolyte is at least 3 wt% (by mass percentage). Step 2: Start the electrochemical workstation using the potential control method. The cathode potential is -0.5V to 1.0V, the initial C / N ratio is set to 4, and aeration is performed. The aeration intensity is 15mL / min to 25mL / min, and each cycle is 6 days. After each cycle, the electrolyte in the reaction chamber is replaced. Step 3: After the denitrification rate stabilizes, adjust the C / N value to 2, keep other parameters unchanged, and continue running for multiple cycles; after the denitrification rate stabilizes again, adjust the C / N value to 0, keep other parameters unchanged, and continue running for multiple cycles until the denitrification rate stabilizes again, then the target bacterial community has been enriched. Step 4: Remove the electrode biofilm from the reaction chamber and prepare it into a bacterial suspension. Perform serial dilutions on the bacterial suspension, and spread the suspensions obtained from each serial dilution on LB medium for culture and purification to obtain the initial screening strain. Step 5: Inoculate the initial screening strain obtained in Step 4 into LB medium for expansion culture. When the absorbance of the bacterial solution at a wavelength of 600 nm reaches 0.5, transfer the bacterial solution into a centrifuge tube, add mineral salt medium, WO3 nanoparticles and sterile paraffin oil, and continue to culture at a constant temperature of 25±5℃ and record the color development. Step 6: When the bacterial culture from Step 5 turns blue, inoculate the bacterial culture into LB medium for further expansion. When the absorbance of the bacterial culture at a wavelength of 600 nm reaches 0.6, perform low-speed freeze centrifugation to obtain bacterial cells. Subsequently, inoculate the bacterial cells into sterilized nitrification medium, denitrification medium, and simultaneous nitrification-denitrification medium, respectively, and culture them at a constant temperature of 25 ℃ and 200 r / min to obtain the salt-tolerant electroactive HNAD target bacteria.

2. The method according to claim 1, characterized in that, In step 1, the electrolyte has the following components: anhydrous sodium acetate 2-3 g / L, NH4Cl 0.5-1.0 g / L, NaCl 10-15 g / L, K2HPO4·3H2O 1-2 g / L, KH2PO4 0.2-0.5 g / L, trace element solution 1-2 ml / L, vitamin solution 1-2 ml / L; and water as the solvent.

3. The method according to claim 1, characterized in that, The trace element solution comprises the following components: 1-2 g / L nitroglycerin, 2-4 g / L magnesium sulfate, 0.1-1.0 g / L manganese sulfate, 1.0-2.0 g / L sodium chloride, 0.1-0.5 g / L ferrous sulfate heptahydrate, 0.01-0.05 g / L copper sulfate pentahydrate, 0.01-0.05 g / L potassium aluminum sulfate dodecahydrate, 0.1-0.5 g / L cobalt chloride hexahydrate, 0.1-0.5 g / L calcium chloride dihydrate, 0.1-0.5 g / L zinc chloride, 0.01-0.05 g / L boric acid, 0.02-0.05 g / L sodium molybdate, 0.02-0.05 g / L nickel chloride hexahydrate, and 0.02-0.05 g / L sodium tungstate dihydrate; the solvent is water.

4. The method according to claim 1, characterized in that, The vitamin solution comprises the following components: biotin 0.1-0.5 g / L, vitamin B6 1-2 g / L, vitamin B12 0.1-0.5 g / L, folic acid 0.1-0.5 g / L, pantothenic acid 0.5-1.0 g / L, nicotinic acid 0.5-1.0 g / L, lipoic acid 0.5-1.0 g / L, P-aminobenzoic acid 0.5-1.0 g / L, thiamine 0.5-1.0 g / L, and riboflavin 0.5-1.0 g / L; the solvent is water.

5. The method according to claim 1, characterized in that, In step 4, the steps for preparing the electrode biofilm into a bacterial suspension are as follows: The removed electrode biofilm is placed under sterile conditions and the surface is rinsed with sterile physiological saline or sterile phosphate buffer to remove loose impurities; then, the electrode biofilm is cut into small pieces and subjected to ultrasonic or oscillation treatment under sterile conditions to fully detach the biofilm and form a uniform bacterial suspension; the bacterial suspension is allowed to stand for 1-2 minutes, and the upper layer is collected to obtain the bacterial suspension; then, the bacterial suspension is serially diluted and plated; the components of LB medium are: 30 g / L NaCl, 10 g / L tryptone, 5 g / L yeast extract powder, and 20 g / L agar powder.

6. The method according to claim 1, characterized in that, In step 5, the amount of mineral salt culture medium added is 1-3 ml / L, the amount of WO3 nanoparticles added is 1%-5% based on the total mass of LB culture medium, and the amount of sterile paraffin oil added is 0.5-1.5 ml / L.

7. A single-chamber microbial electrolysis device as described in claim 1, characterized in that, The device includes a reaction chamber (1), which is a closed structure with an internal cavity; A grid plate (2) is provided horizontally in the reaction chamber. The four edges of the grid plate are fixedly connected to the inner wall of the reaction chamber. The grid plate divides the cavity in the reaction chamber into a cathode area (3) and an anode area (4). The liquid in the cathode area and the liquid in the anode area can flow to each other through the grid plate. Activated carbon particles are filled in the anode area. One end of the anode conductive carbon brush (5) passes through the side wall of the reaction chamber and is inserted into the activated carbon particles. The other end is connected to the electrochemical workstation (6) through a wire. A reference electrode (7) and a working electrode (8) are provided in the cathode area. One end of the reference electrode passes through the side wall of the reaction chamber and extends into the liquid in the cathode area. The other end of the reference electrode is connected to the circuit of the electrochemical workstation. The cathode conductive carbon brush (8) is then placed in the cathode area. The brush is set as the working electrode. One end of the cathode conductive carbon brush passes through the side wall of the reaction chamber and extends into the liquid in the cathode area. The other end of the cathode conductive carbon brush is connected to the circuit of the electrochemical workstation. The cathode conductive carbon brush, the anode conductive carbon brush, and the reference electrode are respectively connected to the circuit of the electrochemical workstation through wires. At least one aeration head (9) is also provided in the cathode area. The aeration head is connected to the aeration device (11) through the aeration pipe (10). The aeration device can aerate the liquid in the cathode area through the aeration head. An outlet (12) is provided in the anode area. The liquid in the anode area can be discharged through the outlet. An overflow port (13) is provided in the cathode area. When the liquid in the cathode area rises to a certain height, it can flow out from the overflow port.

8. The apparatus according to claim 7, characterized in that, The activated carbon particles have a particle size of 2mm-6mm.

9. The apparatus according to claim 7, characterized in that, The reference electrode is an Ag / AgCl electrode.

10. The apparatus according to claim 7, characterized in that, It also includes a gas flow meter (14) for detecting the gas flow rate in the aeration pipe.

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