Gradient domestication and enrichment method for anode biofilms in microbial fuel cells and its application

Abstract

This invention belongs to the fields of bioengineering, environmental engineering, and clean energy technology, and discloses a gradient domestication and enrichment method for anode biofilms in microbial fuel cells and its application. The method includes the following steps: high-temperature treatment of the carrier material for microbial growth; loading a highly active catalyst onto an air cathode; constructing a microbial fuel cell reactor; adding PBS buffer nutrient solution, domestication substrate, and trace element solution to the microbial fuel cell reactor; purging the air from the reactor to induce an anaerobic state; connecting a resistor in series in the external circuit of the reactor; culturing microorganisms and monitoring the voltage across the external resistor in real time. When the output voltage is below 30mV, steps four through seven are repeated, with the proportion of domestication substrate gradually increasing in each cycle. This invention, through a gradient domestication scheme that gradually increases the proportion of domestication substrate, can enrich dominant bacterial communities, resulting in biofilms with complete bacterial structure and high abundance of functional bacteria.

Description

Technical Field

[0001] This invention belongs to the fields of bioengineering, environmental engineering and clean energy technology, and specifically relates to a gradient domestication and enrichment method for anode biofilms in microbial fuel cells and its application. Background Technology

[0002] The petroleum industry generates large quantities of high-concentration, recalcitrant wastewater, known as oilfield wastewater, during extraction, gathering, transportation, and refining processes. The main characteristics of oilfield wastewater include high concentrations of petroleum hydrocarbons, high mineralization, and the presence of biotoxic organic compounds such as benzene compounds and polycyclic aromatic hydrocarbons. These characteristics make traditional activated sludge and physicochemical treatment methods difficult to implement in treating oilfield wastewater, resulting in inhibited microbial activity, inconsistent effluent quality, and high operating costs. Therefore, there is an urgent need to develop new treatment technologies that combine high efficiency and economic benefits.

[0003] Bioelectrochemical systems are a type of technology platform that utilizes electrochemically active microorganisms as catalysts to oxidize organic substrates at the anode and output electrons to an external circuit. Currently, the most commonly used method for enriching anodic biofilms is the enrichment method based on the microbial fuel cell (MFC) model. However, this method has the following significant drawbacks when treating oilfield wastewater systems: Firstly, MFCs rely solely on the self-generated potential difference from microbial metabolism as their driving force, which is typically only 0.3~0.5V. This potential difference is small and fluctuates significantly with substrate concentration and microbial metabolic state. High salt content in oilfield wastewater reduces the fluidity of microbial cell membranes and affects osmotic pressure balance. Furthermore, benzene compounds and polycyclic aromatic hydrocarbons have a significant inhibitory effect on the metabolism of electroactive microorganisms. This weak and unstable driving force is insufficient to overcome the thermodynamic energy barrier of microbial colonization, resulting in system start-up cycles that often last for weeks or even months, severely restricting the feasibility of engineering applications.

[0004] Secondly, under low-potential passive driving conditions, non-electrogenic bacteria can non-specifically attach to the anode surface through ordinary adhesion, competing with functional electrogenic bacteria with extracellular electron transfer capabilities for anode surface attachment sites and organic substrates. This results in a relatively low abundance of functional electrogenic bacteria in mature biofilms, poor overall electron transfer efficiency of the biofilm, and consequently affects pollutant removal efficiency.

[0005] Third, MFCs typically use air cathodes, relying on the reduction reaction of oxygen on the cathode surface as an electron acceptor. However, the oxygen reduction reaction kinetics are sluggish and limited by mass transfer, resulting in high internal resistance in the entire MFC system. This further weakens the metabolic driving force of anodic microorganisms and hinders the rapid accumulation of electroactive microorganisms in highly toxic oilfield wastewater environments.

[0006] Although gradient acclimation strategies have been reported in the field of biological treatment, existing gradient acclimation schemes for oilfield wastewater systems lack organic integration with the electrochemical screening mechanism of MFC systems. Specifically, existing methods fail to fully utilize the inherent electric field selection pressure of MFC systems to enable the gradient concentration stress and electrochemical screening to work synergistically, simultaneously and directionally enriching composite functional bacteria with both power generation and petroleum hydrocarbon degradation functions. Furthermore, the lack of a systematic material replacement ratio scheme designed specifically for the characteristics of oilfield wastewater leads to low acclimation efficiency and unstable targeted screening effects of functional bacteria.

[0007] Therefore, developing a method that can fully leverage the electrochemical advantages of the MFC system and combine it with a gradient domestication synergistic screening strategy to achieve rapid enrichment of oil-resistant and electrogenic biomembranes has significant scientific and engineering application value. Summary of the Invention

[0008] The purpose of this invention is to provide a gradient domestication and enrichment method for anode biofilms in microbial fuel cells and its application, in order to solve the problem that the anode biofilm enrichment method based on the MFC model has obvious limitations in three dimensions—start-up speed, functional bacteria enrichment efficiency, and biofilm activity—when treating high-difficulty systems such as oilfield wastewater, and cannot meet the actual needs of rapid engineering deployment.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: On one hand, this invention provides a gradient domestication and enrichment method for anode biofilms in microbial fuel cells. It includes the following steps: S1. High-temperature pretreated anode carrier material: The carrier material used for microbial growth is subjected to high-temperature heat treatment to remove surface organic impurities and improve its surface hydrophilicity and microbial adhesion ability.

[0010] S2. Preparation of air cathode: A highly active catalyst is loaded onto the carbon cloth substrate of the air cathode to construct an air cathode with high oxygen reduction reaction activity.

[0011] S3. Constructing an MFC reactor: Using pretreated carrier material as the anode and prepared air cathode as the cathode, connect the two electrodes with wires and connect an external resistor to construct a complete MFC reactor.

[0012] S4. Feeding: Add the prepared PBS buffer nutrient solution, acclimatization substrate, and trace element solution to the MFC reactor.

[0013] S5. Create an anaerobic environment: Introduce nitrogen into the reactor for at least 10 minutes, then purge the air from the reactor to allow the system to enter an anaerobic state.

[0014] S6. External resistor: A resistor connected in series in the external circuit of the reactor for current sampling and monitoring of operating status.

[0015] S7. Constant Temperature Culture and Cycle Management: Cultivate microorganisms under constant temperature conditions of 25~35℃ and monitor the voltage across the external resistor in real time. When the output voltage is lower than 30mV, it is determined that a cycle has ended. Repeat the feeding, anaerobic treatment, external resistor operation and constant temperature culture steps. In each cycle, the proportion of PBS buffer nutrient solution is gradually reduced according to the principle of gradient increase, while the proportion of acclimatized substrate is increased at the same time. This allows the microbial community to complete adaptive succession in a gradually increasing substrate stress environment until it uses acclimatized substrate as the only feed and maintains stable operation.

[0016] Preferably, in S1, the carrier is a porous carbon material, which is one of carbon felt, carbon cloth, carbon paper and carbon brush. The high-temperature treatment temperature is 400~700℃ and the treatment time is 30-120 minutes.

[0017] Preferably, in S2, the air cathode is supported on at least one of a commercial Pt / C catalyst, a transition metal (Fe, Co, Ni) / nitrogen-doped carbon catalyst.

[0018] Preferably, in S4, the PBS buffer nutrient solution includes: one of sodium dihydrogen phosphate or potassium dihydrogen phosphate, one of disodium hydrogen phosphate or dipotassium hydrogen phosphate, ammonium chloride, potassium chloride, calcium chloride dihydrate, and a multivitamin solution, and the pH of the prepared PBS buffer is 6.8~7.2.

[0019] Preferably, in S4, the trace element solution is used to provide metal coenzyme factors necessary for microbial growth, and includes at least three of the following elements in a salt solution: boron, cobalt, tungsten, selenium, chlorine, nickel, zinc, molybdenum, etc.

[0020] Preferably, in S6, the resistance value of the external resistor is in the range of 100 Ω to 1000 Ω.

[0021] Preferably, the initial ratio of PBS buffer nutrient solution to acclimatization substrate is 1:1. A higher proportion of PBS is used to provide nutrition in the early stage of acclimatization. As the cycle progresses, the amount of PBS buffer nutrient solution is gradually reduced until only acclimatization substrate is added in the final feeding, so as to achieve targeted screening of oil-tolerant and salt-tolerant microorganisms.

[0022] Preferably, the volume ratio of PBS buffered nutrient solution to acclimatization substrate in the first, second and third cycles is 1:1, 1:2 and 1:4, respectively, and the volume ratio of PBS buffered nutrient solution to acclimatization substrate in the fourth cycle and thereafter is 0:1.

[0023] On the other hand, the present invention provides an application of a gradient domestication and enrichment method for anode biofilms of microbial fuel cells in the treatment of oilfield wastewater.

[0024] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention utilizes an external resistor in the MFC to create a continuous electron outflow channel, constructing a stable anodic oxidation driving force. This allows only electroactive microorganisms with extracellular electron transport capabilities (such as Geobacteraceae) to fully utilize this electrochemical driving force to colonize the anode, competitively inhibiting non-electrogenous bacteria and achieving targeted screening for electrogenic functions. Simultaneously, the gradually increasing concentration of oilfield wastewater forces the microbial community to undergo adaptive succession, selectively retaining high-salt-tolerant and petroleum hydrocarbon-resistant degradation bacteria (such as Thaurea). The combined and synergistic effect of these two screening pressures makes both Geobacteraceae and Thaurea the dominant bacterial groups in the enriched anode biofilm, achieving simultaneous enrichment of electrogenic and petroleum hydrocarbon degradation functions, rather than separately and independently cultivating electrogenic and degradation bacteria. This is the core technological contribution of this invention compared to traditional single cultivation strategies.

[0025] 2. This invention removes organic impurities from the surface of the carbon carrier through high-temperature heat treatment and introduces hydrophilic oxygen-containing functional groups on the carbon fiber surface, which significantly improves the microbial compatibility of the anode surface, which is conducive to the rapid colonization of electroactive microorganisms in the early stage of MFC startup and shortens the overall startup cycle.

[0026] 3. This invention significantly improves the cathode oxygen reduction reaction rate and reduces cathode polarization resistance by loading a highly active catalyst onto the air cathode, resulting in a lower overall internal resistance of the MFC. Lower system internal resistance means stronger anodic oxidation driving force, which is beneficial for maintaining the continuous and efficient metabolic activity of anodic microorganisms in highly toxic oilfield wastewater environments, thereby improving the enrichment quality of functional bacteria.

[0027] 4. This invention only requires the simple construction of an MFC reactor and regular replacement of the culture medium, making it easy to operate; both the anode support and the cathode catalyst are commercially available products; the overall process is simple, the operating cost is low, and it has the foundation for industrial application. Attached Figure Description

[0028] Figure 1 The linear sweep voltammetry curve and power density curve of the MFC reactor in Example 1 of this invention are shown. Figure 2 The electrochemical impedance spectroscopy internal resistance fitting diagram and equivalent circuit diagram of the MFC reactor in Example 1 of this invention are shown. Figure 3 The bar chart shows the COD removal rate and coulombic efficiency of the MFC reactor after 20 and 40 days of cultivation in Example 1 of this invention. Figure 4 The current-time curves for the MFC startup and enrichment process in Embodiment 1 of the present invention are shown. Figure 5The images shown are SEM images of the microbial film enriched on the surface of the carbon brush anode in Example 1 of the present invention, where (a) and (b) are morphology images at different magnifications. Figure 6 The images shown are TEM images and elemental distributions of microorganisms enriched in the anodic biofilm in this embodiment of the invention. (a) is a TEM image, (b) is a high-angle annular dark field image (HAADF), (c) is an energy-dispersive X-ray spectroscopy (EDS) image, and (d~h) are mapping distribution maps of each element. Figure 7 The results show the relative abundance analysis of the anode biofilm microbial community in this embodiment of the invention. Detailed Implementation

[0029] To enable those skilled in the art to better understand the present invention, the technical solutions of specific embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Unless otherwise defined, the technical or scientific terms used in this invention should have the ordinary meaning understood by those skilled in the art.

[0030] Those skilled in the art should understand that, unless otherwise specified, the reagents, materials and instruments used in the following examples are all conventional materials in the art and can be obtained through commercial channels.

[0031] Example 1 This embodiment provides the application of a gradient domestication and enrichment method for anode biofilms in microbial fuel cells in the treatment of oilfield wastewater.

[0032] S1. High-temperature pretreatment of anode carrier material: The carbon brushes were cleaned three times each with deionized water and anhydrous ethanol to remove soluble contaminants adhering to the carbon fiber surface, and then thoroughly dried in a vacuum drying oven. The dried carbon brushes were then placed in a muffle furnace and calcined at 550°C for 60 minutes.

[0033] The mechanism of high-temperature heat treatment is as follows: Under high temperature conditions, the residual organic matter (grease, polymer, etc.) on the carbon brush surface undergoes thermal oxidation and decomposition, transforming into CO2 and H2O that escape, thus fully purifying the carbon fiber surface; at the same time, the high-temperature oxidation process introduces hydrophilic oxygen-containing functional groups such as hydroxyl and carboxyl groups on the carbon fiber surface, which significantly enhances the hydrophilicity of the originally hydrophobic carbon fiber surface, which is conducive to the initial wetting and adhesion of microorganisms on the anode surface, thereby accelerating the formation of biofilm.

[0034] Carbon brushes, as anode materials, have the characteristics of three-dimensional network structure, large specific surface area, excellent conductivity, and good biocompatibility. They can provide sufficient attachment sites for microorganisms, which is conducive to the formation of highly active and high-density three-dimensional biofilm structures.

[0035] S2. Preparation of high-performance air cathode: Cut carbon cloth of appropriate size as the cathode substrate material. First, prepare the diffusion layer: mix carbon black with polytetrafluoroethylene (PTFE) dispersion and deionized water, homogenize by ultrasonication, and then coat it evenly on one side of the carbon cloth with a brush. Heat treat it at 370℃ for 20 minutes, remove it and cool it, then coat it with PTFE again and heat treat it for 10 minutes. Repeat the above steps a total of 3 times to form a hydrophobic gas diffusion layer, which prevents moisture from seeping from the liquid phase side to the air side, while ensuring the effective mass transfer of oxygen from the air side to the catalyst layer.

[0036] Subsequently, a catalyst layer was prepared on the other side of the carbon cloth: a commercial Pt / C catalyst (20 wt% platinum loading) was mixed with 35 μL deionized water, 280 μL Nafion solution (5 wt%), and 140 μL isopropanol in a certain proportion, ultrasonically mixed, and then uniformly coated onto the surface of the carbon cloth. The mixture was then dried with a blower to complete the fabrication of the air cathode. The coating amount of Pt / C catalyst was 10 mg Pt / cm³. 2 The carbon cloth measures 4 cm × 4 cm.

[0037] The core function of the air cathode is to accelerate the oxygen reduction reaction (O2 + 4H+) at the cathode using a supported highly active catalyst. + + 4e - → 2H2O), providing an efficient absorption channel for electrons output by anodic microorganisms, thereby reducing the polarization impedance of the entire MFC system, enhancing the continuous driving force of the system to the external circuit, and thus strengthening the electrochemical screening pressure on the anodic biofilm.

[0038] In addition to commercial Pt / C catalysts, this invention is also applicable to transition metal (Fe, Co, Ni) / nitrogen-doped carbon composite catalysts (MNC catalysts). These non-precious metal catalysts have advantages such as wide availability, low cost, and tunable ORR activity, making them more suitable for low-cost engineering applications.

[0039] S3. Set up the MFC reactor: A carbon brush pretreated at high temperature was used as the anode, and a prepared air cathode was used as the cathode. These were fixed at corresponding positions in the reactor, and the anode and cathode were connected by wires to construct a complete MFC single-chamber air cathode reactor. The reactor's sealing points were treated to ensure good airtightness. The effective volume of the reactor used was 30 mL, and the distance between the anode and cathode was 2.5 cm.

[0040] S4. Feeding: The feed consisted of oilfield wastewater, PBS buffered nutrient solution, and trace element solution. First, 50 mL of PBS buffered nutrient solution was prepared, with the following composition: 15.4 mmol / L sodium dihydrogen phosphate and 32.3 mmol / L disodium hydrogen phosphate, which together constituted a phosphate buffer system. It also included 5.8 mmol / L ammonium chloride to provide the nitrogen source required for microbial growth; 1.7 mmol / L potassium chloride to maintain the ionic strength of the solution; 10 mg / L calcium chloride dihydrate to provide the essential divalent calcium ion nutrient element for microorganisms; and 2 mL / L multivitamin solution to provide the necessary growth cofactors for microorganisms. After the above components were thoroughly mixed, the pH was measured and adjusted to the range of 6.8-7.2, which is consistent with the optimal metabolic pH of most electroactive microorganisms.

[0041] The above-mentioned phosphate components can also be replaced in equal molar amounts with potassium dihydrogen phosphate and dipotassium hydrogen phosphate, with equivalent function.

[0042] Trace element solutions are used to provide metal coenzyme factors necessary for the growth of anodic microorganisms. Their main components include at least three of the following elements in a salt solution: boron (B), cobalt (Co), tungsten (W), selenium (Se), chlorine (Cl), nickel (Ni), zinc (Zn), and molybdenum (Mo).

[0043] The aforementioned trace elements play a crucial coenzyme role in microbial metabolism: cobalt is a vitamin B1+ component. 12 The core metallic elements involved in methyl transfer and isomerization reactions; tungsten and molybdenum are the active centers of many oxidoreductases (such as formate dehydrogenase); nickel participates in the catalysis of hydrogenases and ureases; selenium exists in the form of selenocysteine ​​in many oxidoreductases; zinc is a structural element of many dehydrogenases and transcription factors. Although these trace metal elements are required in extremely small amounts, they are crucial for maintaining normal metabolic activity and extracellular electron transport function in electroactive microorganisms (especially Geobacteraceae) in highly toxic oilfield wastewater environments.

[0044] The dosage of the trace element solution is 1 mL / L, and its formula is as follows: per liter of deionized water, the components are: EDTA 0.5 g, MgSO4·7H2O 3.0 g, MnSO4·H2O 0.5 g, NaCl 1.0 g, FeSO4·7H2O 0.1 g, Co(NO3)2·6H2O 0.1 g, anhydrous CaCl2 0.1 g, ZnSO4·7H2O 0.1 g, CuSO4·5H2O 0.01 g, anhydrous AlK(SO4)2 0.01 g, H3BO3 0.01 g, Na2MoO4·2H2O 0.01 g, anhydrous Na2SeO3 0.001 g, Na2WO4·2H2O 0.01 g, NiCl2·6H2O 0.02 g.

[0045] S5. Construct an anaerobic environment: High-purity nitrogen (≥99.9% purity) is continuously introduced into the sealed MFC reactor for at least 10 minutes. The establishment of an anaerobic environment is crucial for the enrichment of the anodic biofilm. Electroactive microorganisms (such as Geobacteraceae) are strict or facultative anaerobes; the presence of oxygen causes them to preferentially use oxygen as the electron acceptor rather than the anodic biofilm for aerobic respiration, thus losing their electrochemical activity. Simultaneously, the anaerobic environment inhibits the growth of aerobic microorganisms, further increasing the selective pressure on electroactive anaerobic microorganisms. Introducing nitrogen for 10 minutes ensures that dissolved oxygen in the system drops to a negligible level, completing the construction of the anaerobic environment.

[0046] S6. External resistor and real-time monitoring: A 1000Ω resistor is connected in series. This resistor serves two purposes: firstly, as an external load for the MFC, maintaining continuous electron flow in the external circuit and creating a stable electric field selective voltage; secondly, as a current sensor, it measures the voltage across the resistor in real time and calculates the external circuit current using Ohm's law, thus reflecting the metabolic activity and electrogenic level of the anodic biofilm. A data logger continuously acquires the voltage across the resistor in real time, forming a complete current-time curve to determine the biofilm enrichment process and the end of the operating cycle. The choice of the external resistor value has a significant impact on the operating state of the MFC: when the resistance is too large (>1000Ω), the external circuit impedance dominates the total system resistance, hindering electron flow and impeding the continuous electron output of anodic microorganisms, thus weakening the electrochemical driving force for the enrichment of functional bacteria; when the resistance is too small (<100Ω), the external circuit impedance is negligible relative to the internal resistance, the system approaches a short circuit state, the anodic potential is too low, and it may affect the thermodynamic driving force of some electroactive microorganisms. The range of 100~1000Ω provides an effective operating window between these two extreme cases.

[0047] S7. Constant Temperature Incubation and Cycle Management: The MFC reactor was incubated in a 30°C incubator. The output voltage across the external resistor was monitored in real-time using a data logger. Changes in the output voltage dynamically reflected the formation and maturation process of the anolyte biofilm: in the initial inoculation stage, electroactive microorganisms had not yet effectively colonized the anode, resulting in a low output current; as the biofilm gradually formed, the output current gradually increased to its peak, marking the biofilm's entry into the maturation stage; subsequently, as the organic substrate was depleted, substrate limitation led to a gradual decrease in the output current. When the output voltage across the external resistor dropped below 30mV, it was determined that one operating cycle had ended, and the organic substrate was essentially depleted. At this point, fresh culture medium was immediately replaced according to the proportion corresponding to the current acclimatization stage to maintain microbial activity, and the next operating cycle began. This cycle was repeated until acclimatization was complete.

[0048] In the first cycle, the volume ratio of PBS buffer solution to oilfield wastewater was 1:1 to ensure the mixture filled the reactor. This higher proportion of PBS provided sufficient and balanced nutrient support for the electroactive microorganisms in the initial stage before they fully adapted to the toxicity of the oilfield wastewater. Simultaneously, the introduction of the target substrate and initial salinity and toxicity stress with an equal proportion of oilfield wastewater gradually exposed the microorganisms to the selective pressures of the wastewater.

[0049] As the cycle progresses, a gradient increasing strategy is adopted, gradually reducing the proportion of PBS buffer nutrient solution while simultaneously increasing the proportion of oilfield wastewater. The volume ratio of the two is gradually adjusted between 1:1 and 1:4. Specifically, the ratio is 1:1 in the first cycle, 1:2 in the second cycle, 1:4 in the third cycle, and 0:1 in the fourth cycle and thereafter (i.e., using oilfield wastewater as the feed).

[0050] This gradient acclimatization process, through progressively intensified concentration stress, forces the elimination of non-functional bacteria in the microbial community that cannot tolerate the high salinity and toxicity of oilfield wastewater. Simultaneously, the continuous current flow in the MFC external circuit creates a stable electric field selection voltage, enabling electroactive microorganisms capable of directionally transferring electrons generated by intracellular metabolism to the anode to gain a sustained competitive advantage. The combined effect of these two selection pressures leads to Geobacteraceae (electroactive + degradation function) and Thaurea (specific degradation function) simultaneously becoming the dominant bacterial groups in the anode biofilm.

[0051] Test Example 1 This test case is a comprehensive characterization of the system performance after the MFC reactor in Example 1 has been running stably.

[0052] First, linear sweep voltammetry was performed on the MFC reactor using an electrochemical workstation to obtain polarization curves (voltage-current density curves) and power density curves (power density-current density curves). The measured results are as follows: Figure 1 As shown, the anolyte biofilm enriched in this invention enables the MFC reactor to achieve a maximum power density of 1358 mW / m³. 2 This indicates that the anodic biofilm has extremely high electron transfer activity and electrogenic performance, and the electrochemical reaction kinetics at the electrode / biofilm interface are excellent.

[0053] Secondly, an electrochemical impedance spectroscopy test was performed on the system using an electrochemical workstation to fit an equivalent circuit model, such as... Figure 2 As shown in the figure. The measured results show that the ohmic impedance Rs is 42Ω (reflecting the sum of electrolyte resistance, electrode resistance and contact resistance), the polarization impedance Rp is 8.8Ω (reflecting the charge transfer resistance at the biofilm / electrode interface), and the total internal resistance of the system is 50.8Ω, which is at a low level. This indicates that the air cathode and anode biofilms prepared in this invention have excellent electron transfer performance, and the system has low energy loss, which is beneficial for efficient power generation.

[0054] Subsequently, reactor effluent was collected after 20 and 40 days of cultivation, and COD was determined according to the standard potassium dichromate method to calculate the COD removal rate. At the same time, the coulombic efficiency was calculated based on the measured cumulative charge and the theoretical maximum charge.

[0055] Experimental results are as follows Figure 3 As shown, the anodic biofilm enriched by this invention achieves a COD removal rate of over 85% and a coulombic efficiency of over 35% for real oilfield wastewater. The treatment effects of the 20-day and 40-day samples show little difference, indicating that a stable and mature biofilm has been successfully enriched on the anodic carbon brush, and its activity can be maintained over a long period.

[0056] Figure 4 The complete It curve of the MFC startup and enrichment process is shown. The system experienced multiple stable power generation cycles during operation and maintained good operating performance throughout the 50-day continuous monitoring period. The peak current fluctuated little during each cycle, proving that the biofilm structure enriched by this invention is stable and durable, meeting the requirements for long-term engineering applications.

[0057] Figure 5 Images (a) and (b) show SEM images of the biofilm enriched on the carbon fiber surface of the carbon brush anode at different magnifications. The images clearly show that the carbon fiber surface is tightly covered by a dense and uniform microbial film, with various microbial morphologies coexisting, diverse bacterial community morphologies, and a well-developed three-dimensional network structure, indicating that the biofilm is stably attached and has a high coverage.

[0058] Figure 6 TEM images and elemental distribution of functional microorganisms in the anodic biofilm are presented. Figure 6 (a) in the image is a TEM image. Figure 6 (b) in the figure is a HAADF diagram, which clearly shows the ultrastructure of microorganisms. Figure 6 (c) in the figure is the EDS map and (d~h) in the 6 figures are the element mapping diagrams, which further verify the elemental composition characteristics of the bacteria and corroborate the 16S rRNA sequencing results.

[0059] Total DNA was extracted from the biofilm on the surface of the anode carbon brush, and 16S rRNA gene high-throughput sequencing (V3~V4 region) was performed to analyze the community structure of functional microorganisms. Figure 7 The relative abundance analysis of the microbial community showed that Geobacteraceae and Thaurea were the two dominant species in the anodic biofilm.

[0060] Geobacteraceae is the core functional family of bacteria driving MFC (Microbial Fusion) electricity generation. It is one of the microbial groups with the strongest known extracellular electron transport capacity. Through cytochrome c-mediated direct contact electron transport (DMET) and conductive fimbriae (microbial nanowires) mechanisms, it efficiently outputs electrons generated by metabolism to the anode, achieving continuous electricity generation while degrading organic substrates.

[0061] Thauera has been repeatedly identified as a dominant functional bacterial group in various petrochemical / refining wastewater biochemical treatment systems. It has the specific metabolic ability to degrade petroleum hydrocarbon organic pollutants such as benzene series and polycyclic aromatic hydrocarbons under anaerobic or microaerobic conditions. It is one of the key functional bacterial genera for treating oilfield wastewater. Its high abundance accumulation in biofilm is an important microbiological basis for the high efficiency of oilfield wastewater degradation in the biofilm obtained in this invention.

[0062] The simultaneous dominance of two types of functional bacteria in the anodic biofilm is a direct manifestation of the synergistic acclimatization effect of the MFC electric field selection pressure and gradient concentration stress. The electric field screening directionally retains electrogenic bacteria, while the concentration stress directionally retains degradative bacteria. The two types of bacteria highly overlap in the specific application scenario of oilfield wastewater treatment, achieving precise and synchronous enrichment of functional bacterial communities.

[0063] Finally, it should be noted that the described embodiments are merely some, not all, of the embodiments of the present invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the claims and their equivalents; that is, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for gradient domestication and enrichment of a microbial fuel cell anode biofilm, characterized in that, Includes the following steps: S1, a carrier material for microbial growth calcined at 400~700℃ to remove surface impurities and improve surface hydrophilicity; S2. A catalyst is loaded onto an air cathode; S3. Construct a microbial fuel cell reactor, using the pretreated carrier material as the anode and the prepared air cathode as the cathode; S4. Add PBS buffer nutrient solution, oilfield wastewater and trace element solution to the microbial fuel cell reactor; S5. Expel the air from the microbial fuel cell reactor to bring it into an anaerobic state; S6. Connect a resistor in series in the external circuit of the microbial fuel cell reactor; S7. Cultivate microorganisms under constant temperature conditions of 25~35℃, and monitor the voltage across the external resistor in real time. When the voltage across the external resistor is lower than 30mV, repeat steps S4, S5, and S6 and cultivate microorganisms under constant temperature conditions of 25~35℃. In each cycle, gradually reduce the proportion of PBS buffer nutrient solution and simultaneously increase the proportion of acclimatization substrate. Specifically, in the first, second, and third cycles, the volume ratio of PBS buffer nutrient solution to acclimatization substrate is 1:1, 1:2, and 1:4, respectively. In the fourth cycle and thereafter, the volume ratio of PBS buffer nutrient solution to acclimatization substrate is 0:

1. This allows the microbial community to complete adaptive succession in a gradually increasing substrate stress environment until it uses acclimatization substrate as the sole feed and maintains operation.

2. The method according to claim 1, characterized in that, The carrier material in S1 is a porous carbon material, which can be one of carbon felt, carbon cloth, carbon paper or carbon brush; the processing time is 30~120 minutes.

3. The method according to claim 1, characterized in that, The catalyst supported on the air cathode in S2 is one or more of a Pt / C catalyst or a transition metal / nitrogen-doped carbon composite catalyst.

4. The method according to claim 1, characterized in that, S4 contains PBS buffer solution containing sodium or potassium dihydrogen phosphate, disodium or potassium hydrogen phosphate, ammonium chloride, potassium chloride, calcium chloride dihydrate, and a multivitamin solution.

5. The method according to claim 4, characterized in that, S4 contains PBS buffered nutrient solution consisting of: 15.4 mmol / L sodium dihydrogen phosphate, 32.3 mmol / L disodium hydrogen phosphate, 5.8 mmol / L ammonium chloride, 1.7 mmol / L potassium chloride, 10 mg / L calcium chloride dihydrate, and 2 mL / L multivitamin solution.

6. The method according to claim 1, characterized in that, S4 contains trace element solutions that are salt solutions of at least three of the following elements: boron, cobalt, tungsten, selenium, chlorine, nickel, zinc, or molybdenum.

7. The method according to claim 1, characterized in that, In the first, second, and third cycles, the volume ratio of PBS buffered nutrient solution to acclimatization substrate was 1:1, 1:2, and 1:4, respectively. In the fourth cycle and thereafter, the volume ratio of PBS buffered nutrient solution to acclimatization substrate was 0:

1.

8. The method according to claim 1, characterized in that, The calcination temperature in S1 is 550℃, and the calcination time is 60 minutes.

9. The method according to claim 1, characterized in that, The resistance of the external resistor in S6 is 1000Ω.

10. The application of the method as described in any one of claims 1-9 in the treatment of oilfield wastewater.

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