Carbon-based electrode-microorganism self-assembled compound as well as preparation and application thereof

Abstract

The invention discloses a carbon-based electrode-microorganism self-assembled compound, a preparation method thereof and application of the carbon-based electrode-microorganism self-assembled compound in strengthening anaerobic fermentation of agricultural wastes. The compound is formed by self-assembly of direct interspecific electron transfer (DITE) functional microorganisms (anode reduction geobacter and methanobacterium flexural) on the surface of a high-conductivity carbon-based electrode under the induction of a micro electric field, the relative abundance of the anode reduction geobacter is 65-82%, and the relative abundance of the methanobacterium flexural is 18-35%. During preparation, the carbon-based electrode is cleaned by acetone, ethanol and water, then the carbon-based electrode, a reference electrode and a counter electrode form a three-electrode system, and the three-electrode system is induced for 3-5 days in a micro electric field of-0.2-0.2 V to form a compound. 1-5 g / L of the compound is added into a straw-excrement mixed substrate (25-40 g VS / L) subjected to alkali pretreatment, anaerobic fermentation is conducted for 50 d after biogas slurry is inoculated, the pH can be stably maintained at 7.2-8.0, the accumulated biogas yield reaches up to 450-630 mL / g VS, and the volume fraction of methane is 68-82%. The process is simple, the compound is high and stable in activity, and efficient resource utilization of agricultural waste can be achieved.

Description

Technical Field

[0001] The present invention belongs to the field of resource utilization of agricultural waste, and particularly relates to a carbon-based electrode-microorganism self-assembly complex and its preparation and application in strengthening anaerobic fermentation of agricultural waste. Background Art

[0002] Anaerobic fermentation technology, as an important pathway for the resource utilization of agricultural waste, demonstrates significant environmental benefits and resource recycling value. Through three stages: hydrolysis and acidification, hydrogen and acetic acid production, and methane production, this technology efficiently converts biomass wastes such as crop straw and livestock manure into biogas energy, primarily composed of methane. This energy source can replace traditional fossil fuels and directly meet the energy needs of agricultural production. Furthermore, the fermented biogas slurry and residue are rich in nutrients and can be made into high-quality organic fertilizer to enhance soil fertility. However, traditional anaerobic fermentation relies on hydrogen-mediated interspecies electron transport between acid-producing and methanogenic bacteria. This process is susceptible to system acidification due to thermodynamic limitations caused by high hydrogen partial pressure, leading to reaction stagnation and a key bottleneck restricting the efficient resource utilization of this technology. Microbial DIET bypasses the hydrogen generation and transfer process, directly enabling interspecies energy exchange between microorganisms through conductive materials such as cell appendages. This provides a new approach to fundamentally circumventing the inhibitory effects of elevated hydrogen partial pressure on acid-producing bacteria. However, to date, how to effectively utilize DIET in anaerobic fermentation systems and improve methane production efficiency remains an urgent problem to be solved. Summary of the Invention

[0003] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a carbon-based electrode-microbial self-assembly complex and its preparation and application. This method is based on the natural interaction between DIET functional microorganisms and carbon-based electrodes to prepare a highly stable and highly active carbon-based electrode-microbial self-assembly complex, which can directionally enhance the anaerobic fermentation DIET process to improve the methane production efficiency and realize the efficient resource utilization of agricultural waste.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is:

[0005] A method for preparing a carbon-based electrode-microorganism self-assembled complex comprises the following steps:

[0006] Step 1: A carbon-based electrode is used as a working electrode, and together with an Ag / AgCl reference electrode and a platinum counter electrode, a three-electrode reactor is formed. The three-electrode reactor is placed in an anaerobic environment, a buffer solution is added, and DIET functional bacteria are inoculated.

[0007] Step 2: applying a micro-electric field to the three-electrode reactor to directionally load the DIET functional bacteria on the surface of the carbon-based electrode and self-assemble to form a carbon-based electrode-microorganism complex.

[0008] In one embodiment, the carbon-based electrode is one of carbon fiber brush, carbon cloth or carbon felt, and the buffer solution is a phosphate or carbonate buffer solution.

[0009] In one embodiment, the DIET functional bacteria are composed of equal amounts of Geobacter anodireducens and Methanobacterium flexile, and the Geobacter anodireducens and Methanobacterium flexile have been deposited in the China General Microbiological Culture Collection Center (CGMCC), wherein the classification name of Geobacter anodireducens is Geobacter anodireducens, and the preservation number is 1.12536, and the classification name of Methanobacterium flexile is Methanobacterium flexile, and the preservation number is 1.5092.

[0010] The aforementioned Geobacter anodoreductus and Methanobacterium curvatum are both known in the art. For example, Geobacter anodoreductus (CGMCC: 1.12536) has been disclosed in Chinese Patent CN109022319A, and Methanobacterium curvatum (CGMCC: 1.5092) has been disclosed in Chinese Patent CN119019981A. Both can be purchased commercially.

[0011] In one embodiment, in step 1, the total OD of the bacterial solution after inoculation is 600 The value is 0.05~0.20.

[0012] In one embodiment, in step 2, a micro-electric field is applied to the three-electrode reactor through an electrochemical workstation, the micro-electric field is applied in a constant potential mode, the potential of the carbon-based electrode (relative to the reference electrode Ag / AgCl) is set in the range of -0.2 to 0.2 V, and the induction time is 3 to 5 days.

[0013] A micro-electric field was applied to the three-electrode reactor. The specific wiring was as follows: the carbon-based electrode, platinum counter electrode, and Ag / AgCl reference electrode were fixed and led out with titanium wires, and then connected to the corresponding ports on the electrochemical workstation. The potential was applied to the carbon-based electrode through the control software.

[0014] The present invention also claims protection for a carbon-based electrode-microorganism self-assembly complex obtained by the preparation method of the carbon-based electrode-microorganism self-assembly complex, wherein the relative abundance of Geobacter anode-reducens in the complex bacteria is 65-82%, and the relative abundance of Methanobacterium curvatum is 18-35%.

[0015] The present invention also claims the use of the carbon-based electrode-microorganism self-assembly complex for enhancing anaerobic fermentation of agricultural waste.

[0016] The agricultural waste described in the present invention is straw, and the anaerobic fermentation comprises the following steps:

[0017] (1) crushing the straw, pre-treating it with an alkaline solution, and then drying it, mixing it evenly with livestock and poultry manure, and adding water to adjust the volatile solid content of the mixture to obtain a fermentation substrate;

[0018] (2) Anaerobic fermentation enhancement: adding the complex to the fermentation substrate, inoculating the anaerobic fermentation slurry, and performing anaerobic fermentation in a closed reactor.

[0019] In one embodiment, the addition amount of the complex is 1-5 g / L, the pH is maintained at 7.2-8.0 during the fermentation process, and after 50 days of fermentation, the cumulative biogas production in the fermentation system is 450-630 mL / g VS, wherein the methane volume fraction is 68-82%.

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

[0021] (1) The composite provided by the present invention uses carbon-based electrodes as carriers, which have advantages in terms of microbial loading and fermentation enhancement. First, carbon-based electrodes have good biocompatibility and a large specific surface area, making them the preferred growth carriers for DIET functional microorganisms. They can load more functional bacteria than metal-based carriers such as iron. Second, compared with conventional microbial carriers such as biochar, carbon-based electrodes have high conductivity, which can accelerate the efficiency of electron transfer between microorganisms and further improve the efficiency of methane production.

[0022] (2) The microbial cells in the complex provided by the present invention use two DIET functional bacteria, namely, Geobacter anode-reducing and Methanobacterium curvatum. Among them, Geobacter anode-reducing has been proven to be a microorganism with growth advantages on carbon-based electrodes, and has the functions of fatty acid metabolism and electron transfer. Therefore, its presence can not only build an efficient electron transfer network with methanogens, but also consume fatty acids in time to alleviate the acidification problem and improve system stability. In addition, these two functional bacteria have a unique ecological niche in the anaerobic fermentation system. Once they exist, they can occupy a long-term competitive advantage without the need for repeated addition.

[0023] (3) The present invention utilizes the natural tropism of DIET functional bacteria to electric fields and, based on electrochemical principles, provides a method for inducing the self-assembly of DIET functional bacteria and carbon-based electrodes into a complex through micro-electric field induction. Compared with complex physical and chemical binding methods, this method enables DIET functional bacteria to attach specifically and quickly to carbon-based electrodes. The operation is simple, efficient, and does not damage the activity of microorganisms. At the same time, this method can also stimulate the DIET functional bacteria to secrete extracellular polymers (EPS) with adhesive properties, so that the microorganisms are tightly bound to the electrode material, which can effectively avoid the loss of microorganisms during use.

[0024] (4) The carbon-based electrode-microbial self-assembled composite can effectively enhance the DIET process during anaerobic fermentation, avoid system acidification caused by hydrogen accumulation, and reconstruct the anaerobic microbial metabolic network, thereby breaking through thermodynamic limitations and achieving efficient anaerobic methane production. Based on this, the composite provided by the present invention and its method for enhancing anaerobic fermentation are a strategy to fundamentally address the problem of low anaerobic fermentation efficiency, helping to transform agricultural waste into valuable resources and promote the resource utilization of agricultural waste. DETAILED DESCRIPTION

[0025] In order to explain the present invention in detail, the present invention will be further described by the following specific examples. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0026] The present invention discloses a carbon-based electrode-microorganism self-assembly complex, its preparation method, and its application in enhancing anaerobic fermentation of agricultural waste. The complex is formed by self-assembly of direct interspecies electron transfer (DIET)-functional microorganisms on the surface of a highly conductive carbon-based electrode using a micro-electric field. The preparation method can be described as follows:

[0027] (1) Pretreatment of carbon-based electrodes.

[0028] Select a highly conductive carbon-based material (such as a carbon fiber brush, carbon cloth, carbon felt, etc.) as the load electrode. First, soak it in acetone for 24 hours to remove organic impurities, then wash it three times with anhydrous ethanol, rinse it with distilled water, and then place it in an oven to dry. The purpose of this step is mainly to keep the surface of the carbon-based electrode clean to improve the subsequent assembly effect. It is easy to understand that in some embodiments, this step can also be omitted, especially when the carbon-based electrode surface is highly clean.

[0029] (2) Construction of self-assembly system.

[0030] The self-assembly system consists of a three-electrode reactor, a buffer solution and DIET functional bacteria. The carbon-based electrode pretreated in step (1) is used as the working electrode, and an Ag / AgCl reference electrode and a platinum counter electrode are configured to build a three-electrode reactor in an anaerobic reagent bottle covered with a silicone stopper. A phosphate or carbonate solution is used as the buffer solution, and the DIET functional bacteria are inoculated. The DIET functional bacteria in the embodiment of the present invention include equal amounts of Geobacter anodireducens and Methanobacterium flexile, and the initial total OD of the inoculated bacterial solution is 0.05. 600The values ​​are 0.05 to 0.20. Both have been deposited in the China General Microbiological Culture Collection Center (CGMCC), of which the deposition number of Geobacter anodoreducens is 1.12536 and that of Methanobacterium curvatum is 1.5092.

[0031] (3) Self-assembly induced by micro-electric field.

[0032] A micro-electric field is applied to the three-electrode reactor obtained in step (2) using an electrochemical workstation, i.e., the working potential of the carbon-based electrode is set to -0.2 to 0.2 V. After 3 to 5 days of micro-electric field induction, the DIET functional bacteria are naturally loaded on the surface of the carbon-based electrode due to their electric field tropism and self-assemble into a stable carbon-based electrode-microorganism complex. In the carbon-based electrode-microorganism complex prepared by the above method, the relative abundance of Geobacter reductus is 65 to 82%, and the relative abundance of Methanobacterium curvatum is 18 to 35%.

[0033] The present invention also provides a method for enhancing anaerobic fermentation of agricultural waste using the carbon-based electrode-microorganism composite, which mainly comprises the following steps:

[0034] (1) Preparation of fermentation materials.

[0035] Crop straw was crushed to 1-2 cm, mixed with a 4% (w / v) potassium hydroxide solution, sealed and treated at room temperature for 3 days, and then dried for later use. The alkali-pretreated crop straw was then mixed with livestock and poultry manure at a dry weight ratio of 3:1, stirred evenly, and water was added to adjust the volatile solids (VS) content to 25-40 g / L. The mixture was then placed in a sealed container as a fermentation substrate.

[0036] (2) Intensification of anaerobic fermentation.

[0037] The carbon-based electrode-microbial self-assembly complex provided by the present invention is added to the above-mentioned fermentation substrate, and the specific addition ratio is controlled at 1 to 5 g / L, where the mass of the complex is calculated by wet weight. 10% (v / v) anaerobic fermentation slurry is inoculated and fermented under anaerobic conditions for 50 days. During the fermentation process, the pH is maintained at 7.2 to 8.0, and the system stability is high. At the same time, the anaerobic fermentation efficiency is also improved, with a cumulative biogas production of 450 to 630 mL / gVS, of which the methane content is as high as 68 to 82%.

[0038] The process of the invention is simple, the composite material is highly active and stable, and efficient resource utilization of agricultural waste can be achieved.

[0039] The following are several specific embodiments of the present invention.

[0040] Example 1

[0041] (1) Preparation of carbon-based electrode-microorganism self-assembly complex: A carbon fiber brush with a diameter of 2 cm and a length of 3 cm was selected as the carbon-based electrode, and washed and pretreated with acetone, anhydrous ethanol, and distilled water in sequence, and then dried. The dried carbon-based electrode was used as the working electrode, and an Ag / AgCl reference electrode and a platinum counter electrode were configured to construct a three-electrode reactor in a 100 mL anaerobic reagent bottle. After adding phosphate buffer solution to the reactor, the total OD was inoculated according to the OD value. 600 A complete self-assembly system was constructed by incorporating equal numbers of Geobacter anodically reduced and Methanobacterium curvatum into the carbon-based electrode at a ratio of 0.10. Using an electrochemical workstation, a microelectric field was applied to the carbon-based electrode, setting the working potential at -0.1V. After four days of microelectric field induction, the microorganisms were loaded onto the carbon-based electrode surface and self-assembled into a stable carbon-based electrode-microorganism complex. Within this complex, Geobacter anodically reduced comprised 75% of the microorganisms, while Methanobacterium curvatum accounted for 25%.

[0042] (2) Preparation of fermentation materials: After wheat straw was crushed to approximately 1.5 cm, a 4% (w / v) potassium hydroxide solution was used to seal the reaction at room temperature for 3 days, and then dried for use. The wheat straw and cow dung after alkali pretreatment were weighed at a dry weight ratio of 3:1 and mixed and stirred evenly in a 1 L sealed blue-mouth bottle. Water was added to adjust the fermentation substrate concentration to 30 g VS / L. At this point, the volume of the anaerobic fermentation system was 750 mL.

[0043] (3) Strengthening anaerobic fermentation: Weigh 2 g of the carbon-based electrode-microbial self-assembly complex prepared above, add it to the anaerobic fermentation system and mix it evenly. Then, inoculate 10% (v / v) of anaerobic fermentation slurry and ferment it under anaerobic conditions for 50 days. During the entire fermentation process, the pH was always maintained at around 7.5. Ultimately, the system's cumulative biogas production was 520 mL / g VS, of which methane production was 400 mL / g VS, accounting for as much as 77%. This shows that the carbon-based electrode-self-assembly complex provided by the present invention not only helps to maintain the system stability of the anaerobic fermentation system, but also effectively improves the efficiency of fermentation to produce methane.

[0044] Example 2

[0045] (1) Preparation of carbon-based electrode-microorganism self-assembly complex: 2cm×3cm carbon felt was selected as the carbon-based electrode, and it was washed and pretreated with acetone, anhydrous ethanol and distilled water in sequence and then dried. The dried carbon-based electrode was used as the working electrode, and an Ag / AgCl reference electrode and a platinum sheet counter electrode were configured to construct a three-electrode reactor in a 100mL anaerobic reagent bottle. After adding phosphate buffer solution to the reactor, the total OD was inoculated according to the OD value. 600A complete self-assembly system was constructed by incorporating equal numbers of Geobacter anodoreductus and Methanobacterium curvatum into the carbon-based electrode at a ratio of 0.15. Using an electrochemical workstation, a microelectric field was applied to the carbon-based electrode, setting the working potential at -0.1V. After three days of microelectric field induction, the microorganisms were loaded onto the carbon-based electrode surface and self-assembled into a stable carbon-based electrode-microorganism complex. Within this complex, Geobacter anodoreductus accounted for 78% of the microorganisms, while Methanobacterium curvatum accounted for 22%.

[0046] (2) Preparation of fermentation materials: After crushing corn straw to approximately 1 cm, seal the mixture with 4% (w / v) potassium hydroxide solution at room temperature for 3 days and dry it for later use. Weigh the alkaline-pretreated corn straw and cow dung at a dry weight ratio of 3:1 and mix them evenly in a 500 mL sealed blue-mouth bottle. Add water to adjust the fermentation substrate concentration to 35 g VS / L. At this point, the volume of the anaerobic fermentation system is 380 mL.

[0047] (3) Strengthening anaerobic fermentation: Weigh 1.5 g of the carbon-based electrode-microorganism self-assembly complex prepared above, add it to the anaerobic fermentation system and mix it evenly. Then, inoculate 10% (v / v) of anaerobic fermentation slurry and ferment it under anaerobic conditions for 50 days. During the entire fermentation process, the pH was always maintained at around 7.4. Ultimately, the system's cumulative biogas production was 580 mL / g VS, of which methane production was 460 mL / g VS, accounting for as much as 79%. This shows that the carbon-based electrode-self-assembly complex provided by the present invention not only helps to maintain the system stability of the anaerobic fermentation system, but also effectively improves the efficiency of fermentation to produce methane.

[0048] Example 3

[0049] (1) Preparation of carbon-based electrode-microorganism self-assembly complex: 3cm×6cm carbon cloth was selected as the carbon-based electrode, and it was washed and pretreated with acetone-anhydrous ethanol-distilled water in sequence and then dried. The dried carbon-based electrode was used as the working electrode, and an Ag / AgCl reference electrode and a platinum sheet counter electrode were configured to construct a three-electrode reactor in a 250mL anaerobic reagent bottle. After adding phosphate buffer solution to the reactor, the total OD was inoculated according to the OD value. 600 A ratio of 0.08 was used to construct a complete self-assembly system. Equal numbers of Geobacter anodically reduced and Methanobacterium curvatum were added to the carbon-based electrode using an electrochemical workstation, setting the working potential of the carbon-based electrode to 0V and applying a microelectric field. After 5 days of microelectric field induction, the microorganisms were loaded onto the carbon-based electrode surface and self-assembled into a stable carbon-based electrode-microorganism complex. Within this complex, Geobacter anodically reduced comprised 69% of the microorganisms, while Methanobacterium curvatum accounted for 31%.

[0050] (2) Preparation of fermentation materials: After crushing corn straw to approximately 1.5 cm, seal the mixture with 4% (w / v) potassium hydroxide solution at room temperature for 3 days and dry it for later use. Weigh the alkaline-pretreated corn straw and pig manure at a dry weight ratio of 3:1 and mix them evenly in a 2L sealed blue-mouth bottle. Add water to adjust the fermentation substrate concentration to 28g VS / L. At this point, the volume of the anaerobic fermentation system is 1500mL.

[0051] (3) Strengthening anaerobic fermentation: Weigh 4.5 g of the carbon-based electrode-microbial self-assembly complex prepared above, add it to the anaerobic fermentation system and mix it evenly. Then, inoculate 10% (v / v) of anaerobic fermentation slurry and ferment it under anaerobic conditions for 50 days. During the entire fermentation process, the pH was always maintained at around 7.8. Ultimately, the system's cumulative biogas production was 540 mL / g VS, of which methane production was 400 mL / g VS, accounting for as much as 74%. This shows that the carbon-based electrode-self-assembly complex provided by the present invention not only helps to maintain the system stability of the anaerobic fermentation system, but also effectively improves the efficiency of fermentation to produce methane.

[0052] Example 4

[0053] (1) Preparation of carbon-based electrode-microorganism self-assembly complex: A carbon fiber brush with a diameter of 3 cm and a length of 3 cm was selected as the carbon-based electrode, and washed and pretreated with acetone, anhydrous ethanol, and distilled water in sequence, and then dried. The dried carbon-based electrode was used as the working electrode, and an Ag / AgCl reference electrode and a platinum counter electrode were configured to construct a three-electrode reactor in a 100 mL anaerobic reagent bottle. After adding carbonate buffer solution to the reactor, the total OD was inoculated according to the OD value. 600 A complete self-assembly system was constructed by incorporating equal numbers of Geobacter anodically reduced and Methanobacterium curvatum into the carbon-based electrode at a ratio of 0.2. Using an electrochemical workstation, a microelectric field was applied to the carbon-based electrode, setting the working potential at 0.2V. After three days of microelectric field induction, the microorganisms were loaded onto the carbon-based electrode surface and self-assembled into a stable carbon-based electrode-microorganism complex. Within this complex, Geobacter anodically reduced comprised 66% of the microorganisms, while Methanobacterium curvatum accounted for 34%.

[0054] (2) Preparation of fermentation materials: After wheat straw was crushed to approximately 2 cm, a 4% (w / v) potassium hydroxide solution was added to the mixture and sealed for reaction at room temperature for 3 days. The mixture was then dried for later use. The alkali-pretreated wheat straw and pig manure were weighed at a dry weight ratio of 3:1 and mixed uniformly in a 2 L sealed blue-mouth bottle. Water was added to adjust the fermentation substrate concentration to 40 g VS / L. At this point, the volume of the anaerobic fermentation system was 1500 mL.

[0055] (3) Strengthening anaerobic fermentation: Weigh 2 g of the carbon-based electrode-microbial self-assembly complex prepared above, add it to the anaerobic fermentation system and mix it evenly. Then, inoculate 10% (v / v) of anaerobic fermentation slurry and ferment it under anaerobic conditions for 50 days. During the entire fermentation process, the pH was always maintained at around 7.9. Ultimately, the system's cumulative biogas production was 460 mL / g VS, of which methane production was 310 mL / g VS, accounting for as much as 67%. This shows that the carbon-based electrode-self-assembly complex provided by the present invention not only helps to maintain the system stability of the anaerobic fermentation system, but also effectively improves the efficiency of fermentation to produce methane.

[0056] Example 5

[0057] (1) Preparation of carbon-based electrode-microorganism self-assembly complex: 3cm×3cm carbon felt was selected as the carbon-based electrode, and it was washed and pretreated with acetone-anhydrous ethanol-distilled water in sequence and then dried. The dried carbon-based electrode was used as the working electrode, and an Ag / AgCl reference electrode and a platinum sheet counter electrode were configured to construct a three-electrode reactor in a 100mL anaerobic reagent bottle. After adding carbonate buffer solution to the reactor, the total OD was inoculated according to the OD value. 600 A complete self-assembly system was constructed by incorporating equal numbers of Geobacter anodically reduced and Methanobacterium curvatum into the carbon-based electrode at a ratio of 0.1. Using an electrochemical workstation, the carbon-based electrode was set to a working potential of -0.2V to apply a microelectric field. After five days of microelectric field induction, the microorganisms were loaded onto the carbon-based electrode surface and self-assembled into a stable carbon-based electrode-microorganism complex. Within this complex, Geobacter anodically reduced comprised 76% of the microorganisms, while Methanobacterium curvatum accounted for 24%.

[0058] (2) Preparation of fermentation materials: After wheat straw was crushed to approximately 1 cm, a 4% (w / v) potassium hydroxide solution was used to seal the reaction at room temperature for 3 days, and then dried for use. The alkali-pretreated wheat straw and chicken manure were weighed at a dry weight ratio of 3:1 and mixed and stirred evenly in a 1 L sealed blue-mouth bottle. Water was added to adjust the fermentation substrate concentration to 35 g VS / L. At this point, the volume of the anaerobic fermentation system was 800 mL.

[0059] (3) Strengthening anaerobic fermentation: Weigh 3 g of the carbon-based electrode-microbial self-assembly complex prepared above, add it to the anaerobic fermentation system and mix it evenly. Then, inoculate 10% (v / v) of anaerobic fermentation slurry and ferment it under anaerobic conditions for 50 days. During the entire fermentation process, the pH was always maintained at around 7.3. Ultimately, the system's cumulative biogas production was 560 mL / g VS, of which methane production was 450 mL / g VS, accounting for as much as 80%. This shows that the carbon-based electrode-self-assembly complex provided by the present invention not only helps to maintain the system stability of the anaerobic fermentation system, but also effectively improves the efficiency of fermentation to produce methane.

[0060] Example 6

[0061] (1) Preparation of carbon-based electrode-microorganism self-assembly complex: 2cm×4cm carbon cloth was selected as the carbon-based electrode, and it was washed and pretreated with acetone-anhydrous ethanol-distilled water in sequence and then dried. The dried carbon-based electrode was used as the working electrode, and an Ag / AgCl reference electrode and a platinum sheet counter electrode were configured to construct a three-electrode reactor in a 100mL anaerobic reagent bottle. After adding carbonate buffer solution to the reactor, the total OD was inoculated according to the OD value. 600 A ratio of 0.16 was used to construct a complete self-assembly system. Equal numbers of Geobacter anodically reduced and Methanobacterium curvatum were added to the carbon-based electrode using an electrochemical workstation with a working potential of 0V to apply a microelectric field. After four days of microelectric field induction, the microorganisms were loaded onto the carbon-based electrode surface and self-assembled into a stable carbon-based electrode-microorganism complex. Within this complex, Geobacter anodically reduced comprised 74% of the microorganisms, while Methanobacterium curvatum accounted for 26%.

[0062] (2) Preparation of fermentation materials: After crushing corn straw to approximately 1.5 cm, seal and react with 4% (w / v) potassium hydroxide solution at room temperature for 3 days, then dry and set aside. Weigh the alkaline-pretreated corn straw and chicken manure at a dry weight ratio of 3:1 and mix them evenly in a 1L sealed blue-mouth bottle. Add water to adjust the fermentation substrate concentration to 30 g VS / L. At this point, the volume of the anaerobic fermentation system is 780 mL.

[0063] (3) Strengthening anaerobic fermentation: Weigh 3 g of the carbon-based electrode-microbial self-assembly complex prepared above, add it to the anaerobic fermentation system and mix it evenly. Then, inoculate 10% (v / v) of anaerobic fermentation slurry and ferment it under anaerobic conditions for 50 days. During the entire fermentation process, the pH was always maintained at around 7.5. Ultimately, the system's cumulative biogas production was 600 mL / g VS, of which methane production was 490 mL / g VS, accounting for as much as 82%. This shows that the carbon-based electrode-self-assembly complex provided by the present invention not only helps to maintain the system stability of the anaerobic fermentation system, but also effectively improves the efficiency of fermentation to produce methane.

Claims

1. A method for preparing a carbon-based electrode-microorganism self-assembled complex, characterized in that: The following steps are involved: Step 1: A carbon-based electrode is used as a working electrode, and together with an Ag / AgCl reference electrode and a platinum counter electrode, a three-electrode reactor is formed. The three-electrode reactor is placed in an anaerobic environment, a buffer solution is added, and DIET functional bacteria are inoculated. Step 2: applying a micro-electric field to the three-electrode reactor to directionally load the DIET functional bacteria on the surface of the carbon-based electrode and self-assemble to form a carbon-based electrode-microorganism complex.

2. The method for preparing the carbon-based electrode-microorganism self-assembled complex according to claim 1, characterized in that: The carbon-based electrode is one of carbon fiber brush, carbon cloth or carbon felt, and the buffer solution is a phosphate or carbonate buffer solution.

3. The method for preparing the carbon-based electrode-microorganism self-assembled complex according to claim 1, characterized in that: The DIET functional bacteria are composed of equal amounts of Geobacter anodireducens and Methanobacterium flexile. Both Geobacter anodireducens and Methanobacterium flexile have been deposited in the China General Microbiological Culture Collection Center (CGMCC). Geobacter anodireducens is classified as Geobacter anodireducens with a deposit number of 1.12536, and Methanobacterium flexile is classified as Methanobacterium flexile with a deposit number of 1.5092.

4. The method for preparing the carbon-based electrode-microorganism self-assembled complex according to claim 1 or 3, characterized in that: In step 1, the total OD of the bacterial solution after inoculation 600 The value is 0.05~0.

20.

5. The method for preparing the carbon-based electrode-microorganism self-assembled complex according to claim 1, characterized in that: In step 2, a micro-electric field is applied to the three-electrode reactor through an electrochemical workstation. The micro-electric field is applied in a constant potential mode, the potential range of the carbon-based electrode is set to -0.2 to 0.2 V, and the induction time is 3 to 5 days.

6. A carbon-based electrode-microorganism self-assembled complex, characterized in that: Prepared by the method according to any one of claims 1 to 5.

7. The carbon-based electrode-microorganism self-assembled complex according to claim 6, characterized in that: The DIET functional bacteria are composed of equal amounts of Geobacter anodireducens and Methanobacterium flexile. Both Geobacter anodireducens and Methanobacterium flexile have been deposited in the China General Microbiological Culture Collection Center (CGMCC). Geobacter anodireducens is classified as Geobacter anodireducens with a deposit number of 1.12536, and Methanobacterium flexile is classified as Methanobacterium flexile with a deposit number of 1.5092. The relative abundance of Geobacter anodireducens in the composite bacteria is 65-82%, and the relative abundance of Methanobacterium flexile is 18-35%.

8. Use of the carbon-based electrode-microorganism self-assembly complex according to claim 6 for enhancing anaerobic fermentation of agricultural waste.

9. The use according to claim 8, characterized in that The agricultural waste is straw, and the anaerobic fermentation comprises the following steps: (1) crushing the straw, pre-treating it with an alkaline solution, and then drying it, mixing it evenly with livestock and poultry manure, and adding water to adjust the volatile solid content of the mixture to obtain a fermentation substrate; (2) Anaerobic fermentation enhancement: adding the complex to the fermentation substrate, inoculating the anaerobic fermentation slurry, and performing anaerobic fermentation in a closed reactor.

10. The use according to claim 9, characterized in that The addition amount of the complex is 1-5 g / L, the pH is maintained at 7.2-8.0 during the fermentation process, and after 50 days of fermentation, the cumulative biogas production in the fermentation system is 450-630 mL / g VS, wherein the methane volume fraction is 68-82%.