JP2022057634A - Methane fermentation promotion method - Google Patents

Abstract

To provide a methane fermentation promotion method capable of exhibiting efficient and excellent processing capacity even under high load condition and also capable of easy scale-up.SOLUTION: A methane fermentation promotion method includes: (1) a bio-electrochemical chemical treatment process in which a carrier-holding electrode assembled with a carrier at least on a part of an electrode surface is brought into contact with a methane fermentation liquid involving an organic substrate and a microorganism group relating to methane fermentation for methane fermentation treatment of the organic substrate and (2) a fixed film fermentation process in which the fermentation liquid obtained by the bio-electrochemical chemical treatment process is brought into contact with a fixed film involving an electric conductive material. The bio-electrochemical chemical treatment process (1) is run under a controlled electric current of 3 μA/cm2 or under.SELECTED DRAWING: Figure 1

Description

The present invention relates to a method for promoting methane fermentation. In particular, the present invention comprises a methane fermentation promoting method including a two-step step of a bioelectrochemical treatment step and a fixed film fermentation step, a method for producing a fixed film carrying a microbial group, a methane fermentation promoting device, and a microbial group. The present invention relates to an apparatus for producing a fixed film containing a conductive material.

Organic resources (organic waste or organic waste liquid) produced from animals and plants such as wood, paper, food residue, agricultural residue, sewage sludge, human waste sludge, septic tank sludge, and livestock manure are also called biomass. Unlike fossil fuels, this biomass is produced by living organisms from water and carbon dioxide using solar energy, so sustainable renewable resources (biomass energy) such as these are sustainable developments. It is attracting attention as one of the goals (Sustainable Development Goals: SDGs).

As a method for producing biomass energy from these organic resources, methane fermentation, which promotes the decomposition of organic resources, is particularly known.
Methane fermentation means fermentation by a group of environmental microorganisms under anaerobic conditions, and flammable gases such as methane (CH ₄ ) and hydrogen (H ₂ ) can be obtained from the gas (biogas) generated by methane fermentation. Be done. The obtained combustible gas can be used as biomass energy for heat sources, power generation, and the like.
Therefore, it is an urgent task to establish a more efficient methane fermentation technology and improve the production volume (recovery rate) of methane gas in order to build an environment-friendly and clean sound material-cycle society.

As a method for methane fermentation of organic resources, for example, a bioelectrochemical system (bioelectrochemical system) using a group of environmental microorganisms such as methane-fermenting bacteria is known (for example, Patent Document 1).

As a bioelectrochemical system, Patent Document 1 includes a carrier-holding electrode having a hydrophobic carrier on at least a part of the electrode surface, an organic substrate, and a group of microorganisms involved in methane fermentation, and the organic substrate is used. Methane fermentation is performed while contacting with a methane fermentation liquid to be treated with methane fermentation and controlling the potential of the carrier holding electrode to + 0.3V based on the potential at which electrons are supplied from the carrier holding electrode or the potential of a silver or silver chloride electrode. A methane fermentation method characterized by carrying out the treatment is described.
Although the method described in Patent Document 1 can be used to promote methane fermentation, there is a demand for a technique capable of scaling up methane fermentation.

Generally, when a substance is treated by conversion (decomposition, oxidation, reduction, etc.) using a microorganism, the microorganism is subjected to high load conditions, for example, when the concentration of the object to be treated by the microorganism becomes a high concentration of a certain value or more. It is known that the function of the microorganism is reduced and the processing capacity is reduced.
This is no exception in the methane fermentation process, and scaling up the methane fermentation can be difficult, and under high load conditions where the organic matter loading rate or the hydraulic residence time is high, acid depletion of the methane fermentation tank occurs. Therefore, the methane fermentation processing ability may be significantly reduced.
When such a situation occurs, it becomes necessary to regenerate the methane fermentation tank, and the methane fermentation process is delayed. Therefore, it is desired to establish a methane fermentation method capable of continuous treatment without reducing the treatment capacity even under high load conditions.

Japanese Patent No. 587214

The present invention has been made in view of such a demand, and is a method for promoting methane fermentation that can efficiently exhibit excellent processing capacity even under high load conditions and can be easily scaled up. The purpose is to provide.

Another object of the present invention is to provide a method for producing a fixative film containing a conductive material carrying a group of microorganisms that exhibits excellent processing ability even under high load conditions.

That is, the present invention is as follows.
Item 1.
(1) A carrier holding electrode having a carrier on at least a part of the electrode surface,
A bioelectrochemical treatment step containing an organic substrate and a group of microorganisms involved in methane fermentation, and bringing the organic substrate into contact with a methane fermentation liquid to be treated with methane fermentation, and
(2) A methane fermentation promoting method comprising a fixed membrane fermentation step in which a fermented liquid obtained in the bioelectrochemical treatment step and a fixed membrane containing a conductive material are brought into contact with each other.
The above (1) bioelectrochemical treatment step is a method for promoting methane fermentation, which is carried out while controlling the current to 3 μA / cm ² or less.
Item 2.
In the above (1) bioelectrochemical treatment step
The carrier holding electrode is used as a working electrode.
In addition, it is equipped with a counter electrode, a reference electrode, and a potentiostat.
The working electrode, the counter electrode and the reference electrode are connected to the potentiostat, and the working electrode is connected to the potentiostat.
Item 2. The method for promoting methane fermentation according to Item 1, wherein the current of the working electrode is controlled by a three-electrode method.
Item 3.
In the above (2) fixed membrane fermentation step
Item 2. The method for promoting methane fermentation according to Item 1 or 2, wherein the conductive material is hydrophobic.
Item 4.
In the above (2) fixed membrane fermentation step
Item 6. The method for promoting methane fermentation according to any one of Items 1 to 3, wherein the conductive material is carbon fiber.
Item 5.
Item 2. The methane according to any one of Items 1 to 4, wherein the (2) fixed membrane fermentation step can increase methanogens in the fermentation broth obtained in the (1) bioelectrochemical treatment step. Fermentation promotion method.
Item 6.
Item 5. The method for promoting methane fermentation according to Item 5, wherein the methanogens are Methanobacterium sp. And Methanosarcina sp.
Item 7.
(1) A carrier holding electrode having a carrier on at least a part of the electrode surface,
A bioelectrochemical treatment step containing an organic substrate and a group of microorganisms involved in methane fermentation, and bringing the organic substrate into contact with a methane fermentation liquid to be treated with methane fermentation, and
(2) Preparation of a fixed membrane containing a conductive material carrying a group of microorganisms, which comprises a fixed membrane fermentation step in which the fermentation broth obtained in the bioelectrochemical treatment step is brought into contact with a fixed membrane containing a conductive material. It ’s a method,
The above-mentioned (1) bioelectrochemical treatment step is a method for producing a fixative film containing a conductive material carrying a group of microorganisms, which is carried out while controlling the current to 3 μA / cm ² or less.
Item 8.
In the above (1) bioelectrochemical treatment step
The carrier holding electrode is used as a working electrode.
In addition, it is equipped with a counter electrode, a reference electrode, and a potentiostat.
The working electrode, the counter electrode and the reference electrode are connected to the potentiostat, and the working electrode is connected to the potentiostat.
Item 8. The method for producing a fixative film containing a conductive material on which a group of microorganisms is supported, wherein the current of the working electrode is controlled by a three-electrode method.
Item 9.
In the above (2) fixed membrane fermentation step
Item 8. The method for producing a fixative film containing a conductive material on which the microorganism group according to Item 7 or 8 is supported, wherein the conductive material is hydrophobic.
Item 10.
In the above (2) fixed membrane fermentation step
The method for producing a fixed film containing the conductive material on which the microorganism group according to any one of Items 7 to 9 is supported, wherein the conductive material is carbon fiber.
Item 11.
Item 3. The microorganism according to any one of Items 7 to 10, wherein the (2) fixed membrane fermentation step can increase methanogens in the fermentation broth obtained in the (1) bioelectrochemical treatment step. A method for producing a fixed film containing a conductive material on which a group is supported.
Item 12.
Item 2. The method for producing a fixative film containing a conductive material carrying a group of microorganisms according to Item 11, wherein the methanogens are Methanosarcina sp. And Methanosarcina sp.
Item 13.
(1) A carrier holding electrode having a carrier on at least a part of the electrode surface,
A bioelectrochemical treatment unit that contains an organic substrate and a group of microorganisms involved in methane fermentation, and is in contact with the methane fermentation liquid for methane fermentation treatment of the organic substrate, and
(2) A methane fermentation accelerating device including a fixed membrane fermentation unit in which a fermentation broth obtained in the bioelectrochemical treatment unit and a fixed membrane containing a conductive material are brought into contact with each other.
The (1) bioelectrochemical treatment unit is a methane fermentation promoting device that controls a current of 3 μA / cm ² or less.
Item 14.
In the above (1) bioelectrochemical treatment section,
The carrier holding electrode is used as a working electrode.
In addition, it is equipped with a counter electrode, a reference electrode, and a potentiostat.
The working electrode, the counter electrode, and the reference electrode are connected to the potentiostat, and the working electrode, the counter electrode, and the reference electrode are connected to each other.
Item 3. The methane fermentation promoting device according to Item 13, wherein the current of the working electrode is controlled by a three-electrode method.
Item 15.
(1) A carrier holding electrode having a carrier on at least a part of the electrode surface,
A bioelectrochemical treatment unit that contains an organic substrate and a group of microorganisms involved in methane fermentation, and is in contact with the methane fermentation liquid for methane fermentation treatment of the organic substrate, and
(2) Production of a fixed membrane containing a conductive material carrying a group of microorganisms, which comprises a fixed membrane fermented portion in which the fermented liquid obtained in the bioelectrochemical treatment section and a fixed membrane containing a conductive material are brought into contact with each other. It ’s a device,
(1) The bioelectrochemical treatment unit is an apparatus for producing a fixed membrane containing a conductive material carrying a group of microorganisms, which controls a current of 3 μA / cm ² or less.
Item 16.
(1) A carrier-holding electrode having a hydrophobic carrier on at least a part of the electrode surface,
A bioelectrochemical treatment unit that contains an organic substrate and a group of microorganisms involved in methane fermentation, and is in contact with the methane fermentation liquid for methane fermentation treatment of the organic substrate, and
(2) A methane fermentation promotion system including a fixed membrane fermentation unit in which a fermented liquid obtained in the bioelectrochemical treatment unit and a fixed membrane containing a conductive material are brought into contact with each other.
The (1) bioelectrochemical treatment unit is a methane fermentation promotion system that controls a current of 3 μA / cm ² or less.
Item 17.
(1) A carrier-holding electrode having a hydrophobic carrier on at least a part of the electrode surface,
A bioelectrochemical treatment unit that contains an organic substrate and a group of microorganisms involved in methane fermentation, and is in contact with the methane fermentation liquid for methane fermentation treatment of the organic substrate, and
(2) Production of a fixed membrane containing a conductive material carrying a group of microorganisms, which comprises a fixed membrane fermented portion in which the fermented liquid obtained in the bioelectrochemical treatment section is brought into contact with a fixed membrane containing a conductive material. It ’s a system,
(1) The bioelectrochemical treatment unit is a fixing film manufacturing system containing a conductive material carrying a group of microorganisms, which is controlled to a current of 3 μA / cm ² or less.

According to the present invention, excellent methane fermentation processing ability can be exhibited, and the ability can be exhibited even under high load conditions in which a large amount of organic waste or the like is input (scaled up), and the ability can be exhibited for a long period of time. , The methane fermentation process can be performed continuously and efficiently.

Furthermore, according to the method for producing a fixing film containing a conductive material on which a group of microorganisms is supported, a fixing film in which a group of microorganisms useful in methane fermentation treatment is supported on a conductive material can be obtained.

Further, according to the method of the present invention, methanogens can be efficiently increased.

INDUSTRIAL APPLICABILITY According to the present invention, an inexpensive carbon electrode can be used in the bioelectrochemical system in the first step. In addition, since a control method is adopted in which a weak current is passed through the surface of the electrode, energy consumption can be kept low.

FIG. 1 is a cross-sectional view of the experimental device of the present invention used in Example 1. FIG. 2 is a cross-sectional view of the experimental device used in Comparative Example 1. FIG. 3 is a cross-sectional view of the experimental device used in Comparative Example 2. FIG. 4 is a cross-sectional view showing another embodiment of the experimental device in the present invention. FIG. 5 is a diagram showing the amount of gas (CH ₄ , CO ₂ , and H ₂ ) produced in each of the steps of Example 1 and Comparative Example 1. FIG. 6 is a diagram showing the concentrations of lower fatty acids (acetic acid, propionic acid, and butanoic acid) and ethanol obtained in each step of Example 1 and Comparative Example 1. FIG. 7 is a diagram showing the amounts (carbon amounts) of CH ₄ , CO ₂ , SCFA + ethanol, and residual C-SS obtained in each step of Example 1 and Comparative Example 1, and the carbon amount of cellulose. FIG. 8 is a diagram showing the microbial composition in the fermentation broth fractions obtained in each step of Example 1 and Comparative Example 1. FIG. 9 is a diagram showing the amount of gas (CH ₄ , CO ₂ , and H ₂ ) produced in each of the steps of Example 1 and Comparative Example 1. FIG. 10 is a diagram showing the concentrations of lower fatty acids (acetic acid, propionic acid, and butanoic acid) and ethanol obtained in each step of Example 1 and Comparative Example 1. FIG. 11 is a diagram showing the amounts (carbon amounts) of CH ₄ , CO ₂ , SCFA + ethanol, and residual C-SS obtained in each step of Example 1 and Comparative Example 1, and the carbon amount of cellulose. FIG. 12 is a diagram showing the production rates of gases (CH ₄ , CO ₂ , and H ₂ ) obtained in each of the steps of Example 1 and Comparative Example 2. FIG. 13 is a diagram showing the concentrations of lower fatty acids (acetic acid, propionic acid, and butanoic acid) and ethanol obtained in each step of Example 1 and Comparative Example 2. FIG. 14 is a diagram showing the amounts (carbon amounts) of CH ₄ , CO ₂ , SCFA + ethanol, and residual C-SS obtained in each step of Example 1 and Comparative Example 2, and the carbon amount of cellulose.

Hereinafter, the method for promoting methane fermentation of the present invention, the method for producing a fixing film containing a conductive material carrying a group of microorganisms, the device for promoting methane fermentation, and the device for producing a fixing film containing a conductive material carrying a group of microorganisms will be described. It will be explained in detail. Moreover, the embodiment for carrying out the present invention will be described in detail with reference to the drawings as appropriate. The device can be rephrased as a system.

<Methane fermentation promotion method>
The method for promoting methane fermentation of the present invention includes (1) a carrier-holding electrode having a carrier on at least a part of the electrode surface, an organic substrate, and a group of microorganisms involved in methane fermentation, and the organic substrate is methane. A bioelectrochemical treatment step (hereinafter, also referred to as "first step" or "BES") in which the methane fermentation liquid to be fermented is brought into contact, and (2) fermentation obtained in the bioelectrochemical treatment step. The present invention comprises a fixed film fermentation step (hereinafter, also referred to as “second step” or “FFR”) in which a liquid and a fixed film containing a conductive material are brought into contact with each other. In particular, the present invention comprises the bioelectricity. The chemical treatment step is characterized in that it is performed while controlling the current to 3 μA / cm ² or less.
Here, BES means a Bioelectrochemical System. Not using this BES is called NBES (Non-Bioelectrochemical System).
Further, FFR means Fixed Film Reactor, and in the present specification, the second step may be referred to as “FFR”. Not using this FFR is called NFFR (Non-Fixed Film Reactor). The process can be paraphrased as a part or a means.
Hereinafter, the first step and the second step will be described with reference to FIGS. 1 and 4 as appropriate.

First step: Bioelectrochemical treatment step (BES)
The first step (bioelectrochemical treatment step: BES) in the methane fermentation promotion method of the present invention comprises a carrier-holding electrode having a carrier on at least a part of the electrode surface, an organic substrate, and a group of microorganisms involved in methane fermentation. The organic substrate is brought into contact with the methane fermentation liquid to be treated with methane fermentation, and the biological treatment is performed while controlling the current of the carrier holding electrode within the optimum range of the group of microorganisms to be carried. By carrying out the first step, the carrier can be activated by supporting the group of microorganisms to be supported.

As the carrier-holding electrode used in the present invention, the properties of the carrier held on the electrode surface can be appropriately selected and used according to the microbial reaction process. For example, when carrying a microorganism capable of adhering to a hydrophilic carrier, a hydrophilic carrier may be used, and when carrying a microorganism capable of adhering to a hydrophobic carrier, a hydrophobic carrier may be used. Just do it. That is, the properties of the carrier can be appropriately selected without depending on the properties of the electrodes, and the microbial group can be supported according to the microbial reaction process.
Therefore, for example, by holding both the hydrophilic carrier and the hydrophobic carrier on the surface of the electrode, it is easy to support both the microorganisms adhering to the hydrophilic carrier and the microorganisms adhering to the hydrophobic carrier. Can be done.

The hydrophobic carrier is not particularly limited, and examples thereof include plastics such as carbon, graphite (graphite), polyethylene and polypropylene, and metals such as platinum and alumina. The form or shape of the hydrophobic carrier is not particularly limited, and examples thereof include a plate shape (sheet shape, film shape, etc.), a block shape, and the like. Among them, as the hydrophobic carrier, a conductive carrier is preferable, and a carbon plate (carbon sheet, carbon film) and a graphite block are more preferable. The size of the hydrophobic carrier is not particularly limited. The hydrophobic carrier may be used alone or in combination of two or more.

The material of the electrode used for the carrier holding electrode is not particularly limited, and any electrode capable of causing a reduction reaction on the electrode can be used. For example, examples of the carrier holding electrode include a metal such as platinum, carbon, and the like. Among them, as the electrode, a carbon electrode is preferable to a platinum electrode from the viewpoint of cost.

The carrier-holding electrode used in the present invention may be provided with a carrier on at least a part of the electrode surface, preferably on one side of the electrode surface, and is provided on the entire electrode surface. Is more preferable. The higher the carrier holding area on the electrode surface, the easier it is to carry microorganisms. Examples of the method of providing the carrier on the surface of the electrode include, but are not limited to, a method of adhering the carrier with an adhesive and a method of covering the carrier with a bag or a cylinder to cover the electrode.

Here, it is preferable that the carrier capable of supporting the microorganism has a liquid permeability that can ensure contact between the electrode and the methane fermentation liquid. In this case, the microorganism can be sufficiently supported up to the vicinity of the electrode of the carrier, and the controllability of the current in the vicinity of the electrode can be ensured to sufficiently activate the microorganism on the carrier. That is, even if the carrier material is a conductive material such as carbon, if the void ratio is improved in order to increase the amount of microorganisms supported, the conductive performance is significantly reduced and substantially. Although no current flows, if the carrier has a liquid permeability that can ensure contact between the electrode and the methane fermented liquid, the current of the methane fermented liquid that fills the voids of the carrier is controlled and the current environment of the carrier is changed by microorganisms. It can be controlled to the optimum range for.

The organic substrate is not particularly limited as long as it is an organic waste or an organic waste liquid. Examples of organic substrates include wood, paper, paper waste, rice straw, straw such as wheat straw, seaweed, food residue, agricultural residue, sewage sludge, sewage sludge, wastewater treatment sludge, septic tank sludge, and livestock manure (livestock waste). ), Various types of biomass such as garbage. Further, depending on the properties of the organic substrate, methane fermentation treatment can be performed after pretreatment such as crushing and separation, if necessary. Cellulose, which is a component contained in wood, paper, etc., is a persistent substance and has a problem that methane fermentation is difficult. The present invention has an effect of promoting methane fermentation, particularly on an organic substrate containing cellulose.

The group of microorganisms to be supported in the present invention is not particularly limited, and for example, a group of microorganisms capable of performing conversion treatments such as decomposition, oxidation, and reduction on an object to be treated; using a certain source substance. Examples include general groups of microorganisms capable of producing substances.

Specifically, as a group of microorganisms capable of performing conversion treatments such as decomposition, oxidation, and reduction of the object to be treated, for example, decomposition of SS (solid suspended matter) and COD (chemical oxygen demand). A group of known or novel microorganisms carrying known or novel microorganisms having functions such as decomposition and removal, decomposition and removal of TOC (total organic carbon), tetrachlorethylene (PCE), dichloroethylene (DCE) and vinyl chloride (VC) dechlorination treatment, etc. Can be mentioned. The group of microorganisms referred to here is not limited to these.
For example, for the decomposition of SS (solid suspended matter), the decomposition and removal of COD (chemical oxygen demand), and the decomposition and removal of TOC (total organic carbon), a group of microorganisms contained in sludge obtained from a methane fermenter is used. can do.
Further, for dechlorination of tetrachlorethylene (PCE), for example, Desulfitobacterium, Dehalococcoides, Dehalobacter, Geobacter, etc .; for dechlorination of dichloroethylene (DCE) and vinyl chloride (VC), for example, a group of microorganisms such as Dehalococcoides is used. can do.
Examples of the group of microorganisms having the ability to produce a substance using a certain source substance include a group of known or novel microorganisms having a function of producing methane gas, hydrogen, amino acids, and various organic substances.
For example, these microorganisms include hydrogen-utilizing methanogens that produce methane gas from hydrogen and carbon dioxide, acetic acid-utilizing methanogens that produce methane gas from acetic acid, and glucose to ethanol. Examples thereof include Clostridium acetobutylicum, which produces butanol or acetone. The group of microorganisms referred to here is not limited to these.

Examples of hydrogen-utilizing methanogens include methanogens (sometimes referred to as methanogen archaea or methanogens) include Methanogen, Methanobrevibacter, Methanosphaera, Methanothermus, and Methanococcus. Methanolacinia, Methanomicrobium, Methanogenium, Methanospirillum, Methanoculleus, Methanoplanus, Methanosarcina, Methanolobus, Methanococcoides, Methanoregula, Methanolinea, Methanohalophilus, Methanohalobium, etc. In addition, in this embodiment, any bacterium capable of producing methane can be used without being limited to these. Methanogens and various microorganisms that decompose the above-mentioned organic matter can be easily obtained from existing digestion tanks and the like. The present invention, among other things, can increase Methanobacterium sp. And Methanosarcina sp.

Examples of acetic acid-utilizing methanogens include the genus Methanosaeta (Methanosaeta) and the genus Methanosarcina.

Examples of the producing bacteria that produce ethanol, butanol, acetone and the like from glucose include the genus Clostridium such as Clostridium acetobutylicum.

In the present invention, when carrying out methane fermentation treatment or methane gas production, it is preferable to use a hydrophobic carrier.

The methane fermentation broth is not particularly limited as long as it contains an organic substrate and a group of microorganisms involved in methane fermentation, and the organic substrate can be subjected to methane fermentation treatment. For example, a culture solution containing an organic substrate and a group of microorganisms involved in methane fermentation can be used as a methane fermentation solution. As the culture solution, a normal culture solution used for culturing microorganisms may be used, and a substance that becomes an energy source for microorganisms, a substance for controlling the culture environment such as pH, etc. may be appropriately added and used. good. Since the culture solution usually contains monovalent cations such as hydrogen ion (H ⁺ ), sodium ion (Na ⁺ ), and potassium ion (K ⁺ ), the culture solution itself has electrical conductivity. Therefore, the current control of the carrier holding electrode itself can be easily performed.

The method of contacting the carrier holding electrode and the methane fermentation solution (supported microorganism group and culture solution) is not particularly limited. For example, the carrier holding electrode and the carrier target microorganism group are separately put into the culture solution. Alternatively, the microbial community to be supported or the microbial community containing the microbial group to be supported may be previously supported on a carrier in the form of, for example, a biofilm, and this may be placed in a culture solution.
In either case, the carrier of the carrier holding electrode can be supported and activated by supporting the group of microorganisms to be supported.
According to the present invention, it is possible to support and activate a group of microorganisms to be supported, and of course, among the microbial communities including the group of microorganisms to be supported, the group of microorganisms to be supported is supported as a main component. Can be activated. That is, a desired microbial community can be supported on a carrier as a main constituent and activated from a microbial community containing a plurality of types of microbial communities.

The optimum range of the microorganisms to be carried is derived by the experimental method described below. That is, when a group of microorganisms capable of converting a substance to be supported is to be supported, a certain amount of the substance to be treated is added to the culture solution, and the culture solution contains at least a carrier holding electrode and a group of microorganisms to be supported. By contacting with the community, the carrier holding electrode can be controlled to a current for a certain period of time, and the current range in which the amount of the object to be treated contained in the culture solution is significantly reduced can be set as the optimum range.
When a microbial group that produces a product is to be supported, a certain amount of a source substance is added to the culture solution, and the carrier holding electrode and the microbial community containing at least the microbial group to be supported are brought into contact with the culture solution. Therefore, the carrier holding electrode can be controlled to various currents for a certain period of time, and the current range in which the amount of product produced is large can be set to the optimum range.

According to the present invention, a group of microorganisms to be supported can be supported on the carrier of the carrier holding electrode and activated. Further, according to the present invention, it may be possible to efficiently carry out the desired treatment by inactivating or removing a group of microorganisms that inhibit the desired treatment. In the method for promoting methane fermentation of the present invention, the first step (bioelectrochemical treatment) is one of the factors that can be efficiently carried out by demonstrating excellent treatment capacity even when carried out under high load conditions. By the step), it is possible to increase the ratio of the carrier target microorganism group to all the bacteria in the culture solution while supporting and activating the carrier target microorganism group on the carrier, and the carrier holding electrode is included with the culture solution. It is considered that the processing capacity of the entire processing tank is improved. In any case, by adopting the configuration of the present invention, excellent processing capacity can be exhibited. That is, until now, a bioelectrochemical treatment method that controls at a specific potential has been known, but in the present invention, the bioelectrochemical treatment step is an extremely minute current, that is, a current of 3 μA / cm ² or less. It is characterized by being controlled. No specific biochemical electrochemical treatment step performed by controlling the current to 3 μA / cm ² or less has been disclosed so far.

The method and apparatus of the present invention can generate biogas. Biogas is a type of biofuel and means gas generated by fermentation of organic fertilizers, biodegradable substances, sludge, sewage, garbage, energy crops, etc., anaerobic digestion, etc. The main components of biogas are methane and carbon dioxide. Methane can be used as an alternative fuel to fossil fuels for power generation, cogeneration, boilers, vehicle engines, and cooking.

In the present invention, methane fermentation means that organic waste is methane-fermented to reduce its volume, and in the process, biogas containing methane gas is generated and recovered.

As the methane fermentation broth, a methane fermentation broth from a methane fermentation tank in which methane fermentation treatment such as organic waste is performed, or a culture broth to which methane fermentation sludge is added can be used. It is also possible to use a culture solution to which a microbial community in which a microbial community for performing methane fermentation is artificially cultured is added.

As the carrier holding electrode, as described above, an electrode, for example, one provided with a hydrophobic carrier, for example, carbon fiber on at least a part of the surface of a carbon plate, is immersed in the methane fermentation broth.

The current (cathode) of the carrier holding electrode is 3 μA / cm ² or less, preferably 0.01 to 2.9 μA / cm ² , more preferably 0.1 to 2.8 μA / cm ² , and particularly. It is preferably 1 to 2.75 μA / cm ² . The cathode is the electrode on the left side (working electrode), and means the one in which a positive charge flows from the electrode toward the solution (cathode in which a reduction reaction occurs). Further, the anode is the right electrode (counter electrode), and means the direction in which a positive charge flows from the solution toward the electrode (anode in which an oxidation reaction occurs).
In Patent Document 1, the methane fermentation treatment is performed while controlling the potential of the carrier holding electrode to + 0.3 V based on the potential at which electrons are supplied from the carrier holding electrode or the potential of the silver or silver chloride electrode. Although it is described, it is not described that the methane fermentation treatment is performed while controlling the specific current. Moreover, this Patent Document 1 does not disclose that the cathode current is controlled to 0.3 μA / cm ² or less at all.
In the present invention, the step of controlling the current in the first step may be controlled by repeating on / off. That is, the current can be passed for a certain period of time, then stopped, and after a certain period of time has passed, the current can be passed again for a certain period of time.

The reactor used in the first step is not particularly limited, and for example, a closed container, a box-shaped closed container as shown in FIG. 4, an H-shaped container, a U-shaped container, or the like can be used. The material of the reactor is not particularly limited, and for example, a glass container or the like can be used. Here, an ion exchange membrane can be provided in the reactor. The ion exchange membrane is not particularly limited, and known ones can be used.

The size (volume) of the reactor used in the first step is not particularly limited, but for example, the lower limit is preferably 100 mL or more, and more preferably 250 mL or more. The upper limit of the size of the reactor is not particularly limited.

The first step (bioelectrochemical treatment step; BES) in the methane fermentation method of the present invention can be carried out by, for example, the apparatus shown in FIG. 1 or FIG. 4, but is not limited thereto. The apparatus used in the first step of the present invention (the apparatus used in the bioelectrochemical treatment step or the methane fermentation promoting apparatus) is involved in a carrier holding electrode having a carrier on at least a part of the electrode surface, an organic substrate and methane fermentation. It is provided with a bioelectrochemical treatment unit (hereinafter, also referred to as "bioelectrochemical treatment unit") that contains a group of microorganisms and is in contact with a methane fermentation liquid for methane fermentation treatment of the organic substrate.
The carrier holding electrode can be rephrased as a working electrode. Further, the bioelectrochemical treatment unit can be provided with a counter electrode, a reference electrode and the like.
Further, the apparatus (bioelectrochemical processing unit) used in the first step of the present invention may be provided with an electrochemical measuring apparatus (potentiostat or galvanostat).
In the case of galvanostat, it is not necessary to provide a reference electrode. An electrochemical measuring device is a device that measures current in addition to controlling current. An electrical signal is added to a sample cell to be measured to cause a chemical reaction, and vice versa. It is a device for measuring the electrical properties of the sample cell by measuring the response signal.
A potentiostat is generally a device for controlling a cell of three electrodes including a working electrode, a counter electrode, and a reference electrode, and arbitrarily controlling the current level of the electrode, etc., regardless of the state of the electrolytic solution. be.
A galvanostat is a device for accurately controlling the current flowing through an electrode by having a very high internal resistance.

The temperature of the fermentation broth in the reaction of the first step is not particularly limited, and is usually in the range of room temperature (for example, 25 ° C.) to 80 ° C., preferably 30 to 70 ° C., and more preferably 40 to 60 ° C. ℃.

The reaction time in the first step is not particularly limited, and is usually in the range of 1 hour to 1 month, preferably 1 to 20 days, and more preferably 3 to 10 days.

Second step: Fixed membrane fermentation step (FFR)
The methane fermentation method of the present invention further includes, as a second step, a fixed membrane fermentation step in which the fermented liquid obtained in the bioelectrochemical treatment step is brought into contact with a fixed membrane containing a conductive material. In the fixed membrane fermentation step, fermentation is carried out using a fixed membrane reactor (FFR). In the present specification, the second step may be referred to as "fixed membrane fermentation step" or "FFR". The fixed membrane reactor can be rephrased as a packed bed reactor, a fixed membrane fermenter, or an anaerobic digestion tank.

As used herein, the term "fixed membrane reactor" refers to a reactor provided with a fixed membrane, that is, a reactor in which a membrane is fixed therein.
The fixing film may be the conductive material in whole or in part. Further, the conductive material is preferably in a form capable of increasing the surface area as a three-dimensional structure such as a fibrous material, a porous body, and a granular material, and increasing the amount of microorganisms supported.

The conductive material is not particularly limited, and examples thereof include carbon, carbon black, and graphite.

Examples of carbon black include acetylene black, ketjen black, channel black, furnace black, lamp black, thermal black and the like.

Examples of graphite (graphite) include natural graphite and artificial graphite.

Anaerobic microorganisms can be carried on this conductive material. The conductive material preferably has hydrophobicity, and more preferably has pores (for example, porous, fibrous, etc.). The fiber may be a woven fabric (woven fabric) or a non-woven fabric. One type of conductive material may be used alone, or two or more types may be used in combination.

Carbon fiber is preferable as the conductive material. Examples of carbon fibers include not only ordinary carbon fibers but also single-walled carbon nanotubes, multi-walled carbon nanotubes, gas-phase grown carbon fibers and the like. Further, the carbon fiber referred to here may be a woven fabric (woven fabric) or a non-woven fabric.
As the fixing film, for example, a film made of a conductive material can be used, or a film made of a conductive material can be used on one or both sides of a film made of a non-conductive material (for example, polyethylene, polypropylene, etc.). Laminated films can also be used. Here, as a method of laminating, a method of adhering with an adhesive or the like can be mentioned. It is preferable to use a carbon fiber woven fabric as the conductive material and use a film made of the carbon fiber woven fabric as the fixing film.

Since the conductive material has pores (if the porosity or the like is improved), the microorganism group adheres and inhabits, so that the microorganism can be efficiently supported in a short time. Moreover, it has the advantage that the carried microorganisms are not easily exfoliated.

The fermentation broth obtained in the bioelectrochemical treatment step (first step) contains a group of microorganisms. The first step is a step of preparing a flora for more efficiently growing methanogens in the second step. In the second step, it is not necessary to separately culture and prepare a group of microorganisms, and if the fermentation broth obtained in the first step is immersed in the organic substrate, it can be contained in the organic substrate under anaerobic conditions. Microorganisms that exist in their natural state (usually anaerobic mixed microorganisms) can be cultivated on a fixed membrane or conductive material. However, if necessary, a separately cultured anaerobic microorganism may be attached before immersion in the organic substrate.

The fixed membrane used in the present invention is more likely to carry microorganisms as its surface area is increased. Examples of the method of fixing the membrane in the reactor include, but are not limited to, a method of fixing the membrane with a wire or the like.

The present invention is characterized in that the methane fermentation broth obtained in the first step is used in the fixed film fermentation step of the second step by utilizing the effect of the bioelectrochemical system (BES) in the first step. Thereby, it can be scaled up, and the amount of specific methanogens can be further increased by the methane fermentation in the second step. That is, it is possible to change the metabolism of microorganisms and the interaction between microbial species.

The reactor used in the second step is not particularly limited, and for example, not only the usual box-shaped closed container as shown in FIGS. 1 and 4, but also various containers can be used. The material of the reactor is not particularly limited, and examples thereof include glass and the like.

The size (volume) of the reactor (fermenter) used in the second step is not particularly limited, but for example, the lower limit is preferably 100 mL or more, and more preferably 250 mL or more. The upper limit of the size of the reactor is not particularly limited, and the second step can be performed with an industrial scale, for example, a reactor (reactor) of 300 L to 20,000 L). By using the method for promoting methane fermentation of the present invention, it can be easily scaled up.

The second step (fixed membrane fermentation step; FFR) in the methane fermentation method of the present invention can be carried out by, for example, the apparatus shown in FIG. 1 or FIG. 4, but is not limited thereto. The apparatus used in the second step of the present invention (the apparatus used in the fixed membrane fermentation step or the methane fermentation promoting apparatus) brings the fermentation broth obtained in the bioelectrochemical treatment section into contact with the fixed membrane containing the conductive material. It is equipped with a fixed membrane fermentation unit (hereinafter, also referred to as "fixed membrane fermentation unit").

The methane fermentation promoting method and the methane fermentation promoting apparatus of the present invention use, for example, a microorganism that transfers electrons to an electrode or a microorganism that receives electrons from an electrode, wastewater treatment by simultaneous power generation, electrochemical production of hydrogen or methane, and the like. It can also be used for desalination and the like.

The temperature of the fermentation broth in the second step is not particularly limited, and is usually in the range of room temperature (for example, 25 ° C.) to 80 ° C., preferably 30 to 70 ° C., and more preferably 40 to 60 ° C. be.

The fermentation time in the second step is not particularly limited, and is usually in the range of 1 hour to 1 month, preferably 1 day to 20 days, and more preferably 3 days to 10 days.

Examples of the present invention will be described below, but the present invention is not limited to these examples.

(Example 1)
A method for promoting methane fermentation in which the first step is carried out in a bioelectrochemical system (BES) and the second step is carried out in a fixed membrane fermentation step (FFR) will be described below with reference to a cross-sectional view of the experimental apparatus of FIG.
First step (BES)
Device
The first step was methane fermentation in a bioelectrochemical system (BES). Specifically, as shown in FIG. 1, the BES has two carbons in a three-electrode H-type glass reactor having two reactors (acting capacity of each reactor: 250 ml, total acting capacity: 500 mL). Sheets (graphite blocks measuring 2.5 mm × 75 mm × 2 mm) were placed as working electrodes (cathode, left side) and counter electrodes (anode, right side), respectively.
Further, an Ag / AgCl reference electrode (saturated KCl) was inserted on the working electrode (cathode) side (left side). A working electrode and a counter electrode were fixed to each reactor using stainless steel wire. Then, a potentiostat (product name: PS-08, manufactured by Toho Giken) was connected to the working electrode, counter electrode and reference electrode. In addition, each glass reactor was equipped with one gas outlet connected to a gas sampling bag (gas pack).
The current of the working electrode was electrochemically adjusted and controlled to 2.7 μA / cm ² .

In Example 1, the experimental device shown on the left side of FIG. 1 was used as the experimental device for carrying out the first step, but it is also possible to use a device other than the experimental device shown in FIG. As another experimental device, for example, the experimental device shown on the left side of FIG. 4 can be mentioned.
In the bioelectrochemical system (BES) shown in FIG. 4, two carbon sheets (graphite blocks having dimensions of 2.5 mm × 75 mm × 2 mm) are contained in a three-electrode glass box-type sealed reactor (500 mL), respectively. It is arranged as a working electrode (cathode, left side) and a counter electrode (anode, right side). An Ag / AgCl reference electrode (saturated KCl) is inserted on the working electrode (cathode) side (left side), and the working electrode and counter electrode are fixed to each reactor using a stainless steel wire. .. In addition, a potentiostat (product name: PS-08, manufactured by Toho Giken) is connected to the working electrode, counter electrode and reference electrode, and the glass box-type sealed reactor is in a gas sampling bag (gas pack). One connected gas outlet is attached.

The BES in the first step of initiation and operation was inoculated with 500 mL of sludge from a methane fermenter that decomposes 1% glucose at 55 ° C. After inoculation, a predetermined amount of the fermentation broth on the cathode side and the anode side of BES was discharged, and the same amount of fresh artificial medium was added once a day.
The artificial medium was 20 g / L cellulose powder (manufactured by Nakaraitesk Co., Ltd.), 0.1 g / L KH ₂ PO ₄ , 0.2 g / L K ₂ HPO ₄ , 1 g / L yeast in deionized water. Extract, ₂ g / L NaCl ₃ , 1 g / L NH ₄ Cl, 0.1 g / L MgCl 2.6H ₂ O, 0.1 g / L CaCl _{2.2H 2 O} , 0.6 g / L NaCl It contains 10 mL / L trace element solution (Deutsche Sammlung von Mikroorganismen und Zellkulturen [DSMZ] 141 medium) and 1 mL / L vitamin solution (DSMZ 141 medium).

Second step (FFR)
The second step is a fixed membrane fermentation step (FFR) in which methane fermentation is performed using a fixed membrane reactor. Specifically, as a fixed film reactor, a carbon fiber woven fabric [product name: Dona Carbo (registered trademark) paper, Osaka Gas Chemical Co., Ltd.) is contained in a glass reactor (product name: DURAN Co., Ltd., working capacity: 250 mL). (Manufactured); diameter 25.0 mm, height 70.0 mm, thickness 2.4 mm] Two sheets were installed. Further, a gas sampling bag (gas pack) was attached to the glass reactor as in the first step.
The reactor was inoculated with 250 mL of sludge similar to that in the first step. After inoculation, a predetermined amount of the fermented liquid in the reactor was discharged, and the same amount of the above-mentioned waste water (mixture of cathode side and anode side) of the first step (BES) was added once a day.
The contents of the working electrode (cathode) side and the counter electrode (anode) side of the first step (BES) were thoroughly mixed using a magnetic stirrer and then added to the reactor of the second step (FFR).
The temperature of the culture was maintained at 55 ° C. The operation was divided into three times.

(Comparative Example 1)
The first step is carried out in a non-bioelectrochemical system (NBES) and the second step is carried out in a fixed membrane fermentation step (FFR). FIG. 2 shows a cross-sectional view of the experimental device used in Comparative Example 1.
As shown in FIG. 2, Comparative Example 1 was carried out in the same manner as in Example 1 except that the reference electrode was not provided and the current was not passed through the carbon sheet in the glass container in the first step.

(Comparative Example 2)
The first step is carried out in a bioelectrochemical system (BES) and the second step is carried out in a non-fixed membrane fermentation step (NFFR). FIG. 3 shows a cross-sectional view of the experimental device used in Comparative Example 2.
As shown in FIG. 3, Comparative Example 2 was carried out in the same manner as in Example 1 except that the film made of carbon fiber woven fabric (CFT) was not arranged in the glass container in the second step.

Operating conditions Table 1 shows the operating conditions of Example 1, Comparative Example 1 and Comparative Example 2. Each was operated for 17 days with a relatively long hydraulic residence time (HRT) to acclimatize (acclimate), and then operated for 13 days with a relatively short HRT for comparison. The hydraulic residence time (HRT) means the time for replacing all the working volumes in the reactor.

Example 1 and Comparative Example 1 were prepared with a synthetic medium containing cellulose as a main carbon source at a hydraulic residence time (HRT) of 2.5 days and a cellulose loading of 3555.6 mg-carbon (C) / L / day. Was added to the reaction vessel of the first step. During the operation of the first step, the pH at the time of charging (6.1) of Example 1 decreased, and the pH at the time of discharging became 5.2 ± 0.3. In addition, the pH at the time of charging (6.1) of Comparative Example 1 also decreased, and the pH at the time of discharging became 5.2 ± 0.2. Therefore, most of the cellulose remains without being consumed, and the amount of suspended solids (SS) removed after the first step is 11.4 ± 4.3% in Example 1 and 11 in Comparative Example 1. It was 0.6 ± 4.3%.
In the second step FFR [BES → FFR] following the first step (BES) of Example 1 and the second step FFR [NBES → FFR] following the first step (NBES) of Comparative Example 1, the pH at the time of charging is , 6.1 to 7.5 (neutralized), and the pH at discharge was 6.8 ± 0 and 6.4 ± 0.4, respectively.
The following Test Examples 1 to 6 were performed according to the above operating conditions.

<Test Example 1>
Microbial 16S rRNA gene sequence analysis of the microbial community in the reactor
In Example 1 (1st step (BES) → 2nd step (FFR)) and Comparative Example 1 (1st step (NBES) → 2nd step (FFR)), the number of eubacteria at each measurement point shown below is determined. It was measured.
First, the microbial compositions grown in Example 1 and Comparative Example 1 were examined by HRT for 2.5 days and 4.2 days, respectively, as shown in Table 1.
The number of eubacteria (microorganism concentration) was measured by real-time PCR. The suspended fraction of the cathode of the reactor of the first step (BES-cathode), the suspended fraction of the anode (BES-anode), and the suspended fraction of the reactor of the second step (FFR) of Example 1. , And the 16S rRNA gene copy count was determined for the adherent fraction (FFR-C) on the carbon fiber fabric (CFT) membrane of the reactor in the second step.
Similarly, the suspension fraction on the left side of the reactor in the first step (NBES-left side), the suspension fraction on the right side (NBES-right side), and the suspension fraction in the reactor in the second step of Comparative Example 1. The 16S rRNA gene copy count was determined for (FFR) and the adherent fraction (FFR-C) on the CFT membrane of the reactor in the second step. These results are shown in Table 2.

From the result table 2, the following was found.
The suspension fractions of the first step reactor of Example 1 (BES-cathode and BES-anode) and the suspension fractions of the first step reactor of Comparative Example 1 (NBES-left side and NBES-right side) , The microbial concentration was low due to the low pH conditions.
The suspension fraction (FFR) of the reactor of the second step of Example 1 and the suspension fraction (FFR) of the reactor of the second step of Comparative Example 1 are the suspension fractions of the reactor of the first step, respectively. The microbial concentration was increased compared to the minutes (BES-cathode and BES-anode, or BES-cathode and BES-anode).
The concentration of airborne microorganisms in the suspension fraction (FFR) of the second step reactor of Example 1 is relative to the concentration of airborne microorganisms in the suspension fraction (FFR) of the second step reactor of Comparative Example 1. It was expensive.
Further, both the adhered fraction (FFR-C) on the CFT film of the second step of Example 1 and the adhered fraction (FFR-C) on the CFT film of the second step of Comparative Example 1 were applied to the CFT film. The concentration of microorganisms in the attached fraction was high.

<Test Example 2>
In Example 1 (first step (BES) → second step (FFR)) and Comparative Example 1 (first step (NBES) → second step (FFR)), the number of reads, the number of OTUs, the Shannon index, and Faith. The systematic diversity index of was measured as follows.

Number of leads (microbial community analysis)
The V3-V4 region of the prokaryotic 16S rRNA gene was amplified using primers Pro341F (5'-CCTACGGGNBGCWSCAG-3') and Pro805R (5'-GACTACNVGGGTATCTAATCC-3') using genomic DNA as a template.
PCR amplification is performed by Takagi R, Sasaki K, Sasaki D, Fukuda I, Tanaka K, Yoshida K, Kondo A, Osawa R. A single-batch fermentation system to simulate human colonic microbiota for high-throughput evaluation of prebiotics. PLos ONE. 2016; 11: e0160533. It was done by the method described.
Here, N, B, W, and V are denatured nucleotides A / C / G / T, G / T / C, A / T, and each, as described in JP-A-2011-224039, respectively. It corresponds to A / C / G.
The polymerase chain reaction and the preparation of the amplicon pool were carried out according to the manufacturer's instructions (Illumina).
Amplicon was purified using AMPure XP DNA purified beads (Beckman Coulter) and eluted in 25 μL of 10 mM Tris (pH 8.5).
Purified amplicons are quantified using an Agilent 2100 bioanalyzer electrophoresis system equipped with a DNA 1000 chip (Agilent Technologies, Inc.) and a Qubit® 2.0 device (Thermo Fisher Scientific). , Pooled at an equimolar concentration (5 nM).
16S rRNA genes and internal controls (Phix Control v3; Illumina) were subjected to pair-end sequencing using a MiSeq system device (Lumina KK) and MiSeq Reagent Kit, v3 (600 cucles; Illumina). PhiX sequences were removed and paired-end reads with a Q score of ≧ 20 were performed using Automated CASAVA 1.8 paired-end demipultlexed fastq performed according to FASTQ Generation on the Illumina Basespace Sequence Hub (https://bases pace.Illumina.com/). Joined.
The sequence quality control of the sequence data and the construction of the function table were performed in QIIME 2 version 2018.2 using the DADA2 pipeline and corrected. The results are shown in Table 3.

Number of OTUs The number of OTUs (Operational Taxonomic Units) roughly represents the number of species. The taxonomic composition of OTUs was classified via a naive Bayes classifier. This classifier was analyzed on a Greengenes 13_8 99% OTU full-length sequence database. The results are shown in Table 3.

Shannon Index and Faith Systematic Diversity Index The Shannon Index and Faith systematic diversity were used for α-diversity estimation from OTU data. α-diversity refers to the diversity of a sample. In the bacterial flora analysis of soil, intestine, etc., an index such as α-diversity may be used when comparing the diversity of microbial community structures. That is, it is a sample-specific index, and the larger the value, the higher the species diversity. Depending on the index, it depends on whether "the number of observed species" or "each species is observed evenly" is emphasized.
The Shannon Index is Shannon CE. A mathematical theory of communication. Bell Syst Tech J. 1948; 27: 623-56. And Shannon CE. A mathematical theory of communication. Bell Syst Tech J. 1948; 27: 379-423. .It is described in.
The phylogenetic diversity of Faith is described in Faith DP. Conservation evaluation and phylogenetic diversity. Biol Conserv. 1992; 61: 1-10.
Microbial species diversity is expressed in Shannon index, and Faith's systematic diversity correlates with species abundance. The results are shown in Table 3.

From the result table 3, the following was found.
Number of leads
Example 1 (BES-cathode, BES-anode, FFR, and FFR-C) obtained by each sequencing reaction using a combination of a prokaryotic universal primer and a MiSeq platform (Illumina) at the same time, and a comparative example. The number of reads of 1 (NBES-left side, NBES-right side, FFR, and FFR-C) was totaled and averaged to be 196,444 (± 29,391).

Number of OTUs observed The number of OTUs in the suspension fraction (FFR) of the reactor in the second step of Example 1 or the adherent fraction (FFR-C) on the CFT film, and the reaction of the second step of Comparative Example 1. The number of OTUs of the suspension fraction (FFR) or the adhesion fraction (FFR-C) on the CFT film of the vessel depends on the difference in pH conditions, respectively, depending on the difference in the pH conditions, the suspension fraction of the reactor in the first step of Example 1 (FFR-C). It was higher than the number of OTUs of BES-cathode and anode) and the number of OTUs of the suspension fractions (NBES-right side and left side) of the reactor in the first step of Comparative Example 1.

Shannon index The Shannon index of the suspension fraction (FFR) of the reactor of the second step of Example 1 or the adhesion fraction (FFR-C) on the CFT film is the suspension fraction of the reactor of the first step (FFR). Although it showed a value higher than the Shannon index of BES-cathode and anode), it was the suspension fraction (FFR) of the reactor of the second step of Comparative Example 1 or the adhesion fraction (FFR-C) on the CFT film. The Shannon index was lower than the Shannon index of the suspension fractions (NBES-right and left) of the reactor in the first step.
As a result, the values of the Shannon index of the suspension fraction (FFR) of the reactor in the second step of Example 1 or the adherent fraction (FFR-C) on the CFT membrane are the values of the second step of Comparative Example 1, respectively. It was higher than the suspension fraction (FFR) of the reactor or the adhesion fraction (FFR-C) on the CFT membrane.

Faith systematic diversity Value of Faith systematic diversity in the suspension fraction (FFR) or adherent fraction (FFR-C) of the reactor in the second step of Example 1 and Comparative Example 1. Are the values of the suspension fraction (BES-cathode and anode) of the reactor of the first step of Example 1 and the suspension fraction (NBES-right side and left side) of the reactor of the first step of Comparative Example 1, respectively. Was higher than.
That is, in Example 1, the α-diversity value (Shannon index and systematic diversity of Faith) is higher than that of the fraction attached to the CFT membrane (FFR-C), which is the suspension (floating) fraction of the reactor. It showed a value higher than the minute (FFR).

<Test Example 3>
Example 1 (methane fermentation method in which BES is performed as a first step and FFR is performed as a second step; BES → FFR) and Comparative Example 1 (NBES is performed as a first step and FFR is performed as a second step) methane fermentation. Methods; In each of NBES → FFR), the produced gas and the product were analyzed by the following methods after the completion of the first step and after the completion of the second step.
In Example 1, a low-reactivity carbon sheet (that is, a graphite block) is used as an electrode in the first step, and the current flowing through the cathode actuated electrode is adjusted to a low value of 2.7 μA / cm ² . Therefore, it is possible to eliminate the production of hydrogen (H ₂ ) that occurs as the fermentation progresses.
Therefore, by comparing Example 1 and Comparative Example 1, the effect of microorganisms _on CH4 production from organic substrates can be investigated.
The reactor performance on both sides of the H-type reactor in the first step was the same on the cathode side and the anode side of Example 1 and on the left and right sides of Comparative Example 1. Is the average value of the cathode side value and the anode side value in Example 1, and the average value of the left side value and the right side value in Comparative Example 1.

Amount of gas produced (CH ₄ , CO ₂ , and H ₂ )
The amount of produced gas was measured by a water substitution method using a graduated cylinder.
The contents of CH ₄ , CO ₂ and H ₂ in the gas are determined by a thermal conductivity detector (GC390B; manufactured by GL Sciences Co., Ltd.) and a stainless steel column (30/60 mesh; GL Sciences Co., Ltd.) filled with activated charcoal. The measurement was carried out by the same method as described in JP-A-2011-224039, using gas chromatography (manufactured by GL Sciences Co., Ltd.). The results are shown in Table 4 and FIG.

Results From Table 4 and FIG. 5, the production of biogas (CH ₄ , CO ₂ and H ₂ ) is suppressed in the first step (BES) of Example 1 and the first step (NBES) of Comparative Example 1. I understand.
The biogas production rate containing CH ₄ was much higher in Example 1 than in Comparative Example 1.

Amount of product (acetic acid, propionic acid, butanoic acid, and ethanol)
The amount (concentration) of acetic acid, propionic acid, butanoic acid, and ethanol is determined by high performance liquid chromatography equipped with an Aminex HPX-87H column (Bio-Radola Volatries) and a refractive index detector (RID-10A, Shimadzu Corporation). It was measured using a graph (Shimadzu Corporation). The results are shown in Table 5 and FIG.

Results From Table 5 and FIG. 6, most of the products in the fermentation broth after the first step (BES) in Example 1 and the first step (NBES) in Comparative Example 1 are acetic acid, propionic acid, and butane. It was found that it was occupied by short-chain fatty acids (SCFA) such as acid and ethanol.
In addition, the concentrations of SCFA and ethanol after the second step were lower in Example 1 than in Comparative Example 1.

Carbon content (CH ₄ , CO ₂ , SCFA + ethanol, residue C-SS)
In Example 1, the waste liquid (fermentation liquid) after the first step was added to the reaction vessel of the second step. Further, in Comparative Example 1, the waste liquid (fermentation liquid) after the first step was added to the reaction vessel of the second step. These mean that the residual carbon (C) consisting of SS (Suspended Substance) and SCFA + ethanol is added to the second step (FFR).
Since these waste liquids (fermented liquids) are suspensions, they are filtered with a glass fiber filter (0.45 μm) to determine the SS in the culture, and the residue on the fibers is 120 at 105 ° C. After drying for a minute, the dry weight was measured, and the removal rate of SS, the amount of residual carbon (C) in the solid suspended matter (SS), and the yield of CH ₄ were determined as follows. The results are shown in Tables 6, 7 and 7.

<SS removal rate (%)>
The SS removal rate (%) was calculated from the weight difference between before fermentation (at the time of input) and after fermentation (at the time of discharge) as shown in Equation 1 below.

Here, SS _inlet is the load weight of SS at the time of charging, and SS _outlet is the residual weight of SS after fermentation at the time of discharging.

<Amount of residual carbon (C) in solid suspended matter (SS)>
The amount of residual carbon (C) (residual C-SS) in the solid suspended matter (SS) was calculated by multiplying the SS weight by 72/162 [: 6C / (cellulose molecular weight)].

<Yield of CH ₄ >
The yield of CH ₄ was calculated on a gram carbon basis and calculated as in Equation 2 below.

Results As shown in Table 7, during the operation of the second step (FFR), the pH at the time of charging (7.5) of Example 1 decreased, and the pH at the time of discharging became 6.8 ± 0.0. In addition, the pH at the time of charging (7.5) of Comparative Example 1 also decreased, and the pH at the time of discharging became 6.4 ± 0.4.
The SS removal rate in the second step (FFR) of Example 1 was 90.7 ± 2.2%, and the SS removal rate (66.9 ± 1.1%) in the second step (FFR) of Comparative Example 1. ) Much higher (Table 7).
Further, in Example 1, by performing the first step and the second step, the CH ₄ yield was 37.5 ± 1.4%, and the CH ₄ content in the gas was 52.8 ± 0.3 vol%. became. On the other hand, in Comparative Example 1, it was found that the CH ₄ yield was 22.1 ± 1.4% and the CH ₄ content in the gas was 46.8 ± 1.3 vol% (see Table 6 and FIG. 7). ).
As shown in Table 7, a synthetic medium containing cellulose as a main carbon source with a hydraulic residence time (HRT) of 2.5 days was used as an example with a cellulose loading of 3555.6 mg-carbon (C) / L / day. 1 and Comparative Example 1 were added to the reaction vessel of the first step. During the operation of the first step, the pH at the time of charging (6.1) of Example 1 decreased, and the pH at the time of discharging became 5.2 ± 0.3.
On the other hand, when Comparative Example 1 was added, the pH (6.1) also decreased, and when discharged, the pH became 5.2 ± 0.2. Therefore, most of the cellulose remains without being consumed, and the removal rate of suspended solids (SS) after the first step is 11.4 ± 4.3% in Example 1 and 11 in Comparative Example 1. It was 0.6 ± 4.3%.

<Test Example 4>
Relative abundance of microorganisms (%)
In Example 1 and Comparative Example 1, the relative abundance (%) of the microorganisms in the respective fermentation broths (culture broths) and the fractions (microorganisms) attached to the fixing film (carbon fiber woven fabric (CFT) film) was determined. The measurement was carried out by the same method as that described in JP-A-2011-224039.
Specifically, 5000 μL of each fermentation broth (culture broth) was centrifuged at 5000 × g, and the pellets were suspended in 200 μL of Tris-EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). I made it muddy. Alternatively, the attached fraction (microorganism) is vortexed and recovered in 5000 μL Tris-EDTA buffer, and the pelleted material after centrifugation is placed in 200 μL Tris-EDTA buffer as described above. Suspended.
For each of these suspensions, 300 mg of glass beads (diameter: 0.1 mm), 500 μL of Tris-EDTA saturated phenol, 250 μL of lysis buffer, and 50 μL of 10% (w / v) sodium dodecyl sulfate were added to each tube. .. The mixture was then vigorously shaken at 5.0 m / s for 30 seconds using a FatPrep-24 device (MP Biomedicals). The mixture was then centrifuged at 22,000 xg for 5 minutes. The upper aqueous layer was transferred to a clean tube containing 275 μL of isopropyl alcohol and 1/10 volume of 3M sodium acetate and cooled at −20 ° C. for 10-15 minutes. The extracted DNA precipitate was centrifuged at 22,000 × g for 5 minutes, washed with 70% ethanol, and then dried under vacuum. The DNA was then dissolved in Tris-EDTA buffer.

Measurement of microbial composition (real-time PCR analysis)
The microbial composition of Example 1 and Comparative Example 1 was measured using real-time PCR. This real-time PCR was performed using a LightCycler® 96 system (Roche) with universal primer sets (5'-ACTCCTACGGGAGGCAGCAGT-3'and 5'-GTATTACCGCGGCTGCTGGCAC-3') targeting eubacteria. Was carried out to quantify. The results are shown in Table 8 and FIG.

The types of microorganisms in Table 8 are as follows.
A: Methanobacterium sp.
B: Methanothermobacter thermautotrophicus
C: Methanosarcina sp.
D: Anaerolineae; CFB-26
E: Ureibacillus sp.
F: Unclassified Haloplasmataceae
G: Unclassified Clostridiales
H: Unclassified Clostridium
I: Clostridium sp.
J: Thermoanaerobacterium saccharolyticum
K: Lutispora sp.
L: Coprococcus sp.
M: Unclassified Peptococcaceae
N: Unclassified Ruminococcaceae
O: Ruminococcus sp.
P: Syntrophomonas sp.
Q: Unclassified Veillonellaceae
R: Tepidimicrobium sp.
S: Clostridia; MBA08
T: Clostridia; SHA08
U: Others
V: Unclassified bactria

Results As a result, most of the microorganisms are contained in the suspension fraction (BES-cathode and BES-anodide) of the reactor of the first step and the suspension (floating) fraction of the reactor of the second step of Example 1. FFR), and the suspension fraction (NBES-right side and NBES-left side) of the reactor of the first step of Comparative Example 1 and the suspension (floating) fraction (FFR) of the reactor of the second step. Among the fractions (FFR-C) attached to the CFT film of the second step of Example 1 and the fractions (FFR-C) attached to the CFT film of the second step of Comparative Example 1, three lines (gates) It was classified as (Euryarchaeota, Chloroflexi, Firmicutes).
The suspension fractions of the first step reactor (BES-cathode and anode) of Example 1 and the suspension fractions of the first step reactor of Comparative Example 1 (NBES-right and left) are mainly Thermoanaerobacterium. Firmicutes consisting of bacteria related to saccharolyticum (type J of the above bacteria) were dominant.
Bacteria related to Clostridium sp. And Ruminococcus sp. Were the other major microorganisms in the first step of Example 1, and bacteria related to Unclassiified Clostridiales were also the main bacteria in the first step of Comparative Example 1.
Since the pH was low in the first step of Example 1 and the first step of Comparative Example 1, almost no methanogen was detected.
Bacteria related to Clostridium sp. Are the main organisms in the suspension (floating) fraction of the reactor and the fraction attached to the CFT membrane in the second step of Example 1 and the second step of Comparative Example 1. In particular, the suspension (floating) fraction (FFR) of the reactor in the second step of Comparative Example 1 occupies the largest amount, which corresponds to the low diversity of microorganisms.
In the reactor suspension (floating) fraction (FFR) of the second step of Example 1, bacteria associated with Unclassified Clostridiales and Clostridia; MBA08 were the other major microorganisms.
The relative abundance of Methanobacterium sp. (Type of bacterium: A) was 2.585% in the suspension (floating) fraction (FFR) of the reactor in the second step of Example 1, so that comparison was made. It was higher than 1.408% of the suspended (floating) fraction (FFR) of the reactor in the second step of Example 1.
Interestingly, the fraction attached to the CFT film in the second step of Example 1 (FFR-C) was compared with the fraction attached to the CFT film in the second step of Comparative Example 1 (FFR-C). The relative abundance of methanogens (Methanosarcina sp., Bacterial type: C) increased from 0.007% to 5.835%.
The total amount of methanogens (types of bacteria: A to C) was 2.955% in the suspension (floating) fraction (FFR) of the reactor in the second step of Example 1, and the second of Example 1. The fraction attached to the CFT film (FFR-C) in the process was 6.512%. The total amount of methane-producing bacteria (types of bacteria: A to C) in Comparative Example 1 was 1.474% in the suspended (floating) fraction (FFR) of the reactor in the second step, and to the CFT film in the second step. Since the adhered fraction (FFR-C) of was 0.703%, it can be seen that the number of methane-producing bacteria was clearly increased by passing a weak current through the working electrode in the first step.

<Test Example 5>
Recovery test
In Example 1, it is clarified whether or not the deteriorated state of Comparative Example 1 (NBES → FFR) in which the electric current is not applied in the first step is recovered by applying the electric current as the bioelectrochemical treatment in the first step. I tried that.
First, the first step (NBES) and the second step (FFR) after acclimatization were operated at HRT for 2.5 days and 4.2 days, respectively. This corresponds to Comparative Example 1. Next, an electric current was passed through the first step, and the bioelectrochemical treatment (BES) was performed on the 2.5th day and the second step (FFR) was performed on the 4.2th day by the HRT after acclimatization. (BES → FFR). This corresponds to Example 1.
The discharge (outlet) pH is 5.6 ± 0.1 in the first step (NBES) of Comparative Example 1 and 6.3 ± 0.1 in the second step (FFR) of Comparative Example 1. The first step (BES) of Example 1 was 5.2 ± 0.1, and the first step (FFR) of Example 1 was 6.6 ± 0.1.
In the same manner as in Test Example 3, the amount of biogas (CH ₄ , CO ₂ and H ₂ ) produced (mM / day) and the amount of product (acetic acid, propionic acid, butanoic acid, and ethanol) were measured. The amount of carbon (CH ₄ , CO ₂ , SCFA + ethanol, residue C-SS) and the SS removal rate were determined. The results are shown in Tables 9-12 and FIGS. 9-11.

Results The SS removal rate in the first step (NBES) of Comparative Example 1 performed first was 10.0 ± 0.1%. In the second step (FFR) of Comparative Example 1, as expected, only 49.9 ± 4.2% of SS was removed (Table 12).
Interestingly, by passing an electric current (performing a bioelectrochemical treatment) in the first step, the SS removal rate was increased to 82.3 ± 3.0% in the second step (FFR). The SS removal rate in the first step (BES) at this time was 23.2 ± 0.9% (Table 12).
Therefore, in the second step (FFR) of Comparative Example 1 in which no current was passed in the first step (NBES), the biogas production amount was small (total 22.1 ± 3.8 mM / day), but in the first step. In the second step (FFR) of Example 1 in which an electric current was passed (BES), the amount of biogas produced increased (total 103.3 ± 3.9 mM / day) (Table 9, FIG. 9).
Further, in the second step (FFR) of Comparative Example 1, a large amount of SCFA and ethanol was accumulated (total 56.1 ± 1.8 mM), but in the second step (FFR) of Example 1, SCFA and ethanol were accumulated. Decreased (total 16.5 ± 1.9 mM) (Table 10, FIG. 10).
As a result, in Comparative Example 1 (NBES → FFR) in which no current was passed in the first step, the CH ₄ yield was 7.1 ± 1.9%, and the CH ₄ content in the gas was 47.9 ± 1. It was 9.9 vol%. In Example 1 (BES → FFR), the CH ₄ yield was 38.4 ± 2.3% and the CH ₄ content in the gas was 60.8 ± 2.4 vol% by passing an electric current in the first step. (See Table 11 and FIG. 11).
That is, the use of a fixed film (carbon fiber woven fabric (CFT) film) in the second step stabilizes the methane fermentation method of the present invention (first step: bioelectrochemical system, second step: fixed film fermentation step). It can be said that it is necessary for driving.

<Test Example 6>
Necessity of Conductive Material (CFT) Membrane in Second Step (FFR)
The necessity of the conductive material film (fixing film) in Example 1 was confirmed as compared with Comparative Example 2 having no conductive material film (fixing film).
First, the HRT after acclimatization was set to 2.5 days and 4.2 days, respectively (Table 1), and the first step (BES) and the subsequent second step (NFFR) without CFT were carried out (NFFR). Comparative example 2). The apparatus used in Comparative Example 2 is shown in FIG.
A CFT was installed in the second step, and the first step (BES) and the subsequent second step (FFR) were operated in the HRT of 2.5 days and 4.2 days after the adaptation (acclimation). (Example 1).
The pH at discharge was 5.8 ± 0.4 in the first step (BES) of Comparative Example 2, 6.8 ± 0 in the second step (NFFR) of Comparative Example 2, and the first step (BES) of Example 1. ) Was 5.4 ± 0.4, and the second step (FFR) of Example 1 was 6.6 ± 0.1.
In the same manner as in Test Example 3, the amount of biogas (CH ₄ , CO ₂ and H ₂ ) produced (mM / day) and the amount of product (acetic acid, propionic acid, butanoic acid, and ethanol) were measured. The amount of carbon (CH ₄ , CO ₂ , SCFA + ethanol, residue C-SS) and the SS removal rate were determined. The results are shown in Tables 13-16 and FIGS. 12-14.

From the result table 16, in the first step (BES) of Comparative Example 2, the SS removal rate was 11.9 ± 0.6% as in the first step (BES) of Example 1. The SS removal rate in the second step (NFFR) of Comparative Example 2 was only 51.2 ± 1.6%.
Interestingly, the installation of the conductive material (CFT) membrane in the reactor of the second step increased the SS removal rate to 74.9 ± 5.7% in the second step (FFR) of Example 1. In the first step (BES) of Example 1, the SS removal rate was 8.9 ± 1.5%.
In the second step (NFFR) of Comparative Example 2 in which the CFT membrane is not installed in the reactor, the amount of biogas produced is small (total 44.4 ± 5.8 mM / day), but in Example 1 in which the CFT membrane is installed in the reactor. In the second step (FFR), the amount of biogas produced increased (total 91.0 ± 1.8 mM / day) (Table 13, FIG. 12).
In the second step (NFFR) of Comparative Example 2, a large amount of SCFA and ethanol was accumulated (total 51.5 ± 1.9 mM), but in the second step (FFR) of Example 1, SCFA and ethanol decreased (total). 31.4 ± 5.1 mM) (Table 14, FIG. 13).
As a result, in Comparative Example 2 (BES → NFFR), the CH ₄ yield was as low as 13.4 ± 3.1%, and the CH ₄ content in the gas was as low as 48.8 ± 5.8 vol%. In Example 1 (BES → FFR) to which the CFT film was added in the second step, the CH ₄ yield was 30.0 ± 1.4%, and the CH ₄ content in the gas was 56.0 ± 2.6 vol%. (See Table 15 and FIG. 14).

CONCLUSIONS In the first step of the present invention, a low cost carbon sheet was used for the cathode and anode to operate the thermophilic BES in microbial electrolysis cell mode ₍ low current and inanimate H2 production). In the second step of the present invention, a thermophilic FFR containing a film made of CFT, which is a conductive material, was operated. Such a methane fermentation method (BES → FFR) of the present invention includes the method of Comparative Example 1 (NBES → FFR) in which the first step does not have a bioelectrochemical system, and the comparison in which the second step does not have a CFT film. Compared with the method of Example 2 (BES → _NFFR ), it was possible to promote the production of CH4 by promoting the decomposition of cellulose by microorganisms and reducing the accumulation of short-chain fatty acids and ethanol.
The electrochemical CH4 production amount in the _first step was negligible when compared with the (actual) CH4 production amount after the _second step. From these facts, it was found that both the bioelectrochemical system in the first step and the fixed film (CFT film) in the second step are required for the stable operation of the methane fermentation method (BES → FFR) of the present invention. rice field.
Further, in the second step (FFR) of the methane fermentation method of the present invention, the concentration of suspended microorganisms and adhered microorganisms are compared with the second step (FFR) of the method of Comparative Example 1 in which the first step does not have a bioelectrochemical system. It turned out that the variety of.
Bacteria considered to have cellulose-degrading activity and sugar-degrading activity are the first step (BES) of the methane fermentation method of the present invention, the first step (NBES) of the method of Comparative Example 1, and the first step of the methane fermentation method of the present invention. It was present in the second step (FFR) and the second step (FFR) of the method of Comparative Example 1.
Further, in the _second step (FFR) of the methane fermentation method of the present invention, the relative abundance of the methanogens Methanobacterium sp. And Methanosarcina sp. , Each increased as compared with the second step (FFR) of Comparative Example 1. Therefore, the bioelectrochemical treatment of the first step promoted the interspecific H2 transfer in the _second step ₍ FFR) and accelerated the conversion of cellulose to CH4.
From the above results, it was suggested that the methane fermentation method (BES → FFR) of the present invention has a short HRT in the first step (BES) and is an advantageous system for anaerobic digestion of cellulose. The short HRT of the first step (BES) means that the first step (BES) can be operated on a small scale and the second step (FFR) can be operated on a large scale.
Therefore, the present invention can provide a thermophilic methane fermentation method comprising a first step of performing bioelectrochemical treatment and a second step of immobilizing a CFT film in an anaerobic reaction (digestor). .. The methane fermentation method of the present invention promotes the conversion of cellulose to CH ₄ by microorganisms as compared with the case where the bioelectrochemical system is not used in the first step and the case where the CFT film is not used in the second step. be able to.

Claims (8)

(1) A carrier holding electrode having a carrier on at least a part of the electrode surface,
A bioelectrochemical treatment step containing an organic substrate and a group of microorganisms involved in methane fermentation, and bringing the organic substrate into contact with a methane fermentation liquid to be treated with methane fermentation, and
(2) A methane fermentation promoting method comprising a fixed membrane fermentation step in which a fermented liquid obtained in the bioelectrochemical treatment step and a fixed membrane containing a conductive material are brought into contact with each other.
The above (1) bioelectrochemical treatment step is a method for promoting methane fermentation, which is carried out while controlling the current to 3 μA / cm ² or less. In the above (2) fixed membrane fermentation step
The method for promoting methane fermentation according to claim 1, wherein the conductive material is hydrophobic. In the above (2) fixed membrane fermentation step
The method for promoting methane fermentation according to claim 1 or 2, wherein the conductive material is carbon fiber. (1) A carrier holding electrode having a carrier on at least a part of the electrode surface,
A bioelectrochemical treatment step containing an organic substrate and a group of microorganisms involved in methane fermentation, and bringing the organic substrate into contact with a methane fermentation liquid to be treated with methane fermentation, and
(2) Preparation of a fixed membrane containing a conductive material carrying a group of microorganisms, which comprises a fixed membrane fermentation step in which the fermentation broth obtained in the bioelectrochemical treatment step is brought into contact with a fixed membrane containing a conductive material. It ’s a method,
The above-mentioned (1) bioelectrochemical treatment step is a method for producing a fixative film containing a conductive material carrying a group of microorganisms, which is carried out while controlling the current to 3 μA / cm ² or less. In the above (2) fixed membrane fermentation step
The method for producing a fixative film containing the conductive material on which the microorganism group according to claim 4 is supported, wherein the conductive material is hydrophobic. In the above (2) fixed membrane fermentation step
The method for producing a fixative film containing the conductive material on which the microorganism group according to claim 4 or 5 is supported, wherein the conductive material is carbon fiber. (1) A carrier holding electrode having a carrier on at least a part of the electrode surface,
A bioelectrochemical treatment unit that contains an organic substrate and a group of microorganisms involved in methane fermentation, and is in contact with the methane fermentation liquid for methane fermentation treatment of the organic substrate, and
(2) A methane fermentation accelerating device including a fixed membrane fermentation unit in which a fermentation broth obtained in the bioelectrochemical treatment unit and a fixed membrane containing a conductive material are brought into contact with each other.
The (1) bioelectrochemical treatment unit is a methane fermentation promoting device that controls a current of 3 μA / cm ² or less. (1) A carrier holding electrode having a carrier on at least a part of the electrode surface,
A bioelectrochemical treatment unit that contains an organic substrate and a group of microorganisms involved in methane fermentation, and is in contact with the methane fermentation liquid for methane fermentation treatment of the organic substrate, and
(2) Production of a fixed membrane containing a conductive material carrying a group of microorganisms, which comprises a fixed membrane fermented portion in which the fermented liquid obtained in the bioelectrochemical treatment section and a fixed membrane containing a conductive material are brought into contact with each other. It ’s a device,
(1) The bioelectrochemical treatment unit is an apparatus for producing a fixed membrane containing a conductive material carrying a group of microorganisms, which controls a current of 3 μA / cm ² or less.

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