Microorganism, and fuel cell, electrolytic cell and composition comprising same

Abstract

Provided are: a novel microorganism having a higher current-generating ability; a composition for a microbial fuel cell and a composition for a microbial electrolysis cell, each of which contains the microorganism; and a microbial fuel cell and a microbial electrolysis cell, each of which uses the microorganism. The present invention provides: a microorganism of the genus geobacter 60473 strain; a composition for microbial fuel cells, which contains the genus geobacter 60473 strain and an organic material; and a microbial fuel cell in which the genus geobacter 60473 strain is used to decompose an organic material that is a fuel. A composition for microbial electrolysis cells, which contains a strain of genus Geobacterium 60473 and an organic substance, and a microbial electrolysis cell in which the strain of genus Geobacterium 60473 is used to decompose an organic substance that is a fuel.

Description

Technical Field

[0001] The present invention relates to a microorganism used in a microbial fuel cell and a microbial fuel cell using the microorganism. Background Art

[0002] Actively developing a system that produces energy or useful substances by electrochemically controlling the metabolism of microorganisms, namely a microbial electrochemical system (BES), will help solve problems such as global warming and the depletion of natural resources. Examples of microorganisms used in microbial electrochemical systems include electrochemically active bacteria (EAB) that can send and receive electrons between electrodes.

[0003] Regarding microbial electrochemical systems, there are microbial fuel cells (MFC), microbial electrolysis cells (MEC), microbial electrosynthesis systems (tanks) (microbial electrosynthesis systems, MES), etc. Microbial fuel cells are devices that use the metabolic ability of microorganisms to convert fuels such as organic matter (substances that retain chemical energy) into electrical energy. Microbial fuel cells have attracted much attention as devices that process biomass waste while generating electricity or energy-saving wastewater treatment devices (non-patent document 1). Microbial electrolysis cells are devices that use the current generated by electrochemically active bacteria from fuels such as organic matter to reduce protons in water to produce hydrogen or methane (non-patent document 2). Microbial electrosynthesis systems are devices that supply electrons from low-potential electrodes to electrochemically active bacteria to synthesize useful substances from carbon dioxide or nitrogen, etc., and are expected to become a technology that supports humans in the future when fossil fuels are depleted (non-patent document 3). In this way, although various devices are included in the microbial electrochemical system, high performance is expected for the social installation of these devices.

[0004] It is said that in each device of the microbial electrochemical system, the efficiency of electron transfer between electrochemically active bacteria and electrodes, which is evaluated by the current value (current density) per unit electrode area, determines the performance of the device. In fact, there are reports that the electron transmission and reception between electrochemically active bacteria and electrodes is the rate-limiting stage in the microbial electrochemical system (Non-Patent Document 4).

[0005] As a method for enhancing the activity (power generation capacity) of electrochemically active bacteria, it is known that the following method is used: using artificially cultured foreign microorganisms (e.g., electrochemically active bacteria) as a microbial preparation and being put into the microbial ecosystem in the device to improve the performance of the device (bioaugmentation). For example, an example (non-patent literature 5) of starting to generate electric current in advance by putting into a microbial consortium (microbial consortium) comprising electrochemically active bacteria in the microbial fuel cell used for wastewater treatment is reported. However, in the example, the current value after the current rises is the same as the microbial fuel cell that is not bioaugmented, thereby further high performance is expected. In addition, it is known that there is a bioaugmentation by adding sulfur-reducing Geobacter sulfurreducens strain YM18 to the anode electrode of a microbial fuel cell to promote electric current generation and hydrogen generation (non-patent literature 6).

[0006] As a method for increasing the power generation (output energy) of a microbial fuel cell, there is a method of using a compound (electron mediator) that transfers electrons such as 2-hydroxy-1,4-naphthoquinone (2-hydroxy-1,4-naphthoquinone, HNQ), and as an improved method of the method, a method of using electrochemically active bacteria that are active even in a lightless environment and using porphyrin as the electron mediator is reported (Patent Document 1). In addition, a method of adjusting the activity of the electrochemically active bacteria by using a bacteriophage (bacteriophage) that uses the electrochemically active bacteria as a host to enhance the activity of the electrochemically active bacteria is reported (Patent Document 2). However, regarding the use of the mediator, there is the following problem: since it will disappear due to decomposition based on microorganisms, it is necessary to continue to add it artificially, which increases the cost in practical terms. In addition, regarding the use of bacteriophages, there is a problem that the cost is required for the certification of phages that are not genes related to pathogenicity and drug resistance.

[0007] [Prior art literature]

[0008] [Patent Document]

[0009] [Patent Document 1] Japanese Patent Application Publication No. 2010-218690

[0010] [Patent Document 2] Japanese Patent Application Publication No. 2020-14418

[0011] [Non-patent literature]

[0012] [Non-patent document 1] Shohei Yamada and Kazuya Watanabe, "Basics and Applications of Microbial Fuel Cells", Fuel Cells, Fuel Cell Development Information Center (Published), 2020, Vol. 20, pp. 33-38.

[0013] [Non-patent document 2] Kazuta Ochiai et al., "Hydrogen production using microbial electrolyzers", Microbial-based power generation / hydrogen production, CMC Publishing (distribution), 2021, pages 201-206.

[0014] [Non-patent document 3] Yamada et al., Frontiers in Chemistry, 2022, vol. 9, Article 805597.

[0015] [Non-patent document 4] Lu and Ren, Bioresource Technology, 2016, vol. 215, pp. 254-264.

[0016] [Non-patent document 5] Yanuka-Golub et al., mBio, 2021, vol. 12, e03629-20.

[0017] [Non-patent document 6] Ochiai et al., Bioresource Technology, 2023, vol. 386, 129508.

[0018] [Non-patent document 7] Yoshitsu et al., Microorganisms, 2023, vol. 11, 598. Summary of the invention

[0019] [Problems to be solved by the invention]

[0020] In order to improve the performance of each device of the microbial electrochemical system, it is desired to have an electrochemically active bacterium that can achieve a high current density. For example, in the example of bioenhancement reported in non-patent literature 4, the activity of the electrochemically active bacteria in the microbial electrochemical system cannot be fully increased. It can be considered that the reason is that electrochemically active bacteria with sufficiently high electrochemical activity cannot be put into use. In addition, regarding the YM16 strain used in the microbial fuel cell reported in non-patent literature 6, if the power generation time becomes longer, the amount of current generated will decrease. Therefore, it is also sought to have an electrochemically active bacterium that can achieve stable power generation for a longer time.

[0021] An object of the present invention is to provide a novel microorganism having a higher current generating capacity, a composition for a microbial fuel cell and a composition for a microbial electrolytic cell containing the microorganism, and a microbial fuel cell and a microbial electrolytic cell using the microorganism.

[0022] [Technical means to solve the problem]

[0023] The present inventors have repeatedly conducted research efforts to solve the above-mentioned problems, and as a result, found that the novel microorganisms isolated and identified from the mud along the shore of Lake Suwa are novel electrochemically active bacteria with high current generation capabilities, which are suitable for use in microbial fuel cells and microbial electrolytic cells, thereby completing the present invention.

[0024] The microorganism, composition for microbial fuel cell, microbial fuel cell, composition for microbial electrolytic cell, and microbial electrolytic cell of the present invention are as follows.

[0025] [1] A microorganism, which is Geobacter sp. 60473 (NITE BP-03900).

[0026] [2] The microorganism according to [1], which is used in a microbial fuel cell.

[0027] [3] A composition for a microbial fuel cell, comprising Geobacter sp. 60473 (NITE BP-03900) and organic matter.

[0028] [4] The composition for a microbial fuel cell according to [3], further comprising electrochemically active bacteria other than Geobacter 60473 strain.

[0029] [5] The composition for a microbial fuel cell according to [3] or [4], wherein at least a part of the organic matter is sludge.

[0030] [6] A microbial fuel cell, wherein Geobacter sp. 60473 (NITEBP-03900) is used to decompose organic matter as a fuel.

[0031] [7] A microbial fuel cell comprising:

[0032] Liquid, with water containing organic matter as the main component;

[0033] A container for containing the liquid;

[0034] A conductive anode electrode disposed in the liquid;

[0035] A conductive cathode electrode, at least one surface of which is in contact with the liquid;

[0036] a resistor having one end connected to the anode electrode and the other end connected to the cathode electrode and allowing electrons to flow; and

[0037] Geobacter sp. 60473 (NITE BP-03900) decomposes the organic matter contained in the liquid and emits electrons.

[0038] [8] The microbial fuel cell according to [7], wherein the container is divided into a groove in which the anode electrode is arranged and a groove in which the cathode electrode is arranged by a membrane that can selectively allow protons to pass through.

[0039] The organic matter and the Geobacter 60473 strain are contained in the liquid contained in the tank in which the anode electrode is arranged.

[0040] [9] The microbial fuel cell according to [7], wherein the cathode electrode has one surface in contact with the liquid in the container and the other surface in contact with the atmosphere.

[0041]

[10] A microbial fuel cell comprising:

[0042] A container containing organic mud and a liquid with water as a main component;

[0043] A conductive anode electrode is disposed in the organic mud;

[0044] A conductive cathode electrode disposed in or on the surface of the liquid;

[0045] a resistor having one end connected to the anode electrode and the other end connected to the cathode electrode and allowing electrons to flow; and

[0046] Geobacter sp. 60473 (NITE BP-03900) is contained in the organic mud.

[0047]

[11] A microbial fuel cell comprising:

[0048] A conductive anode electrode is disposed in the organic mud;

[0049] A conductive cathode electrode is placed in the water or on the water surface;

[0050] a resistor having one end connected to the anode electrode and the other end connected to the cathode electrode and allowing electrons to flow; and

[0051] Geobacter sp. 60473 (NITE BP-03900), attached to the anode electrode,

[0052] The microbial fuel cell is provided to purify organic mud at the bottom of a river or a lake.

[0053]

[12] The microbial fuel cell according to

[10] or

[11] , comprising a supply pipe for supplying the substrate and / or the Geobacter 60473 strain from the outside to the inside of the organic mud.

[0054]

[13] The microbial fuel cell according to

[12] , wherein the supply pipe branches into the liquid or the water so as to be dispersed in a plurality of portions in a planar direction of the anode electrode for supply.

[0055]

[14] The microbial fuel cell according to any one of [7],

[10] and

[11] , wherein the anode electrode is a conductive metal material having a lower internal resistance than the cathode electrode.

[0056]

[15] The microbial fuel cell according to any one of [7],

[10] and

[11] , wherein the surface area of ​​the cathode electrode is at least 1.1 times larger than the surface area of ​​the anode electrode.

[0057]

[16] A microbial fuel cell according to

[10] or

[11] , wherein the cathode electrode and the anode electrode are arranged in parallel, one end of the cathode electrode and one end of the anode electrode arranged on the same side as the one end are connected to a support extending along the depth direction in the liquid or water, the cathode electrode and the anode electrode are fixedly supported so as to be adjustable while maintaining a spacing corresponding to the depth, and a fixing portion protruding in the depth direction is included on the bottom surface side of the anode electrode.

[0058]

[17] A composition for a microbial electrolytic cell, comprising Geobacter sp. 60473 (NITE BP-03900) and organic matter.

[0059]

[18] The composition for a microbial electrolytic cell according to

[17] , further comprising electrochemically active bacteria other than Geobacter 60473 strain.

[0060]

[19] The composition for a microbial electrolysis cell according to

[17] or

[18] , wherein at least a portion of the organic matter is sludge.

[0061]

[20] A microbial electrolysis cell, wherein Geobacter sp. 60473 (NITEBP-03900) is used to decompose organic matter as a fuel.

[0062]

[21] A microbial electrolysis cell comprising:

[0063] Liquid, with water containing organic matter as the main component;

[0064] A container for containing the liquid;

[0065] A conductive anode electrode disposed in the liquid;

[0066] A conductive cathode electrode, arranged in contact with the liquid;

[0067] a potential control device, one end of which is connected to the anode electrode and the other end of which is connected to the cathode electrode and provides electron flow; and

[0068] Geobacter sp. 60473 (NITE BP-03900) decomposes the organic matter contained in the liquid and emits electrons.

[0069]

[22] The microbial electrolysis cell according to

[21] , wherein the container is divided into a tank in which the anode electrode is arranged and a tank in which the cathode electrode is arranged by a membrane that can selectively allow protons to pass through.

[0070] The organic matter and the Geobacter 60473 strain are contained in the liquid contained in the tank in which the anode electrode is arranged.

[0071]

[23] A microbial electrolysis cell comprising:

[0072] A container containing organic mud and a liquid with water as a main component;

[0073] A conductive anode electrode disposed in the liquid;

[0074] A conductive cathode electrode disposed in the liquid;

[0075] a potential control device, one end of which is connected to the anode electrode and the other end of which is connected to the cathode electrode and provides electron flow; and

[0076] Geobacter sp. 60473 (NITE BP-03900) is contained in the organic mud.

[0077]

[24] The microbial electrolysis cell according to

[23] includes a supply pipe for supplying the substrate and / or the Geobacter 60473 strain from the outside to the inside of the liquid.

[0078]

[25] The microbial electrolysis cell according to

[24] , wherein the supply pipe branches in the liquid so as to be dispersed in a plurality of portions to supply the anode electrode or the cathode electrode.

[0079]

[26] A microbial electrolysis cell according to any one of

[21] ,

[23] and

[24] , wherein the anode electrode is a conductive material having a smaller internal resistance than the cathode electrode.

[0080]

[27] A microbial electrolysis cell according to any one of

[21] ,

[23] and

[24] , wherein the surface area of ​​the cathode electrode is at least 1.1 times greater than the surface area of ​​the anode electrode.

[0081] [Effects of the Invention]

[0082] According to the present invention, a microorganism having a very high current generation capability or hydrogen generation capability can be provided. In addition, by using the microorganism, a microbial fuel cell having a good power generation efficiency and a microbial electrolysis cell having a good electrolysis efficiency can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 This is a schematic diagram of one form of a microbial fuel cell provided with a microorganism supply device.

[0084] Figure 2 The present invention is a schematic diagram of a microbial fuel cell including a support device for facilitating movement.

[0085] Figure 3 It is a schematic diagram of a three-pole and one-tank type microbial electrolysis cell.

[0086] Figure 4 This is a schematic diagram of the structure of the mud cell used to separate 60473 strains in Example 1.

[0087] Figure 5 This is a diagram showing the results of measuring over time the current generated by culturing the 60473 strain and the current generated by culturing the PCA strain in an electrochemical cell using a graphite felt electrode as a working electrode in Example 1.

[0088] Figure 6 This is a graph showing the results of measuring over time the current generated by culturing the 60473 strain and the current generated by culturing the PCA strain in an electrochemical cell using a glassy carbon electrode as a working electrode in Example 1.

[0089] Figure 7 This is a graph showing the results of measuring the fluorescence intensity of the glassy carbon electrode by DAPI staining after the current generation experiment in Example 1.

[0090] Figure 8 This is a diagram showing the phylogenetic relationship between the 60473 strains and closely related strains shown in Example 1 by molecular phylogenetic analysis using the 16S rRNA gene.

[0091] Fig. 9 This is a diagram showing the phylogenetic relationship between the 60473 strains and closely related strains shown in the molecular phylogenetic analysis using the gyrB gene in Example 1.

[0092] Fig.10 This is a graph showing the polarization analysis results of the mud battery in which 60473 plants were added in Example 2.

[0093] Fig.11 This is a diagram showing the polarization analysis results of the mud battery in Example 2 in which 60473 strains were not added.

[0094] Fig.12 It is shown that in Example 2, according to Fig.10 and Fig.11 The maximum power density (P max mW / m 2 )’s picture.

[0095] Fig.13 This is a diagram showing the results of measuring over time the current generated by culturing the 60473 strain, PCA strain, KN400 strain, or YM18 strain in an electrochemical cell using a graphite felt electrode as a working electrode in Example 3.

[0096] Fig.14 This is a diagram showing the results of measuring the maximum current density of the current generated by culturing the 60473 strain, the PCA strain, the KN400 strain, or the YM18 strain in an electrochemical cell using a graphite felt electrode as a working electrode in Example 3.

[0097] Fig.15 This is a graph showing the results of measuring over time the current generated by culturing the 60473 strain, PCA strain, KN400 strain, or YM18 strain in an electrochemical cell using a glassy carbon electrode as a working electrode in Example 3.

[0098] Fig.16 This is a graph showing the results of measuring the maximum current density of the current generated by culturing the 60473 strain, the PCA strain, the KN400 strain, or the YM18 strain in an electrochemical cell using a glassy carbon electrode as a working electrode in Example 3.

[0099] Fig.17 This is a graph showing the results of measuring the affinity (GAI) of the 60473 strain, PCA strain, KN400 strain, and YM18 strain for graphite in Example 3.

[0100] Fig.18 This is a graph showing the results of measuring the cell hydrophobicity (HPI) of the 60473 strain, PCA strain, KN400 strain, and YM18 strain in Example 3.

[0101] Fig.19 This is a graph showing the measurement results of the cytochrome content (CCI) of the 60473 strain, PCA strain, KN400 strain, and YM18 strain in Example 3.

[0102] Fig. 20 This is a diagram showing the results of measuring over time the current generated in the electrolytic cells (MEC3 and MEC4) in which 60473 strains were cultured and the electrolytic cells (MEC1 and MEC2) in which no strains were cultured in Example 4.

[0103] Fig.21 This is a diagram showing an enlarged view of the results of measuring over time the current generated in the electrolytic cells (MEC1 and MEC2) in which 60473 strains were not cultured in Example 4.

[0104] Fig. 22 The graph shows the bacterial composition of the biofilm formed on the surface of the working electrode (anode electrode) of the electrolytic cells (MEC3 and MEC4) in which 60473 strains were cultured and the electrolytic cells (MEC1 and MEC2) in which no strains were cultured after operation in Example 4.

[0105] Fig.23 This is a diagram showing the results of measuring over time the current generated in the electrolytic cells (60473_1 and 60473_2) in which 60473 strains were cultured and the electrolytic cells (NB_1 and NB_2) in which no strains were cultured in Example 5.

[0106] Fig.24These are current-voltage curves obtained by CV (cyclic voltammetry) of the electrolytic cells (60473_1 and 60473_2) in which the 60473 strain was cultured and the electrolytic cells (NB_1 and NB_2) in which the strain was not cultured in Example 5.

[0107] Fig.25 This is a graph showing the results of measuring the voltage (V) of each reactor over time in Example 6.

[0108] Fig.26 This is a diagram showing the polarization analysis results of the MFC reactors (60473_1 and 60473_2) into which 60473 strains were added in Example 6.

[0109] Fig. 27 This is a diagram showing the polarization analysis results of the MFC reactors (NB_1 and NB_2) to which 60473 strains were not added in Example 6.

[0110] Fig.28 It is shown that in Example 6, according to Fig.26 and Fig. 27 The maximum power density (P max mW / m 2 )’s picture.

[0111] Fig.29 These are current-voltage curves obtained by CV of the MFC reactors (60473_1 and 60473_2) in which the 60473 strain was cultured and the MFC reactors (NB_1 and NB_2) in which no strain was cultured in Example 6.

[0112] [Explanation of Symbols]

[0113] 1: Sediment Microbial Fuel Cell

[0114] 2: Bottom mud

[0115] 3: Water

[0116] 4: Anode electrode

[0117] 5: Cathode electrode

[0118] 6: Resistor

[0119] 6a, 6b: Wire

[0120] 6c: External variable resistor

[0121] 7: Voltage measuring device

[0122] 8: Microorganism supply device

[0123] 8a: Microorganism storage container for supply

[0124] 8b: Microbial supply pipe

[0125] 11: Sediment Microbial Fuel Cell

[0126] 12: Bottom mud

[0127] 13: Water

[0128] 14: Anode electrode

[0129] 15: Cathode electrode

[0130] 16: Resistor

[0131] 16a, 16b: Wire

[0132] 16c: External variable resistor

[0133] 17: Support device

[0134] 17a: Anode electrode support

[0135] 17b: Cathode electrode support

[0136] 17c: Connection

[0137] 20: Microbial electrolysis cell

[0138] 21: Containment

[0139] 24: Anode electrode

[0140] 25: Cathode electrode

[0141] 26: Potentiostat

[0142] 26c: Reference electrode

[0143] 101: Data logger for voltage measurement

[0144] 102: External resistor

[0145] 103: Titanium wire

[0146] 104: Water

[0147] 105: cathode (platinum-coated graphite felt)

[0148] 106: Anode (graphite felt)

[0149] 107: Mud DETAILED DESCRIPTION

[0150] In the present invention and the specification of this application, “X1 to X2 (X1 and X2 are natural numbers satisfying X1<X2)” means a numerical range of “not less than X1 and not more than X2”.

[0151] [Microorganisms for microbial fuel cells]

[0152] The microorganism of one embodiment of the present invention is Geobacter sp. 60473 (hereinafter sometimes referred to as "60473 strain"). 60473 strains are novel electrochemically active bacteria isolated and identified from the mud on the shore of Lake Suwa, which decompose organic matter and discharge electrons. As shown in Example 1 described later, according to the results of genome analysis, the known closest species of 60473 strains is the reference strain of Geobacter sulfurreducens, which is commonly used as electrochemically active bacteria, i.e., Geobacter sulfurreducens PCA strain (hereinafter sometimes referred to as "PCA strain"). In addition, the inventors of the present invention and others deposited 60473 strains in the Patent Microorganism Preservation Center of the Independent Administrative Institution for Product Evaluation Technology Foundation (Room 122, 2-5-8, Kazusa Kamashi, Kisarazu City, Chiba Prefecture). The deposit number of Geobacter sp. 60473 strains is NITE BP-03900 (deposit date: June 8, 2023).

[0153] Strain 60473 is a Gram-negative anaerobic bacillus with flagella. It is an iron-reducing bacterium that oxidizes acetic acid to carbon dioxide by reducing iron (III) ions to obtain energy. The culture of strain 60473 can be carried out in the same way as other Geobacter bacteria. For example, strain 60473 can be cultured in a culture medium containing necessary minerals and vitamins at 10°C to 40°C and in an anaerobic environment. The anaerobic environment for culturing strain 60473 is not particularly limited as long as it is an environment with an oxygen concentration of less than 0.01% (v / v). Strain 60473 can be cultured, for example, in an environment where the nitrogen concentration is controlled to be above 99.99% (v / v), preferably 99.999%. Such culture in an anaerobic environment can be implemented by using a culture system for anaerobic bacteria such as AnaeroPackPouch (manufactured by Mitsubishi Gas Chemical Co., Ltd.).

[0154] As a culture medium used for culturing the 60473 strain, any culture medium commonly used in culturing Geobacter bacteria represented by Geobacter sulfurreducens, such as DSM826 culture medium (manufactured by Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ) and its modified culture medium can be used as long as it contains necessary minerals and vitamins. Liquid culture medium or agar culture medium can be used for culturing the 60473 strain. In addition, since Geobacter bacteria are soil bacteria, the 60473 strain can also be cultured in soil or sludge. The 60473 strain can be cultured even in an anaerobic environment rich in organic matter.

[0155] The 60473 strain has a very high power generation capacity, and its electrochemical cell has a higher current generation capacity than the PCA strain. Therefore, by using the 60473 strain as an electrochemically active bacterium, a microbial fuel cell with a high power generation capacity can be manufactured. Furthermore, like other electrochemically active bacteria, the 60473 strain can also be effectively used as an electrochemically active bacterium used in other microbial electrochemical systems other than microbial fuel cells, such as a microbial electrolysis cell or a microbial electrosynthesis system.

[0156] The affinity of strain 60473 for graphite (graphite affinity index (GAI)) is very high. Therefore, strain 60473 is suitable for biofilm formation on the surface of graphite, and is particularly suitable as an electrochemically active bacterium used in a microbial fuel cell or microbial electrolysis cell using a conductive carbon-based anode electrode with carbon or graphite as the main component. The GAI of strain 60473 is 60% to 80%, preferably 65% ​​to 80%. In addition, the GAI of microorganisms represented by strain 60473 can be measured using the method shown in the examples described below.

[0157] The cell hydrophobicity (hydrophobic index (HPI)) of strain 60473 is high. Therefore, it has good adhesion to hydrophobic electrodes such as carbon-based electrodes and easily forms a biofilm on the surface of the hydrophobic electrode. The HPI of strain 60473 is 50% to 90%, preferably 60% to 90%, and more preferably 65% ​​to 90%. In addition, the HPI of microorganisms represented by strain 60473 can be measured by the method shown in the examples described below.

[0158] The cytochrome content index (CCI) per unit protein (mass) of strain 60473 is high. Cytochrome is a general term for proteins that have heme (containing iron atoms in the form of heme groups) that are bound to iron that can be oxidized and reduced. According to the type of heme and the way it is bound, it is roughly divided into cytochrome a, cytochrome b, cytochrome c, cytochrome d, etc. Since cytochrome is a molecule related to the ability to transfer electrons outside the cell, it can be inferred that the high CCI is one of the reasons for the excellent power generation capacity of strain 60473. The CCI of strain 60473 is 0.75 / mg to 1.00 / mg. In addition, the CCI of microorganisms represented by strain 60473 can be measured using the method shown in the examples described below.

[0159] When the 60473 strain is used as an electrochemically active bacterium in a microbial fuel cell, it can be used alone or in combination with other electrochemically active bacteria, and preferably in combination with other microorganisms other than the electrochemically active bacteria. For example, by using anaerobic bacteria having the ability to produce acetic acid in combination, acetic acid that is reduced due to the proliferation of the 60473 strain can be supplied, thereby further extending the culture period for exerting the power generation capacity of the 60473 strain.

[0160] 60473 strains are also suitable as the active ingredient of the microbial preparation added to the microbial fuel cell by the bioenhancement method. The microbial preparation for bioenhancement can be set as the culture of 60473 strains, the bacterial body obtained by centrifuging the culture and precipitating it, or a composition obtained by mixing other additives (such as excipients, etc.) into these as needed, or it can be set as a granular material obtained by further shaping the composition. In addition, it can also be set as a suspension liquid composition in which the bacterial body of 60473 strains is suspended in water or culture medium, etc. and further contains other components as needed. The microbial preparation for bioenhancement can also contain more than one microorganism except 60473 strains. As the microorganism, anaerobic acetic acid producing bacteria can be listed.

[0161] [Composition for microbial fuel cells]

[0162] The composition for microbial fuel cells of this embodiment is an organic matter containing 60473 strains and organic matter, and used as a fuel for a microbial fuel cell or a composition added to the fuel. The composition for microbial fuel cells can also be used as a microbial preparation for bioenhancement. The form of the composition for microbial fuel cells of this embodiment is not particularly limited, and can be a solid substance such as particles and blocks, a paste, a gel, or a suspension.

[0163] As the organic matter contained in the composition for microbial fuel cells of the present embodiment, as long as it is an organic matter that becomes a fuel for a microbial fuel cell, there is no particular limitation, and biomass can be preferably used, but it can also be an organic matter after chemical synthesis, and it can also be a fossil fuel or its processed product. As the organic matter contained in the composition for microbial fuel cells of the present embodiment, it is particularly preferred that the organic matter is organic matter derived from waste-based biomass such as food waste, agricultural residues, livestock feces and urine, sewage sludge, river / lake sediment (organic mud at the bottom), wood-based waste, paper garbage, and wastewater. The organic matter contained in the composition for microbial fuel cells can be a mixture of organic matter derived from multiple biomass.

[0164] The composition for microbial fuel cells of this embodiment may also contain one or more microorganisms other than the 60473 strains. The microorganisms may be electrochemically active bacteria or microorganisms other than electrochemically active bacteria. In addition, as long as the microorganisms can proliferate in an environment where the 60473 strains can proliferate, anaerobic bacteria are preferred, but microaerobic bacteria may also be used. As microorganisms other than the 60473 strains contained in the composition for microbial fuel cells, electrochemically active bacteria and anaerobic acetic acid producing bacteria may be mentioned in particular.

[0165] [Microbial fuel cell]

[0166] The microbial fuel cell of the present embodiment is characterized in that 60473 strains are used for the decomposition of organic matter as a fuel. In the microbial fuel cell of the present embodiment, the microorganisms used to decompose organic matter may be only 60473 strains, or 60473 strains may be used in combination with other microorganisms. By using 60473 strains in combination with anaerobic acetic acid-producing bacteria, the acetic acid required for power generation based on 60473 strains can be stably supplied, thereby further stably performing power generation based on 60473 strains for a long time. In addition, by using various microorganisms that decompose organic matter in combination, the organic matter as a fuel can be efficiently decomposed until the state in which 60473 strains are easily utilized is reached. In addition, the use of electrochemically active bacteria other than 60473 strains in combination can also improve the power generation efficiency for fuel organic matter, so it is preferred.

[0167] In the microbial fuel cell of this embodiment, the amount of 60473 strain used is not particularly limited, and can be appropriately adjusted in consideration of the type or concentration of the organic matter used as the fuel, the type of the anode electrode, the presence or absence of adhesion to the anode electrode, etc. In the case where the 60473 strain is dispersed in a liquid containing organic matter, the amount of 60473 strain per 1 g of the liquid can be, for example, 10,000 cells / g to 100,000,000 cells / g, preferably 100,000 cells / g to 10,000,000 cells / g. In the case where the 60473 strain is attached to the anode electrode, the amount of 60473 strain per 1 cm of the anode electrode can be 10,000 cells / g to 10,000,000 cells / g. 2 The amount of 60473 cells per surface area can be set to, for example, 10,000 cells / g to 100,000,000 cells / g, and preferably 100,000 cells / g to 10,000,000 cells / g.

[0168] In the microbial fuel cell of this embodiment, when the 60473 strain is used in combination with other electrochemically active bacteria, the proportion of the amount of the 60473 strain relative to the total amount of the electrochemically active bacteria in the microbial fuel cell is not particularly limited, but is preferably 1% by mass or more, more preferably 10% by mass or more, and further preferably 30% by mass or more. Since the 60473 strain has a high power generation capacity, even if a small amount of the 60473 strain is added to a microbial fuel cell using conventional electrochemically active bacteria, the effect of increasing power generation can be expected.

[0169] The power generation capacity of 60473 strains decreases as the battery is used for a longer time. Therefore, in the microbial fuel cell of this embodiment, it is preferred to resupply 60473 strains at a time when the power generation capacity of 60473 strains decreases after the battery starts operating. Anaerobic acetic acid-producing bacteria or other microorganisms may be supplied at the same time as 60473 strains are supplied.

[0170] The timing of resupplying 60473 strains can be determined, for example, by monitoring the change in the voltage generated by the microbial fuel cell. In the microbial fuel cell, the generated voltage stabilizes after a period of time from the start of operation. Based on the generated voltage value in the stable state (the generated voltage value when stable) as a reference, 60473 strains are resupplyed at a time point when the generated voltage value is lower than a preset threshold value (for example, 70% of the generated voltage value when stable), thereby enabling long-term stable power generation. 60473 strains can also be resupplyed at specified intervals from the start of the battery operation.

[0171] As the microbial fuel cell of this embodiment, 60473 strains are used. As long as the anode electrode, cathode electrode, resistor connected to the two electrodes and supplying electron flow, and organic matter as fuel are included as the structures necessary for the battery, other structures are not particularly limited. The microbial fuel cell of this embodiment specifically includes: a liquid, with water containing organic matter as the main component; a container, which contains the liquid; a conductive anode electrode, which is arranged in the liquid; a conductive cathode electrode, at least one of which is arranged in contact with the liquid; and a resistor, one end of which is connected to the anode electrode and the other end is connected to the cathode electrode and supplying electron flow. Microorganisms represented by 60473 strains are contained in a liquid configured with an anode electrode. The organic matter as a fuel can be listed as the same organic matter as the organic matter contained in the composition for the microbial fuel cell. In the microbial fuel cell of this embodiment, it is preferably an organic matter containing phosphorus, nitrogen, and sulfide.

[0172] On the anode electrode side, organic matter as fuel is decomposed by microorganisms and generates carbon dioxide, and in the middle of the reaction, electrochemically active bacteria such as 60473 strains release electrons. As the anode electrode, as long as it is an electrode comprising an electroconductive raw material that can receive the electrons produced by the electrochemically active bacteria and is handed over to the cathode electrode by a resistor, it is not particularly limited. In the microbial fuel cell of this embodiment, with regard to the good adhesion of microorganisms, it is preferably a conductive carbon anode electrode with carbon or graphite as the main component, and with regard to the surface area being large, it is more preferably an anode electrode comprising carbon cloth (cloth woven from carbonized fibers obtained by calcining petroleum or acrylic fibers).

[0173] 60473 strains also have a tendency to easily adhere to smooth surfaces and can also adhere to metal surfaces. In addition, the internal resistance of the metal electrode is smaller than that of the graphite electrode, thereby suppressing the reduction of power generation efficiency. Therefore, in the microbial fuel cell of this embodiment, the anode electrode is also preferably a conductive metal material with a smaller internal resistance than the cathode electrode. By using an anode electrode containing metals such as stainless steel, iron, and magnesium and having 60473 strains attached to the surface, a microbial fuel cell with better power generation efficiency can be obtained.

[0174] In the cathode electrode, a reduction reaction is carried out by electrons received from the anode electrode via a resistor, a compound that becomes an oxidant (electron acceptor), and protons diffused from the anode electrode. The cathode electrode can be an electrode in which a catalyst for the reduction reaction such as platinum is coated on a conductive raw material. As the conductive raw material constituting the cathode electrode, a carbon-based raw material with carbon or graphite as the main component is preferably used in terms of corrosion resistance and the like.

[0175] The size of the anode electrode and the cathode electrode is not particularly limited. The size of the two electrodes can be equal or different. If the microbial fuel cell continues to operate, the performance of the cathode electrode will decrease. Therefore, it is preferred that the area of ​​the cathode electrode is larger relative to the area of ​​the anode electrode, and more preferably the area of ​​the cathode electrode is larger than the area of ​​the anode electrode by more than 1.1 times. By increasing the area of ​​the cathode electrode, the operation time of the microbial fuel cell can be increased. In particular, in the case of a sediment microbial fuel cell provided for purifying the bottom mud of a river or lake, the longer the operation time, the more it can promote the proliferation of 60473 strains, thereby facilitating the purification of the bottom mud.

[0176] The resistor having one end connected to the anode electrode and the other end connected to the cathode electrode is not particularly limited as long as it is a component that uses the potential difference between the chemical reaction caused by the microorganisms that generate carbon dioxide from organic matter on the anode electrode side and release electrons and the reduction reaction generated on the cathode electrode side to make electrons flow from the anode electrode to the cathode electrode, and can be appropriately selected and used from the resistors commonly used in batteries. The resistor may have resistance as a whole from the connection part with the anode electrode to the connection part with the cathode electrode, but it is preferred that the part from the connection part with the anode electrode to the connection part with the cathode electrode is set as a conductive member such as titanium wire with a small internal resistance, and an external resistor showing sufficient resistance is set in the middle. In terms of being able to make adjustments corresponding to the state of the fuel or the state of the microorganisms, the external resistor is preferably set as a variable resistor. In addition, the resistor and the external resistor are kept fixed on the cathode electrode and are arranged in a state that is easy to identify on the liquid (liquid surface).

[0177] The microbial fuel cell of this embodiment can be a double-tank microbial fuel cell or a single-tank microbial fuel cell. The structures or components of the double-tank microbial fuel cell and the single-tank microbial fuel cell can use the structures or components used in the known double-tank microbial fuel cell and the single-tank microbial fuel cell.

[0178] In the case of a double-tank microbial fuel cell, a container containing a liquid mainly composed of water containing organic matter is divided into a tank (anode tank) equipped with an anode electrode and a tank (cathode tank) equipped with the cathode electrode by a membrane that can selectively allow protons to pass. As the membrane that can selectively allow protons to pass, a membrane that can selectively allow H + The organic matter and 60473 strains are contained in the liquid contained in the anode tank.

[0179] In the case of a double-tank microbial fuel cell, the amount of oxygen contained in the liquid contained in the anode tank is preferably maintained in a manner that is about 2% (v / v) to 10% (v / v) of the amount of oxygen contained in the liquid contained in the cathode tank. In order to maintain the oxygen concentration gradient, it is preferred that the liquid in the cathode tank also contains an oxidant that reacts with protons as a source of oxygen. As the oxidant, potassium ferricyanide, manganese oxide, etc. can be used.

[0180] In the case of a single-tank microbial fuel cell, the cathode electrode is arranged in a state where one surface is in contact with the liquid in the container and the other surface is in contact with the atmosphere. Oxygen in the atmosphere is taken in from the surface of the cathode electrode in contact with the atmosphere, thereby performing a reduction reaction. For example, the cathode electrode can be arranged to float on the surface of the liquid in the container. Preferably, a water-repellent layer is provided on the side of the cathode electrode in contact with the atmosphere. As the water-repellent layer, a coating of a fluorine-based resin such as polytetrafluoroethylene (PTFE) can be cited. In addition, it is preferred that a catalyst for the reduction reaction is attached to the surface of the cathode electrode in contact with the liquid. For example, the surface side of the cathode electrode in contact with the liquid can be coated with platinum.

[0181] The microbial fuel cell of this embodiment can also be a microbial fuel cell that uses organic mud to replace the liquid containing organic matter. Specifically, the organic mud and the liquid with water as the main component are contained in the container. In the container, the anode electrode is configured in the organic mud, and the cathode electrode is configured in the liquid or the liquid surface of the liquid. In the case where the cathode electrode is floated on the liquid surface of the liquid, the same material as the cathode electrode used in the single-trough microbial fuel cell can be used. On the other hand, in the case where the cathode electrode is configured in the liquid of the liquid, it is preferably a source of oxygen containing an oxidant that reacts with protons, similar to the liquid in the cathode tank in the double-trough microbial fuel cell. In the case of a microbial fuel cell using organic mud, the microbial group originally contained in the organic mud is also used in the microbial fuel cell. The organic mud can contain 60473 strains, and the 60473 strains can be attached to the anode electrode in advance.

[0182] The microbial fuel cell of this embodiment is also preferably a sediment microbial fuel cell for purifying the water environment. The sediment microbial fuel cell is a microbial fuel cell that generates electricity by setting an anode electrode in the sediment (mud) at the bottom of a river or lake and setting a cathode electrode in the water on the sediment. If the sediment microbial fuel cell is operated, the dissolved oxygen content and organic matter in the mud are reduced, and it is expected that the mud quality of the mud will be improved. Therefore, the sediment microbial fuel cell is set to purify the mud of a river or lake. In the case of a sediment microbial fuel cell, the cathode electrode is preferably floating on the liquid surface of the liquid, and the 60473 strains can be directly injected into the mud near the anode electrode, but preferably attached to the anode electrode in advance. In the case of attaching the 60473 strains to the anode electrode, the 60473 strains can be directly connected to the anode electrode to prevent the 60473 strains from moving to a position away from the anode electrode.

[0183] In order to measure the generated voltage, the microbial fuel cell of this embodiment is preferably provided with a voltage measuring device on the resistor. When an external resistor is used, the voltage measuring device can be provided across the external resistor. By measuring the generated voltage over time, the power generation state can be monitored, thereby determining the timing of resupplying 60473 strains or other microorganisms.

[0184] The microbial fuel cell of this embodiment also preferably includes a supply device for resupplying 60473 strains or other microorganisms. In the case where the microbial fuel cell of this embodiment is a sediment microbial fuel cell, it is more important to operate stably for a long time, so it is particularly preferred to be provided with a microbial supply device that is effective in extending the operation time. The supply device may include, for example: a supply microorganism storage container for storing a microbial solution obtained by suspending 60473 strains or other microorganisms in water or a culture medium; and a microbial supply pipe for supplying the microbial solution from the container to the anode electrode.

[0185] Figure 1 FIG. 2 is a schematic diagram showing one embodiment of a microbial fuel cell provided with a microorganism supply device.

[0186] Figure 1 2 shows a state where a microbial fuel cell 1 is connected to a river or lake bottom mud 2 and water 3. The microbial fuel cell 1 includes an anode electrode 4, a cathode electrode 5, a resistor 6, a voltage measuring device 7, and a microorganism supply device 8.

[0187] In the case of the present embodiment, the anode electrode 4 includes graphite felt inoculated with 60473 strains. The anode electrode 4 is arranged in contact with the bottom mud 2. In the case of the present embodiment, the cathode electrode 5 includes graphite felt carrying a catalyst such as platinum. The cathode electrode 5 is arranged in contact with the water 3. In addition to graphite felt, the conductor of the electrode may also be a porous conductor such as foamed titanium or foamed stainless steel.

[0188] A resistor 6 is electrically connected between the anode electrode 4 and the cathode electrode 5. The resistor 6 includes a lead 6a, a lead 6b, and an external variable resistor 6c. The external variable resistor 6c is connected to the anode electrode 4 via the lead 6a. The external variable resistor 6c is connected to the cathode electrode 5 via the lead 6b. A voltage measuring device 7 for measuring a generated voltage is provided across the external variable resistor 6c.

[0189] The microorganism supply device 8 includes a supply microorganism storage container 8a and a microorganism supply pipe 8b. The supply microorganism storage container 8a is arranged outside the water 3. The microorganism supply pipe 8b extends from the supply microorganism storage container 8a toward the water. The front end of the microorganism supply pipe 8b reaches the inside of the bottom mud 2. The microorganism supply pipe 8b is a single pipe at the part extending from the supply microorganism storage container 8a, but the part on the bottom mud 2 side is branched into a plurality of (five in the figure) branch pipes 8c. The plurality of branch pipes 8c are dispersedly arranged in a plurality in the plane direction of the anode electrode 4. The open ends of the plurality of branch pipes 8c are arranged around the anode electrode 4 and inside the anode electrode 4. By loading 60473 strains or substrates (such as acetic acid, etc.) into the supply microorganism storage container 8a, the 60473 strains or substrates can be supplied to the anode electrode 4 at any time through the microorganism supply pipe 8b. By including a plurality of branch pipes 8c, the 60473 strains or substrates can be dispersedly supplied in the plane direction of the anode electrode 4.

[0190] Figure 1 The microbial fuel cell 1 shown includes a microbial supply device 8, and can supply 60473 strains or substrates to the anode electrode 4 at any time. As a result, it can work continuously for a long time. As a specific application, for example, the voltage measurement device 7 of the microbial fuel cell 1 monitors the change of the generated voltage, and at the time point when the generated voltage is lower than the threshold voltage, the substrate or 60473 strains is supplied to the anode electrode 4 through the microbial supply pipe 8b of the microbial supply device 8. The threshold voltage for determining the supply timing is set to a value of 70% of the reference voltage, for example, based on the generated voltage two weeks after the microbial fuel cell 1 starts to operate. Since it takes about two weeks until the microbial fuel cell 1 stabilizes, when the generated voltage after the stable operation is transferred is used as a reference and the voltage decreases by 30% thereafter, it is judged that there is a shortage of substrate or 60473 strains, and replenishment is implemented.

[0191] In the case where the microbial fuel cell of this embodiment is a sediment microbial fuel cell, it is preferably easy to move in the bottom mud (sediment). Through the operation of the sediment microbial fuel cell, the mud quality can be improved by reducing organic matter and increasing the amount of dissolved oxygen in the bottom mud part where the anode electrode is buried, but it becomes an environment that is not conducive to the proliferation of strain 60473. If the sediment microbial fuel cell can be quickly moved to a place where the mud quality has not been improved, it can continue to operate, which is preferred.

[0192] Figure 2 A schematic diagram of a microbial fuel cell including a support device that facilitates movement is shown in FIG. Figure 2 In Figure 1 The same reference numerals are used to denote common components, and detailed description thereof will be omitted.

[0193] Figure 2 The sediment microbial fuel cell 11 shown in FIG. 1 comprises an anode electrode 4 , a cathode electrode 5 , a resistor 6 and a supporting device 17 .

[0194] The support device 17 has two support bodies 17a, 17b and a setting fixing portion 17c fixed to the lower ends of the support bodies 17a and 17b. The support bodies 17a and 17b are rod-shaped or plate-shaped main bodies extending in the vertical direction. The setting fixing portion 17c is, for example, a conical structure with the front end facing the lower side (the lower side shown in the figure) in the depth direction. The angle of the cone front end of the setting fixing portion 17c is preferably an acute angle. The setting fixing portion 17c can be any shape that has a protrusion toward the lower side in the depth direction so that the support device 17 can be easily buried in the part of the bottom mud 2. The setting fixing portion 17c can be, for example, a pyramid shape. By inserting the setting fixing portion 17c toward the bottom mud 2, the sediment microbial fuel cell 11 can be easily inserted into the bottom mud 2 and fixed.

[0195] The anode electrode 4 and the cathode electrode 5 are connected to the two supports 17a and 17b. Figure 2 In the example shown, the anode electrode 4 and the cathode electrode 5 are arranged between the support bodies 17a and 17b which are arranged at intervals in the horizontal direction. The support bodies 17a and 17b are respectively connected to both ends of the anode electrode 4 in the horizontal direction and both ends of the cathode electrode 5 in the horizontal direction. That is, the anode electrode 4 and the cathode electrode 5 are connected and fixed in parallel at the upper part between the two support bodies 17a and 17b, and the cathode electrode 5 is connected and fixed in parallel at the lower part.

[0196] Furthermore, the supporting device 17 may be configured to include only the supporting body 17 a . In this case, one end of the anode electrode 4 and one end of the cathode electrode 5 are respectively connected to the supporting body 17 a .

[0197] The support device 17 includes a position adjustment mechanism that can variably adjust and fix the positions and intervals of the anode electrode 4 and the cathode electrode 5 supported in parallel by the support body 17a and the support body 17b. Thus, the relative positions of the anode electrode 4, the cathode electrode 5, and the setting fixing portion 17c can be adjusted according to the water depth (depth), the setting position in the bottom mud 2, etc.

[0198] according to Figure 2 The sediment microbial fuel cell 11 shown in the invention can be efficiently carried or set up by including a structure in which both the anode electrode 4 and the cathode electrode 5 are supported by a supporting device 17. In addition, when the sediment microbial fuel cell 11 is operated, the dissolved oxygen content and organic matter of the sludge 2 are reduced, so that the mud quality of the sludge 2 can be expected to be improved. However, since the oxygen content and organic matter of the sludge 2 are reduced after the state of the sludge 2 is improved, it becomes an environment that is not conducive to the proliferation of anaerobic bacteria. Therefore, if the battery can be quickly moved to a place where the mud quality is not improved (a place where the dissolved oxygen content and the amount of organic matter are large), it can be efficiently continued to operate. In the sediment microbial fuel cell 11 of this embodiment, since it includes a supporting device 17 that can move the anode electrode 4 and the cathode electrode 5 as a whole and the front end is an acute angle, it can be easily moved to an environment where sludge is easy to proliferate.

[0199] [Composition for microbial electrolytic cell]

[0200] The composition for the microbial electrolysis cell of this embodiment is an organic matter that contains 60473 strains and organic matter, and is used as a fuel for the microbial electrolysis cell or a composition added to the fuel. The composition for the microbial electrolysis cell can also be used as a microbial preparation for bioenhancement. The form of the composition for the microbial electrolysis cell of this embodiment is not particularly limited, and can be a solid substance such as granules and blocks, a paste, a gel, or a suspension.

[0201] The organic matter contained in the composition for a microbial electrolysis cell of the present embodiment is not particularly limited as long as it is an organic matter that serves as a fuel for a microbial electrolysis cell, and the same organic matter as exemplified as the organic matter contained in the composition for a microbial fuel cell can be used.

[0202] The composition for microbial electrolysis cell of this embodiment may also contain one or more microorganisms other than 60473 strains. The microorganisms may be electrochemically active bacteria or microorganisms other than electrochemically active bacteria. In addition, as long as the microorganisms can proliferate in an environment where 60473 strains can proliferate, anaerobic bacteria are preferred, but microaerobic bacteria may also be used. As microorganisms other than 60473 strains contained in the composition for microbial electrolysis cell, electrochemically active bacteria and anaerobic acetic acid-producing bacteria can be mentioned in particular.

[0203] [Microbial electrolysis cell]

[0204] The microbial electrolysis cell of the present embodiment is characterized in that strain 60473 is used for decomposing organic matter as a fuel. In the microbial electrolysis cell of the present embodiment, the microorganism used for decomposing organic matter may be only strain 60473, or strain 60473 may be used in combination with other microorganisms. By using strain 60473 in combination with anaerobic acetic acid-producing bacteria, the acetic acid required for power generation and hydrogen generation based on strain 60473 can be stably supplied, so that power generation and hydrogen generation based on strain 60473 can be stably performed for a longer period of time. In addition, by using various microorganisms that decompose organic matter in combination, the organic matter used as a fuel can be efficiently decomposed until the state in which strain 60473 is easily utilized. In addition, the use of electrochemically active bacteria other than strain 60473 can also improve the hydrogen generation efficiency for fuel organic matter, so it is preferred.

[0205] In the microbial electrolysis cell of the present embodiment, the amount of 60473 strain used is not particularly limited, and can be appropriately adjusted in consideration of the type or concentration of organic matter used as fuel, the type of anode electrode, the presence or absence of adhesion to the anode electrode, etc. In the case where the 60473 strain is dispersed in a liquid containing organic matter, the amount of 60473 strain per 1 g of the liquid can be, for example, 10,000 cells / g to 100,000,000 cells / g, preferably 100,000 cells / g to 10,000,000 cells / g. In the case where the 60473 strain is attached to the anode electrode, the amount of 60473 strain per 1 cm of the anode electrode can be 10,000 cells / g to 10,000,000 cells / g. 2 The amount of 60473 cells per surface area can be set to, for example, 10,000 cells / g to 100,000,000 cells / g, and preferably 100,000 cells / g to 10,000,000 cells / g.

[0206] In the microbial electrolysis cell of the present embodiment, when the 60473 strain is used in combination with other electrochemically active bacteria, the proportion of the 60473 strain relative to the total amount of electrochemically active bacteria in the microbial electrolysis cell is not particularly limited, but is preferably 1% by mass or more, more preferably 10% by mass or more, and further preferably 30% by mass or more. Since the 60473 strain has a high electrolytic capacity, even if a small amount of the 60473 strain is added to a microbial electrolysis cell using conventional electrochemically active bacteria, the effect of increasing the amount of hydrogen generated can be expected.

[0207] The electrolytic capacity of the 60473 strain decreases as the battery is used for a longer time. Therefore, in the microbial electrolysis cell of this embodiment, it is preferred to resupply the 60473 strain when the electrolytic capacity of the 60473 strain decreases after the cell starts operating. Anaerobic acetic acid-producing bacteria or other microorganisms may be supplied at the same time as the 60473 strain is supplied.

[0208] The timing of resupplying 60473 strains can be determined, for example, by monitoring the change in the generated voltage of the microbial electrolysis cell. In the microbial electrolysis cell, the generated voltage stabilizes after a period of time from the start of operation. Based on the generated voltage value in the stable state (the generated voltage value when stable) as a reference, 60473 strains are resupplyed at a time point when the generated voltage value is lower than a preset threshold value (for example, 70% of the generated voltage value when stable), thereby enabling stable power generation and hydrogen generation for a long time. 60473 strains can also be resupplyed at specified intervals from the start of operation of the cell.

[0209] As the microbial electrolysis cell of this embodiment, strain 60473 is used. As long as it includes an anode electrode, a cathode electrode, a potential control device connected to the two electrodes and supplying electron flow, and an organic matter that serves as a fuel, which are necessary structures of the cell, other structures are not particularly limited. The microbial electrolysis cell of this embodiment specifically includes: a liquid, with water containing organic matter as the main component; a container, which contains the liquid; a conductive anode electrode, which is arranged in the liquid; a conductive cathode electrode, at least one of whose surfaces is in contact with the liquid; and a potential control device, one end of which is connected to the anode electrode and the other end of which is connected to the cathode electrode and supplying electron flow. Microorganisms represented by strain 60473 are contained in a liquid configured with an anode electrode. The organic matter used as a fuel can be the same as the organic matter contained in the composition for the microbial electrolysis cell. In the microbial electrolysis cell of this embodiment, organic matter containing phosphorus, nitrogen, and sulfide is preferred.

[0210] On the anode electrode side, organic matter as fuel is decomposed by microorganisms to generate carbon dioxide, and in the middle of the reaction, electrochemically active bacteria such as 60473 strains release electrons. As the anode electrode, as long as it is an electrode containing a conductive raw material that can receive electrons generated by electrochemically active bacteria and transfer them to the cathode electrode through a potential control device, it is not particularly limited. In the microbial electrolysis cell of this embodiment, in terms of good adhesion of microorganisms, it is preferably a conductive carbon-based anode electrode with carbon or graphite as the main component, and in terms of large surface area, it is more preferably an anode electrode containing carbon cloth (cloth woven from carbonized fibers obtained by calcining petroleum or acrylic fibers). In addition, in the microbial electrolysis cell of this embodiment, the anode electrode is also preferably a conductive metal material with a smaller internal resistance than the cathode electrode. Specifically, in the microbial electrolysis cell of this embodiment, it is also preferably an anode electrode containing metals such as stainless steel, iron, and magnesium and having 60473 strains attached to the surface.

[0211] In the cathode electrode, the electrons received from the anode electrode via the potential control device and the protons diffused from the anode electrode are subjected to a reduction reaction, thereby generating hydrogen. The cathode electrode may be an electrode in which a catalyst for the reduction reaction such as platinum is coated on a conductive raw material. As the conductive raw material constituting the cathode electrode, a carbon-based raw material having carbon or graphite as a main component is preferably used in terms of corrosion resistance and the like.

[0212] The size of the anode electrode and the cathode electrode is not particularly limited. The size of the two electrodes may be equal or different. If the microbial electrolysis cell continues to operate, the performance of the cathode electrode will decrease. Therefore, it is preferred that the area of ​​the cathode electrode is larger than the area of ​​the anode electrode, and it is more preferred that the area of ​​the cathode electrode is 1.1 times larger than the area of ​​the anode electrode. By increasing the area of ​​the cathode electrode, the operation time of the microbial electrolysis cell can be increased.

[0213] The potential control device connected to the anode electrode at one end and the cathode electrode at the other end is not particularly limited as long as it is a member that uses the potential difference between the chemical reaction caused by the microorganisms that generate carbon dioxide from organic matter on the anode electrode side and the reduction reaction generated on the cathode electrode side to make the electrons flow from the anode electrode to the cathode electrode. It can be appropriately selected and used from the potential control devices commonly used in microbial electrolysis cells. As the potential control device, for example, a constant potential meter using a working electrode (anode electrode), a counter electrode (cathode electrode) and a reference electrode can be listed. In addition, the entirety of the potential control device from the connection part with the anode electrode to the connection part with the cathode electrode can have a resistor, but it is preferred that the portion from the connection part with the anode electrode to the connection part with the cathode electrode is set as a conductive member such as a titanium wire with a small internal resistance, and an external resistor showing sufficient resistance is provided in the middle. In terms of being able to make adjustments corresponding to the state of the fuel or the state of the microorganism, the external resistor is preferably set as a variable resistor. In addition, the potential control device and the external resistor are kept fixed on the cathode electrode and are arranged in a state that is easy to identify on the liquid (liquid surface).

[0214] The microbial electrolysis cell of this embodiment may be a double tank microbial electrolysis cell or a single tank microbial electrolysis cell. The structures or components of the double tank microbial electrolysis cell and the single tank microbial electrolysis cell may use the structures or components used in the known double tank microbial electrolysis cell and the single tank microbial electrolysis cell.

[0215] In the case of a double tank microbial electrolysis cell, a container containing a liquid mainly composed of water containing organic matter is divided into a tank (anode tank) equipped with an anode electrode and a tank (cathode tank) equipped with the cathode electrode by a membrane that can selectively allow protons to pass. As the membrane that can selectively allow protons to pass, a membrane that can selectively allow H + The organic matter and 60473 strains are contained in the liquid contained in the anode tank.

[0216] In the case of a double tank microbial electrolysis cell, the amount of oxygen contained in the liquid contained in the anode tank is preferably maintained in a manner that is about 2% (v / v) to 10% (v / v) of the amount of oxygen contained in the liquid contained in the cathode tank. In order to maintain the oxygen concentration gradient, it is preferred that the liquid in the cathode tank further contains an oxidant that reacts with protons as an oxygen supply source. Potassium ferrocyanide, manganese oxide, etc. can be used as the oxidant.

[0217] The microbial electrolysis cell of the present embodiment may also be a microbial electrolysis cell that uses organic mud instead of a liquid containing organic matter. Specifically, the organic mud and a liquid with water as the main component are contained in a container. In the container, the anode electrode is arranged in the organic mud, and the cathode electrode is arranged in the liquid. Similar to the liquid in the cathode tank in the double-tank microbial electrolysis cell, the cathode electrode preferably contains an oxidant that reacts with protons as a source of oxygen. In the case of a microbial electrolysis cell using organic mud, the microbial community originally contained in the organic mud is also used in the microbial electrolysis cell. The organic mud may contain 60473 strains, or the 60473 strains may be pre-attached to the anode electrode.

[0218] The microbial electrolysis cell of this embodiment is also preferably a sediment microbial electrolysis cell for purifying water environment. The sediment microbial electrolysis cell is a microbial electrolysis cell as follows: an anode electrode is provided in the sediment (sediment) at the bottom of a river or lake and a cathode electrode is provided in the water on the sediment to generate electricity and hydrogen. If the sediment microbial electrolysis cell is operated, the dissolved oxygen content and organic matter in the sediment are reduced, and it is expected that the mud quality of the sediment can be improved. Therefore, the sediment microbial electrolysis cell can be provided in a river or lake in the same manner as the sediment microbial fuel cell to purify the sediment.

[0219] In order to measure the generated voltage, the microbial electrolysis cell of this embodiment is preferably provided with a voltage measuring device on the potential control device. When an external resistor is used, the voltage measuring device can be provided across the external resistor. By measuring the generated voltage over time, the power generation state can be monitored, thereby determining the timing of resupplying 60473 strains or other microorganisms.

[0220] The microbial electrolysis cell of this embodiment also preferably includes a supply device for resupplying 60473 strains or other microorganisms. In the case where the microbial electrolysis cell of this embodiment is a sediment microbial electrolysis cell, it is more important to operate stably for a long time, so it is particularly preferred to be provided with a microbial supply device that is effective in extending the operation time. The supply device may include, for example: a microbial storage container for supply, which stores a microbial solution obtained by suspending 60473 strains or other microorganisms in water or a culture medium, and a microbial supply pipe, which supplies the microbial solution from the container to the anode electrode.

[0221] Figure 32 shows a schematic diagram of a three-electrode one-tank type microbial electrolysis cell. The microbial electrolysis cell 20 includes an anode electrode 24, a cathode electrode 25, and a constant potential meter 26 in a container 21. The constant potential meter 26 has a reference electrode 26c. In the anode electrode 24, organic matter is decomposed by 60473 strains of microorganisms, and the generated electrons move to the cathode electrode 25 through the constant potential meter 26. In the cathode electrode 25, the electrons react with protons to generate hydrogen.

[0222] [Example]

[0223] Next, the present invention will be described in further detail with reference to Examples and Reference Examples, but the present invention is not limited to the following Examples and the like.

[0224] [Example 1]

[0225] Electrochemically active bacteria with power generation capability that can be used in microbial fuel cells were isolated and identified from mud on the shore of Lake Suwa (Okaya City, Nagano Prefecture).

[0226] (1) Isolation of 60473 strains

[0227] Specifically, first, Figure 4 The mud battery shown [101···data recorder for voltage measurement, 102···external resistor, 103···titanium wire, 104···water, 105···cathode (platinum-coated graphite felt), 106···anode (graphite felt), 107···mud]. The mud battery was operated in an incubator at 30°C while measuring the voltage across the external resistor (10 kΩ) to allow the electric bacteria to grow on the anode. After the voltage rose to 0.5 V or more, a portion of the anode was cut off and placed in a liquid culture medium of DSM826 culture medium (manufactured by Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ) and stirred to suspend the attached matter. The obtained dilution of the suspension was spread on an agar plate of DSM826 medium, placed in AnaeroPackPouch (manufactured by Mitsubishi Gas Chemical Co., Ltd.), and colonies were formed at 30°C. The red colonies formed on the plate were inoculated into a DSM826 liquid medium in an anaerobic small glass bottle after replacing the gas phase with nitrogen (99.999% (v / v)) and cultured. The culture was cultured again on an agar plate of DSM826 medium to form colonies, and the formed colonies were cultured again using DSM826 liquid medium. The above operation was repeated until only colonies of the same shape were formed on the plate. The anaerobic bacteria of the colonies formed on the plate were named strain 60473.

[0228] (2) Evaluation of the current generating ability of strain 60473

[0229] The 60473 strain was cultured in DSM826 liquid medium to obtain a culture. The current generated by the culture in a three-electrode and one-trough electrochemical cell was measured. In the electrochemical cell, the working electrode was a graphite felt (2.00 cm 2 , manufactured by Nippon Carbon Co., Ltd.), the reference electrode was an Ag / AgCl electrode (RE-T8A, manufactured by ECFrontier Co., Ltd.), the counter electrode was a platinum wire (length 12 cm, thickness 0.3 mm, manufactured by Nilaco Co., Ltd.), and the electrolyte was a DSM826 liquid culture medium (65 mL) without fumaric acid. The culture was added to the electrolyte so that the turbidity at 600 nm reached 0.02, the cell was connected to a potentiostat (VMP3, manufactured by Biologic Co., Ltd.), the working electrode potential was set to -0.2V vs. Ag / AgCl electrode, and the current measurement was started. As a comparison object, the current generated by the PCA strain known as a highly capable electric bacteria was measured (n=2).

[0230] The current measurement results of the electrochemical cell in which the 60473 strain was cultivated and the electrochemical cell in which the PCA strain was cultivated are shown in FIG. Figure 5 .like Figure 5 As shown, the maximum value of the current generated by the 60473 strain is about 2 times higher than the maximum value of the PCA strain.

[0231] The working electrode of the electrochemical cell was replaced with glassy carbon (1 cm 2 , manufactured by BAS), and the current was measured in the same manner as described above (n=2). The results are shown in Figure 6 As a result, in the case of the PCA strain, the current value increased only to 0.02 mA / cm 2 However, in the case of 60473 strains, the current value increased to 0.28 mA / cm 2About. The results show that strain 60473 can also efficiently interact electrochemically with the smooth glassy carbon electrode. At this time, the bacteria attached to the electrode surface were stained with a fluorescent dye (4,6-diamidino-2-phenylindole (DAPI; 0.1μg / mL) and observed using a confocal microscope (FV1200 / BX61WI system, manufactured by Olympus). The results confirmed that a large number of cells in strain 60473 were also attached to the smooth electrode. The amount of bacteria attached to the glassy carbon electrode was compared based on the DAPI staining intensity. The results are shown in Figure 7 The 60,473 strains attached to the glassy carbon electrode were nearly three times the number of PCA strains.

[0232] (3) Genome analysis of strain 60473

[0233] The purpose of determining the biological species of 60473 strains from the genome sequence and clarifying the characteristics of the genome was to determine the genome sequence. 60473 strains were cultured in DSM826 medium, and genomic deoxyribonucleic acid (DNA) was extracted from 100mL of the culture according to the method of Fujikawa et al. (Fujikawa et al., FEMS Microbiology Letters, 2021, vol. 368 (17), fnab119.)). It was confirmed that high-molecular genomic DNA could be extracted by agarose gel electrophoresis, and the order of the entire genome was determined using the Sequel lle system (manufactured by Pacific Bio (PacBio)). The results of the analysis of the base sequence of the genome were compared with the PCA strains and are shown in Table 1.

[0234] [Table 1]

[0235]

[0236] In order to clarify the characteristics of the molecular system of strain 60473, the Basic Local Alignment Search Tool (BLAST) analysis of the National Center for Biotechnology Information (NCBI) database was performed using the 16S ribosomal ribonucleic acid (rRNA) gene in the genome. The result showed that the closest relative of strain 60473 was the PCA strain, with a homology (sequence identity) of 99.48%. Therefore, in order to clarify the phylogenetic relationship with the known Geobacter sulfurreducens, a dendrogram ( Figure 8 and Fig. 9 ). The results showed that strain 60473 belonged to the genus Geobacter, but further research was needed to determine whether it belonged to Geobacter sulfurreducens.

[0237] [Example 2]

[0238] The mud battery is an experimental device that simulates the sediment microbial fuel cell (sMFC) as a power generation device in the environment. sMFC is a device that uses organic matter in sediment (mud or bottom mud) as fuel to generate electricity, and can reduce organic matter in the sediment by generating electricity. Therefore, in order to investigate whether the 60473 strains can be used to improve the performance of sMFC, a mud battery was made using the bottom mud of Lake Suwa, and the 60473 strains were tried.

[0239] Mud batteries are used with Figure 4 The mud battery used contained mud (about 400 mL), an anode (graphite felt, area 36 cm 2 ), cathode (platinum-coated graphite felt, area 24cm 2 ), connected to an external resistor of 10 kΩ and operated. One day after the start of operation, 250 mL of the culture solution of 60473 strains cultured in DSM826 liquid medium was centrifuged, and the concentrated bacterial solution was injected into the vicinity of the anode, and the operation was continued. The results of the polarization analysis conducted on the fifth day after the start of operation are shown in Fig.10 and Fig.11 . Fig.10 This is the result of polarization analysis of mud batteries with 60,473 plants (n=2). Fig.11 This is the polarization analysis result of the mud battery without 60473 plants (n=2). Fig.10and Fig.11 The results of the polarization analysis shown here show the calculated maximum power density (P max mW / m 2 ), and the results of the comparison are shown in Fig.12 In the figure, the error bars represent the standard deviation. By adding 60473 strains, the maximum power density of the mud battery was greatly improved. These results show that the performance of the battery can be improved by bio-enhancing the existing microbial fuel cells, especially sMFC, using 60473 strains.

[0240] [Example 3]

[0241] The power generation capacity of strain 60473 was compared with known bacteria of the genus Geobacter. For comparison, the PCA strain used in Example 1, Geobacter sulfurreducens KN400 strain (Kyoto Encyclopedia of Genes and Genomes GENOME, KEGG GENOME: Geobactersulfurreducens KN400), and Geobacter sulfurreducens YM18 strain described in Non-Patent Document 6 were used.

[0242] (1) Evaluation of current generation capability

[0243] Specifically, in the same manner as in "(2) Evaluation of current generation ability of 60473 strain" of Example 1, the current generated by inoculating the evaluation target Geobacter bacteria in a three-electrode one-tank electrochemical cell was measured (n=2).

[0244] The results of measuring the current density over time when graphite felt was used as the working electrode (anode electrode) are shown in FIG. Fig.13 The maximum current density is shown in Fig.14 In addition, the results of measuring the current density over time when glassy carbon is used as the working electrode (anode electrode) are shown in Fig.15 The maximum current density is shown in Fig.16 In. Fig.13 and Fig.14 As shown in the figure, when using graphite felt electrodes, strains 60473, KN400, and YM18 all showed higher current densities than strain PCA, with strain YM18 showing the highest current density. It was also shown that the current density increased more rapidly in the electrochemical cells inoculated with strains 60473 and KN400 than with strain YM18. Fig.15 and Fig.16As shown, in the case of using glassy carbon electrodes, among these strains, strain 60473 produced the highest current, and the maximum current density (J max ) is more than three times that of KN400 and YM18 strains.

[0245] The graphite felt electrode and glassy carbon electrode after the current generation experiment were stained with DAPI in the same manner as in Example 1, and observed using a confocal microscope. As a result, the KN400 strain and the YM18 strain formed a thick biofilm on the graphite fiber of the graphite felt electrode. In addition, the biofilm of the 60473 strain on the graphite felt fiber was thinner than that of the KN400 strain or the YM18 strain, but the cell density of the biofilm of the 60473 strain was the highest. Furthermore, the 60473 strain also formed a high-density biofilm on the glassy carbon electrode, and its density was much higher than that of the biofilms of other strains. On the other hand, the KN400 strain formed an evenly distributed biofilm with low cell density on the glassy carbon electrode, and the biofilm of the YM18 strain only partially covered the surface of the glassy carbon electrode. It was concluded from these observations that the high current density of the 60473 strain was associated with the ability to form a high-density biofilm on the graphite surface.

[0246] (2) Physiological characteristics of Geobacter

[0247] The affinity for graphite (GAI), cell hydrophobicity (HPI), and cytochrome content per unit protein (CCI) of each of the 60473, KN400, YM18, and PCA strains, which are physiological properties believed to be associated with biofilm formation and current generation, were investigated.

[0248] <Measurement of GAI (%)>

[0249] The GAI (%) of each Bacillus species was measured by the following method.

[0250] First, the microorganisms to be tested were cultured anaerobically in DSM826 medium to proliferate, and then washed with DSM826 buffer (DSM826 medium without acetate, fumarate, or mineral solution). 600 The microorganisms to be tested were suspended in 3.5 mL of DSM826 buffer so that OD 0 was 0.3 to prepare a bacterial suspension. 600 The measurement was performed using a digital colorimeter (Miniphoto 518R, manufactured by TAITEC).

[0251] Graphite felt (0.5 cm 2× 5 mm), and stirred at 30°C and 200 rpm. OD was measured using the digital colorimeter 60 minutes after the start of stirring. 600 .

[0252] GAI (%) was calculated based on the obtained measured values ​​and the following formula: Wherein, A1 is the OD of the bacterial suspension after stirring 600 , A0 is the OD of the bacterial suspension before stirring 600 (0.3).

[0253] [GAI (%)] = (1-A1 / A0) × 100

[0254] <Determination of HPI (%)>

[0255] The HPI (%) of each Bacillus species was measured by the following method.

[0256] Optical density 600nm: OD 600 The microorganism to be tested was suspended in 4.8 mL of a sodium chloride aqueous solution (0.15 M) so that OD 0 was 0.3 to prepare a bacterial suspension. 600 The measurement was performed using a digital colorimeter (Miniphoto 518R, manufactured by TAITEC).

[0257] 0.8 mL of hexadecane was added to the prepared bacterial suspension and stirred for 60 seconds using a vortex mixer. The bacterial suspension was then left overnight. After the two phases were completely separated, the upper layer was removed by decantation and the OD value of the lower layer was measured. 600 Carry out determination.

[0258] HPI (%) was calculated from the ratio of cells transferred to the hexadecane phase according to the following formula: Wherein, A2 is the OD of the bacterial suspension in the aqueous phase after the bacterial suspension and hexadecane are mixed. 600 , A0 is the OD of the bacterial suspension before stirring 600 (0.3).

[0259] [HPI (%)] = (1-A2 / A0) × 100

[0260] <Determination of CCI( / mg)>

[0261] The CCI ( / mg) of each Bacillus species was measured by the following method.

[0262] First, the microorganisms to be tested were cultured anaerobically in DSM826 medium to proliferate, and then centrifuged (10,000×g, 15 minutes) to recover the microbial particles. The obtained microbial particles were washed with TE buffer and suspended in TE buffer containing 1 mg / mL lysozyme. The obtained bacterial suspension was homogenized with a homogenizer, incubated at 37°C for 1.5 hours, and then Triton X-100 (1% w / v) was added and incubated at room temperature for 10 minutes.

[0263] The absorption spectrum of the supernatant of the cultured bacterial cell suspension was measured in the range of 700 nm to 350 nm using a spectrophotometer (UH5300, manufactured by Hitachi, Ltd.).

[0264] Next, in order to measure the absorption spectrum of the reduced cytochrome, sodium bisulfite was added to the bacterial cell suspension to a final concentration of 68 mM, and the absorbance of the peak (about 420 nm) of the Soret band of the reduced cytochrome was measured.

[0265] Furthermore, the protein content (μg) of the bacterial suspension was measured using a commercially available bicinchoninic acid (BCA) assay kit (Micro BCA protein assay kit, manufactured by Thermo Fisher Scientific).

[0266] CCI ( / mg) was calculated based on the obtained measured value and the following formula: Wherein, A1 is the OD of the bacterial suspension after stirring 600 , A0 is the OD of the bacterial suspension before stirring 600 (0.3). In the formula, A Soret Protein Content (PC) is the protein content (μg) of the bacterial suspension.

[0267] [CCI( / mg)]=A Soret / PC

[0268] The results of GAI, HPI and CCI measurements of Bacillus species from various regions are shown in Figure 17 to Figure 19. Regarding CCI, the YM18 strain and the 60473 strain were higher than the PCA strain and the KN400 strain. That is, the 60473 strain can generate relatively high current density on both the graphite felt electrode and the glassy carbon electrode, while the YM18 strain only generates a high current density on the graphite felt electrode. The reason why the YM18 strain can form an electrochemically active biofilm (EABF) on the graphite felt electrode is that the cells are physically captured between the graphite felt fibers, and it is considered that the strain has difficulty adhering to a flat surface. On the other hand, it is interesting that the 60473 strain forms a high-density biofilm on the flat and easily slidable surface of the glassy carbon electrode, and it is considered that the high GAI and high HPI characteristics of the 60473 strain promote this behavior.

[0269] [Example 4]

[0270] To evaluate the usefulness of strain 60473 in bioaugmentation of microbial electrolysis cells, the cells were operated using starch as the main anode substrate and the amount of electricity or hydrogen produced was investigated.

[0271] Specifically, soil was inoculated in four single-layer electrolytic cells, and starch was used as the main anode matrix for operation (MEC1 to MEC4). One day after the start of operation, 60,473 strains were taken up in two electrolytic cells (MEC3 and MEC4), and the operation continued. In the remaining two electrolytic cells (MEC1 and MEC2), 60,473 strains were not taken up, and these electrolytic cells continued to operate directly as controls without bio-enhancement. During the operation, the electrolyte was replaced in order to intermittently add the matrix (starch) to the electrolytic cell. The time from the replacement of the electrolyte to the next replacement of the electrolyte was regarded as "1 cycle", and the operation was carried out until the sixth cycle (after the electrolyte was replaced 5 times).

[0272] The results of measuring the power generation amount (current density) of each cell over time are shown in Fig. 20 and Fig.21 . Fig.21 Yes Fig. 20 The vertical axis of the measured data is enlarged. Fig. 20 and Fig.21 In the figure, the time point indicated by the arrow indicates the time point when the electrolyte is replaced.

[0273] After the operation was completed, the biofilm formed on the surface of the working electrode (anode electrode) of each pool was recovered, and the constituent bacteria were classified by genus and the composition was investigated. The amount of each bacteria was measured by quantitative polymerase chain reaction (PCR) method in the same way as in Non-Patent Document 6. The results are shown in Fig. 22In MEC3 and MEC4 bioaugmented with the 60473 strain, 40% or more of the biofilm on the anode electrode surface was composed of Geobacter bacteria, and it was confirmed that the cultured 60473 strain preferentially adhered to the anode electrode surface.

[0274] like Fig. 20 and Fig.21 As shown in Figure 2, MEC3 and MEC4, which were bioenhanced with strain 60473, produced a relatively large current compared to the non-bioenhanced controls (MEC1 and MEC2). Even after repeated replacement of the electrolyte, the maximum current density (peak current density) (J max ) always reaches about 1.5mA / cm 2 In contrast, in MEC1 and MEC2, the 2 The maximum current density of MEC3 and MEC4 bio-augmented with strain 60473 was unexpectedly high, and was comparable to that achieved in a pure culture electrolytic cell using acetic acid as the substrate. These results suggest the possibility that the performance of an electrolytic cell using high polymer fermentable organic matter (HFO) such as starch as the substrate could be improved to the same level as that of an electrolytic cell using acetic acid as the substrate by bio-augmentation with strain 60473.

[0275] In addition, in the electrolytic cell bioenhanced with the YM18 strain, the maximum current density began to decrease in the fourth and fifth cycles (Non-Patent Document 6). Fig.21 As shown in the figure, it was confirmed that the maximum current density of MEC3 and MEC4 was almost unchanged, and did not substantially decrease even in the 6-cycle operation, so that the high current generation capacity could be maintained for a long time. It is speculated that the constant current generation in the electrolytic cell bio-enhanced with 60473 strains is caused by the high affinity of 60473 strains for graphite electrodes.

[0276] Regarding the electrolysis performed in each electrolytic cell, the maximum current density [J max (mA / cm 2 )], daily hydrogen production rate (hydrogen production rate (HPR): [capacity of hydrogen generated (L)] / [capacity of electrolyte solution in the cell (L)] / [day (Day)]) and removal rate of chemical oxygen demand (COD) [COD R:(1-[COD at the end of operation] / [COD at the start of operation])×100(%)]. The results are shown in Table 2.

[0277] [Table 2]

[0278]

[0279] Hydrogen is only produced in MEC3 and MEC4 at a rate of about 0.5L / L / day. In addition, compared with MEC1 and MEC2, the COD of MEC3 and MEC4 is R These results suggest that in the electrolytic cells bioenhanced with strain 60473, most of the starch is decomposed along with the current generation. In addition, the results of Table 2 show that the current generation (e.g., the material combined with acetic acid oxidation) is the rate-limiting stage of starch decomposition in the electrolytic cell, confirming that the enhancement of this stage by introducing strong electrochemically active bacteria is a promising strategy to improve the performance of electrolytic cells for the treatment of high molecular fermentable organic matter.

[0280] [Example 5]

[0281] In order to evaluate the usefulness of 60473 strains in bioaugmentation of microbial electrolysis cells, a three-electrode one-tank electrochemical cell was operated using food waste as an anode substrate, the generated current was measured, and the amount of power generation or hydrogen generation was investigated (n=2).

[0282] As food waste, vegetable waste from actual restaurants was used. Water was added to the crushed food waste, and after being left for several days, the supernatant was recovered by centrifugation. The supernatant was adjusted to pH 8.0 (COD value: about 55,000 ppm) and used as an anode substrate.

[0283] As a three-electrode one-tank electrochemical cell, the working electrode was set as graphite felt (6.00 cm 2The reference electrode was an Ag / AgCl electrode (RE-T8A, manufactured by EC Frontier), the counter electrode was a platinum mesh (0.12 mm thick, manufactured by Nilaco), and the electrolyte was 50 mL. The electrolyte used was a solution containing 12.3 g / L Na2HPO4, 1.9 g / L KH2PO4, 0.26 g / L NH4Cl, 10 mL / L Modified Wolin's mineral solution (Deutsche Sammlung von Mikroorganismen und Zellkulturen) and 10 mL / L Wolin's vitamin solution (Deutsche Sammlung von Mikroorganismen und Zellkulturen) in 0.1 M poly butylene succinate (PBS) (pH 8.0).

[0284] Specifically, after adding 5.5 mL of anode matrix and 5 mL of anaerobic digested sludge as a bacterial source to the electrolyte of the electrochemical cell, the cell was connected to a potentiostat (VMP3, manufactured by Biologic), the working electrode potential was set to -0.2 V vs. Ag / AgCl electrode, and current measurement was started. During operation, in order to intermittently add anode matrix to the electrolytic cell, a new electrolyte containing the anode matrix was replaced. The time from the time of replacing the electrolyte to the time of the next replacement of the electrolyte was regarded as "1 cycle", and the operation was performed until the sixth cycle (after 5 electrolyte replacements).

[0285] For two of the four electrolytic cells (60473_1 and 60473_2), 4 hours after the current value of the first cycle became positive, the OD value of the 60473 strain in the cell was calculated. 600 60473 strains were added to achieve a concentration of 0.04 (bioaugmentation), and 60473 strains were not taken up in the remaining two pools (NB_1 and NB_2), and these apparatuses were operated as controls without bioaugmentation.

[0286] The results of measuring the power generation amount (current density) of each cell over time are shown in Fig.23 .exist Fig.23The upper value indicates "cycle". In the pools (60473_1 and 60473_2) to which the 60473 strain was added, the current density increased significantly. These results confirmed that the effect of bioaugmentation using the 60473 strain was fully obtained even when food waste was used as the anode matrix.

[0287] Furthermore, the removal rate of COD (chemical oxygen demand) [COD R :(1-[COD at the end of operation] / [COD at the start of operation])×100(%)] and coulombic efficiency (%). Coulombic efficiency is the ratio of discharge capacity to charge capacity during discharge expressed as a percentage. The higher the coulombic efficiency of the battery, the more the capacity entered during charging can be used for discharge without loss, so the life is longer. COD is calculated in the same way as in Example 4 R The coulombic efficiency is calculated by the following formula.

[0288] [Coulomb efficiency (%)] = [Coulomb conversion value of the cumulative amount of generated current] / [Coulomb conversion value of the amount of COD removed] × 100 (%)

[0289] The COD of each electrolytic cell R The results of the measurement of the coulombic efficiency of the sixth cycle are shown in Table 3. Compared with the control pools (NB_1, NB_2), the pools (60473_1, 60473_2) to which the 60473 strain was added had a higher COD removal rate and a higher coulombic efficiency. Based on these results, it is clear that even when food waste is used as the anode matrix, it is easier to reduce COD by using the bioaugmentation of the 60473 strain, thereby extending the life of the electrolytic cell.

[0290] [Table 3]

[0291]

[0292] In addition, regarding the electrolysis performed in each electrolytic cell, the daily hydrogen production rate (HPR: [hydrogen volume generated (L)] / [electrolyte solution volume in the cell (L)] / [Day]), the daily methane production rate (methane production rate, MPR: [methane volume generated (L)] / [electrolyte solution volume in the cell (L)] / [Day]) and the methane production amount (L / L / day) were measured by gas chromatography. The results are shown in Table 4.

[0293] [Table 4]

[0294]

[0295] As shown in Table 4, in the pool to which the 60473 strain was added, hydrogen generation was observed at first, but almost no hydrogen was detected after the fourth cycle. This is presumably because the generated hydrogen was consumed by methanogens, etc. On the other hand, no significant difference was found in the amount of methane generated between the pool to which the 60473 strain was added and the control pool.

[0296] Cyclic voltammetry (CV) was performed on each electrolytic cell after the sixth cycle. CV is an electrochemical measurement method that measures the current flowing when the electrode is immersed in a static sample solution and the potential is repeatedly scanned, and is expressed as a current-voltage curve. The CV results of each cell are shown in Fig.24 .

[0297] like Fig.24 As shown, the CV peaks of the pools (60473_1, 60473_2) to which the 60473 strain was added were larger than those of the control pools (NB_1, NB_2). This is presumably because the amount of electrochemically active bacteria attached to the electrodes of the pools to which the 60473 strain was added was larger.

[0298] [Example 6]

[0299] In order to evaluate the usefulness of strain 60473 in bioenhancement of microbial fuel cells (MFC), a single-tank air cathode MFC reactor was operated using food waste as the anode substrate, the generated current was measured, and the power generation was investigated (n=2). As food waste, a substance prepared in the same manner as in Example 5 was used.

[0300] As a single-tank air cathode MFC reactor, the reactor volume was set to 85 mL, and the anode electrode was set to graphite felt (12.56 cm 2 , thickness 3 mm, manufactured by Nippon Carbon Co., Ltd.), and the cathode electrode was coated with a platinum catalyst (TEC10V20E, manufactured by Tanaka Kikinzo Co., Ltd.) (0.64 mg / cm 2 ) of the air cathode (25cm 2 , Non-Patent Document 7), the electrolyte was set to 60 mL. The electrolyte used had the same composition as in Example 5.

[0301] Specifically, the operation was started after adding 5.5 mL of anode matrix and 6.5 mL of anaerobic digested sludge as a bacterial source to the electrolyte of the MFC reactor. The initial resistance was set to 10 kΩ, and the spring pressure was reduced at any time after it was fully increased. During operation, in order to intermittently add anode matrix to the reactor, the new electrolyte containing the anode matrix was replaced. The time from the replacement of the electrolyte to the next replacement of the electrolyte was regarded as "1 cycle", and the operation was carried out until the tenth cycle (after nine electrolyte replacements).

[0302] For two of the four MFC reactors (60473_1 and 60473_2), 4 hours after the current value of the first cycle became positive, the OD of the 60473 strain in the reactor was 600 60473 strains were added so as to be 0.04 (bioaugmentation). 60473 strains were not taken up in the remaining two reactors (NB_1 and NB_2), and these devices were operated as controls without bioaugmentation.

[0303] The results of measuring the voltage (V) of each reactor over time are shown in Fig.25 .exist Fig.25 In the graph, the upper numerical value indicates “cycle.” Table 5 shows the results of measuring the resistance value of each reactor for each cycle over time.

[0304] [Table 5]

[0305]

[0306] like Fig.25 As shown in Table 5, in the reactors (60473_1, 60473_2) to which the 60473 strain was added, a sufficient voltage (about 0.4 V or more) appeared even when the resistance was reduced. Reducing the resistance causes more current to flow, which places a large load on the electrochemically active bacteria. Nevertheless, since the high voltage was maintained, it was confirmed that the effect of bioenhancement using the 60473 strain could be fully obtained even in the MFC using food waste as the anode matrix.

[0307] The results of polarization analysis for each reactor in the ninth cycle are shown in FIG. Fig.26 and Fig. 27 . Fig.26 The results of polarization analysis of the reactors (60473_1, 60473_2) into which 60473 strains were placed (n=2) are shown. Fig. 27 The polarization analysis results of the reactors (NB_1, NB_2) without 60473 strains (n=2) are shown. Fig.26 and Fig. 27 The results of the polarization analysis shown here show the calculated maximum power density (P max mW / m 2 ), and the comparison results are shown in Fig.28 In the figure, the error bars represent the standard deviation. By inputting 60473 strains, such as Fig.28As shown, if the maximum power density obtained by polarization analysis of the ninth cycle is compared, the output power increases significantly in the MFC with 60473 strains added. The same tendency can also be observed in other cycles. According to these results, even when food waste is used as the anode matrix, the performance of the battery can be improved by using the bioreinforcement of 60473 strains in the existing microbial fuel cell.

[0308] Furthermore, regarding the electrolysis performed in each reactor, the COD (chemical oxygen demand) removal rate (COD R The measurement results are shown in Table 6.

[0309] [Table 6]

[0310]

[0311] As shown in Table 6, the COD removal rate and coulomb efficiency of the reactors (60473_1 and 60473_2) to which 60473 strains were added were higher than those of the control reactors (NB_1 and NB_2). These results show that even when food waste is used as the anode matrix, COD can be more easily reduced by using bioaugmentation with 60473 strains, thereby extending the life of the MFC reactor.

[0312] CV was performed on each MFC reactor after the tenth cycle. The CV results of each reactor are shown in Fig.29 .like Fig.29 As shown in the figure, the peaks of CV in the reactors (60473_1 and 60473_2) to which the 60473 strain was added were larger than those in the control reactors (NB_1 and NB_2). This is presumably because the amount of electrochemically active bacteria attached to the electrodes of the cell to which the 60473 strain was added was larger.

[0313] [Accession number]

[0314] NITE BP-03900.

Claims

1. A microorganism, characterized in that It is Geobacter strain 60473, and its deposit number is NITE BP-03900.

2. The microorganism according to claim 1, wherein It is used in microbial fuel cells.

3. A composition for a microbial fuel cell, characterized in that: It contains Geobacter 60473 strain, whose deposit number is NITE BP-03900, and organic matter.

4. The composition for a microbial fuel cell according to claim 3, wherein It also contains electrochemically active bacteria other than Geobacter 60473 strain.

5. The composition for a microbial fuel cell according to claim 3, wherein At least a portion of the organic matter is sludge.

6. A microbial fuel cell, characterized in that: The Geobacter strain 60473, whose deposit number is NITE BP-03900, is used for decomposition of organic matter used as fuel.

7. A microbial fuel cell, characterized in that: include: Liquid, with water containing organic matter as the main component; A container for containing the liquid; A conductive anode electrode disposed in the liquid; A conductive cathode electrode, at least one surface of which is arranged in contact with the liquid; a resistor having one end connected to the anode electrode and the other end connected to the cathode electrode and allowing electrons to flow; and The Geobacter strain 60473, whose deposit number is NITE BP-03900, decomposes the organic matter contained in the liquid and emits electrons.

8. The microbial fuel cell according to claim 7, wherein: The container is divided into a tank where the anode electrode is arranged and a tank where the cathode electrode is arranged by a membrane that can selectively allow protons to pass through. The organic matter and the Geobacter 60473 strain are contained in the liquid contained in the tank in which the anode electrode is arranged.

9. The microbial fuel cell according to claim 7, wherein: The cathode electrode has one surface in contact with the liquid in the container and the other surface in contact with the atmosphere.

10. A microbial fuel cell, characterized in that: include: A container containing organic mud and a liquid with water as a main component; A conductive anode electrode is disposed in the organic mud; A conductive cathode electrode disposed in or on the surface of the liquid; a resistor having one end connected to the anode electrode and the other end connected to the cathode electrode and allowing electrons to flow; and The Geobacter genus strain 60473, whose deposit number is NITE BP-03900, is contained in the organic mud.

11. A microbial fuel cell, characterized in that: include: A conductive anode electrode is disposed in the organic mud; A conductive cathode electrode is placed in the water or on the water surface; a resistor having one end connected to the anode electrode and the other end connected to the cathode electrode and allowing electrons to flow; and Geobacter strain 60473, whose deposit number is NITE BP-03900, is attached to the anode electrode, The microbial fuel cell is provided for purifying organic mud at the bottom of a river or a lake.

12. The microbial fuel cell according to claim 10 or 11, wherein: A supply pipe is included for supplying a substrate and / or the Geobacter 60473 strain from the outside to the inside of the organic mud.

13. The microbial fuel cell according to claim 12, wherein: The supply pipe branches into the liquid or the water so as to be dispersed into a plurality of pipes in the planar direction of the anode electrode for supply.

14. The microbial fuel cell according to any one of claims 7, 10 and 11, wherein The anode electrode is a conductive metal material having a lower internal resistance than the cathode electrode.

15. The microbial fuel cell according to any one of claims 7, 10 and 11, wherein The surface area of ​​the cathode electrode is at least 1.1 times greater than the surface area of ​​the anode electrode.

16. The microbial fuel cell according to claim 10 or 11, wherein: The cathode electrode is arranged in parallel with the anode electrode, and one end of the cathode electrode and one end of the anode electrode arranged on the same side as the one end are connected to a support body extending along the depth direction in the liquid or the water. The cathode electrode and the anode electrode are fixedly supported so as to be adjustable in a manner maintaining a spacing corresponding to the depth, and include a fixed portion protruding in the depth direction on the bottom side of the anode electrode.

17. A composition for a microbial electrolytic cell, characterized in that: It contains Geobacter 60473 strain, whose deposit number is NITE BP-03900, and organic matter.

18. The composition for a microbial electrolytic cell according to claim 17, wherein It also contains electrochemically active bacteria other than Geobacter 60473 strain.

19. The composition for a microbial electrolytic cell according to claim 17, wherein At least a portion of the organic matter is sludge.

20. A microbial electrolysis cell, characterized in that: The Geobacter strain 60473, whose deposit number is NITE BP-03900, is used for decomposition of organic matter used as fuel.

21. A microbial electrolysis cell, characterized in that: include: Liquid, with water containing organic matter as the main component; A container for containing the liquid; A conductive anode electrode disposed in the liquid; A conductive cathode electrode, arranged in contact with the liquid; A potential control device, one end of which is connected to the anode electrode and the other end of which is connected to the cathode electrode and provides electron flow; as well as The Geobacter strain 60473, whose deposit number is NITE BP-03900, decomposes the organic matter contained in the liquid and emits electrons.

22. The microbial electrolysis cell according to claim 21, wherein: The container is divided into a tank where the anode electrode is arranged and a tank where the cathode electrode is arranged by a membrane that can selectively allow protons to pass through. The organic matter and the Geobacter 60473 strain are contained in the liquid contained in the tank in which the anode electrode is arranged.

23. A microbial electrolysis cell, characterized in that: include: A container containing organic mud and a liquid with water as a main component; A conductive anode electrode disposed in the liquid; A conductive cathode electrode disposed in the liquid; a potential control device, one end of which is connected to the anode electrode and the other end of which is connected to the cathode electrode and provides electron flow; and The Geobacter genus strain 60473, whose deposit number is NITE BP-03900, is contained in the organic mud.

24. The microbial electrolysis cell according to claim 23, wherein: A supply pipe is included for supplying a substrate and / or the Geobacter 60473 strain from the outside to the inside of the liquid.

25. The microbial electrolysis cell according to claim 24, wherein: The supply pipe branches in the liquid so as to be dispersed in a plurality of portions and supply the liquid to the anode electrode or the cathode electrode.

26. The microbial electrolysis cell according to any one of claims 21, 23 and 24, wherein: The anode electrode is a conductive material having a lower internal resistance than the cathode electrode.

27. The microbial electrolysis cell according to any one of claims 21, 23 and 24, wherein: The surface area of ​​the cathode electrode is at least 1.1 times greater than the surface area of ​​the anode electrode.

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