Electrochemically active bacterium

Abstract

To provide a novel electrochemically active bacterium.SOLUTION: Provided is a bacterium having a deposit number of NITE BP-04053.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to electrochemically active bacteria. [Background technology]

[0002] A microbial electrochemical system is a system that produces energy, useful substances, etc. by electrochemically controlling the metabolism of microorganisms. Microbial electrochemical systems include microbial fuel cells, microbial electrolysis cells, and microbial electrosynthesis cells (Non-Patent Document 1).

[0003] In microbial electrochemical systems, electrochemically active bacteria that can donate and receive electrons to and from electrodes are important, and electrochemically active bacteria belonging to the genera Shewanella, Geobacter, and Acidithiobacillus have been discovered to date (Non-Patent Document 2). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Roy, S., & Pandit, S. (2019) Microbial electrochemical system: principles and application. In Microbial electrochemical technology (pp. 19-48). [Non-patent document 2] Xiao X, Yu HQ. Molecular mechanisms of microbial transmembrane electron transfer of electrochemically active bacteria. Curr Opin Chem Biol. 2020 Dec; 59: 104-110. doi: 10.1016 / j.cbpa.2020.06.006. Epub 2020 Jul 23. PMID: 32712559. Summary of the Invention [Problem to be solved by the invention]

[0005] There is a possibility that electrochemically active bacteria other than those in the genera mentioned above may exist, and their discovery is expected to increase the availability of electrochemically active bacteria.

[0006] Therefore, the present invention has been made in consideration of the above circumstances, and an object of the present invention is to provide a new electrochemically active bacterium. [Means for solving the problem]

[0007] One aspect of the present invention is a bacterium having accession number NITE BP-04053. [Effects of the Invention]

[0008] According to the present invention, a new electrochemically active bacterium can be provided. [Brief explanation of the drawings]

[0009] [Figure 1] This is a phylogenetic tree showing the molecular phylogenetic relationship between strain ADMFC3 and bacteria representing the family Geovibrionaceae. [Figure 2] 10 is a photograph showing the appearance of colonies formed by the ADMFC3 strain. [Figure 3] 1 is a graph showing the results of current production by strain ADMFC3 in an electrochemical cell.

Mode for Carrying Out the Invention

[0010] Hereinafter, embodiments according to one embodiment of the present invention will be described. The present invention is not limited only to the following embodiments.

[0011] In this specification, "X to Y" indicating a range means "X or more and Y or less". Also, unless otherwise specified, measurements of operations and physical properties are measured under the conditions of room temperature (20 to 25°C) / relative humidity 40 to 50%RH.

[0012] <ADMFC3 strain> One embodiment of the present invention is a bacterium (electrochemically active bacterium) with an accession number of NITE BP-04053. In this specification, the bacterium with an accession number of NITE BP-04053 is also simply referred to as "ADMFC3 strain".

[0013] The ADMFC3 strain was isolated from an anaerobic digester for food waste treatment (Yokohama City, Kanagawa Prefecture) by the following screening method.

[0014] Using the method described in the literature (Yoshizu D, Kouzuma A, Watanabe K. Use of Microbial Fuel Cells for the Treatment of Residue Effluents Discharged from an Anaerobic Digester Treating Food Wastes. Microorganisms. 2023 Feb 27; 11(3): 598. doi: 10.3390 / microorganisms11030598. PMID: 36985172; PMCID: PMC10059938.), a power generation experiment was conducted by placing the fermentation residue from the anaerobic digester into an air-cathode microbial fuel cell. After the power generation experiment, the anode was cut out in the anaerobic chamber and immersed in a liquid medium (e.g., DSM826 liquid medium or modified DSM826 liquid medium described in the Examples), and the attached microorganisms were suspended in the medium using a vortex mixer. This suspension was used to isolate the bacterial strains by the following procedure: (1) A diluted solution of the suspension is applied to a plate of solid medium (e.g., DSM826 agar medium or modified DSM826 agar medium described in the Examples) and allowed to form colonies under anaerobic conditions at 15 to 37°C (preferably 25 to 30°C); (2) Pick up reddish colonies formed on the plate, place them in a liquid medium (e.g., DSM826 liquid medium or modified DSM826 liquid medium described in the Examples), inoculate them in an anaerobic vial in which the gas phase has been replaced with nitrogen gas (e.g., 99.999%), and cultivate the microorganisms at 15 to 37°C (preferably 25 to 30°C) for 1 to 2 weeks; (3) The culture is grown on a solid medium plate in the same manner as in (1) to form colonies; (4) Pick one of the reddish colonies that formed and culture it in liquid medium in the same manner as in (2); (5) Repeat steps (3) and (4) until only colonies with the same shape are formed on the plate; (6) Isolate the strains that form red colonies on the plates.

[0015] The modified DSM826 medium may be a DSM826 medium supplemented with resazurin (1 mg / L), cysteine ​​HCl (0.9 g / L), and yeast extract (0.5 g / L).

[0016] The strains isolated in this way are planted in a three-pole, single-chamber electrochemical cell to confirm the generation of current (see Examples), which allows us to determine that the isolated strains are electrochemically active bacteria capable of generating current.

[0017] The genome sequence of the isolated strain was determined using Sequel IIe (Pacific Biosciences), and the 16S rRNA gene was identified from the genome sequence. The obtained base sequence is shown below.

[0018] [ka]

[0019] A BLAST analysis of the 16S rRNA gene sequence using the NCBI database revealed that the closest related species was Geovibrio thiophilus, with a gene sequence identity of 97.5%. Two species are registered in the genus Geovibrio. The 16S rRNA gene sequence identity with the PAL-1 strain, the type strain of Geovibrio ferrireducens, was 95.4%.

[0020] A phylogenetic tree was constructed using the nearest neighbor method to compare the molecular phylogeny of representative bacteria of the family Geovibrionaceae. The results are shown in Figure 1.

[0021] The phylogenetic tree shown in Figure 1 indicates that the isolated strain is most closely related to Geovibrio thiophilus. However, no Geovibrio bacteria have been reported to be capable of generating electric current.

[0022] To confirm whether the isolated strain represents a new species of Geovibrio, we performed ANI (average nucleotide identity) and dDDH (digital DNA-DNA hybridization) analyses using the genome sequence (SEQ ID NO: 2) of the isolated strain and the closely related species Geovibrio thiophilus AAFu3 strain. The ANI value was 80.5%, and the dDDH value was undeterminable (due to significant sequence differences). Currently, a species is considered to be distinct if its ANI value is 95% or less and its dDDH value is 70% or less (Chun et al., Int JSyst Evol Microbiol, 2018; 68; 461-466). This confirms that the isolated strain represents a new species of Geovibrio.

[0023] Therefore, the isolated strain was determined to be a novel bacterium and was named ADMFC3 strain. The ADMFC3 strain was deposited at the Patent Microorganisms Depositary Center, National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan) on March 1, 2024, and its accession number is NITE BP-04053.

[0024] The mycological properties of the ADMFC3 strain are shown in Table 1 below. The mycological properties were determined according to known techniques and methods described in the literature (Caccavo F Jr, Coates JD, Rossello-Mora RA, Ludwig W, Schleifer KH, Lovley DR, McInerney MJ. Geovibrio ferrireducens, a phylogenetically distinct dissimilatory Fe(III)-reducing bacterium. Arch Microbiol. 1996 Jun;165(6):370-6. doi: 10.1007 / s002030050340. PMID: 8661930.). The mycological properties of bacteria of the closely related genus Geovibrio are also shown.

[0025] [Table 1]

[0026] As shown in Table 1, the ADMFC3 strain has the following properties: (1) They are cocci that do not have flagella and do not grow fermentatively; (2) Acetic acid (salt), formic acid (salt), lactic acid (salt), pyruvic acid (salt), succinic acid (salt), and proline can be used as electron donors; (3) Fumaric acid and an electrode (see Examples) can be used as electron acceptors; (4) The growth temperature is 15 to 37°C, with an optimum temperature of 25 to 30°C; (5) The type of cytochrome is c-type; and (6) Growth occurs only under anaerobic conditions (see screening method above).

[0027] The method for culturing the ADMFC3 strain according to the present invention is not particularly limited, as long as it allows the ADMFC3 strain to grow and proliferate. For example, the medium used to culture the ADMFC3 strain may be either a solid medium or a liquid medium. Examples of media that can be used to culture the ADMFC3 strain include the DSM826 medium and modified DSM826 medium described in the Examples. Culture conditions can be appropriately set based on the properties (4) and (6) above.

[0028] <Mutant strain> One embodiment of the present invention is a mutant strain of the bacterium (ADMFC3 strain) having accession number NITE BP-04053, which has a 16S rRNA gene sequence that has 98.0% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 3 and is capable of generating electric current. In this specification, a bacterium that is a mutant strain of the bacterium (ADMFC3 strain) having accession number NITE BP-04053 is also referred to as a "mutant strain of the ADMFC3 strain."

[0029] The 16S rRNA gene sequence of the mutant strain ADMFC3 may have sequence identity with the nucleotide sequence set forth in SEQ ID NO: 1 of 98.5% or more, 99.0% or more, 99.5% or more, or 100%. Herein, sequence identity can be determined using an analysis program such as BLAST. When using BLAST, the program's default parameters are used.

[0030] The mutant strain of the ADMFC3 strain has the ability to generate electric current. Whether the mutant strain of the ADMFC3 strain has the ability to generate electric current can be determined, for example, by planting the mutant strain of the ADMFC3 strain in a three-pole, single-chamber electrochemical cell and checking whether electric current is generated or not (see Examples).

[0031] The mutant strain of the ADMFC3 strain can preferably use at least one selected from the group consisting of acetic acid (salt), formic acid (salt), lactic acid (salt), pyruvic acid (salt), succinic acid (salt), and proline as an electron donor, and can use at least one selected from the group consisting of fumaric acid and an electrode as an electron acceptor.

[0032] Mutant strains of the ADMFC3 strain can be obtained, for example, by mutagenesis or exogenous gene introduction into the ADMFC3 strain. Mutagenesis and exogenous gene introduction can be performed using known techniques. Methods for introducing mutations include homologous recombination; genome editing techniques using ZFN, TALEN, CRISPR-CAS9, CRISPR-CPF1, etc.; site-directed mutagenesis and other mutagenesis methods; and random mutagenesis. Random mutagenesis methods include irradiation with radiation such as alpha rays, beta rays, gamma rays, and X-rays; treatment with ethyl methanesulfonate (EMS), ethynylnitrosourea (ENU), N-methyl-N'-nitro-N-nitrosoguanidine (NTG), etc.; and treatment with heavy ion beams.

[0033] The mutant strain of the ADMFC3 strain according to the present invention can be cultured by appropriately referring to the method for culturing the ADMFC3 strain.

[0034] <Composition> One embodiment of the present invention is a composition comprising at least one of the ADMFC3 strain and a mutant strain of the ADMFC3 strain. Herein, the ADMFC3 strain and the mutant strain of the ADMFC3 strain are collectively referred to as the "bacterium of the present invention."

[0035] The bacterium of the present invention contained in the composition may be in the form of a culture, which refers to a culture medium in which the bacterium of the present invention is cultured, a residue containing the bacterium obtained by centrifuging the culture medium, or a dried product of the residue.

[0036] The dried residue can be obtained by centrifuging the culture solution and subjecting the bacteria-containing residue to freeze-drying, spray-drying, air-drying or vacuum-drying.

[0037] The bacterium according to the present invention is an electrochemically active bacterium capable of generating electric current, and therefore the composition according to the present invention is preferably used for a microbial electrochemical system.

[0038] <Microbial electrochemical system> One embodiment of the present invention is a microbial electrochemical system comprising at least one of the ADMFC3 strain and a mutant strain of the ADMFC3 strain.

[0039] The ADMFC3 strain and the mutant strain of the ADMFC3 strain according to the present invention are electrochemically active bacteria capable of generating electric current, and therefore can be used in a microbial electrochemical system.

[0040] Microbial electrochemical systems include microbial fuel cells, microbial electrolyzers, microbial electrosynthesizers, and the like.

[0041] Microbial fuel cells (MFCs) are devices that utilize the metabolic capabilities of microorganisms to convert organic fuels (materials that retain chemical energy) into electrical energy. While MFCs have attracted attention as a means of generating electricity while processing biomass waste and as an energy-efficient wastewater treatment system, increasing their efficiency is essential for practical application (Yamada, S., Watanabe, K. (2020) Fundamentals and Applications of Microbial Fuel Cells. Fuel Cells (Fuel Cell Development Information Center) 20:17-22). Furthermore, efforts are being made to develop technologies that utilize MFCs for water purification (Xu, C., Sun, S., Li, Y., Gao, Y., Zhang, W., Tian, ​​L., Li, T., Du, Q., Cai, J., Zhou, L. (2023) Methane emission reduction-oriented extracellular electron transfer and bioremediation of sediment in microbial fuel cells: A review. Sci. Total Environ. 874:162508). These are called sediment microbial fuel cells, and by placing an anode in the sediments of lake or ocean bottoms and a cathode in the water above the sediments, they can generate electricity while decomposing organic matter in the sediments.

[0042] Microbial electrolyzers are devices that generate hydrogen, methane, etc. by reducing protons in water using an electric current generated by electrochemically active bacteria from fuels such as organic matter, but high efficiency is desired for practical use in society (Ochiai Kazuta, Kawaminami Akari, Watanabe Kazuya (2021) Hydrogen production using microbial electrolyzers. Power generation and hydrogen production by microorganisms (CMC Publishing)).

[0043] A microbial electrosynthesis tank is a device that synthesizes useful substances from CO2, nitrogen, etc. by supplying electrons from a low-potential electrode to electrochemically active bacteria, and is expected to become a process that will support humanity in the future when fossil fuels become depleted (Yamada S, Takamatsu Y, Ikeda S, Kouzuma A, Watanabe K. (2022) Towards application of electro-fermentation for the production of value-added chemicals from biomass feedstocks. Front. Chem. 9:805597).

[0044] <Embodiment> The following describes an embodiment of the present invention. [1] Bacteria with accession number NITE BP-04053. [2] A mutant strain of the bacterium described in [1], The mutant strain is a bacterium having a 16S rRNA gene sequence that has a sequence identity of 98.0% or more with the base sequence shown in SEQ ID NO: 1 and having the ability to generate electric current. [3] A composition comprising at least one of the bacteria described in [1] and the bacteria described in [2]. [4] The composition according to [3], which is for use in a microbial electrochemical system. [5] A microbial electrochemical system comprising at least one of the bacteria according to [1] and the bacteria according to [2], and / or the composition according to [4] or [5]. [Example]

[0045] The present invention will be described below using specific examples, but the present invention is not limited to these examples.

[0046] Preparation of DSM826 liquid medium and DSM826 agar medium Each component was dissolved in purified water to obtain the composition shown in Table 2. The pH was adjusted to 6.8 with 6 M HCl, and the mixture was sterilized by filtration to prepare DSM826 liquid medium.

[0047] DSM826 agar medium was prepared by mixing autoclaved 3% Bacto Agar with 2x the concentration of filter-sterilized DSM826 liquid medium.

[0048] [Table 2]

[0049] The mineral solution was prepared by dissolving nitrilotriacetic acid in pure water to obtain the composition shown in Table 3 below, adjusting the pH to 6.5 with KOH, dissolving the other components, and adjusting the pH to 7.0 with 6M HCl.

[0050] [Table 3]

[0051] The vitamin solution was prepared by dissolving each component in purified water to obtain the composition shown in Table 4 below.

[0052] [Table 4]

[0053] Isolation of strain ADMFC3 A power generation experiment was conducted using an air cathode microbial fuel cell containing fermentation residue from an anaerobic digester (Yokohama, Kanagawa Prefecture) for food waste treatment. The experiment was performed according to the method described previously (Yoshizu D, Kouzuma A, Watanabe K. Use of Microbial Fuel Cells for the Treatment of Residue Effluents Discharged from an Anaerobic Digester Treating Food Wastes. Microorganisms. 2023 Feb 27; 11(3): 598. doi: 10.3390 / microorganisms11030598. PMID: 36985172; PMCID: PMC10059938). After the power generation experiment, the anode was removed from the anaerobic chamber, immersed in DSM826 liquid medium, and the attached microorganisms were suspended in the medium using a vortex mixer. A dilution of this suspension was spread onto a DSM826 agar plate, placed in an AnaeroPouch® Kenki (Mitsubishi Gas Chemical Company, Inc.), and incubated at 30°C for colony formation. A reddish colony formed on the plate was picked, placed in DSM826 liquid medium, and inoculated into an anaerobic vial with the atmosphere replaced with nitrogen gas (99.999%). The microorganism was then cultured at 30°C for one week. The culture was then cultured on a DSM826 agar plate in the same manner as above to allow colony formation. One of the colonies was then picked, placed in DSM826 liquid medium, and inoculated into an anaerobic vial with the atmosphere replaced with nitrogen gas (99.999%). The culture was then cultured at 30°C for one week. The culture was then spread onto a DSS826 agar plate to allow colony formation. This procedure was repeated until only colonies of the same shape were formed on the plate. The red colonies formed on the plates were isolated, named ADMFC3 strain, and deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (NITE BP-04053). The appearance of the colonies is shown in Figure 2.

[0054] Evaluation of the current generating ability of ADMFC3 strain A colony of the ADMFC3 strain was placed in DSM826 liquid medium, inoculated into an anaerobic vial in which the gas phase was replaced with nitrogen gas (99.999%), and cultured at 30°C for 72 hours to obtain a culture. The culture was then placed in a three-electrode, single-chamber electrochemical cell, and the generated current was measured. In the electrochemical cell, the working electrode was a graphite felt (1.0 cm 2 The electrode was a 15-cm long Ag / AgCl electrode (RE-T8A, manufactured by EC Frontier Co., Ltd.), the counter electrode was a platinum wire (15 cm long, 0.3 mm thick; manufactured by Nilaco Corporation), and the electrolyte was 15 mL of DSM826 liquid medium without disodium fumarate. The culture was added to this electrolyte to achieve a turbidity of 0.01 at 600 nm. The cell was connected to a potentiostat (VMP3, manufactured by Biologic), and the working electrode potential was set to -0.2 V relative to the reference electrode, and current measurement was initiated. The results are shown in Figure 3. As shown in Figure 3, the ADMFC3 strain was confirmed to be an electrochemically active bacterium.

Claims

1. A bacterial strain with accession number NITE BP-04053.

2. A mutant strain of the bacterium according to claim 1, The mutant strain is a bacterium having a 16S rRNA gene sequence that has a sequence identity of 98.0% or more with the base sequence shown in SEQ ID NO: 1 and having the ability to generate electric current.

3. A composition comprising at least one of the bacteria of claim 1 and the bacteria of claim 2.

4. The composition of claim 3 for use in a microbial electrochemical system.

5. A microbial electrochemical system comprising at least one of the bacteria according to claim 1 and the bacteria according to claim 2.

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