Electrochemically active bacterium

Abstract

To provide a novel electrochemically active bacterium.SOLUTION: Provided is a bacterium having a deposit number of NITE BP-04052.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-04052. [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 the ADMFC2 strain and various representative bacteria of the genus Sulfurospirillum. [Figure 2] 10 is a photograph showing the appearance of colonies formed by the ADMFC2 strain. [Figure 3]It is a graph showing the results of current generation by the ADMFC2 strain 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 made under the conditions of room temperature (20 to 25°C) / relative humidity 40 to 50%RH.

[0012] <ADMFC2 strain> One embodiment of the present invention is a bacterium (electrochemically active bacterium) with an accession number of NITE BP-04052. In this specification, the bacterium with an accession number of NITE BP-04052 is also simply referred to as "ADMFC2 strain".

[0013] The ADMFC2 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 dilution 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 approximately 30°C; (2) Pick reddish-brown 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 approximately 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-brown colonies that has 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 strain from the reddish-brown colonies that formed on the plate.

[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 Sulfurospirillum alkalitolerans HTRB-L1 strain, with a gene sequence identity of 93.8%. Since bacteria with a homology of 95% or more are generally considered to belong to the same genus, this indicates that the isolated strain represents a new genus and species.

[0020] Furthermore, because the bacteria that showed high homology to the isolated strain in the Blast search belonged to the genus Sulfurospirillum, a phylogenetic tree was created using the nearest neighbor method to compare the molecular phylogeny with various type strains within this genus. The results are shown in Figure 1.

[0021] The phylogenetic tree shown in Figure 1 indicates that the isolated strain is relatively closely related to two species currently described as belonging to the genus Sulfurospirillum: Sulfurospirillum alkalitolerans and Sulfurospirillum tamanensis. However, these two species of bacteria are separated at the genus level from other members of the genus Sulfurospirillum, suggesting that they may not belong to this genus (Sorokin DY, Tourova TP, Muyzer G. Isolation and characterization of two novel alkalitolerant sulfidogens from a thiopaque bioreactor, Desulfonatronum alkalitolerans sp. nov., and Sulfurospirillum alkalitolerans sp. nov. Extremophiles. 2013 May; 17(3): 535-43. doi: 10.1007 / s00792-013-0538-4. Epub 2013 Apr 7. PMID: 23564266.). Furthermore, the isolated strains are also thought to be separated at the genus level from the most closely related species, Sulfurospirillum alkalitolerans.

[0022] Therefore, the isolated strain was determined to be a novel bacterium and was named ADMFC2. The ADMFC2 strain was deposited at the Patent Microorganisms Depositary of the 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-04052.

[0023] The mycological properties of the ADMFC2 strain are shown in Table 1 below. The mycological properties were determined according to known techniques and methods described in the literature (Kodama Y, Ha LT, Watanabe K. Sulfurospirillum cavolei sp. nov., a facultatively anaerobic sulfur-reducing bacterium isolated from an underground crude oil storage cavity. Int J Syst Evol Microbiol. 2007 Apr; 57(Pt 4): 827-831. doi: 10.1099 / ijs.0.64823-0. PMID: 17392214.). The mycological properties of bacteria of the closely related genus Sulfurospirillum are also shown.

[0024] [Table 1]

[0025] As shown in Table 1, the ADMFC2 strain has the following properties: (1) Acetic acid (salt), malic acid (salt), lactic acid (salt) and pyruvic acid (salt) can be used as electron donors; (2) Nitric acid (salt), fumaric acid (salt), DMSO, MnO2, and electrodes (see Examples) can be used as electron acceptors; (3) an optimum pH of 8.0; and (4) Maximum salt concentration (M Na + ) is 0.25.

[0026] Furthermore, as described above, the ADMFC2 strain has the following properties: (5) It can grow under anaerobic conditions at approximately 30°C.

[0027] The method for culturing the ADMFC2 strain according to the present invention is not particularly limited, as long as it allows the ADMFC2 strain to grow and proliferate. For example, the medium used to culture the ADMFC2 strain may be either a solid medium or a liquid medium. As the medium used to culture the ADMFC2 strain, for example, the DSM826 medium or modified DSM826 medium described in the Examples can be used. Culture conditions can be set appropriately.

[0028] <Mutant strain> One embodiment of the present invention is a mutant strain of the bacterium (ADMFC2 strain) having accession number NITE BP-04052, which has a 16S rRNA gene sequence that has 95% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1 and is capable of generating electric current. In this specification, a bacterium that is a mutant strain of the bacterium (ADMFC2 strain) having accession number NITE BP-04052 is also referred to as a "mutant strain of the ADMFC2 strain."

[0029] The 16S rRNA gene sequence of the mutant strain of the ADMFC2 strain may have sequence identity with the nucleotide sequence set forth in SEQ ID NO: 1 of 96% or more, 97% or more, 98% or more, 99% or more, or 100%. Herein, sequence identity can be determined using an analysis program such as BLAST. When BLAST is used, the program's default parameters are used.

[0030] The mutant strain of the ADMFC2 strain has the ability to generate electric current. Whether the mutant strain of the ADMFC2 strain has the ability to generate electric current can be determined, for example, by planting the mutant strain of the ADMFC2 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 ADMFC2 strain can preferably use at least one selected from the group consisting of acetic acid, malic acid, lactic acid, and pyruvic acid as an electron donor, and at least one selected from the group consisting of nitric acid, fumaric acid, DMSO, MnO2, and an electrode as an electron acceptor.

[0032] Mutant strains of the ADMFC2 strain can be obtained, for example, by mutagenesis or exogenous gene introduction into the ADMFC2 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 α-rays, β-rays, γ-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 ADMFC2 strain according to the present invention can be cultured by appropriately referring to the method for culturing the ADMFC2 strain.

[0034] <Composition> One embodiment of the present invention is a composition comprising at least one of the ADMFC2 strain and a mutant strain of the ADMFC2 strain. Herein, the ADMFC2 strain and the mutant strain of the ADMFC2 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 subjecting the bacteria-containing residue obtained by centrifuging the culture solution to freeze-drying, spray-drying, air-drying, vacuum drying, or the like.

[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 ADMFC2 strain and a mutant strain of the ADMFC2 strain.

[0039] The ADMFC2 strain and the mutant strain of the ADMFC2 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-04052. [2] A mutant strain of the bacterium described in [1], The mutant strain is a bacterium having a 16S rRNA gene sequence that has 95% or more sequence identity 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 according to [1] and the bacteria according to [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 ADMFC2 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 two weeks. 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 two weeks. 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 reddish-brown colonies that formed on the plates were isolated, named strain ADMFC2, and deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (accession number NITE BP-04052). The appearance of the colonies is shown in Figure 2.

[0054] Evaluation of the current generating ability of ADMFC2 strain A colony of the ADMFC2 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 one week 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 silver / silica (Ret-T8A, manufactured by Nippon Carbon Co., Ltd.), the reference electrode was a silver / silica (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 so that the turbidity at 600 nm was 0.01. 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 ADMFC2 strain was confirmed to be an electrochemically active bacterium capable of generating current.

Claims

1. A bacterium with accession number NITE BP-04052.

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 95% or more sequence identity 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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