JP7212345B2 - Power-generating bacteria, microbial fuel cell using the same, and power generation method - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Law The 24th Okinawa Conference of the Society for Biotechnology, Japan, Kyushu Branch, December 9, 2017 Master's thesis presentation, Department of Applied Biological Sciences, Faculty of Agriculture, University of Miyazaki, January 31, 2018

TECHNICAL FIELD The present invention relates to a power-generating bacterium, a microbial fuel cell using the same, and a power generation method. INDUSTRIAL APPLICABILITY According to the present invention, an excellent microbial fuel cell can be produced and power can be efficiently generated.

Microbial fuel cell (MFC) is a device that uses the metabolic ability of microorganisms to convert chemical energy such as organic matter into electrical energy.It is a new technology that enables energy recovery from waste biomass. is expected as As shown in FIG. 1, the MFC consists of an anode (negative electrode) and a cathode (positive electrode). At the anode, electrons generated by decomposition of organic matter by microorganisms are collected by electrodes. The collected electrons pass through an external circuit to the cathode, where O ₂ , electrons, and H ⁺ (generated during the decomposition of organic matter at the anode) react to form H ₂ O. In MFC, electric current is generated by the potential difference between the anodic reaction and the cathodic reaction, and electric energy can be obtained (Patent Documents 1 and 2).

JP 2017-69019 A JP 2017-188196 A

The present inventor has developed a microbial fuel cell using livestock waste or shochu lees as fuel (Patent Documents 1 and 2). These microbial fuel cells can generate electricity using biomass waste, and are useful as renewable energy. Expected to be developed.
It is an object of the present invention to provide a high power and efficient microbial fuel cell and power generation method.

As a result of intensive research on microbial fuel cells and power generation methods that have high output and are efficient, the present inventors have surprisingly found that, by using Clostridium neuense as a power generating bacterium, high We have found that it is possible to fabricate a microbial fuel cell with output and that efficient power generation is possible.
The present invention is based on these findings.
Accordingly, the present invention provides
[1] A power generation method using a microbial fuel cell, wherein the content of Clostridium neuense relative to the bacteria contained in the microbial fuel cell is 2% or more at the start of power generation.
[2] The power generation method according to [1], which includes the step of adding Clostridium nuens to an electrolyte solution containing an organic substance,
[3] The power generation method according to [1] or [2], wherein the pH of the electrolyte containing organic matter in the microbial fuel cell is 3.9 to 9.0,
[4] The power generation method according to any one of [1] to [3], wherein the organic matter is shochu lees,
[5] The power generation method according to any one of [1] to [4], wherein the Clostridium nuens is a Clostridium nuens containing 16S rDNA having the base sequence represented by SEQ ID NO: 1 or 2;
[6] The power generation method according to any one of [1] to [5], wherein the Clostridium nuens is Clostridium nuens with accession number NITE P-02709;
[7] Clostridium nuens containing 16S rDNA having the base sequence represented by SEQ ID NO: 1 or 2;
[8] Clostridium nuens according to [7], which has accession number NITE P-02709;
[9] A microbial fuel composition containing an organic matter and Clostridium nuens, wherein the content of Clostridium nuens is 2% or more relative to the number of bacteria contained in the fuel composition,
[10] The microbial fuel composition according to [9], wherein the organic matter is shochu lees,
[11] The microbial fuel composition according to [9] or [10], wherein the Clostridium nuens is Clostridium nuens containing 16S rDNA having the nucleotide sequence represented by SEQ ID NO: 1 or 2;
[12] The microbial fuel composition according to [9] or [10], wherein the Clostridium nuens is Clostridium nuens with accession number NITE P-02709;
[13] A microbial fuel cell that uses an organic matter as a fuel to generate electricity using power-generating bacteria, wherein the content of Clostridium nuens is 2% or more relative to the number of bacteria contained in the microbial fuel cell before the start of power generation. , microbial fuel cells,
[14] The microbial fuel cell according to [13], wherein the organic matter is shochu lees,
[15] The microbial fuel cell of [13] or [14], wherein the Clostridium nuens is a Clostridium nuens containing 16S rDNA having the base sequence represented by SEQ ID NO: 1 or 2;
[16] The microbial fuel cell according to any one of [13] to [15], wherein the Clostridium nuens is Clostridium nuens with accession number NITE P-02709;
Regarding.

According to the power generation method of the present invention, power can be generated at high output. Also, by using Clostridium nuens, it is possible to produce a microbial fuel cell with high output, and to efficiently generate power using microorganisms. By containing Clostridium nuens, the microbial fuel composition of the present invention can efficiently generate power as a fuel for a microbial fuel cell. Furthermore, the Clostridium nuens of the present invention is capable of excellent high-output microbial power generation. In addition, by using Clostridium nuens, it is possible to efficiently treat (decompose) acidic waste such as shochu lees.

1 is a diagram showing the principle of a microbial fuel cell; FIG. 1 is a diagram showing a typical single-tank MFC; FIG. 1 is a diagram showing a typical two-vessel MFC; FIG. It is the figure which showed the structure (A) of a cassette electrode, the schematic of cassette electrode MFC, and the photograph (B). It is the graph which showed the change of the power density of the microbial fuel cell using the shochu lees for about 90 days. It is a graph showing pH changes from 0 to 20 days in a microbial fuel cell using shochu lees. FIG. 2B is a photograph (B) showing the growth of the SDL48 strain in the NBGF medium using glucose as an electron donor. (A) shows a negative control in which the SDL48 strain was not inoculated. It is the photograph (A) which showed the microbial fuel cell using SDL48 stock|strain, and the graph (B) which showed the voltage change in the electric power generation using the same. Pellets of shochu lees undiluted solution (1), shochu lees undiluted solution (COD _cr concentration 73 g / L) MFC anode biofilm (2), electrolyte of shochu lees undiluted solution (3), COD _cr concentration 10 g / L anode biofilm ( 4), a graph showing the results of analysis of the microflora of the electrolytic solution (5) with a COD _cr concentration of 10 g/L.

[1] Power Generation Method In the power generation method using the microbial fuel cell of the present invention, the content of Clostridium neuense relative to the bacteria contained in the microbial fuel cell is 2% or more at the start of power generation.

《Microbial fuel cell》
The microbial fuel cell (hereinafter sometimes referred to as MFC) in the present invention is a device that converts chemical energy such as organic matter into electrical energy using the metabolic ability of microorganisms (microbial fuel power generation device). is. The microbial fuel cell used in the power generation method of the present invention is not particularly limited as long as it contains Clostridium nuens as the power generating bacterium.
An example of a typical two-chamber MFC will be described with reference to FIG. In the two-chamber MFC, one of the two tanks (the anode tank) is anaerobic, where electrochemically active bacteria grow and biofilms are formed on the electrodes. Specifically, it contains an electrolyte solution containing an organic matter and power-generating bacteria, and electrons are generated by the decomposition of the organic matter by the power-generating bacteria. The other tank (cathode tank) contains the cathode and the electrolyte is aerated with air (oxygen). The two tanks, the anode tank and the cathode tank, are separated by a proton exchange membrane.
An example of the one-chamber MFC will be described with reference to FIG. A single-chamber MFC uses a membrane-type cathode called an air cathode. Since the air cathode directly reacts with oxygen in the air, there is no need for aeration unlike the two-vessel MFC. In addition, the single-chamber MFC can also be produced without using a proton exchange membrane, and the single-chamber MFC that does not use a proton exchange membrane can have a low internal resistance. Therefore, compared to a two-layer MFC, a higher output can be obtained. For example, as shown in FIG. 3, the cassette electrode has a structure in which a proton exchange membrane and a graphite felt (anode) are attached in order to both electrodes of a flat cathode box (air cathodes are installed on both sides). A shape in which two layered MFCs (two pairs of anodes and cathodes) are combined may be used. Electricity is generated by contacting the anode with an electrolyte solution containing organic matter and transferring electrons generated in the process of decomposition of the organic matter by power generating bacteria to the electrode. The cassette electrodes can generate electricity simply by being inserted into the reactor, and the size, shape, and number can be changed as appropriate. The microbial fuel cell used in the power generation method of the present invention includes small microbial fuel cells to large-scale microbial fuel power generators.

《Clostridium nuens》
Clostridium nuens used in the power generation method of the present invention is not particularly limited as long as it can decompose organic substances and generate electrons. However, Clostridium bacteria containing 16S rDNA having a nucleotide sequence homology of 99% or more with the nucleotide sequence represented by any one of SEQ ID NOs: 1 to 4 are preferred. Bacteria with 16S rDNA homology of 99% or more are generally considered to be of the same species. For example, Clostridium neuense G1 (Zhao et al., 2017) or Clostridium sp. JCM8022 has 99% or more homology to the 16S rDNA nucleotide sequence of the SDL48 strain described below, and the power generation method of the present invention can be used in The 16S rDNA nucleotide sequence of Clostridium neuense G1 is shown in SEQ ID NO:3, and the 16S rDNA nucleotide sequence of Clostridium sp. JCM8022 is shown in SEQ ID NO:4. As used herein, homology refers to the degree of identity between two nucleotides. In addition, Clostridium nuens used in the power generation method of the present invention is preferably Clostridium nuens with accession number NITE P-02709.

<<Content rate of Clostridium nuens>>
The content of Clostridium nuens in the power generation method of the present invention is 2% or more, preferably 3% or more, more preferably 5% or more, and still more preferably 10% or more at the start of power generation. . Furthermore, in certain embodiments, the content of Clostridium nuens is 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more Yes, 80% or more, or 90% or more. In the power generation method of the present invention, high-output and efficient power generation can be achieved by increasing the content of Clostridium nuens, a power-generating bacterium, at the start of power generation.
Although the method for increasing the content of Clostridium nuens is not particularly limited, for example, cultured Clostridium nuens may be added to the microbial fuel cell. As the organic matter of the fuel used for power generation, shochu lees, wine lees, whiskey lees, or livestock waste can be used as described later. These fuels such as shochu lees, wine lees, whiskey lees, and livestock wastes contain bacteria other than power-generating bacteria, but at the start of power generation, the power-generating bacteria Clostridium nuens content increases. By doing so, it is possible to efficiently generate power. In addition, it is also possible to use organic substances that do not contain bacteria (sterilized organic substances, or purified or synthesized organic substances) as fuel, and electricity can be generated by adding Clostridium nuens to such organic substances. can. In addition, the dominance rate of bacteria belonging to the genus Clostridium in shochu lees is 1.1%.

Although the method for measuring the content of Clostridium nuens is not particularly limited, the content of Clostridium nuens can be measured, for example, by a microbiota analysis method. Specifically, the fuel (organic matter) containing the power generating bacteria contained in the microbial fuel cell is sampled, the 16S rRNA gene (16S rDNA), for example, the V4-V5 region (420 bp) is amplified by PCR, and the base sequence is sequence analyzed. determined by By classifying the obtained 16S rDNA base sequences by clustering analysis, the types of bacteria contained can be identified and the content can be determined. In order to improve the accuracy of the percentage content, it is preferable to combine several sampled samples for analysis or to average the data of several samples separately analyzed.

"organic matter"
The organic matter used in the power generation method of the present invention is not particularly limited as long as it contains a compound that can be decomposed by Clostridium nuens.
Although the organic matter is not limited, an organic matter containing an organic acid is preferable. Organic acids can include, but are not limited to, carboxylic acids such as saturated fatty acids (eg, formic acid, acetic acid, propionic acid, or butyric acid), unsaturated fatty acids (eg, oleic acid, linoleic acid, or linolenic acid), dicarboxylic acids (e.g. oxalic acid, malonic acid, or succinic acid), aromatic carboxylic acids (e.g. benzoic acid or phthalic acid), tricarboxylic acids (e.g. anicotic acid), hydroxy acids (e.g. lactic acid , citric acid, or malic acid), oxocarboxylic acids (eg, pyruvic acid).

The said shochu-liquor lees are the shochu-liquor lees generated in the process of manufacturing the shochu-liquor of potato, barley, rice, or buckwheat, for example. The type of shochu lees is not particularly limited, but examples thereof include lees from preparation (raw lees, saccharification lees) and surplus yeast. Among these, raw lees are preferred. Moreover, the shochu lees may be dried.
The brewer's lees is brewer's lees generated in the process of producing beer using malted barley. The type of beer lees is not particularly limited, but examples include brewer's lees (raw lees, saccharified lees), surplus yeast, and the like. Among these, raw lees are preferred. Also, the beer lees may be dried.

The pH of the electrolyte containing organic matter is not particularly limited as long as Clostridium nuens can decompose the organic matter, but it is preferably acidic to neutral. For example, the lower limit of the pH of the electrolytic solution containing organic matter is preferably pH 3.9, more preferably pH 4.0. The upper limit of the pH of the electrolytic solution is not particularly limited, but is, for example, pH 9.0, preferably pH 8.5, more preferably pH 8.0, and still more preferably pH 7.5. Yes, most preferably pH 7.0. Due to the above pH range, Clostridium nuens can efficiently decompose organic matter and generate electrons.

《Addition process》
The power generation method of the present invention may include a step of adding Clostridium nuens to the microbial fuel cell (organic matter). That is, this addition step may increase the content of Clostridium nuens in bacteria in the microbial fuel cell to 2% or more.
Clostridium nuens may be added to the electrolytic cell containing the anode. It can also be added, for example, to an electrolyte solution containing an organic substance that contacts the anode. Alternatively, it can be added to an organic substance to form an electrolytic solution containing the organic substance.
The Clostridium nuens to be added can be previously cultured in a medium and grown. The medium used for culturing Clostridium nuens includes 2xYTG medium or NBAF medium.

《Chemical Oxygen Demand》
In the power generation method of the present invention, it is preferable to maintain COD _Cr (Chemical Oxygen Demand) within a certain range. COD _Cr is obtained by oxidizing the amount of oxidizable substances in a sample with an oxidizing agent under certain conditions and calculating the amount of oxygen required for oxidation from the amount of the oxidizing agent used at that time (unit: mg/L).
For example, a high output can be obtained by using an electrolytic solution having a COD _Cr of 5,000 to 20,000 mg/L. Also, the COD (Chemical Oxygen Demand) removal rate can be maintained at a relatively high level and within a moderate range. COD _Cr is preferably 5,000 to 20,000 mg/L, more preferably 7,000 to 12,000 mg/L.
In order to make the COD _Cr of the electrolytic solution before the start of power generation 5,000 to 20,000 mg/L, the liquid containing the organic matter is diluted or concentrated to obtain a COD _Cr of 5,000 to 20,000 mg/L. An electrolytic solution within the range may be prepared. Diluents can include, but are not limited to, water or organic solvents (eg, alcohols). The electrolytic solution may contain additives as necessary. Additives include, for example, nutrients that favor the growth of microorganisms, pH adjusters, and the like.

In the power generation method of the present invention, it is preferable to maintain COD _Cr at 5,000 to 20,000 mg/L during power generation, but power generation outside this range is also included in the power generation method of the present invention.
To maintain COD _Cr between 5,000 and 20,000 mg/L, organics can be added to the electrolyte or electrolytes containing organics can be replaced. In the microbial fuel cell, the COD _Cr is reduced by the decomposition of organic matter by power-generating bacteria. COD _Cr can be increased by adding or replacing organics to the electrolyte. In some cases, after removing a predetermined amount of electrolyte from the microbial fuel cell (for example, half the amount of fuel in the container), the electrolyte containing a predetermined amount of organic matter (for example, an amount corresponding to the removed fuel) is added to the microbial fuel cell. It may be added to a fuel cell. In this specification, the operation of replacing about half of the electrolytic solution with a new electrolytic solution containing an organic substance during power generation is sometimes referred to as "semi-batch processing".
The electrolytic solution to be added may be prepared using the electrolytic solution taken out from the microbial fuel cell. For example, the electrolytic solution to be added can be prepared by adding an organic substance to the electrolytic solution taken out. As the electrolytic solution to be added, the organic matter itself may be used, or the electrolytic solution containing the organic matter may be used as described above. This semi-batch may be performed once or multiple times, and the number of times is not particularly limited.
As another form of this semi-batch, for example, while operating the microbial fuel cell, the COD _Cr of the electrolyte of the microbial fuel cell is substantially kept within the range of 5,000 to 20,000 mg / L. Alternatively, the electrolytic solution to be added may flow into the microbial fuel cell at a predetermined flow rate while the electrolytic solution is discharged from the microbial fuel cell at a predetermined flow rate.

As used herein, "COD _Cr is substantially kept within a predetermined range during operation" means that COD _Cr is always within a predetermined range during the entire operation period from the start of operation to the stop of operation. It means that COD _Cr is kept within a predetermined range during the period in which high efficiency, which is the effect of the present invention, can be obtained. Specifically, it is preferable that the COD _{Cr is kept within a predetermined range for 80% or more of the operating period, and that the COD Cr is} kept within a predetermined range for 90% or more of the operating period. is more preferable, and it is even more preferable that the COD _Cr is kept within a predetermined range for 100% of the operating period. COD _Cr can be measured, for example, using a commercially available kit (COD kit (TNT822 and DR2800, manufactured by Hach)).

[2] Clostridium nuens The Clostridium nuens of the present invention contains 16S rDNA having the nucleotide sequence represented by SEQ ID NO: 1 or 2, and is preferably Clostridium nuens of accession number NITE P-02709. The Clostridium nuens of the present invention decomposes organic matter to generate electrons, so it can be used as a power-generating bacterium. In addition, Clostridium nuens containing the 16S rDNA having the base sequence represented by SEQ ID NO: 2 proliferates predominantly in the microbial fuel cell by generating power using shochu lees as fuel. Therefore, like the SDL48 strain, the fungus can be isolated according to the examples herein.
Clostridium nuens of the present invention has 99% or more homology to the 16S rDNA nucleotide sequences of Clostridium neuense G1 (Zhao et al., 2017) and Clostridium sp. JCM8022 strain, and is the same species. being classified.
The Clostridium nuens of the present invention can be used as a power-generating bacterium in the "power generation method,""microbial fuel composition," and "microbial fuel cell" of the present invention.

[3] Microbial fuel composition The microbial fuel composition of the present invention is a microbial fuel composition containing organic matter and power generating bacteria, and the content of Clostridium nuens relative to the number of bacteria contained in the fuel composition is 2% or more. The microbial fuel composition of the present invention can be used as a fuel for microbial fuel cells.
The content of Clostridium nuens in the microbial fuel composition of the present invention is 2% or more, preferably 3% or more, more preferably 5% or more, and still more preferably 10% or more. Furthermore, in certain embodiments, the content of Clostridium nuens is 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more Yes, 80% or more, or 90% or more. The microbial fuel composition of the present invention has a high content of Clostridium nuens, which is a power-generating bacterium. Therefore, a microbial fuel cell using the microbial fuel composition of the present invention can generate power with high output and efficiency. Also, in the power generation method using the microbial fuel composition of the present invention, high-output and efficient power generation can be achieved.

In addition, the "power generating bacteria" contained in the microbial fuel composition of the present invention includes Clostridium nuens, but power generating bacteria other than Clostridium nuens may also be included. Examples of power-generating bacteria other than Clostridium nuens include sulfate-reducing bacteria, nitrate-reducing bacteria, sulfur-reducing bacteria, iron-reducing bacteria, manganese dioxide-reducing bacteria, and dechlorinating bacteria. genus power-generating bacteria.

The "Clostridium nuens" in the microbial fuel composition of the present invention is the Clostridium nuens described in the section "[1] Power generation method".
As the “organic matter” in the microbial fuel composition of the present invention, the organic matter described in the section “[1] Electric power generation method” can be used.
The method for increasing the "Clostridium nuens content" or the "measurement method for the Clostridium nuens content" in the microbial fuel composition of the present invention are described in the above "[1] Power generation method" section. can be implemented according to

[4] Microbial fuel cell The microbial fuel cell of the present invention is a microbial fuel cell that generates power using power generating bacteria, and the content of Clostridium nuens relative to the number of bacteria contained in the microbial fuel cell before the start of power generation is 2% or more. The microbial fuel cell of the present invention is the microbial fuel cell described in the section "[1] Power generation method".

The content of Clostridium nuens in the microbial fuel cell of the present invention is 2% or more, preferably 3% or more, more preferably 5% or more, and still more preferably 10% or more. Furthermore, in certain embodiments, the content of Clostridium nuens is 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more Yes, 80% or more, or 90% or more. The microbial fuel cell of the present invention has a high content of Clostridium nuens, which is a power-generating bacterium. Therefore, the microbial fuel cell of the present invention can generate power with high output and efficiency.

The "Clostridium nuens" in the microbial fuel cell of the present invention is the Clostridium nuens described in the section "[1] Power generation method".
As the “organic matter” in the microbial fuel cell of the present invention, the organic matter described in the above “[1] Power generation method” can be used.
The method for increasing the "Clostridium nuens content" or the "Method for measuring the Clostridium nuens content" in the microbial fuel cell of the present invention is described in the above "[1] Power generation method" section. It can be implemented according to
In the microbial fuel cell of the present invention, Clostridium nuens can be added to the microbial fuel cell to increase the content of Clostridium nuens to 2% or more. It can be carried out according to the "Adding step" in the "Method" section.
When power is generated using the microbial fuel cell of the present invention, it is preferable to maintain the chemical oxygen demand within a certain range. amount”.

《Action》
The Clostridium nuens of the present invention and the Clostridium nuens used in the power generation method and the like of the present invention act as power-generating bacteria in, for example, a microbial fuel cell using shochu lees as fuel, and proliferate dominantly over other bacteria. Although the mechanism by which Clostridium nuens proliferates dominantly over other bacteria has not been investigated in detail, it is presumed as follows. However, the present invention is not limited by the following assumptions. In a microbial fuel cell using shochu lees as fuel, the concentration of organic acids such as citric acid is high, resulting in a low pH. Therefore, it is considered that Clostridium nuens, which can grow in a low pH environment, can grow preferentially. Furthermore, in the microbial fuel cell, the anode collects electrons produced by Clostridium nuens by decomposing citric acid or the like and transfers them to the cathode. Clostridium nuens is believed to be able to grow favorably in the environment of a microbial battery in which the electrons produced by itself are recovered at such an anode. Therefore, it is presumed that Clostridium nuens proliferates predominantly in a microbial fuel cell using shochu lees with a low pH.

EXAMPLES The present invention will be specifically described below with reference to Examples, but these are not intended to limit the scope of the present invention.

<<Reference example 1>>
In this reference example, power generation was performed for about three months using a microbial fuel cell using shochu lees.
A microbial fuel cell was fabricated according to the method of Simoyama et al. (Simoyama et al., 2008; Inoue et al., 2013). FIG. 5 shows the structure of the cassette electrode (FIG. 5A) and the schematic diagram and photograph (FIG. 5B) of the cassette electrode MFC.
As shochu lees, shochu lees discharged after distillation in the production of sweet potato shochu at Watanabe Sake Brewery Co., Ltd. (Miyazaki, Japan) was used. The sweet potato shochu lees were stored frozen at -20°C until use in the experiment. The shochu lees undiluted solution had a COD _cr concentration of 73 g/L and a pH of 4.1.

The shochu lees undiluted solution was diluted with ultrapure water and adjusted to have a COD _cr concentration of 0.5, 1, 5, and 10 g/L, respectively, and used for each MFC. The undiluted solution (COD _cr concentration: 73 g/L) was used as it was without diluting. 300 mL each of shochu lees with COD _cr concentrations of 0.5, 1, 5, 1, and 73 g/L was put into the cassette electrode MFC, and the operation was started. Specifically, each 300 mL of shochu lees was put into a 350 mL graduated cylinder in an anaerobic chamber, and an electrode was inserted. The headspace was replaced with a mixed gas of _N2 and _CO2 (80:20) and then sealed. As a seed source, the anode felt of the shochu lees MFC (using 5-fold diluted shochu lees) that had been operated immediately before was suspended in PBS, and the centrifuged pellet was further suspended in PBS to obtain 1.0 mL. added one by one. The electrolytic solution was constantly stirred at 600 rpm and operated at a temperature of 30° C. for about 3 months. The external resistance was initially set at 470 ohms and decreased to 330, 200 ohms, etc. as the voltage changed. Until the 35th day, when the voltage began to drop after rising, semi-batch (half the volume of the electrolytic solution was replaced with new shochu lees solution) was performed. After the 35th day, the semi-batch was performed once every 10 days.
Since the output of 0.5 g/L was remarkably low, it was all replaced with 20 g/L shochu lees on the 24th day.

FIG. 6 shows a graph of the power density over time at each COD _cr concentration. ↓ indicates the point where semi-batch was performed. In the MFC using shochu lees with a COD _cr concentration of 0.5 g/L and 1 g/L, the power density did not exceed 0.001 W/m ³ during operation, and the output was significantly low. For the COD _cr concentration of 0.5 g/L, a batch treatment was performed on the 24th day by replacing all the MFC electrolytic solution with the shochu lees having a COD _cr concentration of 20 g/L, and high power generation was observed. The average power density for 30 days from the start of operation is 0.0002, 0.19, 0.30, 0.22 and 0.08 W/m ³ at COD _cr concentrations of 1, 5, 10, 20 and 73 g/L, respectively. and showed the highest value when the COD _cr concentration was 10 g/L. For the COD _cr concentration of 20 g/L, the average power density from the 24th day to the 54th day was calculated.
In addition, FIG. 7 shows pH changes from 0 to 20 days at each COD _cr concentration. Not shown for a COD _cr concentration of 20 g/L. The start-up pH at all COD _cr concentrations was 4.3±0.14.

<<Example 1>>
In this example, power-generating bacteria were separated from the microbial fuel cell that generated power in Reference Example 1 above.
A 2×YTG agar plate was coated with a 100-fold dilution of the anode biofilm of Reference Example 1, which had been operated at a COD _cr concentration of 73 g/L, with 1×PBS. Bacterial isolation was performed by streaking grown colonies onto separate 2×YTG agar plates and repeating this three times.
The isolated fungus was inoculated in 2xYTG liquid medium. The isolated fungus grew vigorously in 2xYTG medium. Upon growth, the medium became cloudy yellowish white, and air bubbles were observed in the upper part of the medium, probably due to the generation of gas.
The 16S rDNA gene was amplified using the bacterium solution grown in the 2×YTG liquid medium as a template, primers 27F (AGAGTTTGATCMTGGCTCAG; SEQ ID NO: 5) and 1492R (TACGGYTACCTTGTTACGACTT; SEQ ID NO: 6), and subjected to agarose electrophoresis. Since a band was observed around 1,500 bp, it was confirmed that almost the entire length of 16S rRNA could be amplified. Sequence analysis of the 16S rDNA gene of this isolate was performed to determine the 1,465 bp 16S rDNA gene sequence. The isolate was named strain SDL48.

A homology search of the 16S rDNA gene of the SDL48 strain was performed by BLAST, and 99.86% homology with Clostridium neuense G1 was shown. Based on these results, the SDL48 strain was assumed to be of the same species as C. neuense.
The SDL48 strain was deposited on May 14, 2018 at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (NPMD) (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture 292-0818) with the accession number NITE P- 02709.

<<Example 2>>
In this example, a growth test of the SDL48 strain was performed using a medium containing glucose as an electron donor.
2.5% (vol/vol) cysteine in NBGF medium with glucose as the sole electron donor and fumaric acid as the sole electron acceptor, and 3% (vol/vol) SDL48 strain grown in 2x YTG liquid medium. ) and statically cultured at 37° C. for 2 days. Considering the carryover of the components of the 2×YTG liquid medium, the same procedure was repeated three times to test whether the cells could grow only with the components of the NBGF medium. As a result, the OD600 reached 0.88 after 3 days of culture, confirming that the SDL48 strain can grow in the NBGF medium and can use glucose as an electron donor (Fig. 8). Table 1 shows the composition of the NBGF medium.

<<Example 3>>
In this example, a microbial fuel cell was produced using the SDL48 strain to generate power.
A single-chamber MFC (equipped with an air cathode: FIG. 9A) contains 400 mL of FWG medium (containing glucose as an electron donor and no electron acceptor) and 40 mL of strain SDL48 grown in NBGF medium, with an external resistance of 1 ,000Ω and operated at a temperature of 30°C. Construction of MFC was performed in an anaerobic chamber. As a negative control, 40 mL of SDL48 strain grown in NBGF medium was autoclaved at 121° C. for 30 minutes, incubated at 55° C. for 1 hour, and autoclaved at 121° C. for 30 minutes. The composition of FWG medium and the composition of DL Mineral Mix are shown below. FWG medium is the Freshwater medium of Loveley et al. (Lovely and Phillips, 1988) supplemented with glucose (10 mM final concentration). The FWG medium was replaced with a mixed gas of N ₂ ·CO ₂ (80:20=N ₂ :CO ₂ ) prior to autoclaving so that the medium was placed under anaerobic conditions.

As shown in FIG. 9B, in the MFC (blue line) containing live bacteria of the SDL48 strain, the voltage was about 0.15 V immediately after connecting to the data logger, but gradually decreased to about 0.10 V. bottom. After that, as the SDL48 strain grew vigorously, the voltage increased to 0.25 V, and then decreased, probably because the substrate glucose was depleted. At the 50th hour of operation, after all the electrolyte in the MFC was replaced with new medium, the voltage began to rise again, and power generation of maximum 0.30 V (external resistance 1,000Ω) was recorded. In the negative control containing killed SDL48 strain, the voltage was 0.05 V or less throughout. From this result, it was shown that the SDL48 strain can generate electricity in a single-chamber MFC in which the sole electron donor is glucose and the sole electron acceptor is used as an electrode.

<<Example 4>>
In this example, a bacterium was collected from the microbial fuel cell that generated power in Reference Example 1, and a plasmid containing the 16S rDNA of the bacterium was prepared.
The graphite felt anode (8.98 g) after operation for 92 days with the shochu lees undiluted solution in Reference Example 1 was cut out and suspended in 25 mL of a 1×PBS (Phosphate buffered saline) solution. 1×PBS was prepared by diluting 10×PBS with the composition table below. After that, using a VORTEX-GENIE2 Mixer (Scientific Industrial), stirring was performed at 3,000 rpm for 30 sec×2 times. After suspension, centrifugation was performed at 4,000 rpm and 25° C. for 10 minutes, and the supernatant was discarded to collect pellets. Pellets were stored frozen at -20°C until used for experiments.
DNA was extracted from the pellet and the 16S rDNA gene was amplified by PCR. Agarose electrophoresis gave a band at 1,500 bp, suggesting that the 16S rDNA gene could be amplified. The resulting PCR product was introduced into a plasmid to transform E. coli. The resulting E. coli was inoculated into LB medium, plasmid was extracted, and sequence analysis was performed using primers M13F, M13R, 518F, 800R and 800F. The resulting plasmid was named SDL33. A BLAST search of SDL33 showed 99.86% homology with Clostridium neuense G1. Based on this result, the bacterium having the 16S rDNA of SDL33 was assumed to be of the same species as C. neuense.

<<Analysis example 1>>
In this analysis example, microbiota was analyzed.
Pellets (1) of shochu lees undiluted solution, Pellet (2) obtained by suspending and centrifuging the anode biofilm of MFC after 92 days of operation with shochu lees undiluted solution (COD _cr concentration 73 g / L) in Reference Example 1. , electrolyte after 92 days of operation with the undiluted solution (3), pellet (4) obtained by suspending and centrifuging the anode biofilm of MFC after 92 days of operation with shochu lees diluted to a COD _cr concentration of 10 g / L in PBS, The electrolyte solution (5) after operation for 92 days at a COD _cr concentration of 10 g/L was used as a sample, and the microbiota was analyzed by next-generation sequencing.
First, DNA was extracted using Extra Soil DNA Kit Plus ver.2 (Nippon Steel Sumikin Environment). After that, the V4-V5 region of the 16S rRNA gene was PCR amplified using primers U515F (5'-GTGYCAGCMGCCGCGGTA-3') and 926R (5'-CCGYCAATTCMTTTRAGTT-3') (Yu et al., 2018). PCR used TaKaRa Ex Taq polymerase (Takara Bio, Inc., Shiga, Japan) with initial denaturation at 95°C for 9 min, followed by cycles of 95°C for 15 sec, 55°C for 10 sec, 72°C for 30 sec. was performed for 15-35 cycles and finally an extension reaction at 72° C. for 2 minutes. Sequence analysis was performed using MiSeq (Illumina), and about 250 bases from both sides of the PCR amplification product were analyzed (paired-end analysis). The resulting sequence data was subjected to read quality filtering to determine the best sequences for analysis, and the sequences that were available were merged using the paired-end merge script FLASH (Magoc and Salzberg, 2011). let me The nucleotide sequence of the obtained sequence data was checked using QIIME (Quantitative Insights Into Microbial Ecology) pipeline. In addition, sequence homology clustering analysis was performed, and classified into OTUs (Operational Taxonomic Units). A homology search against the 16S rRNA gene database of Greengene (DeSantis et al., 2006) was performed for each representative OTU sequence to deduce the phylogenetic classification.

Next-generation sequence analysis revealed 101,619 reads for shochu lees, 106,451 reads for anode biofilm after operation with a COD _cr concentration of 73 g/L (undiluted solution), and 106,451 reads for electrolyte solution after operation with a COD _cr concentration of 73 g/L. Data were obtained for 152,907 reads, 50,043 reads for the anode biofilm after operation at a COD _cr concentration of 10 g/L, and 53,433 reads for the electrolyte after operation at a COD _cr concentration of 10 g/L. FIG. 10 shows the results of microbiota analysis at the genus level for each sample.
In the undiluted solution of shochu lees, the genus Bacillus was dominant (40.9%), and although not shown in the figure, the genus Clostridium was 1.1%.
Clostridium (75.4% and 65.7%, respectively) dominated the anode biofilm and electrolyte after operation with shochu lees with a COD _cr concentration of 73 g/L, and Rummeliibacillus (11.4%, respectively). %, 20.4%) followed. The genus Clostridium was detected only at 1.1% in the shochu lees undiluted solution, suggesting that the genus Clostridium proliferated during operation with shochu lees having a COD _cr concentration of 73 g/L.
Bacteroides (65.3%), Clostridium (12.1%), and Ruminococcus (4.1%) dominated in the anode biofilm of MFC after operation with shochu lees with a COD _cr concentration of 10 g/L. In the electrolytic solution, Bacteroides (67.9%), Ruminococcus (8.6%) and Clostridium (7.8%) were dominant in that order.
By analyzing the microbiota of this analysis example, it is possible to measure the content of microorganisms contained in the microbial fuel cell.

The Clostridium nuens of the present invention can be used as a power generator for microbial fuel cells. Efficient power generation can be performed by the microbial fuel cell and power generation method of the present invention.

NITE P-02709

Claims (13)

A method for generating electricity by a microbial fuel cell , comprising the step of adding Clostridium nuens to an electrolyte solution containing organic matter ,
The power generation method, wherein the content of Clostridium neuense relative to the bacteria contained in the microbial fuel cell is 2% or more at the start of power generation. 2. The power generation method according to claim 1, wherein the electrolyte containing organic matter in the microbial fuel cell has a pH of 3.9 to 9.0. The power generation method according to claim 1 or 2 , wherein the organic matter is shochu lees. The power generation method according to any one of claims 1 to 3 , wherein the Clostridium nuens is Clostridium nuens containing 16S rDNA having the nucleotide sequence represented by SEQ ID NO: 1 or 2. The power generation method according to any one of claims 1 to 4 , wherein the Clostridium nuens is Clostridium nuens with accession number NITE P-02709. A microbial fuel composition comprising organic matter and Clostridium nuens,
A microbial fuel composition, wherein the content of Clostridium nuens is 2% or more relative to the number of bacteria contained in the fuel composition. The microbial fuel composition according to claim 6 , wherein the organic matter is shochu lees. The microbial fuel composition according to claim 6 or 7 , wherein the Clostridium nuens is Clostridium nuens containing 16S rDNA having the base sequence represented by SEQ ID NO: 1 or 2. 8. The microbial fuel composition of claim 6 or 7 , wherein the Clostridium nuens is Clostridium nuens with accession number NITE P-02709. A microbial fuel cell that uses organic matter as fuel to generate electricity by power-generating bacteria,
A microbial fuel cell, wherein the content of Clostridium nuens is 2% or more with respect to the number of bacteria contained in the microbial fuel cell before starting power generation. 11. The microbial fuel cell according to claim 10 , wherein the organic substance is shochu lees. 12. The microbial fuel cell according to claim 10 , wherein said Clostridium nuens is Clostridium nuens containing 16S rDNA having the base sequence represented by SEQ ID NO: 1 or 2. The microbial fuel cell according to any one of claims 10 to 12 , wherein the Clostridium nuens is Clostridium nuens with accession number NITE P-02709.

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