JP2021193884A - Microorganism mixtures, compositions for methane production and methods for producing methane - Google Patents

Abstract

To provide microorganism mixtures that allow for methane production from compositions of biomaterials by conducting co-culture various kinds of specific microorganisms at normal temperature.SOLUTION: Disclosed is a mixture of microorganisms comprising: at least one microorganism selected from the group consisting of microorganisms belonging to the family Enterobacteriaceae, microorganisms belonging to the family Pseudomonadaceae and microorganisms belonging to the family Clostridiaceae; at least one microorganism selected from the group consisting of microorganisms belonging to the family Methanobacteriaceae and microorganisms belonging to the family Methanosarcinaceae; at least one microorganism selected from the group consisting of microorganisms belonging to the family Aspergillaceae and microorganisms belonging to the family Arthopyreniaceae.SELECTED DRAWING: Figure 6

Description

The present disclosure relates to microbial mixtures, methane-producing compositions, and methane-producing methods.

Activated sludge methods are commonly used to treat domestic and industrial sewage. The activated sludge method is a method of decomposing soluble substances and residual floating substances contained in sewage by adding aerobic microorganisms to sewage and performing aeration. In the process of this decomposition, aerobic microorganisms proliferate remarkably while utilizing nutrients such as organic matter, nitrogen, or phosphorus in the sewage. However, since the efficiency of the decomposition is better when the concentration of suspended solids (that is, aerobic microorganisms) in the sewage is controlled within a certain range, the overgrown aerobic microorganisms are discharged to the outside of the system as excess sludge. There is a need to.
Sewage treatment plants operate day and night throughout the year, which means that surplus sludge is constantly generated nationwide. Therefore, securing a disposal site for excess sludge and increasing disposal costs have become problems. Some sewage treatment plants use an anaerobic digestion method using microorganisms that produce methane (so-called methane fermentation) as a method to effectively utilize excess sludge to produce biogas containing about 60% methane from excess sludge. In addition, the volume of excess sludge is being reduced. However, the volume reduction rate of the surplus sludge is still about 30%, and the remaining about 70% of the surplus sludge remains as "digested sludge" which is difficult to reuse any more. This digested sludge currently accounts for about 40% of industrial waste in Japan.

For example, Non-Patent Document 1 discloses a filamentous fungus capable of hydrolyzing digestive sludge under aerobic conditions of 30 ° C. It has been described that filamentous fungi of the genera Umbelopsis, Penicillium, Cunninghamella, Neosartorya, Fusarium, or Chaetomium have cunninghamella, chitinase, or keratinase activity.

Non-Patent Document 2 describes that hydrolases obtained from bacteria of the genus Penicillium, Cunninghamella, Neosartorya, Fusarium, etc. are added during methane fermentation by methane-producing bacteria under a closed condition at 50 ° C. Has been done.

Patent Document 1 discloses a filamentous fungus capable of hydrolyzing digestive sludge under aerobic conditions of 30 ° C. It has been described that filamentous fungi of the genera Penicillium, Cunninghamella, Neosartorya, or Umbelopsis have xylanase, chitinase, or keratinase activity. Further, it is described that the solid weight of the digested sludge was reduced by about 10% under aerobic conditions of 30 ° C. by inoculating the digested sludge with the filamentous fungus and culturing it.

Japanese Unexamined Patent Publication No. 2014-158433

Fujii, K. et al (2013) Journal of Applied Microbiology 115, 718-726 Sato, H. et al (2016) New Biotechnology 33, 1-6

However, the filamentous fungi described in Non-Patent Document 1 and Patent Document 1 can hydrolyze digested sludge, which is an example of a biological composition, under aerobic conditions, but further methane is obtained from the hydrolyzed digested sludge. In this case, since the methane-producing bacterium is an anaerobic microorganism, the disclosed filamentous bacterium and the methane-producing bacterium cannot be co-cultured. Further, Non-Patent Document 2 does not describe co-culture of filamentous fungi and methane-producing bacteria, and in Non-Patent Document 2, it is necessary to preliminarily treat digestive sludge as a substrate with acid, and There is a cost of continuing to heat the culture tank at 50 ° C. Therefore, in any of the above-mentioned Patent Document 1, Non-Patent Document 1 and Non-Patent Document 2, a composition derived from a living organism is obtained by co-culturing a plurality of types of microorganisms including a degradable microorganism and a methane-producing microorganism at room temperature. No microbial mixture is described that allows methane production from.

The present disclosure has been made in view of the above, and the present disclosure provides a microbial mixture capable of producing methane from a biological composition by co-culturing a plurality of specific microorganisms at room temperature. That is the issue.

Specific means for solving the problem include the following aspects.
<1> At least one microorganism selected from the group consisting of a microorganism belonging to the family Enterobacteriaceae, a microorganism belonging to the family Pseudomonadaceae, and a microorganism belonging to the family Clostridiaceae.
At least one microorganism selected from the group consisting of microorganisms belonging to the family Methanobacteriaceae and microorganisms belonging to the family Methanosarcinaceae, and
A microbial mixture comprising at least one microorganism selected from the group consisting of microorganisms belonging to the family Aspergillaceae and microorganisms belonging to the family Arthopyreniaceae.
<2> Furthermore
A microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1.
A microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 2.
A microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 3
The microbial mixture according to <1> above, which comprises at least one microorganism selected from the group consisting of.
<3> With at least one microorganism that decomposes at least one of cellulose, chitin, and protein.
With at least one hydrogen-producing microorganism,
With at least one methane-producing microorganism
A microbial mixture comprising at least one oxygen consuming microorganism.
<4> Any one of <1> to <3> above, which can produce 10 ml or more of methane by culturing under normal temperature conditions for 1 month in the presence of 1 g of a biological composition as a substrate. The microbial mixture according to one.
<5> A methane-producing composition containing the microbial mixture according to any one of <1> to <4> above, capable of producing methane from a biologically-derived composition.
<6> Inoculating a solution containing a biological composition with the microbial mixture according to any one of <1> to <4> above.
By culturing the inoculated microbial mixture under normal temperature conditions,
A method for producing methane, including.
<7> The methane production method according to <6>, wherein the biological composition is digestive sludge.

According to the present disclosure, it is possible to provide a microbial mixture capable of producing methane from a biological composition by co-culturing a plurality of types of specific microorganisms at room temperature.

It is a graph which shows the result of the temperature-dependent methane production amount when the microbial mixture was subcultured. It is a photograph of the gel after staining showing the result of running an amplicon derived from a microbial mixture by the PCR-DGGE method. It is a phylogenetic tree drawn by estimating the genus species of amplicon derived from eubacteria. The inside of the square frame in the figure shows the amplicon obtained in Example 1. The length of the scale bar in the figure indicates that the base substitution ratio is 0.05 bases per nucleotide. It is a phylogenetic tree drawn by estimating the genus species of archaea-derived amplicon. The inside of the square frame in the figure shows the amplicon obtained in Example 1. The length of the scale bar in the figure indicates that the base substitution ratio is 0.05 bases per nucleotide. It is a phylogenetic tree drawn by estimating the genus species of amplicon derived from fungi or protists. The inside of the square frame in the figure shows the amplicon obtained in Example 1. The length of the scale bar in the figure indicates that the base substitution ratio is 0.05 bases per nucleotide. It is a relationship estimation diagram of hydrolysis, hydrogen production, ammonia production, and methane production of a biological composition by a microbial mixture. It is a graph which shows the measurement result of cellulase, chitinase, and protease activity which a microbial mixture has.

Hereinafter, embodiments according to the present disclosure will be described in detail. However, the present disclosure is not limited to the following embodiments. In the following disclosure, the components (including element steps, etc.) are not essential unless otherwise specified. The same applies to the numerical values and their ranges, and does not limit this disclosure.
In the present disclosure, the term "process" includes, in addition to a process independent of other processes, the process as long as the purpose of the process is achieved even if it cannot be clearly distinguished from the other process. ..
In the present disclosure, the numerical range indicated by using "~" includes the numerical values before and after "~" as the minimum value and the maximum value, respectively.
In the numerical range described stepwise in the present disclosure, the upper limit value or the lower limit value described in one numerical range may be replaced with the upper limit value or the lower limit value of the numerical range described in another stepwise description. .. Further, in the numerical range described in the text, the upper limit value or the lower limit value of the numerical range may be replaced with the value shown in the embodiment.
In the present disclosure, the content of each component in the composition is the total of the plurality of substances present in the composition when a plurality of substances corresponding to each component are present in the composition, unless otherwise specified. It means the content rate.

≪Microbial mixture≫
In the present disclosure, the microbial mixture is preferably a mixture of two or more kinds of microorganisms, for example, a mixture of 3 kinds to 10 kinds, 11 kinds to 20 kinds, or more kinds of microorganisms. good. The abundance ratio of each microorganism in the microbial mixture of the present disclosure is not particularly limited.

<Microorganisms contained in the microbial mixture>
(First embodiment)
The microbial mixture according to the first embodiment of the present disclosure includes at least one microorganism selected from the group consisting of a microorganism belonging to the family Enterobacteriaceae, a microorganism belonging to the family Pseudomonadaceae, and a microorganism belonging to the family Clostridiaceae, and a microorganism belonging to the family Methanobacteriaceae. It contains at least one microorganism selected from the group consisting of microorganisms belonging to the family Methanosarcinaceae and at least one microorganism selected from the group consisting of microorganisms belonging to the family Aspergillaceae and microorganisms belonging to the family Arthopyreniaceae.

At least one microorganism selected from the group consisting of microorganisms belonging to the family Enterobacteriaceae, microorganisms belonging to the family Pseudomonadaceae, and microorganisms belonging to the family Clostridiaceae, and at least one selected from the group consisting of microorganisms belonging to the family Methanobacteriaceae and microorganisms belonging to the family Methanosarcinaceae. A microbial mixture containing at least one microorganism selected from the group consisting of a microorganism and a microorganism belonging to the family Aspergillaceae and a microorganism belonging to the family Arthopyreniaceae shall be cultivated at room temperature (co-cultivating the microorganism contained in the microbial mixture). Allows the production of methane from biological compositions.

The action of the microbial mixture according to the first embodiment of the present disclosure is not clear, but is presumed as follows.
Among the microbial mixtures according to the first embodiment of the present disclosure, at least one microorganism selected from the group consisting of a microorganism belonging to the family Enterobacteriaceae, a microorganism belonging to the family Pseudomonadaceae, and a microorganism belonging to the family Clostridiaceae is subject to normal temperature conditions. At least one of the decomposition of cellulose into glucose, the decomposition of chitin into glucosamine, and the decomposition of protein into amino acids in a biological composition is carried out, and at least one microorganism selected from the above group is at room temperature. Under the conditions, the decomposed glucose, glucosamine, or amino acid is taken up to produce hydrogen.
Among the microbial mixtures according to the first embodiment of the present disclosure, at least one microorganism selected from the group consisting of microorganisms belonging to the family Methanobacteriaceae and microorganisms belonging to the family Methanosarcinaceae is the hydrogen produced and the above-mentioned hydrogen under normal temperature conditions. It takes in carbon dioxide and produces methane.
Among the microbial mixtures according to the first embodiment of the present disclosure, at least one microorganism selected from the group consisting of microorganisms belonging to the family Aspergillaceae and microorganisms belonging to the family Arthopyreniaceae takes up oxygen and proliferates under normal temperature conditions. Can be done. That is, at least one microorganism selected from the group consisting of the microorganism belonging to the family Aspergillaceae and the microorganism belonging to the family Arthopyreniaceae consumes oxygen in the process of growth, so that the anaerobic condition in the culture tank can be increased. Therefore, at least one microorganism selected from the group consisting of the anaerobic microorganisms belonging to the family Methanobacteriaceae and the microorganisms belonging to the family Methanosarcinaceae can more efficiently produce methane in the culture tank.
From the above, when methane is produced using a biological composition as a substrate, methane can be efficiently obtained even at room temperature with a small number of steps by co-culture. When the biological composition contains a persistent substance such as chitin or cellulose, for example, when it is digestive sludge, such an effect is more remarkable, and it is difficult to use it as a substrate in the past. Even the composition of can be used as a substrate to produce methane.
Furthermore, at least one microorganism selected from the group consisting of the microorganisms belonging to the family Aspergillaceae and the microorganisms belonging to the family Arthopyreniaceae can produce ammonia from nitric acid in the biological composition under normal temperature conditions. The ammonia serves as a nitrogen source for the microbial mixture, and the microorganisms contained in the microbial mixture can exert their functions more efficiently.
The present disclosure is not limited to the above-mentioned presumed action.

In the present disclosure, examples of microorganisms belonging to the family Enterobacteriaceae include microorganisms belonging to the genus Cronobacter, microorganisms belonging to the genus Citrobacter, microorganisms belonging to the genus Enterobacter, microorganisms belonging to the genus Escherichia, and microorganisms belonging to the genus Lelliottia. Not limited. Examples of microorganisms belonging to the genus Cronobacter include Cronobacter sakazakii and the like. Examples of microorganisms belonging to the genus Citrobacter include Citrobacter freundii and the like. Examples of microorganisms belonging to the genus Enterobacter include Enterobacter asburiae and Enterobacter tabaci. Examples of microorganisms belonging to the genus Escherichia include Escherichia coli and the like. Examples of microorganisms belonging to the genus Lelliottia include Lelliottia amnigena and the like.
In the present disclosure, the microorganism belonging to the family Enterobacteriaceae is a microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. It is more preferable, a microorganism having a 16SrRNA gene sequence having 98% or more sequence identity is more preferable, and a microorganism having a 16SrRNA gene sequence having 100% sequence identity is further preferable. In the present disclosure, sequence identity can be determined by using the Basic Local Alignment Search Tool (BLAST, National Center for Biotechnology Information (NCBI)) with default parameters.

In the present disclosure, for example, a microorganism having a 16SrRNA gene sequence (or 18SrRNA gene sequence) having 95% or more sequence identity with the base sequence represented by SEQ ID NO: N is a 16SrRNA gene sequence (or 18SrRNA gene sequence) possessed by the microorganism. A microorganism in which a continuous part of the total length of the 18SrRNA gene sequence) has 95% or more sequence identity with respect to the total length of the DNA base sequence represented by SEQ ID NO: N.

In the present disclosure, examples of microorganisms belonging to the family Pseudomonadaceae include, but are not limited to, microorganisms belonging to the genus Pseudomonas. Examples of microorganisms belonging to the genus Pseudomonas include Pseudomonas matsuisoli and the like.
In the present disclosure, the microorganism belonging to the family Pseudomonadaceae is preferably a microorganism having a 16SrRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 8 or SEQ ID NO: 9, and preferably 98% or more. It is more preferably a microorganism having a 16SrRNA gene sequence having identity, and even more preferably a microorganism having a 16SrRNA gene sequence having 100% sequence identity.

In the present disclosure, examples of microorganisms belonging to the family Clostridiaceae include, but are not limited to, microorganisms belonging to the genus Clostridium, microorganisms belonging to the genus Fonticella, and microorganisms belonging to the genus Lutispora. Examples of microorganisms belonging to the genus Clostridium include Clostridium amylolyticum or Clostridium punense. Examples of microorganisms belonging to the genus Fonticella include Fonticella tunisiensis and the like. Examples of microorganisms belonging to the genus Lutispora include Lutispora thermophila and the like.
In the present disclosure, the microorganism belonging to the family Clostridiaceae is preferably a microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. A microorganism having a 16SrRNA gene sequence having 98% or more sequence identity is more preferable, and a microorganism having a 16SrRNA gene sequence having 100% sequence identity is further preferable.

In the present disclosure, examples of microorganisms belonging to the family Methanobacteriaceae include, but are not limited to, microorganisms belonging to the genus Methanobacterium. Examples of microorganisms belonging to the genus Methanobacterium include Methanobacterium espanolae, Methanobacterium subterraneum, and Methanobacterium flexile.
In the present disclosure, the microorganism belonging to the family Methanobacteriaceae is preferably a microorganism having a 16SrRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14, and preferably 98% or more. It is more preferably a microorganism having a 16SrRNA gene sequence having identity, and even more preferably a microorganism having a 16SrRNA gene sequence having 100% sequence identity.

In the present disclosure, examples of microorganisms belonging to the family Methanosarcinaceae include, but are not limited to, microorganisms belonging to the genus Methanosarcina. Examples of microorganisms belonging to the genus Methanosarcina include Methanosarcina spelaei, Methanosarcina horonobensis, Methanosarcina acetivorans and the like.
In the present disclosure, the microorganism belonging to the family Methanosarcinaceae is preferably a microorganism having a 16SrRNA gene sequence having 95% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 15, and having 98% or more sequence identity. A microorganism having a 16SrRNA gene sequence is more preferable, and a microorganism having a 16SrRNA gene sequence having 100% sequence identity is further preferable.

In the present disclosure, examples of microorganisms belonging to the family Aspergillaceae include, but are not limited to, microorganisms belonging to the genus Aspergillus, microorganisms belonging to the genus Penicillium, and microorganisms belonging to the genus Monascus. Examples of microorganisms belonging to the genus Aspergillus include Aspergillus penicillioides and the like. Examples of microorganisms belonging to the genus Penicillium include Penicillium expansum and the like. Examples of microorganisms belonging to the genus Monascus include Monascus purpureus.
In the present disclosure, the microorganism belonging to the family Aspergillaceae is preferably a microorganism having an 18S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 16 or SEQ ID NO: 17, and preferably 98% or more. It is more preferably a microorganism having an 18SrRNA gene sequence having identity, and even more preferably a microorganism having an 18SrRNA gene sequence having 100% sequence identity.

In the present disclosure, examples of microorganisms belonging to the family Arthopyreniaceae include, but are not limited to, microorganisms belonging to the genus Arthopyrenia. Examples of microorganisms belonging to the genus Arthopyrenia include Arthopyrenia salicis and the like.
In the present disclosure, the microorganism belonging to the family Arthopyreniaceae is preferably a microorganism having an 18S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 18 or SEQ ID NO: 19, and has a sequence of 98% or more. It is more preferably a microorganism having an 18SrRNA gene sequence having identity, and even more preferably a microorganism having an 18SrRNA gene sequence having 100% sequence identity.

In the present disclosure, at least one microorganism selected from the group consisting of a microorganism belonging to the family Enterobacteriaceae, a microorganism belonging to the family Pseudomonadaceae, and a microorganism belonging to the family Clostridiaceae is, for example, one kind of microorganism belonging to any one of the above families. It may be a plurality of kinds of microorganisms that belong to any one of the above families and may belong to the same genus or different genera from each other, and each family may belong to a plurality of the above families. It may be a plurality of kinds of microorganisms consisting of one kind of microorganism each, and a plurality of kinds of microorganisms (may belong to the same genus or different genus) for each family of the above-mentioned multiple families. It may be a species of microorganism.
In the present disclosure, at least one microorganism selected from the group consisting of a microorganism belonging to the family Enterobacteriaceae, a microorganism belonging to the family Pseudomonadaceae, and a microorganism belonging to the family Clostridiaceae has 95% or more sequence identity with the base sequence shown in SEQ ID NO: 4. Microorganism having 16SrRNA gene sequence having 16SrRNA gene sequence, microorganism having 95% or more sequence identity with the base sequence shown by SEQ ID NO: 5, and 95% or more sequence identity with the base sequence shown by SEQ ID NO: 6. A microorganism having a 16SrRNA gene sequence having 16SrRNA gene sequence, a microorganism having a sequence identity of 95% or more with the base sequence shown by SEQ ID NO: 7, a microorganism having 95% or more sequence identity with the base sequence shown by SEQ ID NO: 8. Microorganism having 16SrRNA gene sequence having 16SrRNA gene sequence, microorganism having 95% or more sequence identity with the base sequence shown by SEQ ID NO: 9, and 95% or more sequence identity with the base sequence shown by SEQ ID NO: 10. A microorganism having a 16SrRNA gene sequence having 16SrRNA gene sequence, a microorganism having a 16SrRNA gene sequence having 95% or more sequence identity with the base sequence shown by SEQ ID NO: 11, and a sequence of 95% or more with each base sequence shown by SEQ ID NO: 12. It is preferable to include 9 or more kinds of microorganisms, including microorganisms having a 16S rRNA gene sequence having the same identity. The ratio of the sequence identity is more preferably 98% or more, further preferably 100%.

In the present disclosure, at least one microorganism selected from the group consisting of microorganisms belonging to the family Methanobacteriaceae and microorganisms belonging to the family Methanosarcinaceae may be, for example, one kind of microorganism belonging to any one of the above families. It may be a plurality of species of microorganisms that belong to any one of the families and may belong to the same genus or different genera from each other. It may be a plurality of kinds of microorganisms, or even a plurality of kinds of microorganisms consisting of a plurality of kinds of microorganisms (may belong to the same genus or different genera) for each family in a plurality of families. good.
In the present disclosure, at least one microorganism selected from the group consisting of microorganisms belonging to the family Methanobacteriaceae and microorganisms belonging to the family Methanosarcinaceae has a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence shown by SEQ ID NO: 13. Microorganism, microorganism having 16SrRNA gene sequence having 95% or more sequence identity with the base sequence shown by SEQ ID NO: 14, and 16SrRNA gene sequence having 95% or more sequence identity with each base sequence shown by SEQ ID NO: 15. It is preferable to contain three or more kinds of microorganisms including microorganisms having. The ratio of the sequence identity is more preferably 98% or more, further preferably 100%.

In the present disclosure, at least one microorganism selected from the group consisting of microorganisms belonging to the family Aspergillaceae and microorganisms belonging to the family Arthopyreniaceae may be, for example, one kind of microorganism belonging to any one of the above families. It may be a plurality of species of microorganisms that belong to any one of the families and may belong to the same genus or different genera from each other. It may be a plurality of kinds of microorganisms, or even a plurality of kinds of microorganisms consisting of a plurality of kinds of microorganisms (may belong to the same genus or different genera) for each family in a plurality of families. good.
In the present disclosure, at least one microorganism selected from the group consisting of microorganisms belonging to the family Aspergillaceae and microorganisms belonging to the family Arthopyreniaceae has an 18S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence shown by SEQ ID NO: 16. Microorganism, a microorganism having an 18SrRNA gene sequence having 95% or more sequence identity with the base sequence shown by SEQ ID NO: 17, and having an 18SrRNA gene sequence having 95% or more sequence identity with the base sequence shown by SEQ ID NO: 18. It is preferable to include four or more kinds of microorganisms, including a microorganism and a microorganism having an 18SrRNA gene sequence having 95% or more sequence identity with each base sequence represented by SEQ ID NO: 19. The ratio of the sequence identity is more preferably 98% or more, further preferably 100%.

The microbial mixture according to the first embodiment of the present disclosure further comprises a microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the base sequence shown by SEQ ID NO: 1 and a base sequence shown by SEQ ID NO: 2. It is selected from the group consisting of a microorganism having a 16SrRNA gene sequence having 95% or more sequence identity and a microorganism having a 16SrRNA gene sequence having 95% or more sequence identity with the nucleotide sequence shown by SEQ ID NO: 3. It can contain at least one microorganism.

The microbial mixture according to the first embodiment of the present disclosure is represented by SEQ ID NO: 2 and one or more microorganisms having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence shown by SEQ ID NO: 1. One or more microorganisms having a 16SrRNA gene sequence having 95% or more sequence identity with the base sequence, and one or more kinds having a 16SrRNA gene sequence having 95% or more sequence identity with the base sequence represented by SEQ ID NO: 3. It is preferable to contain the microorganisms of.

In the present disclosure, the microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 1 is preferably a eubacteria.
In the present disclosure, the microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 2 is preferably archaea.
In the present disclosure, the microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 3 is preferably archaea.

The ratio of the sequence identity is more preferably 98% or more, further preferably 100%.

The base sequences of SEQ ID NOs: 1 to 19 described in the present disclosure are shown below. The nucleotide sequence represented by SEQ ID NO: 1 to SEQ ID NO: 19 is a sequence of a continuous part of the total length of the 16S rRNA gene sequence (or 18S rRNA gene sequence) possessed by the microorganism.

[SEQ ID NO: 1]
5'-CCTTCGGGTGGACAGGGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCATGTGTTGCCAGCGTAGAGGCGGGCACTCACATGAGACTGCCGGAGACAATTCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGGCCTGGGCCACACACGTGCTACAATGGCCGGTACAGAGGGCTGCGAACCCGCGAGGGGGAGCCAATCCCAAAAAGCCGGCCCCAGTTGGGATCGGAGGCTGCAACTCGCCTCCGTGAACGCGGAGTTGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTG-3 '

[SEQ ID NO: 2]
5'-GGGGACCCATGTGCCACTCTTAACGGGGTGGCTTTTTTAGAGTGTAAAAAGCTTTAGGAATAAGAGCTGGGCAAGACCGGTGCCAGCCGCCGCGGTAA-3'

[SEQ ID NO: 3]
5'-GGGAGCCCCCATGTGCCACTCTTAACGGGGTGGCTTTTTTAGAGTAAAAAGCTTTACGAATAAAAGCTGGGCAAGACCGGTGCCAGCCGCCGCGGTAA-3'

[SEQ ID NO: 4]
5'-CCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTTCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGGGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCATATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCACAAAGTATGTCGTATTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATCGCTAGTAATCGTATATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTG-3 '

[SEQ ID NO: 5]
5'-CCTTCGGGAACTGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTATGGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTG-3 '

[SEQ ID NO: 6]
5'-CCTTCGGGACTCATGATACAGGGCGGCAGGGCTGTCGTCAGCCTCGTTGTTTGGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCAAAAGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGAGGGGATGACGTCAAGTCATCATGGCCCTTACGACTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGACAAGCGAGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTTAATCGTAGATCAAAATGCTACGGTGAATACGTTCCCGGGCCTTG-3 '

[SEQ ID NO: 7]
5'-CCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACTAGTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAAATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTG-3 '

[SEQ ID NO: 8]
5'-CCTTCGGGAACTCAGACACAGGTGCTGCATGGCTGTCGTCAGACTAGCAAAGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGCCGCGAGGGGGAGCTAATCTCACAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTG-3 '

[SEQ ID NO: 9]
5'-CCTTCGGGAACTCAGACACAGGTGCTGCATGGCTGTCGTCAGACTAGCAAAGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGCCGCGAGGGGGAGCTAATCTCACAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTG-3 '

[SEQ ID NO: 10]
5'-CCTTCGGGGCAGGAAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCATTAGTTGCTACCATTAAGTTGAGCACTCTAGTGAGACTGCCACGGTTAACGTGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCTAGGGCTACACACGTGCTACAATGGTGAGTACAAAGAGATGCAATACCGTGAGGTGGAGCCAAACTCAAAAACTCATCCCAGTTCGGATTGTAGGCTGAAACTCGCCTACATGAAGCCGGAGTTGCTAGTAATCGCGAATCAGCATGTCGCGGTGAATACGTTCCCGGGCCTTG-3 '

[SEQ ID NO: 11]
5'-CCTTTAGGGCAAGAAGACAGGTGGGGCATGGGTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAATCCCGCAACCAGCGCAACCCTTATCTTTTTTTGCTACCATTAAATTGAGCACTCTATTGAGACTGCCCCGGTTAACGTGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCTAGGGCTACACACGTGCTACAATGGTGAGTACAAAAAGATGCAATACCGCGAGGTGGAGCCAAACTCAAAAACTCATCCCAGTTCGGATTGTAGGCTGAAACTCCCCTACCTGAAGCCGGAGTTGCTAGTAATCGCGAATCAACATGTCGCGGTGAATACCTTCCCGGGCCTTG-3 '

[SEQ ID NO: 12]
5'-CCTTCGGGACAGGAAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATACTTAGTTGCTAGCATTTGGTTGAGCACTCTAAGTAGACTGCCGGAGACAATTCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTTCTGGGCTACACACGTGCTACAATGGGTATAACAACGGGAAGCGAACCAGTGATGGCAAGCAAATCCCTAAAAAATACTCCCAGTTCAGATTGTTCTCTGCAACTCGAGAACATGAAGTCGGAGTTGCTAGTAATCGCGAATCAGCATGTCGCGGTGAATACGTTCCCGGGCCTTG-3 '

[SEQ ID NO: 13]
5'-GGGGACCCCATGTGCCACTCTTAACGGGGTGGCTTTTCTTATGTGTAAAAGCTTTTGGAATAAGAGCTGGGCAAGACCGGTGCCAGCCGCCGCGGTAA-3'

[SEQ ID NO: 14]
5'-GGGGACCCCATGTGCCTCTCTTAACGGGGTGGCTTTTTTTGAGTGTAAAAGCTTTGAGAATAAGAGCTGGGCAAGACCGGTGCCAGCCGCCGCGGTAA-3'

[SEQ ID NO: 15]
5'-GGGGACGACACCGAGTGCCAATCTCATGTGCTGGGTGTCCGGGTGTGTAATATACACCTGTTAGCGGGGCCGGGCAAGACCGGCCAGCCGCCGCGGTAA-3'

[SEQ ID NO: 16]
5'-TCAAAGATTAAGCCATGCATGTCTAAGTATAAGCAATTTGTACTGTGAAACTGCGAATGGCTCATTAAATCAGTTATCGTTTATTTGATAGTACCTTACTACATGGATACCTGGTAATTCTAGAGCTAATACATGCTAAAAAACCTCGACTTCGGAAGGGGTGTATTTATTAG

[SEQ ID NO: 17]
5'-TCAAAGATTAAGCCATGCATGTTTAAGTATAAGCAATTTGTACTGTGAAACTGCGAATGGCTCATTAAATCAGTTATCGTTTATTTGATAGTACCTTACTACATGGATACCTGGTAATTCTAGAGCTAATACATGCTAAAAAACCTCGACTTCGGAAGGGGTGTATTTATTAG

[SEQ ID NO: 18]
5'-TCAAAAATTAACCCACCCATGTTTAAGTATAACCAATTATACCGTGAAAATGAGAATGGCTCATTAAATCAGTTATCGTTTATTTGATAGTACCCTACTACTTGGATACCCGTGGTAATTCTAGAGCTAATACATGCTAAAAAACCAACTTCGGGAGGGGTGTATTTTAGATA

[SEQ ID NO: 19]
5'-TCAAAAATTAACCCAAGCCAGTTTTAGTATTAACCAATTATACTGTGAAACTGCGAATGGCTCATTAAATCAGTTATCGTTTATTTGATAGTACCTTACTACATGGATACCCGTGGTAATTCTAGAGCTAATACATGCTAAAACCCCAACTTCGGGAGGGGTGTATTTTAGATACCG

(Second embodiment)
The microbial mixture according to the second embodiment of the present disclosure comprises at least one microorganism that degrades at least one of cellulose, chitin, and protein, at least one hydrogen-producing microorganism, and at least one methane-producing. Includes microorganisms and at least one oxygen consuming microorganism.

Includes at least one microorganism that degrades at least one of cellulose, chitin, and protein, at least one hydrogen-producing microorganism, at least one methane-producing microorganism, and at least one oxygen-consuming microorganism. By co-culturing the microbial mixture at room temperature, methane can be produced from the composition derived from the organism.

The action of the microbial mixture according to the second embodiment of the present disclosure is not clear, but is presumed as follows.
Of the microbial mixtures according to the second embodiment of the present disclosure, at least one microorganism that decomposes at least one of cellulose, chitin, and protein is a microorganism of cellulose in a biological composition under normal temperature conditions. It decomposes into glucose, chitin into glucosamine, and protein into amino acids at least one of them. Of the microbial mixture according to the second embodiment of the present disclosure, at least one hydrogen-producing microorganism takes up the decomposed glucose, glucosamine, or amino acid and produces hydrogen under normal temperature conditions. Of the microbial mixture according to the second embodiment of the present disclosure, at least one methane-producing microorganism takes in the produced hydrogen and carbon dioxide under normal temperature conditions and produces methane. Of the microbial mixtures according to the second embodiment of the present disclosure, at least one oxygen-consuming microorganism can take up oxygen and proliferate under normal temperature conditions. Since the oxygen-consuming microorganism consumes oxygen in the process of growth, anaerobic activity in the culture tank can be increased. Therefore, the methane-producing microorganism, which is an anaerobic microorganism, can produce methane more efficiently in the culture tank. From the above, when methane is produced using a biological composition as a substrate, methane can be efficiently obtained even at room temperature with a small number of steps by co-culture. When the biological composition contains a persistent substance such as chitin or cellulose, for example, when it is digestive sludge, such an effect is more remarkable, and it is difficult to use it as a substrate in the past. Even the composition of can be used as a substrate to produce methane.
Furthermore, the at least one oxygen consuming microorganism can produce ammonia from nitric acid in a biological composition under normal temperature conditions. The ammonia serves as a nitrogen source for the microbial mixture, and the microorganisms contained in the microbial mixture can exert their functions more efficiently.
The present disclosure is not limited to the above-mentioned presumed action.

In the present disclosure, the at least one microorganism that decomposes at least one of cellulose, chitin, and protein is not particularly limited as long as it is a microorganism that can decompose at least one of cellulose, chitin, and protein. .. For example, cellulose-degrading microorganisms, chitin-degrading microorganisms, and protein-degrading microorganisms have cellulase activity, chitinase activity, or protease activity, respectively, and cellulose to glucose, chitin to glucosamine, or It is preferably a microorganism that decomposes proteins into amino acids. Moreover, one microorganism may have a plurality of these activities. The environmental conditions under which the activity is exhibited are normal temperature and anaerobic conditions from the viewpoint that co-culture can be used without the need for heating during methane production using a biological composition as a substrate. It is preferably below.
The microorganism that decomposes at least one of cellulose, chitin, and protein in the present disclosure is a microorganism generally known in the art as a microorganism that decomposes at least one of cellulose, chitin, and protein. You may use it. It should be noted that what is generally known in the art means that it is known, for example, by a document, a database, a microorganism species name, or the like in the art.

In the present disclosure, the hydrogen-producing microorganism is not particularly limited as long as it is a microorganism capable of producing hydrogen. For example, a hydrogen-producing microorganism preferably has at least one of an activity of producing hydrogen from glucose, an activity of producing hydrogen from glucosamine, or an activity of producing hydrogen from an amino acid. The environmental conditions under which the activity is exhibited are normal temperature and anaerobic conditions from the viewpoint that co-culture can be used without the need for heating during methane production using a biological composition as a substrate. It is preferably below.
As the hydrogen-producing microorganism in the present disclosure, a hydrogen-producing microorganism generally known in the art may be used.

In the present disclosure, the methane-producing microorganism is not particularly limited as long as it is a microorganism capable of producing methane. For example, methane-producing microorganisms preferably have the activity of producing methane from hydrogen. The environmental conditions under which the activity is exhibited are normal temperature and anaerobic conditions from the viewpoint that co-culture can be used without the need for heating during methane production using a biological composition as a substrate. It is preferably below.
As the methane-producing microorganism in the present disclosure, a methane-producing microorganism generally known in the art may be used.

In the present disclosure, the oxygen-consuming microorganism is not particularly limited as long as it is a microorganism capable of consuming oxygen. For example, an oxygen-consuming microorganism preferably has an activity of taking oxygen into the microorganism. Furthermore, it is preferable to have an activity of producing ammonia from nitric acid. The environmental conditions under which the activity is exhibited are normal temperature and anaerobic conditions from the viewpoint that co-culture can be used without the need for heating during methane production using a biological composition as a substrate. It is preferably below.
The oxygen-consuming microorganism in the present disclosure may be an oxygen-consuming microorganism generally known in the art. The oxygen-consuming microorganism may be a so-called aerobic microorganism generally known in the art. Since the oxygen-consuming microorganisms in the present disclosure preferably consume oxygen under anaerobic conditions as described above, oxygen can be consumed even under anaerobic conditions in which the oxygen concentration exists to a certain extent (that is, conditions in which the oxygen concentration is below a certain level). It is preferably a microorganism that can be consumed.

In the present disclosure, the normal temperature is preferably 5 ° C to 40 ° C, more preferably 10 ° C to 35 ° C, further preferably 15 ° C to 35 ° C, and particularly preferably 30 ° C from the viewpoint that the culture tank does not need to be heated. preferable. In the present disclosure, anaerobic preferably refers to a condition in which the oxygen concentration in the gas phase or the oxygen concentration in the liquid phase of the culture tank is 5% or less from the viewpoint that each microorganism in the microbial mixture can easily exert its function. It is more preferable to refer to the condition that the oxygen concentration in the gas phase or the oxygen concentration in the liquid phase of the culture tank is 3% or less.

In the present disclosure, microorganisms include aerobic microorganisms and anaerobic microorganisms. The aerobic microorganism is a microorganism that requires oxygen to grow, and includes a microaerobic microorganism and an obligate aerobic microorganism. Anaerobic microorganisms are microorganisms that do not require oxygen to grow and include facultative anaerobic microorganisms, obligate anaerobic microorganisms, and oxygen resistant microorganisms.

In the present disclosure, the biological composition is not particularly limited as long as it contains cellulose, chitin, or protein. Examples of the biological composition in the present disclosure include biological sludge (for example, sewage sludge), and more specifically, digestive sludge. Digestive sludge is a sludge residue composed of biodegradable components generated when anaerobic microorganisms such as methane-producing microorganisms are decomposed using excess sludge as a substrate. Digestive sludge includes, for example, chitin derived from the cell wall polysaccharide of microorganisms, proteins derived from the composition of microorganisms, keratin derived from human hair, cellulose derived from human urine, and persistent sugars such as xylan derived from plant cell walls. Etc. are included.

<How to obtain microbial mixture>
In the present disclosure, the term "microbial mixture of the present disclosure" refers to the microbial mixture according to the first embodiment of the present disclosure and the microorganism according to the second embodiment of the present disclosure, unless otherwise specified and there is no contradiction. Includes both mixtures. Further, "in the present disclosure" includes both the first embodiment and the second embodiment unless otherwise specified and there is no contradiction.

The microbial mixture of the present disclosure can be obtained by culturing soil and a biological composition at 30 ° C. for 1 month. Examples of biological compositions include digestive sludge. More specifically, it can be obtained, for example, by the method described below. Soil samples can be taken from surface sediments on the banks of the Kawaguchi River in Hachioji, Tokyo, Japan. 1 g of the soil sample is suspended in 20 ml of sterile water using a vortex mixer, and this is used as an inoculum.

Next, the digestive sludge for culturing the inoculum is prepared. Dehydrated digested sludge (17% solids) can be obtained, for example, from a sewage treatment plant in Yokohama, Kanagawa, Japan. Since polyaluminum chloride, which is an inorganic flocculant used for dehydrating sewage sludge, is toxic to microorganisms, polyaluminum chloride in dehydrated digested sludge is repeatedly washed with tap water to remove it. Specifically, 300 g of dehydrated digested sludge and 600 ml of tap water are placed in a 1000 ml beaker and mixed, and the mixture is passed through a three-layer gauze filter to remove polyaluminum chloride contained in the supernatant. This removal operation is repeated 5 times until the pH of the filter eluate becomes 6.0 or higher. The residue remaining on the filter after washing is completely dried in a dryer FSP450 (ADVANTEC, Tokyo, Japan) at 60 ° C. for 48 hours, pulverized into a powder, and passed through a sieve having a diameter of 1 mm. The digested sludge powder that has passed through the sieve is used as the digested sludge (substrate) in the following test. The carbon, nitrogen, and hydrogen contents in the digested sludge sample were analyzed by the elemental analyzer JM-11 (J-Science Labs, Ltd., Kyoto, Japan). 335.1 mg, 58.1 mg of nitrogen, and 53.2 mg of hydrogen.

Then, the inoculum is cultured in a glass vial having a capacity of 17 ml. Specifically, 100 μl of the inoculum is inoculated into a mixture of 10 ml of sterile water and 100 mg of digested sludge powder. Nitrogen gas flows into the 7 ml gas phase at the top of the vial for 1 minute, and the vial is sealed with a butyl rubber stopper and an aluminum cap. By culturing the inoculum at 30 ° C. for 1 month, the microbial mixture of the present disclosure can be obtained.

It should be noted that the method for obtaining the microbial mixture of the present disclosure described above is an example and is not limited thereto.

Although the microbial mixture can be collected from soil at a specific location, it is not possible to isolate and cultivate each microbial mixture, and a survival test method that covers all the microorganisms contained in the mixture has also been established. It is not possible to deposit to a depositary institution under the current deposit system.
Therefore, at the Kogakuin University Hachioji Campus (location: 2665-1 Nakanomachi, Hachioji-shi, Tokyo, telephone number: 042-622-9291), the microbial mixture was referred to as the microbial mixture No. As Riverbank-A30, it is stored in a state where it can be sold, and can be obtained by a third party as needed.

In addition, the microorganism mixture of the present disclosure may be obtained by any method, and for example, each microorganism isolated or purchased is appropriately mixed so as to be the microorganism mixture of the present disclosure. It may be produced by.

<Function of microbial mixture>
The microbial mixture of the present disclosure can produce 10 ml or more of methane by culturing under normal temperature conditions for 1 month in the presence of 1 g of a biological composition as a substrate.

The microbial mixture of the present disclosure preferably produces 10 ml to 500 ml of methane and 15 ml to 400 ml of methane by culturing under normal temperature conditions for 1 month in the presence of 1 g of a biological composition as a substrate. It is more preferable to produce 20 ml to 300 ml of methane.
The details of the biological composition, digestive sludge, substrate, and room temperature are the same as those described in <Microorganisms contained in the above-mentioned microbial mixture>.

Further, the method for confirming the function of the microbial mixture (microorganism contained in the microbial mixture) is, for example, a method for culturing the microbial mixture under the conditions described in the examples and confirming the growth of microorganisms, the production of methane, and the like. There may be. Specifically, the dehydrated digested sludge can be made into digested sludge (substrate) by washing, drying, pulverizing, and sieving. The carbon, nitrogen, and hydrogen content in the digested sludge is from the perspective of containing a substrate for producing methane and from the perspective of containing a nutrient source for the growth of microorganisms contained in the microbial mixture. , 10 mg to 1000 mg of carbon, 10 mg to 100 mg of nitrogen, and 10 mg to 1000 mg of hydrogen are preferable in 1.0 g of digested sludge.

In the culture tank, the mixing ratio when inoculating the solution containing the biological composition with the microbial mixture is not particularly limited, but from the viewpoint of easy growth of microorganisms, digestive sludge is 0 with respect to 1 part by mass of the microbial mixture. .1 part by mass is preferable, and 1 part by mass is more preferable. From the viewpoint of easy growth of microorganisms, it is preferable to add 10 parts by mass to 1000 parts by mass, and more preferably 100 parts by mass.
When inoculating a solution containing a biological composition with a microbial mixture, one type of microorganism contained in the microbial mixture may be inoculated at different timings, or a plurality of types of microorganisms contained in the microbial mixture may be inoculated. It may be inoculated in multiple doses. Alternatively, all the microbial types contained in the microbial mixture may be inoculated only once at the same time, or all the microbial types contained in the microbial mixture may be inoculated multiple times at the same time. From the viewpoint of ease of operation, it is preferable to inoculate all the microbial types contained in the microbial mixture only once at the same time.

The culture tank further contains organic or inorganic substances contained in a commercially available medium, for example, for promoting or suppressing the growth of each microorganism, which is generally used for culturing microorganisms or culturing cells. It may be.

After inoculation, a stable gas other than oxygen, for example, nitrogen gas is allowed to flow into the gas phase of the culture tank to seal the culture tank, and the cells can be cultured at room temperature for 1 month. The culture period may be 10 to 60 days or 20 to 40 days.

As a method for quantifying methane, for example, the method described in Examples can be used. Specifically, it is preferable to quantify gaseous methane, and the gas phase in the culture tank can be sampled and the gas contained in the gas phase can be quantified using a gas chromatograph.

In general, the optimum pH at which a methane-producing microorganism produces methane is 6.8 to 7.6. Therefore, by lowering the pH to lower than 6.8, methane production can be suppressed. Further, in general, hydrogen-producing microorganisms have resistance to low pH, and can produce hydrogen at pH 4.5 or higher.
Therefore, the microbial mixture of the present disclosure can produce 10 ml or more of hydrogen by culturing 1 g of a biological composition as a substrate under normal temperature conditions and at, for example, pH 4.5 to pH 6.8 for 1 month. can. The production of hydrogen is preferably 10 ml to 2000 ml, more preferably 20 ml to 1800 ml, further preferably 40 ml to 1600 ml, and particularly preferably 80 ml to 1200 ml.

≪Composition for methane production≫
The methane-producing composition of the present disclosure preferably contains a microbial mixture and is capable of producing methane from a biologically-derived composition.

The composition for methane production of the present disclosure is not particularly limited as long as it contains a microbial mixture and can produce methane from a composition derived from an organism. The microbial mixture contained in the methane-producing composition of the present disclosure may be a living microorganism or a suspended microorganism. When the microorganism is a fungus, it may be contained as either a hypha or a spore.

In addition to the microbial mixture of the present disclosure, the methane-producing compositions of the present disclosure include solid media (eg, gelatin, lactose) and liquid media (eg, water, saline), lysis aids, stabilizers, etc. It may contain a tensioning agent or the like. The blending amount of these components is not particularly limited as long as the microbial mixture of the present disclosure can act as a composition for methane production.

The shape of the methane-producing composition of the present disclosure is not particularly limited, and may be, for example, powdery, granular, solid, liquid, frozen, or a shape in which they are encapsulated. These shapes are not particularly limited as long as the microbial mixture of the present disclosure can act as a composition for methane production.
The detailed conditions under which methane can be produced are the same as the method for confirming the function of the microbial mixture described in <Function of the microbial mixture>.

≪Methane production method≫
The methane production method of the present disclosure can include inoculating a solution containing a composition derived from an organism with a microbial mixture and culturing the inoculated microbial mixture under normal temperature conditions.

In the present disclosure, the solution containing the biological composition is not particularly limited as long as it contains the biological composition. In addition to the biological composition, the solution containing the biological composition in the present disclosure may contain, for example, water or physiological saline.

In the present disclosure, inoculating a solution containing a biological composition with a microbial mixture is not particularly limited as long as it includes adding the microbial mixture to the biological composition. As for the detailed conditions for inoculating the microbial mixture, the same conditions as in the case of inoculating the microbial mixture described in the method for confirming the function of the microbial mixture in the above <Function of microbial mixture> can be applied.

In the present disclosure, culturing the inoculated microbial mixture under normal temperature conditions is not particularly limited as long as it includes culturing the inoculated microbial mixture under normal temperature conditions. As for the detailed conditions for culturing the inoculated microbial mixture under normal temperature conditions, the same conditions as those for culturing after inoculation of the microbial mixture described in the method for confirming the function of the microbial mixture in the above <Function of microbial mixture> can be applied. ..

The methane production method of the present disclosure can include inoculating a solution containing digestive sludge with a microbial mixture and culturing the inoculated microbial mixture under normal temperature conditions. For details of inoculation and culture, the description of inoculation and culture in the method for confirming the function of the microbial mixture in the above <Function of microbial mixture> can be referred to.

Hereinafter, the present disclosure will be described in more detail with reference to Examples, but the present disclosure is not limited to the following Examples as long as the gist of the present disclosure is not exceeded. Unless otherwise specified, "part" is based on mass. "%" Is also a mass standard.

In the following, molecular biological reagents were purchased from Toyo Spinning Co., Ltd. (Osaka, Japan), Thermo Fisher Scientific (Waltham, Massachusetts, USA), or MP Biomedical (Santa Ana, California, USA). All other chemicals were purchased from Wako Pure Chemical Industries, Ltd. (Kyoto, Japan). The glass and plastic laboratory equipment used for culturing were purchased from Maruemu Corporation (Osaka, Japan) or AS ONE Co., Ltd. (Osaka, Japan).

<< Example 1 >>
(Collection of microbial mixture)
Soil samples were collected from surface sediments on the banks of the Kawaguchi River in Hachioji, Tokyo, Japan. 1 g of the soil sample was suspended in 20 ml of sterile water using a vortex mixer, and this was used as an inoculum.

(Preparation of digestive sludge)
Dehydrated digested sludge (17% solids) was obtained from a sewage treatment plant in Yokohama, Kanagawa, Japan. Since polyaluminum chloride, which is an inorganic flocculant used for dehydrating sewage sludge, is toxic to microorganisms, polyaluminum chloride in the dehydrated digested sludge was repeatedly washed with tap water to remove it. Specifically, 300 g of dehydrated digested sludge and 600 ml of tap water were placed in a 1000 ml beaker and mixed, and the mixture was passed through a three-layer gauze filter to remove polyaluminum chloride contained in the supernatant. .. This removal operation was repeated 5 times until the pH of the filter eluate was 6.0 or higher. The residue remaining on the filter after washing was completely dried in a dryer FSP450 (ADVANTEC, Tokyo, Japan) at 60 ° C. for 48 hours, pulverized into a powder, and passed through a sieve having a diameter of 1 mm. The digested sludge powder passed through the sieve was used as the digested sludge (substrate) in the following test. The carbon, nitrogen, and hydrogen contents in the digested sludge sample were analyzed by the elemental analyzer JM-11 (J-Science Labs, Ltd., Kyoto, Japan). It was 335.1 mg, 58.1 mg of nitrogen, and 53.2 mg of hydrogen.

(Culturing inoculum)
The inoculum was cultured in a 17 ml volume glass vial. Specifically, 100 μl of the inoculum was inoculated into a mixture of 10 ml of sterile water and 100 mg of digested sludge powder. Nitrogen gas was flowed into the 7 ml gas phase at the top of the vial for 1 minute, and the vial was sealed with a butyl rubber stopper and an aluminum cap. The inoculum was cultured at 30 ° C, 40 ° C, or 50 ° C for 1 month. The inoculum cultured at 30 ° C., 40 ° C., or 50 ° C. for 1 month was used as a microbial mixture below.

(Quantitative hydrogen production and methane production)
The gas phase at the top of the vial was sampled from the microbial mixture cultured at 30 ° C., 40 ° C., or 50 ° C. for 1 month, and hydrogen and methane were quantified.
For the quantification of hydrogen and methane, a gas chromatograph GC-8A (Shimadzu Corporation, Kyoto, Japan) equipped with a thermal conductivity type detector was used, and the column was a Shincarbon ST column 50/80 (2.0 m × 3. Using 0 mm inner diameter, Shinwa Kako Co., Ltd., Kyoto, Japan), the injection amount was 0.5 ml, argon (43.5 ml / min) was used as the carrier gas, and the column temperature was 80 ° C. Hydrogen and methane specimens were purchased from GL Sciences Co., Ltd. (Tokyo, Japan).
The results are shown in FIG. In the figure, the vertical axis shows the amount of methane produced per 1 g of digested sludge (ml), and the horizontal axis shows the number of passages (times). The microbial mixture of the present disclosure produced 20 ml or more of methane per 1 g of digested sludge at a culture temperature of 30 ° C. and 1 to 8 passages. When subcultured four times at a culture temperature of 30 ° C., 20.79 ml of methane was produced per 1 g of digested sludge. At a culture temperature of 40 ° C., about 2.5 ml or more of methane was produced per 1 g of digested sludge regardless of the number of passages. At a culture temperature of 50 ° C., methane production could not be confirmed regardless of the number of passages. From the above, it was shown that the microbial mixture of the present disclosure enables highly efficient methane production, and that the microbial mixture can be stably maintained even after passage by setting the culture temperature appropriately. .. In FIG. 1, one passage means that the digested sludge and the above-mentioned inoculum were cultured for one month. In FIG. 1, the term “2 passages” means that the digested sludge and the one obtained by the 1st passage were cultured for 1 month. The number of passages of 3 times means that the digested sludge and the one obtained by the number of passages of 2 times were cultured for 1 month. The same applies to the number of passages after 4 times.

(Analysis of microbial mixture by PCR-DGGE method)
For the microbial mixture cultured at 30 ° C., 40 ° C., or 50 ° C. for 1 month, PCR-modifier concentration gradient gel electrophoresis (PCR-DGGE method) was carried out as follows.

-PCR-
The cells of the microbial mixture are collected by centrifuging 1 ml of the culture solution of the microbial mixture cultured at 30 ° C., 40 ° C., or 50 ° C. for 1 month under the conditions of 20,000 × g, 10 minutes, and 4 ° C. did. Genomic DNA was extracted from the cells using the Fast DNA SPIN kit for soil samples (MP Biomedical).

For eubacteria, the 16S rRNA gene corresponding to the V6 to V8 regions was amplified by PCR. Heuer et al. (1997) Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel electrophoretic separation in denaturing gradients. Appl Environ Microbiol 63: 3233-3241. ) And R1378 (SEQ ID NO: 21), and KOD Fx Neopolymerase (Toyo Spinning Co., Ltd.) was used as the polymerase. The PCR conditions were 94 ° C. for 2 minutes for 1 cycle, 94 ° C. for 15 seconds, 50 ° C. for 30 seconds, and 68 ° C. for 30 seconds for 34 cycles. Of the following base sequences described together with each SEQ ID NO, the base sequence enclosed in parentheses [] indicates that it is the base sequence of the GC clamp. By the above operation, an amplicon derived from eubacteria was obtained.

[SEQ ID NO: 20] 5'-[CGCCCGGGGCGCGCCCCGGGCGGGGCGGGGGCACGGGGGG] AACGCGAAGAACCTTAC-3'
[SEQ ID NO: 21] 5'-CGGTGTGTACAAGGCCCGGGAACG-3'

For archaea, including methanogens, Zhou et al. (2010) characterization of variation in rumen methanogenic communities under different dietary and host feed efficiency conditions, as determined by PCR-Denaturing gradient gel electrophoresis analysis. Appl Environ Microbiol 76: 3776 As described in -3786., About 800 bp of the 16S rRNA gene containing the V3 region was amplified by PCR using archaeal primers Met86f (SEQ ID NO: 22) and Met915r (SEQ ID NO: 23). Then, the V3 region was amplified by PCR using the amplified DNA as a template and further using primers GC-ARC344f (SEQ ID NO: 24) and 519r (SEQ ID NO: 25) used for archaea including methane-producing bacteria. .. The PCR conditions were 95 ° C. for 5 minutes for one cycle, 95 ° C. for 30 seconds, 56.5 ° C. for 30 seconds, and 68 ° C. for 30 seconds for 30 cycles. Of the following base sequences described together with the SEQ ID NO:, the base sequence enclosed in parentheses [] indicates that it is the base sequence of the GC clamp. By the above operation, an amplicon derived from archaea including methane-producing bacteria was obtained.

[SEQ ID NO: 22] 5'-GCTCAGTAACACGTGG-3'
[SEQ ID NO: 23] 5'-GTGCTCCCCCGCCAATTCCT-3'
[SEQ ID NO: 24] 5'-[CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGG] ACGGGGYGCAGCAGGCGCGA-3'
[SEQ ID NO: 25] 5'-GWATTACCGCGGCKGCTG-3'

In the base sequence of the above-mentioned primer, Y represents C or T, W represents A or T, and K represents G or T.

For fungi or protists, a portion of the 18S rRNA gene, as described in May et al. (2001) Comparative denaturing gradient gel electrophoresis analysis of fungal communities associated with whole plant corn silage. Can J Microbiol 47: 829-841. Was amplified by PCR using primers NS1 (SEQ ID NO: 26) and GCFung (SEQ ID NO: 27). The PCR conditions were 94 ° C. for 2 minutes for 1 cycle, 94 ° C. for 15 seconds, 50 ° C. for 30 seconds, and 68 ° C. for 30 seconds for 34 cycles. Of the following base sequences described together with the SEQ ID NO:, the base sequence enclosed in parentheses [] indicates that it is the base sequence of the GC clamp. By the above operation, an amplicon derived from a fungus or a protist was obtained.

[SEQ ID NO: 26] 5'-GTAGTCATATGCTTGTCTC-3'
[SEQ ID NO: 27] 5'-[CGCCCGCCGCGCCCCGCGCCCGCCGCCGCCCCCGCCCC] ATTCCCCGTTACCCGTTG-3'

-DGGE method-
In the DGGE method, the amplicon obtained by the above PCR was carried out using the NB-1480A DGGE system (Nippon Aido Co., Ltd., Tokyo, Japan) according to the operation manual. About 1 μg of the amplified amplicon was loaded into the wells of each of the following gels. For eubacterial-derived amplicon, a 6% polyacrylamide gel with a linear denaturant concentration gradient of 50% to 70% was used. For archaeal-derived amplicon, a 6% polyacrylamide gel with a linear denaturant concentration gradient of 35% to 45% was used. For fungal or protist-derived amplicon, a 7% polyacrylamide gel with a linear denaturant concentration gradient of 20% to 45% was used. In order to estimate the molecular weight of the amplicon, a molecular weight marker was also loaded in each gel well.
Electrophoresis was carried out at a constant voltage of 50 V at 58 ° C. for 18 hours for eubacterial amplicon and at 60 ° C. for 20 hours for archaeal, fungal and protist amplicon. The gel after electrophoresis was stained with 10 μl of SYBR Green (Life Technologies, Inc.) dissolved in 100 ml of TAE buffer.
The results are shown in FIG. When the microbial mixture is cultured at 30 ° C., eubacteria-derived amplicon C1 (including SEQ ID NO: 10), C2 (including SEQ ID NO: 11), Ue1 (including SEQ ID NO: 1), E2 (containing SEQ ID NO: 4). ), C4 (including SEQ ID NO: 12), E7 (including SEQ ID NO: 5), P1 (including SEQ ID NO: 8), P2 (including SEQ ID NO: 9), E8 (including SEQ ID NO: 6), and E9 (including SEQ ID NO: 6). Amplicon Mb1 (including SEQ ID NO: 13), Ua2 (including SEQ ID NO: 2), Ua3 (including SEQ ID NO: 3), Mb5 (including SEQ ID NO: 14), derived from paleontology (including SEQ ID NO: 7). And Ms1 (including SEQ ID NO: 15) and amplicon R1 (including SEQ ID NO: 18), R2 (including SEQ ID NO: 19), A4 (including SEQ ID NO: 16), and A5 (including SEQ ID NO: 16) derived from fungi or protozoa. (Including number 17) was confirmed. When the microbial mixture was cultured at 40 ° C., eubacterial amplicon C2, Ue1, E2, C4, E7, P2, and E8 and archaeal amplicon Mb1, Mb2, Ua1, Ua2, Ua3, Mb4, Mb5 and Ua4 and amplicon R1, R2, A3, D1, AC1 and AC2 derived from fungi or protists were identified. When the microbial mixture is cultured at 50 ° C., eubacterial amplicon C2, Ue1, E2, C4, E7, P2, and E8, archaeal amplicon Mb1, Mb2, and Ua2, and fungi or protists. The derived amplicon R1, R2, A3, D1, AC1 and AC2 were confirmed. The number of confirmed amplicon corresponds to the number of existing microbial species, and the migration stop position indicates the GC base content peculiar to the microbial species. Further, the amplicon is amplified with a primer containing a GC clamp rich in GC base content for analysis by the PCR-DGGE method, and the base sequence of the amplicon contains the base sequence of the GC clamp. Therefore, in the above, "amplicon X (including SEQ ID NO: Y)" means that the base sequence of amplicon X includes the base sequence represented by SEQ ID NO: Y and the base sequence of the GC clamp.
The microbial mixture of the present disclosure was found to contain at least 10 eubacteria, 5 archaea, and 4 fungi or protists when subcultured at 30 ° C.

-PCR-Sequencing analysis of amplicon obtained by DGGE method-
The gel obtained by the DGGE method was irradiated with light of 470 nm, and the site containing each amplicon when cultured at 30 ° C. for 1 month was cut out from the gel. The excised site was immersed in 20 μl of TE buffer (pH 8.0) at 4 ° C. for 72 hours, and each amplicon was extracted. Next, PCR was performed again using the extracted 1 μl amplicon as a template and a primer containing no GC clamp. The PCR conditions are the same as the PCR conditions in the above-mentioned DGGE method except for the primers. The PCR product was purified using the GeneJET PCR purification kit (Thermo Fisher Scientific) and sequence analysis was performed using BigDye Terminator v3.1 (Thermo Fisher Scientific). The results of the sequence analysis were compared to the sequences of known microbial species registered in the GenBank, EMBL, and DDBJ databases using the BLAST algorithm. Using the maximum likelihood estimation of the MEGA6 program, a phylogenetic tree was created based on the 16S rRNA gene for eubacteria and archaea, and a phylogenetic tree was created based on the 18S rRNA gene for fungi or protists.
FIG. 3 shows a phylogenetic tree showing the taxonomic position (genus species) of amplicon derived from eubacteria. Amplicons C1, C2, and C4 were found to be from the Clostridiaceae family. Amplicons E2, E7, E8, and E9 were found to be from the family Enterobacteriaceae. Amplicons P1 and P2 were found to be derived from the family Pseudomonadaceae. Amplicon Ue1 was found to be derived from unclassified eubacteria.
A phylogenetic tree showing the taxonomic position (genus species) of archaeal-derived amplicon is shown in FIG. Amplicon Ms1 was found to be derived from the family Methanos arcinaceae. Amplicons Mb1 and Mb5 were found to be derived from the family Methanobacteriaceae. Amplicons Ua2 and Ua3 were found to be derived from unclassified archaea.
A phylogenetic tree showing the taxonomic position (genus species) of amplicon derived from a fungus or a protist is shown in FIG. Amplicons A4 and A5 were found to be derived from the Aspergillaceae family. Amplicons R1 and R2 were found to be derived from the Arthopyreniaceae family.

Since Enterobacter asburiae can degrade proteins, eubacteria from which amplicon E8 and E9 are derived can degrade proteins in digestive sludge. Since Citrobacter freundii has cellulase, chitinase, and protease, and Cronobacter sakazakii has chitinase and protease, eubacteria from which amplicons E2 and E7 are derived can hydrolyze carbohydrates and proteins in digestive sludge. .. Since Clostridium amylolyticum and Clostridium punense can produce hydrogen, eubacteria from which amplicon C1, C2, and C4 are derived can produce hydrogen.
Since the families Methanobacteriaceae and Methanosarcinaceae can produce methane, archaea from which amplicon Ms1, Mb1 and Mb5 are derived can produce methane.
Since Penicillium expansum and Monascus purpureus have cellulase, chitinase, and protease, it is considered that the species of the Aspergillaceae family from which amplicons A4 and A5 are derived have cellulase, chitinase, and protease. On the other hand, since the Aspergillaceae family is generally not suitable under anaerobic conditions, the species of the Aspergillaceae family from which amplicon A4 and A5 are derived consume oxygen in the culture medium containing digestive sludge and are anaerobic microorganisms. It is related to other eubacteria and archaea so that they can work efficiently.
As described above, FIG. 6 shows the mechanism by which the microbial mixture in Example 1 produces methane from digested sludge.

(Measurement of enzyme activity)
The microbial mixture cultured at 30 ° C. for 1 month was used for measuring the enzyme activity. 100 μl of the microbial mixture cultured at 30 ° C. for 1 month was inoculated into a new vial containing 10 ml of sterile water and 100 mg of digested sludge powder and subcultured once. 2 ml of the subcultured microbial mixture was centrifuged at 20,000 × g for 10 minutes at 4 ° C., and the supernatant was used for evaluation of enzyme activity. Palmisano et al. (1993) Hydrolytic enzyme activity in landfilled refuse. Appl Microbiol Biotechnol 38: 828-832. and Ramirez et al. (2004) Colloidal chitin stained with Remazol Brilliant Blue R, a useful substrate to select chitinolytic identifiers and to evaluate Using the method described in chitinases. J Microbiol Methods 56: 213-219., Cellulase and chitinase were quantified using cellulose azul and chitin azul purchased from Sigma Aldrich (St. Louis, Missouri, USA). Specifically, 0.2 ml of the supernatant, 0.8 ml of 50 mM citric acid buffer (pH 5.0), and 5 mg of cellulose azul or chitin azul are mixed in a centrifuge tube at 30 ° C. for 1 hour. It was cultured. After culturing, the centrifuge tube was centrifuged at 20,000 × g for 10 minutes at 4 ° C. to collect the supernatant, and the absorbance at 540 nm was measured. In addition, 1 unit of cellulase and chitinase was defined as the amount of enzyme whose absorbance at 540 nm increased by 0.01. Protease activity was quantified using the Pierce Protease Assay Kit (Thermo Fisher Scientific) in terms of activity equivalent to 1 μg trypsin.
The results are shown in FIG. In the figure, the vertical axis shows the enzyme activity (Unit / ml / hour) when the enzyme activity at 540 nm, which increases the absorbance by 0.01, is set to 1 for the measurement of cellulase activity and the measurement of trypsin. Regarding the measurement of the activity, the enzyme activity (trypsin equivalent / ml / hour) is shown when the activity equivalent to 1 μg of trypsin is set to 1. The bar graph in the figure shows the average value, and the error bar shows the standard deviation. The microbial mixture of the present disclosure has a cellulase activity of about 0.18 Unit / ml / hour, a chitinase activity of about 0.056 Unit / ml / hour, and about 0.33 μg trypsin at a culture temperature of 30 ° C. It had considerable / ml / hour protease activity.

From the above, in Example 1, a microbial mixture capable of producing methane from a biological composition, a composition for methane production, and methane production by co-culturing a plurality of specific types of specific microorganisms at room temperature. I was able to provide a method.

The microbial mixture of the present disclosure can generate hydrogen or methane by inoculating a solution containing digestive sludge and culturing at room temperature, which can be used as a fuel. Further, the microbial mixture of the present disclosure can reduce the volume of digestive sludge and can reduce the amount of industrial waste.

Claims (7)

At least one microorganism selected from the group consisting of microorganisms belonging to the family Enterobacteriaceae, microorganisms belonging to the family Pseudomonadaceae, and microorganisms belonging to the family Clostridiaceae.
At least one microorganism selected from the group consisting of microorganisms belonging to the family Methanobacteriaceae and microorganisms belonging to the family Methanosarcinaceae, and
A microbial mixture comprising at least one microorganism selected from the group consisting of microorganisms belonging to the family Aspergillaceae and microorganisms belonging to the family Arthopyreniaceae. Moreover,
A microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 1.
A microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 2.
A microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 3
The microbial mixture according to claim 1, comprising at least one microbial mixture selected from the group consisting of. With at least one microorganism that degrades at least one of cellulose, chitin, and protein,
With at least one hydrogen-producing microorganism,
With at least one methane-producing microorganism
A microbial mixture comprising at least one oxygen consuming microorganism. The invention according to any one of claims 1 to 3, wherein 10 ml or more of methane can be produced by culturing under normal temperature conditions for 1 month in the presence of 1 g of a biological composition as a substrate. Microbial mixture. A composition for producing methane, which comprises the microbial mixture according to any one of claims 1 to 4, and which can produce methane from a composition derived from an organism. Inoculating a solution containing a composition derived from an organism with the microbial mixture according to any one of claims 1 to 4.
By culturing the inoculated microbial mixture under normal temperature conditions,
A method for producing methane, including. The methane production method according to claim 6, wherein the biological composition is digestive sludge.

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