Microbial mixture, composition for methane production, and methane production method

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

Technical Field

[0001] The present disclosure relates to a microbial mixture, a composition for methane production, and a method for methane production.

Background Art

[0002] In general, the activated sludge process is used to treat sewage from households and industries. The activated sludge process is a method of decomposing soluble substances and residual suspended substances contained in sewage by adding aerobic microorganisms to the sewage and performing aeration. In this decomposition process, aerobic microorganisms grow 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 substances (i.e., aerobic microorganisms) in the sewage is controlled within a certain range, it is necessary to discharge the overgrown aerobic microorganisms out of the system as excess sludge. Sewage treatment plants operate day and night throughout the year, that is, excess sludge is constantly generated nationwide. Therefore, securing a disposal site for excess sludge and increasing disposal costs have become problems. In some sewage treatment plants, as a method of effectively using excess sludge, anaerobic digestion (so-called methane fermentation) by microorganisms that produce methane is used to produce biogas containing about 60% methane from excess sludge, and further volume reduction of excess sludge is performed. However, even then, the volume reduction rate of excess sludge is about 30%, and the remaining about 70% of excess sludge remains as "digested sludge" that is difficult to reuse further. This digested sludge currently accounts for about 40% of industrial waste in Japan.

[0003] For example, Non-Patent Document 1 discloses filamentous fungi that can hydrolyze digested sludge under aerobic conditions at 30°C. It is described that filamentous fungi of the genus Umbelopsis, Penicillium, Cunninghamella, Neosartorya, Fusarium, or Chaetomium have xylanase, chitinase, or keratinase activity.

[0004] Non-Patent Document 2 describes that when performing methane fermentation by methane-producing bacteria under sealed conditions at 50°C, hydrolases obtained from bacteria such as the genus Penicillium, the genus Cunninghamella, the genus Neosartorya, or the genus Fusarium are added.

[0005] Patent Document 1 discloses filamentous fungi that can hydrolyze digested sludge under aerobic conditions at 30°C. It is described that filamentous fungi of the genus Penicillium, the genus Cunninghamella, the genus Neosartorya, or the genus Umbelopsis have xylanase, chitinase, or keratinase activity. Furthermore, it is described that by inoculating and culturing the filamentous fungi in the digested sludge, the solid weight of the digested sludge decreased by about 10% under aerobic conditions at 30°C.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0008] However, although the filamentous fungi described in Non-Patent Document 1 and Patent Document 1 can hydrolyze digestion sludge, which is an example of a biologically-derived composition, under aerobic conditions, if methane is to be further obtained from the hydrolyzed digestion sludge, since methanogenic bacteria are anaerobic microorganisms, it is not possible to co-culture the disclosed filamentous fungi and methanogenic bacteria. Furthermore, Non-Patent Document 2 also does not describe co-culturing of filamentous fungi and methanogenic bacteria, and in Non-Patent Document 2, it is necessary to pre-treat the digestion sludge used as a substrate with acid, and there are costs associated with continuously heating the culture tank at 50°C. Therefore, none of Patent Document 1, Non-Patent Document 1, and Non-Patent Document 2 describe a microbial mixture capable of methane production from a biologically-derived composition by co-culturing multiple types of microorganisms including degradable microorganisms and methanogenic microorganisms at normal temperature.

[0009] The present disclosure has been made in view of the above, and an object of the present disclosure is to provide a microbial mixture capable of methane production from a biologically-derived composition by co-culturing multiple types of specific microorganisms at normal temperature.

Means for Solving the Problems

[0010] Specific means for solving the problems 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 a microorganism belonging to the family Methanobacteriaceae and a microorganism belonging to the family Methanosarcinaceae, and at least one microorganism selected from the group consisting of a microorganism belonging to the family Aspergillaceae and a microorganism belonging to the family Arthopyreniaceae, a microbial mixture. <2> Further, a microorganism having a 16S rRNA gene sequence having a sequence identity of 95% or more with the nucleotide sequence represented by SEQ ID NO: 1, A microorganism having a 16S rRNA gene sequence with 95% or more sequence identity to the nucleotide sequence shown by SEQ ID NO: 2, a microorganism having a 16S rRNA gene sequence with 95% or more sequence identity to the nucleotide sequence shown by SEQ ID NO: 3, The microbial mixture according to <1> above, comprising at least one microorganism selected from the group consisting of. <3> At least one microorganism that decomposes 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, a microbial mixture. <4> The microbial mixture according to any one of <1> to <3> above, which can produce 10 ml or more of methane by culturing for 1 month under normal temperature conditions in the presence of 1 g of a biologically-derived composition as a substrate. <5> A composition for methane production, comprising the microbial mixture according to any one of <1> to <4> above and capable of producing methane from a biologically-derived composition. <6> Inoculating a solution containing a biologically-derived composition with the microbial mixture according to any one of <1> to <4> above, culturing the inoculated microbial mixture under normal temperature conditions, A method for producing methane, comprising. <7> The method for producing methane according to <6> above, wherein the biologically-derived composition is digested sludge.

Advantages of the Invention

[0011] According to the present disclosure, it is possible to provide a microbial mixture capable of producing methane from a biologically-derived composition by co-culturing a plurality of specific microorganisms at normal temperature.

Brief Description of the Drawings

[0012]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Mode for Carrying Out the Invention

[0013] 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, its components (including element steps, etc.) are not essential unless otherwise specified. The same applies to numerical values and their ranges, which do not limit the present disclosure. In the present disclosure, the term "step" includes not only a step independent of other steps but also the step even if it cannot be clearly distinguished from other steps as long as the purpose of the step is achieved. In the present disclosure, in the numerical range indicated using "~", the numerical values described before and after "~" are included as the minimum value and the maximum value, respectively. In the numerical ranges 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 of other stepwise descriptions. 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 examples. In the present disclosure, the content rate of each component in the composition means the total content rate of the plurality of substances corresponding to each component in the composition, unless otherwise specified, when there are a plurality of substances corresponding to each component in the composition.

[0014] ≪Microbial mixture≫ In the present disclosure, the microbial mixture is preferably a mixture of two or more types of microorganisms. For example, it may be a mixture of 3 to 10 types, 11 to 20 types, or more types of microorganisms. The presence ratio of each microorganism in the microbial mixture of the present disclosure is not particularly limited.

[0015] <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 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 at least one microorganism selected from the group consisting of microorganisms belonging to the family Aspergillaceae and microorganisms belonging to the family Arthopyreniaceae.

[0016] A microbial mixture 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, and at least one microorganism selected from the group consisting of microorganisms belonging to the family Aspergillaceae and microorganisms belonging to the family Arthopyreniaceae is capable of producing methane from a biologically-derived composition by culturing at normal temperature (co-culturing the microorganisms contained in the microbial mixture).

[0017] Although the action of the microbial mixture according to the first embodiment of the present disclosure is not clear, it is presumed as follows. Among the microorganisms of the microbial mixture according to the first embodiment of the present disclosure, 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 performs at least one of the decomposition of cellulose to glucose, the decomposition of chitin to glucosamine, and the decomposition of protein to amino acids in a biologically-derived composition under normal temperature conditions, and at least one microorganism selected from the group takes up the decomposed glucose, glucosamine, or amino acid and produces hydrogen under normal temperature conditions. Among the microorganisms of the microbial mixture 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 takes up the produced hydrogen and carbon dioxide and produces methane under normal temperature conditions. 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 can take in oxygen and grow under normal temperature conditions. That is, since at least one microorganism selected from the group consisting of microorganisms belonging to the family Aspergillaceae and microorganisms belonging to the family Arthopyreniaceae consumes oxygen during the growth process, the anaerobicity in the culture tank can be increased. Therefore, at least one microorganism selected from the group consisting of microorganisms belonging to the family Methanobacteriaceae and microorganisms belonging to the family Methanosarcinaceae, which are anaerobic microorganisms, can produce methane more efficiently in the culture tank. From the above, when producing methane using a bio-derived composition as a substrate, methane can be efficiently obtained at normal temperature with a small number of steps by co-culturing. When the bio-derived composition contains a hardly decomposable substance such as chitin or cellulose, for example, when it is digested sludge, such an effect is more remarkable, and even a hardly decomposable composition that has been difficult to use as a substrate can be used as a substrate for methane production. Furthermore, at least one microorganism selected from the group consisting of microorganisms belonging to the family Aspergillaceae and microorganisms belonging to the family Arthopyreniaceae can produce ammonia from nitric acid in the bio-derived 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 function more efficiently. It should be noted that the present disclosure is not limited to the above presumed actions at all.

[0018] In the present disclosure, examples of microorganisms belonging to the family Enterobacteriaceae include, but are not limited to, 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. 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 or Enterobacter tabaci and the like. 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 preferably has a 16S rRNA gene sequence having a sequence identity of 95% or more with the nucleotide sequence represented by SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, more preferably has a 16S rRNA gene sequence having a sequence identity of 98% or more, and even more preferably has a 16S rRNA gene sequence having a sequence identity of 100%. In the present disclosure, the sequence identity can be determined by using the Basic Local Alignment Search Tool (BLAST, National Center for Biotechnology Information (NCBI)) with default parameters.

[0019] In the present disclosure, for example, a microorganism having a 16S rRNA gene sequence (or an 18S rRNA gene sequence) having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: N means that, among the entire length of the 16S rRNA gene sequence (or the 18S rRNA gene sequence) possessed by the microorganism, a continuous partial sequence has 95% or more sequence identity with the entire length of the DNA nucleotide sequence represented by SEQ ID NO: N.

[0020] 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 preferably has a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 8 or SEQ ID NO: 9, more preferably has a 16S rRNA gene sequence having 98% or more sequence identity, and even more preferably has a 16S rRNA gene sequence having 100% sequence identity.

[0021] 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, or microorganisms belonging to the genus Lutispora. Examples of microorganisms belonging to the genus Clostridium include Clostridium amylolyticum or Clostridium punense and the like. 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 preferably has a 16S rRNA gene sequence having a sequence identity of 95% or more with the nucleotide sequence represented by SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, more preferably has a 16S rRNA gene sequence having a sequence identity of 98% or more, and even more preferably has a 16S rRNA gene sequence having a 100% sequence identity.

[0022] In the present disclosure, examples of the microorganism belonging to the family Methanobacteriaceae include, but are not limited to, microorganisms belonging to the genus Methanobacterium. Examples of the microorganism belonging to the genus Methanobacterium include Methanobacterium espanolae, Methanobacterium subterraneum, or Methanobacterium flexile. In the present disclosure, the microorganism belonging to the family Methanobacteriaceae preferably has a 16S rRNA gene sequence having a sequence identity of 95% or more with the nucleotide sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14, more preferably has a 16S rRNA gene sequence having a sequence identity of 98% or more, and even more preferably has a 16S rRNA gene sequence having a 100% sequence identity.

[0023] In the present disclosure, examples of the microorganism belonging to the family Methanosarcinaceae include, but are not limited to, microorganisms belonging to the genus Methanosarcina. Examples of the microorganism belonging to the genus Methanosarcina include Methanosarcina spelaei, Methanosarcina horonobensis, Methanosarcina acetivorans. In the present disclosure, the microorganism belonging to the family Methanosarcinaceae preferably has a 16S rRNA gene sequence having a sequence identity of 95% or more with the nucleotide sequence represented by SEQ ID NO: 15, more preferably has a 16S rRNA gene sequence having a sequence identity of 98% or more, and even more preferably has a 16S rRNA gene sequence having a 100% sequence identity.

[0024] In the present disclosure, examples of the microorganism belonging to the family Aspergillaceae include, but are not limited to, microorganisms belonging to the genus Aspergillus, microorganisms belonging to the genus Penicillium, or microorganisms belonging to the genus Monascus. Examples of the microorganism belonging to the genus Aspergillus include Aspergillus penicillioides and the like. Examples of the microorganism belonging to the genus Penicillium include Penicillium expansum and the like. Examples of the microorganism belonging to the genus Monascus include Monascus purpureus and the like. In the present disclosure, the microorganism belonging to the family Aspergillaceae preferably has an 18S rRNA gene sequence having a sequence identity of 95% or more with the nucleotide sequence represented by SEQ ID NO: 16 or SEQ ID NO: 17, more preferably has an 18S rRNA gene sequence having a sequence identity of 98% or more, and even more preferably has an 18S rRNA gene sequence having a 100% sequence identity.

[0025] In the present disclosure, examples of the microorganism belonging to the family Arthopyreniaceae include, but are not limited to, microorganisms belonging to the genus Arthopyrenia. Examples of the microorganism 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 a sequence identity of 95% or more with the nucleotide sequence represented by SEQ ID NO: 18 or SEQ ID NO: 19, more preferably a microorganism having an 18S rRNA gene sequence having a sequence identity of 98% or more, and even more preferably a microorganism having an 18S rRNA gene sequence having 100% sequence identity.

[0026] 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 may be, for example, one species of microorganism belonging to any one of the above families, a plurality of species of microorganisms belonging to any one of the above families and belonging to the same genus or different genera from each other, a plurality of species of microorganisms each consisting of one species of microorganism for each of a plurality of the above families, or a plurality of species of microorganisms each consisting of a plurality of species of microorganisms (which may belong to the same genus or different genera) for each of a plurality of the above families. In the present disclosure, 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 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, a microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 5, a microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 6, a microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 7, a microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 8, a microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 9, a microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 10, a microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 11, and a microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with each nucleotide sequence represented by SEQ ID NO: 12. It is preferable to include nine or more types of microorganisms. The ratio of the sequence identity is more preferably 98% or more, and further preferably 100%.

[0027] 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 species of microorganism belonging to any one of the above families, a plurality of species of microorganisms belonging to any one of the above families, which may belong to the same genus or different genera, a plurality of species of microorganisms each consisting of one species of microorganism for each of a plurality of the above families, or a plurality of species of microorganisms each consisting of a plurality of species of microorganisms (which may belong to the same genus or different genera) for each of a plurality of the above families. 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 preferably includes three or more types of microorganisms, including a microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 13, a microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 14, and a microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with each nucleotide sequence represented by SEQ ID NO: 15. The ratio of the sequence identity is more preferably 98% or more, and even more preferably 100%.

[0028] 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 type of microorganism belonging to any one of the above families, a plurality of types of microorganisms belonging to any one of the above families and belonging to the same genus or different genera from each other, a plurality of types of microorganisms each consisting of one type of microorganism for each of a plurality of the above families, or a plurality of types of microorganisms each consisting of a plurality of types of microorganisms (which may belong to the same genus or different genera) for each of a plurality of the above families. 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 preferably includes four or more types of microorganisms, including a microorganism having an 18S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 16, a microorganism having an 18S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 17, a microorganism having an 18S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 18, and a microorganism having an 18S rRNA gene sequence having 95% or more sequence identity with each nucleotide sequence represented by SEQ ID NO: 19. The ratio of the sequence identity is more preferably 98% or more, and even more preferably 100%.

[0029] The microbial mixture according to the first embodiment of the present disclosure may further contain at least one microorganism selected from the group consisting of a microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 1, a microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 2, and a microorganism having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 3.

[0030] The microbial mixture according to the first embodiment of the present disclosure preferably contains one or more microorganisms having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence represented by SEQ ID NO: 1, one or more microorganisms having a 16S rRNA gene sequence having 95% or more sequence identity with the nucleotide sequence 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 represented by SEQ ID NO: 3.

[0031] 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 eubacterium. 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: 2 is preferably an archaeon. 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: 3 is preferably an archaeon.

[0032] The ratio of the sequence identity is more preferably 98% or more, and even more preferably 100%.

[0033] The nucleotide sequences of SEQ ID NOs: 1 to 19 described in the present disclosure are shown below. The nucleotide sequences represented by SEQ ID NOs: 1 to 19 are partial consecutive sequences of the full length of the 16S rRNA gene sequence (or 18S rRNA gene sequence) possessed by the microorganism.

[0034] [SEQ ID NO:1] 5'-CCTTCGGGTGGACAGGGAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCATGTGTTGCCAGCGTAGAGGCGGGCACTCACATGAGACTGCCGGAGACAATTCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGGCCTGGGCCACACACGTGCTACAATGGCCGGTACAGAGGGCTGCGAACCCGCGAGGGGGAGCCAATCCCAAAAAGCCGGCCCCAGTTGGGATCGGAGGCTGCAACTCGCCTCCGTGAACGCGGAGTTGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTG-3'

[0035] [SEQ ID NO:2] 5'-GGGGACCCATGTGCCACTCTTAACGGGGTGGCTTTTTTAGAGTGTAAAAAGCTTTAGGAATAAGAGCTGGGCAAGACCGGTGCCAGCCGCCGCGGTAA-3'

[0036] [SEQ ID NO:3] 5'-GGGAGCCCCCATGTGCCACTCTTAACGGGGTGGCTTTTTTAGAGTGTAAAAAGCTTTACGAATAAAAGCTGGGCAAGACCGGTGCCAGCCGCCGCGGTAA-3'

[0037] [SEQ ID NO:4] 5'-CCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTTCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGGGGGGATGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCATATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCACAAAGTATGTCGTATTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATCGCTAGTAATCGTATATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTG-3'

[0038] [SEQ ID NO: 5] 5'-CCTTCGGGAACTGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTATGGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTG-3'

[0039] [SEQ ID NO: 6] 5'-CCTTCGGGACTCATGATACAGGGCGGCAGGGCTGTCGTCAGCCTCGTTGTTTGGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCAAAAGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGAGGGGATGACGTCAAGTCATCATGGCCCTTACGACTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGACAAGCGAGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTTAATCGTAGATCAAAATGCTACGGTGAATACGTTCCCGGGCCTTG-3'

[0040] [SEQ ID NO: 7] 5'-CCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACTAGTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTAAATCAGAATGCTACGGTGAATACGTTCCCGGGCCTTG-3'

[0041] [SEQ ID NO: 8] 5'-CCTTCGGGAACTCAGACACAGGTGCTGCATGGCTGTCGTCAGACTAGCAAAGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGCCGCGAGGGGGAGCTAATCTCACAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTG-3'

[0042] [SEQ ID NO: 9] 5'-CCTTCGGGAACTCAGACACAGGTGCTGCATGGCTGTCGTCAGACTAGCAAAGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGCCGCGAGGGGGAGCTAATCTCACAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTG-3'

[0043] [SEQ ID NO: 10] 5'-CCTTCGGGGCAGGAAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCATTAGTTGCTACCATTAAGTTGAGCACTCTAGTGAGACTGCCACGGTTAACGTGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCTAGGGCTACACACGTGCTACAATGGTGAGTACAAAGAGATGCAATACCGTGAGGTGGAGCCAAACTCAAAAACTCATCCCAGTTCGGATTGTAGGCTGAAACTCGCCTACATGAAGCCGGAGTTGCTAGTAATCGCGAATCAGCATGTCGCGGTGAATACGTTCCCGGGCCTTG-3'

[0044] [SEQ ID NO: 11] 5'-CCTTTAGGGCAAGAAGACAGGTGGGGCATGGGTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAATCCCGCAACCAGCGCAACCCTTATCTTTTTTTGCTACCATTAAATTGAGCACTCTATTGAGACTGCCCCGGTTAACGTGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCTAGGGCTACACACGTGCTACAATGGTGAGTACAAAAAGATGCAATACCGCGAGGTGGAGCCAAACTCAAAAACTCATCCCAGTTCGGATTGTAGGCTGAAACTCCCCTACCTGAAGCCGGAGTTGCTAGTAATCGCGAATCAACATGTCGCGGTGAATACCTTCCCGGGCCTTG-3'

[0045] [SEQ ID NO: 12] 5'-CCTTCGGGACAGGAAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATACTTAGTTGCTAGCATTTGGTTGAGCACTCTAAGTAGACTGCCGGAGACAATTCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTTCTGGGCTACACACGTGCTACAATGGGTATAACAACGGGAAGCGAACCAGTGATGGCAAGCAAATCCCTAAAAAATACTCCCAGTTCAGATTGTTCTCTGCAACTCGAGAACATGAAGTCGGAGTTGCTAGTAATCGCGAATCAGCATGTCGCGGTGAATACGTTCCCGGGCCTTG-3'

[0046] [SEQ ID NO: 13] 5'-GGGGACCCCATGTGCCACTCTTAACGGGGTGGCTTTTCTTATGTGTAAAAAGCTTTTGGAATAAGAGCTGGGCAAGACCGGTGCCAGCCGCCGCGGTAA-3'

[0047] [SEQ ID NO: 14] 5'-GGGGACCCCATGTGCCTCTCTTAACGGGGTGGCTTTTTTTGAGTGTAAAAAGCTTTGAGAATAAGAGCTGGGCAAGACCGGTGCCAGCCGCCGCGGTAA-3'

[0048] [SEQ ID NO: 15] 5'-GGGGACGACACCGAGTGCCAATCTCATGTGCTGGGTGTCCGGGTGTGTAATATACACCTGTTAGCGGGGCCGGGCAAGACCGGTGCCAGCCGCCGCGGTAA-3'

[0049] [SEQ ID NO: 16] 5'-TCAAAGATTAAGCCATGCATGTCTAAGTATAAGCAATTTGTACTGTGAAACTGCGAATGGCTCATTAAATCAGTTATCGTTTATTTGATAGTACCTTACTACATGGATACCTGTGGTAATTCTAGAGCTAATACATGCTAAAAACCTCGACTTCGGAAGGGGTGTATTTATTAGATAAAAAACCAACGCCCTTCGGGGCTCCTTGGTGAATCATAATAACTAAGCGAATCGCATGGCCTTGCGCCG-3'

[0050] [SEQ ID NO: 17] 5'-TCAAAGATTAAGCCATGCATGTTTAAGTATAAGCAATTTGTACTGTGAAACTGCGAATGGCTCATTAAATCAGTTATCGTTTATTTGATAGTACCTTACTACATGGATACCTGTGGTAATTCTAGAGCTAATACATGCTAAAAACCTCGACTTCGGAAGGGGTGTATTTATTAGATAAAAAACCAACGCCCTTCGGGGCTCCTTGGTGAATCATAATAACTAAGCGAATCGCATGGCCTTGCGCCG-3'

[0051] [SEQ ID NO: 18] 5'-TCAAAAATTAACCCACCCATGTTTAAGTATAACCAATTATACCGTGAAAATGAGAATGGCTCATTAAATCAGTTATCGTTTATTTGATAGTACCCTACTACTTGGATACCCGTGGTAATTCTAGAGCTAATACATGCTAAAAACCCCAACTTCGGGAGGGGTGTATTTATTAGATAAAAAACCAATGCCCTTCGGGGCTCCTTGGTGAATCATAATAACTAAACGAATCGCATGGCCTTGCGCGG-3'

[0052] [SEQ ID NO: 19] 5'-TCAAAAATTAACCCAAGCCAGTTTTAGTATTAACCAATTATACTGTGAAACTGCGAATGGCTCATTAAATCAGTTATCGTTTATTTGATAGTACCTTACTACATGGATACCCGTGGTAATTCTAGAGCTAATACATGCTAAAAACCCCAACTTCGGGAGGGGTGTATTTATTAGATAAAAAACCAATGCCCTTCGGGGCTCCTTGGTGAATCATAATAACTAAACGAATCGCATGGCCTTGCGCCG-3'

[0053] (Second Embodiment) The microbial mixture according to the second embodiment of the present disclosure includes at least one microorganism that decomposes 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.

[0054] The microbial mixture, which includes at least one microorganism that decomposes 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, enables methane production from a biologically derived composition by co-culturing at room temperature.

[0055] Although the action of the microbial mixture according to the second embodiment of the present disclosure is not clear, it is presumed as follows. Among 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 decomposes at least one of the decomposition of cellulose to glucose, the decomposition of chitin to glucosamine, and the decomposition of protein to amino acids in a biologically derived composition under normal temperature conditions. Among the microbial mixtures according to the second embodiment of the present disclosure, at least one hydrogen-producing microorganism takes in the decomposed glucose, glucosamine, or amino acid and produces hydrogen under normal temperature conditions. Among the microbial mixtures according to the second embodiment of the present disclosure, at least one methane-producing microorganism takes in the produced hydrogen and carbon dioxide and produces methane under normal temperature conditions. Among the microbial mixtures according to the second embodiment of the present disclosure, at least one oxygen-consuming microorganism can take in oxygen and grow under normal temperature conditions. Since the oxygen-consuming microorganism consumes oxygen during the growth process, the anaerobicity 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 producing methane using a biologically derived composition as a substrate, methane can be efficiently obtained even at normal temperature with a small number of steps by co-culture. When the biologically derived composition contains a hardly decomposable substance such as chitin or cellulose, for example, when it is digested sludge, such an effect is more remarkable, and even a hardly decomposable composition that has been difficult to use as a substrate can be used as a substrate for methane production. Furthermore, the at least one oxygen-consuming microorganism can produce ammonia from nitrate in a biologically derived 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 function more efficiently. It should be noted that the present disclosure is not limited to the above presumed actions.

[0056] 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 can decompose at least one of cellulose, chitin, and protein. For example, a microorganism that decomposes cellulose, a microorganism that decomposes chitin, and a microorganism that decomposes protein each have cellulase activity, chitinase activity, or protease activity, and are preferably microorganisms that decompose cellulose into glucose, chitin into glucosamine, or protein into amino acids, respectively. Also, one microorganism may have a plurality of these activities. The environmental conditions under which the activity is exhibited are preferably normal temperature and anaerobic conditions from the viewpoint that heating is not required during methane production using a biologically-derived composition as a substrate and co-culture can be utilized. In the present disclosure, as the microorganism that decomposes at least one of cellulose, chitin, and protein, a microorganism generally known in the art as a microorganism that decomposes at least one of cellulose, chitin, and protein may be used. Here, being generally known in the art means being known, for example, from literature, databases, or microorganism species names in the art.

[0057] In the present disclosure, the hydrogen-producing microorganism is not particularly limited as long as it can produce hydrogen. For example, the hydrogen-producing microorganism preferably has at least one of the activities of producing hydrogen from glucose, producing hydrogen from glucosamine, or producing hydrogen from amino acids. The environmental conditions under which the activity is exhibited are preferably normal temperature and anaerobic conditions from the viewpoint that heating is not required during methane production using a biologically-derived composition as a substrate and co-culture can be utilized. In the present disclosure, as the hydrogen-producing microorganism, a hydrogen-producing microorganism generally known in the art may be used.

[0058] 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, the methane-producing microorganism preferably has an activity of producing methane from hydrogen. The environmental conditions under which the activity is exerted are preferably normal temperature and anaerobic conditions from the viewpoint that heating is not required during methane production using a biologically-derived composition as a substrate and co-culture can be utilized. In addition, as the methane-producing microorganism in the present disclosure, a methane-producing microorganism generally known in the art may be used.

[0059] 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, the oxygen-consuming microorganism preferably has an activity of taking oxygen into the microorganism. Furthermore, it preferably has an activity of producing ammonia from nitrate. The environmental conditions under which the activity is exerted are preferably normal temperature and anaerobic conditions from the viewpoint that heating is not required during methane production using a biologically-derived composition as a substrate and co-culture can be utilized. In addition, 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 microorganism in the present disclosure preferably consumes oxygen under anaerobic conditions as described above, it is preferably a microorganism capable of consuming oxygen even under anaerobic conditions where a certain level of oxygen concentration exists (that is, a condition where the oxygen concentration is only below a certain level).

[0060] In the present disclosure, normal temperature preferably ranges from 5°C to 40°C, more preferably from 10°C to 35°C, still more preferably from 15°C to 35°C, and particularly preferably 30°C, from the viewpoint that it is not necessary to heat the culture tank. In the present disclosure, anaerobic preferably refers to a condition where the oxygen concentration in the gas phase or the liquid phase of the culture tank is 5% or less, more preferably a condition where the oxygen concentration in the gas phase or the liquid phase of the culture tank is 3% or less, from the viewpoint that it is easy for each microorganism in the microorganism mixture to exert its function.

[0061] In the present disclosure, microorganisms include aerobic microorganisms and anaerobic microorganisms. Aerobic microorganisms are microorganisms that require oxygen for growth, including microaerophilic microorganisms and obligate aerobic microorganisms. Anaerobic microorganisms are microorganisms that do not require oxygen for growth, including facultative anaerobic microorganisms, obligate anaerobic microorganisms, and oxygen-tolerant microorganisms.

[0062] In the present disclosure, a composition derived from an organism is not particularly limited as long as it contains cellulose, chitin, or protein. Examples of the composition derived from an organism in the present disclosure include, for example, biological sludge (e.g., sewage sludge), and more specifically, digested sludge and the like. Digested sludge is a sludge residue composed of hardly biodegradable components generated when anaerobic microorganisms such as methane-producing microorganisms decompose excess sludge as a substrate. Digested sludge contains, for example, chitin derived from the cell wall polysaccharides of microorganisms, proteins derived from the composition of microorganisms, keratin derived from human hair, cellulose derived from urine, and hardly decomposable carbohydrates such as xylan derived from the cell walls of plants.

[0063] <Method for obtaining a microbial mixture> In the present disclosure, the "microbial mixture of the present disclosure" includes both the microbial mixture according to the first embodiment of the present disclosure and the microbial mixture according to the second embodiment of the present disclosure, unless otherwise specified and without contradiction. Also, "of the present disclosure" includes both the first embodiment and the second embodiment, unless otherwise specified and without contradiction.

[0064] The microbial mixture of the present disclosure can be obtained by culturing soil and a composition derived from an organism at 30°C for one month. An example of the composition derived from an organism is digested sludge. More specifically, for example, it can be obtained by the method described below. A soil sample can be collected from the surface sediment of the riverbank of the Kawaguchi River in Hachioji City, Tokyo, Japan. 1 g of the soil sample is suspended in 20 ml of sterilized water using a vortex mixer, and this is used as an inoculum.

[0065] Next, prepare digested sludge for culturing the inoculum. The dewatered digested sludge (with a solids content of 17%) can be obtained, for example, from a sewage treatment plant in Yokohama, Kanagawa Prefecture, Japan. Since polyaluminum chloride, an inorganic flocculant used for dewatering sewage sludge, is toxic to microorganisms, the polyaluminum chloride in the dewatered digested sludge is removed by repeatedly washing it with tap water. Specifically, 300 g of dewatered 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 the polyaluminum chloride contained in the supernatant. This removal operation is repeated 5 times until the pH of the filter eluate reaches 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 to form a powder, and passed through a sieve with 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 tests. The carbon, nitrogen, and hydrogen contents in the digested sludge sample were analyzed using an elemental analyzer JM-11 (J Science Lab Co., Ltd., Kyoto, Japan). As a result, in 1.0 g of digested sludge, there were 335.1 mg of carbon, 58.1 mg of nitrogen, and 53.2 mg of hydrogen.

[0066] Then, culture the inoculum in a 17 ml glass vial. Specifically, inoculate 100 μl of the inoculum into a mixture of 10 ml of sterilized water and 100 mg of digested sludge powder. Nitrogen gas is introduced 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. The inoculum can be cultured at 30 °C for 1 month to obtain the microbial mixture of the present disclosure.

[0067] Note that the method for obtaining the microbial mixture of the present disclosure described above is merely an example and is not restrictive.

[0068] Note that the microbial mixture can be collected from the soil at a specific location, but since each microorganism cannot be isolated and cultured, and a survival test method that encompasses all the microorganisms contained in the mixture has not been established, it is impossible to deposit it with a depository institution under the current deposit system. Therefore, at the School Corporation Kogakuin University Hachioji Campus (address: 2665-1 Nakanocho, Hachioji City, Tokyo, phone number: 042-622-9291), the microbial mixture is stored in a sellable state as Microbial Mixture No. Riverbank-A30, and can be obtained by a third party as needed.

[0069] In addition, the microbial mixture of the present disclosure may be obtained by any method. For example, it may be prepared by appropriately mixing each isolated or purchased microorganism so as to form the microbial mixture of the present disclosure.

[0070] <Function of the microbial mixture> The microbial mixture of the present disclosure can produce 10 ml or more of methane by culturing for 1 month under normal temperature conditions in the presence of 1 g of a biologically-derived composition as a substrate.

[0071] The microbial mixture of the present disclosure preferably produces 10 ml to 500 ml of methane, more preferably 15 ml to 400 ml of methane, and even more preferably 20 ml to 300 ml of methane by culturing for 1 month under normal temperature conditions in the presence of 1 g of a biologically-derived composition as a substrate. Note that the details of the biologically-derived composition, digested sludge, substrate, and normal temperature are the same as those described in <Microorganisms contained in the microbial mixture> above.

[0072] In addition, the method for confirming the function of the microbial mixture (the microorganisms contained in the microbial mixture) may be, for example, a method of culturing the microbial mixture under the conditions described in the examples to confirm the growth of the microorganisms, methane production, etc. Specifically, dehydrated digested sludge can be used as digested sludge (substrate) by washing, drying, pulverizing, and sieving. From the perspective that the contents of carbon, nitrogen, and hydrogen in the digested sludge contain a substrate for producing methane and a nutrient source for the growth of the microorganisms contained in the microbial mixture, it is preferable that in 1.0 g of the digested sludge, there are 10 mg to 1000 mg of carbon, 10 mg to 100 mg of nitrogen, and 10 mg to 1000 mg of hydrogen.

[0073] In the culture tank, the mixing ratio when inoculating the microbial mixture into the solution containing the biologically-derived composition is not particularly limited. However, from the perspective that the microorganisms are likely to grow, it is preferably 0.1 to 10 parts by mass of the digested sludge with respect to 1 part by mass of the microbial mixture, and more preferably 1 part by mass. From the perspective that the microorganisms are likely to grow, it is further preferable to add 10 to 1000 parts by mass of water, and more preferably 100 parts by mass. When inoculating the microbial mixture into the solution containing the biologically-derived composition, the microorganisms contained in the microbial mixture may be inoculated one by one at different timings, or the microorganisms contained in the microbial mixture may be inoculated in multiple types in multiple batches. Alternatively, all the microbial species contained in the microbial mixture may be inoculated simultaneously only once, or the simultaneous ingestion of all the microbial species contained in the microbial mixture may be performed multiple times. From the perspective of ease of operation, it is preferable to inoculate all the microbial species contained in the microbial mixture simultaneously only once.

[0074] In the culture tank, there may further be contained, for example, organic substances or inorganic substances contained in commercially available media generally used for culturing microorganisms or cells, in order to promote or inhibit the growth of each microorganism.

[0075] After inoculation, a stable gas other than oxygen, such as nitrogen gas, can be introduced into the gas phase of the culture tank, the culture tank can be sealed, and cultured at room temperature for 1 month. The culture period may be 10 days to 60 days, or may be 20 days to 40 days.

[0076] Regarding the method for quantifying methane, for example, the method described in the examples can be used. Specifically, it is preferable to quantify gaseous methane, 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.

[0077] Generally, the optimal pH for methane-producing microorganisms to produce methane is 6.8 to 7.6. Therefore, by lowering the pH below 6.8, methane production can be suppressed. Also, generally, hydrogen-producing microorganisms have resistance to low pH, and if the pH is 4.5 or higher, hydrogen can be produced. Therefore, the microbial mixture of the present disclosure can produce 10 ml or more of hydrogen by culturing 1 g of the bio-derived composition as a substrate under room temperature conditions and, for example, at pH 4.5 to pH 6.8 for 1 month. The production of the hydrogen is preferably 10 ml to 2000 ml, more preferably 20 ml to 1800 ml, still more preferably 40 ml to 1600 ml, and particularly preferably 80 ml to 1200 ml.

[0078] ≪Composition for Methane Production≫ The composition for methane production of the present disclosure preferably contains a microbial mixture and is a composition capable of producing methane from a bio-derived composition.

[0079] The composition for methane production of the present disclosure is not particularly limited as long as it contains a microbial mixture and is a composition capable of producing methane from a bio-derived composition. The microbial mixture contained in the composition for methane production of the present disclosure may be live microorganisms or dormant microorganisms. When the microorganism is a fungus, it may be contained either as mycelia or spores.

[0080] The composition for methane production of the present disclosure may contain, in addition to the microbial mixture of the present disclosure, solid media (e.g., gelatin, lactose), liquid media (e.g., water, physiological saline), solubilizing agents, stabilizers, isotonic agents, etc. The blending amounts of these components are not particularly limited as long as the microbial mixture of the present disclosure can act as a composition for methane production.

[0081] The shape of the composition for methane production of the present disclosure is not particularly limited, and may be, for example, powdery, granular, solid, liquid, frozen, or in a shape encapsulated in a capsule. These shapes are not particularly limited as long as the microbial mixture of the present disclosure can act as a composition for methane production. Regarding the detailed conditions for producing methane, it is the same as the method for confirming the function of the microbial mixture described in <Function of the Microbial Mixture>.

[0082] ≪Methane Production Method≫ The methane production method of the present disclosure can include inoculating a microbial mixture into a solution containing a biologically derived composition, and culturing the inoculated microbial mixture under normal temperature conditions.

[0083] In the present disclosure, the solution containing a biologically derived composition is not particularly limited as long as it contains a biologically derived composition. The solution containing a biologically derived composition in the present disclosure may contain, in addition to the biologically derived composition, for example, water or physiological saline.

[0084] In the present disclosure, inoculating a microbial mixture into a solution containing a biologically derived composition is not particularly limited as long as it includes adding the microbial mixture to the biologically derived composition. Regarding 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 the Microbial Mixture> can be applied.

[0085] 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. Regarding the detailed conditions for culturing the inoculated microbial mixture under normal temperature conditions, the same conditions as the culturing after inoculation of the microbial mixture described in the method for confirming the function of the microbial mixture in the <Function of the microbial mixture> can be applied.

[0086] The methane production method of the present disclosure can include inoculating a microbial mixture into a solution containing digested sludge and culturing the inoculated microbial mixture under normal temperature conditions. For details of inoculation and culturing, reference can be made to the description of inoculation and culturing in the method for confirming the function of the microbial mixture in the <Function of the microbial mixture>.

Example

[0087] Hereinafter, the present disclosure will be described more specifically with reference to examples. However, the present disclosure is not limited to the following examples as long as it does not exceed the gist thereof. Unless otherwise specified, "parts" are based on mass. The same applies to "%".

[0088] In the following, molecular biological reagents were purchased from Toyobo Co., Ltd. (Osaka, Japan), Thermo Fisher Scientific (Waltham, Massachusetts, USA), or MP Biomedicals (Santa Ana, California, USA). All other chemicals were purchased from Wako Pure Chemical Industries, Ltd. (Kyoto, Japan). Glass and plastic experimental instruments used for culturing were purchased from Maruem Co., Ltd. (Osaka, Japan) or AS ONE Corporation (Osaka, Japan).

[0089] ≪Example 1≫ (Collection of microbial mixture) A soil sample was collected from the surface sediment on the riverbank of the Kawaguchi River in Hachioji City, Tokyo, Japan. 1 g of the soil sample was suspended in 20 ml of sterilized water using a vortex mixer and used as an inoculum.

[0090] (Preparation of digested sludge) The dewatered digested sludge (solid content 17%) was obtained from a sewage treatment plant in Yokohama, Kanagawa Prefecture, Japan. Since polyaluminum chloride, an inorganic flocculant used for dewatering sewage sludge, is toxic to microorganisms, the polyaluminum chloride in the dewatered digested sludge was removed by repeatedly washing it with tap water. Specifically, 300 g of dewatered 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 the polyaluminum chloride contained in the supernatant. This removal operation was repeated 5 times until the pH of the filter eluate reached 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 to form a powder, and passed through a sieve with a diameter of 1 mm. The digested sludge powder that passed through the sieve was used as the digested sludge (substrate) in the following tests. The carbon, nitrogen, and hydrogen contents in the digested sludge sample were analyzed using an elemental analyzer JM-11 (J Science Lab Co., Ltd., Kyoto, Japan). As a result, in 1.0 g of digested sludge, there were 335.1 mg of carbon, 58.1 mg of nitrogen, and 53.2 mg of hydrogen.

[0091] (Cultivation of inoculum) The inoculum was cultured in a 17 ml glass vial. Specifically, 100 μl of the inoculum was inoculated into a mixture of 10 ml of sterilized water and 100 mg of digested sludge powder. Nitrogen gas was introduced 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 the microbial mixture in the following.

[0092] (Quantification of hydrogen production and methane production) For the microbial mixture cultured at 30 °C, 40 °C, or 50 °C for 1 month, the gas phase at the top of the vial was sampled and the quantification of hydrogen and methane was performed. Quantification of hydrogen and methane was performed using a gas chromatograph GC-8A (Shimadzu Corporation, Kyoto, Japan) equipped with a thermal conductivity detector. The column used was a Shincarbon ST column 50 / 80 (2.0 m × 3.0 mm inner diameter, Shinwa Chemical Industries Co., Ltd., Kyoto, Japan). With an injection volume of 0.5 ml and argon (43.5 ml / min) as the carrier gas, the column temperature was set at 80°C. Standard samples of hydrogen and methane were purchased from GL Sciences Inc. (Tokyo, Japan). The results are shown in Figure 1. In the figure, the vertical axis represents the methane production amount (ml) per 1 g of digested sludge, and the horizontal axis represents the number of subcultures (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 with the number of subcultures ranging from 1 to 8 times. When subcultured 4 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, regardless of the number of subcultures, about 2.5 ml or more of methane was produced per 1 g of digested sludge. At a culture temperature of 50°C, no methane production was confirmed regardless of the number of subcultures. From the above, it was shown that high-efficiency methane production is possible with the microbial mixture of the present disclosure, and by appropriately setting the culture temperature, the microbial mixture can be stably maintained even after subculture. In Figure 1, the number of subcultures 1 indicates that the digested sludge and the above-mentioned inoculum were cultured for 1 month. In Figure 1, the number of subcultures 2 indicates that the digested sludge and the product obtained at the number of subcultures 1 were cultured for 1 month. The number of subcultures 3 indicates that the digested sludge and the product obtained at the number of subcultures 2 were cultured for 1 month. The same applies to the number of subcultures 4 and later.

[0093] (Analysis of the microbial mixture by PCR-DGGE method) For the microbial mixture cultured at 30°C, 40°C, or 50°C for 1 month, the PCR-denaturing gradient gel electrophoresis method (PCR-DGGE method) was carried out as follows.

[0094] -PCR- 1 ml of the culture solution of the microbial mixture cultured at 30 °C, 40 °C, or 50 °C for 1 month was centrifuged under the conditions of 20,000×g, 10 minutes, and 4 °C to collect the cells of the microbial mixture. Genomic DNA was extracted from the cells using the FastDNA SPIN Kit for Soil (MP Biomedicals).

[0095] For eubacteria, the 16S rRNA gene corresponding to the V6 - V8 region was amplified by PCR. As described in 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., the primers F984GC (SEQ ID NO: 20) and R1378 (SEQ ID NO: 21) were used, and the polymerase KOD Fx Neo polymerase (Toyobo Co., Ltd.) was used. The PCR conditions were carried out with 1 cycle of 94 °C for 2 minutes and 34 cycles of 94 °C for 15 seconds, 50 °C for 30 seconds, and 68 °C for 30 seconds. Among the following base sequences described together with each SEQ ID NO, the base sequence enclosed in brackets [] represents the base sequence of the GC clamp. By the above operation, amplicons derived from eubacteria were obtained.

[0096] [SEQ ID NO: 20]5'-[CGCCCGGGGCGCGCCCCGGGCGGGGCGGGGGCACGGGGGG]AACGCGAAGAACCTTAC-3' [SEQ ID NO: 21]5'-CGGTGTGTACAAGGCCCGGGAACG-3'

[0097] For archaea containing methanogens, as described in 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 76:3776-3786., an approximately 800-bp 16S rRNA gene containing the V3 region was amplified by PCR using the archaeal primers Met86f (SEQ ID NO: 22) and Met915r (SEQ ID NO: 23). Subsequently, using the amplified DNA as a template, the V3 region was further amplified by PCR using the primers GC-ARC344f (SEQ ID NO: 24) and 519r (SEQ ID NO: 25) used for archaea containing methanogens. The PCR conditions were carried out with 1 cycle of 95°C for 5 minutes and 30 cycles of 95°C for 30 seconds, 56.5°C for 30 seconds, and 68°C for 30 seconds. Among the following base sequences described together with the SEQ ID NOs, the base sequence enclosed in brackets [] represents the base sequence of the GC clamp. By the above operation, amplicons derived from archaea containing methanogens were obtained.

[0098] [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'

[0099] In the base sequences of the above primers, Y represents C or T, W represents A or T, and K represents G or T.

[0100] For fungi or protists, 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., a portion of the 18S rRNA gene was amplified by PCR using primers NS1 (SEQ ID NO: 26) and GCFung (SEQ ID NO: 27). The PCR conditions were carried out with one cycle of 94°C for 2 minutes and 34 cycles of 94°C for 15 seconds, 50°C for 30 seconds, and 68°C for 30 seconds. Among the following base sequences described together with the SEQ ID NOs, the base sequences enclosed in parentheses [] represent the base sequences of the GC clamp. By the above operation, amplicons derived from fungi or protists were obtained.

[0101] [SEQ ID NO: 26] 5'-GTAGTCATATGCTTGTCTC-3' [SEQ ID NO: 27] 5'-[CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCC]ATTCCCCGTTACCCGTTG-3'

[0102] -DGGE method- The DGGE method was carried out according to the operation manual using the NB-1480A DGGE system (Nippon Eido Co., Ltd., Tokyo, Japan) for the amplicons obtained by the above PCR. Approximately 1 μg of the amplified amplicons was loaded into the wells of each gel. For amplicons derived from eubacteria, a 6% polyacrylamide gel with a linear denaturant concentration gradient of 50% - 70% was used. For amplicons derived from archaebacteria, a 6% polyacrylamide gel with a linear denaturant concentration gradient of 35% - 45% was used. For amplicons derived from fungi or protists, a 7% polyacrylamide gel with a linear denaturant concentration gradient of 20% - 45% was used. In addition, molecular weight markers were loaded into the wells of each gel to estimate the molecular weight of the amplicons. Electrophoresis was carried out at a constant voltage of 50 V for 18 hours at 58 °C for amplicons derived from eubacteria, and for 20 hours at 60 °C for amplicons derived from archaea, fungi, and protists. The gel after electrophoresis was stained with 10 μl of SYBR Green (Life Technologies Corporation) dissolved in 100 ml of TAE buffer. The results are shown in Fig. 2. When the microbial mixture was cultured at 30°C, amplicons C1 (including SEQ ID NO: 10), C2 (including SEQ ID NO: 11), Ue1 (including SEQ ID NO: 1), E2 (including 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: 7) derived from eubacteria, amplicons Mb1 (including SEQ ID NO: 13), Ua2 (including SEQ ID NO: 2), Ua3 (including SEQ ID NO: 3), Mb5 (including SEQ ID NO: 14), and Ms1 (including SEQ ID NO: 15) derived from archaea, and amplicons R1 (including SEQ ID NO: 18), R2 (including SEQ ID NO: 19), A4 (including SEQ ID NO: 16), and A5 (including SEQ ID NO: 17) derived from fungi or protists were confirmed. When the microbial mixture was cultured at 40°C, amplicons C2, Ue1, E2, C4, E7, P2, and E8 derived from eubacteria, amplicons Mb1, Mb2, Ua1, Ua2, Ua3, Mb4, Mb5, and Ua4 derived from archaea, and amplicons R1, R2, A3, D1, AC1, and AC2 derived from fungi or protists were confirmed. When the microbial mixture was cultured at 50°C, amplicons C2, Ue1, E2, C4, E7, P2, and E8 derived from eubacteria, amplicons Mb1, Mb2, and Ua2 derived from archaea, and amplicons R1, R2, A3, D1, AC1, and AC2 derived from fungi or protists were confirmed. Note that the number of confirmed amplicons corresponds to the number of existing microbial species, and the migration stop position indicates the GC base content unique to the microbial species. Further, the above amplicons were amplified with primers containing a GC clamp rich in GC base content for analysis by the PCR-DGGE method, and the base sequences of the above amplicons include 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. It was found that the microbial mixture of the present disclosure contains at least 10 types of eubacteria, 5 types of archaea, and 4 types of fungi or protists when subcultured at 30°C.

[0103] -Sequence analysis of amplicons obtained by the PCR-DGGE method- The gel obtained by the DGGE method was irradiated with light at 470 nm, and the sites containing each amplicon were excised from the gel when cultured at 30°C for 1 month. The excised sites were immersed in 20 μl of TE buffer (pH 8.0) at 4°C for 72 hours to extract each amplicon. Next, using 1 μl of the extracted amplicon as a template, PCR was performed again using primers without a GC clamp. The PCR conditions were the same as those of the PCR in the above 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 with 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, phylogenetic trees were created based on the 16S rRNA gene for eubacteria and archaebacteria, and phylogenetic trees were created based on the 18S rRNA gene for fungi or protists. A phylogenetic tree showing the taxonomic positions (genera and species) of amplicons derived from eubacteria is shown in Figure 3. Amplicons C1, C2, and C4 were found to be derived from the family Clostridiaceae. Amplicons E2, E7, E8, and E9 were found to be derived 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 positions (genera and species) of amplicons derived from archaebacteria is shown in Figure 4. Amplicon Ms1 was found to be derived from the family Methanosarcinaceae. Amplicons Mb1 and Mb5 were found to be derived from the family Methanobacteriaceae. Amplicons Ua2 and Ua3 were found to be derived from unclassified archaebacteria. A phylogenetic tree showing the taxonomic positions (genera and species) of amplicons derived from fungi or protists is shown in Fig. 5. Amplicons A4 and A5 were found to be derived from the family Aspergillaceae. Amplicons R1 and R2 were found to be derived from the family Arthopyreniaceae.

[0104] Since Enterobacter asburiae can degrade proteins, the eubacteria from which amplicons E8 and E9 are derived can degrade proteins in digested sludge. Since Citrobacter freundii has cellulase, chitinase, and protease, and Cronobacter sakazakii has chitinase and protease, the eubacteria from which amplicons E2 and E7 are derived can hydrolyze carbohydrates and proteins in digested sludge. Since Clostridium amylolyticum and Clostridium punense can produce hydrogen, the eubacteria from which amplicons C1, C2, and C4 are derived can produce hydrogen. Since the families Methanobacteriaceae and Methanosarcinaceae can produce methane, the archaebacteria from which amplicons Ms1, Mb1, and Mb5 are derived can produce methane. Since Penicillium expansum and Monascus purpureus have cellulase, chitinase, and protease, the species of the family Aspergillaceae from which amplicons A4 and A5 are derived are considered to have cellulase, chitinase, and protease. On the other hand, since the family Aspergillaceae is generally not optimal under anaerobic conditions, the species of the family Aspergillaceae from which amplicons A4 and A5 are derived are related to consuming oxygen in the culture solution containing digested sludge so that other eubacteria and archaebacteria, which are anaerobic microorganisms, can work efficiently. As described above, Fig. 6 shows the mechanism by which the microbial mixture in Example 1 produces methane from digested sludge.

[0105] (Measurement of Enzyme Activity) The microbial mixture cultured at 30°C for 1 month was used for the measurement of 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 sterilized 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 the evaluation of enzyme activity. For the quantification of cellulase and chitinase, Cellulose Azure and Chitin Azure purchased from Sigma-Aldrich (St. Louis, Missouri, USA) were used by the methods described in 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 microorganisms and to evaluate chitinases. J Microbiol Methods 56:213-219. Specifically, 0.2 ml of the supernatant, 0.8 ml of 50 mM citrate buffer (pH 5.0), and 5 mg of Cellulose Azure or Chitin Azure were mixed in a centrifuge tube and cultured at 30°C for 1 hour. After the culture, the centrifuge tube was centrifuged at 20,000×g for 10 minutes at 4°C to recover the supernatant, and the absorbance at 540 nm was measured. One Unit of cellulase and chitinase was defined as the amount of enzyme that increased the absorbance at 540 nm by 0.01. Protease activity was quantified by converting it to the activity equivalent to 1 μg of trypsin using a Pierce Protease Assay kit (Thermo Fisher Scientific). The results are shown in Fig. 7. In the figure, for the measurement of cellulase activity and chitinase activity, the vertical axis indicates the enzyme activity (Unit / ml / hour) when the enzyme activity that causes an absorbance increase of 0.01 at 540 nm is defined as 1. For the measurement of trypsin activity, the vertical axis indicates the enzyme activity (trypsin equivalent / ml / hour) when the activity equivalent to 1 μg of trypsin is defined as 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 had a cellulase activity of about 0.18 Unit / ml / hour, a chitinase activity of about 0.056 Unit / ml / hour, and a protease activity of about 0.33 μg trypsin equivalent / ml / hour at a culture temperature of 30°C.

[0106] From the above, in Example 1, it was possible to provide a microbial mixture capable of producing methane from a biologically-derived composition, a composition for methane production, and a method for methane production by co-culturing a plurality of specific microorganisms at room temperature.

Industrial Applicability

[0107] The microbial mixture of the present disclosure can generate hydrogen or methane by inoculating a solution containing digested sludge and culturing it at room temperature, and these can be used as fuels. Furthermore, the microbial mixture of the present disclosure can reduce the volume of digested sludge and reduce the amount of industrial waste.

Claims

1. Microorganisms belonging to the family Enterobacteriaceae, Pseudomonadaceae, and Clostridiaceae that decompose at least one of cellulose, chitin, and protein and produce hydrogen, Microorganisms belonging to the family Methanobacteriaceae and Methanosarcinaceae, which are methane-producing microorganisms, Microorganisms belonging to the family Aspergillaceae and Arthopyreniaceae, which are oxygen-consuming microorganisms, Microorganisms having a 16S rRNA gene sequence with 95% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 1, Microorganisms having a 16S rRNA gene sequence with 95% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 2, Microorganisms having a 16S rRNA gene sequence with 95% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 3, and The microorganism belonging to the family Enterobacteriaceae is a microorganism having a 16S rRNA gene sequence with 98% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 4, a microorganism having a 16S rRNA gene sequence with 98% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 5, a microorganism having a 16S rRNA gene sequence with 98% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 6, and a microorganism having a 16S rRNA gene sequence with 98% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 7, The microorganism belonging to the family Pseudomonadaceae is a microorganism having a 16S rRNA gene sequence with 98% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 8 and a microorganism having a 16S rRNA gene sequence with 98% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 9, The microorganisms belonging to the family Clostridiaceae are microorganisms having a 16S rRNA gene sequence with a sequence identity of 98% or more to the nucleotide sequence represented by SEQ ID NO: 10, microorganisms having a 16S rRNA gene sequence with a sequence identity of 98% or more to the nucleotide sequence represented by SEQ ID NO: 11, and microorganisms having a 16S rRNA gene sequence with a sequence identity of 98% or more to the nucleotide sequence represented by SEQ ID NO:

12. The microorganisms belonging to the family Methanobacteriaceae are microorganisms having a 16S rRNA gene sequence with a sequence identity of 98% or more to the nucleotide sequence represented by SEQ ID NO: 13 and microorganisms having a 16S rRNA gene sequence with a sequence identity of 98% or more to the nucleotide sequence represented by SEQ ID NO:

14. The microorganisms belonging to the family Methanosarcinaceae are microorganisms having a 16S rRNA gene sequence with a sequence identity of 98% or more to the nucleotide sequence represented by SEQ ID NO:

15. The microorganisms belonging to the family Aspergillaceae are microorganisms having an 18S rRNA gene sequence with a sequence identity of 98% or more to the nucleotide sequence represented by SEQ ID NO: 16 and microorganisms having an 18S rRNA gene sequence with a sequence identity of 98% or more to the nucleotide sequence represented by SEQ ID NO:

17. The microorganisms belonging to the family Arthopyreniaceae are microorganisms having an 18S rRNA gene sequence with a sequence identity of 98% or more to the nucleotide sequence represented by SEQ ID NO: 18 and microorganisms having an 18S rRNA gene sequence with a sequence identity of 98% or more to the nucleotide sequence represented by SEQ ID NO:

19. By culturing for 1 month under normal temperature conditions in the presence of 1 g of digested sludge as a substrate, 10 ml or more of methane can be produced. A microbial mixture for producing methane using digested sludge as a substrate.

2. A methane-producing composition containing the microbial mixture according to Claim 1 and capable of producing methane from a bio-derived composition.

3. Inoculating the microbial mixture according to Claim 1 into a solution containing a bio-derived composition. Culturing the inoculated microbial mixture under normal temperature conditions; A method for methane production, comprising the above.

4. The method for methane production according to claim 3, wherein the composition derived from organisms is digested sludge.

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