WO2017221765A1 - Microorganism, composition for degrading lignocellulose-based biomass, method for producing saccharified liquid and method for producing compound derived from lignocellulose-based biomass - Google Patents

Abstract

A microorganism that has 16S rRNA gene containing any of nucleic acids (a)-(c) and is capable of degrading a lignocellulose-based biomass: (a) a nucleic acid having a base sequence represented by SEQ ID NO: 1 or 2; (b) a nucleic acid having an identity of 95% or more to a base sequence represented by SEQ ID NO: 1 or 2; and (c) a nucleic acid having a base sequence derived from a base sequence represented by SEQ ID NO: 1 or 2 by deletion, substitution or addition of one to several bases. The microorganism is Clostridium sp. A7 strain (NITE BP-02216). The microorganism is Cellulosibacter alkalithermophilus W21-10 strain (NITE BP-02265).

Description

Microorganism, composition for decomposing lignocellulosic biomass, method for producing saccharified liquid, and method for producing compound derived from lignocellulosic biomass

The present invention relates to a microorganism, a composition for decomposing lignocellulosic biomass, a method for producing a saccharified solution, and a method for producing a compound derived from lignocellulosic biomass.
The present application claims priority based on Japanese Patent Application No. 2016-123065 filed in Japan on June 21, 2016, the contents of which are incorporated herein by reference.

In recent years, the use of biomass using plant resources as a raw material has attracted attention from the viewpoint of global warming countermeasures and effective utilization of waste. In general, the use of lignocellulosic biomass as non-edible starch in addition to sugars such as sugar cane and edible starches such as corn as raw materials for producing compounds such as ethanol from biomass has been studied. . Lignocellulose-based biomass contains lignocellulose as a main component. Lignocellulose has a structure in which cellulose, hemicellulose, and lignin are tightly bound, and it is not easy to decompose into pentose or hexose monosaccharides or oligosaccharides that can be used for fermentation.

Normally, when compounds such as ethanol are produced from lignocellulosic biomass, saccharification by enzymes is performed, so that lignocellulose, which is the main component, is decomposed so that the enzyme can come into contact with polysaccharides contained in lignocellulosic biomass. In order to do this, pre-processing is performed. Examples of the pretreatment method include pulverization, explosion, steaming, microwave irradiation, gamma ray irradiation, electron beam irradiation, dilute sulfuric acid treatment, alkali treatment, and solvolysis treatment. These pretreatment methods have problems such as a large amount of energy input, a large loss of sugar due to pretreatment, a large environmental load due to harmful substances, and a fermentation inhibitory substance produced as a by-product.
On the other hand, as a pretreatment method, a method using a white rot fungus which is a microorganism capable of decomposing lignin in lignocellulose is known.

Patent Document 1 discloses a method for pretreating a saccharification step with an enzyme and a fermentation step with a microorganism using a white rot fungus (FERM AP-20591) having a lignocellulose-degrading action or a mutant thereof.

JP 2007-37469 A

However, microorganisms having higher lignocellulose resolution than before have been demanded.

The present invention has been made in view of the above circumstances, and provides a novel microorganism excellent in lignocellulose resolution.

As a result of diligent research to solve the above-mentioned problems, the inventors of the present invention have an excellent resolution with respect to lignocellulose among the strains collected from humus, and the types of lignocellulosic biomass to be decomposed. A strain having no restriction was found and the present invention was completed.

That is, the present invention includes the following aspects.
The microorganism according to the first aspect of the present invention has a 16S rRNA gene containing any one of the following nucleic acids (a) to (c) and has the resolution of lignocellulosic biomass.
(A) a nucleic acid comprising the base sequence shown in SEQ ID NO: 1 or 2,
(B) a nucleic acid having 95% or more identity with the base sequence shown in SEQ ID NO: 1 or 2,
(C) a nucleic acid comprising a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 1 or 2

The microorganism according to the second aspect of the present invention is Clostridium sp. A7 strain (NITE BP-02216).

The microorganism according to the third aspect of the present invention is Cellulosibacter alkaline thermophilus W21-10 strain (NITE BP-02265).

The composition for decomposing lignocellulosic biomass according to the fourth aspect of the present invention includes at least one microorganism according to the first aspect, or an enzyme or gene product derived from the microorganism.
The composition for degrading lignocellulosic biomass according to the fourth aspect further has a 16S rRNA gene containing any one of the following nucleic acids (d) to (f), and the resolution of lignocellulosic biomass, or You may contain at least 1 sort (s) among the microorganisms which have the metabolic capacity of the degradation product of lignocellulosic biomass.
(D) a nucleic acid comprising the base sequence shown in any of SEQ ID NOs: 3 to 11,
(E) a nucleic acid having 90% or more identity with the base sequence shown in any of SEQ ID NOs: 3 to 11,
(F) a nucleic acid comprising a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in any of SEQ ID NOs: 3 to 11

The manufacturing method of the saccharified liquid which concerns on the 5th aspect of this invention is a method provided with the saccharification process of producing | generating a saccharified liquid from lignocellulosic biomass using the composition for lignocellulosic biomass decomposition | disassembly which concerns on the said 4th aspect. .

The method for producing a lignocellulosic biomass-derived compound according to the sixth aspect of the present invention produces a lignocellulosic biomass-derived compound from the saccharified liquid after producing the saccharified liquid using the production method according to the fifth aspect. This is a method including a production process.

According to the above aspect, a novel microorganism having an excellent lignocellulose resolution can be provided.

(A)-(C) are photomicrographs of Clostridium sp. Strain A7. (A) and (B) are photomicrographs of Cellulosibacter alkaline thermophilus W21-10 strain. This is a histogram prepared by comparing the base sequence of the 16S rDNA gene of Clostridium sp. A7 strain and Cellulosibacter alkaline thermophilus W21-10 with the base sequence of the 16S rDNA gene of closely related Bacillus species. 2 is an image showing a state of decomposition in each biomass on the fifth day of culture after inoculation with ISI-3 in Example 1. FIG. 2 is a graph showing the degradation rate of each biomass on the fifth day of culture after inoculation with ISI-3 in Example 1. FIG. It is a graph which shows the decomposition | disassembly rate in each biomass of the culture | cultivation 5th day after inoculating clostridium thermocellum ATCC27405 strain, A7 strain, W21-10 strain, or those combinations in Example 2.

≪Microorganism≫
The microorganism according to one embodiment of the present invention has a 16S rRNA gene containing any of the following nucleic acids (a) to (c), and has the resolution of lignocellulosic biomass.
(A) a nucleic acid comprising the base sequence shown in SEQ ID NO: 1 or 2,
(B) a nucleic acid having 95% or more identity with the base sequence shown in SEQ ID NO: 1 or 2,
(C) a nucleic acid comprising a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 1 or 2

Among the microorganisms of this embodiment, a microorganism having a 16S rRNA gene comprising a nucleic acid having the base sequence shown in SEQ ID NO: 1 or a functionally equivalent nucleic acid is a Neisseria gonorrhoeae belonging to the genus Clostridium, and is an excellent ligno Has cellulose resolution.
Clostridium is a genus of Gram-positive endospore-forming rods.
In addition, among the microorganisms of this embodiment, a microorganism having a 16S rRNA gene containing a nucleic acid having the base sequence shown in SEQ ID NO: 2 or a nucleic acid functionally equivalent to the nucleic acid is classified as a Cellulosibacter alkaline thermophilus species. It is a koji mold to which it belongs and has excellent lignocellulose resolution.
Cellulosibacter alkaline thermophilus is a thermophilic thermophilic anaerobic cellulose-degrading bacterium.
In the present specification, “nucleic acid” includes RNA and DNA.
In the present specification, the “16S rRNA gene” includes RNA of a small subunit of ribosome and DNA encoding the RNA.

In the present specification, the term “lignocellulose resolution” means an activity of mainly decomposing polysaccharides such as cellulose and hemicellulose into monosaccharides, oligosaccharides, or organic acids contained in lignocellulose.

<Clostridium sp. A7 strain>
The base sequence shown in SEQ ID NO: 1 in the above (a) is the base sequence of 16S rRNA gene of Clostridium sp. A7 strain (NITE BP-02216).

Separation and purification of Clostridium sp. A7 strain can be separated and purified by the method described in the Examples below. Further, the obtained strain was identified as a Clostridium bacterium from morphological observation and others, and named A7 strain.

The microbiological properties of Clostridium species A7 strain are as follows. The photomicrographs of Clostridium sp. Strain A7 taken using a scanning electron microscope (SEM) are shown in FIGS.

(Scientific nature)
Has lignocellulose resolution.

(Morphological properties)
(1) The shape of the cell is an elongated rod or cylinder. The length of the cell is about 2.0 to 6.0 μm, and the width is about 0.3 to 0.4 μm.
(2) Gram-positive bacteria and colonies are orange.

(Reproductive style)
(1) The cell grows in a form in which the long axis direction is constant and only the length increases, and when it grows to a certain size, it divides in a form that is almost equally divided at its center.

(Physiological and biochemical properties)
(1) Culture solution: Can be grown on a common medium used for culturing bacteria of the genus Clostridial.
(2) Growth temperature range: 37 ° C to 60 ° C (optimum temperature 55 ° C).
(3) Growth pH range: pH 6.0 to 10.0 (optimum pH 9.0).
(4) Nutrient source: As a carbon source, it can grow on cellulose, cellobiose, or lignocellulose. Further, as a nitrogen source, not only inorganic nitrogen such as sulfuric acid and ammonium nitrate but also organic nitrogen such as yeast extract, peptone and beef extract can be used.
(5) Product: Acetic acid, ethanol, fumaric acid, lactic acid, and succinic acid are produced.

(Taxonomic properties)
The base sequence of the 16S rDNA gene of Clostridium species A7 strain is as shown in SEQ ID NO: 1 in the sequence listing. FIG. 3 is a histogram prepared by comparing the base sequence of the 16S rDNA gene of Clostridium sp. A7 strain and Cellulosibacter alkaline thermophilus strain W21-10 with the base sequence of the 16S rDNA gene of closely related bacterial species. It is.
As a result, Clostridium species A7 strain was judged as a novel strain.

Clostridium sp. A7 strain was established by the National Institute for Product Evaluation and Technology Patent Microorganisms Depositary Center (NPMD) (2-5-8, Kazusa-Kamazu, Kisarazu-shi, Chiba) on March 8, 2016. Deposited internationally under the deposit number NITE BP-02216.

(Culture medium)
In the present embodiment, it is preferable to use a medium when culturing Clostridium sp.
The medium to be used is not limited as long as bacteria belonging to the genus Clostridium grow, and for example, a BMN medium having the composition shown in Table 1 below can be preferably used as such a medium.
Examples of other media that can be used include NB (Nutrient Broth) medium (for example, “Nutrient Broth, Bacto” manufactured by Difco (containing 3 g / L of beef extract and 5 g / L of peptone)), etc. Can be mentioned. Among them, a BMN medium having the composition shown in Table 1 is particularly preferable because it decomposes lignocellulose with high efficiency.

(Culture method)
In the present embodiment, the culturing method for Clostridium species A7 can be performed by a known and commonly used method. In the culture, the above medium can be used.
In the present embodiment, examples of the culture method include a stationary culture method, a shaking culture method, a deep aeration-agitation culture method, and the like.

The pH in the culture can be neutral to basic at 6.0 to 10.0, and is preferably pH 9.0.

The culture temperature can be 37 ° C or higher and 60 ° C or lower, preferably 55 ° C. When the culture temperature is within the above range, the saccharification process and fermentation process using general lignocellulosic biomass are in the same temperature range, so Clostridium species A7 strain efficiently decomposes lignocellulose and saccharifies. A liquid or lignocellulosic biomass-derived compound can be obtained.

In the present embodiment, when the Clostridium species A7 strain is cultured by the above-described culture method, the Clostridium species A7 strain having not only stable growth but also improved lignocellulose resolution is obtained.

In general, “lignocellulose” is a component of a plant cell wall, and is mainly composed of cellulose, hemicellulose, and lignin. The cellulose in lignocellulose has a strong crystal structure in which linear polymers composed of β-1,4 glucose are bundled by hydrogen bonds.
Further, in this specification, “lignocellulose-based biomass” includes lignocellulose as a main component. Examples of lignocellulosic biomass include conifers (e.g., cedar, spruce, larch, black pine, todomatsu, himekomatsu, yew, nezuko, spruce fir, irakimi, inakiki, fir, sawara, togasawara, asunaro, hiba, tsuga, kotsutsuga, hinoki. , Yew, Inugaya, Spruce, Yellow Cedar (Beihiba), Lawson Cypress (Beihi), Douglas Fir (Beimatsu), Sitka Spruce (Beisuhi), Radiata Pine, Eastern Spruce, Eastern White Pine, Western Larch, Western Fur, Western Hemlock, Tamarac and related tree species, etc.), broad-leaved trees (for example, asben, American black cherry, yellow poplar, walnut, cabbage cherry, zelkova, sycamore, silver cherry, Peach, teak, chinese elm, chinese maple, oak, hard maple, hickory, pecan, white ash, white oak, white birch, red oak and related tree species), rice, wheat, corn (especially corn stover) Cores, stems, leaves)), pineapples, oil palm, cassava, sugar cane (especially sugar cane bagasse, sugar cane stems and leaves) and other agricultural products and wastes; kenaf, cotton and other industrial plants and wastes ; Forage crops such as alfalfa and timothy; and include, but are not limited to, Eliensus, bamboo, and Sasa. These lignocellulosic biomasses may be used alone or as a mixture.

The shape of the lignocellulosic biomass to be decomposed by microorganisms of the present embodiment includes, but is not limited to, for example, powder, chip, square, log, flake, and fiber. Especially, it is preferable that it is a powder form, a chip form, flake form, or a fiber form from a viewpoint of performing the decomposition | disassembly by the microorganisms of this embodiment efficiently.

In the present specification, “hemicellulose” includes a so-called pentose having 5 carbons as a structural unit, such as xylose, and a hexose having 6 carbons as a structural unit, such as mannose, arabinose, galacturonic acid, and the like. What is called, and complex polysaccharides such as glucomannan and glucuronoxylan are included. Therefore, when hemicellulose is hydrolyzed, a pentose monosaccharide consisting of 5 carbons, a pentose oligosaccharide composed of a plurality of such monosaccharides, a hexose monosaccharide consisting of 6 carbons, A hexose oligosaccharide in which a plurality of the monosaccharides are linked, and an oligosaccharide in which a plurality of pentose monosaccharides and a hexose monosaccharide are linked.
“Cellulose” includes hexose containing 6 carbons as a structural unit. Thus, when cellulose is hydrolyzed, it produces a six-carbon monosaccharide consisting of six carbons and a hexose oligosaccharide in which a plurality of such monosaccharides are linked.
In general, the composition ratio and the amount of monosaccharide or oligosaccharide produced from hemicellulose or cellulose vary depending on the pretreatment method and the type of lignocellulosic biomass used as a raw material.

In the present specification, “a compound derived from lignocellulosic biomass” means a compound produced by ingesting monosaccharides and oligosaccharides obtained by decomposing lignocellulosic biomass by microorganisms such as yeast. . Specific examples of the lignocellulosic biomass-derived compound include alcohols such as ethanol, butanol, 1,3-propanediol, 1,4-butanediol, and glycerol; pyruvic acid, fumaric acid, succinic acid, malic acid, itacone Examples thereof include organic acids such as acid, citric acid, acetic acid, and lactic acid; nucleosides such as inosine and guanosine; nucleotides such as inosine acid and guanylic acid; and diamine compounds such as cadaverine. When the obtained lignocellulose-based biomass-derived compound is a monomer such as lactic acid, it may be converted into a polymer by polymerization.

<Cellulosibacter alkaline thermophilus W21-10 strain>
The base sequence shown in SEQ ID NO: 2 in (a) above is the base sequence of the 16S rRNA gene of Cellulosibacter alkaline thermophilus W21-10 strain (NITE BP-02265).

Separation and purification of Cellulosibacter / alkaline thermophilus W21-10 can be separated and purified by the method shown in the Examples below. The obtained strain was identified as an alkaline thermophilus species belonging to the genus Cerulosibacter from morphological observation and others, and was named W21-10 strain.

The microbiological properties of Cellulosibacter / alkaline thermophilus W21-10 are as follows. 2A and 2B show micrographs of Cellulosiacter alkaline thermophilus W21-10 photographed using a scanning electron microscope (SEM).

(Scientific nature)
Has lignocellulose resolution.

(Morphological properties)
(1) The shape of the cell is an elongated rod or cylinder. The cell length is about 2.0-3.0 μm and the width is about 0.2-0.3 μm.
(2) Gram-positive bacteria and colonies are yellow.

(Reproductive style)
(1) The cell grows in a form in which the long axis direction is constant and only the length increases, and when it grows to a certain size, it divides in a form that is almost equally divided at its center.

(Physiological and biochemical properties)
(1) Culture solution: It can be grown on a medium for culturing common cellulobacter bacteria.
(2) Growth temperature range: 37 ° C. or more and 65 ° C. or less (optimum temperature 60 ° C.).
(3) Growth pH range: pH 8.0 to 10.0 (optimum pH 9.5).
(4) Nutrient source: As a carbon source, it can grow on cellulose, cellobiose, lignocellulose, starch, pectin, sorbitol, mannitol, or glycerol. Further, as a nitrogen source, not only inorganic nitrogen such as sulfuric acid and ammonium nitrate but also organic nitrogen such as yeast extract, peptone and beef extract can be used.
(5) Product: Acetic acid, ethanol, fumaric acid, lactic acid, and succinic acid are produced.

(Taxonomic properties)
The base sequence of the 16S rRNA gene of Cellulosibacter alkaline thermophilus strain W21-10 is as shown in SEQ ID NO: 2 in the sequence listing. FIG. 3 is a histogram prepared by comparing the base sequence of the 16S rDNA gene of Clostridium sp. A7 strain and Cellulosibacter alkaline thermophilus strain W21-10 with the base sequence of the 16S rDNA gene of closely related bacterial species. It is.
As a result, cellulosibacter alkaline thermophilus W21-10 was determined to be a novel strain.

Cellulosibacter Alkaline Thermophilus W21-10 was established on May 24, 2016, by the National Institute of Technology and Evaluation for Microorganisms (NPMD) (2-5-8 Kazusa Kamashitsu, Kisarazu City, Chiba Prefecture). Has been deposited internationally under the accession number NITE BP-02265 under the regulations of the Putabest Convention.

(Culture medium)
In the present embodiment, it is preferable to use a medium for culturing the Cellulosibacter alkaline thermophilus W21-10 strain.
The medium to be used is not limited as long as the cellulobacter bacterium grows. For example, a BMN medium having the composition shown in Table 1 can be preferably used as such a medium.
Examples of other media that can be used include NB (Nutrient Broth) medium (for example, “Nutrient Broth, Bacto” manufactured by Difco (containing 3 g / L of beef extract and 5 g / L of peptone)), etc. Can be mentioned. Among them, a BMN medium having the composition shown in Table 1 is particularly preferable because it decomposes lignocellulose with high efficiency.

(Culture method)
In the present embodiment, the method for culturing the Cellulosibacter alkaline thermophilus W21-10 strain can be carried out by a known and commonly used method. In the culture, the above medium can be used.
In the present embodiment, examples of the culture method include a stationary culture method, a shaking culture method, a deep aeration-agitation culture method, and the like.

The culture can be performed at a basic pH of 8.0 to 10.0, preferably pH 9.5.

The culture temperature can be 37 ° C or higher and 65 ° C or lower, preferably 60 ° C. When the culture temperature is within the above range, the cellulosibacter alkaline thermophilus strain W21-10 is efficiently used because it is in the same temperature range as the saccharification process and fermentation process using general lignocellulosic biomass. Lignocellulose can be decomposed to obtain a saccharified solution or a lignocellulose-based biomass-derived compound.

In the present embodiment, when the Cellulosibacter alkaline thermophilus W21-10 strain is cultured by the above-described culture method, the cellulosibacter alkaline thermophila not only shows stable growth but also has improved lignocellulose resolution. S21-10 strain is obtained.

The microorganism of this embodiment may have a 16S rRNA gene containing a nucleic acid of the following (b) as a 16S rRNA gene containing a nucleic acid functionally equivalent to the above (a).
(B) a nucleic acid having 95% or more identity with the base sequence shown in SEQ ID NO: 1 or 2.

In order to be functionally equivalent to the 16S rRNA gene containing the nucleic acid (a), it has 95% or more identity. Such identity is preferably 95% or more, more preferably 96% or more, still more preferably 97% or more, particularly preferably 98% or more, and most preferably 99% or more.
Furthermore, the 16S rRNA gene containing the nucleic acid of (b) has lignocellulose resolution.

The microorganism of this embodiment may have a 16S rRNA gene containing a nucleic acid of the following (c) as a 16S rRNA gene containing a nucleic acid functionally equivalent to the above (a).
(C) A nucleic acid comprising a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 1 or 2.

Here, the number of bases that may be deleted, substituted, or added is preferably 1 or more, 15 or less, more preferably 1 or more and 10 or less, and particularly preferably 1 or more and 5 or less.
Furthermore, the 16S rRNA gene containing the nucleic acid of (c) has lignocellulose resolution.

≪Lignocellulosic biomass decomposition composition≫
The composition for decomposing lignocellulosic biomass according to one embodiment of the present invention comprises at least one kind of the above-mentioned microorganisms, or an enzyme or gene product derived from the microorganisms.

According to the composition for lignocellulose-based biomass decomposition of the present embodiment, lignocellulose can be decomposed with high efficiency, and a saccharified solution or lignocellulose-based biomass-derived compound can be obtained in high yield.

Examples of the microorganisms contained in the lignocellulosic biomass decomposing composition of the present embodiment include the same microorganisms as those described in the above-mentioned << microorganism >>. Among them, the microorganism is preferably Clostridium sp. A7 strain or Cellulosiacter alkaline thermophilus W21-10 strain.

The lignocellulosic biomass decomposing composition of the present embodiment may contain an enzyme or gene product derived from the microorganism instead of the microorganism described above.
Examples of the microorganism-derived enzyme include exo-1,4-β-glucanase, endo-1,4-β-glucanase, cellulase (eg, endoglucanase (EG), cellobiohydrolase (CBH) and β-). Polysaccharide degrading enzymes such as glucosidase (BGL), hemicellulase (eg, xylanase, xylosidase, mannanase, pectinase, galactosidase, glucuronidase, arabinofuranosidase, etc.).

Examples of the gene product derived from the microorganism include DNA or RNA encoding the above-described enzyme. The gene product may be in a form incorporated into an expression vector. Examples of expression vectors include plasmids derived from E. coli such as pBR322, pBR325, pUC12, and pUC13; plasmids derived from Bacillus subtilis such as pUB110, pTP5, and pC194; plasmids derived from yeast such as pSH19 and pSH15; bacteriophages such as λ phage; Examples include, but are not limited to, viruses such as adenovirus, adeno-associated virus, lentivirus, vaccinia virus, baculovirus, and vectors modified from these.

The lignocellulosic biomass decomposing composition of the present embodiment may contain one type of microorganism, or an enzyme or gene product derived from the microorganism, or may contain two or more types. Especially, since the composition for lignocellulose-type biomass decomposition | disassembly of this embodiment can decompose | disassemble lignocellulose with high efficiency, it is preferable that 2 or more types of microorganisms or the said enzyme or gene product derived from the said microorganisms are included.

<Other microorganisms>
The composition for degrading lignocellulosic biomass of the present embodiment may further contain at least one of microorganisms having a 16S rRNA gene containing the following nucleic acid (d).
(D) a nucleic acid comprising the base sequence shown in any one of SEQ ID NOs: 3 to 11.

The base sequence shown in SEQ ID NO: 3 in (d) above is the base of the 16S rRNA gene of a bacterial species having 88% identity to the base sequence of the 16S rRNA gene of the Mororella glycerini JW / AS-Y6 strain. Is an array.
The base sequence shown in SEQ ID NO: 4 in the above (d) is the base sequence of a 16S rRNA gene of a bacterial species having 87% identity to the base sequence of the 16S rRNA gene of the Mororella humiferella 64-FGQ strain. is there.
The base sequence shown in SEQ ID NO: 5 in (d) above is the base sequence of a 16S rRNA gene of a bacterial species having 88% identity to the base sequence of the 16S rRNA gene of Tepidanaerobacteracetoxydans Re1 strain It is.
The base sequence shown in SEQ ID NO: 6 in the above (d) is the base sequence of the 16S rRNA gene of a bacterial species having 91% identity to the base sequence of the 16S rRNA gene of the Tepidanaerobacteracetoxydans Re1 strain It is.
The base sequence shown in SEQ ID NO: 7 in the above (d) is the base sequence of the 16S rRNA gene of a bacterial species having 95% identity to the base sequence of the 16S rRNA gene of the Tepidanaerobacteracetoxydans Re1 strain It is.
The base sequence shown in SEQ ID NO: 8 in the above (d) is the base of the 16S rRNA gene of a bacterial species having 96% identity to the base sequence of the 16S rRNA gene of Tepidimicrobium ferriphyllum DSM 16624 strain Is an array.
The base sequence shown in SEQ ID NO: 9 in (d) above is a 16S rRNA of a bacterial species having 99% identity to the base sequence of the 16S rRNA gene of Clostridium thermocellum DSM1313 strain or Clostridium thermocellum ATCC27406 strain It is the base sequence of a gene.
The base sequence shown in SEQ ID NO: 10 in (d) above is the base sequence of a 16S rRNA gene of a bacterial species having 99% identity to the base sequence of the 16S rRNA gene of Thermoanaerobacter mastranii strain A3. is there.
The base sequence shown in SEQ ID NO: 11 in the above (d) is a 16S rRNA of a bacterial species having 94% identity to the base sequence of the 16S rRNA gene of the uncultured microorganism ATB-CK-1492-11 strain. It is the base sequence of a gene.
Morella glycerini is a thermophilic, anaerobic, spore-forming bacterium.
Morella Humiferella is a thermophilic, anaerobic bacterium that can grow through reciprocating electrons between humic acid and iron (III).
Tepidanaerobacter acetatoxidane is an acetic acid oxidation symbiotic microorganism.
Tepididimicrobium ferriphyllum is an anaerobic moderate thermophile.
Clostridium thermocellum is a thermophilic bacterium that has a characteristic enzyme complex called cellulosome and efficiently decomposes cellulose.
Thermoanaerobacter masalani is a thermophilic ethanol-producing bacterium.
Generally, difficult-to-cultivate microorganisms mean microorganisms that have been systematically different from microorganisms that could not be separated and cultured and known microorganism species.

Among these, the lignocellulose-based biomass decomposing composition of the present embodiment preferably contains all nine types of microorganisms having a 16s rRNA gene containing a nucleic acid having the base sequence shown in any of SEQ ID NOs: 3 to 11. Thereby, lignocellulose can be decomposed | disassembled more efficiently and a saccharified liquid or a lignocellulose biomass-derived compound can be obtained with a high yield.

In addition, the lignocellulose-based biomass decomposing composition of the present embodiment comprises a microorganism having a 16s rRNA gene containing the following nucleic acid (e) as a 16S rRNA gene containing a nucleic acid functionally equivalent to the above (d): May be included.
(E) a nucleic acid having 90% or more identity with the base sequence shown in any of SEQ ID NOs: 3 to 11.

In order to be functionally equivalent to the 16S rRNA gene containing the nucleic acid of (d), it has 95% or more identity. Such identity is preferably 95% or more, more preferably 96% or more, still more preferably 97% or more, particularly preferably 98% or more, and most preferably 99% or more.
Further, the 16S rRNA gene containing the nucleic acid (e) has lignocellulose resolution or the ability to metabolize lignocellulosic biomass degradation products.
In the present specification, “the ability to metabolize the degradation products of lignocellulosic biomass” means the ability to metabolize the degradation products of lignocellulosic biomass. Specifically, the metabolic capacity of the degradation product of lignocellulosic biomass, for example, metabolizes monosaccharides, oligosaccharides, or organic acids that are degradation products of lignocellulosic biomass to produce the above-mentioned lignocellulosic biomass-derived compounds Ability to do so.

The composition for degrading lignocellulosic biomass of the present embodiment includes a microorganism having a 16s rRNA gene containing a nucleic acid of the following (f) as a 16S rRNA gene containing a nucleic acid functionally equivalent to the above (d). May be.
(F) A nucleic acid comprising a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in any of SEQ ID NOs: 3 to 11.

Here, the number of bases that may be deleted, substituted, or added is preferably 1 or more, 15 or less, more preferably 1 or more and 10 or less, and particularly preferably 1 or more and 5 or less.
Furthermore, the 16S rRNA gene containing the nucleic acid (f) has lignocellulose resolution or the ability to metabolize lignocellulosic biomass degradation products.

In the composition for degrading lignocellulosic biomass of this embodiment, an enzyme or gene product derived from the microorganism is included instead of the microorganism having the 16S rRNA gene containing any one of the nucleic acids (d) to (f). May be.

Examples of the microorganism-derived enzyme include exo-1,4-β-glucanase, endo-1,4-β-glucanase, cellulase (eg, endoglucanase (EG), cellobiohydrolase (CBH) and β-). Glucosidase (BGL), etc.), hemicellulases (eg, xylanase, xylosidase, mannanase, pectinase, galactosidase, glucuronidase, arabinofuranosidase, etc.) and the like, and further, proteolytic enzymes, organic acid degrading enzymes Etc. may be included.

Examples of the gene product derived from the microorganism include DNA or RNA encoding the above-described enzyme. The gene product may be in a form incorporated into an expression vector. Examples of expression vectors include those similar to those exemplified above.

The lignocellulosic biomass decomposing composition of the present embodiment may have any shape as long as the lignocellulose-decomposing ability of the contained microorganism, or the enzyme or gene product derived from the microorganism is not impaired. It may be in a dry state or in a state of being stirred in a liquid medium or the like.

≪Method for producing saccharified liquid≫
The manufacturing method of the saccharified liquid which concerns on one Embodiment of this invention is a method provided with the saccharification process of producing | generating a saccharified liquid from lignocellulosic biomass using the above-mentioned composition for lignocellulosic biomass decomposition | disassembly.

According to the production method of the present embodiment, a saccharified solution can be obtained from lignocellulosic biomass with high efficiency and high yield.

<Saccharification process>
A saccharified solution is produced from lignocellulosic biomass using the above-described composition for decomposing lignocellulosic biomass.

Examples of lignocellulosic biomass used in the production method of the present embodiment include those similar to those exemplified in the above-mentioned << microorganism >>.

In the production method of the present embodiment, the water content is added by adding about 5 to 500 parts by weight, preferably about 50 to 400 parts by weight, more preferably about 50 to 300 parts by weight with respect to 100 parts by weight of lignocellulosic biomass. Can be adjusted.
Further, in the lignocellulosic biomass, when the above-mentioned composition for decomposing lignocellulosic biomass is a microorganism, salts and nutrients (for example, bran, peptone, corn) required for the growth of the microorganism are included. Steep liquor, yeast extract, meat extract, malt extract, potato extract, rice bran, synthetic inorganic salts, etc.) may be added.
Alternatively, a medium that can be used for the growth of the microorganism may be added instead of moisture, salt, and nutrients. Examples of the medium include the same media as those exemplified above in <Clostridium sp. Strain A7>.
Further, the lignocellulosic biomass may be subjected to a sterilization treatment such as heat sterilization in order to prevent propagation of various bacteria before mixing with the above-described composition for decomposing lignocellulose biomass.

The saccharification temperature is preferably 45 ° C or higher and 70 ° C or lower, more preferably 45 ° C or higher and 65 ° C or lower, and particularly preferably 50 ° C or higher and 60 ° C or lower. The saccharification time is preferably 12 hours to 120 hours, more preferably 24 hours to 96 hours, and even more preferably 24 hours to 72 hours.

In the saccharification step, the above-described saccharification reaction conditions are appropriately set based on the type of microorganism, enzyme, or gene product contained in the above-described lignocellulosic biomass decomposition composition, the target saccharification degree, and the like.

In the production method of the present embodiment, a pretreatment step may be provided before the saccharification step.
Examples of the pretreatment process include pulverization, microwave irradiation, blasting, steaming, irradiation, chemical treatment (for example, solvolysis, ozone, alkali, acid, oxidant, reducing agent, etc. ), Fungus treatment, oxidase treatment, or combined treatment thereof.

By the saccharification step, the lignocellulosic biomass is reduced in molecular weight to produce a monosaccharide or oligosaccharide, and a saccharified solution containing the monosaccharide or oligosaccharide can be obtained.
By performing various fermentation treatments using the monosaccharide or oligosaccharide having a reduced molecular weight as a carbon source, various lignocelluloses as described in << Method for producing lignocellulosic biomass-derived compound >> described below. A biomass-derived compound can be produced.

≪Method for producing compound derived from lignocellulosic biomass≫
The method for producing a lignocellulosic biomass-derived compound according to an embodiment of the present invention produces a lignocellulosic biomass-derived compound from the saccharified solution after producing the saccharified solution using the above-described saccharified solution producing method. A method comprising a production process.

According to the production method of the present embodiment, a lignocellulosic biomass-derived compound can be obtained from a saccharified solution with high efficiency and high yield.

<Production process>
The lignocellulosic biomass-derived compound is produced using the saccharified solution obtained by the above-mentioned << saccharified solution production method >>.

The production process may be any treatment method capable of obtaining a lignocellulosic biomass-derived compound, and examples thereof include fermentation treatment and chemical synthesis treatment. Examples of fermentation include ethanol fermentation, methane fermentation, hydrogen fermentation, acetone-butanol fermentation, lactic acid fermentation, succinic acid fermentation, itaconic acid fermentation, and amino acid fermentation. Known microorganisms and fermentation treatment conditions may be used for these fermentation treatments.

The fermentation treatment may be performed after the saccharification step or may be performed simultaneously with the saccharification step. When the latter fermentation treatment and the saccharification step are performed simultaneously, the above-mentioned lignocellulosic biomass decomposing composition required for the saccharification step and the microorganisms required for the fermentation treatment (for example, yeast, etc.) are allowed to coexist, What is necessary is just to set and incubate on the conditions which both the composition for lignocellulose type | system | group biomass decomposition | disassembly and microorganisms can act or grow effectively.

The microorganism used for the fermentation treatment is not particularly limited as long as the target lignocellulose-based biomass-derived compound can be produced. Specific examples of the microorganism used for the fermentation treatment include yeast and bacteria, and may be a genetically modified microorganism. A genetically modified microorganism refers to a microorganism that does not have an enzyme gene required for conversion to a target lignocellulosic biomass-derived compound, such as alcohol, by introducing these genes using genetic engineering technology. This enables generation of a cellulosic biomass-derived compound. Examples of the genetically modified microorganism include genetically modified Escherichia coli having alcohol fermentability.

Examples of the lignocellulosic biomass-derived compound obtained in the production process include the same compounds as those exemplified in the above-mentioned << microorganism >>.

Moreover, in the manufacturing method of this embodiment, the refinement | purification process may be provided after the production | generation process.
A refinement | purification process is a process for refine | purifying the lignocellulosic biomass compound obtained in the said production | generation process.
As a purification method, when the lignocellulosic biomass compound is an alcohol, for example, a distillation method and the like can be mentioned. Further, when the lignocellulose-based biomass compound is an amino acid, for example, an ion exchange method, a foreign matter adsorption removal method using activated carbon, and the like can be mentioned.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples.

[Example 1]
(1) Selection of biomass-degrading bacteria First, a wet weight of about 2 to 5 g of compost sample containing sugarcane foliage, rice husks, and cow manure, and fiber or corn foliage after squeezing oil palm trunk are ground in a coffee mill. The fiber material was suspended in 20-25 mL of BMN medium. Next, the obtained suspension was sufficiently bubbled with nitrogen (industrial grade), the gas phase was replaced with nitrogen gas, sealed with a butyl rubber stopper, and cultured at 60 ° C. for 3 days to 1 week. went.
The composition of the BMN medium was 1.5 g / L potassium dihydrogen phosphate, 2.9 g / L dipotassium hydrogen phosphate, 2.1 g / L urea, 2 g / L yeast extract (manufactured by Difco). ) 4 g / L sodium carbonate, 0.5 g / L cysteine hydrochloride, 0.2 mL mineral solution (MgCl ₂ .6H ₂ O 5 g; CaCl ₂ .2H ₂ O 0.75 g; FeSO ₄ .6H ₂ O 0.0063 g dissolved in 4 mL of water).

Next, after culturing for about 3 days to 1 week, compared to the above culture solution with no sample added, the color change of the fibrous material due to microbial degradation, the apparent decrease in the amount added, and the viscosity of the powder were clearly observed, clearly A culture solution in which a difference was observed was selected. Subsequently, the selected culture broth was well stirred and suspended, and 0.5 mL of the broth was inoculated into the BMN medium containing the new fiber material, and cultured again under the same conditions. This operation was repeated 2-3 times to accumulate and culture the biomass fiber-degrading bacteria.

Then, after about 4 days of enrichment culture, a culture solution capable of visually reducing the volume of 1% biomass fiber by half or less was selected. A part of the culture solution is inoculated at an appropriate dilution rate into a BMN agar medium containing 1% biomass fiber and 1.5% agar (Difco), and cultured at 60 ° C. for 120 to 144 hours. went.
Only colonies that appeared after 120 hours to 144 hours were selected and again inoculated into BMN liquid medium containing 1% of the biomass fiber, and the resolution was confirmed. Further, this colony separation operation was repeated twice in order to purify the separated cellulose-degrading bacteria.

In order to further purify the degrading bacteria, a part of the culture solution of the separated colonies was inoculated on a BMN agar medium containing 0.5% cellobiose or 1% crystalline cellulose. Next, colonies that appeared on the agar medium containing 0.5% cellobiose were isolated and separated under anaerobic conditions, and the resolution was confirmed with the BMN medium containing biomass fiber, and the colonies showing high degradability were finally high-decomposed. It was selected as a sex bacterium and named ISI-3.

(2) Examination of biomass resolution Next, in order to examine the biomass resolution of ISI-3 separated in (1), the fiber resolution was examined using various biomass fibers. The biomass resolution of ISI-3 was measured using the following six types of biomass fiber.
i) Dried cassava pulp discharged from the starch factory (hereinafter sometimes referred to as “cassava pulp”)
ii) A pulverized product of sugarcane fiber (sugarcane bagasse) which is a sugarcane juice residue (hereinafter, sometimes referred to as “sugarcane bagasse”).
iii) Ground corn stover (corn stover) (hereinafter sometimes referred to as “corn stover”)
iv) Fiber pulverized material obtained from old oil palm trees (hereinafter, sometimes referred to as “palm trunk fiber”)
v) Rice straw ground product (hereinafter sometimes referred to as “cassava pulp”)
vi) A dried fiber pulverized product of wild relative Eliansus (hereinafter sometimes referred to as “Eliansus”).
Each of the six types of biomass was dried at 70 ° C. for 3 days and pulverized using a pulverizing mill (Wonder Blender WB-1 manufactured by Osaka Chemical Co., Ltd.).
The method for measuring biomass resolution is to reduce the volume of appearance by inoculating ISI-3 using a BMN liquid medium containing 1% of the biomass, and finally dry the decomposed product from the weight of each input biomass. The difference in weight was calculated, and the amount of biomass that could be decomposed was calculated by expressing it in a ratio.
More specifically, as a method of calculating the amount of biomass that can be decomposed, first, the BMN liquid medium containing the biomass and each culture solution of ISI-3 were mixed well so that the remaining solids became uniform. . Next, 3 mL of each 3 mL was collected from the culture solution and transferred to a 15 mL Falcon tube whose empty weight was measured in advance. Next, the falcon tube was separated into a supernatant and a precipitate at 8,000 rpm at 4 ° C. The precipitate was suspended in 5 mL of distilled water, centrifuged again, and the supernatant was removed. Subsequently, in order to dry the obtained precipitate, it was dried for 3 days in an incubator at 80 ° C. Next, the weight of the falcon tube containing the dried precipitate was measured, and the dry weight of the precipitate was calculated by subtracting the weight of the empty falcon tube. The resulting dry weight was then divided by 3 to give the biomass dry weight in 1 mL. Furthermore, the biomass degradation rate (ratio of the amount of biomass saccharified and solubilized) was calculated by the following calculation formula. The test was repeated three times to obtain an average value.
Biomass decomposition rate (%)
= [{(Biomass dry weight before decomposition [g / mL])-(Dry weight of residue contained in culture broth after decomposition [g / mL])} / Biomass dry weight before decomposition [g / mL ]] × 100

4 and 5 show the results of ISI-3 biomass resolving test using the six types of biomass fibers. FIG. 4 is an image showing the state of degradation in each biomass on the fifth day of culture after inoculation with ISI-3. In each biomass fiber image, the left culture tube is a control without inoculation with ISI-3, and the right culture tube is a culture with ISI-3 inoculation. FIG. 5 is a graph showing the degradation rate of each biomass on the fifth day of culture after ISI-3 inoculation.

4 and 5, the biomass decomposition efficiency by ISI-3 was about 40 to 60% for 6 types of biomass.

(3) Identification of bacterial species contained in ISI-3 (3-1) Extraction of genomic DNA In order to clarify the taxonomic properties of ISI-3, a base sequence analysis of 16S rRNA was performed. ISI-3 genomic DNA was extracted by the following procedure.
First, ISI-3 was cultured for 4 days using a BMN liquid medium containing the biomass fiber as a carbon source. Subsequently, the culture was centrifuged at 10,000 rpm for 5 minutes at 4 ° C., and the cells cultured with each biomass were collected. Next, in order to lyse the obtained cells, 10% SDS (sodium lauryl sulfate) to a final concentration of 0.5% and proteinase K (1 mg / mL) to a final concentration of 5 μg / mL The solution was added and reacted at 37 ° C. for 1 hour. Next, 10% cetyltrimethylammonium bromide-0.7M sodium chloride solution was added to a final concentration of 1%, and the mixture was reacted at 65 ° C. for 10 minutes. Subsequently, an equal volume of chloroform-isoamyl alcohol solution was added to the culture solution after the reaction, followed by thorough stirring. Next, centrifugation was performed at 15,000 rpm for 5 minutes to obtain an aqueous layer. Next, an equivalent amount of the phenol-chloroform-isoamyl alcohol mixed solution was added to the aqueous layer again and stirred. Next, centrifugation was performed at 15,000 rpm for 5 minutes to obtain an aqueous layer. Subsequently, 0.6-fold volume of isopropanol was added to the obtained aqueous layer to precipitate genomic DNA. Subsequently, centrifugation was performed to prepare genomic DNA. Next, the prepared genomic DNA was washed with 70% ethanol and dried.

(3-2) Amplification of genomic DNA PCR primers for 16S rRNA amplification were 27F oligonucleotide primer (5'-AGAGTTTGATCCTGGCTCAG-3 ': SEQ ID NO: 12) and 1492R oligonucleotide primer (5'-GGCACTCTGTTACGACTTT-3': sequence Number 13) was used. In PCR, 16S rRNA gene was amplified by ExTaq DNA polymerase (Takara Shuzo). PCR was carried out under conditions of 30 cycles of 98 ° C. for 1 minute, 55 ° C. for 1 minute and 72 ° C. for 2 minutes. The obtained PCR product confirmed the amplified band by 0.8% agarose gel electrophoresis, and then purified the amplified PCR product using a QIAGEN PCR purification kit (manufactured by QIAGEN).

(3-3) Analysis of genomic DNA For nucleotide sequence analysis and homology search, the methods described in References 1 and 2 (Reference 1: [Lane, DJ, et al., “In Stackebrandt, E. and Goodfellow, M. (eds.), Nucleic acid techniques in bacterial systematics “, 16S / 23S rRNA sequencing. P115-175, John Wiley & Sons, New York, 1991.], Reference 2: [Takai, K. and Horikoshi , K., “Rapid detection and quatification of members of archaeal community by quantitative PCR using fluorogenic probes”, Appl. Environ. Microbiol., 66, p5066-5072, 2000.]), the GenBank / EMBL / DDBJ database And performed by BLAST (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi).
In order to consider a microbial consortium in each biomass inoculated with ISI-3, molecular phylogenetic analysis was performed using the base sequence of the amplified 16s rRNA gene, and the presence of 12 microorganisms was confirmed. The base sequences of the 16S rRNA genes of these 11 microorganisms are shown in SEQ ID NOs: 1 to 11 in the sequence listing. As a result of the homology search, the microorganism consortium included in ISI-3 is the following 11 microorganisms.
-Unclosed Clostridium sp. Bacterial species having 99% homology to the base sequence of 16S rRNA gene of clone De3176 (SEQ ID NO: 1)
Cell strain having a homology of 99% with the base sequence of the 16S rRNA gene of Cellulosibacterium alkalythermophilus A6 (SEQ ID NO: 2)
-Bacterial species having 88% homology to the base sequence of the 16S rRNA gene of Moorella glycerini strain JW AS-Y6 (SEQ ID NO: 3)
-Species having a homology of 87% to the base sequence of 16S rRNA gene of Moorella humiferrea strain 64-FGQ (SEQ ID NO: 4)
-Three bacterial species having a homology of 88%, 91%, or 95% to the nucleotide sequence of the 16S rRNA gene of Tepidanaerobacteracetoxydan strain strain Re1 (SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7)
A bacterial species having 96% homology to the base sequence of the 16S rRNA gene of Tepidimicrobium ferriphyllum strain DSM 16624 (SEQ ID NO: 8)
A bacterial species having 99% homology to the base sequence of the 16S rRNA gene of Clostridium thermocellum DSM 1313 or Clostridium thermocellum ATCC 27406 (SEQ ID NO: 9)
-A bacterial species having 99% homology to the base sequence of the 16S rRNA gene of Thermoanaerobacter mathranii A3 (SEQ ID NO: 10)
A bacterial species showing 94% homology to the base sequence of the 16S rRNA gene of Uncultured bacteria clone ATB-CK-1492-11 (SEQ ID NO: 11)
In addition, the identification of the bacterial species contained in ISI-3 this time revealed that approximately 2 to 5 bacterial species always coexist in a consortium and efficiently decompose biomass.

Next, in each biomass fiber used as the carbon source, the types of microorganisms in which the ISI-3 microbial consortium exists are shown in Table 2.

In the culture using all the biomass fibers, Uncultured Clostridium sp. It was a bacterial species having 99% homology to the base sequence of the 16S rRNA gene of Clone De3176 and a bacterial species having 99% homology to the base sequence of the 16S rRNA gene of Cellulosibacterium alkalythermophilus A6.

Subsequently, Clostridium thermocellum DSM 1313 was present in the culture using various types of biomass (cassava pulp, sugarcane bagasse, corn stover, palm stem fiber, and Eliansus).

Also, cassava pulp, sugarcane bagasse, corn stover, and palm stem fiber existed in the Tepidanaerobacteracetoxydan strain Re1, 88%, or 91% homology of 96% in the Epidimicrobium ferrifilum 96 strain DS24 It was a bacterial species having 99% homology to Thermoanaerobacter mastranii A3.

In addition, in the culture using corn stover, palm stem fiber, and Elianthus, the bacterial species having 88% homology to Moorella glycerini train JW / AS-Y6, and Uncultured bacteria clone ATB-CK-1492 It was a bacterial species having 94% homology to -11.

Further, in the culture using cassava pulp and palm stem fiber, a bacterial species having 87% homology with Moorella humiferrea strain 64-FGQ was present.

From the above, it was clarified that microbial species whose existence was confirmed made a consortium and efficiently decomposed biomass depending on the type of biomass.

In addition, from the results of this culture test, it was found that Uncultured Clostridium sp. a strain having 99% homology to the base sequence of the 16S rRNA gene of clone De3176, and a strain having 99% homology to the base sequence of the Cellulosibacterium alkalithermophilus A6 16S rRNA gene, a Clostridium bacterium, a Thermoanaerobacter bacterium, It has been clarified that the involvement of Tepidanaerobacter, Tepidimicrobium, and Moorella bacteria is important.
Among them, Unclosed Clostridium sp. With regard to the bacterial species having 99% homology to the base sequence of the Clone De3176 16S rRNA gene, and the bacterial species having 99% homology to the base sequence of the Cellulosibacterium alkalithermophilus A6, particularly in all biomass. The presence was confirmed, suggesting that it plays an important role in all these biomass degradations. Therefore, pure separation from ISI-3 was attempted for the above two types of bacterial species.

(4) Isolation of two kinds of bacterial species From the result of (3), Unclosed Clostridium sp. Bacteria having 99% homology to the base sequence of the 16S rRNA gene of clone De3176 and strains having 99% homology to the base sequence of the Cellulosibacterium alkalythermophilus A6 are strains having high cellulose resolution. It was inferred. Therefore, in order to isolate bacteria having high cellulose resolving ability from ISI-3, a strain capable of forming a halo accompanying cellulose degradation was isolated using a BMN agar medium containing 1% crystalline cellulose. Subsequently, the separated colonies were subjected to a single colony separation operation using a BMN agar medium containing 1% crystalline cellulose, and this operation was repeated three times. Finally, in a BM7 agar medium containing 1% crystalline cellulose (for the composition of the BM7 medium, see Reference: “Japanese Patent Laid-Open No. 2010-51295”), a halo due to crystalline cellulose degradation was observed in 72 to 80 hours. It was confirmed whether it could be formed.
As a result, several strains capable of forming a halo accompanying cellulose degradation were isolated and the base sequence of the 16S rRNA gene was clarified again. Using the obtained DNA sequence data, homology analysis by BLAST was performed using the database of GenBank / EMBL / DDBJ in the same manner as in (3), and the Unclosed Clostridium sp. It was confirmed whether it had 96% homology to Clone De3176 or 99% homology to Cellulosibacterium alkalythermophilus strain A6.

As a result of the homology analysis, Unclosed Clostridium sp. A new thermophilic anaerobic cellulolytic bacterium with 96% homology to Clone De3176 and 99% homology to Cellulosibacterium alkalythermophilus strain A6 was isolated and obtained. In addition, from the results of the base sequence of the 16S rRNA gene, Unclosed Clostridium sp. The bacterial species having 99% homology to the base sequence of the 16S rRNA gene of clone De3176, and the bacterial species having 99% homology to the base sequence of the 16S rRNA gene of Cellulosibacter alkalythermophilus A6 are respectively SEQ ID NO: 1 and SEQ ID NO: 2 was consistent with the nucleotide sequence shown in FIG.
Unclosed Clostridium sp. An isolated strain having 96% homology to Clone De3176 was identified as Clostridium sp. It was named A7 strain (hereinafter sometimes referred to as “A7 strain”) and was deposited at the International Depositary of Patent Microorganisms Depositary (NPMD) as deposit number NITE BP-02216 on March 8, 2016. ing.
A phylogenetic tree analysis using the base sequence of 16S rRNA gene is shown in FIG. Phylogenetic tree analysis revealed that the A7 strain is highly likely to be a new species closely related to Clostridium alkalicumum Z-7026T.
In addition, a thermophilic anaerobic cellulolytic bacterium having 99% homology to Cellulosibacterium alkalythermophilus strain A6 is Cellulosibacterium sp. It was named W21-10 strain (hereinafter sometimes referred to as “W21-10 strain”), and was deposited with the International Depositary of Incorporated Administrative Agency Patent Microorganisms Depositary (NPMD) as of May 24, 2016 with the accession number NITE BP- Deposited as 02265.

The A7 strain had the following morphological characteristics.
(Morphological features of A7 strain)
-Optimum culture temperature: 55 ° C
-Cell morphology: Neisseria gonorrhoeae (width 0.3-0.4 μm x length 2.0-6.0 μm)
-Gram staining: positive-Spore formation: None-Colony color: Orange

In addition, the W21-10 strain had the following morphological characteristics.
(Morphological features of W21-10 strain)
-Optimum culture temperature: 60 ° C
-Cell morphology: Neisseria gonorrhoeae (width 0.2-0.3 μm x length 2.0-3.0 μm)
-Gram staining: Positive-Spore formation: None-Colony color: Yellow

(5) Examination of pH at which A7 strain and W21-10 strain can be grown Further, in order to examine the pH at which growth is possible, a BMN liquid medium containing 0.5% cellobiose was used to adjust pH from 4.0 to pH 10.0. After adjusting the pH of the medium with hydrochloric acid every pH 1.0, growth was measured using the increase in turbidity due to 600 nm as an indicator. For pH 8.0, Tris-HCl buffer was added to the medium to adjust the pH, and for pH 9.0 to 10.0, pH was adjusted using a sodium carbonate buffer. The liquid medium adjusted to each pH was autoclaved, and after confirming whether it had a specified pH using a pH meter, the bacteria were cultured and tested.

As a result, in the A7 strain, growth could be confirmed in the range of pH 6.0 to pH 10.0, and the optimum growth pH was 9.0. In the W21-10 strain, the growth range was slightly narrow and growth could be confirmed in the range of pH 8.0 to pH 10.0, and the optimum growth pH was 9.5.

(6) Carbon source assimilation test of A7 strain and W21-10 strain In order to clarify the physiological characteristics of the A7 strain and W21-10 strain, a carbon source assimilation test was conducted. The assimilation of sugar was measured using a BM7 liquid medium containing 0.5% each of cellulose, xylan, starch, β-glucan, cellobiose, glucose, fructose, arabinose, mannose, and maltose. Cellulose, which is an insoluble substrate, was determined for assimilation by the disappearance of cellulose, and xylan was measured by gas generation accompanying growth. When other soluble substrates were used, the increase in turbidity at 600 nm was measured as described above. Culture was performed for 4 days, and then the presence or absence of growth was measured.

As a result, the A7 strain and the W21-10 strain showed good growth when cellulose, xylan, and cellobiose were used as the carbon source. Further, when glucose, fructose, arabinose, mannose, maltose, and β-glucan were used as the carbon source, growth was observed although it was slow. On the other hand, when starch was used as the carbon source, no assimilation was observed in the A7 strain, but assimilation was observed in the W21-10 strain. Furthermore, the W21-10 strain also had different carbohydrate utilization capabilities such as pectin, sorbitol, mannitol, and glycerol.

[Example 2]
(1) Cultivation of various strains in each biomass It was tested whether the degradation of biomass was promoted in known strains such as clostridium thermocellum ATCC27405 strain, A7 strain, W21-10 strain, or combinations thereof. In the case of culturing only the clostridium thermocellum ATCC27405 strain, culturing at 60 ° C. for 4 days using BM7 medium instead of the BMN medium (for the composition of the BM7 medium, see Reference: “JP 2010-51295 A”). Went. As typical biomass, cassava pulp, bagasse, and corn stover were used as examples. In order to show the decomposition efficiency for each biomass, using the same method as in (2) of Example 1, after culturing, after thoroughly stirring the residue, the supernatant was removed by centrifugation, washed and dried The dry weight was determined and the biomass degradation rate was calculated. The results are shown in FIG. In FIG. 6, Ct represents the clostridium thermocellum ATCC27405 strain, A7 represents the A7 strain, and W21 represents the W21-10 strain.

From FIG. 6, when clostridium thermocellum ATCC27405 strain was inoculated, the decomposition efficiency of biomass as seen from the residue weight was 40%, 35%, and 39% for cassava pulp, bagasse, and corn stover. In particular, cassava pulp has a low decomposition efficiency, which is considered to be a result of most cassava pulp starch remaining without being decomposed.
Further, even when A7 strain was inoculated, the degradation efficiency was particularly low in cassava pulp. This is also because the A7 strain is weak in starch assimilation, so that most of the starch remains alone, so that a large amount of biomass residue remains.
On the other hand, since the W21-10 strain has both starch and cellulose assimilation performance, cassava pulp and other cellulosic biomass alone showed good decomposition efficiency.
In addition, it became clear that the degradation efficiency was dramatically increased by the coexistence of the A7 strain and / or the W21-10 strain, compared with the clostridium thermocellum ATCC27405 strain alone. In particular, in a test using cassava pulp, it was revealed that about 70% can be decomposed and solubilized by coexisting with the A7 strain and the W21-10 strain.
As described above, in the cellulose degradation, the A7 and W21-10 strains are the center, and lignocellulosic biomass is efficiently decomposed in a coordinated and collaborative manner, and crystals with high crystal parts are obtained through the joint work of clostridium thermocellum. It is considered that the cellulose part is completely decomposed.

On the other hand, compared with the decomposition of biomass by ISI-3, decomposition of biomass with a single fungus tends to decrease the decomposition rate, but the bacterial species that do not participate in cellulose decomposition, ie, Thermoanaerobacter genus, Tepidanaerobacter genus It is considered that the oligosaccharides, organic acids, and the like that are produced are consumed by the bacteria, the genus Tepidimicrobium, and the bacteria of the genus Moorella, and the cellulose degrading action in the enzyme group such as cellulase produced by the cellulolytic bacteria is promoted.
Therefore, it became clear that the role of these non-cellulolytic bacteria and the cooperative action are also very important in the degradation of lignocellulosic biomass.

The microorganism of the present embodiment is a novel microorganism excellent in lignocellulose resolution. Moreover, according to the composition for decomposing lignocellulosic biomass containing the microorganism, lignocellulose can be decomposed with high efficiency, and a saccharified solution or lignocellulosic biomass-derived compound can be obtained in high yield.

Claims (8)

1. A microorganism having a 16S rRNA gene containing any of the following nucleic acids (a) to (c) and having a resolution of lignocellulosic biomass.
   (A) a nucleic acid comprising the base sequence shown in SEQ ID NO: 1 or 2,
   (B) a nucleic acid having 95% or more identity with the base sequence shown in SEQ ID NO: 1 or 2,
   (C) a nucleic acid comprising a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 1 or 2
2. Clostridium sp. A7 strain (NITE BP-02216).
3. Cellulosibacter alkaline thermophilus W21-10 strain (NITE BP-02265).
4. A composition for degrading lignocellulosic biomass comprising at least one microorganism according to claim 1, or an enzyme or gene product derived from said microorganism.
5. 5. The microorganism according to claim 4, wherein the microorganism is Clostridium sp. A7 strain (NITE BP-02216) or Cellulosibacter alkaline thermophilus W21-10 strain (NITE BP-02265) Composition for lignocellulosic biomass decomposition.
6. Further, at least one of the microorganisms having a 16S rRNA gene containing any of the following nucleic acids (d) to (f) and having a resolution of lignocellulosic biomass or a metabolic ability of a degradation product of lignocellulosic biomass: The composition for lignocellulose-type biomass decomposition | disassembly of Claim 4 or 5 containing 1 type.
   (D) a nucleic acid comprising the base sequence shown in any of SEQ ID NOs: 3 to 11,
   (E) a nucleic acid having 90% or more identity with the base sequence shown in any of SEQ ID NOs: 3 to 11,
   (F) a nucleic acid comprising a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in any of SEQ ID NOs: 3 to 11
7. A method for producing a saccharified solution comprising a saccharification step for producing a saccharified solution from lignocellulosic biomass using the lignocellulose-based biomass decomposing composition according to any one of claims 4 to 6.
8. A method for producing a lignocellulosic biomass-derived compound comprising a production step of producing a lignocellulosic biomass-derived compound from the saccharified solution after producing a saccharified solution using the production method according to claim 7.

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