Microorganism, composition for use in decomposition of biodegradable plastic, and method for treating biodegradable plastic

Abstract

To provide a new microorganism capable of decomposing biodegradable plastic, especially polylactic acid (PLA) or polybutylene adipate terephthalate (PBAT).SOLUTION: The microorganism according to the present disclosure belongs to the family Bacillaceae, and has been deposited under Accession No. NITE BP-04083.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present disclosure relates to microorganisms, compositions for use in degrading biodegradable plastics, and methods for treating biodegradable plastics. [Background technology]

[0002] In recent years, research and development has been progressing on biodegradable plastics that can be decomposed into CO2 and water by microorganisms in order to solve the problems of marine plastics and global warming.

[0003] Furthermore, with the expansion of the use of biodegradable plastics, research into microorganisms that can decompose biodegradable plastics is underway (Non-Patent Documents 1-2). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Arena, Maria et al. “Degradation of poly (lactic acid) and nanocomposites by Bacillus licheniformis.” Environmental science and pollution research international vol. 18,6 (2011): 865-70. doi:10.1007 / s11356-011-0443-2 [Non-patent document 2] Bonifer, Kyle S et al. “Bacillus pumilus B12 Degrades Polylactic Acid and Degradation Is Affected by Changing Nutrient Conditions.” Frontiers in microbiology vol. 10 2548. 22 Nov. 2019, doi:10.3389 / fmicb.2019.02548 Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, an object of the present disclosure is to provide a novel microorganism capable of degrading biodegradable plastics, particularly polylactic acid (PLA) or polybutylene adipate terephthalate (PBAT), for example. [Means for solving the problem]

[0006] To achieve the above object, the microorganism of the present disclosure belongs to the Bacillaceae family, It has been deposited under accession number NITE BP-04083.

[0007] The microorganisms of the present disclosure are mutant strains of the microorganisms of the present disclosure.

[0008] The microorganism of the present disclosure belongs to the family Bacillaceae and has the following properties (1) to (10): (1) Gram-stain-positive bacilli that form spores; (2) Growth temperature is 30-58°C; (3) Growth pH is 6 to 10; (4) The growth salt concentration is 0-4%; (5) The catalase reaction is positive; (6) The oxidase reaction is positive; (7) have metabolic activity of D-ribose, D-xylose, D-glucose, D-fructose, L-rhamnose, inositol, arbutin, esculin ferric citrate, D-maltose, D-sucrose, D-trehalose, D-melezitose, D-turanose, D-tagatose, potassium 2-ketogluconate, and / or potassium 5-ketogluconate; (8) L-arginine, sodium citrate, L-tryptophan, gelatin, and / or esculin have metabolic activity; (9) Having alkaline phosphatase activity, esterase activity, esterase lipase activity, leucine aryl amidase activity, α-chymotrypsin activity, acid phosphatase activity, naphthol-AS-BI-phosphohydrolase activity, α-glucosidase activity, arginine dihydrolase activity, citrate hydrolysis activity, tryptophan deaminase activity, esculin hydrolysis activity, and / or gelatinase activity. (10) Ability to decompose polylactic acid and / or polybutylene adipate terephthalate.

[0009] The composition for use in degrading biodegradable plastics of the present disclosure comprises the microorganism of the present disclosure.

[0010] The method for treating biodegradable plastics of the present disclosure includes the steps of contacting the microorganisms of the present disclosure with biodegradable plastics and allowing the microorganisms to decompose the biodegradable plastics. [Effects of the Invention]

[0011] According to the present disclosure, for example, it is possible to provide a novel microorganism capable of decomposing biodegradable plastics, in particular, polylactic acid (PLA) or polybutylene adipate terephthalate (PBAT). [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows a phylogenetic tree of komham264 based on the partial base sequence of 16S rRNA in Example 1. [Figure 2] FIG. 2 is a photograph showing the results of morphological observation of komham264 under an optical microscope in Example 1. [Figure 3] FIG. 3 is a photograph showing the results of Gram staining of komham264 in Example 1. [Figure 4] FIG. 4 is a photograph showing the results of the second stage bacterial test using API (registered trademark) 50CH in Example 1. [Figure 5]FIG. 5 is a photograph showing the results of the second stage bacterial test using API® 20E and API® 20NE in Example 1. [Figure 6] FIG. 6 is a photograph showing the results of the second stage bacterial test using API (registered trademark) ZYM in Example 1. [Figure 7] FIG. 7 shows the results of ANI analysis in Example 1. [Figure 8] FIG. 8 is a photograph showing the results of decomposition of polybutylene adipate terephthalate in Example 1. [Figure 9] FIG. 9 is a photograph showing the results of decomposition of polylactic acid in Example 1. [Figure 10] FIG. 10 is a photograph showing the results of decomposition of polylactic acid in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present disclosure will be specifically described below using examples. Unless otherwise specified, each disclosure may incorporate the explanations of other disclosures.

[0014] As a result of extensive research, the present inventors discovered a novel microorganism that has the activity of degrading biodegradable plastics such as PLA and PBAT. The microorganism was isolated and identified, leading to the establishment of the present disclosure. Specifically, the microorganism of the present disclosure was confirmed to belong to the Bacillaceae family through morphological observation, physiological and biochemical property tests, partial 16S rRNA sequence analysis, and average nucleotide identity (ANI) analysis of the genome sequence. However, the microorganism did not belong to any known genera in the Bacillaceae family, and was found to be a new genus. Furthermore, as a result of extensive research, the present inventors discovered that the microorganism exhibits the ability to degrade biodegradable plastics, leading to the establishment of the present disclosure. Therefore, the microorganism of the present disclosure is expected to be useful, for example, in the degradation of biodegradable plastics.

[0015] The microorganism of the present disclosure is a new species of microorganism belonging to the family Bacillaceae. The microorganism of the present disclosure includes, for example, the strain deposited under accession number NITE BP-04083 or its progeny (hereinafter also referred to as "komham264").

[0016] <komham264> The komham264 of the present disclosure or its progeny line has, for example, the following characteristics (1) to (9). (1) Gram-stain-positive bacilli that form spores; (2) Growth temperature is 30-58°C; (3) Growth pH is 6 to 10; (4) The growth salt concentration is 0-4%; (5) The catalase reaction is positive; (6) The oxidase reaction is positive; (7) have metabolic activity of D-ribose, D-xylose, D-glucose, D-fructose, L-rhamnose, inositol, arbutin, esculin ferric citrate, D-maltose, D-sucrose, D-trehalose, D-melezitose, D-turanose, D-tagatose, potassium 2-ketogluconate, and / or potassium 5-ketogluconate; (8) L-arginine, sodium citrate, L-tryptophan, gelatin, and / or esculin have metabolic activity; (9) Having alkaline phosphatase activity, esterase activity, esterase lipase activity, leucine aryl amidase activity, α-chymotrypsin activity, acid phosphatase activity, naphthol-AS-BI-phosphohydrolase activity, α-glucosidase activity, arginine dihydrolase activity, citrate hydrolysis activity, tryptophan deaminase activity, esculin hydrolysis activity, and / or gelatinase activity.

[0017] The komham264 of the present disclosure may have any one, multiple, or all of the characteristics (1) to (9). For example, the komham264 of the present disclosure may have one or more of the metabolic activities in the metabolic activity (7), preferably all of them. For example, the komham264 of the present disclosure may have one or more of the metabolic activities in the metabolic activity (8), preferably all of them. For example, the komham264 of the present disclosure may have one or more of the activities in the metabolic activity (9), preferably all of them.

[0018] The above characteristics (1) to (9) can be measured in accordance with Example 1 (5) to (6) described later.

[0019] The komham264 of the present disclosure may further have, for example, the following characteristic (10): (10) Ability to decompose polylactic acid and / or polybutylene adipate terephthalate.

[0020] An example of komham264 in the present disclosure is the strain (deposited strain) deposited under accession number NITE BP-04083 or its progeny strain. The deposit information is shown below. Type of deposit: International deposit Name of depository institution: National Institute of Technology and Evaluation, Patent Organism Depositary Center Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan (komham264) Accession number: NITE BP-04083 Identification mark: komham264 Entrustment date: February 26, 2024

[0021] The komham264 of the present disclosure includes, for example, a gene having the base sequence of the following (R) as a 16S rRNA gene, and preferably has a gene having the base sequence of the following (R1), i.e., a 16S rRNA gene having the base sequence shown in SEQ ID NO: 1.

[0022] (R) The following base sequence (R1), (R2), or (R3) (R1) the nucleotide sequence shown in SEQ ID NO: 1; (R2) a nucleotide sequence in which one or several nucleotides are deleted, substituted, inserted, and / or added in the nucleotide sequence shown in SEQ ID NO: 1; (R3) A nucleotide sequence having an identity of 98.72% or more to the nucleotide sequence shown in SEQ ID NO: 1.

[0023] In (R1), the nucleotide sequence of SEQ ID NO: 1 is a nucleotide sequence encoding 16S RNA. The nucleotide sequence of SEQ ID NO: 1 can be isolated from, for example, komham264.

[0024] Nucleotide sequence of the 16S rRNA gene of komham264 (SEQ ID NO: 1)

[0025] In (R2), "one or several" may be within a range in which a microorganism having a 16S rRNA gene containing the base sequence of (R2) maintains the characteristics of komham264. Examples of the characteristics of komham264 include the characteristics of (1) to (10), preferably the characteristic of (10) (the same applies below). The "one or several" in (R2) refers to, for example, 1 to 18, 1 to 14, 1 to 10, 1 to 8, 1 to 5, 1 to 3, 1 or 2, or 1 in the base sequence of (R1). In the present disclosure, a numerical range of the number of bases, etc., discloses, for example, all positive integers within that range. For example, the expression "1 to 5" means the disclosure of all of "1, 2, 3, 4, and 5" (the same applies below).

[0026] In (R3), the "identity" may be within a range in which a microorganism having a 16S rRNA gene containing the base sequence of (R3) maintains the characteristics of komham264. The "identity" of (R3) is, for example, 98.72% or more, 98.8% or more, 98.9% or more, or 99% or more in the base sequence of (R1). The "identity" can be determined by aligning two base sequences or amino acid sequences (the same applies below). The alignment can be calculated using, for example, BLAST, FASTA, or the like with default parameters.

[0027] The 16S rRNA gene can be measured by, for example, a known method. Examples of such known methods include PCR (Polymerase Chain Reaction) and sequencing analysis. rRNA, including the 16S rRNA, is present in all living organisms except viruses and is an important molecule involved in protein synthesis. For this reason, the evolutionary rate of rRNA is relatively slow, and it is known to exhibit high homology at the species level. Bacteria are defined as being of the same species if they share 70% or more DNA-DNA hybridization homology with a type strain (Reference 1). Meanwhile, in bacterial species identification using 16S rRNA sequences, an identity of 97% or more indicates a close relationship, and an identity of 98.7% or more is considered highly likely to be the same species (Reference 2). Therefore, in bacterial species identification using 16S rRNA sequences, if there is no species with a similarity of 98.7% or more, it is considered a new species. Specifically, the bacterial species identification is performed by comparing the target bacterial cell with the 16S rRNA base sequence registered in a public gene bank. Examples of the public gene banks include the DNA Data Bank of Japan, GenBank, and EMBL. The comparison of the known base sequences can be carried out using, for example, base sequence analysis software (e.g., BLAST, etc.). Reference 1: Wayne LG, Brenner DJ, Colwell RR, Grimont PAD, Kandier O., Krichevsky MI, Moore LH, Moore WEC, Murray RGE, Stackebrandt E., Starr MP, Truper HG 1987; Report of the Ad Hoc Committee on Reconciliation of Approaches to Bacterial Systematics. Int. J. Syst. Bacteriol. 37:463-464 Reference 2: Stackebrandt, Erko. "Taxonomic parameters revisited: tarnished gold standards." Microbiol. Today 33 (2006): 152-155.

[0028] The 16S rRNA of komham264 of the present disclosure preferably has, for example, less than 98.7%, 98.6% or less, 98.5% or less, or 98.4% or less identity compared to the base sequence of the 16S rRNA gene of a known microorganism.

[0029] Examples of closely related species of komham264 of the present disclosure include bacteria of the genus Bacillus.

[0030] The komham264 of the present disclosure has genomic DNA consisting of the base sequence shown in SEQ ID NO:2, for example.

[0031] Identification of bacterial species based on the genomic DNA of a strain may be performed using, for example, ANI (Average Nucleotide Identity) analysis. The ANI analysis is a method of determining whether the species are similar or different by calculating the identity (ANI value) of the full-length genome sequences or draft genome sequences of a target strain and a comparison strain on a computer. In the ANI analysis, if a value of 95% or higher is shown, it can be determined that the genomes belong to the same bacterial species, and if a value of less than 95% is shown, it can be determined that the genomes belong to different bacterial species. For details on the ANI analysis method, see, for example, Reference 3 below. Reference 3: Rodriguez-R, Luis M., and Konstantinos T. Konstantinidis. The enveomics collection: a toolbox for specialized analyzes of microbial genomes and metagenomes. No. e1900v1. PeerJ Preprints, 2016.

[0032] The ANI value between the genome sequence of komham264 (SEQ ID NO: 2) of the present disclosure and the genome sequence of a known microorganism is preferably, for example, less than 95%, less than 90%, less than 85%, or less than 82%. Examples of the known microorganism include Peribacillus faecalis, Anoxybacillus gonensis, Neobacillus drentensis, Bacillus licheniformis, Bacillus smithii, Bacillus obstructivus, Aeribacillus composti, Compostibacillus humi, Pallidibacillus thermolactis subsp. Kokeshiiformis, Aeribacillus pallidus, and Pallidibacillus thermolactis.

[0033] The komham264 of the present disclosure preferably comprises genomic DNA having an ANI value of, for example, 82% or more, 83% or more, 84% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the base sequence shown in SEQ ID NO: 2.

[0034] The komham264 of the present disclosure includes, for example, a gene consisting of the base sequence (G) below as genomic DNA, and preferably has a gene consisting of the base sequence (G1) below, i.e., genomic DNA consisting of the base sequence shown in SEQ ID NO: 2.

[0035] (G) The following base sequence (G1), (G2), or (G3): (G1) the nucleotide sequence shown in SEQ ID NO: 2; (G2) a nucleotide sequence in which one or several nucleotides are deleted, substituted, inserted, and / or added in the nucleotide sequence shown in SEQ ID NO: 2; (G3) A nucleotide sequence having 80% or more identity to the nucleotide sequence shown in SEQ ID NO: 2.

[0036] In (G1), the nucleotide sequence of SEQ ID NO: 2 is a nucleotide sequence encoding genomic DNA. The nucleotide sequence of SEQ ID NO: 2 can be isolated from, for example, komham264.

[0037] In (G2), "one or several" may be within a range in which a microorganism containing genomic DNA having the base sequence of (G2) maintains the characteristics of komham264. The characteristics of komham264 include, for example, the characteristics of (1) to (10), preferably the characteristic of (10) (the same applies hereinafter). The "one or several" in (G2) means, for example, 1 to 719,454, 1 to 539,590, 1 to 359,727, 1 to 179,863, 1 to 143,890, 1 to 107,918, 1 to 71,945, 1 to 53,959, 1 to 32,972, 1 to 17,986, 1 to 14,389, 1 to 10,791, 1 to 7,194, 1 to 5,395, 1 to 3,297, or 1 to 1,798 in the base sequence of (G1).

[0038] In (G3), the "identity" may be within a range in which a microorganism containing genomic DNA having the base sequence of (G3) maintains the characteristics of komham264. The "identity" of (G3) is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with respect to the base sequence of (G1).

[0039] The taxonomic group of komham264 of the present disclosure may be identified using, for example, bacterial morphological observation, a first-stage bacterial test, and a second-stage bacterial test. Examples of bacterial morphological observation include colony color, cell morphology, Gram staining, and sporulation. Examples of the first-stage bacterial test include colony characteristics, cell morphology, and motility observation, as well as physiological and biochemical property tests such as catalase, oxidase, and O / F tests. Examples of the second-stage bacterial test include tests for carbon source assimilation, oxidation / fermentation, and enzyme activity using a physiological and biochemical property test kit. komham264 of the present disclosure exhibits morphological characteristics and physiological and biochemical properties shown in the results of bacterial morphological observation, a first-stage bacterial test, and a second-stage bacterial test, for example, in Example 1 described below.

[0040] The komham264 of the present disclosure can be passaged, for example, by culturing it according to the culture conditions of Example 1 described below. This makes it possible to obtain progeny lines of the komham264 of the present disclosure.

[0041] <Mutant strain> The microorganism of the present disclosure may be, for example, a mutant strain of komham264 or a progeny lineage thereof.

[0042] The mutant strain of the present disclosure is, for example, a bacterial cell that maintains the taxonomic properties of komham264 as a new species. The properties include, for example, the above-mentioned characteristics (1) to (10). The mutant strain may have, for example, any one, multiple, or all of the characteristics (1) to (10). The description of komham264 of the present disclosure can be applied to the mutant strain of the present disclosure.

[0043] The mutant strain of the present disclosure can be obtained, for example, by mutation of the komham264 strain or by introducing an exogenous gene. The mutation can be induced, for example, by introducing a mutation using a conventional method. The mutation can be introduced, for example, by homologous recombination; genome editing techniques using ZFN, TALEN, CRISPR-CAS9, CRISPR-CPF1, etc. The mutation can also be introduced by a mutation introduction method such as site-directed mutagenesis. The mutation can also be introduced by random mutagenesis. Examples of random mutagenesis include irradiation with α-rays, β-rays, γ-rays, X-rays, etc.; chemical treatment with mutagens such as ethyl methanesulfonate (EMS) or ethynylnitrosourea (ENU); and heavy ion beam treatment.

[0044] The mutant strain contains, as a 16S rRNA gene, a gene consisting of the following nucleotide sequence (R): (R) The following base sequence (R1), (R2), or (R3) (R1) the nucleotide sequence shown in SEQ ID NO: 1; (R2) a nucleotide sequence in which one or several nucleotides are deleted, substituted, inserted, and / or added in the nucleotide sequence shown in SEQ ID NO: 1; (R3) A nucleotide sequence having an identity of 98.72% or more to the nucleotide sequence shown in SEQ ID NO: 1.

[0045] The explanation in komham264 of the present disclosure can be used for (R1) to (R3) above.

[0046] The mutant strain contains, for example, a gene consisting of the base sequence (G) below as genomic DNA, and preferably has a gene consisting of the base sequence (G1) below, i.e., genomic DNA consisting of the base sequence shown in SEQ ID NO: 2.

[0047] (G) The following base sequence (G1), (G2), or (G3): (G1) the nucleotide sequence shown in SEQ ID NO: 2; (G2) a nucleotide sequence in which one or several nucleotides are deleted, substituted, inserted, and / or added in the nucleotide sequence shown in SEQ ID NO: 2; (G3) A nucleotide sequence having 80% or more identity to the nucleotide sequence shown in SEQ ID NO: 2.

[0048] The explanations in komham264 of the present disclosure can be used for (G1) to (G3) above.

[0049] <Method for disposing of biodegradable plastics> In another aspect, the present disclosure provides a method capable of treating biodegradable plastics. The method for treating biodegradable plastics of the present disclosure (hereinafter also referred to as the "treatment method") uses the microorganisms of the present disclosure. The treatment method of the present disclosure includes a step of contacting the microorganisms of the present disclosure with biodegradable plastics and decomposing the biodegradable plastics using the microorganisms (hereinafter also referred to as the "decomposition step"). The treatment method of the present disclosure is characterized by the use of the microorganisms of the present disclosure, and other steps and conditions are not particularly limited.

[0050] In this disclosure, "biodegradable plastic" refers to plastic that is ultimately decomposed into CO2 and water by microorganisms, etc. Examples of biodegradable plastics include polylactic acid (PLA), polybutylene adipate terephthalate (PBAT), polyhydroxybutyrate (PHB), polycaprolactone (PCL), polybutylene succinate (PBS), polyethylene terephthalate (PET), etc., and preferably polylactic acid and / or polybutylene adipate terephthalate. Examples of polylactic acid include lactic acid homopolymers such as poly-L-lactic acid (PLLA) and / or poly-D-lactic acid (PDLA), and copolymers containing structural units derived from lactic acid.

[0051] In the decomposition step, the object to be decomposed needs only to include the biodegradable plastic, and may consist solely of the biodegradable plastic, or may include the biodegradable plastic and other objects to be treated (e.g., food waste, metals, etc.). The biodegradable plastic may be, for example, a substance formed solely from biodegradable plastic, or may contain other substances such as non-biodegradable plastic in addition to the biodegradable plastic. Examples of materials formed from biodegradable plastic include tableware, cutlery, packaging containers, trays, bags, mulch film, and packaging materials.

[0052] In the decomposition step, the contact between the microorganism and the biodegradable plastic can be carried out, for example, by mixing. The mixing can be carried out mechanically, for example, using a stirrer or the like.

[0053] In the decomposition step, the decomposition temperature is, for example, a temperature at which the microorganism of the present disclosure can process biodegradable plastics, and is, for example, 30 to 58°C, preferably 40 to 50°C.

[0054] In the decomposition step, the pH of the decomposition (decomposition pH) is, for example, a pH at which the microorganism of the present disclosure can treat biodegradable plastics. The decomposition pH is, for example, pH 6 to 10, and preferably pH 8 to 9.

[0055] In the decomposition step, for example, oxygen may be supplied. The supply can be carried out, for example, by stirring a mixture of the biodegradable plastic and the microorganism of the present disclosure. The time interval for the supply is, for example, 1 hour to 10 hours, preferably 5 hours to 8 hours, and more preferably 8 hours. The supply time is, for example, 1 minute to 30 minutes, preferably 1 minute to 10 minutes, and more preferably 5 minutes.

[0056] In the decomposition step, the water content of the mixture of the biodegradable plastic and the microorganism of the present disclosure is, for example, 40 to 70%, preferably 50 to 70%, and more preferably 55 to 65%. [Example]

[0057] Next, examples of the present invention will be described. However, the present invention is not limited to the following examples. Commercially available reagents were used according to their protocols unless otherwise specified. Note that "mol / l" may also be abbreviated as "M."

[0058] [Example 1] The novel microorganism disclosed herein has been identified and confirmed to be capable of degrading biodegradable plastics.

[0059] (1) Isolation of komham264 Compost from an organic waste composting facility in Hokkaido, Japan, was collected as a sample. 8 g of hypeptone, 3 g of yeast extract, 1 g of dipotassium phosphate, and 0.25 g of ammonium chloride were dissolved in 900 ml of distilled water. After dissolution, 100 ml of alkaline buffer (sodium carbonate, pH 9.0) was added to prepare PYA medium (pH 9.0). After preparation, the sample was diluted with distilled water and inoculated into the PYA medium. After inoculation, the medium was cultured at 50°C. After culture, colonies grown on the medium were isolated. After isolation, the colonies were inoculated into the PYA medium and cultured repeatedly. Subsequently, isolation and purification were performed, and the novel microorganism, komham264, was isolated and deposited. The presence of this novel microorganism was also confirmed in several other compost samples from Hokkaido.

[0060] (2) Determination of the 16S rRNA sequence of komham264 The 16S rRNA sequence of komham264 isolated in Example 1(1) was analyzed to identify the microorganism. Specifically, DNA was extracted from the isolated komham264. After extraction, the following primer set (manufactured by FASMAC, synthesis requested) and KOD One (registered trademark) PCR Master Mix (manufactured by TOYOBO) were added to the DNA to prepare a reaction solution. After preparation, the 16S rRNA gene of the DNA was amplified using a thermal cycler (T100 Thermal Cycler, manufactured by BIO-RAD). After amplification, DNA sequencing was performed using the obtained PCR product to obtain the 16S rRNA sequence of komham264 isolated in Example 1(1). The nucleotide sequence was the nucleotide sequence represented by SEQ ID NO: 1.

[0061] 27F primer (SEQ ID NO: 3) 5'-AGAGTTTGATCMTGGCTCAG-3' 1492R primer (SEQ ID NO: 4) 5'-GGYTACCTTGTTACGACTT-3'

[0062] (3) Comparison with existing microbial 16S rRNA sequences Using the 16S rRNA base sequence obtained in Example 1 (2), the 16S rRNA of komham264 was compared with the 16S rRNA of existing microorganisms. Specifically, BLAST was used for the comparison. A BLAST homology search was performed to search for microorganisms having the same or similar sequence as the 16S rRNA base sequence of komham264. As a result of the search, a microorganism belonging to the genus Bacillus ( Bacillus It was found that the Bacillus thermolactis (strain Marseille-AA00136) strain of the species Bacillus sp. has a 16S rRNA sequence that is closest to the 16S rRNA sequence of komham264.

[0063] Nucleotide sequence of the 16S rRNA gene of Bacillus thermolactis (strain Marseille-AA00136) (SEQ ID NO: 5)

[0064] (4) Construction of phylogenetic tree Next, based on the results of the homology search in Example 1(3), a phylogenetic tree was constructed based on the partial nucleotide sequence of 16S rRNA, and the phylogenetic position of komham264 was analyzed. The phylogenetic tree was constructed using MEGA (Molecular Evolutionary Genetics Analysis) software, a tool for phylogenetic analysis, and the neighbor-joining method. The results are shown in Figure 1.

[0065] Figure 1 shows the phylogenetic tree of komham264 based on the partial nucleotide sequence of 16S rRNA.

[0066] (5) Examination of taxonomic characteristics 1 Morphological observation and physiological property tests (hereinafter referred to as "first-stage bacterial tests") were conducted on komham264 to examine its taxonomic characteristics. Specifically, komham264 was aerobically cultured at 50°C for 24 hours. After the culture, morphological observation was performed using an optical microscope, and tests were performed on the catalase reaction, oxidase reaction, acid / gas production from glucose, and glucose oxidation / fermentation (O / F) based on the method described in Reference 4. The optical microscope used was a BX50F4 (Olympus). These results are shown in Figures 2 and 3 and Table 1 below. Reference 4: Barrow GI, Feltham RKA. Cowan and Steel's Manual for the Identification of Medical Bacteria. 3rd edition. Cambridge: University Press; 1993.

[0067] [Table 1]

[0068] Figure 2 is a photograph showing the results of morphological observation of komham264 under an optical microscope. As a result of morphological observation under an optical microscope, it was found that komham264 formed colonies as shown in Figure 2.

[0069] Figure 3 is a photograph showing the results of Gram staining of komham264. In Figure 3, the scale bar indicates 10 μm. In Figure 3, (A) shows the results of Gram staining of komham264 cultured on PYA agar medium, and (B) shows the results of Gram staining of komham264 cultured on sporulation-promoting medium. As shown in Figure 3, komham264 was found to be a Gram-positive bacterium.

[0070] (6) Examination of taxonomic characteristics 2 Biochemical property tests (hereinafter also referred to as "second-stage bacterial tests") were conducted on komham264 to examine its taxonomic properties. Specifically, API (registered trademark) 50CH, API (registered trademark) 20E, API (registered trademark) 20NE, and API (registered trademark) ZYM (all manufactured by Biomerieux) were used in the second-stage bacterial tests. The results are shown in Figures 4 to 6 and Tables 2 and 3 below.

[0071] [Table 2]

[0072] Figure 4 is a photograph showing the results of the second stage bacterial test using API® 50CH. Table 2 above is a table showing the results of the second stage bacterial test using API® 50CH. As shown in Figure 4 and Table 2 above, komham264 was found to have metabolic activity for glycerol, D-ribose, D-xylose, D-glucose, D-fructose, L-rhamnose, inositol, arbutin, esculin, ferric citrate, D-maltose, D-sucrose, D-trehalose, D-melezitose, D-turanose, D-tagatose, potassium 2-ketogluconate, and potassium 5-ketogluconate.

[0073] Figure 5 is a photograph showing the results of the second stage bacterial test using API® 20E and API® 20NE. As shown in Figure 5, komham264 was found to have metabolic activity for L-arginine (ADH), sodium citrate (CIT), L-tryptophan (TDA), gelatin (GEL), and esculin (ESC), and to have arginine dihydrolase activity, citrate hydrolysis activity, tryptophan deaminase activity, gelatinase activity, and esculin hydrolysis activity.

[0074] [Table 3]

[0075] Figure 6 is a photograph showing the results of the second-stage bacterial test using API (registered trademark) ZYM. Table 3 above is a table showing the results of the second-stage bacterial test using API (registered trademark) ZYM. As shown in Figure 6 and Table 3 above, komham264 was found to have alkaline phosphatase activity, esterase activity, esterase lipase activity, leucine aryl amidase activity, α-chymotrypsin activity, acid phosphatase activity, naphthol-AS-BI-phosphohydrolase activity, and α-glucosidase activity.

[0076] (7) Determination of the genomic DNA sequence The genomic DNA of komham264 was sequenced. Specifically, genomic DNA from komham264 was first extracted. A Genomictip 20G (QIAGEN) was used for the extraction. After extraction, a library was prepared using the SMRTbell® gDNA Sample Amplification Kit (PacBio) and the SMRTbell® Express Template Prep Kit 20 (PacBio) according to the procedure described in Procedure & Checklist - Preparing HiFi SMRTbell® Libraries from Ultra-Low DNA Input. After preparation, a polymerase complex was formed for the library using the Revio™ Polymerase kit (PacBio). After the formation, sequencing was performed using Revio™ (PacBio). Next, overhang adapter sequences were removed from the obtained sequences using SMRT® Link (ver. 13.0.0.207600) to generate subreads. After this, the subreads were aligned to generate consensus sequences. After this, consensus sequences with an average quality score per read of less than 20 were removed to generate HiFi reads. After this, ultra-low PCR adapters were removed from the HiFi reads using lima (ver. 2.7.1). After this removal, PCR duplicate reads were removed using pbmarkdup (ver. 1.0.3). After this removal, reads shorter than 1000 bases were deleted using Filtlong (ver. 0.2.1). After this removal, HiFi reads longer than 1000 bases were assembled using the default settings of Flye (ver. 2.9.2-b1786). As a result, it was found that the genomic DNA of komham264 consisted of the base sequence shown in SEQ ID NO:2.

[0077] (8) ANI analysis of genome sequences Further, ANI analysis was performed on komham264. Specifically, the ANI value was determined for the base sequence obtained in Example 1(1) above using pyani v0.2.12 and BLAST (Reference 5). The results are shown in Figure 7. Reference 5: Pritchard, Leighton, et al. "Genomics and taxonomy in diagnostics for food security: soft-rotting enterobacterial plant pathogens." Analytical Methods 8.1 (2016): 12-24.

[0078] Figure 7 shows the results of the ANI analysis. As shown in Figure 7, even the genomic DNA with the highest identity to the genomic DNA sequence of komham264 had an identity rate of less than 95%. These results indicated that komham264 is not a microorganism belonging to a known species, but a new species of microorganism.

[0079] (9) Evaluation of the decomposition ability of biodegradable plastic (polybutylene adipate terephthalate) The ability of komham264 to decompose biodegradable plastic (polybutylene adipate terephthalate) was examined. Specifically, komham264 obtained in Example 1(1) above was inoculated into 10 ml of PYA liquid medium (pH 9), and pieces of a plastic bag (NOVAMONT, Mater-Bi) containing biodegradable plastic (PBAT) were added. Shaking culture was performed at 40°C and 160 rpm. Observations were made on the day the shaking culture began (day 0), and on days 4 and 8 after the start of the shaking culture. A control was performed in the same manner except that komham264 was not added. These results are shown in Figure 8.

[0080] Figure 8 is a photograph showing the results of polybutylene adipate terephthalate degradation. From left to right, photographs from days 0, 4, and 8 are shown in Figure 8. As shown in Figure 8, on days 4 and 8 from the start of shaking culture, degradation of polybutylene adipate terephthalate was confirmed in komham264 compared to the control.

[0081] (10) Evaluation of biodegradability of biodegradable plastic (polylactic acid) 1 The ability of komham264 to decompose biodegradable plastic (polylactic acid) was examined. Specifically, komham264 obtained in Example 1(1) above was inoculated into 10 ml of PYA liquid medium (pH 9), and fragments of a straw containing biodegradable plastic (polylactic acid) (product name: Biodegradable Straw, sold by Daiso Industries Co., Ltd.) were added. Shaking culture was performed at 50°C and 160 rpm. Observations were made on the start of the shaking culture (day 0) and on days 17, 31, and 37 after the start of the shaking culture. These results are shown in Figure 9.

[0082] Figure 9 shows photographs showing the results of polylactic acid degradation. From left to right, photographs taken on days 0, 17, 31, and 37 are shown in Figure 9. As shown in Figure 9, degradation of polylactic acid by komham264 was confirmed on days 31 and 37 after the start of shaking culture.

[0083] (11) Evaluation of biodegradability of biodegradable plastic (polylactic acid) 2 The ability of komham264 to decompose biodegradable plastic (polylactic acid) was examined. Specifically, komham264 obtained in Example 1(1) above was inoculated into 10 ml of PYA liquid medium (pH 9), and fragments of a straw containing biodegradable plastic (polylactic acid) (sold by 4Nature Co., Ltd.) were added. Shaking culture was performed at 50°C and 160 rpm. Observations were made on the day the shaking culture began (day 0), and on days 1, 5, 11, and 14 after the beginning of the shaking culture. A control was performed in the same manner, except that komham264 was not added. These results are shown in Figure 10.

[0084] Figure 10 is a photograph showing the results of polylactic acid degradation. From left to right, Figure 10 shows photographs taken on days 0, 1, 5, 11, and 14. As shown in Figure 10, polylactic acid degradation was confirmed in komham264 compared to the control on days 5, 11, and 14 after the start of shaking culture. These results demonstrate that komham264 of the present disclosure can degrade biodegradable plastics. Generally, biodegradable plastics are considered to have low microbial decomposition potential in aqueous environments. komham264 of the present disclosure was found to exhibit excellent biodegradable plastic degradation properties, capable of decomposing polybutylene adipate terephthalate and polylactic acid even in aqueous environments. It is also expected that komham264 of the present disclosure can decompose biodegradable plastics in compost.

[0085] Although the present disclosure has been described above with reference to embodiments and examples, the present disclosure is not limited to the above embodiments and examples. Various modifications that can be understood by a person skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0086] <Additional Notes> Some or all of the above-described embodiments and examples can be described as, but are not limited to, the following supplementary notes. <Deposited microorganism> (Appendix 1) It belongs to the Bacillaceae family, The microorganism is deposited under accession number NITE BP-04083. <Mutant strain> (Appendix 2) 10. A microorganism that is a mutant strain of the microorganism described in Appendix 1. (Appendix 3) The mutant strain is a microorganism according to Appendix 2, which contains, as a 16S rRNA gene, a gene having the following nucleotide sequence (R): (R) The following base sequence (R1), (R2), or (R3) (R1) the nucleotide sequence shown in SEQ ID NO: 1; (R2) a nucleotide sequence in which 1 to 18 nucleotides are deleted, substituted, inserted, and / or added in the nucleotide sequence shown in SEQ ID NO: 1; (R3) A nucleotide sequence having an identity of 98.72% or more to the nucleotide sequence shown in SEQ ID NO: 1. (Appendix 4) The mutant strain is a microorganism according to Appendix 2 or 3, which has at least one characteristic selected from the group consisting of the following (1) to (8) and (9): (1) Gram-stain-positive bacilli that form spores; (2) Growth temperature is 30-58°C; (3) Growth pH is 6 to 10; (4) The growth salt concentration is 0-4%; (5) The catalase reaction is positive; (6) The oxidase reaction is positive; (7) have metabolic activity of D-ribose, D-xylose, D-glucose, D-fructose, L-rhamnose, inositol, arbutin, esculin ferric citrate, D-maltose, D-sucrose, D-trehalose, D-melezitose, D-turanose, D-tagatose, potassium 2-ketogluconate, and / or potassium 5-ketogluconate; (8) L-arginine, sodium citrate, L-tryptophan, gelatin, and / or esculin have metabolic activity; (9) Having alkaline phosphatase activity, esterase activity, esterase lipase activity, leucine aryl amidase activity, α-chymotrypsin activity, acid phosphatase activity, naphthol-AS-BI-phosphohydrolase activity, α-glucosidase activity, arginine dihydrolase activity, citrate hydrolysis activity, tryptophan deaminase activity, esculin hydrolysis activity, and / or gelatinase activity. (Appendix 5) Further, the microorganism described in Appendix 4 has the following property (10): (10) Ability to decompose polylactic acid (PLA) and / or polybutylene adipate terephthalate (PBAT). (Appendix 6) 6. A microorganism according to any one of appendices 2 to 5, wherein the mutant strain comprises genomic DNA having an ANI value of 82% or more as shown in SEQ ID NO: 2. <Microorganisms> (Appendix 7) It belongs to the Bacillaceae family, A microorganism containing a gene consisting of the following base sequence (R) as a 16S rRNA gene: (R) The following base sequence (R1), (R2), or (R3) (R1) the nucleotide sequence shown in SEQ ID NO: 1; (R2) a nucleotide sequence in which 1 to 18 nucleotides are deleted, substituted, inserted, and / or added in the nucleotide sequence shown in SEQ ID NO: 1; (R3) A nucleotide sequence having an identity of 98.72% or more to the nucleotide sequence shown in SEQ ID NO: 1. (Appendix 8) A microorganism according to Appendix 7, having at least one characteristic selected from the group consisting of the following (1) to (9) and (10): (1) Gram-stain-positive bacilli that form spores; (2) Growth temperature is 30-58°C; (3) Growth pH is 6 to 10; (4) The growth salt concentration is 0-4%; (5) The catalase reaction is positive; (6) The oxidase reaction is positive; (7) have metabolic activity of D-ribose, D-xylose, D-glucose, D-fructose, L-rhamnose, inositol, arbutin, esculin ferric citrate, D-maltose, D-sucrose, D-trehalose, D-melezitose, D-turanose, D-tagatose, potassium 2-ketogluconate, and / or potassium 5-ketogluconate; (8) L-arginine, sodium citrate, L-tryptophan, gelatin, and / or esculin have metabolic activity; (9) Having alkaline phosphatase activity, esterase activity, esterase lipase activity, leucine aryl amidase activity, α-chymotrypsin activity, acid phosphatase activity, naphthol-AS-BI-phosphohydrolase activity, α-glucosidase activity, arginine dihydrolase activity, citrate hydrolysis activity, tryptophan deaminase activity, esculin hydrolysis activity, and / or gelatinase activity. (10) Ability to decompose polylactic acid and / or polybutylene adipate terephthalate. (Appendix 9) 9. The microorganism according to claim 7 or 8, comprising genomic DNA having an ANI value of 82% or more relative to the base sequence shown in SEQ ID NO: 2. <Characteristics> (Appendix 10) It belongs to the Bacillaceae family, A microorganism having the following characteristics (1) to (10): (1) Gram-stain-positive bacilli that form spores; (2) Growth temperature is 30-58°C; (3) Growth pH is 6 to 10; (4) The growth salt concentration is 0-4%; (5) The catalase reaction is positive; (6) The oxidase reaction is positive; (7) have metabolic activity of D-ribose, D-xylose, D-glucose, D-fructose, L-rhamnose, inositol, arbutin, esculin ferric citrate, D-maltose, D-sucrose, D-trehalose, D-melezitose, D-turanose, D-tagatose, potassium 2-ketogluconate, and / or potassium 5-ketogluconate; (8) L-arginine, sodium citrate, L-tryptophan, gelatin, and / or esculin have metabolic activity; (9) Having alkaline phosphatase activity, esterase activity, esterase lipase activity, leucine aryl amidase activity, α-chymotrypsin activity, acid phosphatase activity, naphthol-AS-BI-phosphohydrolase activity, α-glucosidase activity, arginine dihydrolase activity, citrate hydrolysis activity, tryptophan deaminase activity, esculin hydrolysis activity, and / or gelatinase activity. (10) Ability to decompose polylactic acid and / or polybutylene adipate terephthalate. (Appendix 11) It belongs to the Bacillaceae family, A microorganism according to Appendix 10, comprising, as a 16S rRNA gene, a gene having the following nucleotide sequence (R): (R) The following base sequence (R1), (R2), or (R3) (R1) the nucleotide sequence shown in SEQ ID NO: 1; (R2) a nucleotide sequence in which 1 to 18 nucleotides are deleted, substituted, inserted, and / or added in the nucleotide sequence shown in SEQ ID NO: 1; (R3) A nucleotide sequence having an identity of 98.72% or more to the nucleotide sequence shown in SEQ ID NO: 1. (Appendix 12) It belongs to the Bacillaceae family, The base sequence shown in SEQ ID NO: 2 12. The microorganism according to claim 10 or 11, comprising genomic DNA having an ANI value of 82% or more. <Composition> (Appendix 13) A composition for use in decomposing biodegradable plastics, comprising a microorganism according to any one of appendices 1 to 12. (Appendix 14) 14. The composition of claim 13, wherein the biodegradable plastic comprises polylactic acid and / or polybutylene adipate terephthalate. <Method> (Appendix 15) A method for treating biodegradable plastics, comprising the steps of contacting a biodegradable plastic with a microorganism according to any one of appendices 1 to 12 and allowing the microorganism to decompose the biodegradable plastic. (Appendix 16) 16. The method of claim 15, wherein the biodegradable plastic comprises polylactic acid and / or polybutylene adipate terephthalate. (Appendix 17) 17. The method according to claim 15 or 16, wherein the decomposition is carried out at a temperature of 25 to 55°C. (Appendix 18) 18. The method according to any one of claims 15 to 17, wherein the decomposition is carried out under conditions of pH 6 to 10. [Industrial Applicability]

[0087] As described above, the present disclosure provides a novel microorganism capable of degrading biodegradable plastics, particularly polylactic acid (PLA) or polybutylene adipate terephthalate (PBAT). Therefore, the present invention can be said to be extremely useful, for example, in fields such as waste disposal.

Claims

1. It belongs to the Bacillaceae family, A microorganism deposited under accession number NITE BP-04083.

2. A microorganism which is a mutant strain of the microorganism according to claim 1.

3. The microorganism according to claim 2, wherein the mutant strain comprises a gene consisting of the following base sequence (R) as a 16S rRNA gene: (R) The following base sequence (R1), (R2), or (R3): (R1) the base sequence shown in SEQ ID NO: 1; (R2) a nucleotide sequence in which 1 to 18 nucleotides are deleted, substituted, inserted, and / or added in the nucleotide sequence shown in SEQ ID NO: 1; (R3) A base sequence having an identity of 98.72% or more in the base sequence shown in SEQ ID NO:

1.

4. The microorganism according to claim 2 or 3, wherein the mutant strain has at least one characteristic selected from the group consisting of the following (1) to (8) and (9): (1) Gram-stain-positive bacilli that form spores; (2) The growth temperature is 30 to 58°C; (3) Growth pH is 6 to 10; (4) The growth salt concentration is 0 to 4%; (5) The catalase reaction is positive; (6) The oxidase reaction is positive; (7) Having metabolic activity of D-ribose, D-xylose, D-glucose, D-fructose, L-rhamnose, inositol, arbutin, esculin ferric citrate, D-maltose, D-sucrose, D-trehalose, D-melezitose, D-turanose, D-tagatose, potassium 2-ketogluconate, and / or potassium 5-ketogluconate; (8) L-arginine, sodium citrate, L-tryptophan, gelatin, and / or esculin metabolic activity; (9) Having alkaline phosphatase activity, esterase activity, esterase lipase activity, leucine aryl amidase activity, α-chymotrypsin activity, acid phosphatase activity, naphthol-AS-BI-phosphohydrolase activity, α-glucosidase activity, arginine dihydrolase activity, citrate hydrolysis activity, tryptophan deaminase activity, esculin hydrolysis activity, and / or gelatinase activity.

5. The microorganism according to claim 4, further having the following characteristic (10): (10) Ability to decompose polylactic acid (PLA) and / or polybutylene adipate terephthalate (PBAT).

6. The microorganism according to claim 2 or 3, wherein the mutant strain comprises genomic DNA having an ANI value of 82% or more as shown in SEQ ID NO:

2.

7. It belongs to the Bacillaceae family, A microorganism comprising, as a 16S rRNA gene, a gene having the following base sequence (R): (R) The following base sequence (R1), (R2), or (R3): (R1) the base sequence shown in SEQ ID NO: 1; (R2) a nucleotide sequence in which 1 to 18 nucleotides are deleted, substituted, inserted, and / or added in the nucleotide sequence shown in SEQ ID NO: 1; (R3) A base sequence having an identity of 98.72% or more in the base sequence shown in SEQ ID NO:

1.

8. The microorganism according to claim 7, having at least one characteristic selected from the group consisting of the following (1) to (9) and (10): (1) Gram-stain-positive bacilli that form spores; (2) The growth temperature is 30 to 58°C; (3) Growth pH is 6 to 10; (4) The growth salt concentration is 0 to 4%; (5) The catalase reaction is positive; (6) The oxidase reaction is positive; (7) Having metabolic activity of D-ribose, D-xylose, D-glucose, D-fructose, L-rhamnose, inositol, arbutin, esculin ferric citrate, D-maltose, D-sucrose, D-trehalose, D-melezitose, D-turanose, D-tagatose, potassium 2-ketogluconate, and / or potassium 5-ketogluconate; (8) L-arginine, sodium citrate, L-tryptophan, gelatin, and / or esculin metabolic activity; (9) Having alkaline phosphatase activity, esterase activity, esterase lipase activity, leucine aryl amidase activity, α-chymotrypsin activity, acid phosphatase activity, naphthol-AS-BI-phosphohydrolase activity, α-glucosidase activity, arginine dihydrolase activity, citrate hydrolysis activity, tryptophan deaminase activity, esculin hydrolysis activity, and / or gelatinase activity. (10) Ability to decompose polylactic acid and / or polybutylene adipate terephthalate.

9. 9. The microorganism according to claim 7 or 8, comprising genomic DNA having an ANI value of 82% or more relative to the base sequence shown in SEQ ID NO:

2.

10. It belongs to the Bacillaceae family, A microorganism having the following characteristics (1) to (10): (1) Gram-stain-positive bacilli that form spores; (2) The growth temperature is 30 to 58°C; (3) Growth pH is 6 to 10; (4) The growth salt concentration is 0 to 4%; (5) The catalase reaction is positive; (6) The oxidase reaction is positive; (7) Having metabolic activity of D-ribose, D-xylose, D-glucose, D-fructose, L-rhamnose, inositol, arbutin, esculin ferric citrate, D-maltose, D-sucrose, D-trehalose, D-melezitose, D-turanose, D-tagatose, potassium 2-ketogluconate, and / or potassium 5-ketogluconate; (8) L-arginine, sodium citrate, L-tryptophan, gelatin, and / or esculin metabolic activity; (9) Having alkaline phosphatase activity, esterase activity, esterase lipase activity, leucine aryl amidase activity, α-chymotrypsin activity, acid phosphatase activity, naphthol-AS-BI-phosphohydrolase activity, α-glucosidase activity, arginine dihydrolase activity, citrate hydrolysis activity, tryptophan deaminase activity, esculin hydrolysis activity, and / or gelatinase activity. (10) Ability to decompose polylactic acid and / or polybutylene adipate terephthalate.

11. It belongs to the Bacillaceae family, The microorganism according to claim 10, comprising a gene consisting of the following base sequence (R) as a 16S rRNA gene: (R) The following base sequence (R1), (R2), or (R3): (R1) the base sequence shown in SEQ ID NO: 1; (R2) a nucleotide sequence in which 1 to 18 nucleotides are deleted, substituted, inserted, and / or added in the nucleotide sequence shown in SEQ ID NO: 1; (R3) A base sequence having an identity of 98.72% or more in the base sequence shown in SEQ ID NO:

1.

12. It belongs to the Bacillaceae family, The base sequence shown in SEQ ID NO: 2 The microorganism according to claim 10 or 11, comprising genomic DNA having an ANI value of 82% or more.

13. A composition for use in decomposing biodegradable plastics, comprising the microorganism according to claim 1 or 2.

14. The composition of claim 13 , wherein the biodegradable plastic comprises polylactic acid and / or polybutylene adipate terephthalate.

15. A method for treating biodegradable plastics, comprising the steps of contacting the microorganism according to claim 1 or 2 with biodegradable plastics and allowing the microorganisms to decompose the biodegradable plastics.

16. 16. The method of claim 15, wherein the biodegradable plastic comprises polylactic acid and / or polybutylene adipate terephthalate.

17. 16. The method according to claim 15, wherein the decomposition is carried out at a temperature of 25 to 55°C.

18. The method according to claim 15, wherein the decomposition is carried out under conditions of pH 6 to 10.

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