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).　The microorganism according to the present disclosure belongs to the family Bacillaceae, and has been deposited under Accession No. NITE BP-04083.

Description

Microorganisms, compositions for use in decomposing biodegradable plastics, and methods for treating biodegradable plastics

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

[0002] In recent years, in order to solve the problems of marine plastics and global warming, the final CO ２ Research and development is also underway into biodegradable plastics that can be decomposed in water.

[0003] Furthermore, with the expansion of the use of biodegradable plastics, research into microorganisms that can decompose biodegradable plastics is progressing (Non-Patent Documents 1 and 2).

[0004] 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 / s11356-011-0443-2Bonifer, 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

[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.

[0006] To achieve the above object, the microorganism of the present disclosure belongs to the family Bacillaceae and 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 Bacillaceae family and has the following characteristics (1) to (10): (1) it is a Gram-stain-positive bacillus and forms spores; (2) it grows at a temperature of 30 to 58°C; (3) it grows at a pH of 6 to 10; (4) it grows at a salt concentration of 0 to 4%; (5) it exhibits a positive catalase reaction; (6) it exhibits a positive oxidase reaction; and (7) it has metabolic activity for 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) having 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 decomposition activity, tryptophan deaminase activity, esculin hydrolysis activity, and / or gelatinase activity; (10) having the 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.

[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).

[0012] FIG. 1 shows a phylogenetic tree of Komham264 based on the partial base sequence of 16S rRNA in Example 1. FIG. 2 is a photograph showing the results of morphological observation of Komham264 using an optical microscope in Example 1. FIG. 3 is a photograph showing the results of Gram staining of Komham264 in Example 1. FIG. 4 is a photograph showing the results of a second-stage bacterial test using API (registered trademark) 50CH in Example 1. FIG. 5 is a photograph showing the results of a second-stage bacterial test using API (registered trademark) 20E and API (registered trademark) 20NE in Example 1. FIG. 6 is a photograph showing the results of a second-stage bacterial test using API (registered trademark) ZYM in Example 1. FIG. 7 shows the results of ANI analysis in Example 1. FIG. 8 is a photograph showing the results of degradation of polybutylene adipate terephthalate in Example 1. FIG. 9 is a photograph showing the results of degradation of polylactic acid in Example 1. FIG. 10 is a photograph showing the results of decomposition of polylactic acid in Example 1.

[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, and 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) A bacillus that is Gram-stain positive and can form spores; (2) The growth temperature is 30 to 58°C; (3) The 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) The bacterium has metabolic activity for 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) The bacterium has metabolic activity for L-arginine, sodium citrate, L-tryptophan, gelatin, and / or esculin; (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 any 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 any 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 any 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 (5) to (6) of Example 1 described later.

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

[0020] An example of komham264 disclosed herein 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 Microorganism 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 Date of deposit: 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), that is, a 16S rRNA gene having the base sequence shown in SEQ ID NO: 1.

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

[0023] In the (R1), the base sequence of SEQ ID NO: 1 is a base sequence encoding 16S rRNA. The base sequence of SEQ ID NO: 1 can be isolated from, for example, Komham264.

[0024]

[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. In other words, for example, the description "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, known methods. Examples of 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 their DNA-DNA hybridization homology with a reference strain is 70% or higher (Reference 1). Meanwhile, in bacterial species identification using 16S rRNA sequences, a homology of 97% or higher is considered to indicate a close relationship, and an identity of 98.7% or higher is considered to be highly likely to be of the same species (Reference 2). Therefore, in bacterial species identification using 16S rRNA sequences, if there is no species with a homology of 98.7% or higher, it is considered a new species. Specifically, 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] 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 species similarity 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, a value of 95% or higher indicates that the genomes belong to the same bacterial species, while a value of less than 95% indicates 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 analyses of microbial genomes and metagenomes. No. e1900v1. PeerJ Preprints, 2016.

[0032] The ANI value between the genome sequence of the 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 of (G) below as genomic DNA, and preferably has a gene consisting of the base sequence of (G1) below, i.e., genomic DNA consisting of the base sequence shown in SEQ ID NO: 2.

[0035] (G) A base sequence of (G1), (G2), or (G3) below: (G1) A base sequence shown in SEQ ID NO: 2; (G2) A base sequence in which one or more bases have been deleted, substituted, inserted, and / or added in the base sequence shown in SEQ ID NO: 2; (G3) A base sequence having 80% or more identity to the base sequence shown in SEQ ID NO: 2.

[0036] In (G1), the base sequence of SEQ ID NO: 2 is a base sequence encoding genomic DNA. The base 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. Examples of the characteristics of komham264 include 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 719454, 1 to 539590, 1 to 359727, 1 to 179863, 1 to 143890, 1 to 107918, 1 to 71945, 1 to 53959, 1 to 32972, 1 to 17986, 1 to 14389, 1 to 10791, 1 to 7194, 1 to 5395, 1 to 3297, or 1 to 1798 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 the komham264 strain 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 observation of colony color, cell morphology, Gram staining, and sporulation ability. Examples of the first-stage bacterial test include observation of colony characteristics, cell morphology, and motility, 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 of carbon source assimilation, oxidation / fermentation, and enzyme activity using a physiological and biochemical property test kit. The komham264 strain 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. In this way, the present disclosure makes it possible to obtain progeny lines of the komham264.

[0041] <Mutant Strain> The microorganism of the present disclosure may be, for example, a mutant strain of komham264 or a descendant strain 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. Examples of the properties include 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, or the like. The mutation can be introduced, for example, by a mutation introduction method such as site-directed mutagenesis. The mutation can also be introduced, for example, by random mutagenesis. Examples of random mutagenesis methods include irradiation treatment with α-rays, β-rays, γ-rays, X-rays, or the like; chemical treatment with mutagens such as ethyl methanesulfonate (EMS) or ethynylnitrosourea (ENU); heavy ion beam treatment; and the like.

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

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

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

[0047] (G) A base sequence of (G1), (G2), or (G3) below: (G1) A base sequence shown in SEQ ID NO: 2; (G2) A base sequence in which one or more bases have been deleted, substituted, inserted, and / or added in the base sequence shown in SEQ ID NO: 2; (G3) A base sequence having 80% or more identity to the base 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 treating biodegradable plastic> In another aspect, the present disclosure provides a method capable of treating biodegradable plastic. The method for treating biodegradable plastic of the present disclosure (hereinafter also referred to as the "treatment method") uses the microorganism of the present disclosure. The treatment method of the present disclosure includes a step of contacting the microorganism of the present disclosure with biodegradable plastic and decomposing the biodegradable plastic with the microorganism (hereinafter also referred to as the "decomposition step"). The treatment method of the present disclosure is characterized by using the microorganism of the present disclosure, and other steps and conditions are not particularly limited.

[0050] In this disclosure, "biodegradable plastic" refers to a plastic that can be decomposed by microorganisms and eventually decomposed into CO ２ Biodegradable plastics refer to plastics that decompose into water or biodegradable materials. Examples of biodegradable plastics include polylactic acid (PLA), polybutylene adipate terephthalate (PBAT), polyhydroxybutyrate (PHB), polycaprolactone (PCL), polybutylene succinate (PBS), and polyethylene terephthalate (PET), with polylactic acid and / or polybutylene adipate terephthalate being preferred. Examples of polylactic acid include homopolymers of lactic acid 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 process, the object to be decomposed may contain the biodegradable plastic, and may consist solely of the biodegradable plastic, or may contain 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 the biodegradable plastic, such as 30 to 58°C, and 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 process the biodegradable plastic. The decomposition pH is, for example, pH 6 to 10, 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 moisture 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%.

[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 of the present disclosure was 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 obtained 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. 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 the novel microorganism was also confirmed in several other composts in Hokkaido.

[0060] (2) Determination of the 16S rRNA Base Sequence of komham264 The 16S rRNA base 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 Corporation, synthesis requested) and KOD One (registered trademark) PCR Master Mix (manufactured by TOYOBO Corporation) 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 resulting PCR product to obtain the 16S rRNA base sequence of komham264 isolated in Example 1(1). The base sequence was the base 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 16S rRNA sequences of existing microorganisms Using the 16S rRNA 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 sequences identical or similar to the 16S rRNA sequence of komham264. As a result of the search, it was found that the microorganism having the sequence represented by SEQ ID NO: 5, Bacillus thermolactis (strain Marseille-AA00136) of the genus Bacillus (Bacillus sp.), has a 16S rRNA sequence closest to the 16S rRNA sequence of komham264.

[0063]

[0064] (4) Creation of Phylogenetic Tree Next, from the results of the homology search in Example 1 (3), a phylogenetic tree based on the partial base sequence of 16S rRNA was created, and the phylogenetic position of komham264 was analyzed. The phylogenetic tree was created using MEGA (Molecular Evolutionary Genetics Analysis) software and neighbor-joining, which are child evolutionary and phylogenetic analysis tools. These results are shown in Figure 1.

[0065] FIG. 1 shows a phylogenetic tree of komham264 based on the partial base sequence of 16S rRNA.

[0066] (5) Taxonomic Characterization 1: Morphological observation and physiological property tests (hereinafter referred to as "first-stage bacterial tests") were performed on Komham 264 to examine its taxonomic characteristics. Specifically, Komham 264 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]

[0068] 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 forms colonies as shown in FIG.

[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 Properties 2 A biochemical property test (hereinafter also referred to as the "second-stage bacterial test") was 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 test. The results are shown in Figures 4 to 6 and Tables 2 and 3 below.

[0071]

[0072] Figure 4 is a photograph showing the results of the second stage bacterial test using API (registered trademark) 50CH. Table 2 above is a table showing the results of the second stage bacterial test using API (registered trademark) 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 (registered trademark) 20E and API (registered trademark) 20NE. As shown in Figure 5, komham264 was found to have metabolic activities 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]

[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 Genomic DNA Sequence The genomic DNA sequence of komham264 was determined. Specifically, komham264 genomic DNA 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 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 of less than 20 per read 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 consists of the base sequence shown in SEQ ID NO:2.

[0077] (8) ANI Analysis of Genomic Sequence ANI analysis was further performed on komham264. Specifically, the ANI value was determined for the nucleotide 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 Biodegradable Plastic (Polybutylene Adipate Terephthalate) Degradation Ability of komham264 The degradability of 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 fragments 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 start day of the shaking culture (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 degradation of polybutylene adipate terephthalate. From left to right in Figure 8, photographs taken on days 0, 4, and 8 were shown. As shown in Figure 8, degradation of polybutylene adipate terephthalate was confirmed in Komham264 compared to the control on days 4 and 8 from the start of shaking culture.

[0081] (10) Evaluation of Biodegradable Plastic (Polylactic Acid) Degradation Ability 1 komham264 was examined for its ability to decompose biodegradable plastic (polylactic acid). 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 day 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 is a photograph 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 from the start of shaking culture.

[0083] (11) Evaluation of Biodegradable Plastic (Polylactic Acid) Degradation Ability 2 komham264 was examined for its ability to decompose biodegradable plastic (polylactic acid). 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 start day of the shaking culture (day 0), and on days 1, 5, 11, and 14 after the start of the shaking culture. Note that a control was performed in the same manner except that komham264 was not added. These results are shown in Figure 10.

[0084] FIG. 10 is a photograph showing the results of polylactic acid degradation. From left to right, FIG. 10 shows photographs taken on days 0, 1, 5, 11, and 14. As shown in FIG. 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 decompose 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. KOMHAM264 of the present disclosure is also expected to be able to 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] This application claims priority based on Japanese Patent Application No. 2024-072323, filed April 26, 2024, the disclosure of which is incorporated herein in its entirety by reference.

[0087] <Appendix> Some or all of the above embodiments and examples can be described as, but are not limited to, the following appendices. <Deposited microorganism> (Appendix 1) A microorganism belonging to the family Bacillaceae and deposited under accession number NITE BP-04083. <Mutant> (Appendix 2) A microorganism that is a mutant of the microorganism described in Appendix 1. (Appendix 3) The microorganism described in Appendix 2, wherein the mutant contains a gene consisting of the nucleotide sequence of (R) below as a 16S rRNA gene: (R) A nucleotide sequence of (R1), (R2), or (R3) below: (R1) The nucleotide sequence shown in SEQ ID NO: 1; (R2) A nucleotide sequence in which 1 to 18 bases have been 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. (Supplementary Note 4) The microorganism according to Supplementary Note 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) it is a Gram-stain-positive bacillus and forms spores; (2) it grows at a temperature of 30 to 58°C; (3) it grows at a pH of 6 to 10; (4) it grows at a salt concentration of 0 to 4%; (5) it shows a positive catalase reaction; (6) it shows a positive oxidase reaction; (7) Having metabolic activity for 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) Having metabolic activity for L-arginine, sodium citrate, L-tryptophan, gelatin, and / or esculin; (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) The microorganism according to Appendix 4, further having the following property (10): (10) having the ability to decompose polylactic acid (PLA) and / or polybutylene adipate terephthalate (PBAT). (Appendix 6) The 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) A microorganism belonging to the family Bacillaceae, comprising a gene consisting of the nucleotide sequence of (R) below as a 16S rRNA gene: (R) a nucleotide sequence of (R1), (R2), or (R3) below: (R1) the nucleotide sequence shown in SEQ ID NO: 1; (R2) a nucleotide sequence in which 1 to 18 bases have been deleted, substituted, inserted, and / or added in the nucleotide sequence shown in SEQ ID NO: 1; (R3) a nucleotide sequence having 98.72% or more identity to the nucleotide sequence shown in SEQ ID NO: 1. (Appendix 8) The microorganism according to Appendix 7, having at least one characteristic selected from the group consisting of the following (1) to (9) and (10): (1) being a Gram-stain-positive bacillus and capable of spore formation; (2) growing at a temperature of 30 to 58°C; (3) growing at a pH of 6 to 10; (4) growing at a salt concentration of 0 to 4%; (5) being positive in catalase reaction; (6) being positive in oxidase reaction; (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) Having 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) Having the ability to decompose polylactic acid and / or polybutylene adipate terephthalate. (Appendix 9) The microorganism according to appendix 7 or 8, comprising genomic DNA having an ANI value of 82% or more with respect to the base sequence shown in SEQ ID NO:2. <Characteristics> (Appendix 10) A microorganism belonging to the family Bacillaceae, having the following characteristics (1) to (10): (1) being a Gram-stain-positive bacillus and capable of spore formation; (2) growing at a temperature of 30 to 58°C; (3) growing at a pH of 6 to 10; (4) growing at a salt concentration of 0 to 4%; (5) being positive in catalase reaction; (6) being positive in oxidase reaction; (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) having 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 decomposition activity, tryptophan deaminase activity, esculin hydrolysis activity, and / or gelatinase activity; (10) having the ability to decompose polylactic acid and / or polybutylene adipate terephthalate. (Appendix 11) A microorganism according to Appendix 10, which belongs to the family Bacillaceae and contains, as a 16S rRNA gene, a gene consisting of the nucleotide sequence of (R) below: (R) a nucleotide sequence of (R1), (R2), or (R3) below: (R1) the nucleotide sequence shown in SEQ ID NO: 1; (R2) a nucleotide sequence in which 1 to 18 bases have been deleted, substituted, inserted, and / or added in the nucleotide sequence shown in SEQ ID NO: 1; (R3) a nucleotide sequence which has an identity of 98.72% or more in the nucleotide sequence shown in SEQ ID NO: 1.(Appendix 12) The microorganism according to Appendix 10 or 11, belonging to the family Bacillaceae, and comprising genomic DNA having an ANI value of 82% or more for the base sequence shown in SEQ ID NO: 2. <Composition> (Appendix 13) A composition for use in decomposing biodegradable plastics, comprising the microorganism according to any one of Appendices 1 to 12. (Appendix 14) The composition according to Appendix 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 the microorganism according to any one of Appendices 1 to 12 with a biodegradable plastic and allowing the microorganism to decompose the biodegradable plastic. (Appendix 16) The treatment method according to Appendix 15, wherein the biodegradable plastic comprises polylactic acid and / or polybutylene adipate terephthalate. (Appendix 17) The treatment method according to appendix 15 or 16, wherein the decomposition is carried out at a temperature of 25 to 55° C. (Appendix 18) The treatment method according to any one of appendices 15 to 17, wherein the decomposition is carried out under conditions of pH 6 to 10.

[0088] 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 in fields such as waste disposal.

Claims

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

2. A microorganism which is a mutant strain of the microorganism described in claim 1.

3. The microorganism according to claim 2, wherein the mutant strain comprises a gene consisting of the nucleotide sequence (R) below as a 16S rRNA gene: (R) the nucleotide sequence (R1), (R2), or (R3) below: (R1) the nucleotide sequence shown in SEQ ID NO: 1; (R2) a nucleotide sequence in which 1 to 18 bases have been 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 in the nucleotide 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) it is a Gram-stain-positive bacillus and forms spores; (2) it grows at a temperature of 30 to 58°C; (3) it grows at a pH of 6 to 10; (4) it grows at a salt concentration of 0 to 4%; (5) it exhibits a positive catalase reaction; (6) it exhibits a positive oxidase reaction; and (7) it has metabolic activity for 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) Having 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. A 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. A microorganism belonging to the Bacillaceae family, which contains a gene consisting of the following base sequence (R) as its 16S rRNA gene: (R) a base sequence of (R1), (R2), or (R3) below: (R1) The base sequence shown in SEQ ID NO: 1; (R2) A base sequence in which 1 to 18 bases have been deleted, substituted, inserted, and / or added in the base 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) being a Gram-stain-positive bacillus and capable of spore formation; (2) growing at a temperature of 30 to 58°C; (3) growing at a pH of 6 to 10; (4) growing at a salt concentration of 0 to 4%; (5) being positive in catalase reaction; (6) being positive in oxidase reaction; (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) having 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 decomposition activity, tryptophan deaminase activity, esculin hydrolysis activity, and / or gelatinase activity; (10) having the ability to decompose polylactic acid and / or polybutylene adipate terephthalate.

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

2.

10. A microorganism belonging to the family Bacillaceae and having the following characteristics (1) to (10): (1) being a Gram-stain-positive bacillus and capable of spore formation; (2) growing at a temperature of 30 to 58°C; (3) growing at a pH of 6 to 10; (4) growing at a salt concentration of 0 to 4%; (5) exhibiting a positive catalase reaction; (6) exhibiting a positive oxidase reaction; (7) having metabolic activity for 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) having 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 decomposition activity, tryptophan deaminase activity, esculin hydrolysis activity, and / or gelatinase activity; (10) having the ability to decompose polylactic acid and / or polybutylene adipate terephthalate.

11. The microorganism according to claim 10, which belongs to the Bacillaceae family and contains, as a 16S rRNA gene, a gene consisting of the nucleotide sequence of (R) below: (R) a nucleotide sequence of (R1), (R2), or (R3) below: (R1) the nucleotide sequence shown in SEQ ID NO: 1; (R2) a nucleotide sequence in which 1 to 18 bases have been 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 in the nucleotide sequence shown in SEQ ID NO:

1.

12. A microorganism according to claim 10 or 11, which belongs to the Bacillaceae family and contains genomic DNA having an ANI value of 82% or more for the base sequence shown in SEQ ID NO:

2.

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 step of contacting the microorganisms according to claim 1 or 2 with biodegradable plastics and allowing the microorganisms to decompose the biodegradable plastics.

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

17. The method of 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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