Plastic-degrading microorganism

Abstract

To provide a plastic-degrading microorganism.SOLUTION: In one aspect, the present disclosure provides a microorganism capable of degrading plastic, belonging to a genus selected from the group consisting of the genus Acinetobacter, the genus Achromobacter, the genus Pseudomonas, the genus Serratia, the genus Amycolatopsis, and the genus Bacillus. In one aspect, the plastic to be degraded comprises at least one selected from polypropylene (PP), polyethylene (PE), polyvinylidene chloride (PVDC), and ethylene-vinyl alcohol copolymer (EVOH).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to microorganisms capable of degrading plastics and uses of such microorganisms. [Background technology]

[0002] Plastics synthesized from petroleum-based components have been used for decades, and waste plastics are mainly disposed of in landfills or incinerated. These plastic wastes are poorly degradable in the natural environment, so once they are released into the natural world, they are difficult to recover and process. Only a few microorganisms are known that can degrade such plastics, so new plastic-degrading microorganisms are needed. Summary of the Invention [Means for solving the problem]

[0003] As a result of intensive research, the present inventors have found a novel microorganism capable of degrading plastic. The present disclosure provides the thus found microorganism and its derivatives, as well as uses thereof. The present disclosure also provides a method for obtaining a microorganism capable of degrading plastic.

[0004] Thus, the present disclosure provides: (Item 1) A microorganism belonging to a genus selected from the group consisting of Acinetobacter, Achromobacter, Pseudomonas, Serratia, Amycolatopsis and Bacillus, said microorganism having the ability to degrade plastic. (Item 2) The microorganism of any of the preceding items, wherein the plastic comprises at least one of polypropylene (PP), polyethylene (PE), polyvinylidene chloride (PVDC) and ethylene-vinyl alcohol copolymer (EVOH). (Item 3) The microorganism according to any of the preceding items, wherein the microorganism is a microorganism of a genus selected from the group consisting of Acinetobacter, Achromobacter and Pseudomonas and has the ability to degrade polypropylene (PP). (Item 4) The microorganism according to any of the preceding items, wherein the microorganism is an Acinetobacter genus microorganism selected from the group consisting of A. beijerinckii, A. junii, A. venetianus, A. haemolyticus, A. halotolerans, A. nosocomialis, A. baumannii, A. seifertii, A. pittii, A. lactucae, A. calcoaceticus, A. soli, and A. baylyi. (Item 5) The microorganism according to any of the preceding items, wherein the microorganism is 2-4-2A (strain with accession number AP-03962), 5-3-1A (strain with accession number AP-03963), 17-5-3D (strain with accession number AP-03964) or a closely related strain thereof. (Item 6) Any of the microorganisms described above, comprising a 16S rDNA having at least 90% sequence identity to any of the sequences set forth in SEQ ID NOs: 1 to 4. (Item 7) The microorganism of any of the preceding items, which is a strain selected from the group consisting of Accession Nos. AP-03962, AP-03963 and AP-03964. (Item 8) The microorganism according to any of the preceding items, wherein the microorganism is a microorganism of the genus Serratia, Acinetobacter, Achromobacter or Pseudomonas and has the ability to degrade polyethylene (PE). (Item 9) The microorganism according to any of the preceding items, wherein the microorganism is TIKK4D (strain with accession number AP-03965), 2-4-2A (strain with accession number AP-03962), 5-3-1A (strain with accession number AP-03963), 17-5-3D (strain with accession number AP-03964) or a closely related strain thereof. (Item 10) Any of the microorganisms described above, comprising a 16S rDNA having at least 90% sequence identity to any of the sequences set forth in SEQ ID NOs: 5 to 6. (Item 11) The microorganism of any of the preceding items, which is a strain selected from the group consisting of accession numbers AP-03965, AP-03962, AP-03963 and AP-03964. (Item 12) The microorganism according to any of the preceding items, wherein the microorganism is a microorganism of the genus Amycolatopsis or Bacillus and has the ability to degrade polyvinylidene chloride (PVDC). (Item 13) The microorganism according to any of the preceding items, wherein the microorganism is 5Acc (strain with accession number AP-03966) or a closely related strain thereof. (Item 14) Any of the microorganisms described in the preceding items, comprising a 16S rDNA having at least 90% sequence identity to any of the sequences set forth in SEQ ID NOs: 7 to 8. (Item 15) The microorganism of any of the preceding items, which is the strain with accession number AP-03966. (Item 16) A plastic decomposition agent comprising a microbial cell, culture supernatant, homogenate, extract thereof or a combination thereof of any of the microorganisms described above. (Item 17) A method for decomposing plastics, comprising: A step of contacting a microorganism, a culture supernatant, a lysate, an extract thereof or a combination thereof of any of the microorganisms described above with a plastic. A method comprising: (Item 18) The method of any of the preceding claims, not including the step of modifying the plastic prior to said contacting step. (Item 19) A method for obtaining plastic-degrading microorganisms, comprising the steps of: Cultivating candidate microorganisms in an environment containing plastic as the only carbon source; and A step of isolating microorganisms from colonies found after cultivation. A method comprising: (Item 20) A method for producing a plastic decomposer, comprising the steps of: A step of preparing a composition using the microorganism obtained by any of the methods described above. A method comprising:

[0005] It is contemplated that the present disclosure may provide one or more of the above features in combinations other than those specifically described. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary. Effect of the Invention

[0006] The microorganisms disclosed herein can decompose plastics and can therefore be used in the treatment of petroleum waste products, thereby reducing the environmental burden. [Brief description of the drawings]

[0007] [Figure 1] Photographs of streak cultures of the PP-degrading bacteria found are shown. [Diagram 2] A cluster diagram of the molecular phylogenetic tree analysis based on the 16S rDNA base sequences of the 2-3-1A, 5-3-1A, 2-4-2A and 6-3-1F strains is shown. [Diagram 3] A cluster diagram of the molecular phylogenetic tree analysis based on the 16S rDNA base sequences of the 13-3-1C, 17-4-2C, 17-4-1F and 17-5-3D strains is shown. [Figure 4] The test results for the PP decomposition ability of each isolated strain are shown below. The incubation periods for each were as follows: _1: 2-3-1A, 17-5-3D, and ctrl were all incubated for 55 days. _2: 2-4-2A was incubated for 9 days, and ctrl was incubated for 63 days. _3: 5-3-1A was incubated for 1 day, and 6-3-1F and ctrl were incubated for 56 days. _4: 13-3-1C was incubated for 2 days, and ctrl was incubated for 7 days. 17-4-1F: incubated for 22 days. _5: 17-4-2C and ctrl were all incubated for 95 days. The vertical axis shows the degradation rate calculated as (weight of PP pieces at the start - weight of PP pieces after incubation) / (weight of PP pieces at the start). [Diagram 5](Left) A photograph of a PP cap piece cultured with the 17-5-3D strain is shown. (Right) A photograph of a PP cap piece cultured under control conditions without the addition of bacteria is shown. [Figure 6] A cluster diagram of molecular phylogenetic analysis based on the 16S rDNA base sequences of the TIKK4A, TIKK4D, and APKK9-2B strains is shown. [Figure 7] The obtained plastic-degrading bacteria were evaluated for their ability to degrade LDPE fragments (top) and HDPE fragments (bottom), and the results are shown together with the average value and standard error of three experiments. [Figure 8] Photographs showing that PE-degrading bacteria are adsorbed onto PE film. (Left) Before washing, (Right) After washing. (Top) TIKK4D, (Interrupted) APKK9-2B, (Bottom) TIKK4A. [Figure 9] The results of FT-IR analysis of a PE film with PE-degrading bacteria adsorbed thereon are shown. [Figure 10] A photograph of a streak culture of PVDC-degrading bacteria 5A is shown. [Figure 11] A cluster diagram of the molecular phylogenetic tree analysis based on the 16S rDNA base sequences of strains 7B, 5A, 7A, and 5C is shown. [Figure 12] A cluster diagram of the molecular phylogenetic tree analysis based on the 16S rDNA base sequence of the 5Acc strain is shown. [Figure 13] The growth ability of the obtained PVDC-degrading bacteria in a medium containing PVDC as the sole carbon source was evaluated based on turbidity. [Figure 14] The growth ability of the obtained plastic-degrading bacteria in a medium containing EVOH as the sole carbon source was evaluated based on turbidity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] The following provides definitions of terms particularly used in this specification and / or explains basic technical content as appropriate.

[0009] (definition, etc.) In this specification, "decomposition" refers to the breaking down of a target substance (e.g., plastic) into smaller molecules. It is not necessary to detect these small molecules in order to determine the decomposition of plastic, but decomposition can be determined by detecting a decrease in the weight of plastic, modification of plastic (oxidation, etc.), growth of microorganisms in an environment where plastic is the only carbon source, etc.

[0010] As used herein, a "closely related strain" preferably includes, but is not limited to, a gene (e.g., 16S rDNA) that includes a region that is substantially homologous to the DNA of the microorganism of interest. For example, a closely related strain has a whole genome sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, at least 99.8% or at least 99.9% identical when compared to the whole genome sequence of the original strain by alignment with a computer homology program known in the art, and / or a 16S rDNA that is at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, at least 99.8% or at least 99.9% identical when compared to the sequence of the 16S rDNA of the original strain. This is a microorganism modified by gene mutation, substitution, deletion and / or addition, and the related strain preferably exhibits the biological function of the original microorganism, although not necessarily to the same degree. For example, gene mutations can be introduced using any known mutagen, UV, plasma, genome editing technology, etc. In this specification, "identity" can be expressed as a value measured by NCBI's BLAST unless otherwise specified. For the algorithm when comparing base sequences with BLAST, Blastp can be used with default settings. The measurement results are quantified as Positives or Identities.

[0011] As used herein, a "purified" biological agent (e.g., a particular strain of organism or intracellular component) refers to a biological agent from which at least a portion of the factors naturally associated with the biological agent have been removed. Thus, the purity of the biological agent in a purified biological agent is usually higher (i.e., concentrated) than the state in which the biological agent is normally found. As used herein, the term "purified" means that preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight of the same type of biological agent (e.g., a particular strain of organism) is present.

[0012] As used herein, the term "about" refers to the indicated value plus or minus 10%, unless otherwise specified. When used in reference to temperature, "about" may refer to the indicated value plus or minus 5°C.

[0013] As used herein, the term "kit" refers to a unit in which the parts to be provided (e.g., a composition containing the microorganism of the present disclosure, additional components, buffer solution, instructions, etc.) are provided, usually in two or more compartments. This kit form is preferred when the purpose is to provide a composition that should not be provided in a mixed state for stability or the like, but is preferably mixed and used immediately before use. Such a kit is preferably provided with instructions or instructions that describe how to use or process the parts to be provided (e.g., a composition containing the microorganism, additional components, etc.). When the term "kit" is used herein, the kit usually includes instructions that describe how to use the microorganism or composition of the present disclosure.

[0014] (Preferred embodiment) The preferred embodiments of the present disclosure are described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that a person skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description in this specification. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.

[0015] (Plastic-degrading microorganisms) In one aspect, the present disclosure provides a new microorganism capable of decomposing plastics. The plastics to be decomposed include polymers formed by radical polymerization of monomers (or a combination of multiple types of monomers) containing carbon-carbon double bonds, such as polypropylene (PP), polyethylene (PE), polyvinylidene chloride (PVDC), and ethylene-vinyl alcohol copolymer (EVOH).

[0016] In one embodiment, the microorganism of the present disclosure is a microorganism of a genus selected from the group consisting of Acinetobacter, Achromobacter, Pseudomonas, Serratia, Amycolatopsis, and Bacillus.

[0017] In one embodiment, the microorganism of the present disclosure is a bacterium of the genus Acinetobacter. Acinetobacter microorganisms are gram-negative bacillus eubacteria that are aerobic, short rod-shaped, non-flagellated, and commonly found in moist environments such as soil. Genus Acinetobacter includes albensis, apis, baumannii, baylyi, beijerinckii, bereziniae, bohemicus, boissieri, bouvetii, brisouii, calcoaceticus, celticus, chengduensis, chinensis, colistiniresistens, courva linii, cumulans, defluvii, dispersus, equi, gandensis, gerneri, guerrae, guillouiae, gyllenbergii, haemolyticus, halotolerans, harbinensis, indicus, johnsonii, junii, kookii, lactucae, lanii, larvae, lw offii, modestus, nectaris, nosocomialis, parvus, piscicola, pittii, populi, portensis, pragensis, proteolyticus, pseudodolwoffii, pullicarnis, pullorum, puyangensis, qingfengensis, radioresistens, rong Includes species such as chengensis, rudis, schindleri, seifertii, shaoyimingii, sichuanensis, soli, tandoii, tianfuensis, tjernbergiae, towneri, ursingii, variabilis, venetianus, vivianii, wanghuae, and wuhouensis.

[0018] A classification of the genus Acinetobacter has been proposed in Jiayuan et al., mSystems. 2021 May; 6(3), which lists A. pittii as the closest relative of A. lactucae, A. calcoaceticus as the next closest relative, and A. baumannii, A. nosocomialis, and A. seifertii as the next closest relatives. The closer a species is to A. lactucae, the more likely it is that similar properties such as 6-3-1F, 17-4-1F, and 17-5-3D will be found. Taking into consideration the results of the Examples of the present application, it is possible to find polypropylene decomposition ability in Acinetobacter microorganisms selected from the group consisting of A. beijerinckii, A. junii, A. venetianus, A. haemolyticus, A. halotolerans, A. nosocomialis, A. baumannii, A. seifertii, A. pittii, A. lactucae, A. calcoaceticus, A. soli, and A. baylyi.

[0019] In one embodiment, the microorganism of the present disclosure is of the genus Acinetobacter, with A. lactucae being the most closely related. The present inventors discovered this by examining the growth ability in a medium containing polypropylene as a carbon source. New microbial strains (6-3-1F, 17-4-1F and 17-5-3D) were identified as Acinetobacter bacteria closely related to A. lactucae, and 17-5-3D was deposited at the National Institute of Technology and Evaluation and received on August 14, 2023. The accession number is AP-03964. In one embodiment, the microorganism of the present disclosure is 17-5-3D (accession number: AP-03964) or a closely related strain thereof. In one embodiment, the microorganism of the present disclosure comprises a whole genome having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to strain 17-5-3D (Accession Number: AP-03964). In one embodiment, the microorganism of the present disclosure comprises a 16S rDNA having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the 16S rDNA of SEQ ID NO:4.

[0020] In one embodiment, the microorganism of the present disclosure is a bacterium of the genus Achromobacter. Achromobacter microorganisms are gram-negative, bacillus species that have flagella, are aerobic, and are commonly found in water and soil. The genus Achromobacter includes species such as arsenitoxydans, cholinophagum, clevelandea, cycloclastes, denitrificans, insolitus, lyticus, marplatensis, mucicolens, obae, piechaudii, ruhlandii, spanius, and xylosoxidans.

[0021] In one embodiment, the microorganism of the present disclosure is a bacterium of the genus Achromobacter. The present inventors identified a new microbial strain 5-3-1A, discovered by examining its growth ability in a medium containing polypropylene as a carbon source, as A. mucicolens, and identified 17-4-2 as a bacterium of the genus Achromobacter closely related to A. mucicolens. Of these, 5-3-1A was deposited at the National Institute of Technology and Evaluation and received on August 14, 2023. The accession number is AP-03963. In one embodiment, the microorganism of the present disclosure is 5-3-1A (accession number: AP-03963) or a closely related strain thereof. In one embodiment, the microorganism of the present disclosure comprises a whole genome having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to strain 5-3-1A (Accession Number: AP-03963). In one embodiment, the microorganism of the present disclosure comprises a 16S rDNA having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the 16S rDNA of SEQ ID NO: 2 or 3.

[0022] In one embodiment, the microorganism of the present disclosure is a bacterium of the genus Pseudomonas. The microorganism of the genus Pseudomonas may have at least one of the following characteristics: rod-shaped, gram-negative, one or several polar flagella that provide motility, aerobic, non-sporulating, catalase test positive, oxidase test positive, non-gas forming in the Hugh-Leifson test with glucose, beta-hemolytic on blood agar, indole test negative, methyl red, Voges-Proskauer test negative, citrate test positive. Genus Pseudomonas includes abietaniphila, acidovorans, aestusnigri, aeruginosa, agarici, alcaligenes, alcaliphila, aminovorans, amygdali, andropogonis, anguil liseptica, antarctica, antimicrobica, argentinensis, arsenicoxydans, asplenii, asturiensis, aurantiaca, aureofaciens, avellanae, avenae, avenae subsp. avenae, avenae subsp. citrulli, avenae subsp. konjaci, azotifigens, azotoformans, baetica, balearica, bauzanensis, beijerinckii, benzenivorans, beteli, borbori, boreopolis, brassicacearum, brassicacearum subsp. brassicacearum, brassicacearum subsp. neoaurantiaca, brenneri, caeni, cannabina, carboxydohydrogena, caricapapayae, caryophylli, cattleyae, cedrina, cedrina subsp. cedrina, cedrina subsp. fulgida, cepacia, chengduensis, chloritidismutans, chlororaphis,chlororaphis subsp. aurantiaca, chlororaphis subsp. aureofaciens, chlororaphis subsp. chlororaphis, chlororaphis subsp. piscium, cichorii, cissicola, citronellolis, cocovenenans, composti, congelans, corrugata, costantinii, cremoricolorata, cuatrocienegasensis, deceptionensis, delafieldii, delhiensis, diminuta, doudoroffii, duriflava, echinoides, elongata, entomophila, extremaustralis, extremorientalis, facilis, ficuserectae, flava, flavescens, flectens, fluorescens, fragi, frederiksbergensis, fulva, fuscovaginae, gelidicola, geniculata, gessardii, gladioli, glathei, glumae, graminis, grimon tii, guangdongensis, guariconensis, guguanensis, guineae, halophila, helmanticensis, hibiscicola, hussainii, huttiensis, indica, indigofera, iners, japonica, jessenii, jinjuensis, kilonensis, knackmussii, koreensis, kunmingensis, kuykendallii, lanceolata, lemoignei, libanensis, lini, litoralis, lundensis, lurida, lutea, luteola, mallei, maltophilia, mandelii, marginalis, marina, marincola, mediterranea, meliae, mendocina, mephitica, meridiana, mesophilica, migulae, mixta, mohnii,monteilii, moorei, moraviensis, mosselii, mucidolens, multiresinivorans, nautica, nicosulfuronedens, nitroreducens, oleovorans, oleovorans subsp. lubricantis, oleovorans subsp. oleovorans, orientalis, oryzihabitans, otitidis, pachastrellae, palleroniana, palleronii, panacis, panipatensis, parafulva, paucimobilis, pelagia, peli, perfectomarina, pertucinogena, phenazinium, pickettii, pictorum, plantarii, plecoglossicida, poae, pohangensis, prosekii, protegens, proteolytica, pseudoalcaligenes, pseudoalcaligenes subsp. citrulli, pseudoalcaligenes subsp. konjaci, pseudoalcaligenes subsp. pseudoalcaligenes, pseudoflava, pseudomallei, psychrophila, psychrotolerans, punonensis, putida, pyrrocinia, radiora, reinekei, resinovorans, rhizosphaerae, rhodesiae, rhodos, rubrilineans, rubrisubalbicans, sabulinigri, saccharophila, salegens, salomonii, saponiphila, savastanoi, segetis, seleniipraecipitans, simiae, solanacearum, spinosa, stanieri, straminea, stutzeri, synxantha, syringae, syringae subsp. savastanoi, syringae subsp. syringae, syzygii, taeanensis, taeniospiralis,Includes species such as taetrolens, taiwanensis, testosteroni, thermotolerans, thivervalensis, tolaasii, toyotomiensis, tremae, trivialis, tuomuerensis, umsongensis, vancouverensis, veronii, vesicularis, viridiflava, vranovensis, woodsii, xanthomarina, xiamenensis, xinjiangensis, zeshuii. ,

[0023] In one embodiment, the microorganism of the present disclosure is a Pseudomonas bacterium closely related to Pseudomonas nicosulfuronedens. The present inventors identified new microbial strains (2-3-1A, 13-3-1C, and 2-4-2A) discovered by examining their growth ability in a medium containing polypropylene as a carbon source as Pseudomonas bacteria closely related to P. nicosulfuronedens, and deposited 2-4-2A of these strains with the National Institute of Technology and Evaluation, which was received on August 14, 2023. The accession number is AP-03962. In one embodiment, the microorganism of the present disclosure is 2-4-2A (accession number: AP-03962) or a closely related strain thereof. In one embodiment, the microorganism of the present disclosure comprises a whole genome having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to strain 2-4-2A (Accession Number: AP-03962). In one embodiment, the microorganism of the present disclosure comprises a 16S rDNA having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the 16S rDNA of SEQ ID NO:1.

[0024] In one embodiment, the microorganism of the present disclosure is a bacterium of the genus Serratia. Serratia microorganisms are gram-negative, non-spore-forming, facultative anaerobic bacilli that have flagella and are found in soil, water, etc. The genus Serratia includes species such as aquatilis, entomophila, ficaria, fonticola, glossinae, grimesii, liquefaciens, marcescens, myotis, nematodiphila, odorifera, plymuthica, proteamaculans, quinivorans, rubidaea, symbiotica, ureilytica, and vespertilionis.

[0025] In one embodiment, the microorganism of the present disclosure is Serratia marcescens belonging to Serratia nematodiphila or Serratia marcescens subsp. sakuensis. In one embodiment, the microorganism of the present disclosure is Serratia marcescens. The present inventors identified new microbial strains (TIKK4A, TIKK4D, APKK9-2B) found by examining their growth ability in a medium containing polyethylene as a carbon source as a closely related species of S. marcescens, and deposited TIKK4D of these strains with the National Institute of Technology and Evaluation, which was received on August 14, 2023. The accession number is AP-03965. In one embodiment, the microorganism of the present disclosure is TIKK4D (accession number: AP-03965) or a closely related strain thereof. In one embodiment, the microorganism of the present disclosure comprises a whole genome having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the strain of TIKK4D (Accession Number: AP-03965). In one embodiment, the microorganism of the present disclosure comprises a 16S rDNA having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the 16S rDNA of SEQ ID NO: 5 or 6.

[0026] In one embodiment, the microorganism of the present disclosure is a bacterium of the genus Amycolatopsis. Microorganisms of the genus Amycolatopsis are gram-positive bacteria with a high GC content. Genus Achromobacter includes acidicola, acididurans, acidiphila, aidingensis, alba, albidoflavacorrig, albispora, alkalitolerans, anabasis, antarctica, arida, australiensis, azurea, balhimycina, bartoniae, benzoatilytica, bullii, camponoti, cappadoca, ciha nbeyliensis, circi, coloradensis, decaplanina, dendrobii, deserti, dongchuanensis, eburnea, echigonensis, endophytica, equina, flava, granulosa, halotolerans, helveola, hippodromi, japonicacorrig, jejuensis, jiangsuensis, jiguanensis, kentuckyen sis, keratiniphila, lactamdurans, lexingtonensis, lurida, magusensis, marina, mediterranei, methanolica, minnesotensis, nigrescens, niigatensis, nivea, oliviviridis, orientalis, palatopharyngis, panacis, pigmentata, pithecellobiicorrig, pittosp ori, plumensis, pretoriensis, regifaucium, rhabdoformis, rhizosphaerae, rifamycinica, roodepoortensis, ruanii, rubida, saalfeldensis, sacchari, samanee, silviterrae, speibonae, stemonae, suaedae, sulphurea, taiwanensis, thailandensis, thermalba,Species include thermoflava, thermophila, tolypomycina, tucumanensis, ultiminotia, umgeniensis, vancoresmycina, vastitatis, viridis, xuchangensis and xylanica.

[0027] The present inventors identified the new microbial strain 5Acc, which was discovered by examining its growth ability in a medium containing polyvinylidene chloride as a carbon source, as a microorganism of the genus Amycolatopsis, and deposited it with the National Institute of Technology and Evaluation, which received it on August 14, 2023. The accession number is AP-03966. 5Acc is closely related to A. halotolerans and A. albidoflavus by clustering based on 16S rDNA. In one embodiment, the microorganism of the present disclosure is 5Acc (accession number: AP-03966) or a closely related strain thereof. In one embodiment, the microorganism of the present disclosure comprises a whole genome having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the strain of 5Acc (Accession Number: AP-03966). In one embodiment, the microorganism of the present disclosure comprises a 16S rDNA having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the 16S rDNA of SEQ ID NO:7.

[0028] In one embodiment, the microorganism of the present disclosure is a bacterium of the genus Bacillus. Bacillus microorganisms are spore-forming, obligately aerobic (and some facultatively anaerobic) Gram-positive rod-shaped bacteria. Genus Bacillus includes acidiceler, acidicola, acidiproducens, acidocaldarius, acidoterrestris, aeolius, aerius, aerophilus, agaradhaerens, agri, aidingensis, akibai, alcalophilus, algicola, alginolyticus, alkalidiazotrophicus, alkalinitrilicus, alkalis ediminis, alkalitelluris, altitudinis, alveayuensis, alvei, amyloliquefaciens, amylolyticus, andreesenii, aneurinilyticus, anthracis, aquimaris, arenosi, arseniciselenatis, arsenicus, aurantiacus, arvi, aryabhattai, asahii, atrophaeus, axarquie nsis, azotofixans, azotoformans, badius, barbaricus, bataviensis, beijingensis, benzoevorans, beringensis, berkeleyi, beveridgei, bogoriensis, boroniphilus, borstelensis, brevis, butanolivorans, canaveralius, carboniphilus, cecembensis, cellulo silyticus, centrosporus, cereus, chagannorensis, chitinolyticus, chondroitinus, choshinensis, chungangensis, cibi, circulans, clarkii, clausii, coagulans, coahuilensis, cohnii, composti, curdlanolyticus, cycloheptanicus, cytotoxicus, daliensis,decisifrondis, discolorationis, deserti, dipsosauri, drentensis, edaphicus, ehimensis, eiseniae, enclensis, endophyticus, endoradicis, farraginis, fastidiosus, fengqiuensis, firmus, flexus, foraminis, fordii, formosus, fortis, fumarioli, funiculus, fusiformis, galactophilus, galactosidilyticus, galliciensis, gelatini, gibsonii, ginsengi, ginsengihumi, ginsengisoli, globisporus, glucanolyticus, gordonae, gottheilii, graminis, halmapalus, haloalkaliphilus, halochares, halodenitrificans, halodurans, halophilus, halosaccharovorans, hemicellu losilyticus, hemicentroti, herbersteinensis, horikoshii, horneckiae, horti, huizhouensis, humi, hwajinpoensis, idriensis, indicus, infantis, infernus, insolitus, invictae, iranensis, isabeliae, isronensis, jeotgali, kaustophilus, kobensis, kochii, kokeshiiformis, koreensis, korlensis, kribbensis, krulwichiae, laevolacticus, larvae, laterosporus, lautus, lehensis, lentimorbus, lentus, licheniformis, ligniniphilus, litoralis, locisalis, luciferensis, luteolus, luteus, macauensis, macerans, macquariensis, macyae, malacitensis, mannanilyticus,marinus, marisflavi, marismortui, marmarensis, massiliensis, megaterium, mesonae, methanolicus, methylotrophicus, migulanus, mojavensis, mucilaginosus, muralis, murimartini, mycoides, naganoensis, nanhaiensis, nanhaiisediminis, nealsonii, neidei, neizhouensis, niabensis, niacini, novalis, oceanisediminis, odysseyi, okhensis, okuhidensis, oleronius, oryzaecorticis, oshimensis, pabuli, pakistanensis, pallidus, pallidus, panacisoli, panaciterrae, pantothenticus, parabrevis, paraflexus, pasteurii, patagoniensis, peoriae, persepolensis, persi cus, pervagus, plakortidis, pocheonensis, polygoni, polymyxa, popilliae, proteolyticus, pseudalcaliphilus, pseudofirmus, pseudomycoides, psychrodurans, psychrophilus, psychrosaccharolyticus, psychrotolerans, pulvifaciens, pumilus, purgationiresistens, pycnus, qingdaonensis, qingshengii, reuszeri, rhizosphaerae, rigui, ruris, safensis, salarius, salexigens, saliphilus, schlegelii, sediminis, selenatarsenatis, selenitireducens, seohaeanensis, shacheensis, shackletonii, siamensis, silvestris, simplex, siralis, smithii, soli, solimangrovi,solisalsi, songklensis, sonorensis, sphaericus, sporothermodurans, stearothermophilus, stratosphericus, subterraneus, subtilis, taeanensis, tequilensis, t hermantarcticus, thermoaerophilus, thermoamylovorans, thermocatenulatus, thermocloacae, thermocopriae, thermodenitrificans, thermoglucosidasius, thermo lactis, thermoleovorans, thermophilus, thermouber, thermosphaericus, thiaminolyticus, thioparans, thuringiensis, tianshenii, trypoxylicola, tusciae, vali Species include dus, vallismortis, vedderi, velezensis, vietnamensis, vireti, vulcani, wakoensis, weihenstephanensis, xiamenensis, xiaoxiensis and zhanjiangensis. ,

[0029] In one embodiment, the microorganism of the present disclosure belongs to the Bacillus cereus group, and the candidate species is Bacillus proteolyticus or Bacillus cereus. The present inventors identified new microbial strains (7A, 7B, 5A, 5C) found by examining their growth ability in a medium containing polyvinylidene chloride as a carbon source as microorganisms of the B. cereus group that may belong to B. proteolyticus or B. cereus. In one embodiment, the microorganism of the present disclosure comprises a 16S rDNA having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity with the 16S rDNA of SEQ ID NO:8.

[0030] In one embodiment, the microorganism of the present disclosure may have polypropylene (PP) decomposition ability. The microorganism of the present disclosure having polypropylene (PP) decomposition ability may be a microorganism of the genus Acinetobacter, Achromobacter, or Pseudomonas. In one embodiment, the microorganism of the present disclosure has polypropylene (PP) decomposition ability, and is cultured by placing a piece of discarded PP cap (cut into about 1 cm after washing with distilled water) and a specific amount (dry weight or cell number, for example, 1% v / v of preculture solution) of isolated bacteria in a test tube containing 5 mL of W medium in a clean bench, and culturing the isolated bacteria with reciprocal shaking (for example, at 28°C, 160 min) for a specific number of days (for example, 1 day, 1 week, 30 days, etc.). -1 ), recovering, washing and drying the PP cap pieces, and measuring the decomposition rate of the PP cap pieces in comparison with a control culture without the addition of bacteria. The microorganism may be a microorganism that achieves a decomposition rate of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% or at least 200% compared to the decomposition rate when the same amount of 2-4-2A, 5-3-1A or 17-5-3D strain is used.

[0031] In one embodiment, the microorganism of the present disclosure may have polyethylene (PE) decomposition ability. The microorganism of the present disclosure having polyethylene (PE) decomposition ability may be a microorganism of the genus Acinetobacter, Achromobacter, Pseudomonas, or Serratia. In one embodiment, the microorganism of the present disclosure has polyethylene (PE) decomposition ability, and is cultured by rotating and shaking (for example, at 30° C., 160 min at 30° C. for ... -1), the PE pieces are recovered, washed and dried, and the decomposition rate of the PE pieces is measured in comparison with that of a control culture without the addition of bacteria. The microorganism can be a microorganism that achieves a decomposition rate of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% or at least 200% compared to the decomposition rate when the same amount of the TIKK4D strain is used.

[0032] In one embodiment, the microorganism of the present disclosure may have polyvinylidene chloride (PVDC) decomposition ability. The microorganism of the present disclosure having polyvinylidene chloride (PVDC) decomposition ability may be a microorganism of the genus Amycolatopsis or Bacillus. In one embodiment, the microorganism of the present disclosure has polyvinylidene chloride (PVDC) decomposition ability, and is cultured by reciprocal shaking (e.g., at 37°C, 160 min, etc.) for a specific number of days (e.g., 28 days, etc.) by placing a piece of Crewrap (Kureha Corporation) and a specific amount (dry weight or cell number, e.g., 1% v / v of preculture solution) of the isolated bacteria in a test tube containing 5 mL of W medium in a clean bench. -1 ), the PVDC pieces are collected, washed and dried, and the decomposition rate of the PVDC pieces is measured in comparison with that of a control culture without the addition of bacteria. The microorganism can be a microorganism that achieves a decomposition rate of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% or at least 200% compared to the decomposition rate when the same amount of the 5Acc strain is used.

[0033] In one embodiment, the microorganism of the present disclosure is a microorganism obtained by the method for obtaining a microorganism described below.

[0034] (Method of obtaining plastic-degrading microorganisms) In one aspect, the present disclosure provides a method for obtaining plastic-degrading microorganisms. This method may include a step of culturing a candidate microorganism in an environment containing plastic as the only carbon source, and a step of isolating a microorganism from a colony found after culturing. The microorganism grown in an environment containing plastic as the only carbon source may have the ability to degrade plastic. The isolated candidate microorganism may be further confirmed to have the ability to degrade plastic. A culture obtained by culturing a candidate microorganism in an environment containing plastic as the only carbon source may be seeded on a plate and cultured to form a colony of the candidate microorganism.

[0035] In one embodiment, a piece of discarded PP cap (washed with distilled water and cut to about 1 cm) and a specific amount (dry weight or cell number, for example, 1% v / v of preculture solution) of the isolated bacteria are placed in a test tube containing 5 mL of W medium in a clean bench, and cultured with reciprocal shaking (for example, at 28°C, 160 min) for a specific number of days (for example, 1 day, 1 week, 30 days, etc.). -1 ), the PP cap pieces are collected, washed and dried, and the decomposition rate of the PP cap pieces is measured in comparison with a control culture without the addition of bacteria. When degradation of the PP cap pieces (the PP decomposition ability of the microorganisms described in this specification) is confirmed, a microorganism capable of decomposing polypropylene (PP) can be obtained.

[0036] 1 In one embodiment, three pieces of a commercial low-density polyethylene (LDPE) plastic bag (Japan Sanipack, thickness 0.03 mm) cut into approximately 3 cm squares or three pieces of a white opaque high-density polyethylene (HDPE) shopping bag (Askul Corporation, thickness 0.022 mm) cut into approximately 3 cm squares and a specific amount (dry weight or cell number, for example, 2% v / v of preculture solution) of the isolated bacteria are placed in a test tube containing 5 mL of W medium in a clean bench, and then cultured with gyratory shaking (for example, at 30°C, 160 min) for a specific number of days (for example, 7 days). -1) and recovering, washing and drying the PE pieces; measuring the decomposition rate of the PE pieces in comparison with a control culture without added bacteria; and confirming the decomposition of the PE pieces (the PE-decomposing ability of the microorganisms described in this specification), a microorganism capable of decomposing PE can be obtained.

[0037] In one embodiment, a piece of Crewrap (Kureha Corporation) cut into approximately 1 cm square and a specific amount (dry weight or cell number, for example, 1% v / v of preculture solution) of the isolated bacteria are placed in a test tube containing 5 mL of W medium in a clean bench, and cultured with reciprocal shaking (for example, at 37°C, 160 min) for a specific number of days (for example, 28 days). -1 ), the PVDC pieces are collected, washed and dried, and the decomposition rate of the PVDC pieces is measured in comparison with a control culture without added bacteria. When decomposition of the PVDC pieces (the PVDC decomposition ability of the microorganisms described in this specification) is confirmed, a microorganism capable of decomposing PVDC can be obtained.

[0038] (Composition containing plastic-degrading microorganisms) In one aspect, the present disclosure provides a composition comprising the microbial cells, culture supernatant, lysate, extract thereof, or a combination thereof of the microbial cells of the present disclosure. The microbial cells of the present disclosure can be produced by culturing them by any suitable method. In one embodiment, the composition is a plastic decomposition agent. In one embodiment, the composition is a decomposition agent for polypropylene (PP), polyethylene (PE), polyvinylidene chloride (PVDC), and / or ethylene-vinyl alcohol copolymer (EVOH).

[0039] In one aspect, the present disclosure provides a method for producing a plastic decomposition agent comprising the microorganisms of the present disclosure, culture supernatant, disruption, extracts thereof, or combinations thereof. The method may include preparing a composition using the microorganisms obtained by the methods described herein. In one embodiment, the method may include carrying out the method for obtaining a plastic decomposition microorganism described herein, and preparing a composition using the obtained microorganism. The composition may be prepared by combining the microorganisms, culture supernatant, disruption, extracts thereof, or combinations thereof with any substance (additional components, carriers, etc. described herein).

[0040] In one embodiment, the plastic degrading agent is a polypropylene (PP) and / or polyethylene (PE) degrading agent containing a microorganism selected from the group consisting of Acinetobacter, Achromobacter, or Pseudomonas. In one embodiment, the plastic degrading agent is a polyethylene (PE) degrading agent containing a microorganism selected from the group consisting of Serratia, ...

[0041] (Applies to) In one embodiment, the microorganism or composition of the present disclosure can be applied to a target containing any type of plastic, such as petrochemical waste. In one embodiment, the microorganism or composition of the present disclosure can be applied to a target containing at least one of polypropylene (PP), polyethylene (PE), polyvinylidene chloride (PVDC) and ethylene-vinyl alcohol copolymer (EVOH). In one embodiment, the target may be subjected to physical disruption (such as cutting), washing, drying, sterilization, etc., before applying the microorganism or composition of the present disclosure. In one embodiment, the target may be subjected to operations such as exposure to UV radiation, high temperature and / or high pressure (such as autoclaving), chemical treatment, etc., before applying the microorganism or composition of the present disclosure, which may promote plastic degradation by microorganisms.

[0042] (Usage form) The form of the microorganism or composition of the present disclosure may be, for example, in a liquid state or a solid state. Examples of the microorganism or composition in a liquid state include a culture solution of the microorganism, and a microorganism collected from the culture solution by centrifugation or the like and then dispersed again in water, a buffer solution, or a culture solution. Examples of the microorganism or composition in a solid state include a dehydrated one by centrifugation or pressing, a paste state or mayonnaise state that is intermediate between a solid and a liquid, and a dried body that has been dried (for example, dried under reduced pressure or freeze-dried). Examples of the solid form include a powder, granules, and tablets. The composition may also be provided in a state in which the microorganism or culture supernatant is fixed to a carrier.

[0043] In one embodiment, the microorganism or composition of the disclosure comprises about 1×10 8 cells / mL, approximately 1×10 7 cells / mL, approximately 1×10 6 cells / mL, approximately 1×10 5 cells / mL, approximately 1×10 4 cells / mL, approximately 1×10 3 cells / mL, approximately 1×10 2 The cells may be used by being dispersed in a carrier (culture medium, immobilizing agent, protective material, or protective agent, etc.) to a density of 10 cells / mL or about 10 cells / mL.

[0044] (Applicable environment) The microorganism or composition of the present disclosure can be used in any suitable environment. In one embodiment, the microorganism or composition of the present disclosure can be used in any temperature environment, such as 0 to 100°C, 5 to 70°C, 10 to 50°C, 15 to 40°C, 20 to 35°C, less than 70°C, less than 60°C, less than 50°C, less than 40°C, less than 30°C, less than 25°C, less than 20°C, less than 15°C, less than 10°C, less than 5°C, less than 0°C, about 70°C, about 60°C, about 50°C, about 40°C, about 30°C, about 25°C, about 15°C, about 10°C, about 5°C, or about 0°C.

[0045] In one embodiment, the microorganism or composition of the present disclosure may be used in any pH environment, such as pH 3-13, pH 4-12, pH 5-11, pH 6-10, pH 7-9, pH 5.5-8.5, about pH 3, about pH 4, about pH 5, about pH 6, about pH 7, about pH 8, about pH 9, about pH 10, about pH 11, about pH 12, or about pH 13.

[0046] In one embodiment, the microorganisms or compositions of the present disclosure may be used in an environment with any salt (e.g., sodium chloride) concentration, such as about 0 g / L, about 0.05 g / L, about 0.1 g / L, about 0.5 g / L, about 0.7 g / L, about 1 g / L, about 1.5 g / L, about 2 g / L, about 2.5 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 7 g / L, or about 10 g / L.

[0047] In one embodiment, the microorganism or composition of the present disclosure may be used in the presence of salt, surfactant, light, ultrasound, electromagnetic waves such as microwaves, electric current, stirring, aeration, organic solvents, or any combination thereof.

[0048] In one embodiment, the microorganism or composition of the present disclosure may be used together with a carrier capable of immobilizing the microorganism. The material of the carrier is not particularly limited as long as it can immobilize the microorganism, and examples thereof include carbon fiber (PAN-based, pitch-based, phenolic resin-based, etc.), polyethylene resin, polypropylene resin, polyurethane resin, polystyrene resin, polyvinyl chloride resin, polyvinyl acetate resin, polyvinyl alcohol resin, polyethylene glycol resin, acrylic resin, gelatin, sodium alginate, carrageenan, dextrin, ceramics, silicon, metal, charcoal, activated carbon, minerals (zeolite, diatomaceous earth, etc.), glass, glass waste, and composites thereof. In order to increase the immobilization rate of the microorganism and the action efficiency of the microorganism, a porous or fibrous carrier can be used. The microorganism may be included in a gel-like carrier. The shape of the carrier may be, for example, a cube, a rectangular parallelepiped, a cylinder, a sphere, a disk, a sheet, a membrane, etc.

[0049] (Additional Ingredients) In one embodiment, the microorganism or composition of the present disclosure may be used in combination with additional components, which may be added to the composition or may be used separately from the microorganism or composition, and if used separately, may be provided as a kit.

[0050] In one embodiment, the additional components include, but are not limited to, components that enhance the activity of the microorganisms used (such as transition metal ions and vitamins), surfactants, drying protection agents, components for maintaining the microorganisms for a long period of time, preservatives, excipients, strengthening agents, antioxidants, dispersants, flocculants, and other microorganisms, and any suitable components can be used. Additional components may include liquids for preserving the microorganisms, culture media, and the like, which may be provided in the composition, may be used separately from the microorganisms or the composition, or may be provided as a kit.

[0051] (Method using microorganisms) In one aspect, the present disclosure provides a method for degrading plastic. In one embodiment, the method includes a step of contacting a target containing plastic with a microbial cell, culture supernatant, lysate, extract thereof, or a combination thereof, or a composition of the present disclosure. The target containing plastic may be any target described herein to which the microorganism or composition of the present disclosure can be applied. In one embodiment, the method for degrading plastic of the present disclosure may not include a step of denaturing plastic before the contacting step. The denaturation may be, for example, denaturation caused by UV irradiation, exposure to high temperature and / or high pressure (such as autoclaving), or chemical treatment. Typically, the modification may be caused by physical treatment of the surface of the article (such as rubbing the surface of the article, applying a force to the surface of the article), plasma irradiation, corona discharge, UV irradiation, exposure to high temperature and / or high pressure (e.g., autoclaving), or chemical treatment (e.g., treatment with a radical source such as transition metal ions, unsaturated fatty acids, surfactants, organic solvents such as chloroform, acids such as sulfuric acid and chromic acid, bases such as sodium hydroxide, hydrogen peroxide, oxidizing agents such as hypochlorous acid, and primer treatment (such as a primer for adhesives)). The method for decomposing plastics of the present disclosure can be carried out in any of the environments described herein in which the microorganism or composition of the present disclosure can be applied. The method for decomposing plastics of the present disclosure can use any of the additional components described herein that can be used in combination with the microorganism or composition of the present disclosure.

[0052] The present disclosure will be described below based on examples, but the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present invention. Therefore, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but is limited only by the claims. EXAMPLES

[0053] (Example 1: Polypropylene (PP) decomposition bacteria) Screening of PP-degrading microorganisms was carried out using W medium containing waste PP caps as the sole carbon source, and the obtained strains were then taxonomically identified by 16S rDNA sequence analysis.

[0054] Reagents and Materials The reagents used in the experiment are shown in the table below. Samples were taken from soil, outdoor rubber, outdoor plastic, etc., and attempts were made to isolate microorganisms. All reagents used were of the highest purity or of the highest grade available on the market unless otherwise specified. [Table 1]

[0055] (Enrichment culture) In order to grow PP decomposition bacteria, we carried out enrichment shaking culture using W medium, which uses waste PP caps as the only carbon source, to grow and isolate specific microorganisms from a mixture of many different types of microorganisms. We also confirmed the growth of the bacteria by measuring turbidity. The procedure for enrichment shaking culture is as follows.

[0056] 5 mL of W medium was placed in a test tube (φ18 × 180 mm), coated with silicone, covered with aluminum foil, and sterilized in an autoclave (ES-315, Tommy Seiko Co., Ltd., Tokyo) at 121°C for 15 minutes. [Table 2] [Table 3]

[0057] In a clean bench, a discarded PP cap (washed with distilled water and cut into approximately 1 cm pieces) was added to 1% (v / v) of the supernatant of the isolated source suspended in distilled water (DW), and the mixture was incubated with reciprocal shaking (37°C, 160 min) for up to 20 days. -1 ) was carried out.

[0058] The turbidity (optical density: OD) at 660 nm was measured using a spectrophotometer, and from the test tube culture liquid where the OD had risen to about 0.05 or more, 1% (v / v) of the bacteria was inoculated into 5 mL of W medium containing one discarded PP cap, and cultured in the same manner as above. This procedure was repeated at least three times (enrichment culture), and the reproducibility of the increase in turbidity of the culture liquid was confirmed.

[0059] (Isolation of PP-degrading bacteria) To isolate PP-degrading bacteria, streaking was performed on samples that showed a reproducible increase in turbidity. The enrichment culture was streaked onto a nutrient agar plate using a platinum loop to isolate the degrading bacteria, and a master plate and glycerol stock (final concentration 20 v / v% glycerol) were prepared for each colony (Figure 1). [Table 4]

[0060] In a clean bench, one discarded PP cap was placed in a 5 mL test tube of W medium, and one platinum loopful of bacteria was inoculated from the monocultured decomposition bacteria master plate, and incubated at 37°C for 160 min. -1 The culture was performed with reciprocal shaking for 1 to 14 days.

[0061] 5 mL of W medium was placed in a test tube, and a discarded PP cap, which had been dried overnight in a vacuum desiccator and weighed on an electronic balance just before being added to the test tube, was placed in the test tube. One platinum loopful of bacteria was inoculated from the decomposition bacteria master plate and incubated at 37°C for 160 min. -1 The culture was performed with reciprocal shaking for a maximum of 58 days. Discarded PP caps were collected from the culture medium using tweezers.

[0062] 20 mL of ultrapure water (UPW) was poured into a 50 mL beaker and stirred with a stirrer. The collected discarded PP caps were placed in this beaker and washed for 5 minutes. This operation was repeated twice. After washing, the discarded PP caps were placed in a PS petri dish, vacuum-dried overnight in a vacuum desiccator containing silica gel, and immediately weighed. The decomposition rate was calculated from the change in weight of the discarded PP caps. This experiment was repeated at least twice.

[0063] (Taxonomic identification of PP-degrading bacteria) The isolated strains were subjected to 16S rDNA sequence analysis to identify the taxonomic group of PP-degrading candidate strains. A simple molecular phylogenetic tree analysis gave the following results (Figs. 2 and 3). The partial 16S rDNA sequence of the 2-3-1A strain showed 99.9% identity to Pseudomonas nicosulfuronedens LAM 1902T (accession number MN007089). The partial 16S rDNA sequence of the 2-4-1A strain showed 99.9% identity to Pseudomonas nicosulfuronedens LAM 1902T (accession number MN007089). The partial 16S rDNA sequence of the 5-3-1A strain showed 99.9% identity to Achromobacter mucicolens R-46662T (accession number HE613446). The partial 16S rDNA sequence of the 6-3-1F strain showed 99.8% identity to Acinetobacter lactucae CCUG68785T (accession number KU921101). The partial 16S rDNA sequence of the 13-3-1C strain showed 99.9% identity to Pseudomonas nicosulfuronedens LAM 1902T (accession number MN007089). The partial 16S rDNA sequence of the 17-4-1F strain showed 99.8% identity to Acinetobacter lactucae CCUG68785T (accession number KU921101). The partial 16S rDNA sequence of the 17-4-2C strain showed 99.8% identity to Achromobacter mucicolens R-46662T (accession number HE613446). The partial 16S rDNA sequence of the 17-5-3D strain showed 99.8% identity to Acinetobacter lactucae CCUG68785T (accession number KU921101).

[0064] Strains 2-4-2A, 5-3-1A and 17-5-3D were deposited and received accession numbers AP-03962, AP-03963 and AP-03964, respectively.

[0065] (PP decomposition test using isolated strains) The PP decomposition ability of each of the identified isolates was tested. The test was carried out in the same manner as the above PP decomposition bacteria screening, using a discarded PP cap (washed with distilled water and cut into approximately 1 cm pieces) in a clean bench. The samples were incubated with reciprocal shaking (28°C, 160 min) for 1 day up to 95 days. -1 As a result, the ability to decompose PP was observed in all the isolated strains (Figure 3).

[0066] In addition, a piece of PP cap (Nichiden Rika Glass, TGK product number 0717-04-10-57, cut to approximately 1 cm) was placed in a 5 mL test tube of 802 medium (Hipolypeptone 10 g / L, yeast extract 2 g / L, MgSO4·7H2O 1 g / L) in a clean bench, and a loopful of bacteria was inoculated from the 17-5-3D master plate that had been made into a single bacteria, and incubated at 37°C for 160 min. -1 The culture was performed with reciprocal shaking for 14 days and compared with the case without the addition of bacteria. As a result, when the PP-degrading bacteria was added, cracks were observed in the PP cap, and it was possible to visually confirm that the PP had been decomposed (Figure 4).

[0067] (PP decomposition by various Acinetobacter bacteria) We also tested whether species of the genus Acinetobacter other than Acinetobacter lactucae had the ability to degrade PP.

[0068] A classification of Acinetobacter species based on genomic analysis has been proposed (Jiayuan et al., mSystems. 2021 May-Jun; 6(3)), and based on this classification, we tested whether species surrounding Acinetobacter lactucae have the ability to degrade PP.

[0069] The following strains were obtained from NBRC: A. calcoaceticus, A. radioresistens, A. baumannii, A. haemolyticus, A. pittii, A. baylyi, A. gyllenbergii, A. parvus, and A. ursingii. [Table 5]

[0070] The 17-5-3D strain, which is closely related to A. lactucae, and the strains obtained from NBRC were subjected to the same screening for PP-degrading bacteria as described above, using 15 mg of PP powder (Seishin Enterprises, PPW-5J). -1 ) was carried out.

[0071] The decomposition experiment was carried out twice, and the decomposition of the PP powder was evaluated according to the following criteria. [Table 6]

[0072] The strains judged as (1), (2), and (3) were judged to have relatively high PP degradation activity, and the strains judged as (1), (2), or (3) in both the results of two experiments were judged to have particularly high PP degradation activity. A. lactucae, A. calcoaceticus, A. pittii, A. baumannii, A. baylyi, and A. haemolyticus met these criteria, while the other strains did not. The other strains (A. ursingii, A. parvus, A. gyllenbergii, and A. radioresistens) also showed at least low levels of PP degradation activity.

[0073] In addition to A. lactucae, excellent PP degradation ability was observed in A. lactucae, A. calcoaceticus, A. pittii, A. baumannii, A. baylyi, and A. haemolyticus.

[0074] Based on this, it is expected that microorganisms with excellent PP degradation ability can be obtained in the range of species including A. beijerinckii, A. junii, A. venetianus, A. haemolyticus, A. halotolerans, A. nosocomialis, A. baumannii, A. seifertii, A. pittii, A. lactucae, A. calcoaceticus, A. soli and A. baylyi, in accordance with the classification of Acinetobacter genus microorganisms proposed by Jiayuan et al., mSystems. 2021 May-Jun; 6(3).

[0075] (Example 2: Polyethylene (PE) decomposition bacteria) The same procedure as for the above PP decomposition bacteria screening was carried out in a clean bench using a piece of commercially available transparent rubber bag (LDPE) (cut into approximately 1 cm square).

[0076] (Isolation of PE-degrading bacteria) To isolate PE-degrading bacteria, streaking was performed on samples that showed reproducible increases in turbidity. The enrichment culture was streaked onto a W medium plate using a platinum loop, and then the plate was overlaid with a commercially available transparent rubber bag and cultured at 28°C. Colonies that grew preferentially around the PE film were picked up and the same procedure was repeated several times. Finally, the plate was streaked onto a normal medium plate to confirm the formation of single bacteria, and a master plate and glycerol stocks (final concentration 20 v / v% glycerol) were prepared for each single colony.

[0077] (Taxonomic identification of PE-degrading bacteria) The isolated strains were subjected to 16S rDNA base sequence analysis to identify the taxonomic group of PE-degrading candidate strains. The following results were obtained by simple molecular phylogenetic tree analysis (Figures 9-10). The partial 16S rDNA sequences of the TIKK4A and TIKK4D strains showed 99.8% identity to Serratia nematodiphila DZ0503SBS1 (accession number EU036987) and Serratia marcescens subsp. sakuensis KRED (accession number AB061685). The TIKK4A and TIKK4D strains were identified as Serratia marcescens that may belong to Serratia nematodiphila or Serratia marcescens subsp. sakuensis. The partial 16S rDNA sequence of the APKK9-2B strain showed 99.7% identity to Serratia marcescens subsp. marcescens NMRC 102204 (accession number AB681729).

[0078] The TIKK4D strain was deposited and received accession number AP-03965.

[0079] (PE decomposition test by isolated strains) In a 500 mL flask, 100 mL of W medium was placed in a clean bench. Three pieces of low-density polyethylene (LDPE) commercial plastic bags (Nihon Sanipack) (cut into approximately 3 cm squares), three pieces of high-density polyethylene (HDPE) milky white shopping bags (Askul Corporation) with a thickness of 0.022 mm, and 2% (v / v) NB culture medium (approximately 10 8 ~10 9 The cells were incubated at a concentration of 1000 cells / mL for 7 days with rotary shaking (30°C, 160 min -1 ) was performed. The LDPE and HDPE pieces were collected from the culture medium using tweezers.

[0080] 5mL of 2% SDS solution, LDPE pieces, and HDPE pieces were added to a test tube and ultrasonically cleaned for 5 minutes. 20mL of ultrapure water (UPW) was poured into a 50mL beaker and stirred with a stirrer. The ultrasonically cleaned LDPE and HDPE pieces were placed in this beaker and washed for 3 minutes. After washing, the LDPE and HDPE pieces were placed in a PS petri dish and vacuum dried for 3 days in a vacuum desiccator containing silica gel, and immediately weighed. The decomposition rate was calculated from the change in weight of the LDPE and HDPE pieces. This experiment was performed three times for each bacterium (Figures 11 and 12).

[0081] As a result, it was found that the strains 2-3-1A, 2-4-2A, 5-3-1A, 17-4-1F, 17-4-2C, 17-5-3D, TIKK4A, and TIKK4D had LDPE decomposition ability, and the strains 2-3-1A, 2-4-2A, 5-3-1A, 6-3-1F, 13-3-1C, 17-4-1F, 17-4-2C, and TIKK4A had HDPE decomposition ability.

[0082] (PE adsorption by PE-degrading bacteria) The same PP decomposition bacteria screening was carried out as above, using a piece of a commercial low-density polyethylene (LDPE) plastic bag (Nihon Sanipack) (cut into approximately 1 cm squares) in a clean bench. The dried LDPE piece was observed under a stereomicroscope and analyzed by FT-IR (Figures 13 and 14). As a result, it was observed that the TIKK4A, TIKK4D and APKK9-2B strains all adsorbed to PE. In particular, it was observed that TIKK4A had high adsorption properties.

[0083] (Example 3: Polyvinyldichlordane (PVDC) decomposing bacteria) The same procedure as for the above PP decomposition bacteria screening was carried out in a clean bench using a piece of Krewrap (Kureha Corporation) (cut into approximately 1 cm square).

[0084] (Isolation of PVDC-degrading bacteria) To isolate PVDC-degrading bacteria, streaking was performed on samples that showed a reproducible increase in turbidity. The enrichment culture was streaked onto a W medium plate using a platinum loop, and then the plate was overlaid with commercially available cling wrap and cultured at 28°C (Figure 15). Colonies that preferred to grow around the periphery of the PVDC film were picked up, and the same procedure was repeated several times. Finally, the plate was streaked onto a normal medium plate to confirm the isolation of single bacteria, and a master plate and glycerol stocks (final concentration 20v / v% glycerol) were prepared for each single bacteria colony.

[0085] (Taxonomic identification of PVDC-degrading bacteria) The isolated strains were subjected to 16S rDNA base sequence analysis to identify the taxonomic group of PVDC-degrading candidate strains. The following results were obtained by simple molecular phylogenetic tree analysis (Figures 16 to 18). The partial 16S rDNA sequence of strain 7A showed 99.9% identity to Bacillus proteolyticus MCCC1A00365 (accession number KJ812418) and Bacillus cereus ATCC14579 (accession number AED16877), and the strain was identified as a microorganism of the B. cereus group that may belong to B. proteolyticus or B. cereus. The partial 16S rDNA sequence of strain 7B showed 99.9% identity to Bacillus proteolyticus MCCC1A00365 (accession number KJ812418) and Bacillus cereus ATCC14579 (accession number AED16877), and the strain was identified as a microorganism of the B. cereus group that may belong to B. proteolyticus or B. cereus. Strain 5A was identified as a member of the Bacillus cereus group based on the partial 16S rDNA sequence. The 5C strain was identified as a member of the B. cereus group based on the partial 16S rDNA sequence. The 16S rDNA partial sequence of the 5Acc strain showed the highest homology to Amycolatopsis halotolerans N4-6 (accession number DQ000196) and Amycolatopsis albidoflavus IMSNU22139 (accession number AJ252832), with the homology rate being 98.9%, suggesting that it may be a new species in the genus Amycolatopsis.

[0086] The 5Acc strain was deposited and received accession number AP-03966.

[0087] (PVDC degradation test by isolated strains) 5 mL of W medium was placed in a test tube, and a piece of Krewrap (Kureha Corporation) was placed in the test tube. The weight of the Krewrap had been measured using an electronic balance just before it was added to the test tube. A loopful of bacteria was inoculated from the decomposition bacteria master plate and incubated at 37°C for 160 min. -1The PVDC pieces were harvested from the culture medium using tweezers.

[0088] 20 mL of ultrapure water (UPW) was poured into a 50 mL beaker and stirred with a stirrer. The collected PVDC pieces were placed in this beaker and washed for 5 minutes. After washing, the PVDC pieces were placed in a PS petri dish and vacuum dried for 2 days in a vacuum desiccator containing silica gel, and immediately weighed. The decomposition rate was calculated from the change in weight of the PVDC pieces. [Table 7]

[0089] As shown in the table, PVDC degradation was confirmed in all of the strains tested, and the top three strains in terms of decomposition ability were strains 7A, 7B, and 5Acc.

[0090] (Growth ability of PVDC-degrading bacteria) Next, the growth ability of the PVDC-degrading strains was tested in a medium containing PVDC as the sole carbon source.

[0091] In a clean bench, a piece of Krewrap (Kureha Corporation) was placed in a 5 mL test tube of W medium, and a loopful of bacteria was inoculated from the master plate of decomposition bacteria that had been isolated into a single bacteria. The bacteria were then incubated at 37°C for 160 min. -1 The strain was cultured with reciprocal shaking at 4°C for 14 days. During the culture, the turbidity (optical density: OD) at 660 nm was measured using a spectrophotometer. As a result, the 5Acc strain grew particularly well in the W medium containing PVDC (Figure 19).

[0092] Example 4: Poly(vinyl alcohol-co-ethylene) (EVOH) Decomposition In addition, the above plastic-degrading bacteria (PP, PE-degrading bacteria) were tested for their EVOH decomposition ability.

[0093] Three pieces of EVOH (Sigma, catalog number 414093-100G) were placed in 5 mL of test tube W medium and sterilized by autoclave (121°C, 15 minutes).

[0094] In a clean bench, one platinum loop of the decomposition bacteria master plate was inoculated into 5 mL of EVOH-added W medium in an autoclave-sterilized test tube, and incubated at 28°C for 160 min. -1 The medium was cultured with reciprocal shaking at 4° C. for 7 days (FIG. 20). During the culture, the turbidity (optical density: OD) at 660 nm was measured using a spectrophotometer.

[0095] These results demonstrated that the 2-3-1A, 2-4-2A, 5-3-1A, 13-3-1C, 17-4-1F and 17-4-2C strains have the ability to decompose EVOH.

[0096] (Note) Although the present disclosure has been illustrated using the preferred embodiments thereof, it is understood that the present invention should be interpreted in scope only by the claims. It is understood that the patents, patent applications, and other documents cited in this specification are incorporated by reference into this specification in the same manner as if the contents themselves were specifically set forth herein. [Industrial Applicability]

[0097] The present disclosure provides microorganisms that degrade plastics, which may enable efficient processing of petroleum waste and reduce the environmental burden. [Accession number]

[0098] 2-4-2A (Receipt number: AP-03962) 5-3-1A (Receipt number: AP-03963) 17-5-3D (Receipt number: AP-03964) · TIKK4D (Receipt number: AP-03965) 5Acc (Receipt number: AP-03966) [Sequence List Free Text]

[0099] SEQ ID NO: 1: Partial 16S rDNA sequences of 2-3-1A, 2-4-2A, and 13-3-1C SEQ ID NO: 2: Partial 16S rDNA sequence of 5-3-1A SEQ ID NO: 3: Partial 16S rDNA sequence of 17-4-2C SEQ ID NO: 4: Partial 16S rDNA sequences of 6-3-1F, 17-4-1F, and 17-5-3D SEQ ID NO: 5: Partial 16S rDNA sequences of TIKK4A and TIKK4D SEQ ID NO: 6: Partial 16S rDNA sequence of APKK9-2B SEQ ID NO: 7: Partial 16S rDNA sequence of 5Acc SEQ ID NO: 8: Partial 16S rDNA sequences of 5A, 5C, 7A and 7B

Claims

1. A microorganism belonging to a genus selected from the group consisting of Acinetobacter, Achromobacter, Pseudomonas, Serratia, Amycolatopsis and Bacillus, which has the ability to degrade plastic.

2. The microorganism of claim 1, wherein the plastic comprises at least one of polypropylene (PP), polyethylene (PE), polyvinylidene chloride (PVDC) and ethylene-vinyl alcohol copolymer (EVOH).

3. The microorganism according to claim 1, wherein the microorganism is a microorganism of a genus selected from the group consisting of Acinetobacter, Achromobacter and Pseudomonas and has the ability to degrade polypropylene (PP).

4. The microorganism according to claim 3, wherein the microorganism is an Acinetobacter genus microorganism selected from the group consisting of A. beijerinckii, A. junii, A. venetianus, A. haemolyticus, A. halotolerans, A. nosocomialis, A. baumannii, A. seifertii, A. pittii, A. lactucae, A. calcoaceticus, A. soli and A. baylyi.

5. The microorganism according to claim 3, wherein the microorganism is 2-4-2A (strain with accession number AP-03962), 5-3-1A (strain with accession number AP-03963), 17-5-3D (strain with accession number AP-03964) or a closely related strain thereof.

6. The microorganism according to claim 3, comprising a 16S rDNA having at least 90% sequence identity to any of SEQ ID NOs: 1 to 4.

7. 4. The microorganism according to claim 3, which is a strain selected from the group consisting of accession numbers AP-03962, AP-03963 and AP-03964.

8. The microorganism according to claim 1, which is a microorganism of the genus Serratia, Acinetobacter, Achromobacter or Pseudomonas and has the ability to degrade polyethylene (PE).

9. The microorganism according to claim 8, wherein the microorganism is TIKK4D (strain with accession number AP-03965), 2-4-2A (strain with accession number AP-03962), 5-3-1A (strain with accession number AP-03963), 17-5-3D (strain with accession number AP-03964) or a closely related strain thereof.

10. The microorganism according to claim 8, comprising a 16S rDNA having at least 90% sequence identity to any of the sequences of SEQ ID NOs: 5 to 6.

11. 9. The microorganism according to claim 8, which is a strain selected from the group consisting of accession numbers AP-03965, AP-03962, AP-03963 and AP-03964.

12. The microorganism according to claim 1 , which is a microorganism of the genus Amycolatopsis or Bacillus and has the ability to degrade polyvinylidene chloride (PVDC).

13. The microorganism according to claim 12, wherein the microorganism is 5Acc (strain with accession number AP-03966) or a closely related strain thereof.

14. The microorganism according to claim 13, comprising a 16S rDNA having at least 90% sequence identity to any of the sequences set forth in SEQ ID NOs: 7 to 8.

15. The microorganism according to claim 13, which is a strain with accession number AP-03966.

16. A plastic decomposition agent comprising the microorganism according to claim 1, a culture supernatant, a disrupted product, an extract thereof or a combination thereof.

17. A method for decomposing plastics, comprising: A process for contacting the microbial cells, culture supernatant, lysate, extract or combination thereof of the microbial cell of claim 1 with plastic. A method comprising:

18. 20. The method of claim 17, which does not include modifying the plastic prior to said contacting step.

19. A method for obtaining plastic-degrading microorganisms, comprising the steps of: Cultivating candidate microorganisms in an environment containing plastic as the only carbon source, and isolating the microorganisms from colonies found after cultivation. A method comprising:

20. A method for producing a plastic decomposer, comprising the steps of:

20. A step of preparing a composition using the microorganism obtained by the method according to claim 19. A method comprising:

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