Novel microorganism for degrading polyethylene and use thereof

Abstract

The present invention relates to a novel microorganism having plastic-degrading activity, a composition for degrading plastic including the microorganism, and a method for degrading plastic using the microorganism. Lysinibacillus macroides JNU01 with accession number KCTC 14428BP and Paenibacillus polyethylenelyticus JNU01 with accession number KCTC 14429BP according to the present invention exhibit excellent effects in catalyzing depolymerization of inert plastics composed solely of carbon, such as polyethylene. The microorganisms according to the present invention enable structural modification required for plastic degradation, and thus can be usefully applied in related industrial fields such as biodegradation treatment of waste plastics and microplastics present in various environments, or pretreatment processes for plastic upcycling.

Description

Novel microorganism for polyethylene degradation and its uses

[0001] The present invention relates to a novel microorganism having polyethylene degradation activity and a method for degrading plastic using the same.

[0002] This invention was carried out with the support of the national research and development project described below.

[0003] [Project ID] 2710018295

[0004] [Sub-project Number] RS-2024-00440681

[0005] [Ministry Name] Ministry of Science and ICT

[0006] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0007] [Project Name] Biomedical Technology Development

[0008] [Project Title] Development of Customized Enzyme-Photocatalyst Hybrid Bioconversion Technology for Recalcitrant Hydrocarbons

[0009] [Name of Project Performing Organization] Konkuk University

[0010] [Research Period] 2024.08.01 ~ 2025.07.31

[0011]

[0012] [Project ID] 2710006002

[0013] [Sub-project Number] RS-2024-00398252

[0014] [Ministry Name] Ministry of Science and ICT

[0015] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0016] [Project Name] Development of Core Technologies in Synthetic Biology

[0017] [Project Title] Development of Innovative Technologies for Gene Circuit Design and Fabrication for Non-Model Useful Strains

[0018] [Name of Project Performing Organization] Pohang University of Science and Technology

[0019] [Research Period] April 1, 2024 ~ December 31, 2028

[0020]

[0021] [Project ID] 2710088992

[0022] [Sub-project Number] RS-2025-24523548

[0023] [Ministry Name] Ministry of Science and ICT

[0024] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0025] [Project Name] Fostering Science and Technology Innovation Talent

[0026] [Project Title] Synthetic Biology Super-regional Convergence Talent Development Project Group

[0027] [Name of Project Performing Organization] Chungnam National University Industry-Academic Cooperation Foundation

[0028] [Research Period] 2025-07-01~2029-12-31

[0029]

[0030] [Project ID] 2710000558

[0031] [Sub-project Number] RS-2023-00208002

[0032] [Ministry Name] Ministry of Science and ICT

[0033] [Name of Project Management (Specialized) Agency] National Research Foundation of Korea

[0034] [Project Name] Individual Basic Research (Ministry of Science and ICT)

[0035] [Project Title] Development of a Synthetic Biology-Based Artificial Enzyme System for Plastic Degradation

[0036] [Name of Project Performing Organization] Industry-Academic Cooperation Foundation, Chonnam National University

[0037] [Research Period] 2023-03-01~2026-02-28

[0038]

[0039] Although plastics were developed for the convenience of daily life, the increasing demand driven by the development of modern society has led to a boomerang effect, causing environmental pollution and destruction through overuse and misuse. Consequently, social interest in the impact of plastics on exposure, the environment, and living organisms is rapidly increasing, and the need for related research is also growing. In particular, microplastics resulting from the weathering of plastics are a primary cause of adverse effects that disrupt the food chain in marine ecosystems. Among plastics, polyethylene (PE), polypropylene (PP), and polystyrene (PS) are the most widely used in daily life, and the demand for recycled materials is also high. Among these plastics, polyethylene (PE) is used in various industrial and everyday products such as packaging, shopping bags, and agricultural film. While it possesses advantages such as low cost and excellent durability, these characteristics simultaneously lead to its accumulation in soil and marine environments, acting as a major cause of exacerbating environmental problems.

[0040] In particular, polyethylene plastic is composed solely of dense carbon, making it structurally very stable and extremely difficult to monomerize biologically or chemically. While several bacteria, fungi, and larvae have been reported to biodegrade polyethylene, these reports merely describe the phenomenon of degradation; research regarding the elucidation of the degradation mechanisms remains severely lacking. In 2016, microorganisms capable of degrading polyethylene terephthalate (PET) and the PETase enzyme were reported in Japan. Since then, research on PETase enzymes has steadily progressed globally, with studies reporting PET degradation within two days (Nature, 2022). Furthermore, Carbios, a French biotechnology company, aims to commercialize PETase enzymes and complete a large-scale plastic recycling plant by 2025. However, as research into the biological and biochemical degradation mechanisms of polyethylene—a major culprit in environmental destruction—remains insufficient, further discovery and application of microorganisms are necessary.

[0041] The objective of the present invention is to provide a novel microorganism for plastic degradation.

[0042] The objective of the present invention is to provide a composition for plastic degradation.

[0043] The objective of the present invention is to provide a method for decomposing plastic.

[0044] 1. A microorganism having plastic-degrading activity, wherein the microorganism is the strain Rhinisibacillus macrolides JNU01 of accession number KCTC14428BP, isolated from landfill soil containing waste plastic; or the strain Paenibacillus polyethylenetiticus JNU01 of accession number KCTC14429BP.

[0045] 2. In the above 1, the plastic is at least one of polyethylene and polypropylene, a microorganism.

[0046] 3. In the above 1, the microorganism wherein the Rhyniscibacillus macrolides JNU01 strain of accession number KCTC14428BP contains the 16S rRNA of sequence number 1.

[0047] 4. In the above 1, the microorganism in which the strain *Paenibacillus polyethylenetiticus* JNU01 of accession number KCTC14429BP contains the 16S rRNA of sequence number 2.

[0048] 5. A microorganism according to 1 above that produces at least one selected from the group consisting of branched alkane, linear alkane, alcohol, ketone, aldehyde, and carboxylic acid.

[0049] 6. A composition for degrading plastic comprising any one of the microorganisms listed in 1 to 5 above or a culture thereof.

[0050] 7. A composition for plastic degradation according to 6, wherein the plastic is a waste plastic or microplastic comprising at least one of polyethylene and polypropylene.

[0051] 8. A plastic degradation method comprising the step of treating plastic or plastic-containing waste with the composition of 6 above.

[0052] 9. A method for decomposing plastic according to 8, wherein the plastic is a waste plastic or microplastic comprising at least one of polyethylene and polypropylene.

[0053] The microorganism according to the present invention has an excellent effect in catalyzing the monomerization of inert plastics composed solely of carbon, such as polyolefins.

[0054] The plastic decomposition composition according to the present invention has an excellent effect of inducing structural deformation for the decomposition of plastics with large molecular weights.

[0055] The plastic decomposition method according to the present invention has an excellent decomposition effect on waste plastics, microplastics, etc.

[0056] The present invention can be usefully applied in related industrial fields, such as pretreatment processes for the decomposition or upcycling of plastics present in various environments.

[0057] Figure 1 is a diagram showing the process of environmental sample collection and polyethylene-degrading microorganism screening.

[0058] Figure 2 shows the results of screening microorganisms growing using polyethylene as a carbon source, represented by the Fold change value.

[0059] Figure 3 is a phylogenetic tree of the polyethylene-degrading candidate microorganism, Rhynisculus macrolides strain.

[0060] Figure 4 is a figure showing the phylogenetic tree of the polyethylene-degrading candidate microorganism, Paenibacillus polyethyleneiticus strain.

[0061] Figure 5 shows the growth curves of the Rhynisibacillus macrolides JNU01 strain in media and a control group at different polyethylene concentrations (10, 30, 50 mg / mL).

[0062] Figure 6 shows the growth curves of the Phenibacillus polyethyleneiticus JNU01 strain in media and a control group at different polyethylene concentrations (10, 30, 50 mg / mL).

[0063] Figure 7 is a chromatogram result obtained by GC-MS analysis of polyethylene media inoculated with Rhinisibacillus macrolides JNU01 strain and Paenibacillus polyethyleneiticus JNU01 strain, respectively.

[0064] Figure 8 is a comparison of the FT-IR spectra of polyethylene powder reacted with the Rhyniscibacillus macrolides JNU01 strain and the control polyethylene powder not treated with the strain.

[0065] Figure 9 is a comparison of the FT-IR spectra of polyethylene powder reacted with the Paenibacillus polyethyleneiticus JNU01 strain and the control polyethylene powder not treated with the strain.

[0066] Figure 10 shows SEM images and contact angle measurements of polyethylene films cultured with Rhinisibacillus macrolides JNU01 and Paenibacillus polyethylenetiticus JNU01 strains, respectively, and a control (Untreated PE film). A and D: SEM images of the control PE film; B and E: SEM images of the PE film reacted with Rhinisibacillus macrolides JNU01 strain; C and F: SEM images of the PE film reacted with Paenibacillus polyethylenetiticus JNU01 strain. As a result of the contact angle measurements, the control is labeled A, the polyethylene film cultured with Rhinisibacillus macrolides JNU01 strain is labeled B, and the polyethylene film cultured with Paenibacillus polyethylenetiticus JNU01 strain is labeled C.

[0067] The present invention provides a novel microorganism having plastic-degrading activity.

[0068] The present invention provides a microorganism having decomposition activity of at least one of polyethylene and polypropylene.

[0069] The present invention provides, as the microorganism, strain Rhinisibacillus macrolides JNU01 of accession number KCTC14428BP, isolated from landfill soil containing waste plastic; or strain Paenibacillus polyethylenetiticus JNU01 of accession number KCTC14429BP.

[0070] In the present invention, "landfill soil containing waste plastic" is soil in which household waste, household waste, industrial waste, urban solid waste, etc., have been landfilled. The landfill soil may be inland landfill soil or coastal landfill soil, and preferably may be inland landfill soil.

[0071] In one embodiment, the landfill soil containing waste plastic may be soil and waste collected from 10 to 20 meters underground in an urban sanitary landfill. From the soil and waste samples and colonies produced in a minimal medium containing polyethylene powder, strains corresponding to Lysinibacillus macroides and strains corresponding to Paenibacillus polyethylenelyticus were identified.

[0072] The microorganism having plastic-degrading activity according to the present invention may be the Rhynichthys bacillus macrolides JNU01 strain or the Paenibacillus polyethylenetiticus JNU01 strain.

[0073] The strain Lysinibacillus macrolides JNU01 was named “Lysinibacillus macroidesJNU01” and deposited at the National Institute of Bioscience and Biotechnology’s Center for Biological Resources on January 5, 2021, and was assigned the accession number KCTC14428BP.

[0074] The Rhyniscibacillus macrolides JNU01 strain contains, but is not limited to, the 16S rRNA of SEQ ID NO. 1. It may contain 16S rRNA having 97% or more, 98% or more, or 99% or more homology with the nucleotide sequence represented by SEQ ID NO. 1.

[0075] The strain Paenibacillus polyethylenelyticus JNU01 was named “Paenibacillus polyethylenelyticusJNU01” and deposited at the National Institute of Biotechnology and Bioengineering’s Center for Biological Resources on January 5, 2021, and was assigned the accession number KCTC14429BP.

[0076] The strain Paenibacillus polyethylenetiticus JNU01 includes, but is not limited to, the 16S rRNA of SEQ ID NO. 2. It may include 16S rRNA having 97% or more, 98% or more, or 99% or more homology with the nucleotide sequence represented by SEQ ID NO. 2.

[0077] The microorganism having plastic-degrading activity according to the present invention can oxidize polyolefin-based polymers.

[0078] In one embodiment, after culturing strains Rhynicibacterium macrolides JNU01 and Paenibacillus polyethyleneiticus JNU01 in a minimal medium containing polyethylene powder, GC-MS analysis revealed the presence of numerous branched alkanes, linear alkanes, and carboxylic acid compounds. Linear and branched alkanes and carboxylic acids are compounds produced when polyolefin-based plastics are degraded. The production of carboxylic acids is attributed to the oxidation of alkanes through oxidation reactions when microorganisms degrade plastics. This can occur during the process in which microorganisms cleave the long carbon chains of plastics and convert them into fatty acids.

[0079] The microorganism having plastic-degrading activity according to the present invention can produce at least one selected from the group consisting of branched alkane, linear alkane, alcohol, ketone, aldehyde, and carboxylic acid.

[0080] The microorganism having plastic-degrading activity according to the present invention can induce structural modifications based on carbon-carbon bonds to degrade polyolefin-based plastics such as polyethylene and polypropylene.

[0081] Polyolefins are polymer compounds produced by the polymerization of olefin monomers, which are compounds containing carbon and hydrogen atoms, and are the raw materials for polyethylene and polypropylene, the most commonly used materials in everyday life. Polyethylene and polypropylene are widely used for various applications due to their excellent properties, such as high strength, flexibility, chemical resistance, and low cost.

[0082] Polyethylene (PE) is a polymer obtained by polymerizing ethylene, an olefin, as a monomer. Along with polypropylene, it is the most produced product globally. Ethylene, the main raw material for polyethylene, is primarily generated from crude oil or natural gas. Because it consists of chemically stable bonds between carbon and carbon, and between carbon and hydrogen, it takes a long time to decompose after use. It is known that commonly used plastic packaging takes 10 to 30 years or more to decompose after landfilling.

[0083] Types of polyethylene include ultra-high molecular weight polyethylene (UHMWPE), high-density polyethylene (HDPE), low-density polyethylene (LDPE), linear low-density polyethylene (LLDPE), and very low-density polyethylene (VLDPE).

[0084] Ultra-high molecular weight polyethylene is a special type of polyethylene with a molecular weight of over one million.

[0085] High-density polyethylene is high-density polyethylene with a linear structure, and is approximately 0.941 g / cm³ based on density. 3 That is all. During the production process, polymerization is performed to achieve a low degree of branching, and the polyethylene chains are densely stacked, resulting in a higher density compared to low-density polyethylene with many chains.

[0086] Low-density polyethylene (LDL) is polyethylene manufactured by free radical polymerization under high-pressure ethylene conditions. During the polymerization process, branching occurs due to radical transfer, forming a structure that is close to radial. It is a material with low density due to a high degree of branching and low stacking between chains; while its tensile strength is low, its ductility is enhanced. Generally, the density is 0.910 g / cm³. 3 Up to 0.940 g / cm³ 3 It is a plastic with a low decomposition rate because it has a low density within the range but a chemically stable structure.

[0087] Linear low-density polyethylene is a linear polymer produced by the copolymerization of ethylene and linear olefins. Olefins such as 1-butene, 1-hectene, and 1-octene are copolymerized with ethylene to impart a linear structure with side chains of constant length. The density range is 0.915 g / cm³. 3 Up to 0.925 g / cm³ 3 am.

[0088] Ultra-low density polyethylene is produced by copolymerizing ethylene and linear olefins using a metallocene catalyst, with a density range of 0.880 g / cm³. 3 Up to 0.915 g / cm³ 3 It is a type of polyethylene. In addition, EVA (Ethylene Vinyl Acetate), produced by copolymerizing ethylene and vinyl acetate, is also sometimes classified as polyethylene. Compared to low-density polyethylene, it has greater transparency and adhesive strength, as well as excellent elasticity and low-temperature heat sealability, making it widely used in daily life.

[0089] Polypropylene (PP) is a representative thermoplastic resin along with polyethylene. It is a polymer produced by polymerizing propylene via Ziegler-Natta polymerization or metallocene-catalyzed polymerization. Polypropylene consists of methyl groups (CH3) attached to every other carbon atom in the polyethylene molecular chain, and it has a structure with regularly arranged short branches. Its density is 0.9 g / cm³. 3 It is the lightest among currently available plastics, and because its melting temperature is high at around 165°C, its range of applications is wider than that of polyethylene (140°C).

[0090] Microorganisms having plastic-degrading activity according to the present invention can degrade waste plastics or microplastics comprising at least one of polyethylene and polypropylene.

[0091] The microorganism having plastic-degrading activity according to the present invention can degrade waste plastic or microplastic comprising at least one of ultra-high molecular weight polyethylene, high-density polyethylene, low-density polyethylene, linear low-density polyethylene, ultra-low density polyethylene, EVA, and polypropylene.

[0092] The present invention provides a composition for degrading plastic comprising a microorganism or a culture thereof having degradation activity of at least one of the polyethylene and polypropylene.

[0093] The above microorganism is the strain Rhinisibacillus macrolides JNU01 of accession number KCTC14428BP, isolated from landfill soil containing waste plastic; or the strain Paenibacillus polyethyleneiticus JNU01 of accession number KCTC14429BP, as described above.

[0094] The composition of the present invention may include any species or subspecies corresponding to Rhynichthys macrolides or Paenibacillus polyethylenetiticus strains.

[0095] In the present invention, the culture may be, but is not limited to, a medium in which a strain of Rhinisibacillus macrolides or a strain of Paenibacillus polyethylenetiticus is cultured, or a culture product obtained therefrom through processing such as concentration, filtration, drying, centrifugation, or crushing. The culture may contain the strain, or may contain only the culture product obtained by filtering the strain. The culture may be a dead cell or a culture of a dead cell. The culture may additionally contain a suitable excipient or carrier.

[0096] The composition of the present invention may further include known substances that enhance the activity of the Rhinisibacillus macrolides JNU01 strain or the Paenibacillus polyethylenetiticus JNU01 strain, or enhance plastic degradation activity.

[0097] The composition of the present invention can oxidize waste plastic or microplastic comprising at least one of polyethylene or polypropylene, and thereby decompose the plastic.

[0098] The composition of the present invention can decompose waste plastic or microplastic comprising at least one of ultra-high molecular weight polyethylene, high-density polyethylene, low-density polyethylene, linear low-density polyethylene, ultra-low density polyethylene, EVA, and polypropylene.

[0099] The composition of the present invention can decompose various forms of plastic, such as liquid, resin, powder, and film.

[0100] The composition of the present invention may be used for the purpose of pretreatment for the decomposition of plastics or plastic recycling. The purpose may be achieved by bringing the composition into contact with plastic, but is not limited thereto.

[0101] The present invention provides a plastic degradation method comprising the step of treating plastic or plastic-containing waste with the plastic degradation composition.

[0102] The method of the present invention can decompose various forms of plastics discarded in daily life, such as liquids, suspensions, films, molding agents, crushed materials, and powders.

[0103] The plastic degradation method of the present invention may include culturing the strain *Rhinicia macrolides* JNU01 or the strain *Paenibacillus polyethylenetiticus* JNU01 in a medium containing plastic or plastic-containing waste. The medium may preferably be either a commonly used solid or liquid medium. Additionally, it may be a basic medium capable of microbial growth and may further include known substances necessary for the cultivation of microorganisms. For example, it may include various vitamins, minerals, and other nutritional components to promote microbial growth.

[0104] The plastic decomposition method of the present invention can oxidize waste plastic or microplastic containing at least one of polyethylene or polypropylene, thereby decomposing the plastic.

[0105] The plastic decomposition method of the present invention can decompose waste plastic or microplastic comprising at least one of ultra-high molecular weight polyethylene, high-density polyethylene, low-density polyethylene, linear low-density polyethylene, ultra-low density polyethylene, EVA, and polypropylene.

[0106]

[0107] The present invention will be described in detail below through examples. However, these are presented as preferred examples of the invention and should not be interpreted as limiting the invention. Details not described herein can be sufficiently technically inferred by those skilled in the art, so such descriptions are omitted.

[0108]

[0109] Preparation of ingredients

[0110] Used reagents, media, and reprecipitation of polyethylene and film preparation

[0111] Luria Bertani Broth (LB) was purchased from MB cell (Seoul, South Korea), and methanol (MeOH) was purchased from Deoksan (Ansan, South Korea).

[0112] For the M9 medium composition, 0.1% of trace element solution (21.8 mg / L CoCl2·6H2O, 21.6 mg / L NiCl2·6H2O, 24.6 mg / L CuSO4·5H2O, 1.62 g / L FeCl3·6H2O, 0.78 g / L CaCl2, and 14.7 mg / L MnCl2·4H2O) was added to M9 minimal medium (6.0 g / L Na2HPO4, 3.0 g / L KH2PO4, 0.5 g / L NaCl, 1.0 g / L NH4Cl, 240.7 mg / L MgSO4, and 11.098 mg / L CaCl2).

[0113] Polyethylene powder and film were prepared by purchasing Polyethylene (average Mw~4,000 by GPC, average Mn~1,700 by GPC, CAS Number: 9002-88-4) from Sigma-Aldrich (St. Louis, MO, USA) and re-precipitating the powder and film.

[0114]

[0115] Example 1. Polyethylene-Degrading Microorganism Screening Process and Results

[0116] Environmental samples (soil and waste) were collected from an urban sanitary landfill located in Gwangju Metropolitan City to find polyethylene-degrading microorganisms (Fig. 1). Soil and waste samples, buried about 20 meters underground for about 11 years, were obtained using drilling equipment.

[0117] Approximately 100g of the collected environmental sample was placed in a 1L beaker, mixed with 100 mL of PBS buffer, and filtered through Whatman paper (pore size 11 μm). The filtered solution was plated onto M9 agar plates containing 1 g / L of polyethylene powder and incubated in a 28°C incubator. After about one week, the grown colonies were streaked onto PE M9 agar plates and incubated in a 28°C incubator. This process was repeated once more, and the grown colonies were inoculated into LB liquid medium and incubated overnight in a shaking incubator at 37°C and 200 rpm. The grown microorganisms were centrifuged and washed using M9 minimal medium. The washed microorganisms were inoculated into M9 minimal liquid medium containing 1 g / L of polyethylene powder and incubated in a 28°C and 200 rpm incubator. The culture was performed for approximately 40 days, and the 40-day OD 600 The value of 0 days OD 600 by The fold change values ​​were calculated by dividing. Looking at the fold change results, the 2GT6 colony and the 2GT37 colony were calculated to be 2.4 times and 2.7 times, respectively (Fig. 2).

[0118]

[0119] Example 2. Identification of polyethylene-degrading microorganisms

[0120] To identify the microorganisms confirmed in Example 1, individual colonies 2GT6 and 2GT37 identified from plate plating were cultured in liquid medium, and DNA extraction was performed using the HiGene™Genomic DNA Prep Kit (BIOFACT, Daejeon, South Korea). 16S rRNA analysis was performed to confirm the bacterial species (Solgent, Daejeon, South Korea). 27F (AGAGTTTGATCCTGGCTCAG; SEQ ID NO. 3) and 1492R (GGTTACCTTGTTACGACTT; SEQ ID NO. 4) were used for sequencing. As a result, the 16S rRNA of SEQ ID NO. 1 was sequenced for the 2GT6 colony, and the 16S rRNA of SEQ ID NO. 2 was sequenced for the 2GT37 colony (Table 1).

[0121] 서열 이름염기 서열 (5'- 3')27F(서열번호3)AGAGTTTGATCCTGGCTCAG1492R(서열번호4)GGTTACCTTGTTACGACTT2GT6(서열번호1)GGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGATACAAACGGTTGCCAACTCGCGAGAGGGAGCTAATCCGATAAAGTCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCGAAGGTGA2GT37(서열번호2)GCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTTCGGGTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTACTACAATGGCCGGTACAACGGGCTGTGAAGCCGCGAGGTGGAACGAATCCTAAAAAGCCGGTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGTCGGAATTGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTTATAACACCCGAAGTCGGTGGGGTAACCGCAAGGAGCCAGCCGCCGA

[0122] The phylogenetic tree between the 16s rRNA gene sequences of strains 2GT6 and 2GT37 was calculated using Molecular Evolutionary Genetics Analysis Version X 10.1 (MEGA X 10.1) to confirm the phylogenetic tree between the strains. As a result, it was confirmed that 2GT6 belongs to the genus Lysinibacillus macroides (Fig. 3), and that 2GT37 belongs to the genus Paenibacillus polyethylenelyticus (Fig. 4). Accordingly, strain 2GT6 was named “Lysinibacillus macroidesJNU01” and strain 2GT37 was named “Paenibacillus polyethylenelyticusJNU01,” and the deposit of biological resources at KCTC was completed.

[0123]

[0124] Example 3. Analysis of microbial growth at various polyethylene concentrations

[0125] Strains Rhynicibacterium macrolides JNU01 and Paenibacillus polyethylenetiticus JNU01 were inoculated into LB liquid nutrient medium, respectively, and cultured overnight in a stirring incubator at 28°C and 200 rpm. After centrifugation at 3800 rpm for 20 minutes, the medium was washed with M9 minimal medium. Subsequently, each strain was inoculated into M9 minimal medium, polyethylene was added to achieve concentrations of 10, 30, and 50 mg / mL, and the mixture was cultured for 30 days in a stirring incubator at 28°C and 200 rpm. Initial OD 600 It is 0.2. Two control groups were used: Control Group 1 consisted only of polyethylene powder in M9 minimal medium, and Control Group 2 consisted only of each strain. OD was measured every 5 days using a UV-Vis Spectrophotometer (SHIMADZU, Kyoto, Japan). 600The absorbance was measured. As a result, it was confirmed that the Rhynisibacillus macrolides JNU01 strain showed no change in absorbance in 10 mg / mL polyethylene powder, but grew in media containing 30 and 50 mg / mL polyethylene powder (Fig. 5). It was confirmed that the Paenibacillus polyethyleneiticus JNU01 strain grew in media containing 10, 30, and 50 mg / mL polyethylene powder (Fig. 6). Both Control 1 and Control 2 showed no change in absorbance.

[0126] Additionally, for the analysis of dissolved oxygen, strains Rhynicibacterium macrolides JNU01 and Paenibacillus polyethylenetiticus JNU01 were inoculated into 60 mL BOD bottles, each containing 60 mL of M9 liquid minimal medium and 30 mg / mL of polyethylene powder. The samples were cultured in an incubator at 28 °C and 200 rpm, and dissolved oxygen was analyzed at 5-day intervals for a total of 10 days using a DO meter (YSI 4100 BOD IDS PROBE & 158 YSI 4010-1W DO meter, YSI Incorporated, Yellow Springs, Ohio, USA). Initial OD 600 ... is 0.2. As a result of the dissolved oxygen analysis, the initial dissolved oxygen levels of Rhinisibacillus macrolides JNU01 and Paenibacillus polyethylenetiticus JNU01 were measured as 9.65 ± 0.035 mg / L and 9.82 ± 0.025 mg / L, respectively; however, after 5 days, they were measured as 0.185 ± 0.010 mg / L and 0.255 ± 0.075 mg / L, confirming that almost all oxygen had been used. This indicates that oxygen was consumed as Rhinisibacillus macrolides JNU01 or Paenibacillus polyethylenetiticus JNU01 grew in a medium using polyethylene as the sole carbon source (Table 2).

[0127] MicroorganismsInitial DO(mg / L)DO after 5 days(mg / L)DO after 10 days(mg / L)Lysinibacillus macroidesJNU019.65 ± 0.0350.185 ± 0.0100.17 ± 0.021Paenibacillus polyethylenelyticusJNU019.82 ± 0.0250.255 ± 0.0750.155 ± 0.007

[0128]

[0129] Example 4. Analysis of microbial decomposition products

[0130] GC-MS analysis was performed to determine whether polyethylene metabolites were produced in M9 minimal medium in which Rhynicibacterium macrolides JNU01 and Paenibacillus polyethylenetiticus JNU01 strains were cultured, respectively. The PE culture medium was extracted in equal volumes with ethyl acetate (EA) at a 1:1 ratio. The 50-fold concentrated and filtered EA layer was analyzed using a GC-MS equipped with an electron impact ionization source (Shimadzu-QP2020 NX, Shimadzu, Kyoto, Japan) and a DB-5MS capillary column (Agilent, Santa Clara, CA, USA). The column temperature was increased from 40°C to 280°C at a rate of 6°C / min, then maintained at 40°C for 3 minutes and at 280°C for 4 minutes. The injector and detector temperatures were set to 300°C and 260°C, respectively. The chemical structures of the products were confirmed using GC-MS and the NIST / WILEY database. GC-MS analysis revealed the presence of numerous branched alkanes, linear alkanes, and carboxylic acid compounds (Fig. 7 and Table 3).

[0131] No.minCompound nameMWFormulaLysinibacillus macroidesJNU01Paenibacillus polyethylenelyticusJNU01Y / NSimilarity (%)Y / NSimilarity (%)15.3233,4,5-Trimethylheptane142C 10 H 22 Y93Y9425.5032,3,3-Trimethylheptane128C9H 20 Y93Y9335.9872,4-Dimethyl-1-heptene126C9H 18 Y96Y96412.013-Ethyl-3-methylheptane142C 10 H 22 Y93Y93513.267n-Dodecane170C 12 H 26 Y92Y92617.8735-Methyl-5-propylnonane185C 13 H 28 Y91Y91719.477n-Tetradecane198C 14 H 30 Y91Y91823.883n-Hexadecane226C 16 H 34 Y91Y91926.08711-Oxododecanoic acid214C 12 H 22 O3N-Y831028.15n-Eicosane282C 20 H 42 Y85Y841128.4Eicosanoic acid312C 20 H 40 O2N-Y861235.921-Tricosanol340C 23 H 48 ON-Y82

[0132]

[0133] Example 5. Chemical change of polyethylene powder treated with microorganisms

[0134] FT-IR analysis was performed to observe chemical changes in polyethylene cultured with the strains Rhinisibacillus macrolides JNU01 or Paenibacillus polyethylenetiticus JNU01, and changes in various functional groups were observed. Compared to the control polyethylene powder not treated with the strains, changes in the -OH (Hydroxyl group) functional group were observed in the polyethylene powder reacted with Rhinisibacillus macrolides JNU01 and Paenibacillus polyethylenetiticus JNU01, indicating that the polyethylene structure was altered by the strains Rhinisibacillus macrolides JNU01 or Paenibacillus polyethylenetiticus JNU01 (Figs. 8 and 9).

[0135]

[0136] Example 6. SEM and contact angle analysis of polyethylene film treated with microorganisms

[0137] Strains Rhynicibacterium macrolides JNU01 and Paenibacillus polyethylenetiticus JNU01 were inoculated into M9 minimal medium and medium containing polyethylene film, respectively, and cultured for 30 days in a shaking incubator at 28°C and 120 rpm. Initial OD 600 The value was 2.0. The polyethylene film after culture was washed with a 2% SDS solution. Platinum coating was performed for 60 seconds at a current of 10 mA to form a thin conductive layer on the polyethylene film. Subsequently, changes in the surface of the polyethylene film before and after strain treatment were observed using an FE-SEM (JSM-7900F, JEOL, Tokyo, Japan) under an acceleration voltage of 2.0 kV (Chonnam National University ECEF, Gwangju, South Korea). Additionally, a contact angle analyzer (Phoenix 300, Suwon, Seoul, South Korea) was used to measure surface roughness and surface hydrophilicity (or hydrophobicity) resulting from changes in chemical structure.

[0138] SEM image results of polyethylene films are shown in Figure 10. The control polyethylene film has a smooth surface (A and D), but the films reacted with the Rhyniscibacillus macrolides JNU01 strain (B and E) and the films reacted with the Paenibacillus polyethylenetiticus JNU01 strain (C and F) showed a melted-looking surface and many pores were observed.

[0139] In addition, the change in contact angle was confirmed through the analysis of the water contact angle of the polyethylene film treated with microorganisms. The contact angles were measured as 87.81°, 59.32°, and 48.33° for the control group, the film treated with the strain Rhinisibacillus macrolides JNU01, and the film treated with the strain Paenibacillus polyethyleneiticus JNU01, respectively. In all cases, the contact angles of the films reacted with the strains were measured to be lower than those of the control group. This is an indirect result showing that the surface of the polyethylene film was altered by the strains, and it implies that the polyethylene film was hydrophilized by the strains.

[0140]

[0141] [Consignment Number]

[0142] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC)

[0143] Trustee Number: KCTC14428BP

[0144] Date of Consignment: 20210105

[0145]

[0146] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC)

[0147] Trustee Number: KCTC14429BP

[0148] Date of Consignment: 20210105

[0149]

[0150]

[0151]

Claims

1. A microorganism having plastic-degrading activity, wherein the microorganism is the strain Rhinisibacillus macrolides JNU01 of accession number KCTC14428BP, isolated from landfill soil containing waste plastic; or the strain Paenibacillus polyethylenetiticus JNU01 of accession number KCTC14429BP.

2. The microorganism of Claim 1, wherein the plastic is at least one of polyethylene and polypropylene.

3. A microorganism according to claim 1, wherein the Rhinisibacillus macrolides JNU01 strain of accession number KCTC14428BP comprises the 16S rRNA of sequence number 1.

4. A microorganism according to claim 1, wherein the strain of Paenibacillus polyethylenetiticus JNU01 of accession number KCTC14429BP comprises the 16S rRNA of sequence number 2.

5. A microorganism according to claim 1 that produces at least one selected from the group consisting of branched alkane, linear alkane, alcohol, ketone, aldehyde, and carboxylic acid.

6. A composition for degrading plastic, comprising a microorganism or a culture thereof according to any one of claims 1 to 5.

7. A composition for plastic degradation according to claim 6, wherein the plastic is a waste plastic or microplastic comprising at least one of polyethylene and polypropylene.

8. A method for decomposing plastic, comprising the step of treating plastic or plastic-containing waste with the composition of claim 6.

9. A method for decomposing plastic according to claim 8, wherein the plastic is a waste plastic or microplastic comprising at least one of polyethylene and polypropylene.

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