Novel microorganism having plastic decomposition activity and use thereof

Abstract

The present invention relates to a novel microorganism capable of decomposing plastic and a method for decomposing plastic using the microorganism. The microorganism can decompose at least one plastic selected from the group consisting of PET, PVC, PS, PP, and PE under appropriate culture conditions and convert the plastic into a low molecular weight substance.

Description

Technical Field

[0001] The present invention relates to a novel microorganism capable of degrading various plastics and a method for degrading plastics by using the microorganism. Background Art

[0002] Plastics are rapidly growing in use due to their lightweight properties, high physical and chemical durability, high processability, and very low price. Global annual plastic production reached 2 million tons in 1950 and approached 370 million tons by 2019. Furthermore, the recent continued spread of COVID-19 has led to a surge in the use of personal protective equipment, such as disposable masks and gloves made primarily of plastic; the rise of takeout and home delivery has led to an increase in the use of disposable tableware; and the preference for online shopping has led to an increase in the use of plastic packaging containers. Consequently, the use of plastics is expected to continue to grow in the future (Trends in Microbial Biodegradable Plastics, BRICView2021-T34).

[0003] The dramatic increase in plastic use and the depletion of petroleum, the raw material for plastics, have created a new demand for recycling plastics that are discarded as waste. Plastics are often collected and recycled together because different types of plastics are used in the same product. This makes it difficult to maintain the complete purity of each plastic as a single material due to impurities from other plastics, resulting in a decrease in quality and an economic value estimated to be approximately 34% lower than that of new plastics. In particular, mixed waste plastics in household waste are difficult to economically separate by material, creating an urgent need for the development of mixed waste plastic sorting technology.

[0004] Meanwhile, technologies for treating plastics contained in wastewater or waste using microorganisms have recently been developed. For example, Korean Patent No. 10-0350928 discloses a novel microorganism, Klebsiella pneumoniae CJ-PVAa (Deposit No. KFCC-11126), that grows well under aerobic conditions and has improved polyvinyl alcohol decomposition capabilities, and a method for treating polyvinyl alcohol-containing wastewater using the microorganism. Furthermore, Korean Patent No. 10-0513931 discloses Microbacterium barkeri LC (Deposit No. KCCM 10507), and a method for biodegrading polyvinyl alcohol using the Microbacterium barkeri LC. Summary of the Invention

[0005] Technical issues

[0006] Under the above circumstances, the inventors of the present invention conducted research to explore microorganisms that can degrade plastics. By feeding polystyrene as food to beetle larvae (super worms) and isolating the microorganisms, they discovered a new type of microorganism that can degrade plastics - Pseudomonas aeruginosa repla1 strain.

[0007] Therefore, the present invention aims to provide a novel microorganism for degrading plastics and a method for degrading plastics using the microorganism.

[0008] Technical Solution

[0009] The invention relates to a Pseudomonas aeruginosa repla1 strain with plastic degradation activity. The strain is preserved with the preservation number of KACC 81230BP.

[0010] In the present invention, the plastic may be one or more types selected from the group consisting of polyethylene terephthalate (PET), polyvinyl chloride (PVC), polystyrene (PS), polypropylene (PP) and polyethylene (PE).

[0011] PET is highly transparent and tasteless, making it the most common plastic beverage bottle on the market. PVC is a strong, rigid or flexible, and wear-resistant thermoplastic. It is used in artificial leather, packaging, pipes, and electrical insulation, and is the oldest plastic known as "vinyl."

[0012] PS, a lightweight, tasteless and odorless thermoplastic, is used in household products, toys, electrical insulators, radio and television casings, and packaging. PP, made from the polymerization of propylene obtained from petroleum, is widely used in bottles, among other products.

[0013] PE is a lightweight and flexible thermoplastic used as a general-purpose plastic in everything from industrial materials to everyday products. High-density polyethylene (HDPE) is impact-resistant and cold-resistant, making it primarily used in shopping bags and pipes. Low-density polyethylene (LDPE) has branched chains, resulting in a lower density than linear HDPE. Its excellent elasticity makes it easier to process.

[0014] In the present invention, "plastic degradation" refers to the decomposition of polymer materials constituting plastics into low molecular weight intermediates or intermediates that can be metabolized by microbial metabolic pathways.

[0015] According to one embodiment of the present invention, the Pseudomonas aeruginosa repla1 strain was isolated from the intestines of beetle larvae fed PS (see Example 1). However, the Pseudomonas aeruginosa repla1 strain can degrade both LDPE and HDPE (see Examples 2-1 and 2-4).

[0016] The present invention also provides a method for degrading plastics, which comprises co-culturing the Pseudomonas aeruginosa repla1 strain deposited with the accession number KACC 81230BP and the plastics.

[0017] After inoculating Pseudomonas aeruginosa repla1 strain into a culture medium containing plastic as a carbon source, the culture is performed at 8 to 40° C. for 7 to 60 days, but the present invention is not limited thereto. Preferably, the culture is performed at 8 to 37° C. for 7 to 30 days.

[0018] In the present invention, the above-mentioned culture medium composition, culture temperature and culture time can vary according to the type of plastic to be degraded. When the Pseudomonas aeruginosa repla1 strain and waste plastic or plastic-containing waste are cultured under conditions in which these conditions are combined into process parameters, specific plastics can be degraded relatively more, and all plastics can also be degraded into reusable small molecule materials.

[0019] To enhance the contact efficiency with the Pseudomonas aeruginosa repla1 strain or the plastic-degrading enzyme secreted by the strain, the plastic added to the culture medium can be in the form of fine fragments (such as discarded vinyl plastic) or films.

[0020] According to one embodiment of the present invention, the Pseudomonas aeruginosa repla1 strain is capable of expressing laccase when plastic is used as a carbon source.

[0021] Laccase is a copper-containing, polycopper blue oxidase found in higher plants, insects, bacteria, and fungi. Laccase utilizes the electrons released during the reduction of oxygen molecules to water to catalyze the oxidation of several substances, particularly phenols and aromatic amines. This property has led to its application in a variety of industrial and environmental applications, including lignin decomposition, decolorization, compound synthesis, and the purification of contaminated water and petroleum.

[0022] Regarding the degradation of plastics as polymer materials, oxidation-induced catalysis can increase the hydrophilicity of the hydrophobic plastic surface, thereby promoting the formation and disaggregation of microbial biofilms (hydrophilic) (References 1 and 2).

[0023] The method for degrading plastics according to the present invention can be applied as a pretreatment process for plastic recycling.

[0024] The present invention also provides a composition for degrading plastics, which comprises the Pseudomonas aeruginosa repla1 strain deposited with the accession number KACC81230BP, a culture of the strain, or a plastic-degrading enzyme derived from the strain.

[0025] As described above, the Pseudomonas aeruginosa repla1 strain expresses laccase and uses plastic as a carbon source, so the strain itself, its culture, and the plastic-degrading enzyme derived from the strain can be used for plastic degradation.

[0026] The enzyme with plastic-degrading activity can be an extracellular or intracellular substance of a microorganism with plastic-degrading activity, and can be produced on a large scale through various gene recombination methods.

[0027] Beneficial effects

[0028] The microorganisms with plastic-degrading activity according to the present invention can, under appropriate culture conditions, degrade one or more plastics selected from the group consisting of polyethylene terephthalate (PET), polyvinyl chloride (PVC), polystyrene (PS), polypropylene (PP), and polyethylene (PE), converting them into small molecule materials. Therefore, the microorganisms with plastic-degrading activity according to the present invention can be used in pretreatment processes for plastic recycling. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The results of confirming whether plastic-degrading microorganisms (hereinafter referred to as repla1) metabolize polyethylene (PE) after culturing repla1 according to an embodiment of the present invention in a basic medium supplemented with PE powder by DCPIP absorbance are shown: the error bars represent the standard deviation of the mean, and the asterisk indicates p < 0.05 (Mann-Whitney U test).

[0030] Figure 2A shows the results of confirming whether repla1 metabolizes metabolites when it was cultured in a medium containing various metabolites: At represents the DCPIP absorbance value of each material changing over time, ACt represents the DCPIP absorbance value of the control (containing no metabolites) changing over time, and the error bars represent the standard deviation of the mean.

[0031] Figure 2B shows the results of confirming whether Escherichia sp. without plastic degradation activity metabolizes carbon sources when it is cultured in a culture medium containing various carbon sources: At represents the DCPIP absorbance value of each material over time, ACt represents the DCPIP absorbance value of the control (no metabolite) over time, and the error bars represent the standard deviation of the mean.

[0032] Figure 3 Shown are the results of confirmation of the number of viable cells after Repla1 culture in minimal medium containing plastic film: error bars represent standard deviation of the mean, and asterisks indicate p < 0.05 (Mann-Whitney U test).

[0033] Figure 4 A and 4B show the results of confirming the formation of microbial biofilm on the surface of plastic film after repla1 was cultured in minimal medium containing plastic film.

[0034] Figure 4C shows the results of confirming the degree of corrosion on the plastic film surface after repla1 was cultured in minimal medium containing the plastic film. Figure 4 D shows the results of confirming the degree of corrosion on the plastic film surface after incubation in a minimal medium containing plastic film without repla1. DETAILED DESCRIPTION

[0035] One or more exemplary embodiments will be described in more detail below through examples. However, these examples are only used to illustrate one or more exemplary embodiments, and the scope of the present invention is not limited to these examples.

[0036] Example 1: Isolation of plastic-degrading microorganisms

[0037] Isolation of plastic-degrading microorganisms

[0038] By feeding beetle larvae (superworms) polyethylene (PE) for two weeks, selective pressure was applied to hydrocarbon-based plastic monomers, ensuring the dominance of the gut microbiota. To isolate pure gut microbes, the larvae were sterilized with 70% ethanol, washed with 0.9% saline, and the head and tail were removed to extract the viscera. The extracted viscera were minced with a sterile blade and mixed with saline. The epithelial cells were then separated from the supernatant by centrifugation.

[0039] A portion of the separated supernatant was added to a minimal culture medium, and a polystyrene (PS) membrane was provided as the sole carbon and energy source. Microorganisms with plastic degradation potential were cultured under aerobic conditions (25°C, 180 rpm) for approximately 60 days. The minimal culture medium composition was as follows: pH 6.51, 0.7g NH2PO4, 0.7g K2HPO4, 0.7g MgSO4·7H2O, per 1L of distilled water.

[0040] 1.0g NH4NO3, 0.005g NaCl, 0.002g FeSO4·7H2O, 0.002g ZnSO4·7H2O, 0.001g MnSO4·H2O. After cultivation, a strain that grows on PS membrane as a carbon source was obtained through purification and isolation.

[0041] 2. Microbial Identification

[0042] To identify the single pure strain obtained from the third passage, 16S rRNA sequencing was performed, confirming that the strain belonged to the genus Pseudomonas. The isolated microorganism was named Pseudomonas aeruginosa repla1 and deposited in the Korean Agricultural Culture Collection (KACC) of the National Institute of Agricultural Science under the accession number KACC 81230BP.

[0043] This isolate is described below as "repla1".

[0044] Example 2: Verification of plastic degradation ability

[0045] 2-1. Verification of direct plastic metabolic activity

[0046] After culturing repla1 in a nutrient medium (Luria-Bertani broth, LB) for 24 hours, cells (10 9 The recovered repla1 cells were washed twice with 0.9% physiological saline to remove the previous culture medium, and then suspended in minimal culture medium.

[0047] 2,6-Dichlorophenol indophenol (DCPIP) oxidant and 8 g / L low-density polyethylene (LDPE; molecular weight: up to 4,000, Sigma-Aldrich) powder were added to the minimal medium. 7 After inoculation with 100 cells / mL (100 μg / mL), cells were cultured and DCPIP absorbance was measured every 24 hours. A portion of the culture was collected every 24 hours, the cells were separated from the suspension by centrifugation, and the absorbance of the supernatant was measured at 600 nm. The DCPIP color change in the experimental group was compared with the following two controls: 1) without PE and with repla1, and 2) with PE and without repla1.

[0048] DCPIP is reduced by receiving electrons from energy generated by microbial metabolic activity, and its color changes from blue to colorless. This allows for indirect assessment of the direct degradation and internal metabolic activity of isolated Repla1 on plastics.

[0049] From the absorbance measurement results, it can be seen that repla1 can use LDPE powder as the only carbon and energy source to generate reducing energy through internal metabolism. This reaction appears about 5 days after the start of culture ( Figure 1The results marked with * indicate statistically significant differences compared with the control inoculated with only repla1 in the basic medium (Mann-Whitney U test, p<0.05).

[0050] 2-2. Verification of internal metabolism of plastic monomers

[0051] Plastics made from metabolizable polymer structures inevitably degrade into low-molecular-weight monomer units (up to 500), which are then metabolized by various microorganisms. Among these monomers, hydrocarbon structures ([CnHn]n) are primarily metabolized via the fatty acid degradation pathway (β-oxidation pathway), with metabolism occurring sequentially in the order of alkanes, alcohols, aldehydes, and finally fatty acids (References 3-6).

[0052] Here, the ability of Repla1 to utilize plastic monomers was assessed.

[0053] In the same manner as in Example 2-1, repla1 was pre-cultured and then suspended in a minimal culture medium. Alkanes, alcohols, aldehydes, fatty acids, or glucose (positive control) were added to the minimal culture medium as the sole energy and carbon source. The repla1 suspension was diluted with water at a ratio of 1 / 100 (10 7 cells / mL) were inoculated into the culture medium and cultured, and the DCPIP absorbance was measured every 24 hours. Part of the culture medium was collected every 24 hours, the cells and the supernatant were separated by centrifugation, and then the absorbance of the supernatant was measured (600nm). The color change of DCPIP in the experimental group was compared with the following two controls: 1) no metabolites + with repla1, 2) with internal metabolites + without repla1. As a result of the absorbance measurement, repla1 was able to utilize all alkanes, alcohols, aldehydes and fatty acids used in the experiment as carbon sources, in descending order of metabolic rate: fatty acids, aldehydes, alkanes, alcohols (Figure 2A). It was confirmed that the Escherichia sp. strain with no plastic degradation ability as a negative control could not metabolize substances other than glucose (Figure 2B).

[0054] 2-3. Verification of expression of plastic-degrading enzymes (exoenzymes)

[0055] There are two main methods for degrading polymeric plastics into monomeric units: physical and chemical methods and biological methods. Biological methods involve enzyme-mediated degradation, and the enzymes involved in plastic degradation include enzymes that oxidize the plastic surface and hydrolases that break down polymer chains. Representative enzymes involved in surface oxidation include laccases (oxidases), while hydrolases include lipases and esterases.

[0056] The enzyme activity of repla1 was confirmed in conventional environments (e.g., nutrient medium) and specialized environments (e.g., plastic medium). Repla1 was cultured in minimal medium containing nutrient medium or plastic film (LDPE and HDPE), and the culture was centrifuged and the supernatant was separated to prepare an enzyme sample.

[0057] Laccase activity is determined based on the amount of enzyme required to oxidize 1 μM 2,6-dimethoxyphenol (DMP) substrate for 1 minute. Add 600 μL of sodium citrate (100 mM sodium citrate, pH 4.0), 400 μL of DMP (10 mM), and 500 μL of enzyme sample and mix. The enzyme activity per minute is calculated by measuring the change in absorbance (468 nm) over 2 minutes.

[0058] Lipase and esterase activities were assessed by measuring the amount of product produced by substrate degradation within 10 minutes. Enzyme samples were reacted with either 4-nitrophenyl palmitate or 4-nitrophenyl butyrate at 28°C for 10 minutes, followed by measuring the absorbance (410 nm) of the reaction solution. The confirmed Repla1 enzyme activity is shown in Table 1.

[0059] [Table 1]

[0060] Culture conditions Laccase lipase Esterase Nutrient medium (LB) X O O Basic culture medium + plastic film O X X

[0061] The experimental results showed that Repla1 uses laccase when it uses plastic as a carbon source.

[0062] 2-4. Observation of the growth of the strain using plastic film as a carbon source The plastic degradation ability of repla1 was further confirmed by observing the number of viable cells in the strain grown using plastic film as the sole carbon and energy source, as well as the microbial biofilm formed on the surface of the plastic film.

[0063] Plastic films (low-density PE and high-density PE) were added to the minimal medium, inoculated with repla1, and cultured with shaking under aerobic conditions (28°C, 130 rpm) for about one month. The number of viable cells of the strain was expressed as colony-forming units (CFU).

[0064] The culture results confirmed that the number of viable cells of repla1 increased significantly after 21 days of culture, and increased to 1.4 to 2.2 times compared with the control after 28 days of culture (p < 0.05) ( Figure 3 ).

[0065] In addition, the microbial biofilm formed on the surface of the plastic film and the corrosion caused by biodegradation were observed using a scanning electron microscope. As a result, after 28 days of cultivation, a microbial biofilm ( Figure 4 A and 4B), and can confirm that the plastic surface is corroded due to biodegradation ( Figure 4 C). In contrast, no microbial biofilm or plastic surface corrosion was observed in the control (without repla1) ( Figure 4 D).

[0066] References

[0067] 1.Stephen M.Jones,Edward I.Solomon.Electron Transfer and ReactionMechanism of Laccases.Cell Mol Life Sci.2015Mar;72(5):869-883.

[0068] 2. Leticia Arregui et al. Laccases: structure, function, and potential application in water bioremediation. Microb Cell Fact. 2019Nov 14;18(1):200.

[0069] 3.Zahra Montazer et al.Challenges with Verifying MicrobialDegradation of Polyethylene.Polymers(Basel).2020Jan;12(1):123.

[0070] 4.Zahra Montazer et al. Microbial degradation of low-densitypolyethylene and synthesis of polyhydroxyalkanoate polymers.Can JMicrobiol.2019Mar;65(3):224-234.

[0071] 5.Hector M.Alvarez.Relationship betweenβ-oxidation pathway and thehydrocarbon-degrading profile in actinomycetes bacteria.InternationalBiodeterioration&Biodegradation Volume 52,Issue1,July 2003,Pages 35-42.

[0072] 6.Masaji Watanabe et al. Computational method for analysis ofpolyethylene biodegradation. Journal of Computational and Applied MathematicsVolume 161,Issue 1,1December 2003,Pages133-144.

[0073] [Collection Information]

[0074] Name of depository institution: Korean Agricultural Culture Collection (KACC) of the National Academy of Agricultural Sciences, Rural Development Administration of Korea

[0075] Accession number: KACC81230BP

[0076] Collection date: September 26, 2022

[0077]

Claims

1. A Pseudomonas aeruginosa repla1 strain having plastic degradation activity, whose deposit number is KACC 81230BP.

2. The strain according to claim 1, wherein the plastic is one or more selected from the group consisting of polyethylene terephthalate (PET), polyvinyl chloride (PVC), polystyrene (PS), polypropylene (PP) and polyethylene (PE).

3. The strain according to claim 3, wherein the Pseudomonas aeruginosa repla1 strain expresses laccase.

4. A method for degrading plastics, comprising co-culturing the Pseudomonas aeruginosa repla1 strain with a deposit number of KACC 81230BP with plastics. 5 . The method according to claim 4 , wherein the plastic is one or more selected from the group consisting of polyethylene terephthalate (PET), polyvinyl chloride (PVC), polystyrene (PS), polypropylene (PP) and polyethylene (PE). The method according to claim 4 , wherein the culturing is performed at 8° C. to 40° C.

7. A composition for degrading plastics, comprising the Pseudomonas aeruginosa repla1 strain with a deposit number of KACC 81230BP, a culture of the strain, or a plastic-degrading enzyme derived from the strain.

8. The composition according to claim 7, wherein the plastic is one or more selected from the group consisting of polyethylene terephthalate (PET), polyvinyl chloride (PVC), polystyrene (PS), polypropylene (PP) and polyethylene (PE). 9 . The composition according to claim 7 , wherein the plastic-degrading enzyme derived from a strain is laccase.

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