Aromatic polyester-degrading bacteria

Abstract

Provided is a microorganism that can decompose aromatic polyester or a breakdown product thereof. Also provided is a method for decomposing aromatic polyester or a breakdown product thereof using said microorganism.　The microorganism that decomposes aromatic polyester or a breakdown product thereof is any one of three strains of microorganisms: Strain No. 2a (accession number NITE BP-03483), Strain No. 7a (accession number NITE BP-03484), or Strain No. 8d (accession number NITE BP-03485).

Description

[Technical Field]

[0001] The present invention relates to a technique for rapidly decomposing waste liquids containing aromatic polyesters such as polyethylene terephthalate (PET) or their decomposition products using microorganisms. [Background technology]

[0002] As awareness of environmental issues grows, standards for industrial wastewater are becoming stricter around the world. When building a new factory, evidence must be presented that wastewater treatment meets the standards, and wastewater treatment is now positioned as an important technology as part of the manufacturing process. Polyethylene terephthalate (PET), used in beverage bottles and polyester fibers, can generate wastewater during its manufacturing and recycling processes. This wastewater contains high concentrations of PET degradation products, making it difficult to adequately decompose using existing activated sludge. Combining the newly discovered aromatic polyester-linked compound-degrading microorganism with activated sludge can resolve this issue.

[0003] For example, wastewater containing BHET discharged in the chemical recycling process of PET is difficult to decompose using existing activated sludge. In such cases, the impact on factory operating costs is significant, as factories must pay to have the wastewater treated by an external company or adjust their operations.

[0004] It has been reported that Enterobacter sp. was isolated as a microorganism capable of degrading BHET, but it was only able to degrade about 30% of 2000 mg / L (approximately 8 mM) of BHET even after 120 hours (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Lequan Qiu et al., J Basic Microbiol. 2020;60:699-711 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a microorganism capable of degrading aromatic polyesters such as BHET or their degradation products, and a method for degrading aromatic polyesters such as BHET or their degradation products using the microorganism. [Means for solving the problem]

[0007] The present inventors conducted extensive research into methods for treating BHET wastewater containing aromatic polyesters such as polyethylene terephthalate (PET) or their degradation products, for example, BHET wastewater discharged during the PET recycling process. The present inventors discovered microorganisms in soil that can rapidly degrade BHET and found that these microorganisms can efficiently degrade BHET. The inventors discovered that using these microorganisms alone or in combination with activated sludge enables rapid treatment of BHET wastewater, which has traditionally been difficult to decompose, and thus completed the present invention.

[0008] That is, the present invention is as follows. [1] Any of the following three strains of microorganisms that degrade aromatic polyesters or their degradation products: (i) Delftia lacustris strain, (ii) a strain of Pseudarthrobacter sp. belonging to the genus Pseudarthrobacter, or (iii) Pseudomonas sp. strains belonging to the genus Pseudomonas. [2] The microorganism according to [1], which is one of the following three strains of microorganisms that degrade aromatic polyesters or their degradation products: (i) Delftia lacustris strain No. 2a (accession number NITE BP-03483), (ii) Pseudarthrobacter sp. strain No. 7a (accession number NITE BP-03484), which belongs to the genus Pseudarthrobacter; or (iii) Pseudomonas sp. strain No. 8d (accession number NITE BP-03485), which belongs to the genus Pseudomonas. [3] A microorganism [1] or [2] that decomposes bis(2-hydroxyethyl) terephthalate (BHET). [4] A microorganism [1] or [2] that decomposes monohydroxyethyl terephthalate (MHET). [5] A microorganism [1] or [2] that decomposes terephthalic acid (TPA). [6] A method for decomposing an aromatic polyester or a degradation product thereof, comprising contacting one or more of the microorganisms according to any one of [1] to [3] with the aromatic polyester or a degradation product thereof. [7] The method according to [6], wherein aromatic polyester or its decomposition products in wastewater obtained in a polyethylene terephthalate (PET) recycling process are decomposed. [8] The method of [6] or [7], wherein the aromatic polyester or its degradation product is bis(2-hydroxyethyl) terephthalate (BHET). [9] A composition comprising one or more of the microorganisms described in [1] to [5].

[10] The composition of [9], which is activated sludge.

[11] The composition of [9], which is a microbial carrier carrying any one of the microorganisms of [1] to [5].

[12] The composition of

[11] , wherein the microbial carrier is selected from the group consisting of resin, activated carbon, and zeolite.

[13] A method for decomposing aromatic polyesters or decomposition products thereof in waste obtained in a polyethylene terephthalate (PET) recycling process, comprising contacting any one of the compositions [9] to

[12] with the waste liquid obtained in the PET recycling process.

[14] The method of

[13] , wherein the aromatic polyester or its degradation product is bis(2-hydroxyethyl) terephthalate (BHET). This specification includes the disclosure of Japanese Patent Application No. 2021-115744, from which this application claims priority. [Effects of the Invention]

[0009] The use of microorganisms capable of decomposing aromatic polyesters such as BHET or their degradation products according to the present invention enables smooth chemical recycling of PET. These microorganisms, either alone or in combination with activated sludge, enable rapid treatment of BHET wastewater, which has traditionally been difficult to decompose. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 shows the results of phylogenetic estimation based on the 16S rDNA base sequence of the 2a strain. [Figure 2-1] FIG. 1 shows the results of a comparison of the partial base sequences of 16S rDNA between strain 2a and Delftia lacustris. [Figure 2-2] FIG. 2 shows the results of a comparison of the partial base sequences of 16S rDNA between strain 2a and Delftia lacustris (continuation of FIG. 2-1). [Figure 3] FIG. 1 shows an image of a colony of the 2a strain. [Figure 4] FIG. 1 shows a Gram stain image of strain 2a. [Figure 5] FIG. 1 shows the results of biochemical properties (first stage bacterial test) of strain 2a. [Figure 6] FIG. 1 shows the results of biochemical properties of the 2a strain (second-stage bacterial test). [Figure 7]FIG. 1 shows the results of biochemical properties of strain 2a (second stage bacterial test (additional test)). [Figure 8] FIG. 1 shows the results of phylogenetic estimation based on the 16S rDNA base sequence of strain 7a. [Figure 9] FIG. 1 shows an image of a colony of strain 7a. [Figure 10] FIG. 1 shows a Gram stain image of strain 7a. [Figure 11] FIG. 1 shows the results of biochemical properties (first stage bacterial test) of strain 7a. [Figure 12] FIG. 1 shows the results of biochemical properties of strain 7a (second-stage bacterial test). [Figure 13] FIG. 1 shows the results of biochemical properties of strain 7a (second stage bacterial test (additional test)). [Figure 14] FIG. 1 shows the results of phylogenetic estimation based on the 16S rDNA base sequence of strain 8d. [Figure 15] FIG. 1 shows an image of a colony of strain 8d. [Figure 16] FIG. 1 shows a Gram stain image of strain 8d. [Figure 17] FIG. 1 shows the results of biochemical properties (first stage bacterial test) of strain 8d. [Figure 18] FIG. 1 shows the results of biochemical properties of strain 8d (second-stage bacterial test). [Figure 19] FIG. 1 shows the results of the biochemical properties of strain 8d (second stage bacterial test (additional test)). [Figure 20] 20A shows the degradation of 8 mM BHET by strains 2a, 7a, and 8d over a period of 0 to 150 hours, with Fig. 20A representing the control, Fig. 20B showing the results for strain 2a, Fig. 20C showing the results for strain 7a, and Fig. 20D showing the results for strain 8d. [Figure 21] 21A shows the degradation of 12 mM BHET by strains 2a, 7a, and 8d over a period of 0 to 150 hours, with Fig. 21A representing the control, Fig. 21B showing the results for strain 2a, Fig. 21C showing the results for strain 7a, and Fig. 21D showing the results for strain 8d. [Figure 22]22A shows the degradation of 16 mM BHET by strains 2a, 7a, and 8d over a period of 0 to 150 hours, with Fig. 22A representing the control, Fig. 22B showing the results for strain 2a, Fig. 22C showing the results for strain 7a, and Fig. 22D showing the results for strain 8d. [Figure 23] 23A shows the degradation of BHET when strains 2a, 7a, and 8d were added to activated sludge, with Fig. 23A representing the control, Fig. 23B showing the results for strain 2a, Fig. 23C showing the results for strain 7a, and Fig. 23D showing the results for strain 8d. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention will be described in detail below.

[0012] In the present invention, % indicating concentration indicates wt%.

[0013] The microorganism of the present invention is an aromatic polyester-degrading bacterium that decomposes aromatic polyesters such as polyethylene terephthalate (PET).

[0014] In the present invention, "polyester" refers to a polymeric substance having an ester bond in the main chain. Furthermore, "aromatic polyester" degradable by the microorganism of the present invention refers to a polyester containing an aromatic component as a repeating unit. The content of the repeating unit is, for example, 50 to 100% by weight, preferably 70 to 100% by weight, more preferably 90 to 100% by weight, and even more preferably 95 to 100% by weight, based on the total compound. Examples of aromatic polyesters include polyethylene terephthalate (PET), and further examples include PET containing 95% or more by weight of ethylene terephthalate repeating units.

[0015] Aromatic polyesters can be produced by polycondensation of dicarboxylic acid and diol components. For example, PET can be produced using terephthalic acid as the dicarboxylic acid component and ethylene glycol as the diol component. Examples of dicarboxylic acid components other than terephthalic acid include aromatic dicarboxylic acids and their derivatives, such as phthalic acid, isophthalic acid, diphenyldicarboxylic acid, diphenoxyethanedicarboxylic acid, and 2,5-naphthalenedicarboxylic acid; and aliphatic dicarboxylic acids and their derivatives, such as succinic acid, adipic acid, azelaic acid, sebacic acid, and decanedicarboxylic acid. Examples of diol components other than ethylene glycol include diethylene glycol, trimethylene glycol, tetramethylene glycol, propylene glycol, pentamethylene glycol, hexamethylene glycol, and decamethylene glycol.

[0016] When aromatic polyesters are decomposed using the aromatic polyester-degrading bacteria of the present invention, the form of the aromatic polyester to be decomposed is not limited, and examples thereof include fibrous, granular, flake, pellet, film, lump, and bottle forms. Mixtures of these forms can also be used.

[0017] The aromatic polyester-degrading microorganisms of the present invention can also decompose monohydroxyethyl terephthalate (MHET) and terephthalic acid (TPA), which are intermediate products of PET degradation. MHET is hydrolyzed to TPA, which is ultimately decomposed to carbon dioxide. That is, decomposition occurs along the pathway of BHET → MHET → TPA → carbon dioxide. By using the microorganisms of the present invention, BHET and MHET, which are intermediate products of PET degradation, can be completely decomposed to ultimately carbon dioxide.

[0018] The aromatic polyester-degrading microorganism of the present invention is a bacterium that decomposes bis(2-hydroxyethyl) terephthalate (BHET), a decomposition intermediate product discharged into wastewater when polyethylene terephthalate (PET) is chemically decomposed and recycled. The aromatic polyester-degrading microorganism of the present invention can also be called a BHET-degrading bacterium.

[0019] PET production and recycling processes can produce wastewater containing high concentrations of PET degradation products. The microorganisms of the present invention can decompose these PET degradation products.

[0020] For example, in chemical recycling of PET, PET is chemically decomposed into the monomer BHET, which is then purified and repolymerized to produce PET. Chemical decomposition methods for PET include methanolysis, glycolysis, and hydrolysis. In these recycling methods, BHET is discharged into the waste liquid.

[0021] By using the microorganism of the present invention, it is possible to decompose BHET in the waste liquid generated in the PET recycling process.

[0022] The microorganisms capable of degrading aromatic polyesters such as BHET or their degradation products of the present invention include three strains: strain 2a, strain 7a (31076-02-B1), and strain 8d. These three strains of microorganisms were isolated from soil.

[0023] As a result of biochemical and genetic identification tests, strain 2a was identified as Delftia lacustris, strain 7a (31076-02-B1) as Pseudarthrobacter sp. belonging to the genus Pseudarthrobacter, and strain 8d as Pseudomonas sp. belonging to the genus Pseudomonas.

[0024] Strain 2a: Delftia lacustris The 16S rDNA base sequence is shown in SEQ ID NO: 1. When compared with known 16S rDNA (16S rRNA gene) base sequences, the highest homology is 99.9%. Figure 1 shows the results of phylogenetic inference based on the 16S rDNA base sequence.

[0025] The 2a strain was deposited on June 17, 2021, at the NITE Patent Microorganisms Depository (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under accession number NITE P-03483 (identification number: 31076-01). It was subsequently transferred to international deposit (request date for transfer to international deposit: May 27, 2022, accession number: NITE BP-03483).

[0026] Strain 7a (31076-02-B1): Pseudarthrobacter sp. The 16S rDNA sequence is shown in SEQ ID NO: 2. The highest homology rate compared with known 16S rDNA sequences is 99.4%. Figure 2 shows the results of phylogenetic inference based on the 16S rDNA sequence.

[0027] The 7a (31076-02-B1) strain was deposited on June 17, 2021, at the NITE Patent Microorganisms Depository (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under accession number NITE P-03484 (identification number: 31076-02-B1). It was subsequently transferred to international deposit (request for transfer to international deposit: May 27, 2022, accession number: NITE BP-03484).

[0028] Strain 8d: Pseudomonas sp. The 16S rDNA sequence is shown in SEQ ID NO: 3. The highest homology rate compared with known 16S rDNA sequences is 99.7%. Figure 3 shows the results of phylogenetic estimation based on the 16S rDNA sequence.

[0029] The 8d strain was deposited on June 17, 2021, at the NITE Patent Microorganisms Depository (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under accession number NITE P-03485 (identification number: 31076-03). It was subsequently transferred to international deposit (request for transfer to international deposit: May 27, 2022, accession number: NITE BP-03485).

[0030] The microorganisms of the present invention can be cultured in any medium that allows the growth of the microorganism and is suitable for degrading aromatic polyesters such as BHET or their degradation products. Examples of such media include, but are not limited to, nutrient agar, MS medium, and MS(+) medium. Nutrient agar can be prepared, for example, by dissolving 5.0 g of meat extract, 10.0 g of peptone, 5.0 g of sodium chloride, and 15.0 g of agar in 1000 mL of deionized water. MS(+) medium contains, for example, yeast extract, ammonium sulfate, and sodium phosphate buffer, and further contains metal salts and hydrates thereof, such as iron sulfate, copper sulfate, zinc sulfate, manganese sulfate, and magnesium sulfate.

[0031] The microorganism of the present invention may be cultured by either static culture or permeation culture under aerobic conditions, but static culture is preferred. When cultured by shaking culture, the shaking speed is, for example, 100 to 400 reciprocations / minute, preferably 200 to 300 reciprocations / minute.

[0032] The culture temperature for the microorganism of the present invention is not limited, but can be, for example, in the range of 20 to 40° C., preferably in the range of 25 to 35° C., and more preferably in the range of 30° C. The pH during culture is also not limited, but can be, for example, in the range of 5 to 9, and preferably in the range of 6 to 8. To adjust the pH within this range, inorganic acids such as hydrochloric acid and sulfuric acid, inorganic bases such as sodium hydroxide and potassium hydroxide, and aqueous solutions thereof, or various buffer solutions such as phosphate buffers may be used.

[0033] The culture period is not limited, and the culture can be carried out for, for example, 1 day or more, preferably 2 days or more, more preferably 5 days or more, and even more preferably 10 days or more, and for example, 5 months or less, preferably 2 months or less, and more preferably 1 month or less. If necessary, it may be effective to add fresh microbial cells during the culture or to replace the medium with a fresh one.

[0034] The present invention includes a method for degrading BHET using the above-mentioned microorganisms alone or in combination.

[0035] When the above microorganisms are used for decomposing BHET, the BHET-degrading ability can be calculated by mixing BHET with the above microorganisms and measuring the OD600 of BHET in the treated solution. Alternatively, BHET can be detected by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), or the like.

[0036] Degradation of aromatic polyesters such as BHET or their degradation products using the above-mentioned microorganisms can be achieved by contacting the aromatic polyesters such as BHET or their degradation products with the above-mentioned microorganisms. Here, "contact" refers to mixing the aromatic polyesters such as BHET or their degradation products with the microorganisms, and then allowing the microorganisms to act on the aromatic polyesters such as BHET or their degradation products to cause a degradation reaction of the aromatic polyesters such as BHET or their degradation products by the microorganisms. The density of the microorganisms used during the degradation reaction can be appropriately set depending on the amount of aromatic polyesters such as BHET or their degradation products to be degraded. The reaction temperature is 20 to 40°C, preferably 25 to 35°C, and more preferably 30°C. The pH during the reaction is around 7.0, preferably 5.0 to 9.0, more preferably 6.0 to 8.0, and particularly preferably 6.5 to 7.5. The reaction time can be appropriately set depending on the amount of aromatic polyesters such as BHET or their degradation products to be degraded. The reaction time ranges from several hours to several months. For long-term treatment, the above-mentioned microorganisms can be added periodically.

[0037] Among the above-mentioned microorganisms, any single strain may be used, or any two strains, i.e., strains 2a and 7a(31076-02-B1), strains 2a and 8d, or strains 7a(31076-02-B1) and 8d, may be used in combination, or three strains, i.e., strains 2a, 7a(31076-02-B1), and strain 8d, may be used in combination. When a single strain is used, it is easier to determine the conditions appropriate for the growth of the strain and / or the degradation of aromatic polyesters such as BHET or their degradation products.

[0038] Among the three strains, strain 8d had the highest decomposition ability, completely degrading not only BHET but also MHET and TPA in 24 hours when 2 mM BHET, MHET, and TPA were treated with one platinum loop of the microorganism. Strain 7a completely degraded BHET in 24 hours and MHET in 96 hours, and strain 2a almost completely degraded BHET, MHET, and PTA in 72 hours.

[0039] When BHET is degraded using the microorganism of the present invention, the BHET concentration is 20 mM or less, preferably 16 mM or less, and more preferably 8 mM or less. That is, when the microorganism of the present invention is used, 8 mM BHET can be completely degraded within 72 hours, preferably within 48 hours, and more preferably within 24 hours. BHET can be degraded by adding the microorganism to a solution containing BHET.

[0040] The microorganism of the present invention is used to treat wastewater containing aromatic polyesters such as BHET or their degradation products. It is preferably used to treat wastewater containing BHET generated during chemical recycling of PET. In chemical recycling of PET, PET is depolymerized using ethylene glycol to produce the intermediate BHET. Next, a purification process (distillation and recrystallization) is performed to obtain highly pure BHET. The wastewater discharged during the recrystallization process contains high concentrations of BHET and related substances such as MHET and TPA.

[0041] By adding a solution containing the microorganism of the present invention to the waste liquid and bringing it into contact with the waste liquid, aromatic polyesters such as BHET or their degradation products can be decomposed.

[0042] Furthermore, waste is typically treated using activated sludge. Activated sludge is a general term for "living" floating organic sludge containing artificially or artificially cultivated aerobic microorganisms, and is widely used in sewage treatment plants, sewage treatment plants, septic tanks, and other facilities as a means of purifying wastewater and sewage. However, activated sludge cannot decompose aromatic polyesters such as BHET or their degradation products, and aromatic polyesters such as BHET or their degradation products remain in waste treated with activated sludge generated during the PET recycling process.

[0043] Therefore, by mixing the microorganism of the present invention into activated sludge and adding and mixing wastewater, which is waste obtained in a PET recycling process, with the activated sludge containing the microorganism of the present invention, aromatic polyesters such as BHET or their degradation products in the waste can be decomposed.

[0044] For example, 1 to 100 mL, preferably 1 to 50 mL, and more preferably about 5 to 10 mL of the microbial culture solution of the present invention is added to 1 L of activated sludge, and the activated sludge containing the microorganisms is added to a wastewater containing BHET and treated at 20 to 40°C for 8 hours to 7 days, preferably 8 hours to 3 days, more preferably 8 hours to 2 days, and even more preferably 8 hours to 12 hours. The treated wastewater is discharged into the sea as treated water that does not exceed environmental standards. If the environmental standards are exceeded, it is treated as industrial waste.

[0045] In this case, a substance that serves as a nutrient for the microorganisms, such as a culture medium, may be added to the activated sludge.

[0046] Furthermore, before the waste obtained in the PET recycling process is mixed with activated sludge and treated, it can be mixed with another carrier holding the microorganism of the present invention and treated. Such a carrier can be a resin capable of adhering and holding microorganisms, such as polyvinyl alcohol (PVA), polypropylene, polyurethane, or a porous material with a large surface area, such as activated carbon or zeolite. An example of a carrier capable of holding PVA microorganisms is Kuraray Poval. TM and Kurageru (registered trademark) (Kuraray Co., Ltd.). The shape of these carriers is not limited, and membrane-like carriers, capsule-like carriers, tubular carriers, gel-like carriers, etc. can be used. Carriers capable of retaining these microorganisms are called microbial carriers.

[0047] Furthermore, wastewater obtained from the PET recycling process can be placed in a dedicated incubator, where the microorganisms of the present invention are allowed to grow in suspension to decompose and remove only the BHET from the wastewater. After that, the remaining wastewater can be passed through an aeration tank using activated sludge to remove organic matter other than BHET, thereby removing BOD (biochemical oxygen demand) and COD (chemical oxygen demand).

[0048] In the above description, a solution containing the microorganism of the present invention, activated sludge containing the microorganism of the present invention, and a microbial carrier holding the microorganism of the present invention are referred to as a composition containing the microorganism of the present invention.

[0049] As described above, the present invention also includes a wastewater treatment facility for treating the wastewater obtained in the PET recycling process. [Example]

[0050] The present invention will be specifically explained by the following examples, but the present invention is not limited to these examples.

[0051] [Example 1] Isolation of BHET-degrading bacteria and activity measurement 1. Purpose In chemical recycling of PET, PET is depolymerized using ethylene glycol to produce the intermediate BHET. Next, a purification process (distillation and recrystallization) is carried out to obtain highly pure BHET. The waste liquid discharged in the recrystallization process contains high concentrations of BHET as well as related substances such as MHET and TPA. However, this waste liquid cannot be adequately treated using the activated sludge method, and a new treatment technology was needed. Therefore, the aim was to obtain bacteria that can efficiently decompose BHET.

[0052] 2. Experimental Materials and Methods 2.1 Soil samples, BHET refined product, and crystallization wastewater Soil was collected from the site of the Kawasaki plant of Japan Environmental Design Co., Ltd. Recycled, refined BHET pellets provided by the company were used as the BHET reagent. Of the three types of crystallization wastewater also provided (Waste A 20190604, Waste B 20191009, and Waste C 20190517), experiments were primarily conducted using Waste A.

[0053] 2.2 Enrichment culture medium and culture method 1 g of each of the 10 types of soil was suspended in 10 mL of physiological saline and allowed to stand at room temperature for 1 hour. Enrichment culture was performed by adding 0.5 mL of the supernatant of the soil suspension to 10 mL of screening medium (0.2% BHET / MS(+) (Table 1) containing 0.2% (8 mM) BHET powder ground in a mortar. The culture was performed in a 20 mm diameter test tube at 30°C. The enrichment culture was repeated several times by subculture of 0.1 mL of the culture medium onto fresh medium every week.

[0054] [Table 1]

[0055] 2.3 Isolation of bacteria After cultivation, bacteria were isolated from the culture medium containing the bacterial cells by the plate dilution method. 3 ~10 9 The mixture was diluted 2x and spread onto 0.2% BHET / MS(+) agar medium (Table 2) and 0.2% BHET / MS(+1 / 10) agar medium (Table 3) containing 1 / 10 the amount of yeast extract, and plate culture was carried out at 30°C.

[0056] [Table 2] The composition of the trace elements is the same as in Table 1.

[0057] [Table 3] The composition of the trace elements is the same as in Table 1.

[0058] 2.4 Measurement of BHET degradation activity of isolated bacteria by HPLC analysis To examine the BHET-degrading activity of the isolated strains, a loopful of bacteria that had been subcultured on 0.2% BHET / MS(+) agar medium was taken and inoculated into 10 mL of 2 mM BHET / MS(+) medium. The culture was then cultured at 30°C for 5 days with shaking, and the culture medium was analyzed by HPLC. The collected culture medium was diluted appropriately with 1 mL of mobile phase (formic acid / acetonitrile / water = 1 / 2 / 7 vol ratio) and further mixed thoroughly. After filtering through a 0.20 μm pre-filter for amphoteric affinity, it was analyzed by HPLC. The HPLC analysis was performed using a Shimadzu LC-2010A HT system and a Nacalai Tesque COSMOCIL 5C column. 18-AR-II (4.5ID x 250mm) and 5C 18 -AR-II guard column was used. The analysis was performed using isocratic elution with a mobile phase (formic acid / acetonitrile / water = 1 / 2 / 7 vol ratio) at a flow rate of 1 mL / min and a column temperature of 40°C, with detection at a wavelength of 254 nm. Peaks for BHET, MHET, and TPA in the culture medium were identified from the elution times, and their peak areas were examined. For BHET, a calibration curve of BHET concentration versus peak area was prepared using an aqueous BHET solution dissolved at 70°C, and quantitative analysis was also performed.

[0059] 3. Results 3.1 Isolation of BHET-utilizing bacteria The following three strains were successfully isolated by plate culture. 2a 7a(31076-02-B1) 8d

[0060] Each strain was identified at Techno Suruga Laboratory. The results are shown below. The results include 16s rDNA sequence analysis, morphological observation, and physiological and biochemical property tests (first-stage bacterial test and second-stage bacterial test).

[0061] 3.2 Identification of isolated microorganisms (1) Identification method (i) Culture conditions Culture medium: Nutrient Agar (Oxoid, GBR) ·Culture temperature: 30℃ ·Culture time: 24~72 hours Other conditions: Aerobic cultivation

[0062] (ii) 16s rDNA partial sequence analysis DNA extraction: Achromopeptidase (FUJIFILM Wako Pure Chemical, Japan) PCR amplification: Tks Gflex DNA Polymerase (Takara Bio, Japan) Cycle sequencing: BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) Primers used (Yasuyoshi Nakagawa et al., Genetic Analysis Method: Determining the Base Sequence of the 16S rRNA Gene, edited by the Japanese Society for Actinomycetes, Classification and Identification of Actinomycetes, Japan Society Administration Center; 2001, pp. 88-117) PCR amplification: 9F, 1510R Sequence (approximately 1,500 bp): 9F, 515F, 1099F, 536R, 926R, 1510R Sequencing: ABI PRISM 3130xL Genetic Analyzer System (Applied Biosystems) Sequencing: ChromasPro 2.1 (Technelysium, AUS) ·BLAST homology search Analysis software: ENKI v3.2 (TechnoSuruga Laboratory, Japan) Database: DB-BA15.0 (TechnoSuruga Laboratory), International Nucleotide Sequence Database (DDBJ / ENA(EMBL) / GenBank) ·Simple molecular phylogenetic analysis Phylogenetic tree estimation: neighbor-joining method Base substitution model: Kimura-2-parameter Reliability assessment of tree structure: Bootstrap method (1,000 iterations)

[0063] (iii) Bacterial Tier 1 Test Colonies were observed using a stereomicroscope, morphological observations were made using a light microscope, and tests were performed on catalase reaction, oxidase reaction, acid / gas production from glucose, and glucose oxidation / fermentation (O / F) based on the methods of Barrow & Feltham (Cowan and Steel's Manual for the Identification of Medical Bacteria, 3rd ed. Cambridge: Cambridge University Press; 1993). Gram staining: Faber G "Nissui" (Nissui Pharmaceutical, Japan) Microscope: Optical microscope BX50F4 (Olympus, Japan) Stereo microscope: SMZ800N (Nikon, Japan)

[0064] (iv) Bacterial Tier 2 Testing The following kits were used for the bacterial second stage test: 2a strain: API 20 NE, API ZYM (bioMerieux, FRA) 7a strain:API CORYNE (bioMerieux, FRA) 8d strain: API 20 NE (bioMerieux, FRA)

[0065] (2) Identification results (i) 2a stock Figure 1 shows the results of phylogenetic inference based on the 16S rDNA base sequence. In Figure 1, the line in the upper left corner represents the scale bar, the numbers at the phylogenetic branch branches represent bootstrap values, the T at the end of the strain name indicates the type strain, and BSL indicates the biosafety level (BSL1* (opportunistic pathogen) or higher). The 16s rDNA base sequence is shown in SEQ ID NO: 1.

[0066] Figures 2-1 and 2-2 (Figure 2-2 is a continuation of Figure 2-1) show the results of a comparison of the partial 16S rDNA base sequences of the 2a strain and Delftia lacustris. In Figure 2, Query represents the sequence of the 2a strain, and Sbjct represents the sequence of Delftia lacustris. Figure 3 shows a colony image of the 2a strain, and Figure 4 shows a Gram stained image of the 2a strain. The biochemical properties of strain 2a are shown in Figure 5 (results of the first stage bacterial test), Figure 6 (results of the second stage bacterial test), and Figure 7 (results of the second stage bacterial test (additional test)).

[0067] A BLAST homology search against DB-BA and the international base sequence database using the microbial identification system "ENKI" revealed that the 16S rDNA partial base sequence of strain 2a showed 99.9% identity with the type strain 332T of Delftia lacustris (accession number EU888308) and D. tsuruhatensis T7T (AB075017).

[0068] In the molecular phylogenetic tree (Fig. 1) analyzed based on the base sequence obtained by homology search against DB-BA, strain 2a was included in the cluster consisting of the genus Delftia, and showed the same molecular phylogenetic position as D. lacustris 332T (EU888308).

[0069] The results of the first stage bacterial test showed that strain 2a was a motile Gram-negative bacillus that did not oxidize glucose and showed positive results in both catalase and oxidase reactions (Figs. 3, 4, and 5). These properties were consistent with those of the genus Delftia.

[0070] A second-stage bacterial test using the API kit showed that the sample reduced nitrate and utilized D-mannitol, potassium gluconate, and n-capric acid, but not glucose or maltose (Figure 6). Further testing showed that the sample grew in 5% NaCl, hydrolyzed casein, and exhibited lipase activity (Tween 80) (Figure 7). The results of each enzyme reaction performed using API ZYM are shown in Figure 7. While these properties were similar to those of D. lacustris, some differences were also observed. In particular, the sample did not utilize L-arabinose or N-acetyl-D-glucosamine, and did not exhibit α-glucosidase or β-glucuronidase activity, which were distinct from D. lacustris.

[0071] Based on the above results and taking into account the results of the 16S rDNA partial base sequence analysis, strain 2a was identified as a D. lacustris strain.

[0072] The 2a strain was deposited on June 17, 2021, at the NITE Patent Microorganisms Depository (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under accession number NITE P-03483 (identification number: 31076-01). It was subsequently transferred to international deposit (request date for transfer to international deposit: May 27, 2022, accession number: NITE BP-03483).

[0073] (ii) 7a strain Figure 8 shows the results of phylogenetic inference based on the 16S rDNA base sequence. In Figure 8, the line in the upper left corner is the scale bar, the numbers at the phylogenetic branch branches are bootstrap values, the T at the end of the strain name indicates the type strain of that species, and BSL indicates the biosafety level (BSL1* (opportunistic pathogen) or higher). The 16s rDNA base sequence is shown in SEQ ID NO: 2. Figure 9 shows a colony image of the 7a strain, and Figure 10 shows a Gram stained image of the 2a strain. The biochemical properties of strain 7a are shown in Figure 11 (results of the first stage bacterial test), Figure 12 (results of the second stage bacterial test), and Figure 13 (results of the second stage bacterial test (additional test)).

[0074] A BLAST homology search against DB-BA and the international base sequence database using the microbial identification system "ENKI" revealed that the 16S rDNA partial base sequence of SIID31076-02-B1 showed 99.4% identity to Pseudarthrobacter equi IMMIB L-1606T (FN673551), 99.1% identity to P. defluvii 4C1-aT (AM409361), and 99.0% identity to P. chlorophenolicus A6T (AF102267).

[0075] In the molecular phylogenetic tree (Fig. 8) analyzed based on the base sequence obtained by homology search against DB-BA, strain 7a was included in the cluster formed by the genus Pseudarthrobacter and formed a cluster with P. chlorophenolicus A6T (AF102267), although there was a distance between the two.

[0076] The results of the first stage bacterial test showed that strain 7a was a non-motile, Gram-positive bacillus, did not form spores, and exhibited a bacillus-coccus life cycle with morphological changes over time (Figs. 9, 10, and 11). It also did not oxidize glucose, and the catalase reaction was positive, while the oxidase reaction was negative (Fig. 11). These properties were consistent with those of the genus Pseudarthrobacter.

[0077] The results of the second stage bacterial test using the API kit showed that the specimen did not reduce nitrate, hydrolyze esculin, did not hydrolyze gelatin, and did not oxidize any sugars (Figure 12). Furthermore, additional tests showed that the specimen did not grow under anaerobic conditions, hydrolyzed casein and tyrosine, but did not hydrolyze starch (Figure 13). While these properties were similar to those of P. chlorophenolicus, which formed the same cluster in 16S rDNA partial base sequence analysis, differences were also identified. In particular, the specimen's lack of motility and inability to hydrolyze gelatin were distinct from those of P. chlorophenolicus. Furthermore, no known species in the genus Pseudarthrobacter was found to match the specimen.

[0078] These results indicate that strain 7a belongs to the genus Pseudarthrobacter and is most closely related to P. chlorophenolicus among known species. However, 16S rDNA sequence analysis and physiological and biochemical characterization results suggest that strain 7a is distinct from P. chlorophenolicus. Finally, strain 7a was identified as Pseudarthrobacter sp.

[0079] The 7a strain was deposited on June 17, 2021, at the NITE Patent Microorganisms Depository (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under accession number NITE P-03484 (identification number: 31076-02-B1). It was subsequently transferred to international deposit (request for transfer to international deposit: May 27, 2022, accession number: NITE BP-03484).

[0080] (iii) 8d shares Figure 14 shows the results of phylogenetic inference based on the 16S rDNA base sequence. In Figure 14, the line in the upper left corner is the scale bar, the numbers at the phylogenetic branch branches are bootstrap values, the T at the end of the strain name indicates the type strain of that species, and BSL indicates the biosafety level (BSL1* (opportunistic pathogen) or higher). The 16S rDNA base sequence is shown in SEQ ID NO: 3. FIG. 15 shows a colony image of strain 8d, and FIG. 16 shows a Gram stained image of strain 8d. The biochemical properties of strain 8d are shown in Figure 17 (results of the first stage bacterial test), Figure 18 (results of the second stage bacterial test) and Figure 19 (results of the second stage bacterial test (additional test)).

[0081] A BLAST homology search against DB-BA and the international base sequence database using the microbial identification system "ENKI" showed that the 16S rDNA partial base sequence of strain 8d was 99.7% identical to Pseudomonas nitroreducens DSM 14399T (AM088474).

[0082] In a molecular phylogenetic tree (Figure 14) analyzed based on the base sequence obtained by a homology search against DB-BA, strain S8d was included in a cluster formed by the genus Pseudomonas and formed a cluster with P. nitroreducens DSM 14399T (AM088474) supported by a high bootstrap value of 99%, indicating that they are closely related, although there was a distance between the two.

[0083] The results of the first stage bacterial test showed that strain 8d was a motile Gram-negative bacillus that oxidized glucose and showed positive results in both catalase and oxidase reactions (Figures 15, 16, and 17). These properties were consistent with those of the genus Pseudomonas.

[0084] The results of the second stage bacterial test using the API kit showed that the specimen reduced nitrate, exhibited arginine dihydrolase activity, did not hydrolyze gelatin, and utilized glucose, potassium gluconate, and n-capric acid, but not L-arabinose or D-mannose (Figure 18). Furthermore, additional tests showed that the specimen did not produce fluorescent pigments on King's A or King's B agar media, and did not hydrolyze starch (Figure 19). These properties were similar to those of P. nitroreducens, which formed the same cluster in the 16S rDNA partial base sequence analysis, but did not match. The lack of fluorescent pigment production was particularly different from that of P. nitroreducens.

[0085] These results indicate that strain 8d is included in the Pseudomonas genus and is most closely related to P. nitroreducens among known species. However, 16S rDNA sequence analysis and physiological and biochemical characterization studies suggest that strain 8d is distinct from P. nitroreducens. Finally, strain 8d was identified as Pseudomonas sp.

[0086] The 8d strain was deposited on June 17, 2021, at the NITE Patent Microorganisms Depository (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under accession number NITE P-03485 (identification number: 31076-03). It was subsequently transferred to international deposit (request for transfer to international deposit: May 27, 2022, accession number: NITE BP-03485).

[0087] 3.3 Time course measurement of decomposition activity of BHET-decomposing bacteria Three different degrading bacteria (2a, 7a, and 8d) were cultured in 2 mM BHET / MS(+) medium. 1 mL of the culture medium was sampled every 24 hours, and the OD600 was measured and then collected. The amount of BHET degraded was then examined by HPLC.

[0088] The decomposition rate of BHET in 2a was slower than that in 7a and 8d, but BHET, MHET, and TPA almost disappeared within 72 h. 7a completely degraded BHET in 24 h and MHET in 96 h. 8d completely degraded BHET, MHET, and TPA within 24 h.

[0089] Since the molecular weight of BHET is 254, 1 mM BHET is 254 mg / L BHET, or 0.0254% BHET, and 8 mM BHET is 2032 mg / L BHET, or 0.2032% BHET.

[0090] 3.4 Decomposition activity under high BHET concentrations To investigate whether higher concentrations of BHET could be degraded in actual crystallization wastewater treatment, we investigated whether bacteria 2a, 7a, and 8d could degrade BHET in culture media containing 8 mM, 12 mM, and 16 mM BHET. A culture media with the same composition but without the addition of BHET-degrading bacteria served as a control for comparison.

[0091] BHET concentrations above 8 mM are insoluble even at 70°C and cannot be sterilized by filtration. The same preparation method as for BHET / MS(+)FT medium cannot be used. Therefore, although BHET decomposes slightly to MHET, the medium was prepared using the following method. BHET pellets were added to water to concentrations of 16 mM, 24 mM, and 32 mM, and completely dissolved by autoclaving. Before the BHET in the aqueous solution precipitated, the mixture was mixed with an equal amount of 2xMS(+), which had also been autoclaved, stirred, and then immediately dispensed into culture tubes.

[0092] One loopful of bacterial cells was taken from the prepared 8mM, 12mM, and 16mM BHET / MS(+) medium and suspended in 1mL of MS(+) medium, and then 80µL of each suspension was inoculated into 10mL of each medium. The culture was cultured with shaking at 30°C, and 1mL of the culture medium was sampled every 24 hours. The culture medium was temporarily stored at -20°C and thawed at a later date. The pH of the culture medium and the amount of BHET decomposition were measured by HPLC. Experiments were performed in duplicate.

[0093] After 4 days of culture, 2a and 7a were confirmed to grow even at 16 mM BHET, while 8d did not grow at 12 mM or higher.

[0094] The results are shown in Figures 20 to 22. 2a and 7a degraded 12 mM BHET over 3 days. 2a had the highest BHET degradation activity at 12 mM, and 7a had the highest activity at 8 mM. 8d had extremely high BHET degradation activity at 8 mM, completely degrading BHET, MHET, and TPA within 24 hours.

[0095] [Example 2] Decomposition of BHET in waste obtained from a PET recycling process using activated sludge containing BHET-degrading bacteria 1. Purpose The purpose of this study was to confirm the improvement in BHET degradation capacity when BHET-degrading bacteria isolated at Keio University were added to activated sludge at the wastewater treatment facility of the Japan Environmental Design Co., Ltd. factory.

[0096] 2. Experimental Materials and Methods 2.1 BHET-degrading bacteria, purified BHET products, and activated sludge The three BHET-degrading bacteria used were 2a, 7a, and 8d, which were isolated at Keio University. The experiments were conducted using BHET manufactured by Pet Refine Technology, a subsidiary of Japan Environmental Design Co., Ltd., and activated sludge collected from the wastewater treatment facility at the Kitakyushu Hibikinada Plant of Japan Environmental Design Co., Ltd.

[0097] 2.2 Experimental Method 100 μl of culture solution containing three strains of BHET-degrading bacteria was added to 5 ml of activated sludge, and the culture solution was adjusted to a total volume of 10 ml, containing 1% trace metals (Table 4), 0.2 wt% BHET, and water as the solvent. A culture solution with the same composition but without the BHET-degrading bacteria served as a control for comparison. The prepared culture solution was cultured with shaking at 30°C, and 100 μl samples were taken after 1, 2, 3, and 5 days.

[0098] [Table 4]

[0099] 2.3 Comparison of BHET degradation activity by HPLC The collected culture medium was diluted 10-fold with 0.1 ml of mobile phase (formic acid / acetonitrile / water = 1 / 2 / 7 vol ratio) and stirred thoroughly, then filtered through a 0.2 μm filter and analyzed by HPLC.

[0100] HPLC analysis was performed using a Shimadzu LC-2010A HT system and a Nacalai Tesque COSMOCIL 5C18-AR-II (4.6 ID x 250 mm) column with a 5C18-AR-II guard column. Analysis was performed using a mobile phase (formic acid / acetonitrile / water = 1 / 2 / 7 vol ratio) at a flow rate of 1 mL / min with isocratic elution at 40 °C. Detection was performed at a wavelength of 254 nm. Peaks for BHET, MHET, and TPA in the culture medium were identified based on elution time, and their peak areas were measured. A calibration curve of BHET concentration versus peak area was prepared using a BHET solution dissolved at 70 °C to verify quantitative analysis.

[0101] 3. Results Three different types of BHET-degrading bacteria (2a, 7a, and 8d) were added to activated sludge to prepare a culture medium. Immediately after preparation of the culture medium, and 1, 2, 3, and 5 days after the start of cultivation, 100 μl samples were taken and analyzed by HPLC to examine the amount of BHET decomposition.

[0102] The results are shown in Figure 23. Compared to the activated sludge control, the BHET degradation rates of BHET-degrading bacteria 2a, 7a, and 8d were all improved. Among them, 8d had the fastest BHET degradation rate, with BHET, MHET, and TPA almost completely disappearing after 5 days.

[0103] [Example 3] Decomposition of BHET in waste obtained from PET recycling process using a carrier immobilizing BHET-degrading bacteria 1. Purpose The purpose of this study was to confirm the BHET decomposition ability of BHET-decomposing bacteria isolated at Keio University when immobilized on a commercially available bacterial immobilization carrier.

[0104] 2. Experimental Materials and Methods 2.1 BHET-degrading bacteria, purified BHET products, and bacterial immobilization carriers The BHET-degrading bacteria used were 8d bacteria isolated at Keio University. The experiments were conducted using BHET manufactured by Pet Refine Technology Co., Ltd., a subsidiary of Japan Environmental Design Co., Ltd. PVA gel (Kuraray) manufactured by Kuraray Co., Ltd. was used as the bacterial immobilization carrier.

[0105] 2.2 Immobilization of BHET bacteria on bacterial immobilization carriers 5 ml of 0.4 wt% BHET and 5 ml of 2xMS(+) were added to a test tube, and 100 μl of a culture medium of BHET-degrading bacteria 8d was added to prepare the culture medium. 50 pieces of seaweed gel, which had been washed and replaced with sterilized saline, were added and the mixture was incubated with shaking at 30°C for 2 days.

[0106] 2.3 Experimental Method 5 ml of 0.4 wt% BHET and 5 ml of 2xMS(+) were added to a test tube, and 50 pellets of immobilized carriers containing BHET-degrading bacteria were added to prepare a culture medium. The culture medium was cultured with shaking at 30°C, and 100 μl of the medium was sampled one day later.

[0107] 2.4 Comparison of BHET degradation activity by HPLC The collected culture medium was diluted 10-fold with 0.1 ml of mobile phase (formic acid / acetonitrile / water = 1 / 2 / 7 vol ratio) and stirred thoroughly, then filtered through a 0.2 μm filter and analyzed by HPLC.

[0108] HPLC analysis was performed using a Shimadzu LC-2010A HT system and a Nacalai Tesque COSMOCIL 5C18-AR-II (4.6 ID x 250 mm) column with a 5C18-AR-II guard column. Analysis was performed using a mobile phase (formic acid / acetonitrile / water = 1 / 2 / 7 vol ratio) at a flow rate of 1 mL / min with isocratic elution at 40 °C. Detection was performed at a wavelength of 254 nm. Peaks for BHET, MHET, and TPA in the culture medium were identified based on elution time, and their peak areas were measured. A calibration curve of BHET concentration versus peak area was prepared using a BHET solution dissolved at 70 °C to verify quantitative analysis.

[0109] 3. Results Immediately after preparation of the culture medium of the immobilized carrier containing the BHET-degrading bacteria and one day after the start of cultivation, 100 μl of the medium was sampled and subjected to HPLC to examine the amount of BHET decomposition.

[0110] The carrier immobilized with BHET-degrading bacteria showed a rapid BHET decomposition rate, with BHET, MHET, and TPA almost completely disappearing after one day. [Industrial Applicability]

[0111] Using the microorganism of the present invention, polyethylene terephthalate can be efficiently recycled. [Accession number]

[0112] NITE BP-03483 NITE BP-03484 NITE BP-03485 All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. Any of the following three strains of microorganisms that degrade aromatic polyesters or their degradation products: (i) Delftia lacustris strain No. 2a (accession number NITE BP-03483); (ii) Pseudarthrobacter sp. No. 7a strain (accession number NITE BP-03484), which belongs to the genus Pseudarthrobacter; or (iii) No. 8d strain (accession number NITE BP-03485), which is a Pseudomonas sp. strain belonging to the genus Pseudomonas.

2. The microorganism according to claim 1, which decomposes bis(2-hydroxyethyl) terephthalate (BHET).

3. The microorganism according to claim 1, which decomposes monohydroxyethyl terephthalate (MHET).

4. The microorganism according to claim 1, which decomposes terephthalic acid (TPA).

5. A method for degrading an aromatic polyester or its degradation products, comprising contacting one or more of the microorganisms of claim 1 with the aromatic polyester or its degradation products.

6. 6. The method according to claim 5, wherein the aromatic polyester or its decomposition products in a waste liquid obtained in a polyethylene terephthalate (PET) recycling process is decomposed.

7. 6. The method of claim 5, wherein the aromatic polyester or degradation product thereof is bis(2-hydroxyethyl) terephthalate (BHET).

8. 7. The method of claim 6, wherein the aromatic polyester or degradation product thereof is bis(2-hydroxyethyl) terephthalate (BHET).

9. A composition comprising one or more of the microorganisms of claim 1.

10. 10. The composition of claim 9, which is activated sludge.

11. The composition according to claim 9, which is a microbial carrier carrying the microorganism according to claim 1.

12. 12. The composition of claim 11, wherein the microbial carrier is selected from the group consisting of resin, activated carbon, and zeolite.

13. A method for decomposing aromatic polyesters or decomposition products thereof in waste obtained in a polyethylene terephthalate (PET) recycling process, comprising contacting the composition according to claim 9 with the waste liquid obtained in the PET recycling process.

14. 14. The method of claim 13, wherein the aromatic polyester or degradation product thereof is bis(2-hydroxyethyl) terephthalate (BHET).

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