Plastic decomposition method and plastic biodegradability test method

Abstract

According to the present invention, a microorganism (e.g., Shwanela sp. BN-3 strain (accession number NITE BP-03961), etc.) belonging to the genus Shewanella and capable of decomposing a polyamide can be used for a plastic decomposition method and a plastic biodegradability test method both for a plastic containing a polyamide. By carrying out the above test method under low-temperature / high-pressure conditions, biodegradability in deep sea environments can be evaluated.

Description

Methods for decomposing plastics and testing methods for biodegradability of plastics

[0001] The present invention relates to a method for decomposing plastics and a method for testing the biodegradability of plastics, and more particularly to a method for decomposing plastics using microorganisms and a method for testing the biodegradability of plastics using microorganisms.

[0002] Against the backdrop of serious plastic pollution in the marine environment, the development of marine biodegradable plastics has been progressing in recent years. These marine biodegradability assessments are generally conducted using seawater-based test methods that simulate the ocean surface and coastal areas. However, it has become clear that a large amount of plastic waste that was released into the marine environment in the past has accumulated on the deep seafloor, but current test methods are not suitable for assessing biodegradability in deep-sea environments.

[0003] Patent Document 1 reports that novel microorganisms capable of degrading biodegradable plastics were discovered by culturing deep-sea sediments under low temperature and high pressure conditions in the presence of plastics. The paper then proposes a method for testing the biodegradability of biodegradable plastics, particularly in the deep sea, using these novel microorganisms belonging to the Moritella or Shewanella genera.

[0004] Japanese Patent Application Laid-Open No. 2010-200697

[0005] Patent Document 1 discloses that a novel microorganism is capable of biodegrading biodegradable plastics that have ester bonds as a repeating unit. However, it does not disclose the biodegradability of polyamide (nylon), a material used in fishing lines and nets that can have a negative impact on marine ecosystems due to ghost fishing and accidental ingestion. The present invention aims to provide a method for decomposing plastics, including polyamide-containing plastics, and a method for testing the biodegradability of plastics.

[0006] To solve the above problems, the present inventors isolated microorganisms capable of degrading plastics, including polyamides, using marine environment samples made from actual seawater and marine sediments, and discovered among them microorganisms that grow under low temperature and high pressure and have the ability to degrade plastics. Based on this finding, the present invention was completed. Specifically, the present invention is as follows.

[0007] <1> A method for decomposing plastics, comprising at least using a microorganism belonging to the genus Shewanella and having polyamide-degrading ability, wherein the plastic contains at least polyamide. <2> The method for decomposing plastics according to <1>, wherein the 16S rDNA of the microorganism contains a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1, or a polynucleotide having 90% or more identity to the base sequence shown in SEQ ID NO: 1.

[0008] <3> The method for decomposing plastics according to <1>, wherein the microorganism is Shewanella sp. strain BN-3 (accession number NITE BP-03961). <4> The method for decomposing plastics according to any one of <1> to <3>, which comprises contacting the microorganism with the plastic in water. <5> The method for decomposing plastics according to any one of <1> to <4>, which comprises contacting the microorganism with the plastic in water at 10°C or less. <6> The method for decomposing plastics according to any one of <1> to <5>, which comprises contacting the microorganism with the plastic in water under pressurized conditions. <7> The method for decomposing plastics according to any one of <1> to <6>, wherein the plastic contains an itaconic acid-derived polyamide.

[0009] <8> A method for testing the biodegradability of plastics, wherein the microorganism at least uses a microorganism belonging to the genus Shewanella and having the ability to decompose polyamide. <9> The test method according to <8>, wherein the 16S rDNA of the microorganism contains a polynucleotide consisting of the base sequence set forth in SEQ ID NO: 1, or a polynucleotide having 90% or more identity to the base sequence set forth in SEQ ID NO: 1. <10> The test method according to <8>, wherein the microorganism is Shewanella sp. BN-3 strain (accession number NITE BP-03961). <11> The test method according to any one of <8> to <10>, which includes contacting the microorganism with the plastic in water. <12> The test method according to any one of <8> to <11>, which includes contacting the microorganism with the plastic in water at 10°C or below. <13> The test method according to any one of <8> to <12>, which includes contacting the microorganism with the plastic in water under pressurized conditions. <14> The test method according to any one of <8> to <13>, which includes measuring at least one of the amount of carbon dioxide generated and the oxygen demand. <15> The test method according to any one of <8> to <14>, wherein the plastic contains an itaconic acid-derived polyamide.

[0010] <16> A method for decomposing plastics, comprising using at least a microorganism belonging to the genus Marinobacter or Sulfitobacter and having the ability to decompose polyamide, wherein the plastic contains at least polyamide. <17> The method for decomposing plastics according to <16>, wherein the microorganism belongs to the genus Marinobacter and whose 16S rDNA contains a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2, or a polynucleotide having 90% or more identity to the base sequence shown in SEQ ID NO: 2. <18> The method for decomposing plastics according to <17>, wherein the microorganism is Marinobacter sp. strain BN-1 (accession number NITE BP-03618). <19> The method for decomposing plastics according to <16>, wherein the microorganism belongs to the genus Sulfitobacter and the 16S rDNA contains a polynucleotide consisting of the base sequence shown in SEQ ID NO: 3, or a polynucleotide having 90% or more identity to the base sequence shown in SEQ ID NO: 3. <20> The method for decomposing plastics according to <19>, wherein the microorganism is Sulfitobacter sp. BN-2 strain (accession number NITE BP-03611).

[0011] The present invention provides a method for decomposing plastics, including polyamide-containing plastics, and a method for testing the biodegradability of plastics. By carrying out the above test method under low-temperature and high-pressure conditions, biodegradability in a deep-sea environment can be evaluated.

[0012] 1 shows the microbial community structure after cultivation under different temperature and pressure conditions in the presence of water-solubilized itaconic acid-derived polyamide. 2 shows the BOD test results when the BN-3 strain was cultivated alone in the presence of water-solubilized itaconic acid.

[0013] The present invention will be described in detail below. The following description of the components may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments.

[0014] <Method for Degrading Plastics> [Microorganisms] The method for degrading plastics of the present invention uses microorganisms. The inventors confirmed that water-solubilized itaconic acid-derived polyamides can be degraded in seawater, and from the microorganisms that accumulated during this process, they identified three strains of microorganisms with the ability to degrade water-solubilized itaconic acid-derived polyamides. As shown in the Examples below, these strains included a strain that could degrade water-solubilized itaconic acid-derived polyamides under low temperature and high pressure. Whole genome analysis of this strain identified three types of polyamide-degrading enzymes. The method for degrading plastics of the present invention uses one of these strains or at least the microorganisms with polyamide-degrading activity described below.

[0015] As used herein, decomposing a polyamide includes decomposing the polyamide into units with a lower degree of polymerization, decomposing the polyamide into monomers, and further decomposing the monomers (e.g., into carbon dioxide). Using the method of the present invention, polyamides can be decomposed into units with lower molecular weights than the monomers.

[0016] One of the strains found by the present inventors (strain BN-3) had a 16S rDNA sequence shown in SEQ ID NO: 1. This sequence was 99.93% identical to the 16S rDNA sequence of Shewanella vesiculosa strain M7 of the class Gammaproteobacteria, and it was found to be classified into the genus Shewanella. The BN-3 strain was deposited on August 8, 2023, under the accession number NITE AP-03961, at the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan) by the Japan Agency for Marine-Earth Science and Technology (2-5 Natsushima-cho, Yokosuka City, Kanagawa Prefecture, Japan), and was requested to be transferred to an international deposit under the Budapest Treaty on May 23, 2024 (accession number NITE BP-03961).

[0017] Another strain discovered by the present inventors (BN-1 strain) has a 16S rDNA base sequence shown in SEQ ID NO: 2 in the sequence listing, and phylogenetic analysis using 16S rDNA has revealed that it is classified as a member of the genus Marinobacter. This strain was deposited by the Japan Agency for Marine-Earth Science and Technology (JAMSTEC) at the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan) on March 2, 2022, under the accession number NITE P-03618, and was requested to be transferred to an international deposit under the Budapest Treaty on May 23, 2024 (accession number NITE BP-03618).

[0018] The remaining strain discovered by the inventors (BN-2 strain) has a 16S rDNA base sequence shown in the sequence number in the sequence listing, and phylogenetic analysis using 16S rDNA has revealed that it is classified as a member of the genus Sulfitobacter. This strain was deposited on February 21, 2022, under the accession number NITE P-03611, at the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan) by the Japan Agency for Marine-Earth Science and Technology, and was requested to be transferred to an international deposit under the Budapest Treaty on May 23, 2024 (accession number NITE BP-03611).

[0019] The above-mentioned microorganisms, BN-1, BN-2, and BN-3 strains, are all bacteria with the following chemical properties: - They form white colonies on Marine Agar 2216 plates (described below). - They can grow using water-solubilized itaconic acid-derived polyamide as the sole energy and carbon source. - The culture conditions are as follows: Culture medium: Heterotrophic medium with a seawater salt concentration, such as Marine Broth 2216 medium (described below). Culture temperature: 25-30°C. Culture period: 3 days. Culture method: Aerobic shaking culture is suitable.

[0020] Microorganisms that can be used in the polyamide degradation method include microorganisms other than the above-mentioned deposited strains that have polyamide-degrading activity. In particular, microorganisms belonging to the genus Shewanella that have polyamide-degrading activity, or microorganisms classified as Marinobacter or Sulfitobacter that have polyamide-degrading activity can be used. Microorganisms classified as Shewanella, Marinobacter, or Sulfitobacter can be identified by methods known in the art, such as morphological observation and 16S rDNA analysis. Polyamide-degrading ability can be confirmed by growth in a medium containing a polyamide, such as a water-solubilized itaconic acid-derived polyamide, as the sole energy and carbon source. In explaining the present invention and embodiments of the present invention, the above-mentioned deposited strains may be used as examples, but the explanations also apply to cases where other strains classified into the genus Shewanella, Marinobacter, or Sulfitobacter are used.

[0021] In a preferred embodiment, a microorganism belonging to the genus Shewanella and having a 16S rDNA consisting of the following (a1) or (b1): (a1) a polynucleotide consisting of the sequence set forth in SEQ ID NO: 1, or (b1) a polynucleotide consisting of a sequence highly identical to the sequence set forth in SEQ ID NO: 1 can be used.

[0022] In another preferred embodiment, a microorganism belonging to the genus Marinobacter and having a 16S rDNA consisting of the following (aa1) or (bb1): (aa1) a polynucleotide consisting of the sequence set forth in SEQ ID NO: 2, (bb1) a polynucleotide consisting of a sequence highly identical to the sequence set forth in SEQ ID NO: 2, or a microorganism belonging to the genus Sulfitobacter and having a 16S rDNA consisting of the following (aa2) or (bb2): (aa2) a polynucleotide consisting of the sequence set forth in SEQ ID NO: 3, (bb2) a polynucleotide consisting of a sequence highly identical to the sequence set forth in SEQ ID NO: 3 can be used.

[0023] High identity means, for example, 90% or more, preferably 95% or more, more preferably 99.0% or more, even more preferably 99.40% or more, particularly preferably 99.70% or more, and most preferably 99.80% or more.

[0024] With respect to base sequences (sometimes simply referred to as sequences), unless otherwise specified, identity refers to the percentage of matching nucleotides shared between two sequences when the two sequences are optimally aligned. Percent identity can be calculated by (number of matching positions / total number of positions) x 100, and can be calculated using commercially available algorithms. Such calculations can also be performed using algorithms or programs well known to those skilled in the art (e.g., BLASTN, BLASTX, ClustalW). When using a program, parameters can be appropriately set by those skilled in the art, or the default parameters of each program can be used. Specific techniques for these analysis methods are also well known to those skilled in the art.

[0025] The microorganisms used in the plastic degradation method of the present invention may consist of multiple microorganisms. For example, the inoculum for plastic biodegradation may contain at least the above-mentioned microorganisms belonging to the genus Shewanella, Marinobacter, or Sulfitobacter and having polyamide-degrading ability. Furthermore, two or more strains of the above-mentioned microorganisms belonging to the genus Shewanella, Marinobacter, or Sulfitobacter and having polyamide-degrading ability may be used simultaneously or in combination, as long as they do not destroy each other's activity. Furthermore, any one or more of the above-mentioned microorganisms belonging to the genus Shewanella, Marinobacter, or Sulfitobacter and having polyamide-degrading ability may be used simultaneously or in combination with other microorganisms, as long as they do not destroy the activity of the microorganism. As the other microorganism, it is preferable to use, for example, a microorganism known to have plastic biodegrading activity. The plastic degradation method of the present invention may also include a plastic degradation reaction using other microorganisms. For example, a plastic degradation reaction by a microorganism belonging to the genus Shewanella, Marinobacter, or Sulfitobacter and having polyamide decomposition ability may be carried out before or after the plastic degradation reaction by another microorganism.

[0026] [Plastics] Plastics that can be decomposed by the plastic decomposition method of the present invention are plastics containing at least polyamide. Polyamides are polymers that have at least a portion where monomer units are bonded by an amide bond, and there are no particular limitations on the molecular weight. Polyamides are generally called nylons. Plastics may also contain polymers that have other bonds, such as ester bonds, in addition to amide bonds. Furthermore, plastics may contain polyamides and other polymers, such as polyesters. Plastics may also be composite materials containing inorganic particles other than polymers.

[0027] The plastics to which the plastic decomposition method of the present invention is applied preferably contain polyamides whose biodegradability has already been confirmed. Examples of polyamides whose biodegradability has been confirmed include polyamide 4, polyamide 6, polyamide 66, and itaconic acid-derived polyamides. The plastics to which the plastic decomposition method of the present invention is applied particularly preferably contain itaconic acid-derived polyamides.

[0028] [Itaconic Acid-Derived Polyamide] Itaconic acid-derived polyamide is a polyamide having a structure obtained by a dehydration condensation reaction (polymerization) of itaconic acid and a diamine. Itaconic acid-derived polyamide can be produced, for example, by using itaconic acid as a dicarboxylic acid and reacting it with a diamine compound.

[0029] Itaconic acid is a type of aliphatic dicarboxylic acid represented by the following formula:

[0030] Itaconic acid can be obtained by thermal decomposition of citric acid. Itaconic acid can also be produced by fermentation using fungi, and bio-derived itaconic acid is supplied stably and relatively inexpensively on an industrial scale. The polyamide decomposed by the method of the present invention does not necessarily have to use bio-derived itaconic acid. However, using bio-derived itaconic acid can realize so-called bio-based engineering plastics, which meet social demands such as long-term fixation of carbon dioxide. Itaconic acid-derived polyamides produced using bio-derived itaconic acid are sometimes called bio-nylons.

[0031] The diamine compound used in the production of itaconic acid-derived polyamides is not particularly limited and can be selected depending on the polymer application. Furthermore, the diamine compound used in the production of itaconic acid-derived polyamides may be a single compound or a mixture of multiple compounds. Examples of diamine compounds include aliphatic diamines and aromatic diamines. The aliphatic diamine may be either a linear alkylenediamine or a branched alkylenediamine, with linear alkylenediamines being preferred. The aliphatic diamine preferably has 2 to 12 carbon atoms, more preferably 2 to 8, and even more preferably 2 to 6, with linear alkylenediamines having 2 to 6 carbon atoms being particularly preferred. Specific examples include hexamethylenediamine, pentamethylenediamine, tetramethylenediamine, trimethylenediamine, and ethylenediamine. Examples of aromatic diamines include p-phenylenediamine, m-phenylenediamine, p-xylylenediamine, m-xylylenediamine, and 4,4'-diaminodiphenyl ether. As the diamine compound, an aliphatic diamine is preferred, and hexamethylenediamine (1,6-diaminohexane) is more preferred.

[0032] Itaconic acid-derived polyamides are polymers that can be produced by polymerizing (dehydration condensing) itaconic acid and a diamine compound in a 1:1 (molar ratio) and linking them through amide bonds. When the diamine compound is composed of multiple diamine compounds, it may be produced in the form of a random copolymer, an alternating copolymer, or a block copolymer. For specific production methods, reference can be made to known production methods for polyamides (nylons).

[0033] The itaconic acid-derived polyamide preferably has a structure in which a pyrrolidone ring (5-membered ring) is formed by a dehydration condensation reaction of the imine produced by aza-Michael addition of the amino group of a diamine compound to the vinyl group of itaconic acid with the carboxyl group farther from the double bond. For example, the method described in JP 2012-107122 A can be referenced as a method for producing such an itaconic acid-derived polyamide. Specifically, the itaconic acid-derived polyamide preferably has a structure represented by the following formula (1):

[0034]

[0035] In formula (1), m is an integer of 2 or greater indicating the degree of polymerization of the polymer, and is, for example, 5,000 or less, preferably 1,000 or less, and more preferably 50 or less. It is preferable that the value of m in a water-solubilized itaconic acid-derived polyamide is small. By water-solubilization, as described below, the value of m in an itaconic acid-derived polyamide represented by formula (1) can be further reduced. For example, in an itaconic acid-derived polyamide before water-solubilization having an m value of 21 or greater, the value of m can be reduced to 20 or less by water-solubilization, and preferably, in an itaconic acid-derived polyamide before water-solubilization having an m value of 25 or greater, the value of m can be reduced to 10 or less by water-solubilization.

[0036] L is a divalent linking group derived from the linking group between two amino groups of a diamine compound. L preferably has any of the following structures, and the m Ls may be the same or different from one another.

[0037]

[0038] In the above formula, n is a natural number, preferably 2 to 12, more preferably 2 to 8, and even more preferably 2 to 6.

[0039] The cyclic amide structure (pyrrolidone ring structure) of itaconic acid-derived polyamides reduces moisture absorption and improves their performance as engineering plastics. Therefore, establishing a method for decomposing itaconic acid-derived polyamides under environmental conditions will enable industrially practical bioremediation of plastics.

[0040] The molecular weight of the itaconic acid-derived polyamide is not particularly limited. For example, the weight-average molecular weight Mw is about 400 to 1,000,000, preferably about 600 to 500,000, and more preferably about 600 to 100,000, and the number-average molecular weight Mn is about 400 to 800,000, preferably about 600 to 500,000, and more preferably about 600 to 300,000. The molecular weight of the water-solubilized itaconic acid-derived polyamide is preferably a weight-average molecular weight Mw of about 400 to 10,000, preferably about 400 to 5,000, and more preferably about 400 to 2,000. The molecular weight can be further reduced by water solubilization. For example, an itaconic acid-derived polyamide having a weight-average molecular weight of 4,000 or more before water solubilization can be reduced to a weight-average molecular weight of less than 4,000, preferably, for example, about 200 to 2,000, by water solubilization.

[0041] The itaconic acid-derived polyamide may be a copolymer produced using other carboxylic acids in addition to itaconic acid as raw materials. Examples of such other carboxylic acids include tetradecanedioic acid. The amount of such other carboxylic acids may be less than 50% by mass, preferably 20% by mass or less, more preferably 10% by mass or less, and even more preferably 5% by mass or less, relative to the total mass of the itaconic acid raw material. The itaconic acid-derived polyamide may also be produced by incorporating other monomers. Examples of such other monomers include aminocarboxylic acids such as 11-aminoundencanoic acid. The amount of such other monomers may be less than 50% by mass, preferably 20% by mass or less, more preferably 10% by mass or less, and even more preferably 5% by mass or less, relative to the total mass of the monomers having the unit structure represented by formula (1). Furthermore, the itaconic acid-derived polyamide may be a composite formed with montmorillonite, titanium oxide, or the like.

[0042] [Water-Solubilized Itaconic Acid-Derived Polyamide] The itaconic acid-derived polyamide is preferably water-soluble. The water-solubilization of the itaconic acid-derived polyamide is promoted, for example, by low-molecular-weight conversion (oligomerization). Water-solubilization is also promoted by the ring-opening reaction of at least some of the pyrrolidone rings in the structure to generate carboxyl groups and additional secondary amino groups. The ring-opening reaction should occur to an extent sufficient to water-solubilize the itaconic acid-derived polyamide.

[0043] The water-solubilized itaconic acid-derived polyamide can be obtained by exposing the itaconic acid-derived polyamide to ultraviolet irradiation or alkaline conditions. For example, the water-solubilized itaconic acid-derived polyamide can be obtained by exposing the itaconic acid-derived polyamide to sunlight (natural light) containing ultraviolet rays. The water-solubilized itaconic acid-derived polyamide may also be one that has been water-solubilized by a physicochemical means such as pressure application.

[0044] In a preferred embodiment, the water-solubilized itaconic acid-derived polyamide degraded by the microorganism of the present invention is an oligomer (itaconic acid-derived polyamide oligomer). The itaconic acid-derived polyamide oligomer is typically an oligomer (dimer to 20-mer) in which m is 2 to 20 in the above formula (1), more preferably an oligomer (dimer to 15-mer) in which m is 2 to 15, even more preferably an oligomer (trimer to 10-mer) in which m is 3 to 10, and most preferably an oligomer (dimer to octamer) in which m is 3 to 8. As described above, in each oligomer represented by formula (1), the pyrrolidone ring structure in some or all of the structural units may be ring-opened.

[0045] Itaconic acid-derived polyamide oligomers can be obtained by the partial hydrolysis of itaconic acid-derived polyamides as described above. Like itaconic acid-derived polyamides, itaconic acid-derived polyamide oligomers can also be produced by a dehydration condensation reaction between itaconic acid and a diamine compound. Therefore, water-solubilized itaconic acid-derived polyamide oligomers can also be produced as a by-product in the production process of itaconic acid-derived polyamides. Therefore, the decomposition method of the present invention can also be used to treat wastewater containing oligomers produced during the production process of itaconic acid-derived polyamides.

[0046] [Method for Decomposing Plastics] In the method for decomposing plastics of the present invention, the decomposition reaction is promoted by bringing plastics and microorganisms into contact with each other. To bring plastics and microorganisms into contact with each other, for example, they may be allowed to coexist in water. It is particularly preferable to bring them into contact with each other in seawater. The seawater may be natural seawater or saltwater artificially prepared to have a composition similar to that of seawater.

[0047] The water in which plastics and microorganisms coexist may contain one or more other substances that are preferably present to promote the decomposition of plastics or for the growth and proliferation of microorganisms. Such substances include nitrogen sources and phosphorus sources. Examples of nitrogen sources include ammonium chloride and yeast extract. Examples of phosphorus sources include disodium hydrogen phosphate (Na ２ HPO ４ ) and potassium dihydrogen phosphate (KH ２ P.O. ４ ) etc.

[0048] Plastics may be subjected to the decomposition method in any form as long as they are in a form that allows them to come into contact with microorganisms. For example, plastics may be in the form of a powder or thin film to promote the decomposition reaction. Furthermore, plastics that become water-soluble when exposed to light or heat may be brought into contact with microorganisms in a water-soluble form. For example, itaconic acid-derived polyamides may be exposed to ultraviolet light in water to become water-soluble itaconic acid-derived polyamides as described above. Water-solubilization makes them more susceptible to decomposition by microorganisms.

[0049] The concentration of the microorganisms during the decomposition reaction (initial concentration) is not particularly limited as long as it is a concentration that maintains the plastic biodegradation activity. ４ The time required for decomposition can be adjusted by adjusting the initial concentration of the microorganisms.

[0050] The temperature conditions in the plastic decomposition method of the present invention are not particularly limited as long as the polyamide decomposition activity of the microorganism is maintained, and can be used within a range of, for example, −10° C. to 45° C., preferably 0° C. to 30° C. The microorganisms of the genus Shewanella that are used in the method of the present invention and have the ability to decompose polyamide are active even at low temperatures, and can be preferably used at, for example, 0° C. to 10° C.

[0051] The pressure conditions for the plastic decomposition method of the present invention are not particularly limited, and may be, for example, 0.1 MPa to 45 MPa. The microorganisms of the genus Shewanella that are used in the method of the present invention and have the ability to decompose polyamides are active even under pressurized conditions, and can be preferably used, for example, under conditions of 10 MPa to 40 MPa.

[0052] The plastic degradation method of the present invention can be carried out in a marine environment. Furthermore, when a microorganism belonging to the genus Shewanella and having the ability to decompose polyamide is used, the method can be carried out under the same low-temperature, high-pressure conditions as those found in the deep sea (e.g., 0°C to 10°C, 10 MPa to 40 MPa), as described above. Therefore, the method of the present invention can promote the biodegradation of plastics under conditions that mimic the marine environment, particularly the low-temperature, high-pressure conditions found in the deep sea.

[0053] The time required for the plastic decomposition method of the present invention is not particularly limited and can be, for example, 30 minutes to several years. The time required for decomposition can be adjusted by adjusting the initial concentration of the microorganisms. At the laboratory level, it is preferable to adjust the initial concentration of the microorganisms and the amount of plastic so that decomposition is completed within 12 hours to 1 month, preferably 1 day to 1 week. Although the method of the present invention uses microorganisms found in seawater, by artificially using microorganisms at higher concentrations than those found in natural environments, plastics can be decomposed in a shorter time, making industrial applications possible.

[0054] In the plastic decomposition method of the present invention, it is preferable to expose the plastic to ultraviolet light in water when or before contacting the plastic with the microorganism in seawater. For example, the polyamide decomposition method of the present invention is preferably carried out in an environment where it can be irradiated with light such as sunlight.

[0055] <Testing Method for Biodegradability of Plastics> The plastic decomposition method of the present invention can be applied to testing the biodegradability of plastics. The biodegradability of plastics can be evaluated by placing the above-mentioned Shewanella microorganisms capable of decomposing polyamide and plastic in an environment in which the microorganisms can exert their polyamide decomposition ability, and then measuring the degree of decomposition of the plastic.

[0056] Specifically, microorganisms are brought into contact with a plastic sample in seawater under the conditions described in the plastic decomposition method, and at least one of the amount of carbon dioxide generated and the oxygen demand is measured, and the measured value can be used as an index of the biodegradability of the plastic. In other words, the greater the amount of carbon dioxide generated or the oxygen demand, the higher the evaluation of biodegradability.

[0057] Preferably, the sample plastic is in the form of a fine powder or film, or includes a step of rendering it into such a form before contacting it with the microorganisms.

[0058] For specific procedures for testing the biodegradability of plastics, please refer to standards related to the marine biodegradability of plastics (e.g., ISO 18830, ISO 196790, ISO 22404, ISO 23799-1, 2).

[0059] By mixing a plastic sample with microorganisms belonging to the genus Shewanella and capable of degrading polyamides under low temperature and high pressure, the marine biodegradability of plastics in the deep sea can be evaluated. For example, a pressure of 10 MPa can simulate deep sea conditions equivalent to a depth of 1,000 m, a pressure of 20 MPa equivalent to a depth of 2,000 m, a pressure of 30 MPa equivalent to a depth of 3,000 m, and a pressure of 40 MPa equivalent to a depth of 4,000 m. Therefore, the plastic biodegradability testing method of the present invention, which uses microorganisms belonging to the genus Shewanella and capable of degrading polyamides, can replace deep sea installation experiments, which have previously been limited in practice due to the enormous cost and system difficulties.

[0060] The present invention will be described in more detail below with reference to examples. The materials, reagents, amounts and proportions of substances, procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention is not limited to the following examples.

[0061] In the examples, the itaconic acid-derived polyamide used was an itaconic acid-derived polyamide (solid) having a weight-average molecular weight of approximately 6,000, which was produced from itaconic acid and hexamethylenediamine as raw materials by the method described in JP 2012-107122 A. The water-solubilized itaconic acid-derived polyamide was prepared by irradiating the itaconic acid-derived polyamide with ultraviolet light (250 to 400 nm, 150 mW / cm) in pure water. ２ ) was artificially irradiated and gelled.

[0062] The media used are as follows: Culture medium 1: Marine Broth 2216 (1.6 mg / L ammonium nitrate, 22.0 mg / L boric acid, 1.8 g / L calcium chloride, 8.0 mg / L disodium phosphate, 0.1 g / L ferrous citrate, 5.9 g / L magnesium chloride, 3.24 g / L magnesium sulfate, 5.0 g / L peptone, 0.08 g / L potassium bromide, 0.55 g / L potassium chloride, 0.16 g / L sodium carbonate, 19.45 g / L sodium chloride, 2.4 mg / L sodium fluoride, 4.0 mg / L sodium silicate, 34.0 mg / L strontium chloride, 1.0 g / L dry yeast extract, pH 7.6 ± 0.2). Culture medium 2: Itaconic acid-derived polyamide medium (2.0 g / L water-solubilized itaconic acid-derived polyamide, 7.7 mg / L potassium dihydrogen phosphate, 200 mg / L ammonium chloride, 100 mg / L sodium nitrate, 33 g / L Tetramarin Salt Pro) Plate medium for pure isolation: Itaconic acid-derived polyamide plates (2.0 g / L water-solubilized itaconic acid-derived polyamide, 7.7 mg / L potassium dihydrogen phosphate, 200 mg / L ammonium chloride, 100 mg / L sodium nitrate, 33 g / L Tetramarin Salt Pro, 0.4 g / L gellan gum)

[0063] Seawater used: Yokosuka seawater, Kanagawa Prefecture. Sediment used: Lake Nakaumi sediment, Shimane Prefecture.

[0064] (1) BOD test A pressure sensor type BOD meter (OxiTop IDS) was set in a 250 mL medium bottle. 50 mL of seawater was diluted with inorganic salt (final concentration 7.7 mg / L potassium dihydrogen phosphate (KH ２ P.O. ４ ), 200 mg / L ammonium chloride (NH ４ Cl), 100 mg / L sodium nitrate (NaNO ３) and 5 g of sediment to activate microbial activity were added to prepare a test solution. 0.5 g of water-solubilized itaconic acid-derived polyamide was added to prepare a polyamide test solution, and a control solution without water-solubilized itaconic acid-derived polyamide was prepared. 0.4 g of Acoma Lime Zero was placed in the bottle top holder as an adsorbent for carbon dioxide generated in the gas phase. A triangular PTFE rotor was inserted to agitate the bottle, and the mixture was continuously stirred at 180 rpm and cultured at 25°C for 14 days. BOD measurements were taken every 28 minutes.

[0065] Test solutions were collected from test plots where the BOD value had increased significantly by this method, and isolation and culture of itaconic acid-derived polyamide-decomposing microorganisms was carried out.

[0066] (2) Isolation and Cultivation of Itaconic Acid-Derived Polyamide-Degrading Microorganisms After the BOD test, 100 μL of the test solution was applied to an itaconic acid-derived polyamide plate and subjected to static incubation at 25°C. After approximately one week, multiple colonies formed on the plate. The only organic nutrient in this medium was itaconic acid-derived polyamide, and these colonies were purified as microorganisms capable of degrading itaconic acid-derived polyamide. The colonies formed on the plate were picked up using a disposable loop and inoculated again onto an itaconic acid-derived polyamide plate by streaking and subjected to static incubation at 25°C. The obtained colonies were inoculated into 5 mL of Marine Broth 2216 medium and incubated with shaking at 25°C for three days. Glycerol was added to a final concentration of 20% and the medium was stored at -80°C.

[0067] Microbial cells were collected from 2 mL of culture medium by centrifugation (12,000 × g, 10 minutes), and DNA was extracted using a commercially available kit. PCR was performed using the 27F (AGAGTTTGATCMTGGCTCAG: SEQ ID NO: 4) / 1492R (GGYTACCTTGTTACGACTT: SEQ ID NO: 5) primer set, and the purified PCR product was sequenced by the Sanger method. The resulting sequence was assembled into a consensus sequence, which was used for a BLAST homology search against the international nucleotide sequence databases (DDBJ / ENA(EMBL) / GenBank).

[0068] Based on 16S rDNA analysis, three strains of different taxonomic classifications were identified as polyamide-degrading microorganisms. One of the strains showed a 99% identity with Gammaproteobacteria species Marinobacter hydrocarbonoclasticus ATCC 49840, and was therefore designated Marinobacter sp. BN-1 and deposited at the National Institute of Technology and Evaluation (NIET) Patent Microorganisms Depositary (Accession Number NITE BP-03618). The other strain showed a 99% identity with Alphaproteobacteria species Sulfitobacter dubius KMM 3554, and was therefore designated Sulfitobacter sp. BN-2 and deposited at the National Institute of Technology and Evaluation (NIET) Patent Microorganisms Depositary (Accession Number NITE BP-03611). Furthermore, the other strain, BN-3, showed 99.93% identity with the Shewanella vesiculosa M7 strain of the Gammaproteobacteria class, and was therefore designated Shewanella sp. BN-3 strain. A glycerol stock was prepared and deposited at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (Accession Number NITE BP-03961).

[0069]

[0070] (3) Whole Genome Analysis. For each strain, microbial cells were collected from 2 mL of culture medium by centrifugation (12,000 × g, 10 minutes). DNA was extracted using a commercially available kit capable of extracting long-chain DNA (Quick-DNA HMW MagBead Kit). Short-read analysis was performed using Illumina's MiSeq and long-read analysis was performed using Oxford Nanopore's MinION. The resulting FASTQ data was used for de novo assembly using a UniCycler to determine the draft whole genome. Annotation was performed using DFAST. For strain BN-3, 4,639,713 base pairs consisting of nine contigs were determined. 3,909 genes were predicted for strain BN-3, and putative genes encoding three polyamide degrading enzymes (NylA [cyclic dimer degrading enzyme], NylB [exo-degrading enzyme], and NylC [endo-degrading enzyme]) were identified (Table 2). These polyamide-degrading enzymes are characterized by their ability to degrade polyamide 4 and polyamide 6, and are capable of degrading many polyamide compounds, not just water-solubilized itaconic acid polyamides. Putative genes encoding the same three types of polyamide-degrading enzymes were identified for the BN-2 strain, and putative genes encoding two types of polyamide-degrading enzymes were identified for the BN-1 strain.

[0071]

[0072] (4) Pressure culture test: 5 mL of seawater was added with an inorganic salt (final concentration 7.7 mg / L potassium dihydrogen phosphate (KH ２ P.O. ４ ), 200 mg / L ammonium chloride (NH ４ Cl), 100 mg / L sodium nitrate (NaNO ３) and 5 g of Nakaumi sediment and 0.05 g of water-solubilized itaconic acid-derived polyamide were added to prepare a test solution, which was placed in a 5 mL tube with the cap removed and sealed with self-adhesive silicone tape. The tube was placed in a pressure-resistant container capable of pressurizing up to 100 MPa, and pressurized to the desired pressure using ultrapure water and a hand pump. The pressure conditions tested were 0.1 (atmospheric pressure), 10, 20, 30, 40, and 50 MPa. For cultures at 25°C, pressurization was performed using room-temperature ultrapure water, while for cultures at 4°C, pressurization was performed using ultrapure water that had been cooled in ice water, with the pressurization device placed in ice water. Each pressurization container was placed in an incubator at 25°C or 4°C, and static culture was performed.

[0073] After incubation, the pressure was slowly reduced, and the 5 mL tube was removed from the pressure culture system. DNA was extracted from the contents using the same method as described above, and microbial community structure analysis was performed. Water samples were filtered through a 0.22 μm membrane filter to recover microbial cells. The membrane filter was then cut into 1-2 mm cubes and placed in a cell disruption tube. Approximately 0.5 g of sediment sample was weighed and placed in a cell disruption tube. DNA was extracted using a commercially available kit (FAST DNA SPIN Kit for SOIL). Using the extracted DNA as a template, the 16S rRNA gene (16S rDNA) was partially amplified by PCR using the U530F (GTGCCAGCMGCCGCGG: SEQ ID NO: 6) / U907R (CCGTCAATTCMTTTRAGTTT: SEQ ID NO: 7) primer set targeting bacteria and archaea. The nucleotide sequence was then determined using the MiSeq Reagent Kit v3 and a MiSeq next-generation sequencer. The data obtained were analyzed using the QIIME2 pipeline to estimate the relative abundance of microbial species in each sample. The results are shown in Figure 1.

[0074] As shown in Figure 1, the microbial community structure varied depending on temperature and pressure. At room temperature and atmospheric pressure, bacteria of the genus Marinobacter, which degrades itaconic acid-derived polyamides, enriched. At low temperature and atmospheric pressure, bacteria of the genus Sulfitobacter, a different type of itaconic acid-derived polyamide-degrading microorganism, enriched. At low temperature and atmospheric pressure, bacteria of the genus Shewanella, isolated in the previous section, enriched. In this pressurized culture, itaconic acid-derived polyamides were added as the sole carbon source, and these enriched microorganisms are thought to have enriched by assimilating itaconic acid-derived polyamides. Therefore, these results demonstrate that Shewanella bacteria can degrade itaconic acid-derived polyamides at low temperature and high pressure. Using Shewanella bacteria, it is possible to evaluate the marine biodegradability of plastics in the deep sea.

[0075] (5) BOD Test Using Shewanella sp. BN-3 Strain To evaluate the ability of Shewanella sp. BN-3 strain alone to degrade itaconic acid-derived polyamides, a BOD test was conducted using this bacterium and itaconic acid-derived polyamides. Shewanella sp. BN-3 strain was cultured in 50 mL of Marine Broth 2216 medium at 25°C for two nights with shaking, and the cells were harvested by centrifugation (12,000 × g, 10 min). The cells were washed three times with artificial seawater, and the washed cells were suspended in artificial seawater containing 0.1 g of water-solubilized itaconic acid-derived polyamide. The test was conducted using a pressure sensor-type BOD meter (OxiTop IDS). The results are shown in Figure 2. The BOD value did not increase in the test area where the washed cells were not suspended, but the BOD value increased rapidly from the start of the test only in the test area where the washed cells were suspended. That is, Shewanella sp. The BN-3 strain is capable of aerobic respiration using itaconic acid-derived polyamides as the sole carbon source.

Claims

1. A method for decomposing plastics, comprising using at least a microorganism belonging to the genus Shewanella and having polyamide-decomposing ability, wherein the plastics contain at least polyamide.

2. The method for decomposing plastics according to claim 1, wherein the 16S rDNA of the microorganism contains a polynucleotide consisting of the base sequence shown in SEQ ID NO:1, or a polynucleotide having 90% or more identity to the base sequence shown in SEQ ID NO:

1.

3. The method for decomposing plastics according to claim 1, wherein the microorganism is Shewanella sp. BN-3 strain (accession number NITE BP-03961).

4. The method for decomposing plastic according to claim 1, which comprises contacting said microorganisms with said plastic in water.

5. A method for decomposing plastics according to claim 1, comprising contacting said microorganisms with said plastics in water at or below 10°C.

6. A method for decomposing plastics according to any one of claims 1 to 5, comprising contacting the microorganisms with the plastics in water under pressurized conditions.

7. A method for decomposing plastics according to any one of claims 1 to 5, wherein the plastics contain polyamide derived from itaconic acid.

8. A method for testing the biodegradability of plastics, which uses at least a microorganism belonging to the genus Shewanella and having the ability to decompose polyamide.

9. The testing method according to claim 8, wherein the 16S rDNA of the microorganism comprises a polynucleotide consisting of the base sequence shown in SEQ ID NO:1, or a polynucleotide having 90% or more identity to the base sequence shown in SEQ ID NO:

1.

10. The testing method according to claim 8, wherein the microorganism is Shewanella sp. BN-3 strain (accession number NITE BP-03961).

11. The test method of claim 8, comprising contacting said microorganism with said plastic in water.

12. The test method of claim 8, which comprises contacting the microorganism with the plastic in water at or below 10°C.

13. The test method of claim 8, comprising contacting said microorganism with said plastic in water under pressurized conditions.

14. The test method according to any one of claims 8 to 13, comprising measuring at least one of the amount of carbon dioxide produced and the amount of oxygen demand.

15. The test method according to any one of claims 8 to 13, wherein the plastic comprises an itaconic acid-derived polyamide.

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