Plastic decomposition method and plastic biodegradability test method

Abstract

To provide a plastic decomposing method that is applied to plastic containing polyamide, as well as a plastic biodegradability test method.SOLUTION: The present invention provides a method for decomposing plastic containing polyamide, using a microorganism (e.g., Shwanela sp. BN-3 strain (accession number NITE AP-03961)) belonging to the genus Shewanella and capable of decomposing polyamide, and a plastic biodegradability test method using the microorganism. By carrying out the test method under low-temperature and high-pressure conditions, biodegradability in deep sea environments can be evaluated.SELECTED DRAWING: None

Description

[Technical field]

[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. [Background technology]

[0002] In recent years, the development of marine biodegradable plastics has been progressing against the backdrop of serious plastic pollution in the marine environment. The marine biodegradability of these plastics is generally evaluated using test methods that use seawater and are designed for the ocean surface and coastal areas. On the other hand, it has been discovered that a large amount of plastic waste that was discharged into the marine environment in the past has accumulated on the deep sea floor, but current test methods are not suitable for evaluating biodegradability in a deep sea environment.

[0003] Patent Document 1 reports that novel microorganisms capable of decomposing biodegradable plastics were discovered by culturing deep-sea sediments under low temperature and high pressure conditions in the presence of plastics. It also proposes a method for testing the biodegradability of biodegradable plastics, particularly in the deep sea, using the novel microorganisms belonging to the Moritella or Shewanella genus. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2010-200697 A Summary of the Invention [Problem to be solved by the invention]

[0005] Patent Document 1 shows that a novel microorganism has the biodegradability of biodegradable plastics that have ester bonds as a repeating structure. However, there is no disclosure about the biodegradability of polyamide (nylon), which is a material for fishing lines and fishing nets that have a negative impact on the marine ecosystem due to ghost fishing and accidental ingestion. The objective of the present invention is to provide a method for decomposing plastics, including plastics containing polyamide, and a method for testing the biodegradability of plastics. [Means for solving the problem]

[0006] In order to solve the above problems, the present inventors isolated microorganisms capable of decomposing plastics including polyamides using marine environment samples made of actual seawater and marine sediments, and among them, found microorganisms that grow under low temperature and high pressure and have the ability to decompose 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 the steps of: At least a microorganism belonging to the genus Shewanella and having polyamide decomposition ability is used, A method for decomposing plastics, wherein the plastics contain at least polyamide. <2> The 16S rDNA of the above microorganisms A polynucleotide consisting of the base sequence shown in SEQ ID NO: 1, or A polynucleotide having 90% or more identity with the base sequence shown in SEQ ID NO:1. Includes <1> The method for decomposing plastics described in the above.

[0008] <3> The above microorganism is Shewanella sp. BN-3 strain (accession number NITE AP-03961). <1> The method for decomposing plastics described in the above. <4> contacting said microorganism with said plastic in water. <1> ~ <3> 13. The method for decomposing plastics according to any one of the preceding claims. <5> contacting the microorganism with the plastic in water at or below 10°C. <1> ~ <4> 13. The method for decomposing plastics according to any one of the preceding claims. <6> contacting said microorganism with said plastic in water under pressurized conditions. <1> ~ <5> 13. The method for decomposing plastics according to any one of the preceding claims. <7> The plastic contains an itaconic acid-derived polyamide. <1> ~ <6> 13. The method for decomposing plastics according to any one of the preceding claims.

[0009] <8> A method for testing the biodegradability of plastics, comprising the steps of: The test method uses at least the microorganism belonging to the genus Shewanella and having polyamide decomposition ability. <9> The 16S rDNA of the above microorganisms A polynucleotide consisting of the base sequence shown in SEQ ID NO: 1, or A polynucleotide having 90% or more identity with the base sequence shown in SEQ ID NO:1. Includes <8> The test method described in. <10> The above microorganism is Shewanella sp. BN-3 strain (accession number NITE AP-03961). <8> The test method described in. <11> contacting said microorganism with said plastic in water. <8> ~ <10> 3. The test method according to claim 1 , <12> contacting the microorganism with the plastic in water at or below 10°C. <8> ~ <11> 3. The test method according to claim 1 , <13> contacting said microorganism with said plastic in water under pressurized conditions. <8> ~ <12> 3. The test method according to claim 1 , <14> Measuring at least one of the amount of carbon dioxide produced and the amount of oxygen demand. <8> ~ <13> 3. The test method according to claim 1 , <15> The plastic contains polyamide derived from itaconic acid <8> ~ <14> 3. The test method according to claim 1 ,

[0010] <16> A method for decomposing plastics, comprising the steps of: At least a microorganism belonging to the genus Marinobacter or Sulfitobacter and having polyamide decomposition ability is used, A method for decomposing plastics, wherein the plastics contain at least polyamide. <17> The above microorganisms belong to the genus Marinobacter and have 16S rDNA A polynucleotide consisting of the base sequence shown in SEQ ID NO: 2, or A polynucleotide having 90% or more identity with the base sequence shown in SEQ ID NO:2 Includes <16> The method for decomposing plastics described in the above. <18> The above microorganism is Marinobacter sp. BN-1 strain (accession number NITE P-03618). <17> The method for decomposing plastics described in the above. <19> The above microorganisms belong to the genus Sulfitobacter and have 16S rDNA A polynucleotide consisting of the base sequence shown in SEQ ID NO:3, or A polynucleotide having 90% or more identity with the base sequence shown in SEQ ID NO:3 Includes <16> The method for decomposing plastics described in the above. <20> The above microorganism is Sulfitobacter sp. BN-2 strain (accession number NITE P-03611). <19> The method for decomposing plastics described in the above. Effect of the Invention

[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-mentioned test method under low-temperature and high-pressure conditions, the biodegradability in a deep sea environment can be evaluated. [Brief description of the drawings]

[0012] [Figure 1]FIG. 1 shows the microbial community structure after cultivation under different temperature and pressure conditions in the presence of water-solubilized itaconic acid-derived polyamide. [Diagram 2] FIG. 1 shows the results of a BOD test when the BN-3 strain was cultured alone in the presence of water-solubilized itaconic acid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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] <How to disassemble plastic> [Microorganisms] The method for decomposing plastics according to the present invention uses microorganisms. The present inventors confirmed that water-solubilized itaconic acid-derived polyamides are decomposed in seawater, and from the microorganisms that accumulate during the decomposition, they identified three strains as microorganisms that have the ability to decompose water-solubilized itaconic acid-derived polyamides. As will be shown in the Examples below, among these strains was a strain that was able to decompose water-solubilized itaconic acid-derived polyamides under low temperature and high pressure. Whole genome analysis of this strain identified three types of polyamide-decomposing enzymes. In the plastic decomposition method of the present invention, any of these strains or at least a microorganism having polyamide decomposition activity described below is used.

[0015] In this specification, 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, it is possible to decompose the polyamide into units having a lower molecular weight than the monomers.

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

[0017] Another strain found by the present inventors (BN-1 strain) has a 16S rDNA base sequence shown in SEQ ID NO: 2 in the sequence table, and phylogenetic analysis using 16S rDNA has revealed that it is classified into the genus Marinobacter. This strain has been deposited by the Japan Agency for Marine-Earth Science and Technology (JAMSTEC) at the Patent Microorganism Depositary of the National Institute of Technology and Evaluation (NITE) on March 2, 2022 under the accession number NITE P-03618.

[0018] The remaining strain (BN-2 strain) found by the present inventors has a 16S rDNA base sequence shown in the sequence number in the sequence table, and phylogenetic analysis using 16S rDNA has revealed that it is classified into the genus Sulfitobacter. This strain has been deposited by the Japan Agency for Marine-Earth Science and Technology (JAMSTEC) at the Patent Microorganism Depositary of the National Institute of Technology and Evaluation (NITE) on February 21, 2022 under the accession number NITE P-03611.

[0019] The above-mentioned microorganisms BN-1, BN-2, and BN-3 strains are all bacteria having the following scientific properties. - Forms white colonies on Marine Agar 2216 plates (described below). - It is possible for the bacteria to grow using water-soluble itaconic acid-derived polyamide as the sole energy and carbon source. The culture conditions are as follows: Culture medium: Heterotrophic medium with seawater salt concentration such as Marine Broth 2216 medium (described below) Culture temperature: 25~30℃ Culture period: 3 days Cultivation method: Aerobic shaking culture is suitable.

[0020] As the microorganisms that can be used in the method for decomposing polyamides, microorganisms having polyamide decomposition activity other than the above-mentioned deposited strains can be used. In particular, microorganisms belonging to the genus Shewanella that have polyamide decomposition activity, or microorganisms classified into the genus Marinobacter or Sulfitobacter that have polyamide decomposition activity can be used. Microorganisms classified into the genus Shewanella, Marinobacter, or Sulfitobacter can be identified based on methods known in the art, such as morphological observation and 16S rDNA analysis. The polyamide decomposition ability can be confirmed by growth in a medium containing a polyamide, such as a water-solubilized itaconic acid-derived polyamide, as the only energy source and carbon source. In explaining the present invention or embodiments of the present invention, the above-mentioned deposited strains may be used as examples. However, the explanations also apply to the use of other strains classified into the Shewanella, Marinobacter, or Sulfitobacter genera.

[0021] In a preferred embodiment, the microorganism belongs to the genus Shewanella and has a 16S rDNA consisting of the following (a1) or (b1): (a1) a polynucleotide consisting of the sequence set forth in SEQ ID NO:1; (b1) A polynucleotide having 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 sequence consisting of the following (aa1) or (bb1): (aa1) a polynucleotide consisting of the sequence set forth in SEQ ID NO:2; (bb1) a polynucleotide having a sequence highly identical to the sequence set forth in SEQ ID NO:2, or A microorganism belonging to the genus Sulfitobacter that has the following 16S rDNA sequence (aa2) or (bb2): (aa2) a polynucleotide consisting of the sequence set forth in SEQ ID NO:3; (bb2) a polynucleotide having a sequence having a high identity 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), identity means the percentage of the number of matching nucleotides shared between two sequences when the two sequences are aligned in an optimal manner, unless otherwise specified. The percent identity can be calculated by (number of matching positions / total number of positions)×100, and can be calculated using a commercially available algorithm. Such calculations can also be performed using algorithms or programs (e.g., BLASTN, BLASTX, ClustalW) well known to those skilled in the art. When using a program, the parameters can be appropriately set by those skilled in the art, and the default parameters of each program may 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 decomposition method of the present invention may be composed of a plurality of 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 decomposition ability. In addition, two or more strains of the above-mentioned microorganisms belonging to the genus Shewanella, Marinobacter, or Sulfitobacter and having polyamide decomposition ability may be used simultaneously or in a mixture, as long as they do not destroy the activity of each other. Furthermore, any one or more microorganisms belonging to the genus Shewanella, Marinobacter, or Sulfitobacter and having polyamide decomposition ability may be used simultaneously or in a mixture with other microorganisms, as long as they do not destroy the activity of this microorganism. As the other microorganisms, it is preferable to use, for example, microorganisms having known plastic biodegradation activity. The plastic decomposition method of the present invention may include a plastic decomposition reaction by other microorganisms. For example, a plastic decomposition reaction by a microorganism belonging to the genus Shewanella, Marinobacter, or Sulfitobacter and having polyamide decomposition ability may be carried out before or after a plastic decomposition reaction by another microorganism.

[0026] [plastic] The plastics that can be decomposed by the plastic decomposition method of the present invention are plastics that contain at least polyamide. The polyamide may be a polymer that has at least a portion where monomer units are bonded by an amide bond, and the molecular weight is not particularly limited. Polyamide is generally called nylon. The plastic may also contain a polymer that has other bonds such as ester bonds in addition to amide bonds. Furthermore, the plastic may contain polyamide and other polymers such as polyester. The plastic may also be a composite material that contains inorganic particles other than polymers.

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

[0028] [Polyamide derived from itaconic acid] The itaconic acid-derived polyamide is a polyamide containing a structure obtained by a dehydration condensation reaction (polymerization) of itaconic acid and a diamine. For example, the itaconic acid-derived polyamide can be produced 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: [ka]

[0030] Itaconic acid can be obtained by thermal decomposition of citric acid. Itaconic acid can also be produced by fermentation using mold, 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, but by using bio-derived itaconic acid, so-called bio-based engineering plastics can be realized, which meets 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 polyamide is not particularly limited and can be selected according to the polymer application. The diamine compound used in the production of itaconic acid-derived polyamide may be one type or a mixture of multiple compounds. The diamine compound may be an aliphatic diamine or an aromatic diamine. The aliphatic diamine may be either a linear alkylenediamine or a branched alkylenediamine, but is preferably a linear alkylenediamine. The carbon number of the aliphatic diamine is preferably 2 to 12, more preferably 2 to 8, and even more preferably 2 to 6, and is particularly preferably a linear alkylenediamine having 2 to 6 carbon atoms. 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 preferable, and hexamethylenediamine (1,6-diaminohexane) is more preferable.

[0032] Itaconic acid-derived polyamide is a polymer that can be produced by polymerizing (dehydration condensation) itaconic acid and a diamine compound in a 1:1 (molar ratio) and linking them with an amide bond. When the diamine compound is composed of a plurality of diamine compounds, it may be produced in any form of a random copolymer, an alternating copolymer, or a block copolymer. For a specific production method, a known production method for polyamide (nylon) can be referred to.

[0033] Itaconic acid-derived polyamides preferably have a structure in which a pyrrolidone ring (5-membered ring) is formed by dehydration condensation reaction of imine generated by aza-Michael addition of amino group of diamine compound to vinyl group of itaconic acid with carboxyl group farther from double bond. For example, the method described in JP2012-107122A can be referred to as a method for producing such itaconic acid-derived polyamides. Specifically, itaconic acid-derived polyamides preferably have a structure represented by the following formula (1).

[0034] [ka]

[0035] In formula (1), m is an integer of 2 or more indicating the degree of polymerization of the polymer, and is, for example, 5000 or less, preferably 1000 or less, and more preferably 50 or less. The value of m in the water-solubilized itaconic acid-derived polyamide is preferably small. The value of m in the itaconic acid-derived polyamide represented by formula (1) can be made smaller by water-solubilization described later. For example, in an itaconic acid-derived polyamide before water-solubilization having a value of m of 21 or more, the value of m can be made 20 or less by water-solubilization, and preferably, in an itaconic acid-derived polyamide before water-solubilization having a value of m of 25 or more, the value of m can be made 10 or less by water-solubilization.

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

[0037] [ka]

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

[0039] Itaconic acid-derived polyamides have the above-mentioned cyclic amide structure (pyrrolidone ring structure), which reduces their hygroscopicity and improves their performance as engineering plastics. Therefore, by establishing a method for decomposing itaconic acid-derived polyamides in the environment, it is possible to realize 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, 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, 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, more preferably about 400 to 2,000. The molecular weight can be made smaller by water-solubilization. For example, in an itaconic acid-derived polyamide before water-solubilization having a weight average molecular weight of 4,000 or more, the weight average molecular weight can be made less than 4,000 by water-solubilization, and preferably, for example, the weight average molecular weight can be made about 200 to 2,000.

[0041] The itaconic acid-derived polyamide may be a copolymer produced by using other carboxylic acids as raw materials in addition to itaconic acid. Examples of the other carboxylic acids include tetradecanedioic acid. The 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, based on the total mass of the raw material itaconic acid. The itaconic acid-derived polyamide may also be produced by including other monomers. Examples of the other monomers include amino carboxylic acids such as 11-amino undencanic acid. The 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, based on the total mass of the monomers having the unit structure represented by formula (1). The itaconic acid-derived polyamide may also be a composite formed with montmorillonite, titanium oxide, or the like.

[0042] [Water-soluble itaconic acid-derived polyamide] It is preferable that the itaconic acid-derived polyamide is water-soluble. The water-solubilization of the itaconic acid-derived polyamide is promoted, for example, by low molecular weight (oligomerization). The water-solubilization is also promoted by the ring-opening reaction of at least a part of the pyrrolidone ring in the structure to generate a carboxyl group and an additional secondary amino group. It is sufficient that the ring-opening reaction occurs to an extent sufficient for the water-solubilization of the itaconic acid-derived polyamide.

[0043] The water-soluble itaconic acid-derived polyamide can be obtained by placing the itaconic acid-derived polyamide under ultraviolet irradiation or alkaline conditions. For example, the water-soluble itaconic acid-derived polyamide can be obtained under irradiation with sunlight (natural light) containing ultraviolet rays. The water-soluble itaconic acid-derived polyamide may be one which has been water-soluble by a physicochemical means such as pressurization of the itaconic acid-derived polyamide.

[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, further 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] The itaconic acid-derived polyamide oligomer can be obtained by the partial hydrolysis of the itaconic acid-derived polyamide as described above. The itaconic acid-derived polyamide oligomer can also be produced by a dehydration condensation reaction between itaconic acid and a diamine compound, similar to the itaconic acid-derived polyamide. Therefore, the water-solubilized itaconic acid-derived polyamide oligomer can also be produced as a by-product in the production process of the itaconic acid-derived polyamide. Therefore, the decomposition method of the present invention can also be used for wastewater treatment containing oligomers produced in the production process of the itaconic acid-derived polyamide.

[0046] [How to disassemble plastic] In the method for decomposing plastics of the present invention, the decomposition reaction is advanced by contacting plastics with microorganisms. In order 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 in seawater. The seawater may be natural seawater or may be salt water 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. Nitrogen sources include ammonium chloride and yeast extract. Phosphorus sources include disodium hydrogen phosphate (Na2HPO4) and potassium dihydrogen phosphate (KH2PO4).

[0048] The plastic may be subjected to the decomposition method in any form as long as it is in a form that can contact the microorganisms. For example, the plastic may be in a powder or thin film form to promote the decomposition reaction. Furthermore, for plastics that are rendered water-soluble by light or heat, the plastic may be brought into contact with the microorganisms in a water-soluble form. For example, itaconic acid-derived polyamide may be exposed to ultraviolet light in water to become a water-soluble itaconic acid-derived polyamide as described above. The water-solubilization makes the plastic more susceptible to decomposition by the microorganisms.

[0049] The concentration of the microorganisms during the decomposition reaction (initial concentration) is not particularly limited as long as it is a concentration at which the plastic biodegradation activity is maintained. 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 at that temperature, and can be used within the range of, for example, −10° C. to 45° C., preferably 0° C. to 30° C. The microorganisms belonging to the genus Shewanella and having polyamide decomposition ability used in the method of the present invention 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 belonging to the genus Shewanella and having polyamide decomposition ability used in the method of the present invention are active even under pressurized conditions, and can be preferably used, for example, under conditions of 10 MPa to 40 MPa.

[0052] The plastic decomposition method of the present invention can be carried out in a marine environment. When a microorganism belonging to the genus Shewanella and having polyamide decomposition ability is used, the method can be carried out under the same conditions as those of the low-temperature, high-pressure deep sea as described above (for example, conditions of 0°C to 10°C and 10 MPa to 40 MPa). Therefore, the method of the present invention can promote biodegradation of plastics under conditions simulating a marine environment, particularly the low-temperature, high-pressure 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 about 1 day to 1 week. Although the method of the present invention uses microorganisms found in seawater, by artificially using microorganisms at a higher concentration than in the natural environment, plastic can be decomposed in a shorter time, making industrial application possible.

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

[0055] <Testing methods for biodegradability of plastics> The plastic decomposition method of the present invention can be applied to a method for testing the biodegradability of plastics. The above-mentioned Shewanella microorganism having polyamide decomposition ability and plastics are placed in an environment in which the microorganism can exert its polyamide decomposition ability, and the degree of decomposition of the plastic is measured, thereby enabling the biodegradability of the plastic to be evaluated.

[0056] Specifically, the microorganisms are brought into contact with a plastic sample in seawater under the conditions explained 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 higher the amount of carbon dioxide generated or the oxygen demand, the higher the biodegradability can be evaluated.

[0057] Preferably the sample plastic is in fine powder or film form or includes a step of rendering it in such form prior to contacting 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., ISO18830, ISO196790, ISO22404, ISO23799-1, 2).

[0059] By mixing the plastic sample with the microorganisms belonging to the Shewanella genus and capable of decomposing polyamide under low temperature and high pressure, the marine biodegradability of the plastic in the deep sea can be evaluated. For example, a pressure condition of 10 MPa can simulate a deep sea condition equivalent to a depth of 1000 m, a pressure condition of 20 MPa can simulate a deep sea condition equivalent to a depth of 2000 m, a pressure condition of 30 MPa can simulate a deep sea condition equivalent to a depth of 3000 m, and a pressure condition of 40 MPa can simulate a deep sea condition equivalent to a depth of 4000 m. Therefore, the method for testing the biodegradability of plastics of the present invention using the microorganisms belonging to the Shewanella genus and capable of decomposing polyamide can replace deep sea installation experiments, which have been limited in practice due to the enormous cost and difficulty of the system. EXAMPLES

[0060] The present invention will be described in more detail below with reference to examples. The materials, reagents, amounts of substances and their ratios, operations, etc. shown in the following examples can be appropriately changed 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 about 6000 produced by the method described in JP-A-2012-107122 using itaconic acid and hexamethylenediamine as raw materials. The water-soluble itaconic acid-derived polyamide was prepared by subjecting the itaconic acid-derived polyamide to 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 dried yeast extract, pH 7.6±0.2) Culture medium 2: Itaconic acid-derived polyamide medium (2.0 g / L water-soluble 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 plate (2.0 g / L water-soluble 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 from Kanagawa Prefecture Sediment used: Lake Nakaumi, Shimane Prefecture

[0064] (1) BOD test A pressure sensor type BOD meter (OxiTop IDS) was set in a 250 mL medium bottle. The test solution was prepared by adding inorganic salts (final concentration 7.7 mg / L potassium dihydrogen phosphate (KH2PO4), 200 mg / L ammonium chloride (NH4Cl), 100 mg / L sodium nitrate (NaNO3)) and 5 g of sediment to activate microbial activity to 50 mL of seawater. 0.5 g of water-soluble itaconic acid-derived polyamide was added to prepare a polyamide test solution, and a control solution without water-soluble itaconic acid-derived polyamide was prepared. 0.4 g of ACOMALIME ZERO was set in the holder on the bottle top as an adsorbent for carbon dioxide generated in the gas phase. A triangle-shaped PTFE rotor was placed in the bottle to stir it continuously at 180 rpm, and the culture was performed at 25°C for 14 days. BOD was measured at 28-minute intervals.

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

[0066] (2) Isolation and culture of microorganisms that degrade itaconic acid-derived polyamides 100μL of the test solution after the BOD test was applied to an itaconic acid-derived polyamide plate and cultured at 25℃. After about one week, multiple colonies were formed on the plate. The only organic nutrient salt in this medium was itaconic acid-derived polyamide, and these colonies were purified as microorganisms with the ability to decompose itaconic acid-derived polyamide. The colonies formed on the plate were picked up with a disposable loop, inoculated again onto an itaconic acid-derived polyamide plate by streaking, and cultured at 25℃. The obtained colonies were inoculated into 5mL of Marine Broth 2216 medium and cultured with shaking at 25℃ for three days, glycerol was added to a final concentration of 20%, and the medium was stored at -80℃.

[0067] Microbial cells were collected from 2 mL of culture medium by centrifugation (12,000 × g, 10 min), DNA was extracted using a commercially available kit, PCR was performed using the 27F (AGAGTTTGATCMTGGCTCAG: sequence number 4) / 1492R (GGYTACCTTGTTACGACTT: sequence number 5) primer set, and the nucleotide sequence of the purified PCR product was decoded by the Sanger method. The consensus sequence obtained by assembling the nucleotide sequence was used to perform a BLAST homology search against the international nucleotide sequence database (DDBJ / ENA (EMBL) / GenBank).

[0068] As a result of 16S rDNA analysis, three strains of different taxonomic groups were identified as polyamide-degrading microorganisms. One of the strains was 99% identical to Gammaproteobacteria-class Marinobacter hydrocarbonoclasticus ATCC 49840, and was named Marinobacter sp. BN-1 and deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary (Accession No. NITE P-03618). The other strain was 99% identical to Alphaproteobacteria-class Sulfitobacter dubius KMM 3554, and was named Sulfitobacter sp. BN-2 and deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary (Accession No. NITE P-03611). Furthermore, the other BN-3 strain was 99.93% identical to the Gammaproteobacteria Shewanella vesiculosa M7 strain, so it was named Shewanella sp. BN-3 strain, and a glycerol stock was prepared and deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Accession Number NITE AP-03961).

[0069] [Table 1] JPEG2025025826000005.jpg118170

[0070] (3) Whole genome analysis For each strain, microbial cells were collected from 2 mL of culture by centrifugation (12,000 × g, 10 min), and DNA was extracted using a commercially available kit (Quick-DNA HMW MagBead Kit) capable of extracting long-chain DNA. Short-read analysis was performed using Illumina's MiSeq and long-read analysis was performed using Oxford Nanopore's MinION. The obtained FASTQ data was used for de novo assembly using a UniCycler to determine the draft whole genome. Annotation was performed using DFAST. For the BN-3 strain, 4,639,713 base pairs consisting of nine contigs were determined, and 3,909 genes were predicted for the BN-3 strain, and putative genes encoding three types of 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 limited to water-solubilized itaconic acid polyamides. Putative genes encoding the same three types of polyamide-degrading enzymes were also identified for the BN-2 strain, and putative genes encoding two types of polyamide-degrading enzymes were also identified for the BN-1 strain.

[0071] [Table 2]

[0072] (4) Pressure culture test Inorganic salts (final concentration 7.7 mg / L potassium dihydrogen phosphate (KH2PO4), 200 mg / L ammonium chloride (NH4Cl), 100 mg / L sodium nitrate (NaNO3)), 5 g of Nakaumi sediment, and 0.05 g of water-soluble itaconic acid-derived polyamide were added to 5 mL of seawater to prepare a test solution, which was then placed in a 5 mL tube with the lid removed and the tube was capped with self-adhesive silicone tape. The tube was placed in a pressure-resistant container capable of being pressurized up to 100 MPa, and pressurized to an arbitrary 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 the culture at 25°C, pressure was applied using ultrapure water at room temperature, and for the culture at 4°C, the pressurization device was placed in ice water and pressure was applied using ultrapure water that had been cooled in the ice water. Each pressurized container was placed in an incubator at 25°C and 4°C, and static culture was performed.

[0073] After the cultivation was completed, the pressure was slowly reduced, the 5 mL tube was removed from the pressurized cultivation apparatus, and DNA was extracted from the contents in the same manner as above to perform microbial community structure analysis. The water sample was filtered using a 0.22 μm pore size membrane filter to recover microbial cells, and the membrane filter was cut into 1-2 mm squares and placed in a cell disruption tube. Approximately 0.5 g of the sediment sample was weighed and placed in a cell disruption tube. A commercially available kit (FAST DNA SPIN Kit for SOIL) was used for DNA extraction. Using the extracted DNA as a template, the 16S rRNA gene (16S rDNA) was partially amplified by PCR using the U530F (GTGCCAGCMGCCGCGG: sequence number 6) / U907R (CCGTCAATTCMTTTRAGTTT: sequence number 7) primer set targeting bacteria and archaea, and the base sequence was deciphered using the MiSeq Reagent Kit v3 and the next-generation sequencer MiSeq. The data obtained were analyzed using the QIIME2 pipeline to estimate the relative abundance of microbial species in each sample, and the results are shown in Figure 1.

[0074] As shown in Figure 1, the microbial community structure differed depending on temperature and pressure. At room temperature and pressure, bacteria of the genus Marinobacter, which is an itaconic acid-derived polyamide decomposing microorganism, accumulated, at low temperature and normal pressure, bacteria of the genus Sulfitobacter, which is a different itaconic acid-derived polyamide decomposing microorganism, accumulated, and at low temperature and high pressure, bacteria of the genus Shewanella isolated in the previous section accumulated. In this pressurized culture, itaconic acid-derived polyamide was added as the only carbon source, and it is considered that these accumulated microorganisms accumulated by assimilating itaconic acid-derived polyamide. Therefore, this result showed that bacteria of the genus Shewanella can decompose itaconic acid-derived polyamide at low temperature and high pressure. By using bacteria of the genus Shewanella, it is possible to evaluate the marine biodegradability of plastics in the deep sea.

[0075] (5) BOD test using BN-3 strain To evaluate the ability of Shewanella sp. BN-3 strain to degrade itaconic acid-derived polyamides by itself, a BOD test was conducted using this bacterium and itaconic acid-derived polyamides. Shewanella sp. BN-3 strain was shake-cultured in 50 mL of Marine Broth 2216 medium at 25°C for two nights, and the cells were collected 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 to which 0.1 g of water-soluble itaconic acid-derived polyamide had been added. 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. In other words, Shewanella sp. BN-3 strain is capable of aerobic respiration using itaconic acid-derived polyamides, which are the only carbon source.

Claims

1. A method for decomposing plastics, comprising the steps of: At least a microorganism belonging to the genus Shewanella and having polyamide decomposition ability is used, A method for decomposing plastics, wherein the plastics contain at least polyamide.

2. The 16S rDNA of the microorganism is A polynucleotide consisting of the base sequence shown in SEQ ID NO: 1, or A polynucleotide having 90% or more identity with the base sequence shown in SEQ ID NO:

1. The method for decomposing plastics according to claim 1, comprising:

3. 2. The method for decomposing plastics according to claim 1, wherein the microorganism is Shewanella sp. BN-3 strain (Accession No. NITE AP-03961).

4. 2. The method for decomposing plastics according to claim 1, comprising contacting said microorganisms with said plastics in water.

5. 2. The method for decomposing plastics according to claim 1, comprising contacting the microorganisms with the plastics in water at 10°C or lower.

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. The 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, comprising the steps of: A test method using at least a microorganism belonging to the genus Shewanella and having polyamide decomposition ability.

9. The 16S rDNA of the microorganism is A polynucleotide consisting of the base sequence shown in SEQ ID NO: 1, or A polynucleotide having 90% or more identity with the base sequence shown in SEQ ID NO:

1.

9. The method of claim 8, comprising:

10. 9. The test method according to claim 8, wherein the microorganism is Shewanella sp. BN-3 strain (Accession No. NITE AP-03961).

11. 9. The method of claim 8, comprising contacting the microorganism with the plastic in water.

12. 9. The method of claim 8, further comprising contacting the microorganism with the plastic in water at 10° C. or less.

13. 9. The method of claim 8, comprising contacting the microorganism with the plastic in water under pressure.

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.

16. A method for decomposing plastics, comprising the steps of: At least a microorganism belonging to the genus Marinobacter or Sulfitobacter and having polyamide decomposition ability is used, A method for decomposing plastics, wherein the plastics contain at least polyamide.

17. The microorganism belongs to the genus Marinobacter and has a 16S rDNA sequence A polynucleotide consisting of the base sequence shown in SEQ ID NO: 2, or A polynucleotide having 90% or more identity with the base sequence shown in SEQ ID NO:2 The method for decomposing plastics according to claim 16, comprising:

18. The method for decomposing plastics according to claim 17, wherein the microorganism is Marinobacter sp. BN-1 strain (accession number NITE P-03618).

19. The microorganism belongs to the genus Sulfitobacter and has a 16S rDNA sequence A polynucleotide consisting of the base sequence shown in SEQ ID NO:3, or A polynucleotide having 90% or more identity with the base sequence shown in SEQ ID NO:

3. The method for decomposing plastics according to claim 16, comprising:

20. The method for decomposing plastics according to claim 19, wherein the microorganism is Sulfitobacter sp. BN-2 strain (accession number NITE P-03611).

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