Microorganisms capable of degrading water-soluble itaconic acid-derived polyamides and their applications

Abstract

To provide a method for degrading water-soluble itaconic acid-derived polyamide, while elucidating the biodegradability of itaconic acid-derived polyamide.SOLUTION: Provided are microorganisms with an ability to degrade water-soluble itaconic acid-derived polyamide, represented by Marinobacter sp. BN-1 (accession number NITE AP-03618) or Sulfitobacter sp. BN-2 (accession number NITE AP-03611); a method of degrading water-soluble itaconic acid-derived polyamide using the microorganism; and a method for degrading itaconic acid-derived polyamide, comprising exposing itaconic acid-derived polyamide to ultraviolet rays in water to obtain a water-soluble itaconic acid-derived polyamide and degrading this using the above method.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a microorganism having a water-soluble itaconic acid-derived polyamide-degrading ability. The present invention also relates to a method for degrading a water-soluble itaconic acid-derived polyamide using the microorganism.

Background Art

[0002] Plastic waste pollution in the marine environment is serious. Fishing lines and fishing nets made of existing nylon products are lost in the marine environment, causing adverse effects on the marine ecosystem due to ghost fishing and accidental ingestion. Against this background, the development of biodegradable plastics has been promoted. However, many biodegradable plastics are excellent in degradability in soil such as compost, and many have no or very slow degradability in the marine environment. In addition, biodegradable plastics that are easily decomposed have problems of deterioration during use and are difficult to put into practical use. Therefore, the development of stimulus-responsive plastic materials has been promoted.

[0003] A polyamide produced from itaconic acid and a diamine compound (hereinafter sometimes referred to as "itaconic acid-derived polyamide") is one of such plastic materials, and it has been reported that it disappears in water by being exposed to ultraviolet rays in water (Non-Patent Documents 1 and 2).

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] Itaconic acid-derived polyamides become hydrophilic and dissolve in water when exposed to a certain amount of ultraviolet light, but the biodegradability of the water-soluble product was unknown. The object of this invention is to elucidate the biodegradability of itaconic acid-derived polyamides and to provide a method for degrading water-soluble itaconic acid-derived polyamides. [Means for solving the problem]

[0006] To solve the above problems, the inventors conducted decomposition experiments using marine environmental samples and demonstrated the decomposition of water-soluble itaconic acid-derived polyamide, finding that specific microorganisms in seawater accumulate during this process. Based on this finding, the present invention was completed. Specifically, the present invention is as follows:

[0007] <1> Microorganisms belonging to the genera *Marinobacter* or *Sulfitobacter* that possess the ability to degrade water-soluble itaconic acid-derived polyamides. <2> It belongs to the genus Marinobacter, and its 16S rDNA is A polynucleotide consisting of the base sequence shown in Sequence ID No. 1, or Polynucleotides having more than 90% identity with the base sequence shown in Sequence ID No. 1 including <1> The microorganisms listed. <3> Consists of Marinobacter sp. BN-1 strain (receipt number NITE AP-03618) <2> The microorganisms listed. <4> It belongs to the genus Sulfitobacter, and its 16S rDNA is A polynucleotide consisting of the base sequence shown in Sequence ID No. 2, or Polynucleotides having more than 90% identity with the base sequence shown in Sequence ID No. 2 including <1> The microorganisms listed. <5> Consists of Sulfitobacter sp. BN-2 strain (receipt number NITE AP-03611) <4> The microorganisms listed. <6> <1> ~ <5> A method for decomposing water-soluble itaconic acid-derived polyamide using a microorganism described in any of the above. <7> The process involves exposing itaconic acid-derived polyamide to ultraviolet light in water to obtain water-soluble itaconic acid-derived polyamide, and The above water-soluble itaconic acid-derived polyamide <6> Decompose by the method described above. A method for decomposing itaconic acid-derived polyamides, including those containing itaconic acid. [Effects of the Invention]

[0008] The present invention provides a microorganism capable of degrading water-soluble itaconic acid-derived polyamides. By using the microorganism of the present invention, it is possible to degrade water-soluble itaconic acid-derived polyamides, and by using this microorganism, bioremediation is possible to degrade itaconic acid-derived polyamides that have leaked into the environment. [Brief explanation of the drawing]

[0009] [Figure 1] These are photographs showing the changes in itaconic acid-derived polyamide before and after exposure to natural light. [Figure 2] The images show the mass spectrometry results of itaconic acid-derived polyamide before and after exposure to natural light in seawater. The top three rows (from top to bottom: filtered seawater, natural seawater, and seawater with added inorganic salts) show the results 14 days after exposure, while the bottom three rows (from top to bottom: filtered seawater, natural seawater, and seawater with added inorganic salts) show the results at the start of the treatment. [Figure 3] This figure shows the microbial community structure of itaconic acid-derived polyamide before and after exposure to natural light in seawater. [Figure 4] This figure shows the BOD test results for polyamide test solutions with 0.1g or 0.5g of water-soluble itaconic acid-derived polyamide added, and a control solution without water-soluble itaconic acid-derived polyamide. [Figure 5] The images show the mass spectrometry results of the test solution and control solution after the BOD test. The top row shows the results for the control solution, and the bottom row shows the results for the test solution with added water-soluble itaconic acid-derived polyamide. [Figure 6] This figure shows the microbial community structure of the test solution and control solution after the BOD test. [Figure 7] This is a photograph showing a plurality of colonies formed on a plate after inoculating a test solution onto a polyamide plate derived from itaconic acid and culturing.

Embodiments for Carrying Out the Invention

[0010] Hereinafter, the present invention will be described in detail. The description of the constituent elements described below may be made based on typical embodiments or specific examples, but the present invention is not limited to such embodiments.

[0011] <Microorganism> The present inventors confirmed that polyamide derived from water-soluble itaconic acid decomposes in seawater, and from the microorganisms that accumulate at that time, two novel strains were confirmed as microorganisms having the ability to decompose polyamide derived from water-soluble itaconic acid. Using such microorganisms, it is possible to decompose polyamide derived from water-soluble itaconic acid.

[0012] In this specification, decomposing polyamide derived from water-soluble itaconic acid means decomposing polyamide derived from water-soluble itaconic acid into units with a lower degree of polymerization, decomposing it into monomers, and further decomposing the monomers (for example, decomposing them into carbon dioxide). Using the microorganism of the present invention, polyamide derived from water-soluble itaconic acid can be decomposed into units with a lower molecular weight than monomers.

[0013] One of the novel strains discovered by the present inventors, the BN-1 strain, has a base sequence shown in SEQ ID NO: 1 in the sequence listing for 16S rDNA, and from phylogenetic analysis using 16S rDNA, it was found that it is classified into the genus Marinobacter. This strain has been deposited as Accession No. NITE AP-03618 on March 2, 2022 at the Patent Microorganism Depositary, National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamashima, Kisarazu City, Chiba Prefecture, Japan) by the National Institute of Advanced Industrial Science and Technology (2-5 Natsushima-cho, Yokosuka City, Kanagawa Prefecture, Japan).

[0014] Another novel strain, the BN-2 strain, discovered by the inventors, has a nucleotide sequence of 16S rDNA shown in SEQ ID NO: 2 in the Sequence Listing, and was found to be classified into the genus Sulfitobacter from phylogenetic analysis using 16S rDNA. This strain was deposited on February 21, 2022, under the accession number NITE AP-03611 at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamashima, Kisarazu City, Chiba Prefecture, Japan) by the National Institute of Advanced Industrial Science and Technology (2-5 Natsushima-cho, Yokosuka City, Kanagawa Prefecture, Japan).

[0015] Both of the above-mentioned microorganisms, the BN-1 strain and the BN-2 strain, are bacteria having the following scientific properties. ·Form white colonies on Marine Agar 2216 plates (described below). ·Can grow using polyamide derived from water-soluble itaconic acid as the sole energy source and carbon source. ·The culture conditions are as follows. Medium: Heterotrophic medium with seawater salt concentration such as Marine Broth 2216 medium (described below). Culture temperature: 25 to 30 °C Culture period: 3 days Culture method: Aerobic shaking culture is suitable.<了

[0016] As microorganisms that can be used in the method for degrading water-soluble itaconic acid-derived polyamides, microorganisms other than the deposited strains described above that have the activity to degrade water-soluble itaconic acid-derived polyamides can be used. In particular, microorganisms classified as belonging to the genera Marinobacter or Sulfitobacter that have the activity to degrade water-soluble itaconic acid-derived polyamides can be used. Microorganisms classified as belonging to the genera Marinobacter or Sulfitobacter can be identified based on methods known in the art, such as morphological observation and 16S rDNA analysis. When describing the present invention and embodiments of the present invention, the above-described deposited strains may be used as examples, but the descriptions also apply to cases where other strains classified as belonging to the genera Marinobacter or Sulfitobacter are used.

[0017] In a preferred embodiment, Microorganisms belonging to the genus Marinobacter that possess 16S rDNA consisting of either (a1) or (b1) below: (a1) A polynucleotide consisting of the sequence described in Sequence ID No. 1, (b1) A polynucleotide consisting of a sequence having high identity with the sequence described in Sequence ID No. 1, or Microorganisms belonging to the genus Sulfitobacter that possess 16S rDNA consisting of either (a2) or (b2) below: (a2) A polynucleotide consisting of the sequence described in Sequence ID No. 2, (b2) A polynucleotide consisting of a sequence having high identity with the sequence described in Sequence ID No. 2, You can use it.

[0018] 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.

[0019] With respect to base sequences (sometimes simply referred to as sequences), identity, unless otherwise specified, refers to the percentage of matching nucleotides shared between two sequences when they are aligned in the most optimal manner. Identity % can be calculated as (number of matching positions / total number of positions) × 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, BLASTP, BLASTX, ClustalW). When using a program, the parameters can be appropriately set by those skilled in the art, or the default parameters of each program may be used. The specific methods for these analysis methods are also well known to those skilled in the art.

[0020] <Method for decomposing water-soluble itaconic acid-derived polyamide> The present invention relates to a method for decomposing water-soluble itaconic acid-derived polyamide, which utilizes microorganisms capable of degrading water-soluble itaconic acid-derived polyamide. Specifically, in the present invention's method for decomposing itaconic acid-derived polyamide, water-soluble itaconic acid-derived polyamide and the above-mentioned microorganisms are coexisted in water. The inventors have found that by using the microorganisms of the present invention, water-soluble itaconic acid-derived polyamide is decomposed to the point where compounds corresponding to its monoamide units can no longer be detected.

[0021] [Itaconic acid-derived polyamide] Itaconic acid-derived polyamides are polyamides containing a structure obtained by a dehydration condensation reaction (polymerization) of itaconic acid and a diamine. Itaconic acid-derived polyamides can be produced, for example, by using itaconic acid as the dicarboxylic acid and reacting it with a diamine compound.

[0022] Itaconic acid is a type of aliphatic dicarboxylic acid represented by the following formula. [ka]

[0023] Itaconic acid can be obtained by the 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. While the polyamide decomposed by the method of this invention does not necessarily have to be bio-derived itaconic acid, using bio-derived itaconic acid enables the realization of so-called bio-based engineering plastics, which meet social demands such as long-term carbon dioxide sequestration. Itaconic acid-derived polyamide produced using bio-derived itaconic acid is sometimes called bio-nylon.

[0024] The diamine compound used in the production of itaconic acid-derived polyamides is not particularly limited and can be selected according to 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. Aliphatic diamines may be either linear alkylenediamines or branched alkylenediamines, but linear alkylenediamines are preferred. Aliphatic diamines preferably have 2 to 12 carbon atoms, more preferably 2 to 8, and even more preferably 2 to 6 carbon atoms; linear alkylenediamines with 2 to 6 carbon atoms are 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, aliphatic diamines are preferred, and hexamethylenediamine (1,6-diaminohexane) is more preferred.

[0025] Itaconic acid-derived polyamides are polymers that can be produced by polymerizing (dehydrating condensation) itaconic acid and a diamine compound in a 1:1 (molar ratio) and linking them by amide bonds. When the diamine compound consists 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, refer to known production methods for polyamide (nylon).

[0026] It is preferable that the itaconic acid-derived polyamide has a structure in which a pyrrolidone ring (5-membered ring) is formed by a dehydration condensation reaction between the imine, which is generated by the addition of the amino group of the diamine compound to the vinyl group of itaconic acid, and the carboxyl group, which is furthest from the double bond. For example, the method described in Japanese Patent Application Publication No. 2012-107122 can be used to describe how such an itaconic acid-derived polyamide is produced. Specifically, it is preferable that the itaconic acid-derived polyamide has a structure represented by the following formula (1).

[0027] [ka]

[0028] In formula (1), m is an integer of 2 or more that indicates the degree of polymerization of the polymer, for example, 5000 or less, preferably 1000 or less, and more preferably 50 or less. It is preferable that the value of m in water-soluble itaconic acid-derived polyamide be small. Through water solubility described later, the value of m in the itaconic acid-derived polyamide represented by formula (1) can be made even smaller. For example, in an itaconic acid-derived polyamide before water solubility with a value of m of 21 or more, the value of m can be reduced to 20 or less by water solubility, and preferably, in an itaconic acid-derived polyamide before water solubility with a value of m of 25 or more, the value of m can be reduced to 10 or less by water solubility.

[0029] L is a divalent linking group derived from the linking group of two amino groups in a diamine compound. Preferably, L has one of the following structures, and m Ls may be the same or different from each other.

[0030] [ka]

[0031] In the above formula, n is a natural number, preferably between 2 and 12, more preferably between 2 and 8, and even more preferably between 2 and 6.

[0032] Itaconic acid-derived polyamides have a cyclic amide structure (pyrrolidone ring structure) as described above, which reduces their hygroscopicity and improves their performance as engineering plastics. Therefore, establishing a method for the environmental degradation of itaconic acid-derived polyamides will enable bioremediation of plastics that can be industrially put into practical use.

[0033] 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-soluble itaconic acid-derived polyamide is preferably about 400 to 10,000, preferably about 400 to 5,000, more preferably about 400 to 2,000. The molecular weight can be reduced further by water solubility. For example, in an itaconic acid-derived polyamide before water solubility with a weight-average molecular weight of 4,000 or more, the weight-average molecular weight can be reduced to less than 4,000 by water solubility, preferably to a weight-average molecular weight of, for example, about 200 to 2,000.

[0034] Furthermore, the itaconic acid-derived polyamide may be a copolymer produced using itaconic acid and other carboxylic acids as raw materials. Examples of other carboxylic acids include tetradecanedioic acid. The other carboxylic acid may be present in amounts of 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 raw material itaconic acid. In addition, the itaconic acid-derived polyamide may be produced containing other monomers. Examples of other monomers include aminocarboxylic acids such as 11-aminoundencanoic acid. The other monomer may be present in amounts of 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 monomer of the unit structure represented by formula (1). Furthermore, the itaconic acid-derived polyamide may be composited with montmorillonite, titanium dioxide, or the like.

[0035] [Water-soluble itaconic acid-derived polyamide] The itaconic acid-derived polyamide can be degraded by the microorganisms of the present invention by making it water-soluble. The water-solubilization of the itaconic acid-derived polyamide is promoted, for example, by demolecular-weight reduction (oligomerization). Furthermore, water-solubilization is also promoted by the ring-opening reaction of at least some of the pyrrolidone rings in the structure, which generates carboxyl groups and additional secondary amino groups. The ring-opening reaction only needs to occur to a degree sufficient to make the itaconic acid-derived polyamide water-soluble.

[0036] Water-soluble itaconic acid-derived polyamide can be obtained by placing itaconic acid-derived polyamide under ultraviolet irradiation or alkaline conditions. Water-soluble itaconic acid-derived polyamide can be obtained under sunlight (natural light) irradiation including ultraviolet light.

[0037] In a preferred embodiment, the water-soluble itaconic acid-derived polyamide degraded by the microorganisms of the present invention is an oligomer (itaconic acid-derived polyamide oligomer). The itaconic acid-derived polyamide oligomer is typically an oligomer (2-20 units) in formula (1) above, more preferably an oligomer (2-15 units) in which the value of m is 2-20, even more preferably an oligomer (3-10 units) in which the value of m is 3-10, and most preferably an oligomer (2-8 units) in which the value of m is 3-8. As described above, in each oligomer represented by formula (1), the pyrrolidone ring structure in some or all structural units may be open.

[0038] Itaconic acid-derived polyamide oligomers can be obtained by the partial hydrolysis of itaconic acid-derived polyamide as described above. Like itaconic acid-derived polyamide, itaconic acid-derived polyamide oligomers can also be produced by a dehydration condensation reaction between itaconic acid and a diamine compound. Therefore, water-soluble itaconic acid-derived polyamide oligomers can also be produced as byproducts in the production process of itaconic acid-derived polyamide. Accordingly, the decomposition method of the present invention can also be used in the treatment of wastewater containing oligomers generated during the production process of itaconic acid-derived polyamide.

[0039] <Method for decomposing itaconic acid-derived polyamide> Water-soluble itaconic acid-derived polyamide is obtained by exposing itaconic acid-derived polyamide to ultraviolet light in water. Therefore, the decomposition method using microorganisms capable of degrading water-soluble itaconic acid-derived polyamide according to the present invention can be used as a decomposition method for itaconic acid-derived polyamide in environments where light such as sunlight may be irradiated. That is, itaconic acid-derived polyamide can be decomposed by the microorganisms of the present invention by becoming water-soluble itaconic acid-derived polyamide by exposure to ultraviolet light in water. Therefore, the method of the present invention can be applied to the bioremediation of itaconic acid-derived polyamide released into the environment. Although the microorganisms of the present invention were found in seawater, as shown in the examples, the rate of decomposition of water-soluble itaconic acid-derived polyamide oligomers in the natural environment is very slow. By artificially using the microorganisms of the present invention at a higher concentration than in the natural environment, itaconic acid-derived polyamide can be decomposed in a shorter time, enabling industrial applications. [Examples]

[0040] The present invention will be described in more detail below with reference to examples. The materials, reagents, amounts and proportions of substances, and procedures shown in the following examples can be modified as appropriate, as long as they do not depart from the spirit of the present invention. Therefore, the scope of the present invention is not limited to the following examples.

[0041] In the examples, the itaconic acid-derived polyamide used was an itaconic acid-derived polyamide (solid) with a weight-average molecular weight of approximately 6000, produced using itaconic acid and hexamethylenediamine as raw materials by the method described in Japanese Patent Application Publication No. 2012-107122. As a water-soluble itaconic acid-derived polyamide, the above-mentioned itaconic acid-derived polyamide is exposed to ultraviolet light (250-400 nm, 150 mW / cm²) in pure water. 2 They used a gel-like substance that was artificially irradiated with ) and then transformed into a gel.

[0042] The culture 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)

[0043] 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 tetramarine salt pro) Culture plate medium: Marine Agar 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, 15.0 g / L agar powder, pH 7.6±0.2)

[0044] Pure isolation plate medium: 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)

[0045] Seawater used: Yokosuka seawater, Kanagawa Prefecture Sediment used: Nakaumi sediment from Shimane Prefecture

[0046] <Natural light exposure treatment> Plate-shaped (2-3 mm thick) polyamide derived from itaconic acid was cut into 1 cm squares and placed in a 100 mL medium bottle with 50 mL of natural seawater. The bottle was left standing on its side outdoors in sunlight for 14 days. As a control, filtered seawater filtered through a 0.22 μm pore size membrane filter was used. After the treatment was complete, the test solution was collected, 15 mL was used for mass spectrometry, and the remainder was used for microbial community structure analysis.

[0047] <Bio-Oxygen Demand (BOD) Test> A pressure-sensor type BOD meter (OxiTop IDS) was used, set in a 250 mL medium bottle. A 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. A polyamide test solution was prepared by adding 0.1 g or 0.5 g of water-soluble itaconic acid-derived polyamide, and a control solution without water-soluble 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 stir the contents of the bottle, and the mixture was continuously stirred at 180 rpm and incubated at 25°C for 14 days. BOD measurements were taken at 28-minute intervals.

[0048] <Mass spectrometry> The solution after the BOD test was diluted 10-fold with ultrapure water, mixed with 5 mg / mL CHCA, and analyzed using a MALDI-8020 laser desorption / ionization time-of-flight mass spectrometer. The measurement conditions were as follows: Laser light source: solid-state laser (355 nm), detected ion: positive ion mode, flight mode: linear mode.

[0049] <Microbial community structure analysis> DNA extraction was performed from the test solution after exposure to natural light, from seawater and sediment before use in BOD testing, and from the test solution and sediment after BOD testing. Microbial cells were collected from the water samples by filtering with a 0.22 μm pore size membrane filter, and the membrane filter was cut into 1-2 mm squares and placed in cell disruption tubes. Approximately 0.5 g of the sediment sample was weighed out and placed in cell disruption tubes. 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: SEQ ID NO: 3) / U907R (CCGTCAATTCMTTTRAGTTT: SEQ ID NO: 4) primer set targeting bacteria and archaea, and the base sequence was decoded using MiSeq Reagent Kit v3 and the MiSeq next-generation sequencer. The obtained data was analyzed using the QIIME2 pipeline to estimate the relative amounts of microbial species contained in each sample.

[0050] <Pure Separation> 100 μL of the test solution after the BOD test was spread onto an itaconic acid-derived polyamide plate and incubated statically at 25°C. After about one week, colonies formed on the plate were picked using a disposable loop and inoculated again onto an itaconic acid-derived polyamide plate by streaking, and incubated statically at 25°C. The obtained colonies were inoculated into 5 mL of Marine Broth 2216 medium, incubated with shaking at 25°C for three days, glycerol was added to a final concentration of 20%, and the culture was stored at -80°C.

[0051] <Systematic classification of isolates> 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, and PCR was performed using the 27F(AGAGTTTGATCMTGGCTCAG: SEQ ID NO: 5) / 1492R(GGYTACCTTGTTACGACTT: SEQ ID NO: 6) primer set. The nucleotide sequence of the purified PCR product was decoded by Sanger sequencing. The consensus sequence assembled from the obtained nucleotide sequences was used for a BLAST homology search against international nucleotide sequence databases (DDBJ / ENA(EMBL) / GenBank).

[0052] <Result> When the polyamide specimens derived from itaconic acid were exposed to natural light in seawater for a certain period (6 hours in the example in Figure 1), water solubilization began from the surface, and after the test, they were so altered that they were unrecognizable (Figure 1). This water solubilization reaction did not require any biological reaction, and the reaction was also observed in the filtered seawater control group (Figure 1). Mass spectrometry of the test solution after the test revealed signals corresponding to the mass of tetramers to decamers, confirming that these were dissolved in an oligomeric state with these as the main components (Figure 2). Similar results were obtained in filtered seawater, natural seawater, and seawater with added inorganic salts, suggesting that microorganisms were not involved in oligomerization. Furthermore, an examination of the microbial community structure of the test solution after exposure to natural light revealed an increase in microbial species belonging to the class Gammaproteobacteria in the test solution (Figure 3).

[0053] BOD test: Higher BOD values ​​were measured in proportion to the amount of itaconic acid-derived polyamide added, and a gradual increase in BOD was observed in the control group without itaconic acid-derived polyamide (Figure 4). The degradation rate calculated from the BOD values ​​was approximately 50%. Mass spectrometry using the solution after the BOD test showed that oligomers of 50-mer or more, which were detected after exposure to natural light, had disappeared (Figure 5). Furthermore, the microbial community structure in the test solution after the BOD test confirmed the accumulation of species closely related to the genus Pseudohongiella of the class Gammaproteobacteria (Figure 6).

[0054] Purification Isolation: After BOD testing, oligomers of itaconic acid-derived polyamide were degraded, and specific microbial species accumulated in the microbial community structure. Therefore, the test solution was inoculated onto an itaconic acid-derived polyamide plate and cultured. After culturing, multiple colonies formed on the plate (Figure 7). The only organic nutrients in this medium were water-soluble itaconic acid-derived polyamides, and these colonies were purified as microorganisms capable of degrading water-soluble itaconic acid-derived polyamides. 16S rDNA analysis identified two strains with different classifications as itaconic acid polyamide-degrading microorganisms. One strain was 99% identical to Marinobacter hydrocarbonoclasticus ATCC 49840 of the Gammaproteobacteria class, and was named Marinobacter sp. BN-1. A glycerol stock was prepared, and it was deposited with the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (NITE AP-03618). The other strain was 99% identical to Sulfitobacter dubius KMM 3554 of the Alphaproteobacteria class, and was named Sulfitobacter sp. BN-2. A glycerol stock was prepared, and it was deposited with the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation (NITE AP-03611).

[0055] [Table 1] [Sequence Listing Free Text]

[0056] Sequence ID 1: 16S rDNA sequence of Marinobacter sp. BN-1 strain (receipt number NITE AP-03618) Sequence ID 2: 16S rDNA sequence of Sulfitobacter sp. BN-2 strain (Receipt No. NITE AP-03611) Sequence IDs 3-6: Primer sequences

Claims

1. A microorganism having the ability to degrade water-soluble itaconic acid-derived polyamides, It belongs to the genus Marinobacter, and its 16S rDNA is A polynucleotide consisting of the base sequence shown in Sequence ID No. 1, or A polynucleotide having 99.40% or more identity with the base sequence shown in Sequence ID No.

1. Includes or, It belongs to the genus Sulfitobacter and has 16S rDNA. A polynucleotide consisting of the base sequence shown in Sequence ID No. 2, or A polynucleotide having 99.40% or more identity with the base sequence shown in Sequence ID No.

2. Microorganisms that include [this].

2. The microorganism according to claim 1, comprising Marinobacter sp. BN-1 strain (receipt number NITE AP-03618).

3. The microorganism according to claim 1, comprising Sulfitobacter sp. BN-2 strain (receipt number NITE AP-03611).

4. A method for degrading water-soluble itaconic acid-derived polyamide using a microorganism belonging to the genus Marinobacter or Sulfitobacter that has the ability to degrade water-soluble itaconic acid-derived polyamide.

5. The microorganism belongs to the genus Marinobacter and has 16S rDNA A polynucleotide consisting of the base sequence shown in Sequence ID No. 1, or Polynucleotides having more than 90% identity with the base sequence shown in Sequence ID No. 1 The method according to claim 4, including the method described in claim 4.

6. The method according to claim 4, wherein the microorganism consists of Marinobacter sp. BN-1 strain (receipt number NITE AP-03618).

7. The microorganism belongs to the genus Sulfitobacter and has 16S rDNA A polynucleotide consisting of the base sequence shown in Sequence ID No. 2, or Polynucleotide having more than 90% identity with the base sequence shown in Sequence ID No. 2 The method according to claim 4, including the method described in claim 4.

8. The method according to claim 4, wherein the microorganism consists of Sulfitobacter sp. BN-2 strain (receipt number NITE AP-03611).

9. The process involves exposing itaconic acid-derived polyamide to ultraviolet light in water to obtain water-soluble itaconic acid-derived polyamide, and Decompose the water-soluble itaconic acid-derived polyamide by the method described in any one of claims 4 to 8. A method for decomposing itaconic acid-derived polyamides, including those containing itaconic acid.

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