Method for decomposing halogenated hydrocarbon resin and microorganism capable of decomposing halogenated hydrocarbon resin

Abstract

To provide a technique for decomposing halogenated hydrocarbon resins using a microorganism.SOLUTION: Provided is a method for decomposing a halogenated hydrocarbon resin that includes a step for bringing a microorganism, and / or a treated microbial body thereof, which belongs to Pholiota genus and which is capable of decomposing halogenated hydrocarbon resins, into contact with a halogenated hydrocarbon resin.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to a method for decomposing halogenated hydrocarbon resins, a microorganism having an ability to decompose halogenated hydrocarbon resins, a culture supernatant and a treated cell product of the microorganism, a halogenated hydrocarbon resin-decomposing enzyme derived from the microorganism, and a resin composition containing them. More specifically, the present disclosure relates to a method for decomposing halogenated hydrocarbon resins using a halogenated hydrocarbon resin-decomposing microorganism. [Background technology]

[0002] In response to concerns about marine pollution by microplastics, the importance of addressing the problem of environmental pollution caused by plastic waste is increasing, and there is a need to develop a technology for decomposing and recycling plastic waste. In relation to the present disclosure, for example, Patent Document 1 proposes a method for decomposing polyvinyl alcohol, which includes contacting microbacterium bacteria with polyvinyl alcohol.

[0003] Recycling technologies for plastic waste include material recycling, which mainly reuses a single type of plastic through physical processing, and chemical recycling, which mainly recycles plastic mixtures through chemical processing. As recycling methods for polyvinyl chloride (PVC), a representative halogenated hydrocarbon resin, material recycling is the main method for products that have a system in place to narrow down the items to be discharged, reduce the risk of foreign matter contamination, and ensure a certain amount of collection at the same time, while chemical recycling is the main method for products that are highly contaminated or difficult to collect individually. Among these, chemical recycling methods for PVC include the blast furnace feedstock process and the gasification melting furnace method, but both of these processes involve the generation of hydrochloric acid at high temperatures, which is a high environmental load, and this is an issue. In addition, in general chemical recycling technologies for plastics, PVC contamination in the processed material generates hydrochloric acid, which leads to corrosion of the processing tank, so it is avoided to mix PVC into the plastic being processed. In this context, a low environmental load processing technology is desired for halogenated hydrocarbon resins such as PVC. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2006-042612 A Summary of the Invention [Problem to be solved by the invention]

[0005] A primary object of the present disclosure is to provide a technique for decomposing halogenated hydrocarbon resins using microorganisms. [Means for solving the problem]

[0006] In order to solve the above problems, this disclosure provides the following [1]-

[13] . [1] A method for decomposing halogenated hydrocarbon-based resins, comprising a step of contacting a microorganism belonging to the genus Pholiota and having the ability to decompose halogenated hydrocarbon-based resins, a treated cell product thereof, and / or a culture supernatant thereof with a halogenated hydrocarbon-based resin. [2] The decomposition method according to [1], wherein the microorganism belongs to Pholiota highlandensis. [3] The method of decomposing microorganisms according to [1], wherein the microorganism belongs to the genus Pholiota and has a spacer region (ITS1 region) between 18S rRNA and 5.8S rRNA, the spacer region being made up of a base sequence having 80% or more sequence identity with Pholiota highlandensis MCI2037 strain. [4] The decomposition method according to any one of [1] to [3], wherein the halogenated hydrocarbon resin is a chlorinated hydrocarbon resin. [5] The method for decomposing the chlorinated hydrocarbon resin according to [4], wherein the chlorinated hydrocarbon resin is polyvinyl chloride or polyvinylidene chloride. [6] The method for decomposing the chlorinated hydrocarbon resin according to [4], wherein the chlorinated hydrocarbon resin is polyvinyl chloride.

[0007] [7] A method for producing a halogenated hydrocarbon resin decomposition product, comprising a step of contacting a microorganism belonging to the genus Pholiota and having the ability to decompose halogenated hydrocarbon resins, a treated cell product thereof, and / or a culture supernatant thereof with a halogenated hydrocarbon resin. [8] A method for producing an organic acid, comprising a step of contacting a microorganism belonging to the genus Pholiota and having the ability to decompose a halogenated hydrocarbon resin, a treated cell product thereof, and / or a culture supernatant thereof with a halogenated hydrocarbon resin.

[0008] [9] Pholiota highlandensis strain MCI2037 (received number: NITE ABP-03620).

[10] A microorganism belonging to the genus Pholiota, having an ITS1 region consisting of a base sequence that has 80% or more sequence identity with Pholiota highlandensis strain MCI2037, and having the ability to degrade halogenated hydrocarbon resins.

[11] A culture supernatant or treated cell culture product of the microorganism described in [9] or

[10] .

[12] A halogenated hydrocarbon resin-decomposing enzyme derived from the microorganisms described in [9] or

[10] .

[13] A nucleic acid encoding the enzyme of

[12] , an expression vector containing the nucleic acid, or a recombinant microorganism expressing the enzyme. Effect of the Invention

[0009] The present disclosure provides a technique for decomposing halogenated hydrocarbon resins using microorganisms. [Brief description of the drawings]

[0010] [Figure 1] Shows polyvinyl chloride film degraded by Pholiota highlandensis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] A preferred embodiment for carrying out the present disclosure will be described below. Note that the embodiment described below is an example of a typical embodiment of the present invention, and the scope of the present invention is not to be interpreted narrowly by this embodiment.

[0012] [Halogenated hydrocarbon resin] The present disclosure provides a method for decomposing a halogenated hydrocarbon-based resin, the method including a step of contacting a microorganism having an ability to decompose a halogenated hydrocarbon-based resin, a treated cell product thereof, and / or a culture supernatant thereof with the halogenated hydrocarbon-based resin. In the present disclosure, the halogenated hydrocarbon resin refers to a polymer compound produced by a polymerization reaction of a halogenated alkyl monomer. The degree of polymerization of the halogenated hydrocarbon resin may be any, and is not particularly limited. The type of halogen contained in the halogenated hydrocarbon resin is not particularly limited, but is preferably chlorine. In addition, the type of the halogenated alkyl monomer is not particularly limited, but is preferably vinyl chloride (chloroethylene) or vinylidene chloride (1,1-dichloroethylene), and more preferably vinyl chloride. Polyvinyl chloride (PVC) refers to a polymer compound produced by the polymerization reaction of vinyl chloride monomer. The degree of polymerization of polyvinyl chloride may be any and is not particularly limited. In addition, in the present disclosure, polyvinyl chloride may be a copolymer of vinyl chloride monomer and other monomers, and may broadly include polymeric compounds containing vinyl chloride monomer units in the polymer chain (preferably in the main chain). The copolymerized monomers include ethylene, propylene, vinyl acetate, methyl methacrylate, alkyl vinyl ethers, and vinyl propionate. Polyvinyl chloride may contain a plasticizer, a reinforcing agent, a stabilizer, a lubricant, an emulsifier, etc. Examples of the plasticizer include di-2-ethylhexyl phthalate, di-n-butyl phthalate, diisononyl phthalate, diisodecyl phthalate, di-2-ethylhexyl adipate, diisononyl adipate, tri-2-ethylhexyl trimellitate, and tricresyl phosphate.

[0013] The form of the halogenated hydrocarbon resin is not particularly limited, and may be a powder, a film, a pellet, an emulsion, or the like.

[0014] [Halogenated hydrocarbon resin decomposition microorganisms] The halogenated hydrocarbon resin-decomposing microorganism according to the present disclosure is a microorganism that belongs to the genus Pholiota, which is a genus of Basidiomycetes, and has the ability to decompose halogenated hydrocarbon resins.

[0015] Species belonging to the genus Pholiota include Pholiota highlandensis (burning mushroom), Pholiota adiposa (slimy mushroom), Pholiota terrestris (ground mushroom), Pholiota agrocybiformis, Pholiota chocenensis, Pholiota spumosa, Pholiota marangania, Pholiota polychroa, Pholiota populnea, Pholiota velaglutinosa, Pholiota veosperma, Pholiota baeosperma, and Pholiota sp.

[0016] The halogenated hydrocarbon resin-decomposing microorganism is, in particular, Pholiota highlandensis or a subspecies belonging to said species. The strain is Pholiota highlandensis MCI2037. Examples include strains.

[0017] The MCI2037 strain has been internationally deposited at the National Institute of Technology and Evaluation, Patent Microorganism Depositary (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture), which is a depository institution under Article 27-2 and 3 of the Enforcement Regulations of the Patent Act and an international depositary authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms. The accession number of the above strain is NITE ABP-03620, and the date of accession is March 11, 2022.

[0018] The halogenated hydrocarbon resin-decomposing microorganism may be a closely related strain belonging to the same species as the above strain, such as a microorganism belonging to the genus Pholiota, preferably Pholiota highlandensis, having an ITS1 region consisting of a base sequence having 80% or more sequence identity with the Pholiota highlandensis MCI2037 strain. These closely related species are expected to have the same ability to degrade halogenated hydrocarbon resins as the above strains.

[0019] In terms of retaining halogenated hydrocarbon resin decomposition activity, the above-mentioned closely related strains have an ITS1 region consisting of a base sequence that shows sequence identity of 85% or more, 90% or more, preferably 95% or more, 98% or more, more preferably 99% or more, or 99.5% or more to the base sequence of the ITS1 region of Pholiota highlandensis MCI2037 strain. Halogenated hydrocarbon resin-decomposing microorganisms can be obtained by selecting candidate microorganisms from among microorganisms belonging to the above-mentioned taxonomic genera or species based on sequence identity of the base sequence of the ITS1 region, and evaluating the halogenated hydrocarbon resin-decomposing ability of the candidate microorganisms.

[0020] For the analysis and identification method of the ITS1 region, for example, "Japanese Pharmacopoeia, 16th Edition: Rapid Identification Method of Microorganisms by Genetic Analysis" can be referred to. Similar closely related species can also be specified by the homology of the ITS2 region (spacer region between 5.8S rRNA and 28S rRNA) or LSU region instead of the homology of the ITS1 region.

[0021] The ability of a microorganism to decompose a halogenated hydrocarbon resin can be evaluated by contacting the halogenated hydrocarbon resin with the microorganism, a treated bacterial cell product, or a culture supernatant to cause a reaction, and visually confirming the decomposition of the halogenated hydrocarbon resin.

[0022] [Treatment of bacterial cells] The above-mentioned halogenated hydrocarbon resin-decomposing microorganism may be used as a culture solution as it is, or the cells obtained by a procedure for collecting the cells from the culture solution (such as centrifugation) or a processed product thereof may be used. Examples of the treated bacterial cells include bacterial cells treated with acetone, toluene, etc., freeze-dried bacterial cells, disrupted bacterial cells, cell-free extracts, and crude or purified enzymes obtained by extracting enzymes from these. As the treated bacterial cells, enzymes (halogenated hydrocarbon resin-decomposing enzymes) are particularly preferred.

[0023] [Recombinant microorganisms] The halogenated hydrocarbon resin-decomposing enzyme may be isolated and purified from the above-mentioned microorganisms, or may be expressed in a recombinant microorganism using a conventionally known molecular biology technique and purified. The recombinant microorganism is produced by introducing a nucleic acid encoding a halogenated hydrocarbon resin-decomposing enzyme into a general host vector system and transforming the microorganism with the vector system. In addition to the above-mentioned marine microorganisms, examples of the host include bacteria such as Escherichia coli, Rhodococcus, Pseudomonas, Corynebacterium, Bacillus, Streptococcus, and Streptomyces, yeasts such as Saccharomyces, Candida, Shizosaccharomyces, and Pichia, and filamentous fungi such as Aspergillus. Among these, the use of Escherichia coli is particularly convenient and efficient, and is therefore preferred.

[0024] [Method of decomposing halogenated hydrocarbon resins] Halogenated hydrocarbon resins can be decomposed by contacting halogenated hydrocarbon resins with halogenated hydrocarbon resin-decomposing microorganisms, recombinant microorganisms, or treated cells or culture supernatants thereof. The decomposition of the halogenated hydrocarbon resin can be carried out by contacting the halogenated hydrocarbon resin with the microorganism, a treated bacterial cell product or a culture supernatant to cause a reaction, and visually confirming the decomposition of the halogenated hydrocarbon resin.

[0025] The step of contacting the halogenated hydrocarbon resin-decomposing microorganisms and the like with the halogenated hydrocarbon resin may be carried out in a suitable solvent, which is usually an aqueous solvent such as a buffer solution.

[0026] The reaction time, temperature, pH, amount of halogenated hydrocarbon resin-decomposing microorganisms, etc. to be added are not particularly limited and may be appropriately adjusted. The reaction temperature and time are usually 10-60° C. for 1 hour to 1 week, preferably 20-50° C. for 1 day or more, and more preferably 30-40° C. for 3 days or more. The pH condition for the reaction is, for example, in the range of pH 4 to 10, and preferably pH 5.0 to 9.0. The amount of halogenated hydrocarbon resin-decomposing microorganisms and the like added is, for example, in the case of an enzyme, 0.001-20% (w / w) relative to the halogenated hydrocarbon resin, preferably 0.01-10% (w / w), more preferably 0.1-5% (w / w).

[0027] The resulting decomposition products of the halogenated hydrocarbon resin may be recovered and used as raw materials for polymers such as halogenated hydrocarbon resins, polyesters, and polyethylenes.

[0028] [Method of producing organic acid] The above-mentioned method for decomposing a halogenated hydrocarbon resin produces an organic acid as a decomposition product of the halogenated hydrocarbon resin. Therefore, the present disclosure also provides a method for producing an organic acid, which includes a step of contacting a halogenated hydrocarbon resin with a microorganism having an ability to decompose a halogenated hydrocarbon resin, a treated cell product thereof, and / or a culture supernatant thereof. The specific steps of the method for producing an organic acid are the same as those of the method for decomposing a halogenated hydrocarbon resin.

[0029] The organic acid obtained is not particularly limited, and examples thereof include linear saturated monocarboxylic acids having 1 to 20 carbon atoms (formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, etc.); linear saturated dicarboxylic acids having 1 to 20 carbon atoms (oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, undecane diacid, dodecane diacid, tetradecane diacid, pentadecanedioic acid, thapsic acid, heptadecanedioic acid, octadecanedioic acid, etc.); linear unsaturated monocarboxylic acids having 3 to 20 carbon atoms and a degree of unsaturation of 1 to 10 (acrylic acid, isocrotonic acid, trans-crotonic acid, bicarbonate, etc.). Nylacetic acid, 2-pentenoic acid, 3-pentenoic acid, 4-pentenoic acid, 2-hexenoic acid, 3-hexenoic acid, 4-hexenoic acid, 5-hexenoic acid, 2-heptenoic acid, 3-heptenoic acid, 4-heptenoic acid, 5-heptenoic acid, 6-heptenoic acid, 2-octenoic acid, 3-octenoic acid, 4-octenoic acid, 5-octenoic acid, 6-octenoic acid, 7-octenoic acid, 2-nonenoic acid, 3-nonenoic acid, 4-nonenoic acid , 5-nonenoic acid, 6-nonenoic acid, 7-nonenoic acid, 8-nonenoic acid, 2-decenoic acid, 3-decenoic acid, 4-decenoic acid, 5-decenoic acid, 6-decenoic acid, 7-decenoic acid, 8-decenoic acid, 9-decenoic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, etc.); linear unsaturated dicarboxylic acids having 4 to 20 carbon atoms and a degree of unsaturation of 1 to 8 (maleic acid, fumaric acid, 2-pentenedioic acid, etc.). EXAMPLES

[0030] [Example 1: Search for halogenated hydrocarbon resin decomposition microorganisms] PDB liquid medium (potato dextrose 24 g / L) was sterilized and dispensed in 1 ml aliquots into 15 ml round-bottom tubes, and basidiomycetes belonging to the genera Amauroderma, Fomitopsis, Ganoderma, Mycoleptodonoides, Pholiota, Polyporus, and Psathyrella were inoculated and cultured at 25°C and 250 rpm for 7 days. The culture medium was transferred to a 2 ml tube and centrifuged at 12,000 rpm for 10 minutes to remove the supernatant, and the cells were collected. The collected cells were washed with 1 ml of Basal MSM (the composition is shown in Table 1), and centrifuged at 12,000 rpm for 10 minutes to remove the supernatant.

[0031] [Table 1]

[0032] The cells were resuspended in 1 ml of Basal MSM and transferred to a 15 ml round-bottom tube. Two or three pieces of PVC nanofiber film (MEC, prepared using Sigma-Aldrich polyvinyl chloride, catalog number 346764) measuring approximately 5 mm square were added to the tube, and the reaction was carried out at 25°C and 250 rpm for 14 days with shaking. A control without the addition of cells was also reacted at the same time. After the reaction was completed, the appearance of the PVC nanofiber film was visually observed, and bacteria that were observed to decompose the PVC film were selected in comparison with the control. Pholiota highlandensis was selected as a candidate strain for the PVC film degradation test in Example 2.

[0033] [Example 2: PVC film decomposition test] PDB liquid medium (potato dextrose 24 g / L) was sterilized, and 1 ml of the medium was dispensed into a 15 ml round-bottom tube, inoculated with Pholiota highlandensis, and cultured at 25°C and 250 rpm for 7 days. The culture medium was transferred to a 2 ml tube and centrifuged at 12,000 rpm for 10 minutes to remove the supernatant, and the cells were collected. The collected cells were washed with 1 ml of Basal MSM, and centrifuged at 12,000 rpm for 10 minutes to remove the supernatant.

[0034] The cells were transferred to a 200 ml Erlenmeyer flask containing 30 ml of Basal MSM. Approximately 200 mg of PVC film was added to the flask, and the reaction was allowed to proceed by shaking at 30°C and 140 rpm. A control without the addition of cells was also reacted at the same time. The weight of the PVC film was measured at the start of the reaction, and on the 14th or 57th day after the start of the reaction. The shape of the film was also observed on the 14th day after the start of the reaction.

[0035] The results are shown in Table 2 and FIG. In the case of Pholiota highlandensis, the weight of the film on the 14th day after the start of the reaction was reduced by 0.58% compared to before the reaction, and the decomposition of the PVC film was confirmed. In Figure 1, it can be seen that the four corners of the PVC film on the 14th day after the start of the reaction with Pholiota highlandensis are rounded.

[0036] [Table 2]

Claims

1. A method for decomposing a halogenated hydrocarbon-based resin, comprising a step of contacting a halogenated hydrocarbon-based resin with a microorganism belonging to the genus Pholiota and having the ability to decompose a halogenated hydrocarbon-based resin, a treated cell product thereof, and / or a culture supernatant thereof.

2. The decomposition method according to claim 1 , wherein the microorganism belongs to Pholiota highlandensis.

3. The degradation method according to claim 1, wherein the microorganism belongs to the genus Pholiota and has a spacer region (ITS1 region) between 18S rRNA and 5.8S rRNA consisting of a base sequence having 80% or more sequence identity with Pholiota highlandensis MCI2037 strain.

4. A method for producing a halogenated hydrocarbon resin decomposition product, comprising a step of contacting a halogenated hydrocarbon resin with a microorganism belonging to the genus Pholiota and having the ability to decompose halogenated hydrocarbon resins, a treated cell product thereof, and / or a culture supernatant thereof.

5. A method for producing an organic acid, comprising a step of contacting a microorganism belonging to the genus Pholiota and having the ability to decompose a halogenated hydrocarbon resin, a treated cell product thereof, and / or a culture supernatant thereof with a halogenated hydrocarbon resin.

6. Pholiota highlandensis strain MCI2037 (Accession number: NITE ABP-03620).

7. A microorganism belonging to the genus Pholiota, having an ITS1 region consisting of a base sequence having 80% or more sequence identity with Pholiota highlandensis MCI2037 strain, and having the ability to degrade halogenated hydrocarbon resins.

8. A culture supernatant or a treated product of the microorganism according to claim 6 or 7.

9. The halogenated hydrocarbon resin-decomposing enzyme derived from a microorganism according to claim 6 or 7.

10. A nucleic acid encoding the enzyme according to claim 9, an expression vector containing said nucleic acid, or a recombinant microorganism expressing said enzyme.

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