JP2005163004A - Method for biodegradation treatment of styrenic polymer and microorganism used for the method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for treating styrenic polymers such as foaming styrene and the like by biodegradation. <P>SOLUTION: Presence of polystyrene decomposing microorganism is experimentally found by adding a solution of polystyrene beads to a culture solution of styrene trimer resistant microorganism and detecting polystyrene degradation ability. Foaming polystyrene is found to be decomposed by bringing a solution of the same to contact with the microorganism. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for decomposing styrenic resin using microorganisms. The present invention can be used, for example, for disposal of discarded polystyrene foam.

  The expanded polystyrene is obtained by expanding polystyrene with a foaming agent such as butane. Depending on the manufacturing method and application, it is classified into expanded polystyrene (EPS), polystyrene paper (PSP), and extruded polystyrene (XPS). EPS is mainly used for agricultural and fishery containers and buffer packaging materials. Is mainly used for food trays, XPS is mainly used for heat insulation building materials. Styrofoam is excellent in heat insulation, shock absorption, etc., and it is easy to process, so it is expected that consumption will continue in the future, but after completion of the mission, styrofoam is discharged as waste in industry and household . Under these circumstances, as with other plastics, the solution to the waste disposal problem of expanded polystyrene is becoming increasingly important, and there is a need for efforts to recycle expanded polystyrene with the aim of achieving zero waste. .

  Materials recycling methods include material recycling and thermal recycling. The material recycling of expanded polystyrene is a recycling method in which expanded polystyrene is reduced with a volume reducing agent / dissolving agent, the volume reducing agent is separated, and then the purified polystyrene resin is reused. Thermal recycling is a recycling method in which combustion heat generated when incinerated styrene foam is used as a heat resource. In FY2002, the polystyrene recycling rate in Japan was 39.1% for material recycling and 25.6% for thermal recycling, and only about 65% when both were combined. The rest was disposed of by landfill or simple incineration.

  The landfill treatment not only makes it difficult to secure land for the future, but also has the problem that the polystyrene stays in the soil semipermanently because microorganisms that directly decompose the polystyrene are not known. From the polystyrene foam in the soil, endocrine disrupting substances such as styrene dimers and styrene trimers suspected of so-called environmental hormones are eluted by heat and organic solvents, and styrene monomers that are carcinogenic are pointed out. The landfill process cannot deny the danger of secondary environmental pollution. Moreover, incineration treatment including thermal recycling does not generate dioxin due to the expanded polystyrene itself, but there remains a problem of increasing dioxin generation due to simultaneous combustion with vinyl chloride or the like. In addition, incinerator exhaustion due to high temperature treatment to avoid dioxin generation, heat generation, and carbon dioxide emission problems. These treatment methods result in the loss of human health, promote global warming and lead to energy waste. Nevertheless, one of the reasons why polystyrene foam is landfilled or incinerated is that it cannot be subjected to conventional material recycling due to dirt, odor and color.

  By the way, as one of the waste treatment methods, a biodegradation method using microorganisms has already been put into practical use. For example, techniques for composting organic waste from primary industries, food processing industries, etc. with microorganisms are known. On the other hand, although the recycling rate and processing technology of a polystyrene foam are progressing every year, the method of biodegrading a polystyrene foam is not known. This is because there is no known microorganism capable of directly decomposing polystyrene produced after dissolving polystyrene foam or polystyrene. When styrene foam is treated with a volume-reducing agent, styrene monomer, styrene dimer, styrene trimer, and polystyrene are produced. Microorganisms that decompose styrene monomer include the genus Pseudomonas (Non-patent Documents 1, 2, 3) and the genus Xanthobacter (Non-patent Document 4). However, there are no reports of microorganisms that show resistance or degradation to styrene dimers, styrene trimers, and polystyrene.

Niall D. O'Leary, Kevin E. O'Connor, Wouter Duetz and Alan D. W. Dobson (2001) .Transcriptional regulation of styrene degradation in Pseudomonas putida CA-3.Microbiology.147: 937-979. Ana Velasco, Sergio Alonso, Jose L. Garcia, J. Perera, and Eduardo Diaz. (1998) Genetic and Functional Analysis of the Styrene Catabolic Cluster of Pseudomonas sp. Strain Y2. J. Bacteriol. 180: 1063-1071. Pedro Miguel Santos, Janet Martha Blatny, Ilaria Di Bartolo, Svein Valla, and Elisabetta Zennaro. (2000) Physiological Analysis of the Expression of Styrene Degradation Gene Cluster in Pseudomonas fluorescens ST. Appl.Environ. Microbiol. 66: 1305-1310. Hartmans, S., Smits, JP, van der Werf, MJ, Volkering, F. and de Bont, JAM (1989) Metabolism of styrene oxide and 2-phenylethanol in the styrene degrading Xanthobacter strain 124X. Appl. Environ. Microbiol. 55 : 2850-2855. O 'connor K. E., Dobson A. D. W., and Hartmans S. (1997). Indigo formation by microorganisms expressing styrene monooxygenase activity. Appl. Environ. Microbiol. 63: 4287-4291. Sambrook, J., Fritsch. E. F. and Maniatis, T .: Molecular Cloning, 2nd Edition, Cold Spring Harbor Laboratory Press. 1989. Taro Yoshikura, Masaaki Kitano, Takayuki Nishio, Hidehito Morishita, Katsuhiro Ikeda, Keito Niizuma (1991) Separation of 2-MIB-degrading bacteria from advanced treatment process and 2-MIB decomposition, water and wastewater, 33: 463 -470. Ameur Cherif, Sara Borin, Aurora Rizzi, Hadda Ouzari, Abdellatif Boudabous, and Daniele Daffonchio. (2003) Bacillus anthracis Diverges from Related Clades of the Bacillus cereus group in 16S-23S Ribosomal DNA.Intergenic Transcribed Spacers Containing tRNA Genes. Microbiol. 69: 33-40. Shoichi Yamada, Eiji Ohashi, Norio Agata, and Kasthuri Venkateswaran. (1999) Cloning and Nucleotide Sequence Analysis of gyrB and Bacillus cereus, B. thuringiensis, B. mycoides, and B. anthracis and Their Application to the Detection of B. cereus in Rice. Appl. Environ. Microbiol. 65: 1483-1490.

  This invention is made | formed in view of such a condition, The objective is to provide the method of processing styrene-type polymers, such as a polystyrene foam, by biodegradation. Moreover, the microorganisms which enable biodegradation, such as a polystyrene foam, are provided.

  In order to solve the above-mentioned problems, the present inventors can discover a microorganism capable of decomposing polystyrene, which accounts for the majority when the expanded polystyrene is dissolved, so that the expanded polystyrene can be decomposed into microorganisms and finally recycled such as composting. I thought. Therefore, the present inventors dissolved solid polystyrene beads (monomer polymerization degree 3000) with dichloromethane as a solubilizing agent, and made the same state as that obtained by dissolving expanded polystyrene with dichloromethane, and then isolated styrene-oxidizing microorganisms isolated from the environment. It was added to a culture solution of styrene trimer resistant microorganisms, and polystyrene resolution was analyzed by high performance liquid chromatography. As a result, it was experimentally confirmed that some styrene-oxidizing microorganisms and styrene trimer-resistant microorganisms have polystyrene decomposability, and it was found that the styrene foam decomposition treatment can be performed using the microorganisms, thereby completing the present invention.

Therefore, the present invention provides a method for decomposing styrene foam using a microorganism containing a styrene-decomposing microorganism or a polystyrene-degrading microorganism, and more specifically, the microorganism.
(1) A method for decomposing a styrenic polymer, comprising a step of contacting a microorganism having polystyrene decomposability with a styrenic polymer,
(2) The method for decomposing a styrenic polymer according to the above (1), wherein the microorganism having polystyrene degradability is a microorganism belonging to any of Arthrobacter, Xanthomonas, Sphingobacterium-Like, and Bacillus.
(3) Microorganisms having polystyrene resolution are Arthrobacter woluwensis PSD-1 strain, Xanthomonas sp. PSD-2 strain indicated by accession number FERM P-19584, Sphingobacterium-Like sp. PSD- indicated by accession number FERM P-19583 A method for decomposing a styrenic polymer according to either (1) or (2) above, comprising 6 strains and any of the Bacillus thuringiensis STR-YO strains indicated by accession number FERM P-19582,
(4) Microorganisms having polystyrene degradability belonging to any of Arthrobacter genus, Xanthomonas genus, Sphingobacterium-Like, and Bacillus genus,
(5) a microorganism having a polystyrene resolution according to the above (4), which is represented by any one of accession number FERM P-19584, accession number FERM P-19583, accession number FERM P-19582,
(6) A method for isolating polystyrene-degrading microorganisms, comprising a step of screening microorganisms using styrene trimer resistance as an index.
(7) A method for decomposing styrene, comprising a step of bringing styrene into contact with a microorganism having styrene-decomposing ability belonging to the genus Bacillus.
(8) The method for decomposing styrene as described in (7) above, wherein the microorganism having styrene decomposability is the Bacillus thuringiensis STR-YO strain represented by accession number FERM P-19582,
(9) A microorganism belonging to the genus Bacillus and having a styrene-decomposing ability,
(10) A microorganism having a styrene-decomposability described in the above (9), which is represented by an accession number FERM P-19582,
(11) A styrenic polymer or a microbial preparation used for styrene decomposition, comprising the microorganism according to any one of (4), (5), (9), and (10),
(12) A method for decomposing a styrenic polymer, wherein a lysate obtained by dissolving the styrenic polymer with a volume reducing agent is contacted with a microorganism having polystyrene decomposability,
(13) A method of decomposing a styrenic polymer by mixing a lysate obtained by dissolving a styrenic polymer with a volume reducing agent and a culture solution of a microorganism having polystyrene resolving ability, A method for decomposing a styrenic polymer having the ability to release the suspended state of the volume reducing agent floating on the surface when the lysate and the culture solution are mixed;
(14) The method for decomposing a styrenic polymer according to (12) or (13), wherein the volume reducing agent is limonene,
(15) The styrenic polymer according to any one of (12) to (14) above, wherein the microorganism having polystyrene decomposability is a Bacillus thuringiensis STR-YO strain represented by accession number FERM P-19582. Disassembly method,
(16) The method for decomposing a styrene polymer according to the above (12), wherein the volume reducing agent is an organic solvent,
(17) The method for decomposing a styrenic polymer according to (16), wherein the organic solvent is dichloromethane,
(18) The styrenic system according to any one of (15) to (17) above, wherein the microorganism having polystyrene decomposability is a microorganism belonging to any one of Arthrobacter, Xanthomonas, Sphingobacterium-Like, and Bacillus. Polymer decomposition method,
(19) The microorganism having the polystyrene decomposability is an Arthrobacter woluwensis PSD-1 strain, a Xanthomonas sp. PSD-2 strain indicated by accession number FERM P-19584, and a Sphingobacterium-Like sp. -6 strain, the method for decomposing a styrenic polymer according to any one of (15) to (17) above, comprising any of the Bacillus thuringiensis STR-YO strains represented by the accession number FERM P-19582,
(20) A microorganism having a polystyrene resolution used in the method for decomposing a styrenic polymer according to any one of (12) to (19) above,
(21) A microorganism preparation containing the microorganism according to (20) above,
Is to provide.

  According to the present invention, a method for biodegrading a styrenic polymer including styrene foam has been provided. In the polystyrene foam waste treatment according to the method of the present invention, EPS that has been unsuitable for material recycling can be treated without depending on incineration treatment or landfill treatment. For this reason, effective use of resources, prevention of global warming by reducing CO2, and the absence of a combustion process and low heat generation can provide beneficial effects on the global environment, such as extending the life of incinerators. In the future, if a system that can uniquely dissolve and compost EPS limonene at the individual or business level, such as garbage disposal using a commercially available processor, will be established. It is thought that it is possible to reduce the amount of styrofoam waste generated from business entities. Furthermore, if EPS microbial degradation products can be used as compost for plant fertilizers, it will help to circulate nitrogen and carbon, making it possible to achieve “zero emission treatment”.

  The present invention provides a method for decomposing a styrenic polymer such as expanded polystyrene or polystyrene, which comprises a step of bringing a microorganism having polystyrene decomposability into contact with a styrenic polymer. This is because the present inventors specifically found a microorganism having a polystyrene resolution.

  In the present invention, the styrenic polymer means a polymer in which two or more styrenes are polymerized or copolymerized with other compounds. For example, styrene dimer, styrene trimer, and polystyrene are included in the present invention. Moreover, the styrenic polymer in the present invention can take the form of beads, pellets, ingots, gels, molded products, pulverized products, polystyrene foam, regenerated foamed styrenic resins, and dissolved products thereof. For example, a styrene foam dissolved product is included in the styrene-based polymer in the present invention, and refers to a material obtained by dissolving expanded styrene in an organic solvent, and is generally a mixture of styrene monomer, styrene dimer, styrene trimer, polystyrene and the like. In the styrenic polymer in the present invention, other substances may be mixed, and for example, there may be a styrofoam coating agent or a deposit such as food.

  The microorganism used in the present invention may have the resolution of other substances as long as it has a polystyrene resolution. For example, microorganisms having both polystyrene and styrene resolution, such as the Bacillus thuringiensis STR-YO strain described later, are intended for the decomposition treatment of a polystyrene foam lysate that is a mixture of styrene monomer, styrene dimer, styrene trimer, polystyrene, etc. In some cases, it is a more preferred option. A microorganism having polystyrene resolution is, for example, cultivating a microorganism for several days in a liquid medium to which a polystyrene solution is added, as in the examples described later, and using a high-performance liquid chromatograph or the like to determine the polystyrene concentration in the culture solution. It can be confirmed by quantifying and observing changes in the amount of polystyrene.

  Specific examples of microorganisms having polystyrene resolution include microorganisms belonging to any of Arthrobacter, Xanthomonas, Sphingobacterium-Like, and Bacillus. More specifically, Arthrobacter woluwensis PSD-1 strain, Xanthomonas sp. PSD-2 strain, Sphingobacterium-Like sp. PSD-6 strain, Bacillus thuringiensis STR-Y-O strain can be mentioned. These strains are the first microorganisms that have been confirmed to have polystyrene resolution, and have been isolated by the present inventors and classified as new strains from the analysis of 16s-rDNA.

The present inventors deposited these strains with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology. The contents specifying the deposit are described below.
(I) Depositary institution: National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (Location: 1st, 1st, 1st East, Tsukuba, Ibaraki, Japan, postal code 305-8566)
(B) Deposit date: November 12, 2003 (c) Deposit number:
Xanthomonas sp. PSD-2 strain (Accession number FERM P-19584)
Sphingobacterium-Like sp. PSD-6 strain (Accession number FERM P-19583)
Bacillus thuringiensis STR-YO strain (Accession number FERM P-19582)

  In order to isolate the polystyrene-degrading microorganism, the present inventors can use a method in which the polystyrene-degrading microorganism is specifically isolated for the first time. That is, a method for screening microorganisms using styrene trimer resistance as an index, completed by the present inventors. Conventionally, since there has been no report of isolation of polystyrene-degrading microorganisms, there is no known method for isolating polystyrene-degrading microorganisms. Polystyrene is solid at room temperature, but it is difficult to dissolve in general solvents, and it is difficult to dissolve unless a special solvent is used. If polystyrene is dissolved in a special solvent and added to the culture medium in order to investigate the resolution of microorganisms, the influence of the solvent on the microorganisms increases and the possibility that polystyrene resolution cannot be evaluated is high. Therefore, the present inventors focused on styrene trimers that are easier to handle than polystyrene, and constructed an isolation method. As in the examples, after the step of screening microorganisms using styrene trimer resistance as an index, a step of further confirming polystyrene resolution can be carried out for more reliable isolation.

  Whether or not the isolated microorganism belongs to the Arthrobacter genus, Xanthomonas genus, Sphingobacterium-Like, Bacillus genus, whether or not the 16S ribosomal (r) DNA base sequence determination for classification and identification of the microorganism (non-patent Judgment can be made by performing literature 6). Furthermore, homology search for classification of microorganisms can be performed at the National Center for Biotechnology Information (NCBI) on the web, http://www.ncbi.nlm.nih.gov:80/BLAST/.

  Microorganisms with polystyrene resolution can be isolated from soil. The type of soil is not limited, but if it is a soil that may contain styrene or polystyrene by elution from industrial products such as EPS, plastic, rubber, etc., there is a high possibility that the microorganism of the present invention can be isolated. For example, the soil along the road is suitable for collecting microorganisms used in the present invention because some of the tires use styrene rubber for automobile tires. In addition, the microorganism used in the present invention may be one obtained by transforming a microorganism isolated from nature as long as it has a polystyrene resolution.

  The step of bringing a microorganism having polystyrene resolution into contact with a styrene polymer can be carried out, for example, by introducing a styrene polymer to be decomposed into a microorganism culture tank into which the microorganism is introduced. The microorganism culture tank is not particularly limited as long as it has an environment capable of effectively decomposing styrene-based polymers, but preferably has a mechanical temperature control function, a stirring function, and the like. Alternatively, a method may be used in which a styrene polymer is charged or passed through an apparatus in which microorganisms are immobilized.

  A pretreatment may be performed before the step of bringing the microorganism having polystyrene resolution into contact with the styrenic polymer. For example, it is possible to decompose a styrenic polymer, which is a molded product, by directly contacting the microorganism of the present invention, but direct degradation of the molded product may be inefficient. In that case, it is possible to perform a pulverization process for improving the efficiency of the decomposition or a dissolution process using an organic solvent. In addition, for the expanded polystyrene, before carrying out the decomposition method of the present invention, a mixture of dibasic acid esters such as limonene, ester-based dimethyl succinate, dimethyl glutarate, dimethyl adipate, or petroleum-based n-paraffin and xylene. Volume reduction may be performed with the mixed solution.

  Furthermore, this process and the decomposition process of compounds other than polystyrene may proceed in parallel. For example, when decomposing a tire or rubber, or when decomposing a mixture of polystyrene, styrene monomer, etc., by mixing a microorganism having polystyrene decomposability with a microorganism having other compound decomposing ability, The compound decomposition step may proceed in parallel.

  Microorganisms with polystyrene resolution may be used as a single type, or as long as microorganisms with polystyrene resolution can live while demonstrating resolution, they can coexist with microorganisms with other polystyrene resolution and other compound-degrading microorganisms. May be used. For example, an existing microorganism having styrene decomposability may be mixed.

  The present invention also provides a microorganism belonging to the genus Bacillus and having a styrene-degrading ability and a method for decomposing styrene using the microorganism. Conventionally known styrene-degrading bacteria include the genus Pseudomonas and the genus Xanthobacter, which are based on the fact that the present inventors have newly identified a styrene-degrading microorganism belonging to the genus Bacillus.

  Among known styrene monomer-degrading microorganisms, microorganisms belonging to the genus Pseudomonas have also been clarified in the initial stage of degradation (Non-patent Document 1). In the early stage of the degradation pathway of styrene monomer by these microorganisms, styrene monomer is oxidized by styrene monooxygenase (SMO) enzyme encoded by styA (large subunit) and styB (small subunit) genes of styrene operon to epoxy styrene. Converted. Thereafter, it is isomerized to phenylacetaldehyde by epoxy styrene isomerase encoded by styC gene, and further converted to phenylacetic acid by phenylacetaldehyde dehydrogenase enzyme encoded by styD. After this, another gene is involved, and it is thought that H2O and CO2 are finally decomposed and utilized through the degradation pathway leading to phenylacetyl-CoA.

  A report by O'connor et al. (Non-Patent Document 5) reveals that styrene monooxygenase (SMO), which works in the early stages of styrene decomposition, can also catalyze the reaction of indole (colorless) to indigo blue (blue). ing. The selection of styrene-degrading microorganisms can be performed by using the above knowledge and easily identifying the colonies as blue colonies on the styrene-added agar medium smeared with indole.

  Any microorganism having the ability to degrade styrene belonging to the genus Bacillus can be used in the styrene decomposition method of the present invention, and more specifically, Bacillus thuringiensis STR-Y-O strain can be mentioned. The Bacillus thuringiensis STR-Y-O strain was discovered and deposited as a microorganism having polystyrene and styrene resolution by the present inventors. The information specifying the deposit is as described above. In addition to the Bacillus thuringiensis STR-YO strain, the present inventors, as will be described later in the Examples, have SMO activity as Arthrobacter woluwensis PSD-1 strain, Xanthomonas sp. PSD-2 strain, Sphingobacterium-Like sp. PSD-6 strain, etc. These strains are also considered to be usable in the styrene decomposition method.

  The present invention provides a styrenic polymer or a microbial agent for decomposing styrene, comprising the above-described microorganism. The above-mentioned microorganisms can be used alone or mixed with other microorganisms to obtain a microorganism preparation as it is or after being processed. Microorganisms may be used immobilized on a carrier such as zeolite or resin. Examples of the form of the microbial preparation include, but are not limited to, a culture solution, powder, lyophilized cells, and the like.

  The microbial preparation of the present invention can be put into a microorganism culture tank or the like and used for styrene-based polymer decomposition treatment, and a method of using the microbial preparation by spraying on soil for the purpose of styrene-based polymer decomposition in soil is also conceivable.

  In the present invention, the expanded polystyrene is dissolved with a volume reducing agent such as limonene or an organic solvent such as dichloromethane, and the resulting dissolved product is brought into contact with microorganisms to decompose the expanded polystyrene.

  As the microorganism in this case, the above-mentioned styrene polymer or a microorganism capable of decomposing styrene is used.

  The volume reducing agent is not limited to limonene, but may be any one used for processing such as recycling of foamed polystyrene or capable of dissolving foamed polystyrene. Further, the organic solvent is not limited to dichloromethane, and any organic solvent can be used as long as it can dissolve the expanded polystyrene.

Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
[Example 1] Isolation of Styrene Oxidizing Microorganism Soil was collected from the shoulder of National Highway No. 4 in Sendai city or the field of Yamamori Daimon Yamori and used as a sample for isolation of styrene oxidizing bacteria. A 20 g soil sample was suspended in physiological saline and centrifuged at 30 ° C. for 10 minutes. 500 μl of the supernatant after centrifugation was planted in 50 ml of a basic inorganic salt medium (non-patent document 7) containing 0.001% (w / v) yeast extract containing styrene monomer added with various concentrations as a carbon source. As for styrene, 10μl (1.74mM), 30μl (5.24mM), 50μl (8.72mM), 100μl (17.4mM), 300μl (52.4mM) and 500μl (87.2mM) were added to the stock solution (the final concentration in parentheses) . The culture solution added with styrene was transferred to a 100 ml Erlenmeyer flask with a stopper, and cultured with shaking at 30 ° C., 130 rpm for 7 days. After confirming the presence of growth, 100 μL of a culture solution of a sample to which 10 μl of styrene was added was smeared on a basic inorganic salt agar medium, followed by stationary culture in a dark place at room temperature. On the agar medium, 100 μl of 100 mM indole (dissolved in N, N-dimethylformamide) was applied in advance. After culturing for about 7 days, blue-colored colonies were streaked on the same agar medium with platinum ears, and further cultured at room temperature for about 7 days. A single blue colony formed on the agar medium was planted in 10 ml of LB medium (Non-patent Document 8) and cultured with shaking at 30 ° C. for 1-2 days. An equal amount of 60% glycerol was added to the culture and stored frozen at -85 ° C.

[Example 2] Isolation of styrene trimer-resistant microorganisms and SMO activity test It is not clear that polystyrene-degrading bacteria possess SMO activity, and cannot be isolated using SMO activity as an indicator, so they are isolated as polystyrene-resistant bacteria. Tried. Soil was collected from the root of the street along the 6-chome industrial road in Wakabayashi-ku, Sendai, and used as a sample for isolating polystyrene-degrading bacteria. A 20 g soil sample was suspended in physiological saline and centrifuged at 3,000 rpm for 5 minutes at room temperature. 500 μl of the supernatant after centrifugation was planted in a basic inorganic salt medium containing 50 ml of yeast extract 0.001% (w / v) to which various concentrations of styrene trimer were added as a carbon source. Styrene trimer (10μg / ml toluene solution, Kanto Chemical Co., Ltd.) is the stock solution of 10μl (1.56μM), 30μl (4.68μM), 50μl (7.8μM), 100μl (15.6μM), 300μl (46.8μM), 500μl (78μM). ) Was added (final concentration in parentheses). The culture solution added with styrene trimer was transferred to a 100 ml Erlenmeyer flask with a stopper, and cultured with shaking at 30 ° C., 130 rpm for 7 days. After the turbidity was confirmed, 500 μl of the culture medium of the sample added with 10 μl, 30 μl, or 50 μl of styrene trimer was transferred to a new medium having the same composition and further cultured for 7 days. The same operation was performed once again. 100 μl of this enriched culture solution was smeared on a basic inorganic salt agar medium and left to stand for 7 days in the dark at room temperature. On the agar medium, 10 μl, 30 μl and 50 μl of styrene trimer were smeared in advance. The appearing colonies were streaked on the same agar medium with platinum ears, and further cultured in the dark at room temperature for 3 days. A single colony from each plate was inoculated into 10 ml of LB medium and cultured with shaking at 30 ° C. for 1-2 days. An equal amount of 60% glycerol was added to the culture and stored frozen at -85 ° C.

  In the SMO activity test of polystyrene-resistant bacteria, in the same manner as the isolation experiment of styrene-oxidizing bacteria, the polystyrene-resistant bacteria stored in glycerol stock were planted in basic inorganic salt agar coated with indole. This was done by observing the blue coloration.

[Example 3] Antibiotic resistance test Antibiotic resistance performance of styrene-oxidizing bacteria and polystyrene-resistant bacteria was examined for four types of antibiotics. Bacteria that had been glycerol stocked at -85 ° C were streaked on 1 x LB agar medium containing various antibiotics at a final concentration of 50 µg / ml and cultured at 30 ° C for 2-7 days. Antibiotics used were ampicillin, kanamycin, and tetracycline. Resistance was determined from the ability to form colonies.

Example 4 Determination of 16S Ribosomal DNA Base Sequence Total DNA from styrene-oxidizing bacteria and polystyrene-resistant bacteria was prepared by a conventional method using SDS and proteinase K. Cloning of 16S ribosomal DNA (16S-rDNA) by PCR from total DNA was performed using known universal primers. PCR primers are classified by using the known 520F primer 5'-GCCACG (AC) GCCGCGGT-3 '(SEQ ID NO: 1) and 1400R primer 5'-ACGGGCGGTGTGT (GA) C-3' (SEQ ID NO: 2). Appropriate genetic information can be obtained. In particular, the following primers were used for classification of Bacillus thuringiensis STR-YO strains isolated in this development. Primer ITS-Af 5'-CCTTGTACACACCGCCCGT-3 for amplifying 16S-rDNA and 23S-rDNA spacer regions, which has been shown by (Non-patent Document 8) to be effective in classifying species of the genus Basillus '(SEQ ID NO: 3) and ITS-Ar 5'-AAAATAGCTTTTTGGTGGAG-3' (SEQ ID NO: 4) and (Non-patent Document 9) show that they are effective for classifying Bacillus sereus and Bacillus thuringiensis. Primers BC1 5'-ATTGGTGACACCGATCAAACA-3 '(SEQ ID NO: 5) and BC2r 5'-TCATACGTATGGATGTTATTC-3' (SEQ ID NO: 6) for the Bacillus sereus DNA gyrase gene gyrB, and Bacillus thuringiensis DNA gyrase gene gyrB Primers BT1 5′-ATCGGTGATACAGATAAGACT-3 ′ (SEQ ID NO: 7) and RT2r 5′-CCTTCATACGTATGAATATTATTT-3 ′ (SEQ ID NO: 8) were used.

  The DNA base sequence was determined using 310 Genetic Analyzer (ABI), and DNA homology analysis was performed using genetic information analysis software Genetyx-MAC ver.9.

[Example 5] Measurement of styrene resolution Styrene degradation experiments were conducted on SD-10 strain and STR-YO strain among 6 isolated styrene-oxidizing bacteria. Styrene-oxidizing bacteria stored in a glycerol solution at -85 ° C were streaked on an LB agar medium and cultured at 30 ° C overnight. The next day, a single colony was scraped with a platinum loop, transferred to 50 ml of M9 minimal medium (Non-patent Document 6), and cultured overnight at 30 ° C. using a 100 ml Erlenmeyer flask with a stopper. 500 μl of the culture solution was transferred to a new 50 ml M9 minimal medium and continued to be cultured. Only M9 minimal medium was used as a control. When the absorbance OD600 nm reached 0.2, 200 μl (18 μg) of a styrene monomer solution diluted with hexane and having a concentration of 0.1 μl / ml was added. Thereafter, shaking culture was continued at 30 ° C. for 8 days. 4 ml of the culture solution was taken every 24 hours and mixed with 2 ml of hexane. The mixed solution was centrifuged, and the hexane layer was recovered. The collected hexane solution was transferred to a vial with a lid and stored at 4 ° C. until immediately before gas chromatographic analysis. Of this, 5 μl was used for gas chromatograph (GC) analysis to measure the styrene concentration. The GC-9A Shimadzu GC-9A was used. The column used was FFAP (Shimadzu). The GC analysis conditions were as follows: the carrier gas was He, the column temperature was 130 ° C, and the detector temperature was 250 ° C.

[Example 6] Measurement of polystyrene resolution Polystyrene degradation experiments were conducted using PSD-, except for the PSD-4 and PSD-5 strains identified as the same strain as the PSD-3 strain among microorganisms classified by the base sequence of 16S-rDNA. 1 strain, PSD-2 strain, PSD-3 strain, PSD-6 strain, and styrene-oxidizing bacteria STR-YO strain were carried out. Strains stored in glycerol solution at -85 ° C were streaked on LB agar medium and statically cultured at 30 ° C for 1-2 days. A single colony was scraped with a platinum loop, inoculated into 15 ml of LB medium, and cultured with shaking at 30 ° C. overnight in a 50 ml Erlenmeyer flask. 250 μl of the culture broth was inoculated into 4 pieces of 25 ml each of the M9 medium, and cultured at 30 ° C. using a 100 ml Erlenmeyer flask with a stopper. Only M9 medium was used as a control. When the absorbance OD 600 nm reached 0.2, 200 μl (60 μg) of a 0.3 mg / ml polystyrene bead solution (polymerization degree n≈3,000) dissolved in dichloromethane was added. Thereafter, shaking culture was continued at 30 ° C. for 8 days. 25 ml of the culture solution on days 0, 2, 5, and 8 was transferred to a 50 ml glass centrifuge. 2.5 g NaCl was added and mixed well to dissolve. Subsequently, 2 ml of dichloromethane was added and mixed well. The mixed solution was centrifuged at 3,000 rpm for 5 minutes at room temperature, and the dichloromethane layer was transferred to a 2 ml microtube. 0.1 g of anhydrous Na2SO4 was added and mixed well, and allowed to stand at room temperature for 10 minutes. Flush centrifuge and transfer the supernatant to a vial with a lid and store at 4 ° C. until analysis. 10 μl of this was used for high performance liquid chromatographic analysis (LC) to measure the polystyrene concentration. The LC device used was Shimadzu LC-10AD. As the column, Shim-pack GPC-803 (Shimadzu) was used. The LC analysis was performed under the conditions that the mobile phase was tetrahydrofuran, the flow rate was 1 ml / min, the column temperature was 30 ° C., and the detection wavelength was 254 nm.

[Example 7] Example of classification results of styrene-oxidizing bacteria Bacteria having the ability to oxidize 10 or more indole (colorless) to indigo blue (blue) from the environment could be isolated. The SD-1 to SD-10 strains are bacteria isolated from the national road along Sendai, and the STR-YO strain is a bacteria isolated from the field of Yamagata City. Table 1 shows the results of classification based on 16S-rDNA nucleotide sequences of 6 strains of the isolated bacteria. The SD-1, SD-4 and SD-9 strains all showed 99.3% homology with Aureobacterium resistens and Microbacterium nematophilum, and these strains were designated as unidentified strains because the two genus names overlapped. The SD-2 strain showed 99.8% with Arthrobacter woluwensis, and the SD-10 strain showed 99.9% homology with Pseudomonas sp. From these results, the SD-2 strain and the SD-9 strain were named Arthrobacter woluwensis SD-2 strain and Pseudomonas sp. SD-10 strain, respectively. The strain STR-YO showed 99.9% homology with Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis. Here, since Bacillus anthracis is a pathogen known as anthrax, the base sequence of the spacer region between the base sequences of 16S-rDNA and 23S-rDNA is determined according to the method of Ameur et al. It was confirmed whether it was anthrax. As a result, 3 bases of about 400 bases were not present in Bacillus anthracis, and only bases present in B. cereus and B. thuringiensis were confirmed. Subsequently, as a result of PCR on the gyrB gene according to the method of Yamada et al. (Non-patent document 9), when a primer capable of amplifying only B. thuringiensis was used, a DNA band was detected in the result of agarose gel electrophoresis, It was not detected when PCR primers capable of amplifying only B. cereus were used. From this result, the STR-YO strain was identified as B. thuringiensis and was named B. thuringiensis STR-YO strain.

[Example 8] Example of classification results of polystyrene-resistant bacteria
Bacteria that were resistant to 10 strains of styrene trimer could be isolated. As a result of classification based on the 16S-rDNA base sequence, PSD-1, PSD-3 and PSD-4 strains showed 99.8% homology with Arthrobacter woluwensis (Table 1). I think these strains may be the same strain as the SD-2 strain of styrene-oxidizing bacteria isolated along the road in Sendai City. From these results, PSD-1, PSD-3 and PSD-4 strains were named Arthrobacter woluwensis PSD-1 strain, PSD-3 and PSD-4, respectively. In addition, PSD-2 strain showed 99.1% homology with Xanthomonas cynarae, Xanthomonas gardneae and Xanthomonas campestris. From this result, the PSD-2 strain was not identified until the species, and was named Xanthomonas sp. PSD-2 strain. The PSD-6 strain showed homology with Sphingobacterium-Like sp. And Sphingobacterium comitans, which was 94.3% and 94.2%, respectively. From this result, PSD-6 strain was named Sphingobacterium-Like sp PSD-6 strain as an unidentified strain.

[Example 9] Observation of antibiotic resistance and SMO activity
Table 2 shows the results of examining the resistance to three types of antibiotics and the results of examining the presence or absence of styrene monooxygenase (SMO) activity. Styrene-oxidizing bacteria and polystyrene-resistant bacteria are kanamycin-resistant except for STR-YO strains, all styrene-oxidizing bacteria are ampicillin-resistant, and polystyrene-oxidizing bacteria are tetracycline resistant except for PSD-2 strain. . It has become clear that all three strains (Non-patent Documents 1, 2, and 3) that have been identified so far have sty operons including styA and styB genes that encode SMO. Yes. The homology between these three strains of styA gene is 92% and has been cloned by PCR (Non-patent Document 1). All strains also showed SMO activity in the isolated polystyrene-resistant bacteria (Table 2). PCR primers were synthesized for the existing styA gene, and PCR was performed using the total DNA from the seven isolated strains of styrene and polystyrene-degrading bacteria as a template to confirm the location of the styA gene. As a result, clones having no homology with the styA gene were not obtained, although no data was shown.

[Example 10] Example of polystyrene and styrene resolution experiment results A polystyrene resolution experiment was attempted using three polystyrene-resistant bacteria and STR-YO strains in which styrene degradation was clarified. As a result, all four strains showed polystyrene degradation, and the PSD-1, strain-2, PSD-6 and STR-YO strains were 47%, 24%, 43% and 56% on the 8th day of culture, respectively ( 34 μg) was shown (FIG. 1). Foamed polystyrene is made by blowing air into polystyrene peas and expanding it to 50 times the volume. The STR-YO strain had the highest polystyrene resolution among the bacteria isolated with styrene resolution. In addition, Fig. 2 shows the results of an experiment of styrene resolution using two strains of styrene-oxidizing bacteria. The SD-10 strain showed 50% (9 μg) degradation of the initial concentration on day 8 of culture, and the STR-YO strain showed 40% (7.2 μg) degradation of the initial concentration.

  So far, there are no reports of bacteria that degrade polystyrene, and elucidation of the polystyrene degradation mechanism of the isolated bacteria is a future issue. However, since SMO activity was observed in all polystyrene-degrading bacteria, styrene was oxidized to form styrene oxide in the same way as the existing sty operon (Non-patent Document 1), and then phenylacetyl- May have a degradation pathway leading to CoA. On the other hand, styA could not be cloned by PCR based on DNA homology in the above-mentioned experiment, so the possibility of a novel SMO and the mechanism for reducing the molecular weight by reacting with the side chain of polystyrene is unknown. For some reason, it is necessary to analyze the structure of the entire sty operon and compare it with existing data.

  The styrene-degrading bacteria known so far were the genus Pseudomonas and the genus Xanthobacter, but it was possible to newly identify microorganisms belonging to the genus Bacillus. In addition, it was possible to classify bacteria related to Arthrobacter woluwensis, Xanthomonas genus and Sphingobacterium genus that degrade polystyrene, indicating the diversity of polystyrene degrading bacteria. At the same time, we were able to show that the soil along the road, which is thought to be rich in styrene and polystyrene, due to the wear of automobile tires, is one of many different bacterial habitats.

[Example 11] Microbial degradation of EPS reduced in volume with limonene and dichloromethane
(1) Culture of degrading microorganisms
The PSD-1 strain, PSD-2 strain, PSD-6 strain and STR-YO strain that had been stored in the glycerol solution at -85 ° C were streaked with platinum ears on the LB agar medium (Non-patent Document 6), 30 ° C And left overnight. The next day, a single single colony was scraped off with a toothpick, inoculated into 10 ml of LB liquid medium, and cultured with shaking at 37 ° C. overnight. Four 100 ml Erlenmeyer flasks with a stopper were prepared in advance, and 25 ml of M9 liquid medium (Non-patent Document 6) was added to each flask. 250 μl of the culture solution was transferred to a prepared M9 liquid medium and cultured with shaking at 30 ° C. When the absorbance OD ₆₀₀ nm indicating the degree of growth reached 0.2, 200 μl (60 μg) of 0.3 mg / ml styrofoam solution (EPS diluted with limonene or dichloromethane) was added, and the mixture was cultured with shaking at 30 ° C. On the 0th, 2nd, 5th, and 8th days of the culture time, a styrene foam lysate was extracted from a total of 25 mL of one flask by the following dichloromethane extraction method. 10 μl of this was used for high performance liquid chromatographic analysis (LC) to measure the concentration of styrene foam lysate. The lysate was quantified using the lysate with the highest peak detected at a retention time of 5.4 minutes as an index. The LC device used was Shimadzu LC-10AD. As the column, Shim-pack GPC-803 (Shimadzu) was used. The conditions of LC analysis were as follows: the mobile phase was tetrahydrofuran, the flow rate was 1 ml / min, the column temperature was 35 ° C., and the detection wavelength was 254 nm. As a control, only M9 liquid medium was used.

(2) Dichloromethane extraction of EPS lysate
0.4 g NaCl was added to a glass centrifuge tube with a 50 ml stopper, and then 25 ml of the culture solution was transferred and mixed well using a vortex mixer. 3 ml of dichloromethane was added and mixed well using a vortex mixer for 2 minutes. Thereafter, centrifugation was performed at 3,000 rpm for 5 minutes at room temperature. The dichloromethane layer (lower layer) was taken using a Pasteur pipette and transferred to a 2 ml microtube to which 0.1 g of anhydrous sodium sulfate was added in advance. Mix well with vortex mixture and let stand at room temperature for 10 minutes. After flash centrifugation, the supernatant was transferred to a glass vial with a lid and stored at 4 ° C. until HPLC analysis.

(3) Experimental results and discussion The decomposition experiment of the polystyrene foam volume reduced with limonene was attempted with PSD-1, PSD-2, PSD-6 and STR-YO strains. The result is shown in FIG. Thus, when the STR-YO strain was used, about 67% (40 μg) of degradation was shown by the 8th day. Data not shown, but no degradation by other strains was observed.

  On the other hand, the decomposition results of the expanded polystyrene reduced with dichloromethane showed the decomposition of the expanded polystyrene solution in all strains. In particular, the degradation results of PSD-6 strain and STR-Y-O strain are shown in FIG. The STR-Y-O strain showed degradation of about 61% (37 μg) of the initial concentration by day 8.

  Degradation was observed only in the STR-Y-O strain when the volume was reduced with limonene, and one of the reasons why no degradation was observed in the other strains was presumed as follows. First, all microorganisms to which limonene foamed styrene solution was added grew. However, changes were observed during the culture for the added limonene. Limonene is sometimes known as an essential oil component of plants, and it can be observed that it floats when it is added to the culture solution and covers the entire interface (surface). In the STR-Y-O strain, as the number of days of culture progressed, the limonene floating on the interface (surface) disappeared, and it became almost unobservable by the eye by the 8th day. As a result, it is suggested that limonene does not inhibit the growth of microorganisms, but inhibits the decomposition of foamed polystyrene. It was also speculated that the STR-Y-O strain decomposed limonene and made it possible to decompose the foamed polystyrene. We have not tried to decompose limonene in the STR-Y-O strain, but if limonene degradation can be confirmed, it is a particularly useful microorganism for microbial degradation of EPS dissolved in limonene.

  Furthermore, when polystyrene-degrading microorganisms other than the STR-Y-O strain are used for the microbial degradation treatment of EPS, it is necessary to consider a method for solving the inhibition of limonene degradation.

  As a method for solving the inhibition of limonene decomposition, a method of simultaneously adding a microorganism having the ability to decompose limonene can be considered.

Using the microorganisms Arthrobacter woluwensis PSD-1 strain, Xanthomonas sp. PSD-2 strain, Sphingobacterium-Like sp PSD-6 strain and Bacillus thuringiensis STR-YO strain isolated in the present invention, polystyrene degradation experiments with a polymerization degree of 3000 were conducted. It is a figure which shows the result. It is a figure which shows the result of having conducted styrene monomer decomposition | disassembly experiment using microorganism Pseudomonas sp. SD-10 strain | stump | stock isolated by this invention, and Bacillus thuringiensis STR-Y-O strain | stump | stock. It is a figure which shows the time-dependent change of decomposition | disassembly of EPS reduced by limonene. It is a figure which shows the decomposition daily change of EPS reduced by the dichloromethane.

Claims (21)

A method for decomposing a styrenic polymer, comprising a step of bringing a microorganism having polystyrene resolution into contact with a styrenic polymer. The method for decomposing a styrenic polymer according to claim 1, wherein the microorganism having polystyrene decomposability is a microorganism belonging to any of Arthrobacter, Xanthomonas, Sphingobacterium-Like and Bacillus. Microorganisms having polystyrene resolution are Arthrobacter woluwensis PSD-1 strain, Xanthomonas sp. PSD-2 strain indicated by accession number FERM P-19584, Sphingobacterium-Like sp. PSD-6 strain indicated by accession number FERM P-19583, The method for decomposing a styrenic polymer according to any one of claims 1 and 2, comprising any one of Bacillus thuringiensis STR-YO strains represented by accession number FERM P-19582. A microorganism having polystyrene degradability belonging to any of Arthrobacter genus, Xanthomonas genus, Sphingobacterium-Like, and Bacillus genus. The microorganism having polystyrene resolution according to claim 4, which is represented by any one of accession number FERM P-19584, accession number FERM P-19583, and accession number FERM P-19582. A method for isolating a polystyrene-degrading microorganism comprising a step of screening a microorganism using styrene trimer resistance as an index. A method for decomposing styrene, comprising a step of bringing styrene into contact with a microorganism belonging to the genus Bacillus and having styrene-decomposing ability. The method for decomposing styrene according to claim 7, wherein the microorganism having styrene decomposability is Bacillus thuringiensis STR-Y-O strain represented by accession number FERM P-19582. A microorganism belonging to the genus Bacillus and having styrene-degrading ability. The microorganism having styrene-decomposing ability according to claim 9, which is represented by an accession number FERM P-19582. A styrenic polymer or a microbial preparation used for decomposing styrene, comprising the microorganism according to any one of claims 4, 5, 9, and 10. A method for decomposing a styrenic polymer, wherein a lysate obtained by dissolving a styrenic polymer with a volume reducing agent is contacted with a microorganism having polystyrene decomposability. A method of decomposing a styrenic polymer by mixing a lysate obtained by dissolving a styrenic polymer with a volume reducing agent and a culture solution of a microorganism having polystyrene degradability,
The method for decomposing a styrenic polymer, wherein the microorganism has an ability to release the suspended state of the volume reducing agent floating on the surface when the lysate and the culture solution are mixed. The method for decomposing a styrenic polymer according to claim 12 or 13, wherein the volume reducing agent is limonene. The method for decomposing a styrenic polymer according to any one of claims 12 to 14, wherein the microorganism having a polystyrene resolution is a Bacillus thuringiensis STR-Y-O strain represented by an accession number FERM P-19582. The method for decomposing a styrenic polymer according to claim 12, wherein the volume reducing agent is an organic solvent. The method for decomposing a styrenic polymer according to claim 16, wherein the organic solvent is dichloromethane. The method for decomposing a styrenic polymer according to any one of claims 15 to 17, wherein the microorganism having polystyrene decomposability is a microorganism belonging to any of Arthrobacter, Xanthomonas, Sphingobacterium-Like, and Bacillus. The microorganism having polystyrene decomposability is Arthrobacter woluwensis PSD-1 strain, Xanthomonas sp. PSD-2 strain indicated by accession number FERM P-19584, Sphingobacterium-Like sp. PSD-6 strain indicated by accession number FERM P-19583 The decomposition method of the styrenic polymer as described in any one of Claims 15-17 containing any of Bacillus thuringiensis STR-YO strain | stump | stock shown by accession number FERM P-19582. A microorganism having polystyrene decomposability used in the method for decomposing a styrenic polymer according to any one of claims 12 to 19. A microorganism preparation comprising the microorganism according to claim 20.

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