Microorganisms capable of degrading thermoplastic polyurethane, methods for degrading thermoplastic polyurethane, and methods for selecting microorganisms capable of degrading thermoplastic polyurethane.
Pseudomonas hibiscicola MS4102 and Sinomonas atrocyanea ES2231 strains efficiently degrade thermoplastic polyurethane, overcoming its recalcitrance through soil burial selection and culture methods, allowing for effective material recycling and reducing marine pollution.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- NIHON PLAST CO LTD
- Filing Date
- 2022-03-30
- Publication Date
- 2026-07-07
Smart Images
Figure 0007886168000013 
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Abstract
Description
Technical Field
[0001] The present invention relates to a microorganism having the ability to decompose thermoplastic polyurethane (TPU), a method for decomposing thermoplastic polyurethane, and a method for selecting a microorganism having the ability to decompose thermoplastic polyurethane.
Background Art
[0002] Polyurethane is a polymer having urethane bonds and is also called urethane resin. Polyurethane has material properties that are prone to degradation of physical properties due to hydrolysis by moisture or the influence of ultraviolet rays, heat, microorganisms, etc. However, polyurethane has been modified so that it is difficult to decompose, and the material has evolved into a structure that is resistant to hydrolysis and less affected by microorganisms, and is still used in various fields.
[0003] Since polyurethane has a crosslinked structure, it cannot be melted and reused as a material like thermoplastic resins, and most of it is landfilled, causing environmental problems. Currently, in order to avoid this problem, research is being conducted in various fields on the reuse as a material and biodegradability by utilizing the decomposition action of environmentally friendly microorganisms. However, it takes a lot of time and effort to find effective bacteria, and there are almost no cases leading to practical application.
[0004] As a method for decomposing polyurethane by utilizing the decomposition action of microorganisms, for example, the methods described in JP-A-2010-220610 (Patent Document 1) and JP-A-2015-128407 (Patent Document 2) are known. Patent Document 1 describes using strain C13a (actinomycetes) belonging to the genus Streptomyces as a microorganism having urethane-decomposing ability. Further, Patent Document 2 describes a method for decomposing polyurethane having a step of pretreating a material to be treated containing urethane with an unsaturated fatty acid such as oleic acid and a step of allowing a microorganism having urethane-decomposing ability (for example, C13a of the genus Streptomyces) to act on the material to be treated pretreated with an unsaturated fatty acid. [Prior art documents] [Patent Documents]
[0005] [Patent Document 1] Japanese Patent Publication No. 2010-220610 [Patent Document 2] Japanese Patent Publication No. 2015-128407 [Overview of the project] [Problems that the invention aims to solve]
[0006] As described above, the methods described in Patent Documents 1 and 2 allow for the efficient decomposition of flexible foamed polyurethane by utilizing the decomposition action of microorganisms. Therefore, the present inventors attempted to decompose thermoplastic polyurethane (TPU) using the methods described in Patent Documents 1 and 2. However, thermoplastic polyurethane is difficult to decompose, and microbial decomposition hardly progressed. Furthermore, there was no change in the surface condition of the thermoplastic polyurethane, so no progress in decomposition was observed. For comparison, when the weight loss was compared by decomposing polyether-based foamed urethane using the same method, a weight loss of slightly less than 4% occurred, and the formation of micropores in the closed-cell foam was observed, confirming that decomposition was progressing.
[0007] Therefore, the present invention aims to provide microorganisms capable of efficiently degrading even recalcitrant thermoplastic polyurethanes, a method for degrading thermoplastic polyurethanes using these microorganisms, and a method for selecting microorganisms that have the ability to degrade thermoplastic polyurethanes. [Means for solving the problem]
[0008] A microorganism according to one embodiment of the present invention is Pseudomonas Generally Belonging to, This is the Pseudomonas hibiscicola MS4102 strain identified by accession number NITE BP-03612. Having the ability to decompose thermoplastic polyurethanes containing urea bonds, It is a microorganism. Furthermore, microorganisms according to another embodiment of the present invention are It belongs to the genus Sinomonas, and is identified as Sinomonas atrocyanea ES2231 strain with accession number NITE BP-03613, and has the ability to degrade thermoplastic polyurethanes containing urea bonds. It is a microorganism.
[0009] A method for decomposing thermoplastic polyurethane according to one embodiment of the present invention is: The process involves reacting a thermoplastic polyurethane containing a urea bond with a microorganism according to the above embodiment of the present invention. This is a method for decomposing thermoplastic polyurethane.
[0010] A method for selecting microorganisms capable of degrading thermoplastic polyurethanes according to one embodiment of the present invention is: A process of burying a mixed sample of thermoplastic polyurethane containing urea bonds and polyurethane without urea bonds in the soil, The buried mixed sample is subjected to infrared spectroscopy to confirm the decomposition of urethane, followed by a step of collecting microorganisms adsorbed on the mixed sample. Having, This is a method for selecting microorganisms capable of degrading thermoplastic polyurethanes. [Effects of the Invention]
[0011] According to the present invention, it is possible to provide microorganisms capable of efficiently degrading even recalcitrant thermoplastic polyurethanes, a method for degrading thermoplastic polyurethanes using these microorganisms, and a method for selecting microorganisms that have the ability to degrade thermoplastic polyurethanes. [Brief explanation of the drawing]
[0012] [Figure 1] Figure 1 shows the spectrum obtained from infrared spectroscopy analysis 14 months after the mixed sample was buried in the soil in the example. [Figure 2] Figure 2 shows the spectrum obtained from infrared spectroscopy analysis 17 months after the mixed sample was buried in the soil in the example. [Figure 3]Figure 3 is a graph showing the weight loss rate when a novel degrading bacterium acts on thermoplastic polyurethane without pretreatment in the examples. [Figure 4] Figure 4 is a graph showing the weight loss rate when a novel degrading bacterium acts on thermoplastic polyurethane after plasma treatment in the examples. [Figure 5] Figure 5 is a graph showing the weight loss rate when a novel degrading bacterium acts on thermoplastic polyurethane after plasma treatment and oleic acid treatment in the examples. [Figure 6] Figure 6 is a graph comparing the pore numbers of thermoplastic polyurethane after the action of each novel degrading bacterium in the examples. [Figure 7] Figure 7 is a photograph showing the result of observing the surface of thermoplastic polyurethane by SEM after the action of each novel degrading bacterium in the examples. [Figure 8] Figure 8 is a spectrum showing the result of infrared spectroscopic analysis of thermoplastic polyurethane after the action of each novel degrading bacterium in the examples. [Figure 9] Figure 9 is a graph comparing the melting characteristics of thermoplastic polyurethane after the action of each novel degrading bacterium in the examples. [Figure 10] Figure 10 is a photograph showing the melting characteristics of thermoplastic polyurethane when the novel degrading bacterium strain MS2231 acts without pretreatment in the examples. [Figure 11] Figure 11 is a photograph showing the ash residue after heating thermoplastic polyurethane when the novel degrading bacterium strain MS2231 acts without pretreatment to 700 °C in the examples. [Figure 12] Figure 12 is a photograph showing the melting characteristics of a molded product of thermoplastic polyurethane. [Figure 13] Figure 13 is a photograph showing the melting characteristics of the raw material powder of thermoplastic polyurethane. [Figure 14] Figure 14 is a diagram showing a schematic of the structure of hardly degradable thermoplastic polyurethane.
Embodiments for Carrying Out the Invention
[0013] The inventors of this invention have conducted extensive research into why the degradation of recalcitrant thermoplastic polyurethane hardly progresses even when exposed to unsaturated fatty acids or microorganisms. As a result, they have concluded that the reason why thermoplastic polyurethane hardly decomposes when exposed to unsaturated fatty acids or microorganisms is due to the molecular structure of the thermoplastic polyurethane.
[0014] As shown in Figure 14, persistent thermoplastic polyurethane has a structure in which hard segments and soft segments are phase-separated (sea-island structure). Polyurethane containing urea bonds is used as the raw material for the hard segments, and polyether-based polyurethane is used as the raw material for the soft segments.
[0015] As shown in equation (1) below, the urea bond has a symmetrical conjugated structure with a carbonyl group (=CO) in between, and as shown in equation (2) below, the intermolecular bonding force increases when it is polarized (see equation (3) below), making it difficult to decompose. Furthermore, against external attacks, as shown in equation (4) below, valence electrons move and a relaxation effect occurs, making it difficult to decompose. Furthermore, the polyether-based polyurethane in the soft segment contains polyester groups with high cohesive force. During the melt-solidification process, the terminal groups hydrogen-bond with the urethane groups, partially cross-linking and creating a stronger, more degradable structure. Thermoplastic polyurethane is being highlighted as a problematic substance (microplastic) that causes marine pollution. Therefore, if a technology is developed that allows for the material recycling of thermoplastic polyurethane, the total amount of plastic can be reduced, leading to a reduction in marine pollution. Furthermore, because thermoplastic polyurethane possesses stable material properties that make it resistant to decomposition, as described above, it is used as a surface material for automotive instrument panels produced by slush molding. In particular, due to its good feel and durability, it has a proven track record of being used in luxury cars.
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[0020] Thermoplastic polyurethane is molded using a powder slush molding method with a raw material of approximately 100 μm. The raw material powder is placed in a mold and heated to approximately 180°C, where it is uniformly melted and formed into a sheet (see Figure 13). During the melt-solidification process, the molded thermoplastic polyurethane sheet undergoes a cross-linking reaction at its ends, as described above. Therefore, even when heated again to approximately 300°C, the material does not melt uniformly and instead carbonizes and decomposes (see Figure 12). For this reason, thermoplastic polyurethane cannot be recycled as a thermoplastic resin even when waste material is melted, and is currently disposed of as thermal recycling.
[0021] Therefore, the inventors of the present invention have conducted extensive research on methods for selecting microbial strains that have a high decomposition capacity for thermoplastic polyurethane, which is difficult to decompose, in order to reuse waste thermoplastic polyurethane as a material. As a result, they have found that the method for selecting microorganisms that have a decomposition capacity for thermoplastic polyurethane according to the following embodiment of the present invention is effective.
[0022] <Method for selecting microorganisms capable of degrading thermoplastic polyurethanes> A method for selecting microorganisms capable of degrading thermoplastic polyurethane according to an embodiment of the present invention comprises the steps of: burying a mixed sample of thermoplastic polyurethane containing urea bonds and polyurethane without urea bonds in the soil; and, after confirming urethane degradation by infrared spectroscopy analysis of the buried mixed sample, collecting the microorganisms adsorbed on the mixed sample. Each step is described in detail below.
[0023] (The process of burying the mixed sample in the soil) To select bacteria with high degradability of recalcitrant thermoplastic polyurethane, it is necessary to collect as many potential bacteria as possible and select from among them. As mentioned above, recalcitrant thermoplastic polyurethane contains recalcitrant urea bonds, so first, novel degrading bacteria with high adsorption to urea bonds should be selected. However, even if recalcitrant thermoplastic polyurethane is buried in the soil as a sample, the number of bacteria that accumulate may be limited, and it may not be possible to select effective bacteria.
[0024] Therefore, in the method for selecting microorganisms capable of degrading thermoplastic polyurethane according to the embodiment of the present invention, a mixed sample is prepared by mixing a general-purpose polyurethane that is easily degraded without urea bonds and a thermoplastic polyurethane that is difficult to degrade. This mixed sample is then used as a specimen and buried in the soil. This creates an environment that easily attracts bacteria that readily adsorb to urea bonds. The polyurethane without urea bonds and the thermoplastic polyurethane containing urea bonds are not particularly limited; for example, sheet-like waste materials can be used, and the mixed sample can be prepared by adjusting their sizes as appropriate and mixing them. In the soil, it is preferable to attract as many bacteria as possible to the surface of the mixed sample and select bacteria from among them that can degrade the thermoplastic polyurethane containing the difficult-to-degrade urea bonds. For this purpose, it is preferable that the mixed sample contains a large amount of polyurethane without urea bonds. The mixing ratio of polyurethane without urea bonds and thermoplastic polyurethane containing urea bonds is not particularly limited, but from the above viewpoint, for example, a ratio of about 70:30 by mass is sufficient.
[0025] (Process for collecting microorganisms) Next, the mixed sample buried in the soil is periodically collected and analyzed to check whether urethane decomposition has progressed. Signs of urethane decomposition can be identified, for example, by examining the spectral waveform using infrared spectroscopy, which shows the presence of ether bonds (COC) at 1100 Kaiser (cm²). -1 This can be done by checking whether a change is observed in the absorption peak of ) in the spectral waveform. -1 If the absorption peak of ) decreased, it means that the ether bond decreased, i.e., an indication of urethane decomposition was observed. When signs of urethane degradation are observed in the mixed sample, bacteria adsorbed onto the mixed sample are collected and selected as candidate strains capable of degrading urethane (primary selection).
[0026] (The process of culturing microorganisms) The bacteria collected as described above may include bacteria capable of degrading recalcitrant polyurethane containing urea bonds. Therefore, further selection (secondary selection) will be performed to determine their degrading capabilities. Specifically, the bacteria collected as described above are cultured with shaking in an inorganic salt medium containing a simple standard substance having a urea bond, which is the target of degradation, as a carbon source and a nitrogen source, and the bacteria with high growth potential are further narrowed down and selected. As a simple standard substance having a urea bond, for example, 1,3-dimethylurea (DMU) (see formula (5) below) or 1,3-diethylurea (DEU) (see formula (6) below) can be used. Because DMU and DEU have simple structures, bacteria can act on the urea bond without being affected by other functional groups, making it easier to select bacteria that have the ability to decompose thermoplastic polyurethanes containing urea bonds.
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[0029] Bacteria grown in inorganic salt media containing DMU and DEU are microorganisms that can decompose urea bonds and use them as a carbon and nitrogen source for growth; therefore, they possess the ability to decompose polyurethane containing urea bonds.
[0030] When isolating superior bacterial strains with higher urethane degrading ability from the bacteria selected as described above, it is advisable to establish quantitative criteria for determining the state of bacterial attachment to thermoplastic polyurethane and urethane degrading ability. For example, the following method can be used to select the bacterial strain with the highest urethane degrading ability.
[0031] -Analysis by infrared spectroscopy- By performing infrared spectroscopy on thermoplastic polyurethane after treatment with novel degrading bacteria, it is possible to evaluate the urethane degradation ability of these bacteria. The 740 Kaiser peak in the infrared spectroscopy spectrum represents a characteristic peak of the first amine produced when the urea bond is degraded. When a resin containing a urea bond undergoes hydrolysis, it is presumed that the nitrate bond of the secondary amine is decomposed by the nitrate bond, as shown in equation (7) below, generating the primary amine R-NH2. When the urea bond is decomposed, infrared spectroscopy analysis reveals that the primary amine exhibits a peak while the secondary amine does not. Therefore, by reading the peak fluctuation around 740 Kaiser, the progress of urethane decomposition can be estimated.
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[0033] As described above, by focusing on the peak ratio around 740 Kaiser in the spectrum obtained by infrared spectroscopy, a correlation with the weight loss rate of thermoplastic polyurethane can be obtained. Therefore, the polyurethane decomposition activity of novel degrading bacteria can be accurately judged from the perspective of changes in the molecular structure of thermoplastic polyurethane, and superior strains can be selected.
[0034] -Identification of pore count using SEM observation and Imagej- (SEM observation of the surface of thermoplastic polyurethane) By photographing the surface layer of thermoplastic polyurethane treated with novel degrading bacteria at 2000x magnification using a scanning electron microscope (SEM), the attachment status of the novel degrading bacteria can be evaluated.
[0035] (Measurement of pore count) The particle analysis mode of the image processing software ImageJ is used to measure the number of pores in thermoplastic polyurethane treated with a novel degrading bacterium. By quantitatively analyzing the state of the pores (number of pores, total pore size area, total area ratio, etc.) appearing on the surface of the thermoplastic polyurethane after degradation by the novel degrading bacterium, it is possible to grasp the characteristics of the novel degrading bacterium in terms of its ability to degrade polyurethane. This allows for the selection of bacterium suitable for material recycling of thermoplastic polyurethane. For measurement conditions, for example, the threshold can be set to a recognizable value of pores from 0 to around 60 to 100, and the pore size can be set to 10 to infinity pixels (1 μm = 7.97 pixels).
[0036] -Evaluation of melting properties by thermal analysis- By performing thermal analysis on thermoplastic polyurethane treated with novel degrading bacteria, and analyzing the softening initiation temperature, melting endpoint temperature, boiling point, and decomposition initiation temperature, it is possible to evaluate properties that are advantageous for reuse as thermoplastic resin. Thermal analysis can be performed using a thermal analyzer (TG-DTA).
[0037] <Microorganisms capable of degrading thermoplastic polyurethanes> The microorganisms having the ability to degrade thermoplastic polyurethanes according to the embodiments of the present invention belong to the genera Pseudomonas or Sinomonas and have the ability to degrade thermoplastic polyurethanes containing urea bonds. More specifically, the microorganisms according to the embodiments of the present invention are preferably Pseudomonas hibiscicola MS4102 strain, which was deposited with the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation on February 24, 2022, under accession number NITE P-03612, or Sinomonas atrocyanea ES2231 strain, which was deposited with the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation on February 24, 2022, under accession number NITE P-03613. These microorganisms are selected from soil by performing the microorganism selection method for thermoplastic polyurethane-degrading agents according to the embodiment of the present invention described above, as will be explained later.
[0038] <Method for decomposing thermoplastic polyurethane> A method for decomposing thermoplastic polyurethane according to an embodiment of the present invention comprises the step of reacting a thermoplastic polyurethane containing urea bonds with microorganisms having the ability to decompose thermoplastic polyurethane according to the above embodiment of the present invention. One method for allowing microorganisms to act on thermoplastic polyurethane is to add the thermoplastic polyurethane to a culture medium for microorganisms. When the thermoplastic polyurethane is in sheet form, it is preferable to process it in small pieces of approximately 1 cm x 1 cm. This makes it easier to reuse the thermoplastic polyurethane after it has been treated with microorganisms. Methods for reusing the thermoplastic polyurethane after it has been treated with microorganisms include, for example, melting it in a twin-screw kneader and processing it into pellets for injection molding for reuse.
[0039] The microorganisms having the ability to decompose thermoplastic polyurethanes, the method for decomposing thermoplastic polyurethanes, and the method for selecting microorganisms having the ability to decompose thermoplastic polyurethanes according to the present invention include the embodiments described below. (1) A species belonging to the genera Pseudomonas or Sinomonas, which has the ability to decompose thermoplastic polyurethanes containing urea bonds. Microorganisms. (2) The microorganism is Pseudomonas hibiscicola MS4102 strain, identified by accession number NITE P-03612. The microorganisms described in (1) above. (3) The microorganism is Sinomonas atrocyanea strain ES2231, identified by accession number NITE P-03613. The microorganisms described in (1) above. (4) A thermoplastic polyurethane containing a urea bond, comprising the step of reacting it with a microorganism described in any one of the above items (1) to (3), Methods for decomposing thermoplastic polyurethane. (5) A step of burying a mixed sample of thermoplastic polyurethane containing urea bonds and polyurethane without urea bonds in the soil, After analyzing the buried mixed sample to confirm urethane decomposition, the process involves collecting microorganisms adsorbed on the mixed sample. Having, A method for selecting microorganisms capable of decomposing thermoplastic polyurethanes. (6) The method further comprises culturing the collected microorganisms in an inorganic salt medium containing 1,3-dimethylurea, 1,3-diethylurea, or 1,3-dimethylurea and 1,3-diethylurea. A method for selecting microorganisms that have the ability to decompose thermoplastic polyurethane as described in (5) above. [Examples]
[0040] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. [Examples] (First selection round) As a first-stage selection, a mixed sample was prepared and buried in the soil. The mixed sample was prepared by mixing a thermoplastic polyurethane containing persistent urea bonds with a general-purpose polyurethane that does not contain urea bonds, in a mass ratio of 30:70. The mixed sample was also prepared in the form of a sheet approximately 1 cm x 1 cm in size. Mixed samples buried in the soil were periodically collected, and the presence or absence of signs of decomposition was checked by examining the spectral waveform using infrared spectroscopy. As a result, as shown in Figure 1, no change was observed in the spectral waveform for 14 months after the mixed sample was buried in the soil. However, as shown in Figure 2, after 17 months (approximately 1.5 years), a change in the 1100 Kaiser (cm²) spectrum was observed, indicating the presence of ether-bound COC. -1 A decrease in the absorption peak was observed, indicating signs of decomposition. This suggested that urethane decomposition by soil bacteria was progressing, so bacteria adsorbed on the mixed sample were sampled to search for new decomposing bacteria. In Figures 1 and 2, "Spectrum 1" in the middle panel shows the spectrum obtained by measuring the mixed sample from the surface, and "Spectrum 2" in the bottom panel shows the spectrum obtained by measuring the mixed sample from the back.
[0041] (Second selection round) In the second selection stage, the microorganisms collected in the first selection stage were cultured in an inorganic salt medium containing 1,3-dimethylurea (DMU) or 1,3-diethylurea (DEU) with urea bonds. This allowed for the selection of highly proliferative bacteria from the microorganisms selected in the first stage that could grow using DMU or EDU as a carbon and nitrogen source. Culturing using DMU-containing medium resulted in the selection of 36 isolated bacteria (DMU-utilizing bacteria). Subsequently, by evaluating the growth potential of each isolated bacteria, the number of useful strains was narrowed down to 11, as shown in Table 1 below. Furthermore, by evaluating the growth potential in DEU-containing medium, the number of strains effective in both conditions was narrowed down to 5.
[0042] [Table 1] R2A medium: Standard medium DMU medium: A medium containing DMU in an inorganic salt medium. DEU medium: A medium containing DEU in an inorganic salt medium.
[0043] For the R2A medium, we used commercially available R2A Agar (BD Difco™) or R2A Broth DAIGO (Fujifilm Wako Pure Chemical Corporation). Furthermore, the evaluation of growth capacity shown in Table 1 is a subjective relative assessment of the growth after spreading bacterial cells on an agar plate and culturing them, and the evaluation criteria are as follows. + : Some of the smeared bacteria proliferate and form colonies. ++: Approximately half of the smeared bacterial cells proliferate and form colonies. +++: The entire smeared bacterial cells proliferate and form colonies. ++++: The entire smear of bacterial cells shows vigorous growth and colony formation. - : The smeared bacterial cells do not proliferate or form colonies. -+ : The growth and colony formation of the smeared bacterial cells are unclear (i.e., some of the smeared bacterial cells appear to have grown and formed colonies, but the growth and colony formation are not clearly discernible).
[0044] Furthermore, when cultured using DEU-containing medium, 44 isolated bacteria (DEU-assimilating bacteria) were selected. Subsequently, by evaluating the growth potential of each isolated bacterium in R2A medium and DEU medium, four useful strains were narrowed down to four, as shown in Table 2 below.
[0045] [Table 2]
[0046] The evaluation of proliferation ability shown in Table 2 was performed in the same manner as the evaluation of proliferation ability shown in Table 1.
[0047] By evaluating the growth capacity as described above, nine superior strains were selected (five DMU-utilizing strains and four DEU-utilizing strains) as shown in Table 3 below. By narrowing down the useful strains through growth capacity evaluation, the search time required to identify the selected strains was significantly reduced. This method allows for quick acquisition of information on the characteristics and harmfulness of the bacteria, enabling efficient implementation of studies toward practical application.
[0048] [Table 3] Database used: ·Ribosomal Database Project (RDP) ·National Center for Biotechnology Information (NCBI) ·DNA Data Bank of Japan (DDBJ)
[0049] From the nine degrading bacteria strains narrowed down in the second selection, the following two strains were selected for DMU and DEU respectively, due to their high adsorption and growth potential.
[0050] ·MS4102 strain (Pseudomonas hibiscicola) Genus Pseudomonas Domain: Bacteria Phylum: Proteobacteria Class: Gammaproteobacteria Order: Pseudomonadales Family: Pseudomonadaceae Genus: Pseudomonas
[0051] ·MS4111 strain (Brevundimonas olei) Genus Pseudomonas Domain: Bacteria Phylum: Proteobacteria Class: Alphaproteobacteria Order: Caulobacterales Family: Caulobacteraceae Genus: Brevundimonas
[0052] ·ES1243 strain (Rhizobium nepotum) Genus Pseudomonas Domain: Bacteria Phylum: Proteobacteria Class: Alphaproteobacteria Order: Rhizobiales Family: Rhizobiaceae Genus: Rhizobium
[0053] ·ES2231 strain (Sinomonas atrocyanea) Genus Pseudomonas Domain: Bacteria Phylum: Actinobacteria Class: Actinobacteria Order: Micrococcales Family: Micrococcaceae Genus: Ainomonas
[0054] (Evaluation of the degradability of thermoplastic polyurethanes) The four isolated bacterial strains selected as described above were each directly reacted with thermoplastic polyurethane, and their urethane degradation properties were evaluated using the following method. -Preparation- • Molding of thermoplastic polyurethane (1cm x 1cm x 0.5mm thickness) • Weight measurement of thermoplastic polyurethane (weight immediately before culturing: α) -culture- • Pre-culture of isolated bacteria • Collection of pre-cultured bacteria, washing of thermoplastic polyurethane • Main culture in a culture medium supplemented with thermoplastic polyurethane (30°C, 3 weeks, 100 rpm). -measurement- • Gravimetric measurement of thermoplastic polyurethane (weight after culture: β) • SEM observation of thermoplastic polyurethane Image analysis of thermoplastic polyurethanes using ImageJ (counting the number of pores).
[0055] The weight loss rate was calculated using the following formula based on the weight of thermoplastic polyurethane measured before and after treatment with each isolated bacterium. Weight reduction rate (%)=(α-β) / β×100
[0056] The isolated bacteria were cultured at 30°C, a temperature at which bacterial growth is high, and the pH was set to 7. The composition of the culture medium (inorganic salt liquid medium) used is shown in Table 4 below.
[0057] [Table 4]
[0058] Furthermore, the effects of pretreatment before treating thermoplastic polyurethane with isolated bacteria were also investigated. As pretreatment to promote the decomposition of thermoplastic polyurethane, a chemical treatment method using oleic acid, which was effective in the invention described in Japanese Patent No. 6489542, and a physical method using plasma treatment, which was effective in the invention described in Japanese Patent Application Publication No. 2021-161338 were used. Each case was then compared with an untreated sample.
[0059] The conditions for plasma treatment were as follows: -Plasma emission irradiation conditions- Chamber pressure: 40 Pa RF power supply: 100V Bias voltage: 600V Sample height: 23 mm Source gas: Atmosphere Plasma irradiation time: 120 seconds
[0060] The conditions for oleic acid treatment were as follows: Oleic acid (manufactured by Wako Pure Chemical Industries, Ltd., grade: Wako Grade 1) was diluted to a 10% (w / w) concentration with 99% ethanol and added to a 100 mL Erlenmeyer flask. Approximately 4g to 5g of thermoplastic polyurethane was placed in the 100mL Erlenmeyer flask mentioned above, completely immersing the polyurethane, and the flask was covered with aluminum foil and treated at room temperature for 1 hour. After the processing time had elapsed, the Erlenmeyer flask was washed with tap water and distilled water, and then further washed with distilled water using ultrasound. The thermoplastic polyurethane was removed from the Erlenmeyer flask and washed again with distilled water. After that, it was thoroughly dried at 40°C (overnight) and sterilized at 121°C for 20 minutes.
[0061] Furthermore, metal ions act as catalysts for plastics, accelerating the auto-oxidation reaction of polymers. This is called a redox reaction. Among metal ions, copper ions are particularly sensitive to this reaction (copper damage). In particular, the presence of carboxyl groups (R0H) or carbonyl groups (RCO) makes the redox reaction more likely to proceed. Therefore, we also investigated whether the degradation of thermoplastic polyurethane by redox reactions of metal ions was accelerated when isolated bacteria were applied to it. Specifically, we evaluated the results by comparing them with culture media in which the concentrations of Cu and Fe ions were increased tenfold.
[0062] Figure 3 shows the weight loss rate when each isolated bacterium was applied to thermoplastic polyurethane (untreated TPU) without pretreatment. The graph on the left of Figure 3 shows the results when the concentrations of Cu and Fe ions in the culture medium for the isolated bacterium were reduced to a constant value, while the graph on the right shows the results when the concentrations of Cu and Fe ions in the culture medium for the isolated bacterium were increased tenfold. As shown in Figure 3, the novel degrading bacterium, strain ES2231, exhibited high degradability of thermoplastic polyurethane, which could not be degraded by conventional c13a actinomycetes, even without pretreatment, and showed a weight reduction rate of over 7%. Furthermore, regarding the acceleration of decomposition by redox reactions of metal ions, evaluation was conducted by increasing the concentrations of Cu and Fe ions tenfold, but no signs of accelerated decomposition were observed.
[0063] Figure 4 shows the weight loss rate when each isolated bacterium was applied to thermoplastic polyurethane (plasma-treated TPU) that had undergone plasma treatment as a pretreatment. The graph on the left of Figure 4 shows the results when the concentrations of Cu ions and Fe ions in the culture medium for the isolated bacterium were reduced to a constant value, while the graph on the right shows the results when the concentrations of Cu ions and Fe ions in the culture medium for the isolated bacterium were increased tenfold. As shown in Figure 4, the novel degrading bacterium, strain ES2231, exhibited the highest degradation rate, similar to the case without pretreatment. However, the weight reduction rate indicating degradation was approximately 7%, which was slightly lower than the case without pretreatment, and no degradation acceleration due to changes in ion concentration was observed.
[0064] Figure 5 shows the weight loss rate when each isolated bacterium was applied to thermoplastic polyurethane (plasma-oleic acid treated TPU) that had undergone plasma treatment and oleic acid treatment as pretreatment. The graph on the left of Figure 5 shows the results when the concentrations of Cu ions and Fe ions in the culture medium for the isolated bacterium were reduced to a constant value, while the graph on the right shows the results when the concentrations of Cu ions and Fe ions in the culture medium for the isolated bacterium were increased tenfold. As shown in Figure 5, pretreatment with oleic acid significantly reduced the weight loss rate, inhibiting the decomposition process by new degrading bacteria. Furthermore, no decomposition-promoting effect was observed due to changes in ion concentration.
[0065] <Measuring the number of pores> Next, the number of pores in thermoplastic polyurethane after treatment with each isolated bacterium was measured. By quantitatively analyzing the state of the pores appearing on the surface of the decomposed thermoplastic polyurethane (number of pores, total pore size area, total area ratio, etc.), it is possible to grasp the characteristics of the bacterium's ability to decompose polyurethane, and as a result, select a bacterium suitable for material recycling. The pore count was measured using the particle analysis mode in ImageJ. The measurement conditions were as follows: Measurement conditions: Set the threshold to a value between 0 and a recognizable pore size (usually around 60-100). Pore size set to 10 to ∞ pixels (1 μm = 7.97 pixels)
[0066] The results are shown in Figure 6. As shown in Figure 6, the increase in pore number tended to be greater with strain ES2231 than with the novel degrading bacterium strain MS4102. From the SEM images shown later, spherical, micron-order bacteria were adsorbed onto the surface of the thermoplastic polyurethane, and it is thought that the degree of adsorption affected the pore number.
[0067] The meanings of the abbreviations shown in Figure 6 are as follows: Untreated ×1: No pretreatment was performed, and the metal ion concentration was adjusted to a constant value. Untreated × 10: No pretreatment was performed, and the metal ion concentration was increased tenfold. P×1: Plasma treatment is performed as a pretreatment, and the metal ion concentration is set to a constant multiple. P×10: Plasma treatment is performed as a pretreatment, and the metal ion concentration is increased tenfold. PO×1: Pre-treated with plasma and oleic acid to achieve a constant metal ion concentration. PO×10: Pre-treated with plasma and oleic acid to increase the metal ion concentration tenfold. Control: Sample without the application of the novel degrading bacteria (comparative example)
[0068] <Surface SEM observation> The surface of thermoplastic polyurethane was photographed at 2000x and 8000x magnification using a SEM (Miniscope TM3030-HITACHI) after treatment with the novel degrading bacteria strains MS4102 or ES2231. The state of adsorption of the novel degrading bacteria to the surface was observed and evaluated. The results are shown in Figure 7. The amount of newly adsorbed degrading bacteria was highest in the untreated sample, followed by the P-treated sample, and then the PO-treated sample, which correlated with the weight reduction rate indicating degrading ability. Oleic acid treatment resulted in low adsorption and a decrease in degrading ability. "Unprocessed" means that no pre-processing has been performed. "P treatment" refers to a process that has undergone plasma treatment as a pretreatment. "PO treatment" refers to a process that has undergone plasma treatment and oleic acid treatment as pretreatment.
[0069] <Infrared spectroscopy> Infrared spectroscopy was performed on thermoplastic polyurethanes treated with novel degrading bacteria strains ES1243, ES2231, or MS4102, and the peak intensity of the 740 Kaiser was compared. The results are shown in Figure 8. No pretreatment was performed on the thermoplastic polyurethanes before treating them with the novel degrading bacteria. In Figure 8, "Control" refers to samples that were not treated with the novel degrading bacteria (comparative example). As shown in Figure 8, the control group, which did not undergo pretreatment, showed double peaks around 740 and 700 Kaiser. This indicates the presence of a CH2 methylene group. On the other hand, when treated with the novel degrading bacterium ES2231, the 700 Kaiser peak disappeared, and the ratio of the 740 Kaiser peak increased. As mentioned above, the 740 Kaiser peak is a peak that exhibits characteristics of the primary amine, and it is presumed that it newly appeared due to the decomposition of the urea bond, and that this caused the double peak of methylene to disappear. As shown in Figure 8, the weight loss rate of thermoplastic polyurethane correlated with the peak ratio of 740 Kaiser, which indicates the decomposition of urea bonds, confirming that the novel degrading bacteria contribute to the urethane decomposition process.
[0070] As described above, the evaluation of the urethane degradation ability of the novel degrading bacteria showed that the highest degradation ability was achieved without any pretreatment step before degrading thermoplastic polyurethane. Therefore, the novel degrading bacteria can degrade thermoplastic polyurethane using a degradation method that omits the pretreatment step, and a significant reduction in the cost required for degradation can be expected.
[0071] <Melting properties> Thermal analysis was performed on thermoplastic polyurethane treated with novel degrading bacteria, and the melting properties of the material were evaluated as follows: A) softening initiation temperature, B) melting endpoint temperature, C) boiling point, D) decomposition initiation temperature, E) melting sensitivity (BA), and F) melting stability (CB). These characteristic values can be used to determine the advantages of the material for reuse as a thermoplastic resin. Melt sensitivity represents the temperature difference from the start of softening to the endpoint temperature of complete melting, and is an indicator of how easily a material melts. A smaller melt sensitivity value indicates a faster melting rate and higher melt sensitivity. When reusing thermoplastic polyurethane after treatment with new degrading bacteria as new thermoplastic polyurethane, higher melt sensitivity results in more stable moldability. Melt stability indicates the temperature difference between the melting endpoint temperature and the boiling point, and is an indicator of the stability of the material's molten state. The higher the melt stability value, the more stable the molten state becomes, and the improved the material properties as a thermoplastic resin. Thermal analysis was performed using a Rigaku Corporation "Thermo Plus EVO2" (thermogravimetric differential thermal analyzer (TG-DTA)). Specifically, each sample was heated at a heating rate of 10°C / min while supplying air at a rate of 200 ml / min. The weight change up to 700°C, as well as the thermal changes associated with physical and chemical changes, were detected as a function of temperature compared with a reference substance (Al2O3). The results are shown in Table 5 below. The measured melting sensitivity and melting stability results are shown in Figure 9.
[0072] [Table 5]
[0073] In Table 5 and Figure 9, "Untreated" means that the thermoplastic polyurethane was not pretreated before being treated with the novel degrading bacteria. Similarly, "Plasma" in Table 5 and Figure 9 means that the thermoplastic polyurethane was plasma-treated before being treated with the novel degrading bacteria, and "Plasma + Oleic Acid" means that both plasma treatment and oleic acid treatment were performed. In addition, "Control" in Table 5 and Figure 9 means that no novel degrading bacteria were treated (comparative example).
[0074] Furthermore, Figure 10 shows a photograph illustrating the state of thermoplastic polyurethane after heating following treatment with the novel degrading bacterium, strain ES2231. When thermoplastic polyurethane was decomposed using the novel degrading bacterium strain ES2231, it was confirmed that the hydrogen bonds at the ends of the urea bonds were acted upon, weakening the bonding force, and that the application of thermal energy made molecular displacement more likely, resulting in complete melting at around 200°C, similar to the case of the powdered raw material (see the photograph on the right in Figure 10).
[0075] In particular, thermoplastic polyurethane treated with novel degrading bacteria without pretreatment exhibited the highest thermal stability and transformed into a material with properties suitable for material recycling as a thermoplastic resin. Furthermore, Figure 11 shows a photograph comparing the amount of ash when the temperature was raised to 700°C to completely decompose the resin. As shown in Figure 11, while the powdered raw material and molded waste material of thermoplastic polyurethane retained ash and carbonized material, the thermoplastic polyurethane treated with the new decomposing bacteria decomposed almost completely, leaving virtually no residue, resulting in environmentally friendly material properties. In Figure 11, "TPU raw material" refers to the powdered raw material of thermoplastic polyurethane. Similarly, "TPU molded product" refers to waste material of molded thermoplastic polyurethane, and "TPU decomposed product" refers to thermoplastic polyurethane after being treated with the novel decomposing bacterium, strain ES2231. Furthermore, as mentioned above, the molded thermoplastic polyurethane products that were not treated with the novel degrading bacteria developed a rigid molecular structure due to partial crosslinking during melting and solidification, preventing complete melting and resulting in carbonization as the temperature rose, making them unsuitable for reuse as thermoplastic resins (see Figure 12).
[0076] As shown above, the selected novel degrading bacteria demonstrated high degradability against recalcitrant thermoplastic polyurethane. In particular, strain ES2231 showed high degradability under conditions without pretreatment of thermoplastic polyurethane, suggesting a significant reduction in the degradation process for thermoplastic polyurethane. Furthermore, conventionally, molded thermoplastic polyurethane products formed a strong cross-linked structure during the melt-solidification process, making uniform melting impossible and thus unsuitable for reuse as thermoplastic resins. However, by applying a decomposition treatment with novel decomposing bacteria, the melting properties were changed to those of a reusable thermoplastic resin. The melting characteristics showed the highest melt stability when thermoplastic polyurethane was not pretreated, demonstrating properties suitable for use as a thermoplastic resin. Therefore, simplification of the process can be expected even when reusing thermoplastic polyurethane as a thermoplastic resin. Furthermore, the thermoplastic polyurethane treated with the selected novel decomposition bacteria decomposed completely upon heating, leaving almost no decomposition residue (ash, carbon, etc.), thus possessing environmentally friendly material properties. [Explanation of Symbols]
[0077] 141 Special Structure A (Highly Cohesive Polyester) 142 Characteristic Structure B (End Group)
Claims
1. It belongs to the genus Pseudomonas, and is identified as Pseudomonas hibiscicola MS4102 strain with accession number NITE BP-03612, and has the ability to degrade thermoplastic polyurethanes containing urea bonds. Microorganisms.
2. A strain of Sinomonas atrocyanea ES2231 belonging to the genus Sinomonas, identified by accession number NITE BP-03613, which has the ability to degrade thermoplastic polyurethanes containing urea bonds. Microorganisms.
3. The process involves reacting a thermoplastic polyurethane containing a urea bond with the microorganism described in claim 1 or claim 2. Methods for decomposing thermoplastic polyurethane.
4. A process of burying a mixed sample of thermoplastic polyurethane containing urea bonds and polyurethane without urea bonds in the soil, The buried mixed sample is analyzed and urethane decomposition is confirmed, followed by a step of collecting microorganisms adsorbed on the mixed sample. Having, A method for selecting microorganisms capable of decomposing thermoplastic polyurethanes.
5. The method further comprises the step of culturing the collected microorganisms in an inorganic salt medium containing 1,3-dimethylurea, 1,3-diethylurea, or 1,3-dimethylurea and 1,3-diethylurea. A method for selecting microorganisms having the ability to decompose thermoplastic polyurethanes as described in claim 4.