Method for decomposing plastic

Abstract

To provide a method for decomposing plastic in which biodegradable plastic is degraded by using a microorganism that can be raised even in a low temperature region.SOLUTION: Biodegradable plastic is degraded by using a microorganism that belongs to the genus Glomerella having plastic degrading ability or an extract thereof. Further, biodegradable plastic is degraded by using a microorganism that belongs to the genus Fusarium having plastic degrading ability or an extract thereof together with the microorganism that belongs to the genus Glomerella or an extract thereof. Furthermore, biodegradable plastic is degraded by using a microorganism that belongs to the genus Aspergillus having plastic degrading ability or an extract thereof together with the microorganism that belongs at least to the genus Glomerella or an extract thereof.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] The present invention relates to a technology for decomposing plastics, and particularly to a method for decomposing plastics using microorganisms.

Background Art

[0002] In recent years, marine pollution caused by plastics has become a problem. That is, most of the plastics that have flowed out into the environment flow into rivers and finally reach the sea, so plastics have a great adverse impact on the marine ecosystem. In addition, the impact of plastics on the ocean also damages industries, resulting in large economic losses. Plastics become microplastic particles in the sea, but general plastics do not naturally decompose even when they are broken down, and it is considered that they will continue to remain for more than several hundred years. In such a situation, instead of conventional plastics, the use of biodegradable plastics that can be naturally decomposed has been spreading.

[0003] However, even when biodegradable plastics are used, it takes time for them to naturally decompose, and the plastics will remain in the sea for a long time. Therefore, it is difficult to solve the problem of marine pollution by this. By the way, some kinds of microorganisms have the ability to decompose plastics. If plastics incorporating such plastic-decomposing microorganisms can be developed in the future, there is a possibility that plastics can be rapidly decomposed by the power of microorganisms and marine pollution can be suppressed.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0005] By the way, microorganisms that can grow in a high temperature range (around 50°C) or a room temperature range (around 25°C) such as compost have been used as plastic decomposition materials. However, these microorganisms have a problem that they are not suitable for growth at low temperatures. On the other hand, in order to achieve rapid natural decomposition of plastics by microorganisms in the ocean, it is desirable to be able to use microorganisms that can grow at various temperatures including low temperature ranges. Therefore, the present inventors have conducted intensive research and found that microorganisms that can grow even in a low temperature range, particularly microorganisms belonging to the genus Glomerella, have plastic-degrading ability, and completed the present invention.

[0006] Here, Patent Documents 1 to 3 describe decomposing biodegradable plastics using various microorganisms. However, decomposing biodegradable plastics using microorganisms belonging to the genus Glomerella is not described. In addition, no other documents describing the matter were found.

[0007] The present invention has been made in view of the above circumstances, and an object thereof is to provide a method for decomposing plastics that decomposes biodegradable plastics using microorganisms that can grow even in a low temperature range.

Means for Solving the Problems

[0008] To achieve the above object, the method for decomposing plastics of the present invention is a method for decomposing biodegradable plastics using microorganisms belonging to the genus Glomerella having plastic-degrading ability or an extract thereof. In addition, the method for decomposing plastics of the present invention is preferably a method for decomposing biodegradable plastics using microorganisms belonging to the genus Fusarium having plastic-degrading ability or an extract thereof together with the microorganisms belonging to the genus Glomerella or an extract thereof. Furthermore, the method for decomposing plastics of the present invention preferably uses a microorganism belonging to the genus Aspergillus having plastic-degrading ability or an extract thereof together with the microorganism belonging to the genus Glomerella or an extract thereof to decompose biodegradable plastics.

[0009] Also, the method for decomposing plastics of the present invention preferably has an ester bond in its chemical structure. Furthermore, the method for decomposing plastics of the present invention preferably uses the biodegradable plastic as polycaprolactone. Also, the method for decomposing plastics of the present invention preferably uses the extract as esterase.

Advantages of the Invention

[0010] According to the present invention, it is possible to provide a method for decomposing biodegradable plastics using microorganisms that can grow even in a low-temperature range.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

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Figure 4

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Figure 6

Embodiments for Carrying Out the Invention

[0012] Hereinafter, embodiments of the plastic decomposition method of the present invention will be described in detail. However, the present invention is not limited to the specific contents of the following embodiments and the examples described later.

[0013] The plastic decomposition method of this embodiment is characterized by decomposing biodegradable plastic using a microorganism belonging to the genus Glomerella having plastic-degrading ability or an extract thereof. As the microorganism belonging to the genus Glomerella, for example, Glomerella cingulate (NBRC107001) or Glomerella cingulate (NBRC107004) can be preferably used.

[0014] Also, as the extract, esterase extracted from a microorganism belonging to the genus Glomerella is used. The extraction method can be, for example, a method in which the microorganism is statically cultured in the dark for a predetermined number of days, then the cultured cells and the culture filtrate are separated and recovered respectively, and the obtained culture filtrate is used as the extract-containing solution.

[0015] The biodegradable plastics decomposed by the plastic decomposition method of this embodiment may be those having an ester bond in their chemical structure, and are not particularly limited. For example, polycaprolactone, polylactic acid, polylactic acid / polycaprolactone copolymer, polyglycolic acid, polylactic acid / polyether copolymer, butanediol / long-chain dicarboxylic acid copolymer, polybutylene adipate / terephthalate copolymer, polytetramethylene adipate-co-terephthalate, polyethylene terephthalate succinate, polybutylene succinate, polybutylene succinate adipate, polyvinyl alcohol, etc. can be mentioned.

[0016] Here, as a method for confirming whether plastics are decomposed by microorganisms, conventionally, there has been a method of preparing an agar medium containing plastics dissolved in an appropriate organic solvent, culturing microorganisms, and confirming the presence or absence of clear zone formation derived from plastic decomposition around the microorganisms. However, this method is effective for cell-dividing microorganisms such as bacteria and yeasts because the formation of clear zones is easy to confirm. However, in the case of filamentous fungi (molds) accompanied by hyphal elongation, depending on the degree and color of hyphal elongation, it may be difficult to confirm the formation of clear zones.

[0017] Therefore, in order to confirm the effect of the plastic decomposition method of this embodiment, a culture medium containing a biodegradable plastic dissolved as a carbon source and an undissolved biodegradable plastic mass is prepared, and using this, microorganisms are cultured to detect whether the biodegradable plastic mass is decomposed.

[0018] As shown in FIG. 1, such a culture medium can be formed based on an agar medium or the like, and an undissolved biodegradable plastic mass can be formed in the medium. This biodegradable plastic mass is usually formed in a state of sinking to the bottom of the medium. This culture medium can be obtained, for example, by adding polycaprolactone (PCL) to a Czapek agar medium, stirring while heating to suspend it, then adding agar, and autoclaving.

[0019] Next, the decomposition of the biodegradable plastic mass will be described based on the image shown in Fig. 2. The left photo in Fig. 2 shows the formation of a biodegradable plastic mass (polycaprolactone mass) in the culture medium, and shows the state after culturing the filamentous fungus (Aspergillus flavus (NBRC6343)) in this culture medium for 8 days. Hyphae are extending from the colony of the filamentous fungus. The upper right photo in the figure shows the biodegradable plastic mass, and the lower right photo in the figure shows the state where the biodegradable plastic mass has been decomposed after culturing the filamentous fungus for 20 days. In the examples described later, the effect of the plastic decomposition method is confirmed using such a culture medium.

[0020] Also, in the confirmation of the effect of the plastic decomposition method, the change in the size of the biodegradable plastic mass can be calculated based on microscopic photos to detect the decomposition of the biodegradable plastic mass. That is, since the biodegradable plastic mass is usually formed at the bottom of the culture medium, for example, when culturing in a container such as a transparent petri dish, its state can be observed from the bottom side of the container. In addition, the state of the biodegradable plastic can be visually observed from the bottom side of the container to confirm the presence or absence of decomposition of the biodegradable plastic mass.

[0021] Furthermore, when the microorganism is a filamentous fungus, the decomposition area of the biodegradable plastic mass can be calculated based on the following formula to quantitatively measure the plastic degradation ability of each microorganism. Decomposition area = (Area of the biodegradable plastic mass - Area of the biodegradable plastic mass after the number of culture days from the hyphae reaching) / Number of culture days from the hyphae reaching

[0022] In addition, in the method for decomposing plastics of the present embodiment, it is preferable to decompose biodegradable plastics using a microorganism belonging to the genus Fusarium having plastic-degrading ability or an extract thereof together with a microorganism belonging to the genus Glomerella or an extract thereof. As the microorganism belonging to the genus Fusarium, for example, Fusarium oxysporum (NBRC9971) and Fusarium sacchari (JCM9676) can be preferably used. Regarding the extract and the extraction method, they are the same as those in the case of using a microorganism belonging to the genus Glomerella.

[0023] As shown in Test 1 described later, such microorganisms belonging to the genus Glomerella and the genus Fusarium can grow in the room temperature range of about 25°C to the low temperature range of about 4°C.

[0024] In addition, in the method for decomposing plastics of the present embodiment, it is preferable to decompose biodegradable plastics using a microorganism belonging to the genus Aspergillus having plastic-degrading ability or an extract thereof together with a microorganism belonging to the genus Glomerella or an extract thereof. Furthermore, in the method for decomposing plastics of the present embodiment, it is also preferable to decompose biodegradable plastics using a microorganism belonging to the genus Aspergillus having plastic-degrading ability or an extract thereof together with a microorganism belonging to the genus Glomerella or an extract thereof and a microorganism belonging to the genus Fusarium having plastic-degrading ability or an extract thereof.

[0025] As the microorganism belonging to the genus Aspergillus, for example, Aspergillus flavus (NBRC6343) can be preferably used. Regarding the extract and the extraction method, they are the same as those in the case of using a microorganism belonging to the genus Glomerella.

[0026] As shown in Test 1 described later, the microorganism belonging to the genus Aspergillus can grow in the high temperature range of about 45°C. Therefore, if a microorganism belonging to such Aspergillus is used together with a microorganism belonging to Glomerella or a microorganism belonging to Fusarium, it becomes possible to grow at least any one of these microorganisms in a wide temperature range from a high temperature range to a low temperature range.

[0027] As described above, according to the method for decomposing plastic of the present embodiment, it is possible to decompose biodegradable plastic using a microorganism belonging to Glomerella or a microorganism belonging to Fusarium that can grow even in a low temperature range. Further, by further using a microorganism belonging to Aspergillus together with these, it becomes possible to grow at least any one of these microorganisms in a wide temperature range from a high temperature range to a low temperature range.

Example

[0028] Hereinafter, tests conducted to confirm the effects and the like of the method for decomposing plastic according to the embodiment of the present invention will be described. [Test 1] A test was conducted to confirm the growth ability of the microorganisms used in the method for decomposing plastic according to the present embodiment at various temperatures. Specifically, as microorganisms, filamentous fungi of Glomerella (Glomerella cingulate (NBRC107004)), Fusarium (Fusarium oxysporum (NBRC9971)), and Aspergillus (Aspergillus flavus (NBRC6343)) were prepared. Also, as culture media, a plurality of petri dishes having a PDA (Potato Dextrose Agar) medium were created.

[0029] Then, the filamentous fungi of Glomerella, Fusarium, and Aspergillus were statically cultured in the dark at 45°C, 25°C, and 4°C, respectively, in three petri dishes, and it was determined whether growth was possible by checking whether colonies were formed on the 21st day of culture.

[0030] As a result, as shown in Fig. 3, it was confirmed that the filamentous fungi of the genus Glomerella did not grow at 45°C, but grew at 25°C and 4°C. In addition, it was confirmed that the filamentous fungi of the genus Fusarium also did not grow at 45°C, but grew at 25°C and 4°C. On the other hand, it was confirmed that the filamentous fungi of the genus Aspergillus grew at 45°C and 25°C, but did not grow at 4°C.

[0031] [Test 2] A test was conducted to confirm the decomposition of biodegradable plastic masses by culturing microorganisms in a culture medium that can be used to confirm the effects of the plastic decomposition method according to this embodiment. Specifically, in a sucrose-free Czapek medium prepared in advance (dissolving 3 g of NaNo 3 3, 1 g of K 2 HPO 4 1, 0.5 g of MgSO 4 ·7H 2 O, 0.5 g of KCL, and 0.01 g of FeSO 4 ·7H 2 O in order into 1000 ml of purified water, with a pH of 7.3), polycaprolactone (PCL) was added as a single carbon source to a concentration of 0.5% (w / v). Using a heat stirrer (set at around 280°C), heat stirring was performed, and while crushing the pellets with a spatula, suspension was carried out to obtain a liquid medium. Next, agar was added to the liquid medium to a concentration of 1.5% (w / v), autoclaved, dispensed into petri dishes with a diameter of 90 mm, and solidified at room temperature to obtain an agar medium. Biodegradable plastic masses were present in the obtained agar medium.

[0032] In addition, filamentous fungi of the genus Glomerella (Glomerella cingulate (NBRC107004)), the genus Fusarium (Fusarium oxysporum (NBRC9971)), and the genus Aspergillus (Aspergillus flavus (NBRC6343)) were each cultured in a PDA (Potato Dextrose Agar) medium, and cell disks with a diameter of 5 mm were punched out from the grown colonies using a cork borer. Next, the mycelial disks were brought into contact with the agar medium, and those inoculated with filamentous fungi of the genus Glomerella were designated as Example 1, those inoculated with filamentous fungi of the genus Fusarium were designated as Example 2, and those inoculated with filamentous fungi of the genus Aspergillus were designated as Example 3.

[0033] Then, static culture was started at 30°C in the dark, and the state of the polycaprolactone mass was observed microscopically over time. Fig. 4 shows photographs depicting the state of decomposition of the polycaprolactone mass after a predetermined number of culture days in each example. As shown in the figure, it was confirmed that the polycaprolactone mass was decomposed on the 13th day or the 20th day of culture by the filamentous fungi of the genus Glomerella, the genus Fusarium, and the genus Aspergillus.

[0034] [Test 3] A test was conducted to confirm the esterase activity by culturing microorganisms in a culture medium that can be used to confirm the effect of the plastic decomposition method according to the present embodiment. That is, in the plastic decomposition method according to the present embodiment, microorganisms decompose biodegradable plastics by hydrolyzing ester bonds in the chemical structure of biodegradable plastics with esterase. Therefore, the presence or absence of esterase activity was confirmed for the extract of plastic-decomposing microorganisms.

[0035] Specifically, filamentous fungi of the genus Glomerella (Glomerella cingulate (NBRC107004)), the genus Fusarium (Fusarium oxysporum (NBRC9971)), and the genus Aspergillus (Aspergillus flavus (NBRC6343)) were each cultured in a PDA (Potato Dextrose Agar) medium, and mycelial disks punched out with a 5-mm diameter cork borer from the grown colonies were prepared.

[0036] Moreover, the pre-prepared sucrose-free Czapek medium was dispensed into three flasks. Then, the cell disks were each placed into the liquid medium, and the one with the filamentous fungus of the genus Glomerella added was designated as Comparative Example 1, the one with the filamentous fungus of the genus Fusarium added was designated as Comparative Example 2, and the one with the filamentous fungus of the genus Aspergillus added was designated as Comparative Example 3.

[0037] Also, in the same manner as in Test 2, PCL was added as a single carbon source to the pre-prepared sucrose-free Czapek medium to a concentration of 0.5% (w / v), and heat stirring was performed using a heating stirrer. While crushing the pellets with a spatula, it was suspended to obtain a liquid medium. The liquid medium was dispensed into three flasks.

[0038] Furthermore, filamentous fungi of the genus Glomerella (Glomerella cingulate (NBRC107004)), the genus Fusarium (Fusarium oxysporum (NBRC9971)), and the genus Aspergillus (Aspergillus flavus (NBRC6343)) were each cultured in a PDA (Potato Dextrose Agar) medium, and cell disks with a diameter of 5 mm punched out from the grown colonies using a cork borer were each placed into the liquid medium. The one with the filamentous fungus of the genus Glomerella added was designated as Example 4, the one with the filamentous fungus of the genus Fusarium added was designated as Example 5, and the one with the filamentous fungus of the genus Aspergillus added was designated as Example 6.

[0039] Then, using these liquid media, static culture was carried out at 25°C in the dark for 17 days. After that, the cultured cells and the culture filtrate were each separated and recovered. The culture filtrate was used as a crude enzyme solution and subjected to an esterase activity measurement test.

[0040] The esterase activity in the crude enzyme solution was measured using p-Nitrophenyl butylate (pNPB, C4) as a substrate. The esterase hydrolyzes pNPB to produce 4-nitrophenol, which can be spectrophotometrically measured at 405 - 410 nm. The pNPB substrate solution was prepared by mixing pNPB in isopropanol to a concentration of 10 mM. After mixing 890 μl of buffer (50 mM Tris-HCL, pH 7.5) and 100 μl of the crude enzyme solution, 10 μl of the pNPB substrate solution was added. Then, it was allowed to stand for 60 minutes, and the absorbance of 1 ml of the reaction solution at 405 nm was measured at room temperature using a spectrophotometer (Nanodrop 2000C, manufactured by Thermo Fisher Scientific Co., Ltd.).

[0041] As a result, as shown in Figure 5, the esterase activities of Comparative Example 1 was 0.11, Comparative Example 2 was 0.00, Comparative Example 3 was 0.02, Example 4 was 0.68, Example 5 was 0.85, and Example 6 was 0.19. No or low esterase activity was observed in the comparative examples, while esterase activity was confirmed in the examples. Therefore, it was confirmed that an esterase capable of degrading biodegradable plastics was produced and secreted by filamentous fungi of the genus Glomerella, Fusarium, or Aspergillus, which are capable of degrading biodegradable plastics.

[0042] [Test 4] A test was conducted to culture microorganisms in a culture medium that can be used to confirm the effect of the plastic degradation method according to the embodiment of the present invention and quantitatively measure the plastic degradation ability. Specifically, in the same manner as in Test 2, multiple Petri dishes with an agar medium having a PCL concentration of 0.5% (w / v) were obtained. Then, PCL blocks with a visually confirmable diameter of about 500 μm present in the agar medium were selected and marked on the back of the Petri dish.

[0043] In addition, filamentous fungi of the genus Glomerella (Glomerella cingulate (NBRC107001), Glomerella cingulate (NBRC107004)), the genus Aspergillus (Aspergillus flavus (NBRC6343)), the genus Fusarium (Fusarium oxysporum (NBRC9971), Fusarium sacchari (JCM9676)), and the genus Alternaria (Alternaria alternate (NBRC106339)) were each cultured on a PDA medium, and mycelial disks with a diameter of 5 mm were punched out from the grown colonies using a cork borer.

[0044] Next, the mycelial disks were brought into contact with the center of the surface of the agar medium, and after static culture at 30°C in the dark for a predetermined number of days, the biodegradable plastic in the culture medium was photographed using a microscope equipped with a CCD camera. Then, the degradation area of polycaprolactone per day (μm 2 ) was calculated according to the following formula. Degradation area = (area of the biodegradable plastic mass - area of the biodegradable plastic mass after the number of culture days from mycelium arrival) / number of culture days from mycelium arrival

[0045] The results are shown in Fig. 6. In each example, the minimum and maximum values of the degradation area are shown based on the calculation results of the culture of microorganisms and the degradation area individually performed a predetermined number of times. The number of Petri dishes tested in each example was 3 in Examples 7, 8, 10, and 11, and 2 in Example 9. The degradation area of polycaprolactone by the filamentous fungus of the genus Glomerella (Glomerella cingulate (NBRC107001)) in Example 7 was 624 - 1082, and the degradation area of polycaprolactone by the filamentous fungus of the genus Glomerella (Glomerella cingulate (NBRC107004)) in Example 8 was 6092 - 17863. In addition, the degradation area of polycaprolactone by the filamentous fungus of the genus Aspergillus (Aspergillus flavus (NBRC6343)) in Example 9 was 41156 - 41241.

[0046] Furthermore, the degradation area of polycaprolactone by the filamentous fungus of the genus Fusarium (Fusarium oxysporum (NBRC9971)) in Example 10 was 13,816 to 25,236, and the degradation area of polycaprolactone by the filamentous fungus of the genus Fusarium (Fusarium sacchari (JCM9676)) in Example 11 was 7,574 to 15,773.

[0047] Thus, according to the method for degrading plastics of the present embodiment, it has been found that biodegradable plastics can be suitably degraded using filamentous fungi of the genus Glomerella, the genus Fusarium, or the genus Aspergillus. In addition, it has become clear that biodegradable plastics can be degraded using filamentous fungi of the genus Glomerella that can grow particularly in a low temperature range.

[0048] The present invention is not limited to the above embodiments and examples, and it goes without saying that various modifications can be made within the scope of the present invention. For example, as the microorganism, it is possible to appropriately change such as adding and using other filamentous fungi in addition to the above filamentous fungi.

Industrial Applicability

[0049] The present invention can be suitably used for degrading biodegradable plastics.

Claims

**Claim 1** A method for decomposing plastics, characterized by decomposing biodegradable plastics having an ester bond in their chemical structure using a microorganism belonging to the genus Glomerella having plastic-degrading ability or an extract thereof containing esterase. **Claim 2** The method for decomposing plastics according to claim 1, characterized by decomposing the biodegradable plastics using a microorganism belonging to the genus Fusarium having plastic-degrading ability or an extract thereof containing esterase together with the microorganism belonging to the genus Glomerella having plastic-degrading ability or an extract thereof containing esterase. **Claim 3** The method for decomposing plastics according to claim 1 or 2, characterized by decomposing the biodegradable plastics using a microorganism belonging to the genus Aspergillus having plastic-degrading ability or an extract thereof containing esterase together with the microorganism belonging to the genus Glomerella having plastic-degrading ability or an extract thereof containing esterase. **Claim 4** The method for decomposing plastics according to any one of claims 1 to 3, characterized in that the biodegradable plastic is polycaprolactone.

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