JP5504440B2 - Novel microorganism and method for testing biodegradability of biodegradable plastic using the microorganism - Google Patents

Description

  The present invention relates to a novel microorganism and a method for testing the biodegradability of a biodegradable plastic in the marine environment, particularly in the deep sea, using the microorganism.

  Plastics are widely used because of their lightness, robustness, stability, and ease of molding. However, due to the characteristics of conventional plastics that do not rust or rot, the problem has arisen that plastics that are not collected after use and remain in the natural environment forever. Therefore, with the growing interest in the environment, efforts have been made to completely recover the plastic after use. However, agricultural materials, fishery materials, and civil engineering materials (especially river and marine materials) are likely to flow out into the natural environment due to their usage, and once out into the natural environment. It is very difficult and not practical to completely recover the plastic product that has been lost. Accordingly, plastics that are decomposed by microorganisms or the like when they flow into the natural environment without being collected, so-called biodegradable plastics, have attracted attention and have been widely used.

  In order to develop a better biodegradable plastic, a method for evaluating and testing the biodegradability of the developed plastic is important. At present, such biodegradability test methods include, for example, JIS K 6950: 2000 (corresponding to ISO 14851: 1999), JIS K 6951: 2000 (corresponding to ISO 14852: 1999), JIS K 6953: 2000. (Corresponding to ISO 14855: 1999) and JIS K 6955: 2006 (corresponding to ISO 17556: 2003) are defined.

  JIS K 6950 and K 6951 stipulate a method for determining the aerobic ultimate biodegradation level in plastic aqueous cultures by measuring the amount of oxygen consumed or the amount of generated carbon dioxide. The activated sludge and the soil are mentioned, and the activated sludge from the sewage treatment plant is mentioned as a suitable one, and the desirable culture temperature is 20-25 ° C. ± 1 ° C. JIS K 6953 defines a method for determining the aerobic ultimate biodegradation and disintegration of plastics under controlled composting conditions by measuring the amount of generated carbon dioxide. It describes that composting of organic components of solid waste waste is used, and that culturing is performed at a constant temperature of 58 ± 2 ° C. JIS K 6955 defines a method for determining the aerobic ultimate biodegradation in soil by measuring oxygen consumption or carbon dioxide generated using a plastic respirometer. The preferred culture temperature is 20-25 ° C. ± 1 ° C. All of these standards are premised on culture under atmospheric pressure.

  As described above, all of the methods currently known as test methods for biodegradability of plastics are soils that are buried in the soil in the terrestrial environment, or in which the plastic that is submerged in the shallow water exists in the terrestrial environment. It was a method for testing how biodegradation is caused by microorganisms in water and sludge. Therefore, all biodegradable plastics developed using these test methods as indicators are only confirmed to be degraded by microorganisms in soil and sludge in such terrestrial environments. there were.

  As an attempt to predict the biodegradability of biodegradable plastics without using living microorganisms from the terrestrial environment, the physical property values of biodegradable plastics are measured by instrumental analysis to predict enzyme degradability This method is disclosed in Japanese Patent Application Laid-Open No. 2008-222758. However, even with such a prediction method, the basis for the prediction is the same as the conventional biodegradability test method, because it is an enzyme derived from soil and sludge microorganisms present in the terrestrial environment. As a result, what can be predicted from the physical property values by instrumental analysis of biodegradable plastics is limited to biodegradation of soil and sludge in the land environment by microorganisms.

  On the other hand, when agricultural materials, civil engineering materials, etc. flow out into the natural environment due to their use, even if they flow out on land, they are carried to the sea by rain and rivers, and many of them result. It is thought that it will flow into the ocean. In addition, fishery materials, civil engineering materials used in rivers and oceans, etc. flow out into the ocean as they are. In this way, the plastic that flows into the ocean sinks by gravity and accumulates in the deep ocean, polluting the natural environment and permanently destroying the marine ecosystem. Many such wastes have been confirmed in actual deep sea surveys.

  Furthermore, for example, fishing gear made of synthetic material (especially fishing nets) that has flowed into the ocean continues to capture and kill many fish without any intention. The damage to marine organisms caused by such spilled fishing gear is called ghost fishing, and there is no benefit to humankind, and it is estimated that some fish species cause damage exceeding the amount of commercial landing. It is becoming a threat to human food resources.

JP 2008-222758 A

  As described above, there is an urgent need to develop a biodegradable plastic having excellent biodegradability even when it flows into the marine environment including the deep sea floor.

  However, as described above, the methods for evaluating and testing the biodegradability of conventional biodegradable plastics that are currently known are all established using microorganisms that are seed sources from the terrestrial environment. Is. On the other hand, the marine environment, particularly the deep sea floor environment, is at a lower temperature than the land environment at normal temperature and normal pressure, and is also several times to several hundred times higher than atmospheric pressure. It is almost impossible to imagine that normal microorganisms that grow in a terrestrial environment at normal temperature and pressure grow in such an environment and decompose biodegradable plastics. Although it was thought that it was completely different from the microbial phase of the environment, knowledge on microorganisms that propagate and decompose biodegradable plastics in such marine environments, especially in the deep sea under low temperature and high pressure environments, has been obtained so far. It was not.

  Therefore, conventional biodegradable plastics have not been evaluated at all for marine environment, particularly biodegradability in the deep sea, and conventional biodegradable plastic biodegradability evaluation and testing methods are In particular, biodegradability in the deep sea could not be evaluated.

  Therefore, in order to enable the development of biodegradable plastics having excellent biodegradability in the marine environment, a test method capable of evaluating the biodegradability of the biodegradable plastics in a marine environment completely different from the land environment, and A microorganism as a seed source that can be used in the test method is required.

  Accordingly, an object of the present invention is to provide microorganisms that are actually present in the marine environment, particularly in the deep sea of low temperature and high pressure, and have the ability to degrade the biodegradable plastic, and the biodegradable plastic in the marine environment using the microorganism. It is to provide a method for testing biodegradability.

  The present inventors collect deep sea sediments using a special deep sea boat that can withstand an ultra-high pressure environment, and remove the microflora contained therein under a low temperature and high pressure using a special pressure vessel. In culture. Then, a plurality of strains having biodegradable plastic resolution were isolated and identified from the cultured microorganisms, and further, these strains were confirmed to exist widely in the deep sea. The wide distribution of these microorganisms in the deep sea means that these microorganisms are natural seeding sources for biodegradation in the marine environment. The present inventors have found that the above object can be achieved using these microorganisms (strains), and have completed the present invention.

  All of the strains identified by the present inventors were novel and further included new species of microorganisms. Thus, microorganisms that can grow in a special environment under low temperature and high pressure and can decompose biodegradable plastics have never been known. The fact that such microorganisms are widely distributed in the deep sea is also a finding made clear by the present inventors for the first time.

Therefore, the present invention includes the following [1] to [7].
[1]
A microorganism belonging to the genus Moritella or genus Schwannella, which has the resolution of a biodegradable plastic.
[2]
The microorganism belonging to the genus Moritella is the following (A) or (B):
(A) DNA comprising the base sequence set forth in SEQ ID NO: 5
(B) DNA having 95% or more identity with the base sequence set forth in SEQ ID NO: 5,
The microorganism according to [1], which has a 16S rRNA gene comprising
[3]
The microorganism belonging to the genus Moritella is the following (C) or (D):
(C) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 8
(D) DNA having 95% or more identity with the base sequence set forth in SEQ ID NO: 8,
The microorganism according to [1], which has a 16S rRNA gene comprising
[4]
The microorganism belonging to the genus Shuwanella is the following (E) or (F):
(E) DNA comprising the base sequence set forth in SEQ ID NO: 1
(F) DNA having 95% or more identity with the base sequence set forth in SEQ ID NO: 1,
The microorganism according to [1], which has a 16S rRNA gene comprising
[5]
The microorganism according to [1] or [2], wherein the microorganism belonging to the genus Moritella is Moritera avicii CT12 strain (accession number: FERM P-21744) or a variant thereof.
[6]
Microorganisms belonging to the genus Moritella sp. The microorganism according to [1] or [3], which is JT01 strain (accession number: FERM P-21745) or a mutant thereof.
[7]
The microorganism according to [1] or [4], wherein the microorganism belonging to the genus Shuwanella is a Schwannelaventica CT01 strain (accession number: FERM P-21743), or a variant thereof.

Furthermore, the present invention also includes the following [8] to [13].
[8]
A biodegradable plastic biodegradation accelerator comprising the microorganism according to any one of [1] to [7].
[9]
The biodegradation accelerator according to [8], which is a biodegradation accelerator for low temperature and high pressure environments.
[10]
The biodegradation accelerator according to any one of [8] to [9], wherein the biodegradable plastic is a biodegradable plastic having an ester bond as a repeating structure.
[11]
A seed source for biodegradability testing of plastic, comprising the microorganism according to any one of [1] to [7].
[12]
The seeding source according to [10], which is a seeding source for a low-temperature and high-pressure environment.
[13]
The seeding source according to any one of [11] to [12], wherein the plastic is a biodegradable plastic having an ester bond as a repeating structure.

Furthermore, the present invention also includes the following [14] to [17].
[14]
Adding the microorganism according to any one of [1] to [7] to a biodegradable plastic;
A method for degrading biodegradable plastics, comprising:
[15]
Adding the microorganism according to any one of [1] to [7] to a plastic;
A method for testing the biodegradability of plastics, comprising:
[16]
The method according to any one of [14] to [15], comprising a step of culturing the microorganism according to any one of [1] to [7] under conditions of low temperature and high pressure.
[17]
The method according to any one of [14] to [16], wherein the plastic is a biodegradable plastic having an ester bond as a repeating structure.

Furthermore, the present invention also includes the following [18] to [24].
[18]
The biodegradation accelerator according to [9], the seeding source according to [11], or the method according to [14], wherein the low temperature is a temperature in the range of 1 to 12 ° C.
[19]
The biodegradation promoter according to any one of [9] to [10], the seeding source according to any one of [12] to [13], wherein the high pressure is a pressure in the range of 0.1 to 100 MPa, or [16] The method according to any one of [17].
[20]
Use of the microorganism according to any one of [1] to [7] for degrading a biodegradable plastic.
[21]
Use of the microorganism according to any one of [1] to [7] for promoting degradation of a biodegradable plastic.
[23]
Use of the microorganism according to any one of [1] to [7] for testing biodegradability of plastics.
[24]
The use according to any one of [20] to [23], wherein the plastic is a biodegradable plastic having an ester bond as a repeating structure.

Furthermore, the present invention is also the following [25].
[25]
DNA consisting of the base sequence described in any of SEQ ID NOs: 1 to 11, or
DNA having 95% or more identity with the base sequence described in any one of SEQ ID NOs: 1 to 11,
A microorganism belonging to the genus Moritella or genus Schwannella, which has a 16S rRNA gene comprising

  Furthermore, the present invention is also the invention according to the above [8] to [24], wherein the microorganism described in [25] is used instead of the microorganism described in any of [1] to [7].

  The present invention provides a novel microorganism capable of growing and breeding and degrading biodegradable plastics in a marine environment, particularly in a low temperature and high pressure environment of the deep sea bottom. Therefore, if the microorganism of the present invention is used as a seeding source, the biodegradability of the biodegradable plastic in a marine environment, particularly a low temperature and high pressure environment can be tested. That is, the present invention makes it possible for the first time to develop a biodegradable plastic having excellent biodegradability in such an environment.

  As described above, according to the present invention, a biodegradable plastic having ensured biodegradability in a marine environment, particularly in a low temperature and high pressure environment can be obtained, so that the marine environment is released from a heavy environmental load and ghost fishing is prevented. Can protect fisheries resources.

  In addition, when the microorganism of the present invention is used, the biodegradable plastic can be decomposed under a low temperature and high pressure environment. Accordingly, the biodegradation of the biodegradable plastic in such an environment can be promoted by previously containing the microorganism of the present invention in the biodegradable plastic or by spraying it on the outflow site. That is, the present invention makes it possible for the first time to promote biodegradation of biodegradable plastics in such an environment.

  As described above, according to the present invention, it becomes possible to promote the biodegradation of plastics in the marine environment, particularly in a low temperature and high pressure environment, so that the marine environment is released from a heavy environmental load, and ghost fishing is prevented to save marine resources. Can be protected.

FIG. 1 is a diagram showing a screening method for a biodegradable plastic-degrading bacterium from a deep-sea bottom mud sample. FIG. 2 is a diagram showing a phylogenetic tree based on the 16sRNA gene base sequence of the isolate. FIG. 3A is a diagram showing the results on the 0th day of the PCL degradation evaluation test using flow cytometry. FIG. 3B is a diagram showing the results of the seventh day of the PCL degradation evaluation test using flow cytometry. FIG. 3C is a diagram showing the results of the 14th day of the PCL degradation evaluation test using flow cytometry. FIG. 3D is a diagram showing the results on the 28th day of the PCL degradation evaluation test using flow cytometry.

  Hereinafter, the present invention will be described in detail with reference to embodiments of the present invention. The present invention is not limited to the specific embodiments exemplified below.

  The microorganism of the present invention is a microorganism belonging to the genus Moritella or Schwannella, which has the resolution of a biodegradable plastic.

As a suitable microorganism,
DNA consisting of the base sequence described in any of SEQ ID NOs: 1 to 11, or
DNA having 95% or more identity with the base sequence described in any one of SEQ ID NOs: 1 to 11,
And a microorganism belonging to the genus Moritella or genus Schwannella, which has a 16S rRNA gene comprising

  In a preferred embodiment, the identity (or homology) of the base sequence is preferably 95% or more, more preferably 96% or more, further preferably 97% or more, more preferably 98% or more, and more preferably 99% or more, more preferably 99.2% or more, more preferably 99.4% or more, more preferably 99.5% or more, more preferably 99.6% or more, more preferably 99.7% or more, Preferably it is 99.8% or more, More preferably, it can be 99.9% or more.

In one preferred embodiment, as a suitable microorganism,
DNA consisting of the base sequence described in any of SEQ ID NOs: 1 to 11, or
DNA consisting of a base sequence in which one or several bases are added, substituted and / or deleted from the base sequence described in any of SEQ ID NOs: 1 to 11,
And a microorganism belonging to the genus Moritella or genus Schwannella, which has a 16S rRNA gene comprising

  In one embodiment of suitable implementation, one or several of the bases is, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 9, and further Preferably 1 to 8, more preferably 1 to 7, more preferably 1 to 6, more preferably 1 to 5, further preferably 1 to 4, more preferably 1 to 3, and still more preferably One or two, more preferably one.

  In one preferred embodiment, the microorganisms belonging to the genus Moritella or genus Schwanella having the resolution of biodegradable plastics include CT01 strain, CT12 strain, three strains of JT01 strain, and CT03 strain disclosed herein, There are 8 strains including CT05 strain, CT06 strain, CT07 strain, CT08 strain, CT13 strain, JT02 strain, and JT04 strain.

In one embodiment of the preferred embodiment, the following three strains disclosed in the present specification can be given as examples of microorganisms belonging to the genus Moritella or genus Schwannella having the ability of biodegradable plastics.
Shewanella benthica CT01:
Accession Number FERM P-21743
Moritella abyssi CT12 strain:
Accession Number FERM P-21744
New species of genus Moritella JT01 (Moritella sp. JT01):
Accession Number FERM P-21745

  In a preferred embodiment, the microorganism of the present invention may be a mutant (variant) of the aforementioned strain as long as it has a biodegradable plastic resolution.

  Mutant strains can be obtained without mutation treatment with ethyl methanesulfonic acid, a commonly used mutation agent, other chemical treatments such as nitrosoguanidine, methylmethanesulfonic acid, ultraviolet irradiation, or treatment with a mutation agent. It can also be obtained by so-called spontaneous mutation.

  For example, such a mutant strain has a resolution of biodegradable plastic. For example, a deep-sea sediment collected from various places is put in a test tube containing a medium containing a film of polycaprolactone, and the temperature is 4 ° C. and 30 MPa. , And applied to an agar plate containing granulated polycaprolactone to obtain bacteria in which a clear zone was formed around the colony due to the decomposition of the plastic.

  Bacteriological properties indicating that it is a genus of Moritella or Schwanella, for example, BERGEY'S MANUAL OF Systematic Bacteriology (Volume 1, 1984, Vol. 1986) Year, volume 3, 1989, volume 4, 1989).

  As the medium used for the growth of the microorganism of the present invention, for example, a medium capable of growing a microorganism belonging to the genus Moritella or Schwanella can be used. Specifically, an MB medium can be mentioned, but is not limited thereto. It is not a thing. The medium used for the growth of the microorganism of the present invention contains a carbon source that can be assimilated by the microorganism of the present invention, such as glucose, and a nitrogen source that can be assimilated by the microorganism of the present invention. For example, ammonium sulfate, ammonium chloride and the like can be contained. Further, if desired, it comprises a cation such as sodium ion, potassium ion, calcium ion and magnesium ion and an anion such as sulfate ion, chlorine ion and phosphate ion. The concentration of the carbon source is about 0.01 to 5%. The concentration of inorganic salts is, for example, about 0.001 to 1%.

  The microorganism of the present invention has an ability to decompose plastics in a marine environment, particularly in a deep sea of low temperature and high pressure. Therefore, the use of the microorganism of the present invention can promote the biodegradation of plastics in the marine environment, particularly in the deep sea of low temperature and high pressure. Moreover, if the microorganism of the present invention is used, plastic biodegradation can be performed under marine environment, particularly low temperature and high pressure conditions such as deep sea of low temperature and high pressure, or plastic biodegradation can be promoted.

  In addition, the biodegradation of plastics by the microorganisms of the present invention is equivalent to the biodegradation of plastics actually performed in the marine environment, particularly in the deep sea of low temperature and high pressure. It is possible to test and evaluate the biodegradability of plastics in the high pressure deep sea. Therefore, the microorganism of the present invention can be used as a seed source for the biodegradability test of such plastics.

  In one preferred embodiment, the plastic biodegradable by the microorganism of the present invention may include a so-called biodegradable plastic. Examples of such a biodegradable plastic include, for example, in the molecular structure of the plastic. Examples thereof include an ester bond, and specific examples include polybutylene succinate, polybutylene succinate-co-adipate, and polyethylene succinate.

  In a preferred embodiment, the plastic biodegradability test using the microorganism of the present invention is performed, for example, according to JIS K 6950: 2000 (corresponding to ISO 14851: 1999), JIS K 6951: 2000 (ISO 14852: 1999), JIS K 6953: 2000 (corresponding to ISO 14855: 1999), JIS K 6955: 2006 (corresponding to ISO 17556: 2003), etc., inoculating the microorganism of the present invention This can be done by adding as a source.

  In a preferred embodiment, the biodegradability test of plastics using the microorganism of the present invention is performed under conditions under low temperature and high pressure. Specifically, for example, as shown in the embodiments of the present invention.

  Testing the biodegradability of plastics using the microorganisms of the present invention is performed using flow cytometry in a preferred embodiment. Specifically, for example, as shown in the embodiments of the present invention.

  The microorganism of the present invention is used by adding the cultured strain together with the culture medium, in addition to powders obtained by freeze-drying the cells by a conventional method, the powder and various vitamins and minerals, necessary nutrient sources such as yeast extract, casamino acid Further, it can be used as a preparation in a solid form such as a tablet tableted after blending peptone or the like, and the strain can also be used in the form of activated sludge and compost.

  The plastic to be decomposed may be added, for example, as granules in a liquid medium, in the form of powder, or as a lump such as a film or a pellet. In addition, as for the input amount of the plastic with respect to a culture medium, 0.01 to 10 weight% is desirable. Although the amount of microorganisms to be added may be extremely small, it is preferably 0.1% by weight or more (wet weight) with respect to the plastic in consideration of decomposition efficiency. Further, the plastic used for decomposition may be one kind or plural kinds.

  Hereinafter, the present invention will be described more specifically with reference to examples. The present invention is not limited to these examples.

[Microbe screening]
[Create granule]
The preparation of granules of polycaprolactone was performed according to the following method. 2 g of polycaprolactone was dissolved in 100 ml of acetone and added to 1000 ml of distilled water containing 30% acetone to form granules. Thereafter, acetone was volatilized by using an evaporator.

[Preparation of medium]
As a medium, Marine Broth 2216 medium (Difco, CatNo. 279110) diluted twice with artificial seawater, and a solid medium with the above medium added with agar (15 g / l, Wako, CatNo. 010-15815) Using.

[Execution of screening]
About 1 g of seabed sediment collected from the Kuril Trench and Japan Trench was placed in a 2 ml plastic tube, and a 1 cm square polycaprolactone film was added thereto, followed by static culture at 4 ° C. and 30 MPa. One week later, 1 L of Marine Broth medium diluted twice with artificial seawater and 10 1 cm square polycaprolactone films were added, and cultured at 4 ° C. and 30 MPa for another week. Then, it was apply | coated to the agar plate which added the polycaprolactone granule to Marine Broth culture medium diluted 2 times with artificial seawater. Bacteria in which a clear zone was formed around the colony due to plastic degradation were obtained. The screening procedure for biodegradable plastic-degrading bacteria is shown in FIG. FIG. 1 is a diagram showing a screening method for a biodegradable plastic-degrading bacterium from a deep-sea bottom mud sample.

  As a result of searching biodegradable plastic-degrading microorganisms using sediment collected from the Kuril and Japan Trench, three strains (CT01, CT12 and JT01) were selected as excellent strains.

[Identification of microorganisms]
Microorganisms selected by screening were identified as follows. Physiological tests were performed according to general methods. Identification was based on Bergey's Manual of Systematic Bacteriology, Baltimore: WILLIAMS & WILKINS Co., (1984). A microorganism identification system manufactured by BIOLOG, USA was also used. The 16S rRNA gene was amplified by direct PCR, and the primers used were 27F and 1492R primer sets capable of amplifying almost the entire length of the bacterial 16S rRNA gene. Sequence analysis was performed using a model 3100 DNA Sequencer from Perkin-Elmer / Applied Biosystems Co. From these analysis results and chromosomal DNA homology test, it was found that the isolate CT01 was identified as Shewanella ventica, CT12 was identified as Moritera avicii, and JT01 was a new species of the genus Moritella.

[Morphological and physiological properties test]
Table 1 shows the results of various morphological and physiological properties tests on the three biodegradable plastic-degrading strains. These isolated strains were aerobic bacilli and other physiological properties were as shown in Table 1 below.

[Bacterial fatty acid composition]
The cell membrane fatty acid composition of the strains isolated using the HPLC method was identified. Was characterized as a strain producing highly unsaturated fatty acids. Table 2 below shows the fatty acid composition data.

[Comparison of base sequences of 16S rRNA gene]
Chromosomal DNA was prepared from each isolate, and the full-length 16S rRNA gene of each strain was amplified by direct PCR using this as a template, and its nucleotide sequence was determined. A homology search was performed based on the obtained sequence information, and a phylogenetic tree was created using the neighborhood joining method (CLUSTAL W). Fig. 2 shows the obtained phylogenetic tree. FIG. 2 is a diagram showing a phylogenetic tree based on the 16sRNA gene base sequence of the isolate. However, FIG. 2 includes, in addition to the CT01 strain, CT12 strain, and JT01 strain, the novel strains described later isolated by the present inventors.

  From the above results, it was found that the isolate CT01 was identified as Shewanella ventica, CT12 was identified as Moritella abyssi, and JT01 was a new species of the genus Moritella. The nucleotide sequences of 16S rRNA genes of the above three strains (CT01 strain, CT12 strain, JT01 strain) are shown in the sequence listing.

The above three isolates were obtained from the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center on December 9, 2008 (Tsukuba Center Chuo 6th, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan) Deposited. The accession numbers are as follows.
Shewanella benthica CT01:
Accession Number FERM P-21743
Moritella abyssi CT12 strain:
Accession Number FERM P-21744
New species of genus Moritella JT01 (Moritella sp. JT01):
Accession Number FERM P-21745

[Plastic disassembly test]
Using various biodegradable plastic materials, a degradation test using the isolated strain was performed as follows. As a result, the isolated strain was found to degrade polycaprolactone (PCL) well.

[Test strain]
Moliterra abishiai CT12 strain, Moliterra sp. JT01 strain, and Shewanella ventica CT01 strain isolated as biodegradable plastic degrading bacteria from deep-sea bottom mud were used.

[Medium and reagents]
In the experiment, the above-described screening medium was used for cultivation. All reagents used in the experiment were Wako Pure Chemicals special grades or equivalent.

[Decomposition test]
The decomposition activity of various plastics was granulated to have a particle size of about 1 to 10 μm, and was identified on an agar plate containing these various plastics. The final concentration of various plastics in the form of granules in the agar medium was adjusted to 0.5 g / l. Each plate was inoculated and cultured at 8 ° C. for 4 weeks, and the amount of degradation was determined by the amount of clear zone formed around the colony. As a result, it was shown that all three isolated strains degraded polycaprolactone (PCL) well. The results of the decomposition test of various plastic materials are shown in Table 3 below. Table 3 is a table | surface which shows the result of the various plastic degradation activity test in three isolate | separated strains. Three strains of CT12, JT01 and CT01 had excellent degradation activity against PCL (polycaprolactone), PBSA (polybutylene succinate adipate) and PHB / V (polyhydroxybutyrate / valerate). .

[Plastic decomposition evaluation using flow cytometry]
As a result of various plastic degradation tests, it was shown that the isolated strains degraded PCL well. Therefore, we conducted a plastic degradation evaluation test under high pressure and low temperature conditions using PCL as a substrate. The decomposition evaluation method of was established.

[Test strain]
The Mortera sp. JT01 strain obtained as a bacterium that degrades polycaprolactone (PCL) well was used.

[Medium and reagents]
In the experiment, the above-described screening medium was used for cultivation. All reagents used in the experiment were Wako Pure Chemicals special grades or equivalent.

[Decomposition test]
For the degradation activity, a liquid medium in which polycaprolactone granules adjusted to a particle size of 1 to 10 μm were added to Marine Broth medium diluted twice with artificial seawater to a final concentration of 1 mg / ml was used. The liquid medium was inoculated with 0.2% (v / v) seed (about 2.0 × 10 6 cells / ml) and cultured at 8 ° C. and 30 MPa for a maximum of 4 weeks. In the culture experiment, the culture solution is dispensed into each 2 ml tube, and sealed with parafilm, placed in a pressurized container (manufactured by Shintech Co., Ltd., a small pressurized container), placed in a water tank cooled to 8 ° C with 30 MPa water pressure Carried out. Sampling is carried out every other week, and the culture is treated with DAPI stain (final concentration 0.01mg / ml) and the microorganism part is fluorescently stained. It was used for the measurement (Nippon Becton Dickinson, model FACS Aria). As detection conditions for flow cytometry, the laser light used a Point Source Violet Solid State, passed through a filter with a wavelength of 407 nm, and detected purple fluorescence. The amount of degradation was determined by the decrease in the number of polycaprolactone granule particles detected by flow cytometry. In addition to DAPI, microorganisms and PCL particles can be separately measured using a nucleic acid staining reagent such as PI or Cyber Green as necessary in addition to DAPI.

  FIG. 3 (FIGS. 3A, 3B, 3C, and 3D) shows the results of testing the degradation activity against polycaprolactone using the method described above. In this strain, the number of particles of polycaprolactone granules decreased in 2 to 4 weeks of culture, and most of them were degraded in about 1 month.

  FIG. 3 (FIGS. 3A, 3B, 3C, and 3D) is a PCL degradation evaluation test using flow cytometry. Sampling was performed on the 0th day (FIG. 3A), the 7th day (FIG. 3B), the 14th day (FIG. 3C), and the 28th day (FIG. 3D), and the process of PCL decomposition was measured. In each figure on the 0th day (FIG. 3A), the 7th day (FIG. 3B), the 14th day (FIG. 3C), and the 28th day (FIG. 3D), the P1 region in the upper diagram is bacteria, and the P2 region is PCL particles are shown. In each figure, the left mountain in the lower figure shows PCL, and the right mountain shows the amount of bacteria. It was shown that PCL was degraded with the growth of bacteria over time. In the figure, the vertical axis shows the complexity of the internal structure of the particles (SSC: equivalent to the size of the particles), the horizontal axis shows the fluorescence intensity (violet-A) by DAPI staining, and the upper figure shows each unit time per unit time. Particle spots are shown, and the figure below is a plot of the distribution.

[Acquisition and identification of another isolate]
Other isolates were obtained and identified in the same manner as the above procedure for obtaining the above three strains (CT01 strain, CT12 strain, JT01 strain). Eight newly acquired strains were named CT03 strain, CT05 strain, CT06 strain, CT07 strain, CT08 strain, CT13 strain, JT02 strain, and JT04 strain.

  As with the above three strains (CT01 strain, CT12 strain, JT01 strain), 8 new strains (CT03 strain, CT05 strain, CT06 strain, CT07 strain, CT08 strain, CT13 strain, JT02 strain, JT04 strain) are also directly used. Nearly full length of 16S rRNA gene of each strain was amplified by PCR, and its base sequence was determined. A homology search was performed based on the obtained sequence information, and a phylogenetic tree was created using the neighborhood joining method (CLUSTAL W). FIG. 2 shows the phylogenetic tree thus obtained. The nucleotide sequences of 16S rRNA genes of 8 new strains (CT03 strain, CT05 strain, CT06 strain, CT07 strain, CT08 strain, CT13 strain, JT02 strain, JT04 strain) are shown in the Sequence Listing.

  For the above 3 strains (CT01 strain, CT12 strain, JT01 strain) and 8 new strains (CT03 strain, CT05 strain, CT06 strain, CT07 strain, CT08 strain, CT13 strain, JT02 strain, JT04 strain) Homology (%) was compared and the results are shown in Table 4 below. Table 4 is a table | surface which shows the result of the homology comparison of 11 isolate | separated isolates.

  In Table 4, 4 strains of JT01, CT05, JT02, and JT04 are Moritella sp. Nov., 4 strains of CT13, CT06, CT08, and CT12 are Moritella abyssi, and 3 strains of CT01, CT07, and CT03 are , Identified as Shewanella benthica.

  As with the above three strains (CT01 strain, CT12 strain, JT01 strain), 8 new strains (CT03 strain, CT05 strain, CT06 strain, CT07 strain, CT08 strain, CT13 strain, JT02 strain, JT04 strain) The biodegradation activity of the degradable plastic was evaluated by a plate test. The evaluation test was performed by determining the size of the halo after one week at 4 ° C. and atmospheric pressure. Regarding the above three strains (CT01 strain, CT12 strain, JT01 strain) and 8 new strains (CT03 strain, CT05 strain, CT06 strain, CT07 strain, CT08 strain, CT13 strain, JT02 strain, JT04 strain) The results of tic decomposition activity evaluation are shown in Table 5 below. Table 5 is a table | surface which shows the result of biodegradable plasticstic activity evaluation of 11 isolate | separated isolates.

  As shown in Table 5, all the 11 isolates showed excellent degradation activity against PCL (polycaprolactone), PBSA (polybutylene succinate adipate), and PHB / V (polyhydroxybutyrate / valerate). I had it. Furthermore, as a result of conducting a plastic degradation evaluation test using PCL as a substrate and using flow cytometry under high pressure and low temperature conditions, it was confirmed that the new 8 strains also have excellent degradation activity.

  The present invention provides a novel microorganism capable of growing and breeding and degrading biodegradable plastics in a marine environment, particularly in a low temperature and high pressure environment of the deep sea bottom. Furthermore, if the microorganism of the present invention is used as a seeding source, the biodegradability of the biodegradable plastic can be tested in a marine environment, particularly in a low temperature and high pressure environment. According to the present invention, the marine environment and marine resources can be protected, and the present invention is an industrially useful invention.

Claims (6)

Is separated from the sediment of deep sea, a microbial that have a resolution of biodegradable plastic, Mori Terra Avecia Lee CT12 strain (Accession Number: FERM P-21744). It is separated from the sediment of deep sea, a microbial that have a resolution of biodegradable plastic, Moritera sp. JT01 strain (Accession number: FERM P-21745) . Is separated from the sediment of deep sea, a microbial that have a resolution of biodegradable plastic, Schwarz Ne Raven Tikka CT01 strain (Accession Number: FERM P-21743). A seed source for biodegradability testing of plastics, comprising the microorganism according to any one of claims 1 to 3 . Adding the microorganism according to any one of claims 1 to 3 to a plastic;
For testing the biodegradability of biodegradable plastics.   The following microbial species:
  (A) Moritera abyssi,
  (B) Moritera sp. The same species as JT01 strain (Accession number: FERM P-21745), and
  (C) Schwanelaventica
Adding to the plastic a microorganism having a biodegradable plastic resolution, which is selected from the group consisting of:
For testing the biodegradability of biodegradable plastics.