WO2006090913A1

WO2006090913A1 - Method for degrading aromatic polyester using microorganism

Info

Publication number: WO2006090913A1
Application number: PCT/JP2006/304041
Authority: WO
Inventors: Kiyotsuna Toyohara; Kohei Oda; Kazumi Hiraga; Yoshiharu Kimura
Original assignee: Teijin Limited; Kyoto Institute Of Technology
Priority date: 2005-02-24
Filing date: 2006-02-24
Publication date: 2006-08-31
Also published as: JP4753210B2; JPWO2006090913A1

Abstract

It is intended to provide a microorganism which belongs to the genus Pigmentiphaga and has an ability to specifically degrade terephthalic acid; a microorganism which belongs to the genus Bacillus and has an ability to produce an extracellular polysaccharide with an ability to adhere to an aromatic polyester; a microorganism which belongs to the genus Pseudomonas and has an ability to specifically degrade bishydroxyethyl terephthalate; a microorganism which belongs to the class Alphaproteobacteria and has an ability to accelerate the ability to degrade an aromatic polyester; a microorganism which enhances the ability of Rhizobium sp. OKH-3 to degrade an aromatic polyester; and a method for degrading an aromatic polyester using a group of these microorganisms.

Description

Decomposition method of aromatic polyester using microorganisms

Technical field

The present invention relates to a method for decomposing aromatic polyester using microorganisms.

Background art

Light

In recent years, aliphatic polyesters have been produced by known enzymes such as common soil microorganisms and lipases.

Therefore, it is being researched and developed as a biodegradable polymer. Even if the polyester has an aromatic component such as terephthalic acid or p-hydroxybenzoic acid, if the amount of the aromatic component is small or the heat resistance is significantly reduced, It is known to be biodegradable by microorganisms and known enzymes in general soil and activated sludge. However, there are few known microorganisms and polymers that degrade aromatic polyesters that contain aromatic components as the main component, such as polyethylene terephthalate (hereinafter sometimes abbreviated as PET). Although it has been proposed that surface treatments such as improving hydrophilicity be performed by treating PET fibers or PET woven fabrics with enzymes, it has been proposed (Special Table 2000- 502412) and Special Table 2001. -502014), there is no data that clearly shows that PET has been decomposed.

For this reason, when PET is decomposed, a heat treatment method is generally used under strong basicity as typified by a high concentration aqueous sodium hydroxide solution.

Disclosure of the invention

An object of the present invention is to provide a microorganism that activates a microorganism having the ability to decompose an aromatic polyester, and a method for decomposing an aromatic polyester using the microorganism group.

In order to achieve the above object, the present inventors have made aromatic polyesters by biological functions. As a result of diligent investigations on the method of disassembling the lysozyme s p. OKH— 03 (FERM P— 19483), it is possible to dramatically improve the lysozyme s p. As a result, the present inventors have succeeded in isolating and identifying bacteria capable of realizing the degradation of aromatic polyester, thereby completing the present invention.

That is, according to the present invention, the above object of the present invention is

A microorganism belonging to the genus Pigmenthifera, a microorganism belonging to the genus Bacillus, a microorganism belonging to the genus Syudumonas, and a microorganism belonging to the class of alphaproteobacteria, Rhizopium sp. This is achieved by microorganisms and their microbes that have the ability to accelerate the degradation of aromatic polyesters.

Furthermore, according to the present invention, the above object of the present invention is

Rhizobium s p. OKH- 03 (FERM P— 19483), Pigmenty Fager s p. (NI TE P— 65), Bacillus Megaterum (NI TE P— 67), Siyudomonas s p. (NI TE P— 64) Decomposing an aromatic polyester, characterized by decomposing the aromatic polyester by contacting the aromatic polyester with at least one microorganism selected from the group consisting of NITE P-66 Achieved by the method. Brief Description of Drawings

FIG. 1 is an overall photograph of the film after the decomposition experiment according to Example 1.

Fig. 2 is a photograph (500x magnification) of the surface of the PET film finally obtained by the operation of Comparative Example 1 taken with a scanning electron microscope ("SEM-2400" manufactured by Hitachi, Ltd.). BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

In the present invention, the aromatic polyester to be decomposed is a polyester containing 50% by weight or more of a repeating unit comprising an aromatic dicarboxylic acid and an aliphatic diol component. Tell. Particularly preferred are aromatic polyesters containing 85% by weight or more of ethylene terephthalate repeat units. The components that may be copolymerized at this time include, for example, fragrances such as phthalic acid, isophthalic acid, diphenyldicarboxylic acid, diphenoxy dicarboxylic acid, and 2,6-naphthalenedicarboxylic acid as dicarboxylic acid components other than terephthalic acid. And aliphatic dicarboxylic acids such as succinic acid, adipic acid, azelaic acid, sebacic acid, and decanedicarboxylic acid, and derivatives thereof.

Examples of diol components other than ethylene glycol include diethylene glycol, trimethylene glycol, tetramethylene glycol, propylene glycol, pentamethylene glycol, hexamethylene glycol, decamethylene glycol and the like.

The aromatic polyester may have any form such as a fiber, a film, a lump, or a mixture thereof at the time of decomposition.

The microorganism of the present invention may be any microorganism belonging to the genus Pigmentifaga, Bacillus, Syudomonas, and Alphaproteobacteria, and having the ability to improve the degradation ability of the aromatic polyester of Rhizobium sp. 〇KH-03. Anything is acceptable. In particular, Pigmenti Ferga sp. (N ITE P—65), Bacillus Megaterium (NI TE P—67), Shumonas sp. (NI TE P_64), Alpha Proteobacteria (NI TE P-66) ) Is preferred.

The above strain group is a strain newly isolated by the present inventors from soil in Japan and has the following mycological properties. table 1

Further, the chemical taxonomic properties of the liposomal DNA are as shown in the attached SEQ ID Nos. 1, 2, 3, and 4.

The bacteriological properties of Rhizobium sp. OKH-03 are shown in Table 2, and the chemical taxonomic properties of the liposomal DNA are as shown in the attached SEQ ID NO: 5. Table 2

Based on the properties shown in Table 1, the results of comparison with Purge Aids, Manual, Ob, Systematic Bacteriology, etc., are the microorganisms belonging to the genus Bacillus, Pigmentifa genus, Syudomonas, and Alphaproteobacteria, respectively. However, these strains were not identified as known strains belonging to the same genus, and these strains were designated as new strains as of January 14, 2005. center one (Chiba Kisarazu巿Kazusa legs bowed in ² - 5-8) deposited in each been received (accession number, NI TE P- 65, NI TE P- 67, NI TE P- 64, NI TE P — 66.) Since these microorganisms are at biosafety level I and are not pathogenic, it is possible to work safely biologically by using this strain.

In addition, about Rhizobium sp. OKH-03 as of August 1, 2003, National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (Tsukuba Ito, Ibaraki Pref. 1 1 1 1 1 Central 6) (Accession number is FERM P—19483 is there).

Each of the fungi of the present invention can enhance its degradability by complementing the ability to degrade aromatic polyester of Rhizobium sp. OKH-03, but, for example, Pigmentifera sp. (NITEP- 65) ) Has the ability to decompose oligomers and monomers, especially terephthalic acid, which are generated during decomposition, so that the resolution of polyester can be increased.

Next, a method for isolating microorganisms according to the present invention that significantly improves the ability to specifically degrade the aromatic polyester of lysozyme sp.O KH-03 will be described.

Microorganisms that specifically degrade aromatic polyesters are isolated by sampling soil, rainwater, etc., and selecting them appropriately by known methods. The soil, rainwater, etc. used at this time are particularly preferably sampled from a waste bin or the like where the aromatic polyester waste is collected.

Next, a medium containing an aromatic polyester as a sole carbon source is used as the medium (hereinafter, this medium may be referred to as an aromatic polyester medium). Examples of other nitrogen sources and mineral sources include inorganic ammonium salts such as ammonium sulfate and ammonium nitrate, metal salts such as iron sulfate, copper sulfate, zinc sulfate, manganese sulfate, and magnesium sulfate, and hydrates thereof. Can be used.

Examples of the culture method include shaking culture and stationary culture, but shaking culture is preferable for obtaining specific microorganisms from a mixed system of microorganisms. It is particularly preferable to use the methods together.

A sample sampled from soil or the like is cultured in the medium, and the medium is changed every predetermined period to accumulate useful bacteria.

The culture period is not particularly limited, but for example, about 1 to 2 months is preferable. Then, collect the aromatic polyester in the enriched culture solution and evaluate the aromatic polyester degradation activity of the microorganisms. The evaluation method is not particularly limited, but for example, surface observation with a scanning electron microscope is preferable because it is simple and highly reliable.

The above sample with the ability to decompose aromatic polyester is diluted as appropriate and applied to LE agar medium (medium in which LE agar is further added to agar), etc. to form colonies and isolated F

(Primary sorting).

Next, from the primary selection strains, a strain involved in the resolution of lysozyme sp. OKH-03 aromatic polyester is selected (secondary selection). In order for Rhizobium sp.OK H—03 to degrade aromatic polyesters, 1) bacteria adherence to aromatic polyesters, 2) aromatic polyester chain degradation, 3) aromatic polyester oligomers and monomers It is presumed that it goes through the process of decomposition. At each of these stages, a strain that supports the degradation ability of Rhizobium sp. OKH-03 can be obtained by secondary selection.

As a strain of the present invention, Pigmentiferga sp. (NI TE-65) grows the primary selected strain to the logarithmic growth phase, and collects a large amount of the collected cells in a bishydroxichtil terephthalate medium. Alternatively, after culturing by a method such as inoculation in a terephthalic acid medium, the bishydroxyterephthalate resolution or terephthalic acid resolution can be confirmed.

The strains of the present invention, Bacillus megateleum (NITE P-67), Syudomonas sp. (N ITE P-64), and Alphaproteobacteria (NITE P-66) are grown to the logarithmic growth phase. After cultivating a large number of collected cells in an aromatic polyester medium by inoculating with Rhizobium sp. OKH-03, etc., by confirming their biofilm-producing ability, Rhizobium s p. A strain capable of improving the ability of decomposing aromatic polyester of OKH-03 can be obtained.

The decomposition method of the present invention uses a group of microorganisms in combination with one or more strains of the present invention for a strain having an aromatic polyester resolving power, so that the aromatic polyester can be safely and even reduced to carbon dioxide. It can be reliably decomposed at a high speed. In the decomposition method of the present invention, it is sufficient that the microorganism group and the aromatic polyester are reliably in contact with each other and the state in which the microorganism group is maintained is formed. In particular, in the aqueous solution in which the microorganism group of the present invention exists. This is preferably performed by immersing the aromatic polyester to be decomposed.

As the aqueous solution, only aromatic polyester is essentially the only organic nutrient source. It is preferable to use such a medium, and there is no particular restriction. It is preferable to use LE medium in which the amount of organic nutrients other than aromatic polyester is 0.2% by weight or less, and it is more preferable to use a medium in which an inorganic compound is added to this LE medium. The LE medium here is a medium consisting of lettuce and egg yolk extract, which can be prepared by the following method. 1 3 g of lettuce leaves dried at 10 ° C for 5 hours and 3 g of boiled egg yolk are separately decocted with 1 L of ion-exchanged water for 10 minutes, cooled to room temperature, and filtered through filter paper. The mixture of these filtrates is used as LE medium.

Examples of the inorganic compound added here include inorganic ammonium salts such as ammonium sulfate and ammonium nitrate, metal salts such as iron sulfate, copper sulfate, zinc sulfate, manganese sulfate, and magnesium sulfate, and hydrates thereof.

The temperature at which the microorganism and the aromatic polyester are brought into contact is preferably in the range of 20 to 37 ° C, more preferably 25 to 35 ° C (: particularly preferably 30 ° C).

Further, the pH when contacting is preferably in the range of 5-9. In order to adjust pH within this range, for example, when an aromatic polyester is immersed in an aqueous solution and brought into contact with the aqueous solution, an inorganic acid such as hydrochloric acid or sulfuric acid, sodium hydroxide, potassium hydroxide is added to the aqueous solution. It is preferable to use an inorganic base such as sulfur and an aqueous solution thereof, and it is also preferable to use various buffer solutions such as a phosphate buffer solution.

Most preferably, the microorganism of the present invention is contacted with an aromatic polyester in LE medium having a temperature of 20 to 37 ° C and a pH of 5 to 9. However, other methods can be used as long as the microorganisms can be brought into contact with the aromatic polyester and the aromatic polyester can be decomposed.

Further, when the microorganism of the present invention is brought into contact with the aromatic polyester, it is preferable to adsorb the microorganism to the aromatic polyester to form a biofilm on the surface of the aromatic polyester. The biofilm here is a layered substance consisting of microorganisms and their discharges, and this forms a place where the microorganisms adhere firmly to the aromatic polyester and decompose the aromatic polyester. It will be. Further, the period for contacting the microorganism of the present invention with the aromatic polyester may be at least 24 hours, but any period can be set according to the target amount of degradation of the aromatic polyester. If the contact period lasts for more than 2 weeks, it is desirable to replace the aqueous solution with a new one every two weeks.

By such a method, the aromatic polyester, which hardly decomposes in nature, can be decomposed in a short period of about 0.5 to 4 months.

As described above, if a microorganism having the ability to specifically decompose the aromatic polyester of the present invention is used, the aromatic polyester can be decomposed safely and inexpensively under mild conditions. Example

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

Example 1

The PET film was immersed in a 0.1 N aqueous hydrochloric acid solution for 3 hours, then immersed in a 70% by weight aqueous ethanol solution for 12 hours or more, and dried under aseptic conditions. A sterilized PET film having dimensions of 1.4 cm × 2.0 cm and a weight of 68.6 mg was prepared. 10 ml of aqueous solution medium with pH 7.0 consisting of the ingredients listed in Table 3, Rhizopium spp. OKH-03 (deposit number FERM P— 1948 3), Pigmenty Ferga sp. (NI TE P—65), Combined with lml culture medium containing Bacillus megaterium (NITE P-67), Shudomonas sp. (NI TE P_64), and alpha proteobacteria (NI TE P-66), a silicon stopper was attached. It was enclosed in a test tube with an inner diameter of 18 mm.

While maintaining the aerobic condition, shake at 30 ° C, 300 strokes / min using a horizontal shaking incubator, and replace the solution in the test tube with a new one every 2 weeks. Shaking culture was performed for 35 days.

Remove the PET film from the test tube and sonicate it in a 70 wt% aqueous ethanol solution for 20 minutes. Was removed.

Next, after the PET film was dried at room temperature under vacuum for 24 hours or more, the weight was measured. As a result, the weight of the PET film after the decomposition treatment was 35.7 mg, and the weight loss rate was 48.0%. The degradation rate was 0.34 mg / cm ² · day based on the area of one side.

Table 3

Example 2

Isophthalic acid copolymer PET film was immersed in 0.1 N aqueous hydrochloric acid solution for 3 hours, then immersed in 70% ethanol aqueous solution for 12 hours or more, and dried under aseptic conditions. A 10% isophthalic acid copolymer PET film having dimensions of 1.2 cm × l. 5 cm and a weight of 66.3 mg was prepared. 10 ml 1 aqueous solution medium with pH 7.0 consisting of the components listed in Table 2, Rhizobium sp. OKH-03 (Deposit Number FERM P-19483), Pigmenti Ferga sp. (NITE P-65), Bacillus Combine with 1 ml of culture medium containing Megaterium (NI TE P-67), Shudomonas sp. (NI TE P-64), and Alpha Proteobacteria (NI TE P-66). The sample was sealed in a test tube having an inner diameter of 18 mm.

While maintaining aerobic conditions, use a side shaker incubator at 30 ° C, 300 straws The mixture was shaken under the condition of the sample and shake culture was performed for a total of 30 days, replacing the solution in the test tube with a new one every two weeks.

Isophthalic acid copolymerized PET film was taken out from the test tube and sonicated in 70% by weight ethanol aqueous solution for 20 minutes, leaving behind the cells and cells discharged from the film surface.

Next, this isophthalic acid copolymer PET film was dried at room temperature under vacuum for 24 hours or more, and then the weight was measured. As a result, the weight of the film after the decomposition treatment was 40.6 mg, and the weight loss rate was 39%. The degradation rate based on the area of one side of the film was 0.48 mg / cm ² · day.

Example 3

Medium containing 4.8mM terephthalic acid disodium salt 1m1 culture medium containing Pigmenty Ferga sp. (NI TE P-65) in 1ml of Table 2 The test tube was sealed. While maintaining the aerobic condition, the mixture was shaken at 30 T: 300 stroke / min using a side-shaking shaker and cultured for 2 days.

When the culture solution was taken out of the test tube and terephthalic acid was detected using TLC, no spot showing terephthalic acid was found.

Comparative Example 1

In Example 1, using a PET film with dimensions of 1.4 cmX 2. O cm, an initial weight of 60.7 mg, and containing only Rhizobium sp. OKH-03 (deposit number FE RM P-19483) When the same operation was performed except that 1 m 1 was added, the weight of the PET film was 56.2 mg in 55 days, the weight loss rate was 7.4%, based on the area of one side of the film The degradation rate was 0.029 mg / cm ² · day.

Comparative Example 2

In Example 1, the same operation was performed except that the dimensions were 1.4 cmX 2. O cm, PET film having an initial weight of 60.7 mg was used, and no culture medium containing bacteria was added. However, the weight of the PET film is 60.7 mg No significant weight loss was observed. In addition, as shown in Fig. 2, it was confirmed by visual observation with an electron microscope that the surface was not decomposed.

Comparative Example 3

In Example 3, the same operation was carried out except that Pigmentia ferga was not included, and TLC detected the same concentration of terephthalic acid as in the initial culture.

Claims

The scope of the claims

1. A microorganism belonging to the genus Pigmenthiferga and having the ability to improve the ability to degrade aromatic polyester of Rhizobium sp. OKH-03 (FERM P-19483) and the ability to degrade terephthalic acid.

2. The microorganism according to claim 1, wherein the microorganism belonging to the genus Pigmentifera has been deposited under the accession number N I TE P—65.

3. A microorganism belonging to the genus Bacillus and capable of improving the ability to degrade aromatic polyester of Rhizobium sp.OKH-03.

4. The microorganism according to claim 3, wherein the microorganism belonging to the genus Bacillus is deposited under the accession number N I TE P-67.

5. A microorganism belonging to the genus Pseudomonas and capable of improving the ability to degrade aromatic polyesters of Rhizopium sp.OKH-03.

6. The microorganism according to claim 5, wherein the microorganism belonging to the genus Pseudomonas is deposited under the receipt number N I TE P-64.

7. A microorganism belonging to the class of alphaproteobacteria and capable of improving the ability of lysobium sp. OKH-03 to degrade aromatic polyester.

8. The microorganism according to claim 7, wherein the microorganism belonging to the class Alphaproteobacteria is deposited under the receipt number N I TE P-66.

9. Rhizobium s p. OKH-03 (FERM P—19483), and Claims: A method for decomposing an aromatic polyester, comprising decomposing the aromatic polyester by contacting at least one microorganism selected from the group consisting of the microorganisms described in! To 8 with the aromatic polyester.

0. The decomposition method according to claim 9, wherein the aromatic polyester is a polyester containing 95% by weight or more of ethylene terephthalate repeating units.

11. The degradation method according to claim 9, wherein the microorganism and the aromatic polyester are contacted in LE medium.

12. The decomposition method according to claim 9, wherein the contact between the microorganism and the aromatic polyester is performed in a range of 20 ° (: to 37 ° C).

13. The decomposition method according to claim 9, wherein the contact between the microorganism and the aromatic polyester is carried out in the range of pH 5-9.

14. The decomposition method according to claim 9, wherein the contact between the microorganism and the aromatic polyester is carried out by adsorbing the microorganism to the aromatic polyester to form a biofilm.