JPH09252791A - Polybutylene succinate-based resin decomposing bacterium, biological decomposition of polybutylene succinate-based resin and production of 4-hydroxy-n-butyric acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide both a method for biologically and directly decomposing a polybutylene succinate-based resin and a plastic containing the resin and a method for biologically producing 4-hydroxy-butyric acid from a polybutylene succinate-based resin and to obtain a bacterium useful for these methods. SOLUTION: This Amycolatopsis mediterranei HT-6 (FERM P-15,023) is capable of decomposing a polybutylene succinate-based resin. An actinomycetes is brought into contact with a polybutylene succinate-based resin to decompose the resin. The actinomycetes is brought into contact with the polybutylene succinate-based resin to produce 4-hydroxy-n-butyric acid.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a microorganism that decomposes a polybutylene succinate resin, a method for biologically degrading a polybutylene succinate resin, and a 4-hydroxy-based resin containing the polybutylene succinate resin as a raw material. n-
It relates to a microbiological production method of butyric acid.

[0002]

2. Description of the Related Art Recently, the treatment of plastic waste has become a problem. As a method of treating waste, incineration and landfill have been mainly performed so far, but incineration promotes global warming, and landfill has problems such as reduction of landfill sites. Therefore, as a means for solving the problem of such plastic waste treatment, a biological treatment method has recently attracted attention, and so-called biodegradable plastics that are decomposed by the action of microorganisms have been developed. Polybutylene succinate-based resins are drawing attention as a material for biodegradable plastics that replace general-purpose plastics that are difficult to decompose in natural environments, and are being developed for various uses as next-generation plastics. It is fully expected that with the increase in the amount, the problem of waste treatment will be highlighted as well as the plastics currently used in the near future.

[0003]

It is known that polybutylene succinate resins are generally not easily decomposed by ester degrading enzymes because of their high melting points. Also,
Until now, there has been no technique for directly biologically degrading polybutylene succine resin and its waste. Further, there is no known technique for recovering and reusing the polybutylene succinate resin.

Therefore, an object of the present invention is to provide a method for biologically directly decomposing polybutylene succinate resins and plastics containing them, and to obtain 4-hydroxy-n-butyric acid from polybutylene succinate resins. To provide a method for producing the same and microorganisms useful for those methods.

[0005]

Means and Actions for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found actinomycetes having an activity of degrading polybutylene succinate resin, and It came to completion.

That is, according to the present invention: 1) Amycolatopsis mediteranei ( Amycolatopsis me ) having a polybutylene succinate resin decomposing ability
diterranei ) HT-6 (FERM P-15023), 2) Polybutylene succinate-based resin is contacted with actinomycetes and decomposed. Topushisu (Amycolatopsis) method degradation of polybutylene succinate-based resin of the 2, wherein the bacteria belonging to the genus, 4) actinomycetes Amycolatopsis media Teller Nei (Am
ycolatopsis mediterranei ) The method for degrading the polybutylene succinate-based resin according to 2 above, 5) The actinomycete is Amycolatopsis mediterranei ( Am
ycolatopsis mediterranei ) HT-6 (FERM P-15023)
The method for decomposing a polybutylene succinate resin according to the above item 2, wherein 6) a method for producing 4-hydroxy-n-butyric acid, which comprises contacting the polybutylene succinate resin with an actinomycete, 7) an actinomycete Is a bacterium belonging to the genus Amycolatopsis (8), and the method for producing 4-hydroxy-n-butyric acid according to the above 6, 8) The actinomycetes is Amycolatopsis mediterranei ( Am
ycolatopsis mediterranei ) and the method for producing 4-hydroxy-n-butyric acid as described in 6 above. And 9) the actinomycete is Amycolatopsis mediterranei ( Am
ycolatopsis mediterranei ) HT-6 (FERM P-15023)
The method for producing 4-hydroxy-n-butyric acid according to 6 above is provided.

The polybutylene succinate resin is an aliphatic polyester resin composed of an aliphatic glycol and an aliphatic dicarboxylic acid, and the aliphatic glycol moiety is 1,4-
Butanediol, other than polybutylene succinate homopolymer in which the aliphatic dicarboxylic acid moiety is succinic acid, part of the 1,4-butanediol moiety in polybutylene succinate homopolymer can be converted to other aliphatic glycol such as ethylene glycol. The polybutylene succinate-based resin includes a copolymer in which a part of the succinic acid moiety is replaced with another aliphatic dicarboxylic acid such as adipic acid. Further, those obtained by extending the chain length of these resins with diisocyanate or the like are also included in the polybutylene succinate resin. In addition, the form to be actually used may be a pure product or a blend with another resin. Further, it may contain a plasticizer, a pigment, an organic filler such as starch or cellulose, or an inorganic filler such as talc.

The present invention makes it possible to decompose polybutylene succinate resin by contacting it with actinomycetes under aerobic conditions and is useful as a polymer raw material or the like. It is possible to recover 4-hydroxy-n-butyric acid. As an example of the microorganism used in the present invention, Amycolatopsis mediterranei HT-6 (FE
RM P-15023). The microorganism will be described below.

The present inventors collected soil, river and lake water from Tsukuba City, Ibaraki Prefecture, and separated and obtained aerobic microorganisms that decompose polybutylene succinate resin from these samples through the operations described in detail below. did.

1 liter of basic medium shown in Table 1 (pH 7)
1000 mg of polybutylene succine-based resin was emulsified in to prepare an agar plate medium containing 1.5% agar. 1 g of each sample was suspended in 5 ml of sterilized water, and 10 ³ to 10 ³
After diluting ⁵ times, 0.2 ml was taken out, applied on the agar plate medium prepared by the above method, and cultured in an incubator at 30 ° C. Among the colonies grown on the medium, those having a transparent region around the colonies were used as the degrading bacteria of the polybutylene succinate resin, and the degrading bacteria were isolated by catching the colonies with a platinum loop.

[0011]

TABLE 1 Basic medium composition table (distilled water 1 liter) Ingredient amount _{Νa 2 MoO 4 · 2H 2 O} 0.5mg Na 2 WΟ 4 · 2H 2 O 0.5mg MnSO 4 0.5mg FeSO 4 · 7Η 2 O 10mg NaCl 10 mg CaCl ₂ .2Η ₂ O 20 mg (N ₄ ) ₂ SO ₄ 1000 mg MgS Ο ₄ .7 Η ₂ O 200 mg K ₂ HPO ₄ 1600 mg KH ₂ PO ₄ 200 mg Surfactant 100 mg Yeast extract 100 mg

Among the 43 samples, there were several samples in which clear areas were confirmed around the colonies grown on the medium. The colony was picked up with a platinum loop and purely separated using the same medium to obtain a strain (FERM P-15023) having a high polybutylene succinate resin decomposing ability.

The isolated strain was inoculated on a YM agar medium to form a colony, and the properties of the obtained bacterial cell were observed with a microscope and the biochemical properties were examined. The results are shown in Table 2 below.

[0014]

[Table 2]  Colony morphology: Spherical, cream, white aerial hypha Gram stainability: + Sporulation ability: -Mobility: -Aerial mycelium: + Cell wall composition: MesoDAP + mycolic acid-Catalase production: + OF test: + OF test:- (Oxidized form) Acid production: Glucose + Maltose + Cellobiose + Raffinose + Rhamnose-Fructose + Xylose + Sucrose + Inulin + Adonitol-Inositol-Mannitol + Sodium azide 0.01% Growth: -In the presence of sodium chloride 7% Growth: -Growth in the presence of 0.1% phenol: -Growth in the presence of rifampicin: + Growth in the presence of neomycin: + Growth in the presence of arbutin: -Growth in the presence of xanthine:-Growth in the presence of allantoin:-

The results shown in Table 2 are shown in Bergey's Manual of Det.
When referring to erminative Bacteriology 9th edition, etc., the above strains were identified as Amyctopsis mediterranei ( Amyc
olatopsis mediterranei ) is similar in properties to the bacteria belonging to olatopsis mediterranei ), but differs from other known strains in that it has strong degrading activity for high molecular weight polybutylene succinate resin. For example, the above-mentioned strain decomposes a high molecular weight polybutylene succinate resin (average molecular weight 5.74 × 10 ⁴ ) by 90% or more after 6 days of culturing, and has a very strong polybutylene succinate resin decomposing ability. Therefore, this strain is Amycolatopsis mediterranei ( Amycolat
HT-6 was recognized as a new strain belonging to opsis mediterranei ).
It has been deposited with the Institute of Biotechnology and Industrial Technology, Institute of Industrial Technology as No. 15023 (FERM P-15023).

Next, the method for treating the polybutylene succinate resin in the present invention will be described in detail. In the method of the present invention, the microorganism used to treat the polybutylene succinate resin is an actinomycete having a polybutylene succinate resin decomposing ability, preferably an actinomycete contained in the genus Amycolatopsis. Yes , more preferably Amycolatopsis mediterranei , more preferably Amycolatopsis mediterranei.
olatopsis mediterranei ) (FERM P-15023) strain.
Further, by using the FERMP-15023 strain as the original strain by natural or induced mutation, a mutant strain having an enhanced property of degrading the polybutylene succinate resin possessed by the FERM P-15023 strain is obtained and used in the present invention. Can be done. As a method for preparing these mutant strains, a conventionally known conventional method, for example, artificial mutation by irradiating the original strain with ultraviolet rays or a drug such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) is used. Treated and spread on an agar medium containing skim milk etc., the clear zone formed around the colony from the growing strains, selected larger colonies, excellent in polybutylene succinate resin decomposing ability A method of selecting a strain can be used. It is also possible to ligate a gene having the properties applicable to the present invention with an appropriate self-replicating DNA, transform an appropriate host cell using this, and improve the polybutylene succinate resin decomposing ability by the gene amplification effect. it can. In treating the polybutylene succinate-based resin, these microorganisms may be used alone, or may be used as a group of microorganisms containing these microorganisms.

The above-mentioned microorganism or a group of microorganisms including the microorganism is, if necessary, a culture solution grown and cultured in a medium, cells separated from the culture solution by a method such as centrifugation, or a method such as freeze-drying the cells. Powder obtained by drying with, powder containing various vitamins and minerals and necessary nutritional sources,
The powder is made into granules, or tablets or the like are used for treatment of the polybutylene succinate resin.

The medium used for culturing the microorganism used in the method of the present invention uses, for example, ammonium sulfate, ammonium phosphate, ammonium carbonate as a nitrogen source, and other inorganic salts such as monopotassium phosphate and phosphoric acid. A culture source that uses dipotassium, magnesium sulfate, sodium chloride, ferrous sulfate, sodium molybdate, sodium tungstate, manganese sulfate, etc. and is used for culturing ordinary microorganisms such as yeast extract and peptone as an organic nutrient source. Is used. The pΗ during the culture is 4.0 to 10.0, preferably 5.0 to 8.0. The culture temperature is 10 to 36 ° C, preferably 2
It is 0 to 35 ° C.

The biodegradation treatment of the polybutylene succinate resin according to the present invention is carried out by adding a culture medium, the polybutylene succinate resin to be treated,
It is desirable to add powders, tablets, culture solutions and the like containing the above microorganisms or a group of microorganisms containing the microorganisms, but the treatment method is not limited to this. For example, it may be carried out by a method of introducing the above-mentioned microorganism or microorganisms into an activated sludge tank used for wastewater treatment or the like, or by introducing it into a waste composting (composting) treatment apparatus. The amount of polybutylene succinate resin added to the basic medium is preferably 0.01% by weight to 10% by weight. The amount of microorganisms to be added may be extremely small, but is preferably 0.01% or more with respect to the polybutylene succinate resin because the amount of the added microorganisms does not affect the processing time. The conditions for the biological decomposition treatment of the polybutylene succinate-based resin in the method of the present invention are: p Η 4.0 to 10.0, temperature 1
It is 0 to 75 ° C.

Next, a method for obtaining 4-hydroxy-n-butyric acid from a polybutylene succinate resin will be described. 4-Hydroxy-n-butyric acid is produced by utilizing the above microorganism or its culture solution and decomposing the polybutylene succinate resin by the method described above. The biological processing conditions for producing 4-hydroxy-n-butyric acid are pΗ 4.0 to 10.0, preferably 5.0.
~ 8.0. The temperature is 10 to 50 ° C, preferably 20 to 35 ° C.

It is also possible to recover the monomer obtained as a product of the above-mentioned strain decomposing the polybutylene succinate resin and utilize it again in the synthesis of the polymer.

[0022]

EXAMPLES Next, examples will be described, but the present invention is not limited to the following examples. Example 1 100 mg of polybutylene succinate (PBS) having an average molecular weight of 5.74 × 10 ⁴ was suspended in 100 ml of a basic medium, and HT-6 strain (FERM P-15023 strain) was inoculated into the suspension and cultured. Amount of undegraded polybutylene succinate, amount of cells, amount of water-soluble total organic carbon (TOC), 4-
The amount of hydroxy-n-butyric acid, pH, etc. were measured over time. The results are shown in FIGS. 1, 2 and 3. After 4 days of culture, polybutylene succinate was decomposed by 80%, and on the 12th day of culture, the decomposition rate of polybutylene succinate was 95%.
% Has been reached. During the culture period, 1,4-butanediol,
Succinic acid, 4-hydroxy-n-butyric acid was produced. Four
The amount of hydroxy-n-butyric acid produced was 17 m after 4 days of culture.
g.

Example 2 Polybutylene succinate was treated by a heat press method to form a film (thickness: 30 to 40 μm).
HT-6 strain (FERM P-1
5023 strain) was cultured. The surface of the film before and after the culture was observed with an electron microscope. The result is shown in FIG.
~ 4 (d). On day 2 of culture, small cracks were seen on the surface of the film (4 (b)), and on day 4 of culture the cracks were larger, which led to the collapse of the film (4 (c)). On the 6th day of culture, it was confirmed that a hemispherical hole having a diameter of 16 to 20 μm was formed on the surface of the film fragment and the decomposition proceeded (4 (d)).

[0024]

The method for degrading a microbiological polybutylene succinate resin according to the present invention does not generate an exhaust gas as in the existing incineration method and does not cause a disposal site problem such as a case of landfill treatment. It is a technology and an extremely valuable method for treating polybutylene succinate resin waste.
By using the treatment method of the present invention in a composting facility, it is possible to convert the polybutylene succinate resin into a useful substance such as an organic acid or compost. Also, 4-hydroxy-n-butyric acid produced according to the present invention can be used again for polymer synthesis.

[Brief description of drawings]

FIG. 1 shows polybutylene succinate (P
(BS) Degradation Method Example, Cell Weight and Recovered P
It is a graph which shows the daily change of the amount of BS.

FIG. 2 is a water-soluble total organic carbon (TOC), 1,
4-butanediol, succinic acid and 4-hydroxy-
It is a graph which shows the daily change of the amount of n-butyric acid.

FIG. 3 is a graph showing the change of pH with time.

FIG. 4a is an electron micrograph of a polybutylene succinate film before treatment according to the method of the present invention.

FIG. 4b is an electron micrograph of a polybutylene succinate film 2 days after treatment by the method of the present invention.

FIG. 4c is an electron micrograph of a polybutylene succinate film 4 days after treatment by the method of the present invention.

FIG. 4d is an electron micrograph of a polybutylene succinate film 6 days after treatment by the method of the present invention.

[Procedure amendment]

[Submission date] October 1, 1996

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0023

[Correction method] Change

[Correction contents]

Example 2 Polybutylene succinate was treated by a heat press method to form a film (thickness: 30 to 40 μm).
And using it as a substrate, the HT-6 strain (FERM
P-15023 strain) was cultured. The surface of the film before and after the culture was observed with an electron microscope. as a result
Is shown in FIGS . On day 2 of culture, small cracks were seen on the surface of the film ( Fig. 5 ), and on day 4 of culture the cracks were larger, which led to the collapse of the film ( Fig. 6 ). On the 6th day of culture, it was confirmed that hemispherical holes having a diameter of 16 to 20 μm were formed on the surface of the film fragment and the decomposition proceeded ( FIG. 7 ). ─────────────────────────────────────────────────── ───

[Procedure amendment]

[Submission date] October 1, 1996

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Brief description of drawings

[Correction method] Change

[Correction contents]

[Brief description of drawings]

FIG. 1 shows polybutylene succinate (P
(BS) Degradation Method Example, Cell Weight and Recovered P
It is a graph which shows the daily change of the amount of BS.

FIG. 2 is a water-soluble total organic carbon (TOC), 1,
4-butanediol, succinic acid and 4-hydroxy-
It is a graph which shows the daily change of the amount of n-butyric acid.

FIG. 3 is a graph showing the change of pH with time.

FIG. 4 is an electron micrograph of a polybutylene succinate film before treatment by the method of the present invention.

FIG. 5 is an electron micrograph of a polybutylene succinate film 2 days after treatment by the method of the present invention.

FIG. 6 is an electron micrograph of a polybutylene succinate film 4 days after treatment by the method of the present invention.

FIG. 7 is an electron micrograph of a polybutylene succinate film 6 days after treatment by the method of the present invention.

[Procedure amendment 2]

[Document name to be amended] Drawing

[Correction target item name] Fig. 4

[Correction method] Change

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FIG. 4

[Procedure 3]

[Document name to be amended] Drawing

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[Figure 5]

[Procedure amendment 4]

[Document name to be amended] Drawing

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[Correction method] Change

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[Procedure Amendment 5]

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FIG. 7

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical display location C12R 1:01) (C12N 1/20 C12R 1:01) (72) Inventor Hideo Tanaka Tsukuba, Ibaraki Prefecture 1-1-1, Tennodai, Ichi, University of Applied Biochemistry, University of Tsukuba (72) Inventor Haldanin Pranamda 1-1-1, Tennodai, Tsukuba, Ibaraki Prefecture, Department of Applied Biochemistry, University of Tsukuba

Claims (9)

[Claims] 1. A poly-butylene succinate-based resin capable of degrading Amycolatopsis mediterranei ( Amycol)
atopsis mediterranei ) HT-6 (FERM P-15023). 2. A method for decomposing a polybutylene succinate resin, which comprises contacting an actinomycete with the polybutylene succinate resin and decomposing it. 3. The actinomycete is Amycolatopsis ( Amycolatop).
The method for degrading a polybutylene succinate resin according to claim 2, which is a bacterium belonging to the genus sis ). 4. The actinomycete is Amycolatopsis mediterranei.
A method for decomposing a polybutylene succinate-based resin as described. 5. The actinomycete is Amycolatopsis mediterranei ( Amycolatopsis mediterranei ) HT-6 (FERM
P-15023), The method for decomposing a polybutylene succinate resin according to claim 2. 6. A 4-hydroxy-n-characterized by contacting actinomycetes with a polybutylene succinate resin.
Butyric acid production method. 7. The actinomycete is Amycolatopsis ( Amycolatop).
The method for producing 4-hydroxy-n-butyric acid according to claim 6, which is a bacterium belonging to the genus sis ). 8. The actinomycete is Amycolatopsis mediterranei ( Amycolatopsis mediterranei ).
The method for producing 4-hydroxy-n-butyric acid described. 9. The actinomycete is Amycolatopsis mediterranei ( Amycolatopsis mediterranei ) HT-6 (FERM
P-15023), which is 4-hydroxy-n-.
Butyric acid production method.