JPH11346763A - Microorganism capable of decomposing olefin (co)polymer and rosin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain novel microorganisms belonging to the genus Acinetobacter, nonpathogenic to crops, having olefin (co) polymer-degrading and rosin decomposability, capable of enhancing resistance to environmental pollutions and useful for agricultural fields, etc. SOLUTION: This microorganism belongs to the genus Acinetobacter such as Acinetobacter radioresistens FASH-3 (FERM P=16819), etc., and decomposes olefin (co)polymers such as polyethylenes and rosin such as hydrogenated rosin. It is preferable to decompose compositions containing one or more species of olefin (co)polymers and rosin.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microorganism belonging to Acinetobacter which can efficiently decompose both an olefin polymer or copolymer (hereinafter, referred to as an olefin (co) polymer) and a rosin resin. . The present invention also relates to a method for decomposing a composition containing one or more of an olefin (co) polymer and a rosin resin using these microorganisms. According to the present invention, olefin (co) polymers and rosins contained in coating films of coated fertilizers, paints, printing inks, adhesives, industrial additives, waste liquids such as pulp, and a wide range of other packaging materials and molding materials Resin can be efficiently biodegraded.

[0002]

2. Description of the Related Art Chemical fertilizers are usually water-soluble and are therefore quick-acting. However, the loss due to runoff of fertilizer components is large. If fertilizer is applied in large quantities at one time, it may cause concentration problems on agricultural crops or cause river pollution. There was a fear. As a means for improving this, a method has been proposed in which a fertilizer is coated with various resins to impart sustained release properties. Olefin (co) polymers such as polyethylene and polypropylene, which are currently used as coating materials, have high stability in these soils, so that the elution stability of fertilizer components is ensured. After that, there is a problem that the membrane material remains in the soil. In order to increase the disintegration rate of this membrane material even slightly, polycaptolactone, aliphatic ester copolymer, polylactic acid, biodegradable resin such as polyvinyl alcohol or cellulose,
There is known a method of mixing a compound or a substance having a high decomposition rate such as a natural polymer such as starch and rosin into a coating film. These high molecular compounds and substances are
Olefin (co) present in the composition containing the polymer
It decomposes faster than the polymer and promotes the disintegration of the composition. Above all, rosin resin is a compound derived from a natural product, has excellent biodegradability, and is relatively inexpensive.
When used as a film material for coated fertilizer, it can be said that it is one of suitable materials.

[0003] Olefin (co) known so far
As the polymer-decomposing microorganism, as a polyethylene-only decomposing bacterium, JP-A-50-126791, a bacterium belonging to Acinetobacter calcoacetics or Kaningamera described in JP-A-52-56175, a genus Aspergillus, Microorganisms such as molds belonging to the genus Resorbes and the like, as degrading bacteria of rosin resin alone, Can.J.Microbiol.
Vol. 43 (599-611) 1997, described in JP-A-7-313143, bacteria of the genus Bacillus, Pseudomonas,
There are microorganisms such as mold and mold of the family Ketomium.
To the inventors' knowledge, the genus Acinetobacter, more specifically Acinetobacter radioresistence, is not known as a rosin resin-degrading bacterium. To the knowledge of the present inventors, there is no known microorganism capable of decomposing both the olefin (co) polymer and the rosin resin. When a composition containing an olefin (co) polymer and a rosin resin is used as a coating film of a coated fertilizer, biodegradation of a rosin resin having a high decomposition rate and biodegradation of an olefin (co) polymer are performed by the same microorganism. Then, the decomposition of the coating film is efficient, and a conjugation effect is expected. In addition, microorganisms that can coexist with crops when used in agricultural land are more desirable from the viewpoint of environmental impact.

[0004]

SUMMARY OF THE INVENTION The present invention has been made in view of such circumstances, and an object thereof is to provide a microorganism having an ability to degrade an olefin (co) polymer and a rosin resin. Things. A further object of the present invention is to provide an olefin (co)
An object of the present invention is to provide a method for efficiently decomposing a composition containing at least one of a polymer and a rosin resin.

[0005]

Means for Solving the Problems In order to solve the above problems, the present inventors have searched for microorganisms having olefin (co) polymer resolution and rosin resin resolution.
First, a method of obtaining microorganisms that have the ability to degrade olefin (co) polymers, which are difficult to decompose and have a low decomposition rate, and then select microorganisms that can degrade rosin resin from them. I took it. Specifically, soil from healthy farmland using olefin (co) polymer-based fertilizer is collected from 154 locations throughout Japan,
After extensive research, Acinetobactor ( Acinetobac
A microorganism belonging to ter) that is effective not only for decomposing olefin (co) polymers but also for decomposing rosin resin was successfully obtained, and completed the present invention.

That is, the present invention relates to a microorganism belonging to Acinetobacter and having the ability to degrade olefin (co) polymer and rosin resin. The present invention also relates to a method for decomposing a composition containing at least one of an olefin (co) polymer and a rosin resin using the microorganism. The present invention relates to a method for decomposing a composition containing at least a rosin resin, and more preferably a composition containing a rosin resin and an olefin (co) polymer. As the microorganism in the present invention, specifically, Acinetobacter radioresistence ( Acinetob) having the ability to degrade olefin (co) polymer and rosin resin
acte r radioresistens ). More specifically, Acinetobacter radioresistens FKSH-3 (FERMP-1681)
9). The present invention is not limited to these microorganisms, and has the ability to degrade olefin (co) polymer and the ability to degrade rosin resin ( Acinetobacte).
r) , more specifically, Acinetobacter r radioresisten
Any microorganism may be used as long as it belongs to s ).

The microorganism according to the present invention was isolated by the following method. Prepared aseptically, ultrasonic dispersion (70-80
Polyethylene wax (Mw / Mn = 720/7)
Inorganic salt medium having the composition of Table 1 wherein
An appropriate amount of an extract of healthy soil collected from 154 locations throughout Japan using olefin (co) polymer-based coating fertilizer as a separation source for olefin (co) polymer-degrading microorganisms was added at 28 ° C. The cells were subjected to shaking culture at 125 rpm. Those in which the growth of the microorganism was observed were subjected to subculture several times using the same medium. As a result, nine types of microorganisms were obtained.

[0008]

[Table 1] Basic medium composition Table component name (in 1 liter of distilled water) NH ₄ NO ₃ 5 g K ₂ HPO ₄ 1 g KCl 1 g MgSO ₄ .7H ₂ O 0.2 g CaCl ₂ .2H ₂ O 0.01 g FeSO ₄・ 7H ₂ O 0.01g MnCl ₂・ 4H ₂ O 0.01g ZnSO ₄・ 7H ₂ O 0.01g Price Surf A210-G (Daiichi Kogyo Pharmaceutical) 0.3 g Yeast extract (DIFCO) 0.1 g pH 7.0-7.2

Next, rosin resin-degrading bacteria were selected by the following method. By subculturing the nine microorganisms into an inorganic salt medium having the composition shown in Table 1 using a rosin resin as a carbon source prepared aseptically and ultrasonically dispersed (70-80 ° C., 15 minutes). The obtained nine kinds of microorganisms were added and cultured with shaking. The culture solution was sampled over time, and the viable cell count of the microorganism at each culture time was measured by a dilution plate method using an ordinary agar medium (manufactured by Eiken Chemical Co., Ltd.) to confirm the growth of one type of microorganism. The microorganism thus obtained was inoculated on an agar medium to form a colony, and the microorganism was isolated and named FKSH-3. Microscopic observation of the properties of the obtained microorganisms, and examination of culture characteristics, biochemical characteristics and genetic characteristics,
FKSH-3 exhibited the following properties.

(A) Morphological properties Morphology of the bacterium: short rod-shaped spores:-(B) Growth state in the medium Growth temperature range: The optimal growth temperature range is 20-30 ° C. It grows at 37 ° C but not at 45 ° C. Form white colonies. (C) Physiological properties (+: positive-: negative) Gram stain:-Mobility:-Anaerobic growth:-Oxidase production:-Catalase production: + Tychrome oxidase:-Acid production from glucose:- OF test: glucose-(alkaline)

Utilization: (1) Capric acid, adipic acid, malic acid, citric acid, phenylacetic acid, lactic acid, histidine, (2) Glucose, arabinose, mannose, mannitol, N-acetylglucosamine, maltose, glucone Does not grow on acids, malonic acid, arginine. Nitrate reduction:-Indole production:-Arginine dihydrolase:-Urease:-Esculin hydrolysis:-Gelatin hydrolysis:-β-galactosidase:-Simmon's citric acid test: + (alkaline) Hemolysis:-γ- Glutamyltransferase 12:-(4 hours reaction) β-xylosidase:-(4 hours reaction) (D) Genetic properties 16SrRNA : Approximately 98% identical to Acinetobacter radioresistens .

Regarding the results shown above, MIDI Labs, Inc.
(125 Sandy Drive Newark, DE 19713)
Due to the 98% homology, strain FKSH-3 was isolated from Acinetobacter radioresisten
s ), identified as a new strain belonging to Acinetobacter radioresistens, and submitted to FERM P-
Deposited as 16819.

The olefin (co) polymer decomposed in the present invention is a high-density or low-density polyethylene and a petroleum wax (paraffin wax, microclestalin wax, petrolatum wax) which is generally referred to as a wax having a low molecular weight. ), Synthetic hydrocarbon waxes (Fischer-Tropsch wax, polyethylene wax, etc.) and polypropylene, polyisoprene, poly-1
-Butene and others, ethylene-1-octene copolymer,
Ethylene-vinyl acetate copolymers and other olefinic copolymers in which part of the skeleton of the olefin (co) polymer has been partially oxidized in soil, It also includes a partially oxidized skeleton of the olefin (co) polymer under one or more complex conditions. The molecular weight of the olefin (co) polymer that can be biodegraded by the microorganism can be degraded from low molecular weight to high molecular weight, but the lower the molecular weight, the faster the decomposition rate. When the microorganism is used alone, the preferred number average molecular weight of the olefin (co) polymer is less than 10,000, more preferably less than 2,000. High-molecular-weight substances having a number average molecular weight of 10,000 or more are used in combination with a photo-oxidation catalyst,
After the number average molecular weight is reduced to less than 10,000, it can be rapidly decomposed using the microorganism.

The rosin resin decomposed in the present invention includes rosin acids such as abietic acid, neoabietic acid, dehydroabietic acid, pimaric acid, repopimaric acid, parastolic acid, isopimaric acid, and sandaracopimaric acid, and rosinates thereof. Rosin acid ester, hydrogenated rosin, polymerized rosin, disproportionated rosin, rosin oxide, rosin amine, maleic acid-added rosin, rosin-modified phenolic resin, rosin-modified maleic resin, rosin-modified fumaral, which is a simple substance and chemically modified derivatives Acid resin and the like. When the microorganism is used alone, preferred rosin resins include inorganic salts such as abietic acid, neoabietic acid, parastolic acid, and levopimaric acid, modified forms of these simple substances with maleic acid, and ester copolymers thereof with polyfunctional alcohols. Coalescence. Examples of the type of the inorganic salt include a calcium salt and a zinc salt. Examples of compositions containing these olefin (co) polymers and / or rosin resins include synthetic polymers such as polyester resins, polyurethane resins, epoxy resins, alkyd resins, terpene resins, starch, and cellulose, and natural polymers. It is also possible to use a mixture with at least one of the above, and a mixture containing an inorganic salt or an inorganic pigment such as talc, silica, calcium carbonate, or clay in the mixture. More specifically, it can be used as a coated fertilizer coated with a film material containing an olefin (co) polymer and / or wax and a rosin-based resin. It can also be used for biodegradation of a composition containing a substance that can be assimilated by the microorganism.

In order to apply the microorganism of the present invention to the decomposition of olefin (co) polymer and / or rosin resin, usually,
Microorganisms are cultured by tank culture, shaking culture, etc., and the culture solution is sprayed or sprayed as it is or appropriately diluted, microorganisms separated from the culture solution by centrifugation or the like,
Powders obtained by drying microorganisms by freeze-drying, powders containing various vitamins and minerals and necessary nutrients in the powders, granulated powders, and tableted tablets I do. If necessary, commonly used agricultural or industrial additives may be added to these culture solutions. Conversely, olefin (co)
The polymer and rosin resin may be added directly.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION

EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (1) Olefin (co) polymer-degrading microorganism
Screening Collect 30 healthy soil samples collected from 154 locations in Japan using olefin (co) polymer-based coating fertilizer, add 10 times the amount of physiological saline to each, and mix well. The extract was used as a microorganism source. Next, the inorganic salt medium having the composition shown in Table 1 sterilized for 15 minutes under heating under pressure (temperature: 120 ° C., pressure: 1 kgf / cm ² ) was cooled to 70-80 ° C., and polyethylene wax (Mw / Mn = 720/700)
After adding 0.5% and ultrasonically dispersing (70-80 ° C, 15 minutes), aseptically put into a Sakaguchi culture flask, add an appropriate amount of the soil extract prepared at the beginning, and add 7 to 28 ° C at 125 rpm. The cells were cultured with shaking for 14 days. Those in which the growth of the microorganism was observed were subjected to subculture several times using the same medium. The microorganism thus obtained was transplanted to an agar plate medium, cultured at 28 ° C. to form a colony, and nine microorganisms were isolated. Microscopic observation, culture characteristics, biochemical characteristics and genetic characteristics of the obtained microorganisms were examined.

(2) decomposing the olefin (co) polymer and
Selection of microorganisms that degrade rosin resin Inorganic salt medium having the composition shown in Table 1 and sterilized for 15 minutes under pressure and heating (temperature: 120 ° C., pressure: 1 kgf / cm ² ) at 70-80 ° C.
Rosin resin (calcium abietic acid, trade name: lime resin No.1 (Arakawa Chemical Co., Ltd.)) 0.5%
After adding and ultrasonically dispersing (70-80 ° C., 15 minutes), the mixture was aseptically placed in a Sakaguchi culture flask, and the above-mentioned 9 types of cells were added to 10
^The cells were added at a concentration of ³ cells / ml and cultured with shaking at 28 ° C. and 125 rpm for 13 days. The culture solution was sampled over time, and the viable cell count of each of the above 9 strains at each culture time was measured by a dilution plate method using an ordinary agar medium (manufactured by Eiken Chemical Co., Ltd.). Was. The growth of the other eight strains was not confirmed. Table 2 shows the growth rate of the cells whose growth was confirmed. When the cells were identified as described above, it was confirmed that the cells were new strains belonging to Acinetobacter radioresistence. This strain was transformed into Acinetobacter radioresistence ( Actinetobacter ra
dioresistens ) FKSH-3 and deposited with the National Institute of Advanced Industrial Science and Technology as described above (accession number FERM P-16
819).

[0018]

[Table 2]

[0019]

Example 2 Heating under pressure (temperature: 120 ° C, pressure: 1 kgf /
cm ²⁾ mineral salts medium having the composition shown in Table 1 were sterilized for 15 minutes at then cooled to 70-80 ° C., polyethylene wax (trade name: Porirettsu made 90SZ (Chugokuseiyu Corporation) was added 0.5% Super After sonication (70-80 ° C, 15 minutes), the mixture was aseptically placed in a Sakaguchi culture flask, and Acinetobacter radioresistens FKSH-3 was added.
The strain (FERM P-16819) was added at a concentration of 10 ⁷ cells / ml, and cultured with shaking at 28 ° C. and 125 rpm for 18 days. After that, the residual polyethylene wax was recovered by filtration from the medium, extracted with a solvent, and the weight loss rate and the change in molecular weight distribution were measured by GPC. The results are shown in Tables 3 and 4. According to the GPC measurement result, since the polyethylene wax was decomposed sequentially from the low molecular weight substance, the molecular weight distribution of the remaining polyethylene shifted to the high molecular weight substance side and the weight was reduced.

[0020]

[Table 3]

[0021]

[Table 4]

[0022]

Example 3 Heating under pressure (temperature: 120 ° C, pressure: 1 kgf /
The mineral salts medium having the composition shown in Table 1 were cm ²⁾ 15 minutes sterilized and cooled to 70-80 ° C., rosin resin (dehydroabietic acid pentaerythritol ester, trade name: Super Ester T-125 (Arakawa Chemical (Co. )) And ultrasonic dispersion (70-80 ° C, 15 minutes), aseptically put into a Sakaguchi culture flask, and strain Acinetobacter radioresistens FKSH-3
(FERM P-16819) to 10 ³ cells / ml and add 28
Shaking culture was performed at 125 ° C. and 125 rpm for 13 days. The culture solution is sampled over time, and Acinetobacter radioresistens FKSH at each culture time is sampled.
The number of viable cells of Strain-3 (FERM P-16819) was measured by a dilution plate method using a normal agar medium (manufactured by Eiken Chemical Co., Ltd.), and growth was confirmed. Table 5 shows the results.

[0023]

[Table 5]

[0024]

Example 4 (1) Preparation of crushed sample of olefin (co) polymer and rosin resin-containing composition Into a 100 ml beaker, 45 g of perchlorethylene was poured and heated on a hot plate to boil. Add 1.5g of polyethylene (trade name: Suntech M2270 made by Asahi Kasei) and 1.0g
Rosin resin (trade name: Lime Resin No. 1, manufactured by Arakawa Chemical) was added and dissolved. Thereafter, 2.5 g of talc was charged and dispersed. This solution was taken with a dropper, cast on a glass plate so that the film thickness after drying was about 10 μm, placed in a hot air circulating drier, and dried at 70 ° C. for 5 minutes.
The film is peeled off from the glass plate in water and dried under reduced pressure (50 ℃ × 3
Time, 10 mmHg). The membrane was cut to a suitable size, placed in a mortar, poured with liquid nitrogen and ground with a pestle. This is 2
A 00 mesh mesh screen was used, and the ground material passed through the mesh was used as a biodegradability test sample of the olefin (co) polymer and rosin resin-containing composition. (2) An inorganic salt medium having the composition shown in Table 1 and sterilized for 15 minutes under pressure and heat (temperature: 120 ° C., pressure: 1 kgf / cm ² ) was used for 70-80.
° C, and the ground olefin according to (1) above.
After 0.5% of the (co) polymer and the rosin resin-containing composition were added and ultrasonically dispersed (70-80 ° C, 15 minutes), the mixture was aseptically placed in a Sakaguchi culture flask, and A. radioresistensFKSH-3 was added to the flask.
The mixture was added at a concentration of 0 ³ cells / ml and cultured with shaking at 28 ° C and 125 rpm for 14 days. The culture solution was sampled over time, and the number of viable cells at each culture time was measured by a dilution plate method using a normal agar medium (manufactured by Eiken Chemical Co., Ltd.) to confirm growth. Table 6 shows the results.

[0025]

[Table 6]

[0026]

Example 5 (1) Preparation of a ground sample of a composition containing an olefin (co) polymer and a rosin resin 45 g of perchlorethylene was poured into a 100 ml beaker, and heated to boiling on a hot plate. Add 3.0g of polyethylene wax (trade name: Polyletz 90SZ, made by China Essential Oil) and 2.0g of rosin resin (trade name: Superester T
-125, manufactured by Arakawa Chemical Co., Ltd.) and dissolved. This solution is
Take it with a dropper, cast it on glass so that the film thickness after drying becomes about 10μ, put it in a hot air circulating dryer, and put it at 70 ° C.
For 5 minutes. Peel the membrane from the glass plate in water,
It was dried under reduced pressure (50 ° C × 3 hours, 10 mmHg). The membrane was cut to a suitable size, placed in a mortar, poured with liquid nitrogen and ground with a pestle. This was sieved with a 200 mesh wire mesh, and the pulverized material passed through the mesh was used as a biodegradability test sample of the olefin (co) polymer and rosin resin-containing composition. (2) An inorganic salt medium having the composition shown in Table 1 and sterilized for 15 minutes under pressure and heat (temperature: 120 ° C., pressure: 1 kgf / cm ² ) was used for 70-80.
° C, and the ground olefin according to (1) above.
After adding 0.5% of the (co) polymer and the composition containing the rosin resin and ultrasonically dispersing (70-80 ° C., 15 minutes), the mixture was aseptically placed in a Sakaguchi culture flask, and A. radioresistensFKSH-3 was added to the flask for 10 minutes.
^The cells were added at a concentration of ³ cells / ml and cultured with shaking at 28 ° C. and 125 rpm for 14 days. The culture solution was sampled over time, and the number of viable cells at each culture time was measured by a dilution plate method using a normal agar medium (manufactured by Eiken Chemical Co., Ltd.) to confirm growth. Table 7 shows the results.

[0027]

[Table 7]

[0028]

According to the present invention, Acinetobacter (A) having the resolution of olefin (co) polymer and rosin resin
microorganisms belonging to C. cinetobacter ) can be provided. According to the microorganism of the present invention, a coating film and a container of a coated fertilizer comprising a composition containing at least one kind of an olefin (co) polymer and a rosin resin remaining in soil after completion of elution, a wide range of other packaging materials, It can biodegrade olefin (co) polymers and rosin resins contained in waste liquids such as paints, printing inks, adhesives, industrial additives, and pulp. In particular, since the microorganism of the present invention has not been reported to be pathogenic to crops and is considered to be highly safe, it is also suitable for use on agricultural lands using coated fertilizer.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI // (C12N 1/20 C12R 1:01) (C12N 1/26 C12R 1:01) (72) Inventor Shizue Hayashi Kawasaki, Kanagawa Asahi Kasei Kogyo Co., Ltd., 1-3-1, Yokko, Kawasaki-ku, Ichi

Claims (4)

[Claims] 1. A microorganism belonging to Acinetobacter and having olefin (co) polymer resolution and rosin resin resolution. 2. Acinetobacter radioresistence
The microorganism according to claim 1, which belongs to (Acinetobacter radioresistens ). 3. Acinetobacter radioresistence
(Acinetobacter radioresistens ) FASH-3 (FERM P-1681
The microorganism according to claim 2, which is 9). 4. A method for decomposing a composition containing at least one of an olefin (co) polymer and a rosin resin using the microorganism according to claim 1.