JPH0739369A - Isolation of plastic-degradative bacteria - Google Patents

Abstract

PURPOSE:To efficiently isolate the subject bacteria for biodegradable plastics compared to such conventional methods as to take samples randomly. CONSTITUTION:The objective isolation is conducted using samples taken from the soil or water having been brought into contact with plastics. Specifically, (1) a sample taken from plastic-embedded soil or plastic-set water is put to enrichment culture in a medium containing the plastic alone as carbon source; (2) as necessary, the above enrichment culture is repeated to thicken the aimed plastic-degradative bacteria; (3) the resultant culture fluid is spread on a solid medium to conduct an isolation culture; and (4) for the colonies obtained by the isolation culture, the degradation of the plastics is confirmed and the objective plastic-degradative bacteria is obtained.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for efficiently isolating microorganisms that decompose and assimilate biodegradable plastics.

[0002]

2. Description of the Related Art Demand for light and strong plastics has been growing more and more in recent years, and the production volume is 10 million tons per year in Japan alone.
It is increasing to exceed. Along with that, the amount of discarded plastics has increased sharply, and is attracting attention as one of the new social problems.

The main methods currently used for disposal of plastics are landfill and incineration. With regard to landfill treatment, the plastic is stable and is not decomposed by microorganisms, which leads to environmental deterioration due to accumulation. In addition, since it is bulkier than its weight and does not decay even when it is landfilled, it requires a large area for landfilling, which causes a problem of landfill shortage.

Further, plastic has a high heat of combustion per weight, which damages the furnace when incinerated. Furthermore, it is said that chlorine-based plastics such as vinyl chloride and vinylidene chloride, which are frequently used at home, produce dioxin, which is highly carcinogenic when burned, and have been regarded as a problem.

Therefore, it is desired to recycle plastics. Although waste plastics in factories whose emission sources are relatively clear have been actively used in recent years, plastic products once on the market are collected and transported.・ It requires a lot of labor and cost for sorting and cleaning, and most of them are not recycled at present. In addition, it is important to create a social system for recycling successfully, but the society is not mature enough to accept this.

Biodegradable plastics have been receiving attention as one of the countermeasures in recent years. Biodegradable plastic is a material that can be decomposed by microorganisms and is rapidly decomposed in an appropriate decomposition environment.
It is a material. Among them, poly (polyester), which is an aliphatic polyester in which microorganisms accumulate in its body as its own energy source,
(3-Hydroxybutyric acid) (hereinafter referred to as P3HB) has been attracting attention as a substitute material for general-purpose plastics due to its thermoplasticity and excellent biodegradability. In particular, P (3HB-3HV), which is an improvement of the hard and brittle property of P3HB, is disclosed in
-293385 and JP-A-63-269989) are being actively developed as new biodegradable materials.

Such biodegradable plastics are decomposed by microorganisms in soil or water even if they are discarded as they are. Isolation of degrading bacteria is useful for further promoting decomposition and elucidating the decomposition mechanism. Determining whether a new biodegradable material can be used is not only due to the mechanical properties of the material, it is necessary to know what it will look like when it is broken down. It is necessary to obtain a degrading enzyme secreted by the degrading bacteria outside the body. In addition, by obtaining a degrading bacterium, it is possible to apply it in advance by treating the plastic with the degrading bacterium when disposing of the plastic to further promote the decomposition in the natural world.

[0008]

[Problems to be Solved by the Invention] Generally, the search for bacteria is
Soil, fresh water, or seawater in which the target strain is likely to live is randomly collected, and the target strain is isolated by using a screening method according to the properties of the target strain. However, the strains are not universally inhabited and are not always present in the soil, freshwater, or seawater at a level where they can be isolated. In other words, whether or not the target strain can be collected largely depends on trial and error, and the effort often ends in complete waste. This is especially true when a degrading bacterium is isolated from a plastic such as polycaprolactone (hereinafter referred to as PCL) that is not produced in nature and is produced by chemical synthesis. An object of the present invention is to provide a method for solving the above problems in the isolation of plastic-degrading bacteria and efficiently isolating bacteria that degrade biodegradable plastics.

[0009]

[Means for Solving the Problems] In order to efficiently isolate plastic degrading bacteria, the present inventors have collected soil in which a specific plastic is buried in advance or water in which plastic is installed and collect the degrading bacteria from this. Once isolated,
It was found that a decomposing bacterium can be obtained with a very high probability.

The gist of the present invention is to isolate a bacterium capable of degrading the plastic by using a sample collected from soil or water which has been previously contacted with the plastic for a certain period of time. It is a separation method. This isolation method specifically includes the following steps.

A sample collected from soil in which plastic is buried or water in which plastic is installed is subjected to accumulation culture in a medium containing only the plastic as a carbon source. If necessary, this accumulation culture is repeated to decompose the plastic. The bacteria are concentrated, the culture solution of the enrichment culture is expanded on a solid medium, and the culture is separated. The colonies obtained by the separation culture are confirmed to decompose the plastic to obtain plastic-degrading bacteria.

Further, in the above method, when the medium used for the integration culture has the plastic in the form of film or powder as the sole carbon source and contains only inorganic salts as the other nutrient source, or is used for the separation culture. When the medium contains the plastic or the monomer or oligomer constituting the plastic as the sole carbon source and contains only inorganic salts as the other nutrient source, the isolation can be performed more efficiently.

[0013]

The decomposing bacterium to which the method of the present invention can be applied is not particularly limited as long as it is a plastic decomposing bacterium generally called biodegradable plastic. In order to isolate a degrading bacterium by the method of the present invention, a sample randomly collected as in the conventional method is not used, but soil or water which has been previously contacted with a plastic to be a substrate of the degrading bacterium to be isolated is used. It uses the sample collected from. Here, the contact specifically includes burying the target plastic in the soil in advance or putting the plastic in water. As a method for burying the plastic in the soil, it is desirable to bury a film or a powdery one having a large surface area because microbial decomposition of the plastic proceeds from the surface of the plastic.

The contact with such a plastic is 1 to 6
After continuing for about a month, collect a sample. Regarding the collection of samples from the soil in which the plastic is buried, if the decomposition of the plastic is sufficiently progressing, the soil in which the plastic is buried is collected, and if it is in the process of being decomposed, the soil containing the plastic is collected as it is. To do. In this case, it is most efficient to collect the soil from the surface of the plastic.

When the plastic is collected from the water in contact therewith, the water in the vicinity of the place where the plastic is installed may be collected, but it is desirable to collect the water together with the plastic in a state where the plastic is not completely decomposed.

When the collected sample is soil, the supernatant obtained by suspending it in sterilized water, stirring, and leaving still is used for the next accumulation culture. If the sample is water, use the supernatant as is.

The culture medium used for the enrichment culture is a culture medium containing the plastic as the sole carbon source in order to avoid the growth of bacteria other than the target plastic degrading bacteria. As the other nutrient sources, it is preferable to use only inorganic salts. An example of the composition of the inorganic salts is shown below, but the composition can be changed according to the growth situation of the microorganism. The medium for the integration culture is usually a liquid medium.

Example of Medium for Integrated Culture [Liquid medium A] Ammonium nitrate 1.0 g Dipotassium hydrogen phosphate 1.0 g Sodium dihydrogen phosphate 1.0 g Magnesium sulfate heptahydrate 0.2 g Ferrous sulfate heptahydrate 0.01 g Sulfuric acid Manganese heptahydrate 0.01 g Zinc sulfate heptahydrate 0.01 g Calcium chloride dihydrate 0.01 g Ion-exchanged water 1 L PH 7.1 The form of plastic added as a carbon source to the culture medium for enrichment culture is the same as that for soil burial. A film or powder is preferable. The film may be formed by hot pressing or solvent casting, but the thinner the film, the better. Also, the finer the particle size of the powder, the better.

Sterilization of the medium is usually carried out in an autoclave (120
However, if a plastic with a low melting point is included, add the appropriate amount of plastic powder to the inorganic salt medium for autoclave sterilization (in this case, leave the film thin on the water surface). A film is formed), or the plastic sterilized with alcohol may be added after sterilization with an inorganic salt medium.

The culture conditions for the integrated culture vary depending on the characteristics of the microorganism, but generally the culture temperature is 20 to 40 ° C.,
Around 30 ° C is suitable. The pH is preferably around neutral, but may be about 6-9.

As the culture method, shaking culture, static culture,
A method such as aeration and agitation culture method is possible, and this can also be selected according to the characteristics of the microorganism. The period of one accumulation culture may be determined according to the progress of the decomposition of the plastic, but usually 5 to 7 days is suitable. The number of times of enrichment culture varies depending on the concentration of degrading bacteria, but usually 4 to
About 6 times is appropriate.

The composition of the solid medium in the separation culture is not particularly limited, but it is desirable not to add unnecessary nutrients in order to avoid the growth of bacteria other than the target plastic degrading bacteria. That is, it is desirable to use a composition in which the plastic or the monomer or oligomer constituting the plastic is used as the sole carbon source, and only the inorganic salts are contained except for solid components such as gellan gum and agar for forming a solid medium. An example of the medium composition other than the carbon source is shown below.

Example of composition of solid medium for separation culture [solid medium B] ammonium nitrate 1.0 g dipotassium hydrogen phosphate 1.0 g sodium dihydrogen phosphate 1.0 g magnesium sulfate heptahydrate 0.2 g ferrous sulfate heptahydrate 0.01 g Manganese sulfate heptahydrate 0.01 g Zinc sulfate heptahydrate 0.01 g Calcium chloride dihydrate 0.01 g Ion-exchanged water 1 L Gellan gum 8.5 g PH 7.1 The above composition is an example, and the microorganism to be isolated Naturally, it may change depending on the characteristics of the above, and some inorganic salts do not need to be added depending on the type. Agar can be used as the solid component in addition to gellan gum. For agar
Addition of about 15 g is desirable.

As the carbon source to be added to the separation medium, water-soluble monomers and oligomers constituting the plastic are particularly suitable, and the optimum concentration is about 0.05 to 0.5%. Further, a method of adding plastic in powder is also conceivable. Of course, normal solid medium for bacteria (eg SC
D agar medium) can also be used. Regarding the culture conditions for the separation culture, the temperature is usually 20 to 40 ° C., and the period may be determined by observing the state of colony formation, but it is usually about 4 to 10 days.

Separation culture can be carried out by developing the culture solution obtained by the integration culture on the above-mentioned solid medium and culturing, and collecting the obtained colonies. In general, plastic degrading bacteria have weak fertility, so it is necessary to collect bacteria from relatively small colonies. Further, depending on the case, it is necessary to carry out enrichment culture again from the obtained colony. The colony obtained by the separation culture is confirmed to decompose the plastic in the same medium as used in the enrichment culture to obtain a plastic-degrading bacterium.

[0026]

EXAMPLES The present invention will be described in more detail below with reference to examples. [Comparative Example] Soil collected at the locations shown in Table 1 was suspended in 10 cm ³ of sterilized water, sufficiently stirred, and then allowed to stand. One drop (about 0.05 cm ³ ) of the obtained soil suspension was used as a film of P (3HB-3HV) and PCL (dimensions: 10w × 10L × 0.06t, weight 7).
-8 mg) was added to each of the liquid medium A (6 cm ³ ) and the mixture was cultured at 30 ° C for 10 days (accumulation culture). The results are shown in Table 2.

[Example] In the place shown in Table 1, P
The powders of (3HB-3HV) and PCL were embedded, and after 3 months, the soil collected from the same place was suspended in 10 cm ³ of sterilized water, sufficiently stirred, and then allowed to stand. One drop (about 0.05 cm ³ ) of the obtained soil suspension was used as a film of P (3HB-3HV) and PCL (dimensions: 10w x 10L x 0.06t, weight 7-8mg).
To the liquid medium A (6 cm ³ ) containing
Cultivated for 10 days (accumulation culture). The results are shown in Table 2.

[0028]

[Table 1]

[0029]

[Table 2]

As is clear from the results shown in Table 2, the presence of degrading bacteria was confirmed in all the samples collected in the examples. In the comparative example, P (3HB-3HV) -degrading bacteria were observed to some extent, but PCL-degrading bacteria were obtained at a very low probability.

From the culture broth obtained by the enrichment culture of the above example, plastic degrading bacteria were further isolated by the following procedure. That is, the supernatant 1 of the culture solution of the sample obtained from the soil A
The drop was added to the liquid medium A containing the above film, and the accumulation culture under the same conditions as above was repeated 5 times to concentrate the plastic degrading strain.

After one platinum loop of the culture solution obtained by this enrichment culture was diluted with sterilized water, in the case of P (3HB-3HV) degrading bacteria, 3
In the case of a sodium salt of hydroxyacetic acid and a PCL-degrading bacterium, the cells were spread on a solid medium B containing 0.2% of ε-caprolactone and cultured at 30 ° C for 5 days. The appearance such as shape and color of the resulting colonies was visually identified to obtain dozens of strains. Each of these strains was inoculated into a medium A to which a film was added, and the culture was continued at 30 ° C to obtain P (3HB-3H
V) and PCL were examined for their degradability, and a bacterium having the ability to decompose and assimilate P (3HB-3HV) and PCL was isolated. The mycological properties of P (3HB-3HV) and PCL degrading bacteria isolated by the method of the present invention are described below.

Mycological properties of P (3HB-3HV) -degrading bacterium (Pseudomonas testosteroni 2601 strain) (a) Morphology (1) Cell shape and size: bacillus, (1.0 to 1.2) × (2 to 4) ( μm) (2) Motility and flagella settling status: Yes, extremely hairy (3) (3) Gram stainability: Cathode (b) Acid production test from various sugars (1) Glucose: − (2 ) Fructose: − (3) Galactose: − (4) Mannose: − (5) Rhamnose: − (6) Xylose: − (7) Lactose: − (8) Sucrose: − (9) Malnose: − (10) Mannitol:-(c) Various sugar and organic acid assimilation test (1) Glucose:-(2) L-arabinose:-(3) D-mannose:-(4) D-mannite:-(5) N- Acetyl-D-glucosamine:-(6) Maltose:-(7) Potassium gluconate: + (8) n-Capric acid: + (9) Adipic acid: + (10) dl-Malic acid: + (11) Quench Acid Natri Um:-(12) Phenyl acetate:-(d) o-Nitrophenyl-β-D-galactopyracid:-(e) Nitrate reduction:-(f) N ₂ gas generation:-(g) Oxidase: + (h) Arginine dehydrolase:-(i) Lysine decarboxylase:-(j) Ornithine decarboxylase:-(k) Hydrolysis test (1) Urea: + (2) Esculin:-(3) Tween 80:-(4) Starch:-(5) Gelatin:-(6) Acetamide:-(l) Growth on various media (1) SS medium:-(2) MacConkey medium: + (3) 6.5% NaCl:-(4) Cetrimide medium :-(M) Utilization of citric acid:-(n) Growth in PH (1) pH 4.5: + (2) pH 3.6: -Mycological properties of PCL-degrading bacterium (Pseudomonas testosteroni 2665 strain) (a) Morphology ( 1) Cell shape and size: Bacillus, 1 × (2-3) (μm) (2) Motility and flagella epiphytic status: Yes, extremely monochryte (3) Cell polymorphism and Child presence: None (4) Gram stainability: Cathode (b) Acid production test from various sugars (1) Glucose: − (2) Fructose: − (3) Galactose: − (4) Mannose: − (5) Rhamnose: − (6) Xylose: − (7) Lactose: − (8) Sucrose: − (9) Marnose: − (10) Mannitol: − (c) Various sugar and organic acid assimilation tests (1) Glucose: -(2) L-arabinose :-( 3) D-mannose :-( 4) D-mannitol :-( 5) N-acetyl-D-glucosamine :-( 6) Maltose :-( 7) Potassium gluconate :-( 8) n-capric acid :-( 9) adipic acid :-( 10) dl-malic acid: + (11) sodium citrate :-( 12) phenyl acetate :-( d) o-nitrophenyl- β-D-galactopyracid: − (e) Nitrate reduction: − (f) N ₂ gas generation: − (g) Oxidase: + (h) Arginine diha Idrolase:-(i) Lysine decarboxylase:-(j) Ornithine decarboxylase:-(k) Hydrolysis test (1) Urea:-(2) Esculin:-(3) Tween 80: + (4) Starch:- (5) Gelatin:-(6) Acetamide:-(l) Growth in various media (1) SS medium:-(2) MacConkey medium: + (3) 6.5% NaCl:-(4) Cetrimide medium:-( m) Utilization of citric acid :-( n) Growth in PH (1) pH 3.6 :-( o) Accumulation of poly-β-hydroxybutyrate (P3HB): +

[0034]

As described above, according to the method of the present invention, a microorganism which decomposes and assimilates biodegradable plastic can be isolated very efficiently as compared with the conventional trial-and-error method. it can. For example, almost 100% regardless of sampling location
With the probability of, it is possible to collect plastic degrading bacteria.

Claims (4)

[Claims] 1. A method for isolating a plastic-degrading bacterium, which comprises isolating a bacterium capable of degrading the plastic using a sample collected from soil or water which has been previously contacted with the plastic for a certain period of time. 2. The method for isolating a plastic degrading bacterium according to claim 1, which comprises the following steps. A sample collected from soil in which plastic is embedded in advance or water in which plastic is installed is subjected to accumulation culture in a medium containing only the plastic as a carbon source. If necessary, this accumulation culture is repeated to concentrate the plastic degrading bacteria. Degradation of the plastic is confirmed for colonies obtained by the isolation culture, and plastic degrading bacteria are obtained. 3. The medium used for enrichment culture uses the plastic in film or powder form as the sole carbon source,
The method according to claim 2, wherein the other nutrient source contains only inorganic salts. 4. The method according to claim 2 or 3, wherein the medium used for the separation culture has the plastic or the monomer or oligomer constituting the plastic as the sole carbon source, and the other nutrient sources include only inorganic salts. .