JPH11155562A - Microorganism having low-density polyethylene decomposing ability - Google Patents

Abstract

PROBLEM TO BE SOLVED: To decompose a low-density polyethylene(LDPE) as a scarcely decomposable polymer in a short period of several months, by treating the low density polyethylene with a bacterium belonging to the genus Bacillus, capable of specifically decomposing the low-density polyethylene(LDPE). SOLUTION: A LDPE waste buried in soil or a LDPE waste, etc., in a solution containing a nitrogen source or an inorganic salt necessary for growing a decomposing bacterium is treated with a bacterium belonging to the genus Bacillus, capable of specifically decomposing the low-density polyethylene(LDPE), preferably Bacillus circulans No. 14 strain (FERM P-16,400), Bacillus brevis No. 19 strain (FERM P-16,401) or Bacillus sphaericus No. 20 strain (FERM P-16,402).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a microorganism having the ability to degrade low-density polyethylene (hereinafter referred to as "LDPE"), which is a hardly decomposable polymer.

[0002]

2. Description of the Related Art In recent years, as the problem of plastic waste incineration of refuse and the environment has become more serious, its disposal has become a problem. In particular, it is said that hardly degradable polymers such as polyethylene are not decomposed by microorganisms when left in nature.

[0003] Polyethylene is consumed by as much as 2 million tons per year, and not all of them are in waste disposal systems. For example, agricultural LDPE films are more often left in the wild than when they are collected. These wastes are affected by sunlight, rain, temperature and the like, and are degraded and decomposed, but progress very slowly. For example, it takes about 300 years for LDPE to be completely decomposed.

[0004] Regarding waste treatment, at present, recycling and incineration are the mainstream of plastic treatment. Also,
Attempts have also been made to develop biodegradable plastics and to degrade biologically using the biodegradability of microorganisms. However, when incinerated, problems such as environmental pollution occur, and at present, it is not possible to collect and treat all waste by recycling or incineration alone. Also, the industrial waste treatment plant is full and it is difficult to secure a new treatment plant.
On the other hand, examples of alternatives with biodegradable plastics include:
LDPE with high biodegradability, for example, LD with added starch
PE (Ecostar ^™ , Ecostar International) is commercially available. In addition, the present inventors have found that the biodegradability of LDPE is enhanced by the addition of oxidized oil or metal salts of organic acids, UV irradiation, etc. ( Nikka, 1996, No. 10, p85).
3-860 ). However, the decomposition rate of these biodegradable plastics cannot be said to be sufficient, and the development of a system and the like that can perform more rational and stable treatment is desired.

Therefore, biological treatment, ie, LDP
There is a strong demand for isolation of microorganisms that degrade various hardly degradable polymers such as E. The present inventors have clarified that a multi-film deteriorated in a field or the like undergoes whitening peculiar to biological deterioration and that small holes are generated in some places. LDP that has been buried in soil for more than 30 years
As a result of analyzing and investigating E, it has been clarified that, unlike urea resin and vinyl chloride, a decrease in the molecular weight occurs due to the involvement of microorganisms in soil. That is, the presence of colonies presumed to be bacteria was confirmed at the decomposition site, and the presence of -C = C- bonds in the main chain and hydroperoxide, which are characteristic of biodegradation by microscopic FT-IR analysis,
A remarkable increase in the absorption of OH groups derived from alcohol has been confirmed. ( Biodegradability of LDPE 10th report, p78
~ 83, 1995 ). This suggests that LDPE-degrading bacteria exist in the soil.

[0006]

However, LDP
In the method in which E is buried in the soil and decomposed by microorganisms in the soil, the effect of improving the decomposition rate is very small. A method for decomposing LDPE under mild conditions at normal temperature and normal pressure has not been known so far.

Accordingly, an object of the present invention is to provide a microorganism having the ability to specifically degrade LDPE.

[0008]

Means for Solving the Problems The present inventors have made intensive studies to achieve the above object, and as a result, succeeded in isolating a microorganism having the ability to degrade LDPE and completed the present invention.

That is, the present invention belongs to the genus Bacillus,
It is intended to provide a microorganism having an ability to specifically degrade DPE.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION LDPE is a kind of polyethylene which is a polymer of ethylene and has the lowest crystallinity among polyethylenes. The oxidation reaction proceeds relatively easily. In general, it is used for agricultural multi-films, laminated films for coating food containers, polybuckets, and the like.

The microorganism of the present invention belongs to the genus Bacillus,
Any microorganism can be used as long as it has the ability to degrade DPE, but it is preferably Bacillus circulans, Bacillus braves, or Bacillus sphaericus, and Bacillus circulans No. 14 strain (FERM P- 16400), Bacillus Braves No. 19 strain (FERM P-16401), or Bacillus sphaericus No. 20 (FERM P-164)
02) is particularly preferable because of its strong LDPE decomposition ability.

The above three strains are strains newly isolated from the soil in Japan by the present inventors and have the following mycological properties.

[0013]

[Table 1]

[0014]

[Table 2]

[0015]

[Table 3]

Based on the above mycological properties,
Bergey's Manual of Determinative Bacteriolog
y) Referring to the eighth edition, as a result of comparative study, it was confirmed that the strain was Bacillus sp., but no bacteria having LDPE degradability have been confirmed so far. On September 3, 1997, they were deposited with the National Institute of Bioscience and Human Technology, National Institute of Advanced Industrial Science and Technology (accession numbers are FERM P-16400 and FERMP-164, respectively).
01 and FERM P-16402. ).

Next, a method for isolating a microorganism having the ability to degrade LDPE will be described. Microorganisms that specifically degrade LDPE have been suggested to be present in soil, and may also be present elsewhere. Therefore, if the soil, the ocean, and the like are sampled and selected by a known method, a microorganism that specifically degrades LDPE can be isolated. In particular, it is preferable to sample from a field or a trash can where deterioration of polyethylene has been confirmed.

As the medium, a medium containing LDPE as a sole carbon source (hereinafter referred to as "LDPE medium") is used.
In particular, it is preferable to use a freeze-crushed fine powder of LDPE that has been thermally degraded. Other nitrogen sources, mineral sources, and the like can be appropriately selected from commonly used ammonium sulfate, sodium chloride, and the like. Culture methods include enrichment culture, shaking culture, and stationary culture. Enrichment culture is particularly preferable for isolating a specific microorganism from a mixed system of microorganisms.

A sample sampled from soil or the like is
The cells are cultured in an LDPE medium, and the medium is added at predetermined intervals to accumulate useful bacteria. The culture period is not particularly limited, but is preferably, for example, about 1 to 6 months. Next, LDPE in the enriched culture solution is collected, and the LDPE degrading activity of the microorganism is evaluated. Although there is no particular limitation on the evaluation method, for example, FT-
A method of confirming the presence of a -C = C- bond and absorption of an OH group by IR analysis, observation by a scanning electron microscope, and the like are preferable because they are simple and highly reliable.

The above-mentioned sample having the ability to degrade LDPE is appropriately diluted and applied to an LDPE agar medium (a medium obtained by adding agar to an LDPE medium) to form a colony. Further, the operation of culturing by culling from the colony is repeated as appropriate to perform isolation. These LDPE degrading activities are evaluated, and colonies (cells) having a degrading ability are selected (primary sorting).

From the primary selected strain, L
A strain having a high DPE utilization ability is selected (secondary selection). That is, the above cells are grown to a logarithmic growth phase, and a large amount of the collected cells is cultured by a method such as inoculation into an LDPE medium, and then the LDPE assimilation ability is confirmed.
A strain having DPE-degrading ability can be obtained.

The use of strains having LDPE resolution alone or in combination makes it possible to decompose LDPE safely and at low cost. As a decomposition method, for example, L
A method of burying DPE waste in soil to cause degrading bacteria to act, a method in which LDPE waste is contained as a carbon source in a solution to which nitrogen sources and inorganic salts necessary for the growth of degrading bacteria are appropriately added to act on the degrading bacteria And the like. By such a method, the decomposition of LDPE, which took about 300 years in nature, can be performed in a short period of about 3 months.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

Test Example 1 Searching for Microorganisms Having LDPE Degradability Samples obtained from the separation sources shown in Table 4 were accumulated and cultured in the LDPE medium shown in Table 5 by the following method. Note that the fine powder LDPE in Table 5 is a freeze-crushed fine powder of LDPE that has been thermally degraded at 70 ° C. for 3 days.

[0025]

[Table 4]

[0026]

Table 5 LDPE medium composition Ammonium sulfate 1.0 g Potassium dihydrogen phosphate 0.2 g Dipotassium hydrogen phosphate 1.6 g Magnesium sulfate heptahydrate 0.2 g Sodium chloride 0.1 g Calcium chloride dihydrate 0.02 g Iron sulfate heptahydrate 0.01 g Sodium molybdate dihydrate 0.5 mg Sodium tungstate dihydrate 0.5 mg Manganese sulfate 0.5 mg Calcium pantothenate 0.4 mg Inositol 2.0 mg Niacin 0.4 mg p- Aminobenzoic acid 0.2 mg Pyridoxine hydrochloride 0.4 mg Thiamine 0.4 mg Riboflavin 0.4 mg Biotin 2.0 μg Vitamin B12 0.5 μg Fine powder LDPE 1 g pH 7.5 Purified water 1000 ml

Preparation of LDPE culture medium Into a high-pressure sterilized 500 mL Sakaguchi flask, 100 ml of the sterilized basic medium is aseptically filtered, and then 0.1 g of heat-degraded LDPE frozen and crushed fine powder is added as the sole carbon source to prepare the culture medium. did.

Separation Sources For the separation samples A, B and C, 10 ml of each culture was inoculated. For samples D, E and F for separation, 3
Each g was inoculated. For separation samples G and H, one piece (2 to 3 cm square) of an LDPE sheet to which microorganisms are considered to be attached was put.

Culture conditions Shaking culture was performed at 25 ° C. and 120 spm (stroke / minute). Three to four weeks later, 10 ml of the culture was collected and subcultured to a fresh medium. Thereafter, the same operation is repeated, and the LDPE
Useful bacteria that degrade E. coli were accumulated up to the fourth generation.

Evaluation of LDPE resolution LDPE was separated from the culture solution with a filter paper. Then LPD
In order to remove proteins adhering around E, the cells were treated with bacterial lytic enzymes. The enzymatic reaction was performed at 37 ° C. for 3 hours after adding a sufficient amount of N-acetylmuramidase, achromopeptidase and lysozyme, and
Was collected by a filter paper, pronase was added in a sufficient amount, and reacted at 37 ° C. for 16 hours. After washing with purified water,
After reaction at 100 ° C. for 10 minutes with% SDS, filtration and washing were performed. For the obtained product, the presence of a -C = C- bond, the absorption of an OH group was confirmed by microscopic FT-IR analysis,
The LDPE resolution was evaluated by observation with a scanning electron microscope.

As a result, in samples C, E and H, O
Since the absorption of the H group was observed, the presence of a microorganism having LDPE resolution was confirmed in the C, E, and H samples.

Test Example 2 Primary Selection of Microorganisms Having LDPE Degradation Pure microorganisms were isolated from samples C, E, and H in which the LDPE resolution was confirmed in Test Example 1. The sample used was the fourth generation of the enriched culture solution.

Medium 15 g of agar was added to the LDPE medium used in Test Example 1,
LDPE agar medium was prepared. Considering that mold may be contaminated even after the enrichment culture, nystatin is added
LDPE-N agar medium supplemented with 00 U / ml was also prepared in parallel.

Inoculation Samples C, E and H for separation were diluted in 5 steps from 10 ³ to 10 ⁷ times and spread on LDPE agar medium and LDPE-N agar medium.

Culture conditions After inoculation, cultivation was performed at 28 ° C. for 2 weeks, the colonies formed were picked, and again LDPE agar medium and LDPE-N
Isolation was performed by repeating the operation of culturing on an agar medium.

Evaluation of LDPE Resolution The LDPE resolution was evaluated in the same manner as in Test Example 1. As a result, LDPE agar medium isolate 2 was obtained from sample C for isolation.
6 strains and 29 strains of LDPE-N agar medium, 11 strains of LDPE agar medium and 11 strains of LPE
Forty-five DPE-N agar medium isolates were used for the sample H for isolation.
, 27 LDPE agar medium isolates and 37 LDPE-N agar medium isolates were selected as primary isolates.

Test Example 3 Secondary Selection of Microorganisms Having LDPE Degradability Sorting of Primary Selected Isolates Based on Colony Properties A total of 175 primary isolates obtained in Test Example 2 were LDP
All the colonies grown on the E agar medium were picked, and the characteristics of those colonies were observed on the LDPE agar medium, and found to be very similar.
Therefore, considering that the LDPE agar medium is an oligotrophic medium and the characteristics of the colonies may not be clearly expressed, the LDPE agar medium is grown on a normal agar medium shown in Table 6 where the characteristics of the colonies are easily expressed. , And colony characteristics. The culture was performed at 28 ° C. for 72 hours, and the emerged colonies were classified according to the characteristics of the colonies and observed with a microscope. From both results, 175 strains considered to be the same were aggregated to obtain 21 strains as secondary isolates.

[0038]

Table 6 Composition of ordinary agar medium Meat extract 5 g Peptone 10 g Sodium chloride 5 g Agar 15 g Purified water 1000 ml

Test Example 4 The bacteriological properties of the 21 strains were confirmed in accordance with the Burger manual (see Tables 7 to 20). As a result, one strain of Bordetella bacterium and one species of Neisseria (Ne
isseria) 2 bacteria, Moraxella bacteria 3
Strain, Flavobacterium genus bacteria 2
A strain, one strain of Acinetobacter and three strains of bacteria belonging to the genus Bacillus were identified.

[0040]

[Table 7]

[0041]

[Table 8]

[0042]

[Table 9]

[0043]

[Table 10]

[0044]

[Table 11]

[0045]

[Table 12]

[0046]

[Table 13]

[0047]

[Table 14]

[0048]

[Table 15]

[0049]

[Table 16]

[0050]

[Table 17]

[0051]

[Table 18]

[0052]

[Table 19]

[0053]

[Table 20]

Test Example 5 Obtaining microorganisms capable of assimilating LDPE The 21 strains obtained in Test Example 3 were inoculated by the following two methods and subjected to an assimilation test. In addition, assimilation tests were similarly performed on the combinations of the strains shown in Table 21.

[0055]

[Table 21]

Inoculation method One colony growing on a plate medium was picked, and
Inoculate the DPE liquid medium. The cells grown to a logarithmic growth phase in an ordinary liquid medium are aseptically washed by centrifugation, and a large amount of the collected cells is inoculated into an LDPE liquid medium.

The inoculated cells were vigorously shaken at 28 ° C., and the cells were allowed to react with LDPE for 4 weeks. Thereafter, when the degradation activity was evaluated in the same manner as in Test Example 1, three strains having high degradation activity (strains Nos. 14, 19, and 20) were selected.

Test Example 6 Identification of LDPE Degrading Bacteria No. obtained in Test Example 5 14 strains, no. 19 strains and The biochemical properties of the 20 strains were confirmed using two Bacillus biochemical property research kits (API50CHB and API20, manufactured by BioMerieux Biotech Japan). That is, each bacterium was first pure-cultured on an agar medium, and then a colony was scraped off and suspended in 1 ml of sterile physiological saline to prepare a concentrated bacterial suspension. This was further diluted with a sterilized physiological saline solution and dispensed into an API 50CHB medium and an API 20 medium. Thereafter, the cells were cultured at 30 ° C., and after 24 hours and 48 hours, judgment was made based on a change in the color of the medium (indicator) in each well. As a result, no. 14 strains are Bacillus Circulance, No. 19 strains are Bacillus Braves, No. 20
The strain was identified as Bacillus sphaericus. These three strains were deposited on September 3, 1997 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology.

Test Example 7 Confirmation of LDPE Resolution The three strains selected in Test Example 5 were subjected to assimilation tests for three types of LDPE shown in Table 22. In addition, assimilation tests were also performed on the mixed strains of the three strains.

[0060]

[Table 22]

Normally, the cells grown to a logarithmic growth phase in a liquid medium are aseptically centrifuged, washed, and collected.
The three types of LDPE were individually inoculated into an LDPE liquid medium to which LDPE was independently added. This was shaken vigorously at 28 ° C. to react the cells with LDPE. In addition, half of the medium was replaced every two weeks to prevent degradation of the decomposition reaction due to lack of inorganic salts and the like.

LDPE was collected from the culture on the days shown in Table 23, and the degradation activity of each was evaluated in the same manner as in Test Example 1. The culture period up to the evaluation is as shown below.

[0063]

[Table 23]

As a result, the body mark of the bacterium was confirmed from all LDPEs, and was particularly remarkable in the No. 14 strain.
In addition, cloudiness of the culture solution was confirmed 4 months after the culture. Bacillus circulance No. A scanning electron micrograph of LDPE degraded by the 14 strains is shown in FIG. White Bacillus circulans No. LDP around 14 strains
It is confirmed that E is decomposed.

[0065]

The microorganism of the present invention having the ability to degrade LDPE can be used to decompose LDPE, which is a difficult-to-degrade biopolymer, under mild conditions safely, inexpensively and relatively quickly. Can be.

[Brief description of the drawings]

FIG. It is a scanning electron micrograph of LDPE decomposed | disassembled by 14 stocks.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12R 1:09) (C12N 1/20 C12R 1:07) (C12N 1/20 C12R 1:08) (71) Applicant 000006884 shares Yakult Honsha Co., Ltd. 1-11-1 Higashi-Shimbashi, Minato-ku, Tokyo (72) Inventor Yoshito Ohtake 5878-11 Tonuma, Nogi-cho, Shimotsuga-gun, Tochigi Prefecture Address: Hagiwara Kogyo Co., Ltd. (72) Inventor Hitoshi Asabe 1-4, Mizushima Nakadori, Kurashiki-shi, Okayama Prefecture Hagiwara Kogyo Co., Ltd. No. 3 within ITOCHU Corporation (72) Inventor Hidenori Makino 4-3-1 Kutaro-cho, Chuo-ku, Osaka-shi, Osaka-shi Within ITOCHU Corporation (72) Inventor Hiroto Omura 1-1-19 Higashi-Shimbashi, Minato-ku, Tokyo Yakult Honsha (72) Inventor Tomiko Nagai 1-11-1 Higashi-Shimbashi, Minato-ku, Tokyo Yakult Honsha (72) Inventor Nobuo Murakami 1-5-1, Otsuka, Inzai City, Chiba Prefecture, Japan Takenaka Corporation Technical Research Institute (72) Inventor Akira Hirose 1-5-1, Otsuka, Inzai City, Chiba Prefecture, Japan Takenaka Corporation Technical Research Institute (72) Inventor Kiyoaki Okuda 8-11-1, Ginza, Chuo-ku, Tokyo Inside the Tokyo head office of Takenaka Corporation (72) Inventor Shinji Takakawa 4-1-1-13 Honcho, Chuo-ku, Osaka-shi, Osaka Inside the Osaka head office of Takenaka Corporation

Claims (7)

[Claims] 1. A microorganism belonging to the genus Bacillus and capable of specifically degrading low-density polyethylene. 2. The microorganism according to claim 1, wherein the bacterium belonging to the genus Bacillus is Bacillus circulans . 3. The microorganism according to claim 1, wherein the bacterium belonging to the genus Bacillus is Bacillus brevis . 4. The microorganism according to claim 1, wherein the Bacillus bacterium is Bacillus sphaericus . 5. The bacterium belonging to the genus Bacillus is designated as Bacillus circulans strain No. 14, and is referred to as FERM P-164.
3. The microorganism according to claim 1, which has been deposited as No. 00. 6. The bacterium of the genus Bacillus is Bacillus braves No. 19 strains and FERM P-16401
The microorganism according to claim 1, wherein the microorganism is deposited as a microorganism. 7. The bacterium belonging to the genus Bacillus sp. 20 strains and FERM P-1640
5. The microorganism according to claim 1, which has been deposited as No. 2.