JPH0655153A - Soil restoring method - Google Patents

Abstract

PURPOSE:To effectively restore soil contaminated with phenolic compds. or buran compds. by restoring soil by allowing bacteria originating from the intestines of a termite to act on soil. CONSTITUTION:Bacteria simultaneously decomposing phenol, cresol and furan in soil and excellent in antibiotic resistance and sugar utilization properties are found out of the intestines of a termite and the bacteria (Pseudomonas sepacia) originating from the intestines of a termite are allowed to act on soil to restore the soil. This method is one example of environment restoring technique and utilizes the functions of bacteria in soil to decompose a pollutant and reinforces the self-purifying capacity of an ecological system to promote the decomposition of the pollutant. This technique is effective for restoring soil contaminated with phenolic compds. or furnans.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a soil remediation method using a termite intestinal microorganism.

[0002] In recent years, environmental pollutants due to the detection of many types of harmful and persistent biochemicals have been highlighted in environmental surveys, and there is concern about their impact on the ecosystem. Therefore, it is strongly desired to establish the technology to prevent the spread of pollution and to regenerate the polluted environment. As an example of environmental restoration technology, there is a technology that decomposes pollutants and makes them pollution-free by utilizing the function of microorganisms in soil, aiming at promoting the decomposition of pollutants by strengthening the self-cleaning ability of the ecosystem. There is. The technology of the present invention is a soil contaminated with phenolic compounds, for example, soil contaminated with phenolic compounds such as gas production plant site, refinery contaminated soil, petroleum refinery site, fuel base site, pulp factory site, etc. It can be applied to repair of Further, the technique of the present invention is also effective for the repair of furan-contaminated soil, for example, furan compounds such as tetrahydrofuran, furfural, furfuryl alcohol, and coumaran are used for various organic solvents such as surface coatings, adhesives, dyes, etc. Since furfural, in particular, is a poisonous substance like general aldehydes, it can be applied to remediate soil pollution such as the site of these chemical factories.

[0003]

2. Description of the Related Art Decomposition means of phenol include heat and ozone. However, considering the cost, operability, and the fact that it is in the soil, the decomposition by microorganisms is strongly suggested. Pseudomonas (Pseudomonas)
Genus, bacterium of Bacillus genus, Acinetobacter genus, etc., Aureobasidium
Genus, Fusarium, and other fungi,
And the genus Trichosporon,
Yeasts such as the genus Candida are known. These situations are detailed in "Conversion of Organic Compounds by Microorganisms" (Academic Publishing Center). In particular, examples of known Pseudomonas bacteria related to the present invention include Putida species. Also with regard to cresol, there are almost no examples isolated with microorganisms capable of degrading cresol. Pseudomonas QT3 as a resistant strain
1 strain (C. Muscue et al., Biotechnology Letters, Vol. 9, No. 9, 655-660, 1987)
Only a small number of bacteria belonging to the genus Pseudomonas have been reported.

On the other hand, a technique for decomposing the above-mentioned various furans with a single microorganism is not known, and there are only a few reports on furfural. S. Morimo
to, M.M. Murakami (1967) J. Ferm
ent. Technol. Example, or Acetoact
er, Achromobacter, Brevibac
terium, Flavobacterium, Mic
rococcus. For example, there are only patents of the former Soviet Union regarding the conversion of furfural into 2-furancarboxylic acid. (Conversion of organic compounds by microorganisms; Academic Publishing Center)

[0005]

As described above, it is not unknown that a bacterium capable of decomposing phenol and furfural is not known, but at the actual contamination site, contamination with only one of these compounds is rather rare. Yes, and often contains other phenolic substances and furans. Moreover, from the perspective of satisfying practical conditions such as being required for activity in a special environment in soil and having sufficient resolution, it cannot be said that the current range of known bacterial species is sufficient, Seed acquisition is required. Naturally, new bacterial species differ from known bacterial species in growth conditions and the like, which means that the application range and usage form of the microorganism will be rich. Examples include antibiotic resistance and sugar availability. Assuming the treatment of soil containing the compounds mentioned above, the bacteria used for the treatment are not only decomposable but also less susceptible to damage and can grow in a poor environment, that is, they are resistant to many antibiotics and some sugars. On the other hand, those who have assimilation capacity are more likely to grow better and can maintain a high level of resolution. Thus, from a practical point of view, it is strongly required to find a new bacterium that possesses a high ability that the conventional bacterium could not have.

[0006]

Means for Solving the Problems From the above viewpoints, the present inventors searched for a new bacterial species that decomposes phenols and furans, and as a result, from the termite intestine, phenol in soil,
The present invention has been completed by discovering a bacterium that simultaneously decomposes cresol and furans and is excellent in antibiotic resistance and sugar utilization. The termites used in the present invention are the scorpion termites (Nasutiters takasagoens).
is) and is distributed in Okinawa and the Yaeyama Islands.

The isolation of the bacterium of the present invention was carried out as described in Example 1 below, and the mycological properties of the strain obtained by the isolation are as follows. A. Morphological properties (1) Gram stain: negative (2) Cell size and shape: length 1.0 to 2.0 μ
bacilli having a width of about 0.5 m and a width of about 0.5 μm (3) Motility: Yes (4) Flagella: B. Growth status in various media C. Physiological properties (1) Distinction between aerobic and anaerobic Oblique aerobic (2) Degradation mode of sugar Oxidized type (3) Generation of oxidase + (4) Reduction of nitrate + (5) Generation of hydrogen sulfide − (6 ) Indole formation- (7) Urease formation- (8) Gelatin liquefaction- (9) Arginine hydrolysis- (10) Lysine decarboxylation + (11) Ornithine decarboxylation- (12) Use of citric acid + (13) Methylcarbinol acetyl reaction (VP reaction)-(14) Detection of tryptophan deaminase- (15) ONPG- (16) Utilization of carbohydrates Glucose + Fructose + Maltose + Galactose + Xylose + Mannitol ± Sucrose- Lactose + Esculin-Inosit-Sorbit-Rhamnose-Meribiose-Amygdalin-L + Arabinose + or more From various properties, it is clear that this strain belongs to the genus Pseudomonas, and it was confirmed that it is suitable to belong to Pseudomonas cepacia.

Pseudomonas cepacia is known to be resistant to many antibiotics.

However, as is clear from the examples described below, this bacterium also has an excellent ability to decompose phenol / cresol and furan. Since such a bacterium does not exist in the previously known Pseudomonas cepacia, it was identified as a new strain and Pseudom
It was named as onas cepacia KKOI and deposited at the Institute of Microbial Technology, Institute of Industrial Science and Technology. (Deposit number: P
No. 12869) Mutant strains obtained by mutating this bacterium naturally or by artificial means are all included in the present invention. Cultivation of the present bacterium may be carried out in a medium usually used for culturing Pseudomonas. As a carbon source, phenol alone is sufficient for growth, but glucose or the like can be appropriately used. As the nitrogen source, for example, yeast extract, peptone or the like can be used alone or in combination. In addition, potassium dihydrogen phosphate, ammonium chloride and the like can be added if necessary. The culture can be performed under aerobic conditions, and may be liquid or solid. The culture temperature is preferably around 30 degrees.

[0010]

【Example】

Example 1 Isolation of Pseudomonas cepacia KKOI from termite gut-derived microorganisms 1) Nasitermest
About 30 larvae termites among akasagoensis) are placed in a petri dish.

2) Pour ethyl alcohol (95%) into a Petri dish to sterilize the termite surface.

3) Wash the termites with the M9 solution to remove ethyl alcohol. (Twice) 4) Crush the termites in the M9 solution and aseptically collect the filtrate.

5) A part of the above solution is used as a culture solution (0.05%).
Inoculate with phenol, 0.05% yeast extract, M9) 3
Incubate at 0 degrees.

6) The above culture solution was added with 0.05% phenol,
1.2% agar and M9 are applied to an agar medium containing phenol as the sole carbon source and cultured at 30 ° C.

7) Pseudomonas cepacia KK01 colonies that grew well were obtained.

Example 2 Restoration of Phenolic Compound-Contaminated Soil by Termite Enterobacteria 1) Nasutertermest
About 30 larvae termites among akasagoensis) are placed in a petri dish.

2) Pour ethyl alcohol (95%) into a Petri dish to sterilize the termite surface.

3) Wash the termites with the M9 solution to remove ethyl alcohol. (Twice) 4) Crush the termites in the M9 solution and filter to obtain a solution.

5) 10 ml of the above solution was added to the test soil (15
00ppm phenol, 500ppm o-cresol,
An 80 ml aqueous solution containing 500 ppm p-cresol and 500 ppm cresol was inoculated into 500 g of sterilized soil), stirred, and then statically cultured.

6) HPL containing phenolic compounds in soil
It was quantified by the C method, and the removal rate of the phenolic compound was set to 1.0 for the total compound at the start of the experiment, and the ratio to that was determined. The result is shown in FIG. This display method is the same from FIG. 2 to FIG. Example 3 Restoration of Phenolic Compound-Contaminated Soil with Pseudomonas cepacia KKOI A single colony of Pseudomonas cepacia KKOI was used in 5 ml of liquid medium (0.05% phenol, 0.05%).
% Yeast extract, M9) and inoculated at 30 ° C. O. D. When it exceeded 0.7, the culture solution was poured into the test soil used in 5) of Example 2 and stirred, and then static culture was performed at room temperature. Phenolic compounds in soil were quantified by the HPLC method, and the removal rate was calculated daily. The result is shown in FIG. Example 4 Restoration of Phenolic Compound Contaminated Soil by Using Termite Enterobacteria and Wood Flour Together 1) 10 ml of the solution obtained in 4) of Example 2 was used as a test soil (1
80 ml of an aqueous solution containing 500 ppm phenol, 500 ppm o-cresol, 500 ppm p-cresol, 500 ppm cresol and 60 mesh beech wood flour 5
0 g was dispersed in 500 g of sterilized soil), stirred, and then statically cultured.

2) HPL containing phenolic compounds in soil
The removal rate of the phenolic compound was determined by the method C. The result is shown in FIG. Example 5 Restoration of Phenolic Compound Contaminated Soil Using Pseudomonas cepacia KKOI with Wood Flour A single colony of Pseudomonas cepacia KKOI was mixed with 5 ml of liquid medium (0.05% phenol, 0.05%).
% Yeast extract, M9) and inoculated at 30 ° C. O. D. When it exceeded 0.7, the culture solution was poured into the test soil prepared in the same manner as in Example 4, and the mixture was stirred and then statically cultured at room temperature. HPLC analysis of phenolic compounds in soil
The removal rate was determined daily by the method. The result is shown in FIG. Example 6 Restoration of furan-based compound-contaminated soil by termite intestinal bacteria 1) 10 ml of the solution obtained in 4) of Example 2 was added to test soil (tetrahydrofuran, furfural, furfuryl alcohol, coumarin, 20 ml each of 1000 ppm aqueous solution) and sterilized soil 500 g (Diffused in 1) and stirred, and then statically cultured. 2) The furan compounds in the soil were quantified by the HPLC method to determine the removal rate. The result is shown in FIG. Example 7 Restoration of Furan Compound-Contaminated Soil with Pseudomonas cepacia KKOI A single colony of Pseudomonas cepacia KKOI was inoculated into 5 ml of a liquid medium (0.05% yeast extract, M9) and cultured at 30 degrees. I did. O. D. When it exceeded 0.7, the culture solution was poured into the test soil prepared in the same manner as in Example 6, and the mixture was stirred and then statically cultured at room temperature. The furan compounds in the soil were quantified by the HPLC method to determine the removal rate. The result is shown in FIG. Example 8 Restoration of a furan-based compound-contaminated soil by termite intestinal bacteria using wood flour together 1) 10 ml of the solution obtained in 4) of Example 2 was used as a test soil (tetrahydrofuran, furfural, furfuryl alcohol, coumaran each 1000 ppm aqueous solution) 20 ml and 6
50 g of 0 mesh beech wood powder was inoculated into 500 g of sterilized soil), stirred, and then statically cultured.

2) The furan compounds in soil were quantified by the HPLC method to obtain the removal rate. The result is shown in FIG. 7. Example 9 Restoration of Furan Compound-Contaminated Soil by Pseudomonas cepacia KKOI Using Wood Flour Combined with a Single Colony of Pseudomonas cepacia KKOI in 5 ml of Liquid Medium (0.05% Yeast Extract, M9) Then, the culture was performed at 30 degrees. O. D. When it exceeded 0.7, the culture solution was poured into the test soil prepared in the same manner as in Example 8 and the mixture was stirred, and then static culture was performed at room temperature. The furan compounds in the soil were quantified by the HPLC method, and the removal rate was calculated daily. The result is shown in FIG.

[0023]

INDUSTRIAL APPLICABILITY The present invention enables the restoration of contaminated soil with a phenol compound or a furan compound, which has been impossible in the past.

[Brief description of drawings]

FIG. 1 is a diagram showing the removal rate of a phenolic compound in Example 2 over time.

FIG. 2 is a diagram showing the removal rate of a phenolic compound in Example 3 over time.

FIG. 3 is a graph showing a day-to-day removal rate of a phenolic compound using a wood component together in Example 4.

FIG. 4 is a diagram showing the rate of removal of a phenolic compound in combination with a wood component in Example 5 over time.

FIG. 5 is a diagram showing a furan-based compound removal rate in Example 6 over time.

FIG. 6 is a graph showing the rate of furan-based compound removal in Example 7 over time.

FIG. 7 is a graph showing a day-to-day removal rate of a furan-based compound using a wood component together in Example 8.

FIG. 8 is a graph showing a day-to-day removal rate of a furan-based compound using a wood component together in Example 9.

Claims (5)

[Claims] 1. A method for soil rehabilitation, which comprises allowing a termite intestine-derived microorganism to act on soil. 2. The method according to claim 1, wherein the microorganism derived from the termite intestine is Pseudomonas cepacia. 3. Pseudomonas cepacia is known as Pseudomonas cepacia KKOI (FERM P-1286).
The method according to claim 2, which is 9). 4. The method according to claim 1, wherein the action is carried out in the presence of a wood component. 5. The method according to claim 4, wherein the wood is beech.