JP2014088548A - Clarifier of oil contaminated soil - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a clarifier of oil contaminated soil which can clarify soil polluted by petroleum components efficiently in a short time.SOLUTION: A clarifier of oil contaminated soil contains NG007 strain (NITE P-1233) of Pestalotiopsis genus. It is preferable that the clarifier further contains S133 strain (NITE P-461) belonging to basidiomycete of Polyporus genus and D7 strain (NITE P-01703) of Trametes genus.

Description

  The present invention relates to a purification agent for petroleum-contaminated soil using microorganisms.

  Development of a treatment method (bioremediation) using microorganisms has been studied as a method for safely and inexpensively treating soil contamination caused by petroleum components.

  For example, Patent Literature 1 and Patent Literature 2 disclose novel heavy oil-degrading bacteria having remarkable degradation activity or degradation acceleration activity against heavy oil such as C heavy oil, which is particularly problematic in oil pollution. Yes. Among these, in Example 1 of Patent Document 1, a novel strain Kaurobacter sp. Inoculate 0.1 mL of a culture of the strain 2B-2 and a strain belonging to the genus Alcanibolax which is a known hydrocarbon-degrading microorganism, into 5 mL of a medium obtained by adding 5 mg / mL C heavy oil to an NSW basic medium, It is described that C heavy oil could be decomposed by 44.7% when cultured with shaking at 30 ° C. for 10 days. In Example 1 of Patent Document 2, a novel strain, Halomonas sp. Inoculate 0.1 mL of a culture of the 6B strain and a strain belonging to the genus Alcanibolax, a known hydrocarbon-degrading microorganism, into 5 mL of a medium obtained by adding 5 mg / mL C heavy oil to an NSW basic medium, and It is described that C heavy oil was able to be decomposed 44.0% when cultured with shaking for 10 days.

  The applicant of the present application also discloses S133 strain (NITE P-461) belonging to Polyporus basidiomycetes in Patent Document 3 as a microorganism having extremely excellent resolution of petroleum components. In Example 4 of Patent Document 3, the decomposition efficiency when using high-concentration C heavy oil contaminated soil (C heavy oil concentration 15000 ppm) was examined. In Experimental Example 12 in which Kapok pulp was added to the S133 strain, the amount of C heavy oil was It is disclosed that (the amount of total petroleum hydrocarbon TPH) could be efficiently decomposed to about 76% after 30 days of culture and to about 93% after 60 days of culture (Table 3). Further, in Experimental Example 12, about 83 to 96 of each component constituting C heavy oil, that is, hydrocarbon (aklane fraction), aromatic compound, nitrogen-sulfur-oxygen-containing compound (NSO), and asphaltene. % Of NSO and asphalt, which were particularly difficult to decompose, disclosed that about 83% could be decomposed 60 days after culturing (Table 3 to Table 6). 6).

JP 2000-83651 A JP 2000-139446 A JP 2009-166027 A

  As described above, if the S133 strain described in Patent Document 3 is used, it is possible to decompose petroleum components at a high decomposition rate, but purification of new petroleum-contaminated soil containing microorganisms having even higher resolution. It is desired to provide an agent.

  This invention is made | formed in view of the said situation, The objective is to provide the purification agent of the petroleum contaminated soil which can purify | clean the soil contaminated with the petroleum component efficiently in a short time.

  The oil-contaminated soil purifier according to the present invention that has solved the above-mentioned problems has a gist in that it includes the NG007 strain (NITE P-1233) belonging to the genus Pestaliopsis.

  In a preferred embodiment of the present invention, the oil-contaminated soil purifier further comprises S133 strain (NITE P-461) belonging to Polyporus basidiomycetes.

  In a preferred embodiment of the present invention, the oil-contaminated soil purifier contains the D7 strain (NITE P-01703) belonging to the genus Trametes in addition to the NG007 strain and the S133 strain.

  According to the present invention, soil contaminated with petroleum components can be efficiently purified in a short time. The oil-contaminated soil purifier according to the present invention can efficiently purify soil contaminated with high-concentration petroleum components, soil contaminated with hard-to-decompose asphalt, among other petroleum components. Is extremely useful.

FIG. 1 is a graph showing the results of asphalt degradation rate (left axis) and various enzyme activities (right axis) of asphalt-contaminated soil (1000 ppm) when NG007 strain and S133 strain are co-cultured at a predetermined ratio (medium No nutritional sources added to). The upper diagram in FIG. 1 shows the results for 30 days after the culture, and the lower diagram in FIG. 1 shows the results for 15 days after the culture. FIG. 2 shows the results of asphalt degradation rate (left axis) and various enzyme activities (right axis) of asphalt-contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when NG007 strain and S133 strain were co-cultured at a ratio of 1: 1. (The number of culture days is 30 days, and no nutrient source is added to the medium). FIG. 3 is a graph in which the influence of nutrient sources added to the culture medium is examined for the asphalt degradation rate of asphalt-contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when the NG007 strain and the S133 strain are co-cultured at a ratio of 1: 1. (The culture days are 15 days and 30 days). The left figure of FIG. 3 shows the result without the addition of the nutrient source, and the right figure of FIG. 3 shows the result with the addition of the nutrient source. FIG. 4 shows A heavy oil degradation rate (left axis) and various enzyme activities (right axis) of A heavy oil contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when NG007 strain and S133 strain were co-cultured at a ratio of 1: 1. Is a graph showing the results of (without addition of a nutrient source to the medium). The upper diagram in FIG. 4 shows the results for 30 days of culture, and the lower diagram in FIG. 4 shows the results for 15 days in culture. FIG. 5 shows C heavy oil degradation rate (left axis) and various enzyme activities (right axis) of C heavy oil contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when NG007 strain and S133 strain were co-cultured at a ratio of 1: 1. Is a graph showing the results (no addition of nutrients to the medium, 15 days of culture). Fig. 6 shows the effects of adding nutrient sources to the medium for the A heavy oil degradation rate of A heavy oil contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when NG007 and S133 were co-cultured at a ratio of 1: 1. (The culture days are 15 days and 30 days, and some are further 60 days and 120 days). The left figure of FIG. 6 shows the result without the addition of the nutrient source, and the right figure of FIG. 6 shows the result with the addition of the nutrient source. Fig. 7 shows the effect of nutrient source addition to the medium on the C heavy oil degradation rate of C heavy oil contaminated soil (1000ppm, 15000ppm, 30000ppm) when NG007 and S133 were co-cultured at a ratio of 1: 1. (The culture days are 15 days and 30 days). The left diagram of FIG. 7 shows the results without the addition of the nutrient source, and the right diagram of FIG. 7 shows the results with the addition of the nutrient source. FIG. 8 shows components constituting C heavy oil of C heavy oil contaminated soil (1000 ppm) [aliphatic hydrocarbon part (aliphatic), aromatic carbonization when NG007 strain and S133 strain are co-cultured at a ratio of 1: 1]. It is the graph which investigated the influence of the nutrient source addition to a culture medium about the decomposition | disassembly rate of a hydrogen part (Aromatic), an asphaltene part (Asphaltene), and NSO] (the culture | cultivation days are 15 days and 30 days). FIG. 9 shows components constituting C heavy oil of C heavy oil-contaminated soil (15000 ppm) [aliphatic hydrocarbon part (Aliphatic), aromatic carbonization when NG007 strain and S133 strain are co-cultured at a ratio of 1: 1]. It is the graph which investigated the influence of the nutrient source addition to a culture medium about the decomposition | disassembly rate of a hydrogen part (Aromatic), an asphaltene part (Asphaltene), and NSO] (the culture | cultivation days are 15 days and 30 days). FIG. 10 shows the components constituting the C heavy oil in the C heavy oil contaminated soil (30000 ppm) [aliphatic hydrocarbon part (aliphatic), aromatic carbonization when the NG007 strain and the S133 strain were co-cultured at a ratio of 1: 1. It is the graph which investigated the influence of the nutrient source addition to a culture medium about the decomposition | disassembly rate of a hydrogen part (Aromatic), an asphaltene part (Asphaltene), and NSO] (the culture | cultivation days are 15 days and 30 days). FIG. 11 is a graph in which the influence of nutrient sources added to the culture medium is examined for the asphalt degradation rate of asphalt-contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when the NG007 strain and the S133 strain are co-cultured at a ratio of 1: 1. (The culture days are 15, 30, 60, and 120 days). The left diagram in FIG. 11 shows the result without the addition of the nutrient source, and the right diagram in FIG. 11 shows the result with the addition of the nutrient source. FIG. 12A shows the components constituting the asphalt of asphalt-contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when the NG007 strain and the S133 strain are co-cultured at a ratio of 1: 1 [aliphatic hydrocarbon part (Aliphatic), aroma It is the graph which investigated the decomposition rate of the aromatic hydrocarbon part (Aromatic), the NSO part, and the asphaltene part (Asphaltene)]. The culture days were 15, 30, 60, and 120 days, and no nutrient source was added to the medium. FIG. 12B shows the components constituting the asphalt of asphalt-contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when the NG007 strain and the S133 strain are co-cultured at a ratio of 1: 1 [aliphatic hydrocarbon part (aliphatic), aroma It is the graph which investigated the decomposition rate of the aromatic hydrocarbon part (Aromatic), the NSO part, and the asphaltene part (Asphaltene)]. The culture days were 15, 30, 60, and 120 days, and a nutrient source was added to the medium. Fig. 13 shows the effect of nutrient addition to the medium on the C heavy oil degradation rate of C heavy oil contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when NG007 and S133 were co-cultured at a ratio of 1: 1. (The culture days are 15, 30, 60, and 120 days). The left figure of FIG. 13 shows the result without the addition of the nutrient source, and the right figure of FIG. 13 shows the result with the addition of the nutrient source. FIG. 14A shows components constituting C heavy oil in C heavy oil contaminated soil (1000 ppm) [aliphatic hydrocarbon part (Aliphetic), aromatic carbonization when NG007 strain and S133 strain are co-cultured at a ratio of 1: 1]. It is the graph which investigated the influence of the nutrient source addition to a culture medium about the decomposition rate of a hydrogen part (Aromatic), an asphaltene part (Asphaltene part, NSO part)] (the culture days are 15, 30, 60, 120 days) . FIG. 14B shows components constituting C heavy oil of C heavy oil-contaminated soil (15000 ppm) [aliphatic hydrocarbon part (Aliphatic), aromatic carbonization when NG007 strain and S133 strain are co-cultured at a ratio of 1: 1]. It is the graph which investigated the influence of the nutrient source addition to a culture medium about the decomposition rate of a hydrogen part (Aromatic), an asphaltene part (Asphaltene part, NSO part)] (the culture days are 15, 30, 60, 120 days) . FIG. 14C shows the components constituting C heavy oil in C heavy oil contaminated soil (30000 ppm) [aliphatic hydrocarbon part (Aliphatic), aromatic carbonization when NG007 strain and S133 strain were co-cultured at a ratio of 1: 1]. It is the graph which investigated the influence of the nutrient source addition to a culture medium about the decomposition rate of a hydrogen part (Aromatic), an asphaltene part (Asphaltene part, NSO part)] (the culture days are 15, 30, 60, 120 days) . FIG. 15 shows the influence of nutrient source addition to the medium on the degradation rate of A heavy oil in soil contaminated with heavy oil A (1000 ppm, 15000 ppm, 30000 ppm) when NG007 and S133 were co-cultured at a ratio of 1: 1. (The culture days are 15, 30, 60, and 120 days). The upper diagram of FIG. 15 shows the results without the addition of nutrient sources, and the lower diagram of FIG. 15 shows the results with the addition of nutrient sources. FIG. 16A shows the components constituting the asphalt of A heavy oil-contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when the NG007 strain and the S133 strain are co-cultured at a ratio of 1: 1 (aliphatic hydrocarbon part (Aliphatic), It is the graph which investigated the decomposition rate of an aromatic hydrocarbon part (Aromatic), asphaltene part (Asphaltene), and NSO part]. The culture days were 15, 30, 60, and 120 days, and no nutrient source was added to the medium. FIG. 16B shows the components constituting the asphalt of heavy oil-contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) [aliphatic hydrocarbon part, aroma, fragrance, when NG007 strain and S133 strain were co-cultured at a ratio of 1: 1. It is the graph which investigated the decomposition rate of an aromatic hydrocarbon part (Aromatic), asphaltene part (Asphaltene), and NSO part]. The culture days were 15, 30, 60, and 120 days, and a nutrient source was added to the medium. FIG. 17A shows the components constituting the A heavy oil of the A heavy oil contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when the NG007 strain, the S133 strain, and the D7 strain are co-cultured at a ratio of 1: 1: 1. It is a graph which shows the result at the time of no nutrient source addition to a culture medium about the decomposition rate of a hydrogen part (Aliphatic), an aromatic hydrocarbon part (Aromatic), an asphaltene part (Asphaltene), and NSO part (the number of culture days is 15) Day, 30th, 60th). FIG. 17B shows the components constituting the A heavy oil of the A heavy oil contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when the NG007 strain, the S133 strain, and the D7 strain are co-cultured at a ratio of 1: 1: 1. It is a graph which shows the result at the time of a nutrient source addition to a culture medium about the decomposition | disassembly rate of a hydrogen part (Aliphatic), an aromatic hydrocarbon part (Aromatic), an asphaltene part (Asphaltene), an NSO part] (culture days are 15) Day, 30th, 60th). FIG. 18A shows components constituting C heavy oil of C heavy oil-contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when NG007 strain, S133 strain and D7 strain are co-cultured at a ratio of 1: 1: 1. It is a graph which shows the result at the time of no nutrient source addition to a culture medium about the decomposition rate of a hydrogen part (Aliphatic), an aromatic hydrocarbon part (Aromatic), an asphaltene part (Asphaltene), and NSO part (the number of culture days is 15) Day, 30th, 60th). FIG. 18B shows components constituting C heavy oil of C heavy oil contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when NG007 strain, S133 strain, and D7 strain are co-cultured at a ratio of 1: 1: 1. It is a graph which shows the result at the time of a nutrient source addition to a culture medium about the decomposition | disassembly rate of a hydrogen part (Aliphatic), an aromatic hydrocarbon part (Aromatic), an asphaltene part (Asphaltene), an NSO part] (the number of culture days is 15) Day, 30th, 60th). FIG. 19A shows components constituting the asphalt of asphalt-contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when the NG007 strain, the S133 strain, and the D7 strain are co-cultured at a ratio of 1: 1: 1 [aliphatic hydrocarbon part. (Aliphatic), aromatic hydrocarbon part (Aromatic), asphaltene part (Asphaltene), NSO part] is a graph showing the results when no nutrient source is added to the medium (the number of culture days is 15 days, 30th, 60th). FIG. 19B shows components constituting the asphalt of asphalt-contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when the NG007 strain, the S133 strain, and the D7 strain are co-cultured at a ratio of 1: 1: 1. (Aliphatic), aromatic hydrocarbon part (Aromatic), asphaltene part (Asphaltene), NSO part] is a graph showing the results when the nutrient source is added to the medium (culture days are 15 days, 30th, 60th). FIG. 20 shows the nutrient sources for the medium for the A heavy oil degradation rate of A heavy oil contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when the NG007 strain, S133 strain, and D7 strain were co-cultured at a ratio of 1: 1: 1. It is the graph which investigated the influence of addition (the culture | cultivation days are 15 days, 30 days, 60 days). The left figure of FIG. 20 shows the result without the addition of the nutrient source, and the right figure of FIG. 20 shows the result with the addition of the nutrient source. FIG. 21 shows the nutrient source for the medium for the C heavy oil degradation rate of C heavy oil contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when NG007 strain, S133 strain and D7 strain were co-cultured at a ratio of 1: 1: 1. It is the graph which investigated the influence of addition (the culture | cultivation days are 15 days, 30 days, 60 days). The left figure of FIG. 21 shows the result without the addition of the nutrient source, and the right figure of FIG. 21 shows the result with the addition of the nutrient source. FIG. 22 shows the asphalt degradation rate of asphalt-contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when NG007 strain, S133 strain, and D7 strain were co-cultured at a ratio of 1: 1: 1. It is the graph which investigated the influence (the culture | cultivation days are the 15th, 30th, 60th). The left diagram of FIG. 22 shows the results without the addition of the nutrient source, and the right diagram of FIG. 22 shows the results with the addition of the nutrient source (culture days are 15, 30, and 60 days), respectively.

  The present inventors have disclosed the above-mentioned S133 strain as a microorganism excellent in petroleum component resolution, and in particular, soil contaminated with a high concentration of petroleum components, asphalt that is hardly decomposable among petroleum components, and the like. We have been studying microorganisms that can be purified more efficiently.

  As a result, it was found that the intended purpose can be achieved by using the NG007 strain (NITE P-1233) belonging to the genus Pestalotiopsis. Furthermore, if two types of the NG007 strain and the aforementioned S133 strain are used in combination, the purification efficiency of soil contaminated with high-concentration petroleum components is increased, and various petroleum components can be more efficiently and efficiently removed. The present invention was completed by finding that it can be purified. Furthermore, if these three types of NG007 strain and S133 strain and the newly discovered D7 strain (details will be described later) are used in combination, the purification efficiency of highly concentrated petroleum component-contaminated soil is further increased. As a result, it has been found that various petroleum components can be purified even more efficiently, and the present invention has been completed.

  In the present specification, the petroleum component is not limited to those derived from crude oil or petroleum as long as it can be contained in crude oil or petroleum. In general, oil before refining is particularly called crude oil, but in the present invention, oil and crude oil are not particularly distinguished. The petroleum component also includes petroleum products using crude oil (for example, heavy oil, gasoline, kerosene, light oil, etc.).

(1) About the purification agent and the purification method of the oil-contaminated soil containing the NG007 strain As described above, the purification agent of the oil-contaminated soil according to the present invention is characterized in that it contains the NG007 strain (NITE P-1233) of the genus Pestaliopsis. is there.

  The NG007 strain is a novel strain found by the present inventors as described in detail below. Regarding the NG007 strain, it was found that the crude enzyme derived from the strain has a strong azo dye resolution, and Japanese Patent Application No. 2012-053269 (hereinafter referred to as the prior application) discloses an azo dye-degrading agent containing the strain. Yes. However, at the time of filing of the previous application, it was not recognized that the NG007 strain had a high petroleum component resolution.

  The NG007 strain is a novel filamentous fungus isolated from dead wood in Matsuyama City, Ehime Prefecture, Japan, and is a non-white rot fungus. The NG007 strain was classified into the genus Pestalotipsis based on the following characteristics.

  The aerial mycelium is white and has each wall, its surface is smooth and has a diameter of about 1 to 3 μm. The conidial pattern is brown to dark black and is formed upright on the aerial hyphae, and its surface is smooth and has a stick-like shape. The conidia are spindle-shaped or oval-shaped, the number of cells constituting the conidia is 5 cells (including 3 colored cells), and are filamentous fungi having three attached yarns. The size excluding the attached thread is length: about 10 to 25 μm, width: 5 μm to 10 μm, the size of the attached thread of the tip cell is about 5 μm to 15 μm, and the size of the petite of the rear cell is about 2 μm to 6 μm.

  The NG007 strain grows as a white mycelium on the medium, and forms brown to dark conidia after about 2-3 weeks. When the NG007 strain was cultured on the following malt extract agar medium, catechol 1,2-dioxygenase activity was observed in the medium.

Malt extract agar medium (1L)
Malt extract 20g
Glucose 20g
1g peptone
Agar 20g
Sterile water 1L
pH 4.5

  NG007 strain was deposited on February 14, 2012 at 2-5-8, Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, and the Japan Patent Evaluation Microorganism Depositary, National Institute of Technology and Evaluation (Reception number NITE AP-1233). It was entrusted on March 23, 1983 (Accession number NITE P-1233).

  As shown in the examples described later, NG007 strain is an asphalt (one of crude oil distillation residue having a composition similar to that of crude oil) described in Patent Document 3 which is a hardly decomposable substance in crude oil. It was found that it can be decomposed more efficiently in a shorter time than S133 strain. Therefore, high concentration asphalt contaminated soil can be purified by using the above strain.

  As described above, the NG007 strain according to the present invention has an extremely excellent petroleum component resolution, so that the NG007 strain is added to the petroleum-contaminated soil without slurrying the petroleum-contaminated soil or extracting the petroleum components in the contaminated soil. Thus, petroleum components can be decomposed only by bringing the NG007 strain into contact with petroleum-contaminated soil.

  And according to this invention, the contaminated soil which contains the various petroleum components mentioned above in high concentration can be decomposed | disassembled efficiently and can be purified. The preferred concentration of petroleum-contaminated soil that can be treated according to the present invention is about 20,000 to 50,000 ppm. The concentration can be further increased, for example, by culturing the NG007 strain preferably in combination with a nutrient source or surfactant described later, or by extending the culture period of the strain.

  The oil-contaminated soil purifier according to the present invention includes all forms including the NG007 strain. Specifically, as described in detail below, the purification agent of the present invention may be a liquid medium containing the NG007 strain, or a fungus bed such as wood chips on which the NG007 strain is grown. Moreover, a nutrient source, surfactant, etc. may be included.

  Further, the oil-contaminated soil purifier according to the present invention may be one in which the NG007 strain is sufficiently grown on a liquid medium or a fungus bed, or the one that has been grown is refrigerated and stored. Also good.

  Next, a method for purifying petroleum-contaminated soil using a purifier containing the NG007 strain will be described. The said purification | cleaning method should just contact NG007 stock with petroleum contaminated soil according to a conventional method.

  Specifically, for example, a method of bringing a culture of the NG007 strain obtained by culturing the NG007 strain in a liquid medium or the like into contact with the oil-contaminated soil is mentioned. Examples of the medium used include Czapek-Dox agar medium, potato-dextrose agar (PDA) medium, and the like in addition to the aforementioned malt extract agar medium. It is preferable to adjust the liquid medium of NG007 strain sprayed on petroleum-contaminated soil to about 4.0 to 8.5 according to the optimum pH of NG007 strain. In addition, it is recommended that the liquid medium be appropriately diluted so that the pH is reached.

  Alternatively, since the NG007 strain is a filamentous fungus, a culture of the NG007 strain cultured using a plant residue that can become a fungal bed may be sprayed on petroleum-contaminated soil. Examples of plant residues that can be used include wood chips, sawdust, sawdust, rice bran, okara, sake lees, soybean meal, paper pulp, sludge, and the like. The plant residue may contain the above-mentioned medium components (for example, malt extract).

  It is preferable to add a nutrient source or a surfactant for the NG007 strain to the medium or fungus bed for obtaining the culture of the NG007 strain described above. Nutrient sources can activate the growth of the NG007 strain and thus activate the resolution of petroleum components. In addition, the surfactant has an action of accelerating the decomposition by detaching petroleum components having high fat solubility and low hydrophilicity from the solid soil to make them easily present in the growth environment of the NG007 strain.

  Examples of nutrient sources for the NG007 strain include: carbon sources such as glucose; nitrogen sources such as polypeptone; trace element salts such as magnesium salt or manganese salt; or trace element sources such as manganese salt; ) And other plant materials. What is marketed as a nutrient source of a filamentous fungus may be used as a nutrient source, for example, “Shitake no Sato” manufactured by Showa Sangyo Co., Ltd. and the like.

  The addition amount of the nutrient source may be appropriately adjusted according to the amount of the NG007 strain, etc. In general, the greater the amount of the nutrient source, the better the growth of the NG007 strain and the higher the decomposition efficiency of the petroleum components. . On the other hand, if there are too many nutrient sources such as a carbon source and a nitrogen source excluding natural materials of plant materials, there is a concern about an adverse effect on the environment. Considering the above, it is preferable that the content is approximately 5 to 30% by mass with respect to the total mass of the medium.

  The surfactant to be used is not particularly limited, such as a cationic surfactant, an anionic surfactant, a zwitterionic surfactant, and a nonionic surfactant. However, since there is little adverse effect on the NG007 strain, it is a nonionic surfactant. Agents are preferred. Examples of the nonionic surfactant include polyoxyethylene alkyl ether, polyoxyethylene alkylphenyl ether, alkyl glucoside, polyoxyethylene fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester and the like. More specifically, Tween20, Tween60, and Tween80 can be used.

  The surfactant can improve the dispersibility of petroleum components in the growth environment of the NG007 strain, and can also promote the growth of the NG007 strain, for example, as a carbon source. However, if the amount of the surfactant is too large, the growth of the NG007 strain may be adversely affected. Therefore, the addition amount of the surfactant is preferably about 0.05% by mass or more and 1.0% by mass or less with respect to the total weight of the medium, preferably 0.1% by mass or more and 0.75%. It is more preferable to make it about mass% or less.

  You may add pH adjusters, such as a citric acid, to the said culture medium. The preferable pH of the medium is generally 4.0 to 8.5 from the viewpoint of growth of the NG007 strain.

  In the culturing step, the above medium is inoculated with NG007 strain and cultured until the mycelium has spread. The inoculation amount and culture conditions of the NG007 strain in the culture medium may be appropriately adjusted according to the contamination state of the contaminated soil to be treated (such as the type and content of petroleum components). For example, the preferable culture temperature is about 15 to 40 ° C.

  Oil-contaminated soil to be treated may be mined and inserted into a container, and NG007 strain or nutrient source may be added to adjust the temperature and humidity so that the NG007 strain can grow well. However, soil contamination is often widespread, and if the soil to be treated is mined one by one, the treatment efficiency can be lowered. Therefore, you may add NG007 stock | strain as it is to petroleum contaminated soil. In addition, since the NG007 strain grows sufficiently at 25 ° C., it is considered that the NG007 strain can decompose petroleum components at room temperature without adjusting the temperature in Japan. However, since the humidity should be kept relatively high, it is preferable to spray water or the like in a timely manner.

  According to the findings by the present inventors, during the treatment of petroleum-contaminated soil with the NG007 strain, it is more efficient to shield it from light. Therefore, after adding the NG007 strain to petroleum-contaminated soil, it is preferable to suppress the amount of irradiation light by covering with a blue sheet or the like.

(2) About the purification agent and purification method of the oil-contaminated soil further containing the S133 strain The oil-contaminated soil purification agent according to the present invention may further include the S133 strain (NITE P-461) belonging to the basidiomycetes of the genus Polyporus. preferable. By using the S133 strain in combination with the above-described NG007 strain, the resolution of the petroleum component is significantly improved as compared with the case where each strain is used alone (see Examples described later).

  The S133 strain is a novel strain found by the present inventors, and details thereof are described in detail in Patent Document 3. The S133 strain also has an excellent oil resolving power. For example, oil-contaminated soil contaminated with 15000 ppm of C heavy oil can be efficiently decomposed in a relatively short period of about two months. Further, among the components constituting C heavy oil, NSO and asphalt, which were particularly difficult to decompose, can be decomposed efficiently.

  Surprisingly, however, it has been found that the use of the S133 strain in combination with the NG007 strain further promotes the high petroleum component resolution of the S133 strain. The details are shown in the examples described later. For example, oil-contaminated soil contaminated with a higher concentration of 30000 ppm C heavy oil can be efficiently decomposed to 58% in a relatively short period of about one month. I was able to.

  Details such as the method for culturing the S133 strain are described in Patent Document 3, so Patent Document 3 may be referred to.

  The mixing ratio of the NG007 strain and the S133 strain [the mixing ratio of the mycelium suspension obtained by culturing each bacterium in an appropriate medium for a certain period (7 days) and then homogenizing the whole culture solution] It may differ depending on the type and concentration of the component, and may be appropriately determined. However, the volume ratio (capacity ratio) is generally in the range of NG007 strain: S133 strain = 10-90: 90-10. The NG007 strain: S133 strain is more preferably within the range of 75-25: 25-75.

  The method for purifying petroleum-contaminated soil using the purification agent containing the above mixture may be performed in the same manner as the method for purification using the purification agent containing the NG007 strain described above. This is because both the NG007 strain and the S133 strain overlap with the types of culture media and fungi that are preferably used, and the types of nutrients and surfactants that are preferably added. Specifically, for example, a mixture of a culture solution of NG007 strain and a culture solution of S133 strain cultured in each appropriate medium for a predetermined period at the above-mentioned preferable mixing ratio is obtained by the above-described method. It may be added to. Alternatively, since both strains are preferably cultured on a malt extract agar medium, each fungus is mixed at an appropriate ratio with a fungus bed grown with a plant residue containing malt extract etc. On the other hand, about 5 to 30% (mass ratio with respect to dry soil)] may be added to petroleum-contaminated soil. A preferable culture temperature at this time is approximately 15 to 40 ° C.

(3) About the purification agent and the purification method of the oil-contaminated soil containing the NG007 strain, the S133 strain, and the D7 strain The oil-contaminated soil purification agent according to the present invention is the above-mentioned (2), and further, It is preferable to include a total of three strains including the D7 strain (NITE P-01703) belonging to the above. By combining the S133 strain and the NG007 strain with the D7 strain as described above, the resolution of the petroleum components is greater than when using each strain alone or when using the two types of the S133 strain and the NG007 strain. (See the examples below).

  Here, the D7 strain is a novel strain found by the present inventors, and details thereof are as follows.

  The D7 strain is a new basidiomycete isolated from dead wood in the suburbs of Iyo City, Ehime Prefecture, Japan, and is a white rot fungus. The D7 strain was classified into the genus Trametes based on the following characteristics.

  The surface has a grayish white semicircular umbrella, the width is 2-7 cm, the thickness is 2-5 mm, and a large number of it grows. The color of the spore is white, and the size is 5-7 μm in length and 2-3 μm in width. The D7 strain grows as a white mycelium on the medium, and a clamp connection is seen in this mycelium.

  When the D7 strain was cultured on the following malt extract agar medium, manganese peroxidase activity and laccase activity were observed in the medium.

Malt extract agar medium (1L)
Malt extract 20g
Glucose 20g
1g peptone
Agar 20g
Sterile water 1L
pH 4.5

  The D7 strain was deposited on 2-6-8, Kazusa Kamashishi, Kisarazu City, Chiba Prefecture, on September 6, 2013 at the National Institute of Technology and Evaluation of Microorganisms (Receipt number NITE AP-01703, date of receipt) (September 6, 2013), and was entrusted (accession number NITE P-01703, entrustment date September 6, 2013).

  The D7 strain is useful as a purification agent for petroleum-contaminated soil. In particular, by using the D7 strain in combination with the NG007 strain and the S133 strain described above, it is possible to more efficiently remove high-concentration asphalt-contaminated soil in a shorter time than when the NG007 strain and the S133 strain are used in combination. It turns out that it can be decomposed. For example, as shown in Table 9 to be described later, by using the above-mentioned three kinds of mixtures, each of oil-contaminated soils of heavy oil A, heavy oil C and asphalt contaminated at a high concentration of 30000 ppm can be obtained for a relatively short period of time of about one month. Thus, it was possible to efficiently decompose to 84% (A heavy oil), 82% (C heavy oil), and 69% (asphalt) (all results with the addition of nutrients). Furthermore, about 2 months after the culture, the above-mentioned decomposition rates tended to be further improved, and were 91% (A heavy oil), 89% (C heavy oil), and 76% (asphalt).

  Similar to the S133 strain described above, the D7 strain can be decomposed by simply adding it to the petroleum-contaminated soil and bringing the D7 strain into contact with the petroleum-contaminated soil. The concentration of petroleum-contaminated soil that can be treated by the strain D7 can be increased by using the strain D7, preferably in combination with nutrients or surfactants described later, or by increasing the culture period of the strain. In general, about 20,000 to 50,000 ppm of contaminated soil can be treated.

  The method of bringing the D7 strain into contact with the oil-contaminated soil may follow a conventional method. For example, when the D7 strain is cultured in a liquid medium, the culture solution may be sprayed on petroleum-contaminated soil. Alternatively, since the D7 strain is a basidiomycete, the D7 strain cultured using a plant residue that can become a fungal bed may be sprayed on petroleum-contaminated soil.

  The liquid medium of D7 strain to be sprayed on the oil-contaminated soil may be diluted. The pH of the liquid medium is preferably adjusted to about 4.5 to 6 in accordance with the optimum pH of the D7 strain.

  Examples of plant residues used for culturing the D7 strain include wood chips, sawdust, sawdust, rice bran, okara, oil cake, soybean meal, and the like.

  When the D7 strain is added to petroleum-contaminated soil, it is preferable to add the nutrient source and surfactant of the D7 strain. Nutrient sources can activate the growth of the D7 strain, which in turn can activate the resolution of petroleum components. In addition, the surfactant has an action of accelerating the decomposition by detaching petroleum components having high fat solubility and low hydrophilicity from the solid soil to make them easily present in the growth environment of the D7 strain.

  As a nutrient source for the D7 strain, commercially available ones such as “Shiitake no Sato” manufactured by Showa Sangyo Co., Ltd. may be used, a carbon source such as glucose; a nitrogen source such as polypeptone; magnesium Trace element sources such as salts and manganese salts; pH adjusting agents such as citric acid may be appropriately selected and used. In addition, plant materials are also preferably used as a nutrient source for the S133 strain, and representative examples thereof include plant fiber crushed material (pulp). This is because the D7 strain is a member of the genus Trametes belonging to white rot fungi and has the ability to decompose lignin in wood such as plants. The raw material of the pulp is not particularly limited as long as white rot fungi can be used, and examples thereof include wood such as conifers and hardwoods, cotton, hemp, kenaf, bagasse and the like. Specifically, for example, kapok pulp using kapok trees, linter pulp using short hair (cotton litter) attached to cotton seeds, rug pulp using fibers from cotton spinning (cotton boro), hemp A typical example is linen pulp as a raw material. In addition, waste paper pulp (recycled paper) may be used.

  The addition amount of the nutrient source may be adjusted as appropriate according to the amount of the D7 strain, etc. In general, the greater the amount of the nutrient source, the better the growth of the D7 strain and the higher the decomposition efficiency of the petroleum components. . On the other hand, if there are too many nutrient sources such as a carbon source and a nitrogen source excluding natural materials of plant materials, there is a concern about an adverse effect on the environment. Considering the above, it is preferable that the amount of the nutrient source to be added is about 5 to 30% by mass with respect to the petroleum-contaminated soil to be treated.

  The surfactant to be used is not particularly limited, such as a cationic surfactant, an anionic surfactant, a zwitterionic surfactant, and a nonionic surfactant. However, since the adverse effect on the D7 strain is small, the nonionic surfactant is used. Agents are preferred. Examples of the nonionic surfactant include polyoxyethylene alkyl ether, polyoxyethylene alkylphenyl ether, alkyl glucoside, polyoxyethylene fatty acid ester, sucrose fatty acid ester, sorbitan fatty acid ester and the like. More specifically, Tween20, Tween60, and Tween80 can be used.

  In addition to improving the dispersibility of petroleum components in the growth environment of the D7 strain, the surfactant may promote the growth of the D7 strain, for example, as a carbon source. However, if the amount of the surfactant is too large, it can adversely affect the growth of the D7 strain. Therefore, the addition amount of the surfactant is preferably about 0.05% by mass or more and 1.0% by mass or less with respect to petroleum-contaminated soil to be treated, and is 0.1% by mass or more and 0.75% by mass. It is more preferable to make it about% or less.

  Oil-contaminated soil to be treated may be mined and inserted into a container, and D7 strain or nutrient source may be added to adjust the temperature and humidity so that the D7 strain can grow well. However, soil contamination is often widespread, and if the soil to be treated is mined one by one, the treatment efficiency can be lowered. Therefore, you may add S133 stock | strain as it is to petroleum contaminated soil. In addition, since D7 strain | stump | stock grows sufficiently at 25 degreeC, if it is in Japan, D7 strain | stump | stock is considered to be able to decompose | disassemble a petroleum component at normal temperature, without adjusting temperature. However, the humidity should be kept relatively high, so water should be sprayed in a timely manner.

  What is necessary is just to adjust the addition amount of D7 stock | strain suitably according to the contamination state of contaminated soil, etc.

  According to the knowledge of the present inventors, it is more efficient to shield the light during the treatment of the oil-contaminated soil with the D7 strain. Therefore, after adding strain D7 to petroleum-contaminated soil, it is preferable to suppress the amount of irradiation light by covering with a blue sheet or the like.

  In the present invention, this D7 strain is used in combination with the aforementioned NG007 strain and S133 strain. Specifically, the mixing ratio of these three strains [the mixing ratio of the mycelial suspension obtained by homogenizing the entire culture after culturing each bacterium in a suitable medium for a certain period (7 days)] is: It may differ depending on the type and concentration of the petroleum component to be decomposed, and may be determined appropriately. However, in terms of volume ratio (capacity ratio), NG007 shares: S133 shares: D7 shares = 1-6: It is preferable to be within the range of 1 to 3: 1 to 3, more preferably within the range of NG007 strain: S133 strain: D7 strain = 1-2: 1-2: 1-2.

  The method for purifying petroleum-contaminated soil using the purification agent containing the above mixture may be performed in the same manner as the method for purification using the purification agent containing the NG007 strain described above. This is because both the NG007 strain and the S133 strain overlap with the types of culture media and fungi that are preferably used, and the types of nutrients and surfactants that are preferably added. Specifically, for example, the above-described method in which the culture solution of the NG007 strain, the culture solution of the S133 strain, and the culture solution of the D7 strain, which have been cultured in each appropriate medium for a predetermined period, are mixed at the above-mentioned preferable mixing ratio. Therefore, it can be added to oil-contaminated soil. Alternatively, since both strains are preferably cultured in a malt extract agar medium, each fungus is mixed with an appropriate ratio of a fungus bed grown with a plant residue containing malt extract etc. On the other hand, about 5 to 30% (mass ratio with respect to dry soil)] may be added to petroleum-contaminated soil. A preferable culture temperature at this time is approximately 15 to 40 ° C.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited by the following examples, and can be implemented with modifications within a range that can meet the purpose described above and below. They are all included in the technical scope of the present invention.

  In the following, “%” is “% by mass” unless otherwise specified.

Example 1
In this example, the NG007 strain and the S133 strain were used alone or in combination, and the purification ability for the soil contaminated with asphalt was examined.

  First, the NG007 strain and the S133 strain used in the experiment were isolated as follows.

(1) Isolation of NG007 strains 82 types of mushrooms and decayed wood collected from the campus of the Faculty of Agriculture, Ehime University, and their relatives were used as samples. In the case of mushrooms, they were cut into approximately 5 mm squares, added to the aforementioned malt extract agar medium, and cultured at 25 ° C. for 2 weeks in the dark. In the case of decayed wood, about 1.2 g (about 1 g in dry weight) was suspended in sterilized water (9 mL), stirred, allowed to stand for 10 minutes, and then each supernatant (1 mL) was described above. It was added to a malt extract agar medium and cultured at 25 ° C. for 2 weeks in the dark. 72 types of bacteria that had grown on the malt extract agar medium were collected, and the malt extract agar medium (specifically, 1000 ppm of asphalt dissolved in dichloromethane was applied on the malt extract agar medium). Thereafter, dichloromethane was evaporated, and the mixture was cultured at 25 ° C. for 7 days in a malt extract agar medium on which asphalt was placed. Among these, NG007, a filamentous fungus that was particularly active, was obtained by collecting 10 species of fast-growing bacteria and culturing them on a malt extract agar medium overlaid with asphalt 15000 ppm for 7 days at 25 ° C. The strain was isolated.

  When the obtained NG007 strain was observed with a microscope, it was white and had respective walls, the surface was smooth, and the diameter was about 1 to 3 μm. The conidial pattern was brown to dark black and formed upright on the aerial hyphae, and its surface was smooth and pin-shaped. The conidia were spindle-shaped or elliptical, the number of cells constituting the conidia was 5 cells (including 3 colored cells), and were filamentous fungi having 3 attached threads. The size excluding the attached thread is length: about 10 to 25 μm, width: 5 μm to 10 μm, the size of the attached thread of the tip cell is about 5 μm to 15 μm, and the size of the petite of the rear cell is about 2 μm to It was 6 μm. Since these are the morphological characteristics of the genus Pestaliopsis, the NG007 strain was identified as the genus Pestalotiopsis.

(2) Isolation of S133 strain Malt (20 g / L), glucose (20 g / L), polypeptone (1 g / L) and agar (20 g / L) were added to distilled water (100 mL). Furthermore, chloramphenicol (6 mg) or benimil (6 mg) was added to the mixture (20 ml) in order to suppress the propagation of miscellaneous bacteria and yeasts to prepare a malt extract agar medium. Separately, a 1% chrysene solution of dimethylformamide containing 1% Tween 80 was prepared. On the malt extract agar medium (20 ml), the chrysene solution (1 ml) was added and spread uniformly.

  124 kinds of soils (1 g in dry weight) collected from the campus of the Faculty of Agriculture, Ehime University and its immediate vicinity were suspended in sterilized water (9 mL), stirred and allowed to stand for 10 minutes. Next, each supernatant (1 mL) was added to the malt extract agar medium and cultured at 25 ° C. for 2 weeks in the dark. 87 types of bacteria grown on the medium were collected and cultured at 25 ° C. for 7 days on the same malt extract agar medium. Seven species of fast-growing bacteria were collected, and the same operation was repeated once more to isolate the S133 strain, a basidiomycete that was particularly active in the presence of chrysene.

  When the obtained S133 strain was observed with a microscope, it had a flat, white umbrella with a dent in the center, and brown scales on the surface. The handle was central, and the spores were white and oblong. Since these are morphological characteristics of Polyporus spp. (Ikuseki Rokuya et al., “Japanese Mushrooms” Mountain and Valley Company, pages 450-451 (1988); and Thomas Laessoe, “DORLIG KINDERSLEY- HANDBOOKS-MUSHROMS ”, Dorling Kindersley Ltd., LONDON, 204, pp. 202-210 (1998)), and the S133 strain was identified as a Polyporus genus.

(3) Preparation of Asphalt Petroleum-Contaminated Soil In this example, soil contaminated with asphalt, which is a crude oil distillation residue, was prepared with asphalt concentrations in the soil of 1000 ppm, 15000 ppm, and 30000 ppm.

First, the soil collected from the farm attached to the Faculty of Agriculture, Ehime University was sieved with a 3 mm mesh and sterilized at 120 ° C. for 2 hours. Separately, asphalt (3 g) having the following composition was dissolved in dichloromethane (100 mL) to prepare a 3% asphalt solution. The asphalt solution (1 mL) was added to the sterilized soil (30 g in dry weight) to adjust the asphalt concentration in the soil to 1000 ppm.
(Asphalt composition)
Aliphatic hydrocarbon (aliphatic fr.) (32%)
Aromatic hydrocarbons (aromatic fr.) (44%)
Asphaltene part (asphaltene fr.) (14%)
Compound containing nitrogen, sulfur and oxygen (NSO part, NSO fr.) (10%)

  Furthermore, the asphalt density | concentration in soil prepared 15000 ppm and 30000 ppm by adjusting the addition amount of the said asphalt solution.

(4) Purification treatment of asphalt petroleum-contaminated soil Each of the strains NG007 and S133 was cultured on the above-described malt extract agar medium at room temperature (25 ° C.) for 7 days, and the culture solution was homogenized at 5000 rpm, 10 A mycelium suspension homogenized for a minute was prepared. The mycelial suspension of each strain thus obtained is NG007 strain: S133 strain = 0: 100, 25:75, 50:50, 75:25, 100: 0) in volume ratio. Adjusted.

(4-1) When no nutrient source is added to the medium The culture of each volume ratio obtained as described above is 13.3% with respect to the petroleum-contaminated soil prepared in (3) above. After mixing well, the mixture was cultured at 25 ° C. in the dark.

(4-2) When nutrient sources are added to the medium For the petroleum-contaminated soil prepared in (3) above, glucose and shiitake villages are 10% and 15% (for dry soil) as nutrient sources, respectively. (Mass ratio). Next, each volume ratio culture obtained as described above was added to the oil-contaminated soil at 13.3% (mass ratio with respect to dry soil), mixed well, and then in the dark. Cultured at 25 ° C.

(5) Measurement of Asphalt Degradation Rate Asphalt degradation rate (purification rate) was measured according to the method of Misra et al. Current Microbiology, Vol. 43, 328-335 (2001)).

  The outline of the measurement method is as follows. First, a part of the soil was collected after 15 days and 30 days from the start of culture, and extracted sequentially with hexane, dichloromethane, and chloroform. Each extract was combined and concentrated to obtain an extract. On the other hand, a part of the asphalt-contaminated soil (without addition of strain) was collected, and in the same manner as described above, the extracts extracted sequentially with hexane, dichloromethane, and chloroform were combined and concentrated to obtain an extract.

  Hexane was added to the above extract to separate it into a hexane soluble part and an insoluble part. Of these, the hexane-soluble part was subjected to silica gel column chromatography, and eluted sequentially with hexane, toluene, chloroform: methanol (1: 1), and the weight of each elution part was measured. The hexane elution part contains an aliphatic hydrocarbon part, the toluene elution part contains an aromatic hydrocarbon part, the chloroform: methanol (1: 1) elution part contains an NSO part, and the hexane insoluble part contains an asphaltene part. . By measuring the weight of each elution part and comparing the weight of oil-contaminated soil before and after purification treatment, the decomposition rate of aliphatic hydrocarbon part, aromatic hydrocarbon part, NSO part and asphaltene part (purification Rate) was calculated respectively. In this example, the sum of the decomposition rates of the components thus obtained was defined as the decomposition rate of A heavy oil or C heavy oil.

(6) Measurement of enzyme activity In this example, the enzyme activities of the following enzymes (a) to (e) (crude enzyme) were measured for reference, as in the previous application. As described in the prior application, these enzymes are thought to contribute to the azo dye decomposing action. In this example, the enzyme activities of various enzymes were measured for reference.

  Specifically, a part (5 g) of the petroleum-contaminated soil prepared in (3) above was collected, distilled water (30 mL) was added, and the mixture was homogenized (5000 rpm, 10 minutes) at room temperature (25 ° C.). The supernatant was collected.

  The activity (U / L or U / l) of each enzyme in the supernatant thus obtained was measured by the following method.

(A) Manganese peroxidase (MnP) activity was determined by the method of Brown et al. (J. Bacteriology, vol. 172 (6), pp. 3125-3130 (1990)) (molar absorbance coefficient ε: 49600 mol ⁻¹ cm ^{− 1} ).
(I) Lignin peroxidase (LiP) activity was determined by the method of Tien and Kirk (Methods in Enzymology, Vo 1.161, pp. 238-249 (1988)) (molar absorbance coefficient ε: 9300 mol ⁻¹ cm ⁻¹ ).
(C) laccase (Laccase, Lac) activity, LeonoWicz and Grzywnouicz method (Enzyme and Microbial Technology, Vol.3, pp, 55-58 (1981)) by the determined (molar extinction coefficient ε: 6500mol ^-1 cm ^{- 1} )
(D) The catechol 1,2-dioxygenase (C120) activity was determined by the method of Nakazawa and Nakazawa (Methods in Enzymology, Vo 1.17, pp, 5181522 (1970)) (molar absorbance coefficient ε: 16000 mol ⁻¹ cm ^-1 )
(E) Catechol 2,3-dioxygenase (C230) activity was determined by the method of Nakazawa and Nakazawa (Methods in Enzymology, Vol. 17, pp, 518-522 (1970)) (molar absorbance coefficient ε: 44000 mol ^{− 1} cm ^-1 ).

  These results are shown in FIGS.

  First, refer to FIG. FIG. 1 is a graph showing the results of asphalt degradation rate (left axis) and various enzyme activities (right axis) of asphalt-contaminated soil (1000 ppm) when NG007 strain and S133 strain were co-cultured at the ratio shown in FIG. Yes (no nutrient source added to the medium). The upper diagram in FIG. 1 shows the results for 30 days after the culture, and the lower diagram in FIG. 1 shows the results for 15 days after the culture.

  From FIG. 1, it can be seen that the combination of the NG007 strain and the S133 strain increases the asphalt purification rate compared to the case where each of them is used alone. For example, when the purification rates on the 30th day after cultivation (upper figure in FIG. 1) were compared, the asphalt purification rate of the NG007 strain alone was 67%, and the asphalt purification rate of the S133 bacterium alone was 59%. The asphalt cleanup rate when combined at a ratio of 1 increased to 89%.

  Reference is now made to FIG. FIG. 2 shows the results of asphalt degradation rate (left axis) and various enzyme activities (right axis) of asphalt-contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when NG007 strain and S133 strain were co-cultured at a ratio of 1: 1. (The number of culture days is 30 days, and no nutrient source is added to the medium).

  From FIG. 2, it was found that even contaminated soil with a high asphalt concentration can be purified to 68% (asphalt concentration of 15000 ppm) and 63% (asphalt concentration of 30000 ppm) by treatment for one month.

  Reference is now made to FIG. FIG. 3 is a graph in which the influence of nutrient sources added to the culture medium is examined for the asphalt degradation rate of asphalt-contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when the NG007 strain and the S133 strain are co-cultured at a ratio of 1: 1. (The culture days are 15 days and 30 days). The left figure of FIG. 3 shows the result without the addition of the nutrient source, and the right figure of FIG. 3 shows the result with the addition of the nutrient source.

  FIG. 3 shows that the purification rate of asphalt is promoted by adding a nutrient source during co-culture.

  From the above experimental results, it was found that according to the present invention, asphalt, which is a hardly decomposable substance in crude oil, can be efficiently purified in a short time.

Example 2
In this example, the purifying ability for A heavy oil contaminated soil and C heavy oil contaminated soil when NG007 strain and S133 strain were used alone or in combination was examined. Heavy oil is classified into 1 type (A heavy oil), 2 types (B heavy oil), and 3 types (C heavy oil) according to kinematic viscosity. In addition to light oil, 90% or more of C heavy oil is residual oil, and A heavy oil is a mixture of 90% of light oil and a small amount of residual oil.

First, the soil collected from the farm attached to the Faculty of Agriculture, Ehime University was sieved with a 3 mm mesh and sterilized at 120 ° C. for 2 hours. Separately, A heavy oil or C heavy oil (3 g) having the following composition was dissolved in dichloromethane (100 mL) to prepare a 3% A heavy oil solution or C heavy oil solution. Each said heavy oil solution (1 mL) was added to the said sterilized soil (30 g by dry weight), and each heavy oil concentration in soil was adjusted to 1000 ppm.
(A heavy oil composition)
Aliphatic hydrocarbon (aliphatic fr.) (75%)
Aromatic hydrocarbons (aromatic fr.) (9%)
Asphaltene part (asphaltene fr.) (13%)
NSO part (NSO fr.) (3%)
(C heavy oil composition)
Aliphatic hydrocarbon (aliphatic fr.) (44%)
Aromatic hydrocarbon (aromatic fr.) (31%)
Asphaltene part (asphaltene fr.) (17%)
NSO part (NSO fr.) (8%)

  Further, by adjusting the amount of each heavy oil solution added, those having a concentration of 15000 ppm and 30000 ppm of each heavy oil in the soil were prepared.

  Subsequently, in the same manner as in Example 1 described above, each contaminated soil was purified, and the decomposition rate of A heavy oil and the decomposition rate of C heavy oil were measured with reference to the method of Misra et al.

  Specifically, first, a part of the soil was collected after 15 days and 30 days from the start of the culture, and extracted sequentially with hexane, dichloromethane, and chloroform. Each extract was combined and concentrated to obtain an extract. On the other hand, a portion of A heavy oil contaminated soil or C heavy oil contaminated soil (without addition of strain) is collected, and in the same manner as above, extracts extracted sequentially with hexane, dichloromethane, and chloroform are combined and concentrated for extraction. I got a thing. Hexane was added to the resulting extract to separate it into a hexane-soluble part and an insoluble part, and the hexane-soluble part was subjected to silica gel column chromatography and eluted sequentially with hexane, toluene, chloroform: methanol (1: 1). By measuring the weight of each elution part and comparing the weights before and after the treatment, the respective decomposition rates of the aliphatic hydrocarbon part, aromatic hydrocarbon part, NSO part and asphaltene part were calculated. In this example, the sum of the decomposition rates of the components thus obtained was defined as the decomposition rate of A heavy oil or C heavy oil.

  These results are shown in FIGS. 4 to 10 and Tables 1 to 5. Table 1 corresponds to FIG. 4 and shows the result of each enzyme activity. Table 2 corresponds to FIG. 5 and shows the results of each enzyme activity. Table 2 also shows the results on the 30th day after the start of processing. Tables 3 to 5 correspond to FIG. 7 and show the results of the respective enzyme activities. Among these, Table 3 shows the results when the C heavy oil contaminated soil is 1000 ppm, Table 4 shows the results when the C heavy oil contaminated soil is 15000 ppm, and Table 5 shows the results when the C heavy oil contaminated soil is 30000 ppm.

  First, referring to FIG. FIG. 4 shows A heavy oil degradation rate (left axis) and various enzyme activities (right axis) of A heavy oil contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when NG007 strain and S133 strain were co-cultured at a ratio of 1: 1. Is a graph showing the results of (without addition of a nutrient source to the medium). The upper diagram in FIG. 4 shows the results for 30 days of culture, and the lower diagram in FIG. 4 shows the results for 15 days in culture. In FIG. 4, HOA means A heavy oil contaminated soil.

  As a result, even if it is contaminated soil with a high concentration of A heavy oil, it can be purified to 67% (A heavy oil concentration 15000 ppm) and 49% (A heavy oil concentration 30000 ppm) by treatment for 15 days; , 78% (A heavy oil concentration 15000 ppm), 68% (A heavy oil concentration 30000 ppm) was found to be purified.

  Reference is now made to FIG. FIG. 5 shows C heavy oil degradation rate (left axis) and various enzyme activities (right axis) of C heavy oil contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when NG007 strain and S133 strain were co-cultured at a ratio of 1: 1. Is a graph showing the results (no addition of nutrients to the medium, 15 days of culture). In FIG. 5, HOC means C heavy oil contaminated soil.

  As a result, it was found that even contaminated soil with a high C heavy oil concentration can be purified to 64% (C heavy oil concentration 15000 ppm) and 58% (C heavy oil concentration 30000 ppm) after 15 days of treatment.

  Reference is now made to FIG. Fig. 6 shows the effects of adding nutrient sources to the medium for the A heavy oil degradation rate of A heavy oil contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when NG007 and S133 were co-cultured at a ratio of 1: 1. It is a graph. The left figure of FIG. 6 shows the result without the addition of the nutrient source, and the right figure of FIG. 6 shows the result with the addition of the nutrient source.

  As a result, it turns out that the purification rate of A heavy oil is accelerated | stimulated by adding a nutrient source at the time of co-culture.

  Reference is now made to FIGS. These figures constitute the C heavy oil decomposition rate (FIG. 7) and C heavy oil of C heavy oil contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when the NG007 strain and S133 strain are co-cultured at a ratio of 1: 1. The result of the decomposition rate (FIGS. 8-10) of each component [aliphatic hydrocarbon part (Aliphetic), aromatic hydrocarbon part (Aromatic), asphaltene part (Asphaltene), NSO part is shown, respectively. 8 to 10, FIG. 8 shows the result when C heavy oil contaminated soil (1000 ppm), FIG. 9 shows the result when C heavy oil contaminated soil (15000 ppm), and FIG. 10 shows C heavy oil contaminated soil (30000 ppm). The results are shown respectively. 8 to 10, □ indicates the result without addition of the nutrient source, and ■ indicates the result with addition of the nutrient source.

  As a result, it is understood that the purification rate of C heavy oil is promoted by adding a nutrient source during co-culture (see FIG. 7).

  From the above experimental results, it was found that according to the present invention, not only asphalt in crude oil but also soil contaminated with C heavy oil or A heavy oil can be efficiently purified.

Example 3
In this example, in order to investigate in more detail the purification ability against asphalt-contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) when the NG007 strain and the S133 strain were co-cultured at a ratio of 1: 1, the above-mentioned Example 1 (culture days) 15 days and 30 days), the culture days were extended to 60 days and 120 days, and the purification rate of asphalt was measured in the same manner as in Example 1. The result is shown in FIG. The left diagram in FIG. 11 shows the result without the addition of the nutrient source, and the right diagram in FIG. 11 shows the result with the addition of the nutrient source.

  From FIG. 11, when no nutrient source was added, the purification rate of asphalt was hardly changed from the case of 30 days of culture, even if the culture days were extended to 60 days and 120 days, regardless of the concentration of asphalt. (See the left figure in Fig. 11) On the other hand, when nutrient sources are added, the asphalt purification rate is further improved when the asphalt concentration is high (15000 ppm, 30000 ppm), and an extremely high purification rate of about 90% is obtained. Could be achieved.

  For reference, Table 6A (1000 ppm), Table 6B (15000 ppm), and Table 6C (30000 ppm) show enzyme activities (U / l) when using asphalt-contaminated soils of various concentrations. These enzyme activities were examined in the same manner as in Example 2 described above, and the bold portions in parentheses in each table indicate enzyme activities after addition of a nutrient source. Nutrient sources were added on the 15th, 30th, 60th, and 90th days after culture. For example, in C12O of Table 6A, the numerical value in parentheses (920 U / l) described in the column of 30-day culture is the value when the nutrient source is added on the 15th day after cultivation and further cultured for 15 days (total 30 days). Yes; the value (9525 U / l) described in the column for 60-day culture is the value when the nutrient source is added on the 15th and 30th days after the cultivation and further cultured for 30 days (60 days in total); 120 days The numerical value (10425 U / l) described in the column of culture is a value when the nutrient source is added on the 15th, 30th, 60th, and 90th days after the cultivation and further cultured for 30 days (120 days in total). is there.

  Further, in the same manner as in Example 2 described above, the decomposition rate of each component constituting the asphalt [aliphatic hydrocarbon part (Aliphatic), aromatic hydrocarbon part (Aromatic), NSO part, asphaltene part (Asphaltene)] was examined. It was. The result is shown in FIG. FIG. 12A shows the result without the addition of the nutrient source, and FIG. 12B shows the result with the addition of the nutrient source. The results of 15 days, 30 days, 60 days, and 120 days after culturing are shown in order from the left for each concentration (1000 ppm, 15000 ppm, 30000 ppm) of each component in FIGS. 12A and 12B. As a result, in any of the components, the purification rate was promoted by adding a nutrient source at the time of co-culture, and the purification rate tended to be further improved particularly at high concentrations (15000 ppm, 30000 ppm).

Example 4
In this example, C heavy oil obtained by extending the culture days to 60 days and 120 days in the same manner as in Example 3 described above, except that C heavy oil contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) was used as the contaminated soil. Purification rate (FIG. 13), purification rate of each component constituting C heavy oil (FIGS. 14A to 14C), and enzyme activity (Tables 7A to 7C). As a result, as shown in FIG. 13 and FIG. 14A to FIG. 14C, the purification rate of C heavy oil tends to increase with the addition of nutrient sources, and particularly when C heavy oil is at a high concentration (15000 ppm, 30000 ppm) A purification rate of about 80% at 30000 ppm and about 100% at 150,000 ppm could be achieved 120 days after culturing in the supplemented medium.

Example 5
In this example, A heavy oil when the number of culture days was extended to 60 days and 120 days in the same manner as in Example 3 except that A heavy oil contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) was used as the contaminated soil. The purification rate (FIG. 15), the purification rate of each component constituting the A heavy oil (FIGS. 16A to 16C), and the enzyme activity (Tables 8A to 8C) were measured. As a result, as shown in FIG. 15 and FIG. 16A to FIG. 16C, the purification rate of heavy fuel oil A tends to increase with the addition of nutrient sources, especially when the heavy fuel oil A has a high concentration (15000 ppm, 30000 ppm). On the 120th day after culturing in the supplemented medium, all concentrations were able to achieve a purification rate close to about 100%.

Example 6
In this example, the purifying ability for petroleum-contaminated soil (1000 ppm, 15000 ppm, 30000 ppm) was examined when the NG007 strain, the S133 strain, and the D7 strain were co-cultured at a ratio of 1: 1: 1. Specifically, in the same manner as in Example 1 and Example 2 described above, the purification rate of heavy oil A, heavy fuel oil C, and asphalt in the medium with and without the addition of nutrients (culture days 15 days, (30 days, 60 days), the purification rate of each component constituting these (culture days 15 days, 30 days, 60 days) was examined. For reference, the enzyme activities of each of A heavy oil, C heavy oil, and asphalt were examined in the same manner as in Example 2 described above (culture days 15 days, 30 days, and 60 days).

  Of the three strains used in this example, the NG007 strain and the S133 strain were isolated in the same manner as in Example 1 described above.

  Moreover, the isolation method of D7 stock | strain is as follows.

  Twelve kinds of mushrooms were collected from rotten trees in the suburbs of Iyo City, Ehime Prefecture. These were cut into approximately 5 mm squares, added to the aforementioned malt extract agar medium containing the pigment RBBR (Remazo-Brilliant Blue R) (100 ppm), and cultured at 25 ° C. in the dark for 2 weeks. Twelve types of bacteria that have grown on the malt extract agar medium were collected, and a malt extract agar medium overlaid with 1000 ppm C heavy oil (specifically, 1000 ppm C heavy oil dissolved in dichloromethane on the malt extract agar medium) Then, dichloromethane was evaporated, and the mixture was cultured at 25 ° C. for 7 days in a malt extract agar medium with C heavy oil placed thereon. Among these, basidiomycetes that were particularly vigorously grown by collecting three species of fast-growing bacteria and further culturing them at 25 ° C. for 7 days in a malt extract agar medium overlaid with 15,000 ppm C heavy oil. D7 strain was isolated.

  When the obtained D7 strain was observed with a microscope, the surface had a grayish white semi-circular umbrella, and the surface was covered with coarse hairs, representing a ring pattern. Its width is 2 to 7 cm and thickness is 2 to 5 mm. The spores are white, and the size is 5-7 μm long × 2-3 μm wide. Since these are morphological features of the genus Trametes, the D7 strain was identified as the genus Trametes.

  Each of the three strains described above was cultured on the aforementioned malt extract agar medium at room temperature (25 ° C.) for 7 days, and a hyphae suspension was prepared by homogenizing the culture with a homogenizer at 5000 rpm for 10 minutes. The mycelial suspension of each strain thus obtained was adjusted so that the volume ratio was NG007 strain: S133 strain: D7 strain = 1: 1: 1, and the above experiment was performed.

  The results when using heavy oil A are shown in FIGS. In detail, while the purification rate of each component which comprises A heavy oil was shown to FIG. 17A and FIG. 17B, the purification rate of A heavy oil was shown in FIG. Table 10 shows the result of each enzyme activity.

  The results when C heavy oil is used are shown in FIGS. In detail, while the purification rate of each component which comprises C heavy oil was shown to FIG. 18A and FIG. 18B, the purification rate of C heavy oil was shown in FIG. Table 11 shows the result of each enzyme activity.

  The results when using asphalt are shown in FIG. 19, FIG. 22, and Table 12. Specifically, the purification rate of each component constituting the asphalt is shown in FIGS. 19A and 19B, and the purification rate of asphalt is shown in FIG. Table 12 shows the result of each enzyme activity.

  Furthermore, Table 9 summarizes the results of the purification rate when using A heavy oil, C heavy oil, and asphalt.

  When the results obtained using the three types of mixture (NG007 strain + S133 strain + D7 strain) are compared with the results obtained using the above-mentioned two types of mixture ((NG007 strain + S133 strain)), three types of mixtures are used. As a result, the tendency of improving the purification rate of oil-contaminated soil was observed, particularly when using heavy oil A and heavy oil C. For example, in the case of heavy oil A, the three types of 15) and FIG. 15 (two kinds of mixtures), the three kinds of mixtures can be obtained on the 15th, 30th, and 60th days after culturing regardless of the presence or absence of the nutrient source and the concentration of heavy oil A. When used, the purification rate of heavy oil A was improved, and the same tendency was observed when heavy oil C was used, comparing Fig. 21 (three types of mixtures) and Fig. 13 (two types of mixtures). Then, the presence or absence of nutrient sources and C heavy oil Regardless of the concentration, 15 days after culture, 30 days, in any of the 60 days, who when using three kinds of mixture, with improved purification rate of C heavy oil.

  From the results of Examples 1 to 6, the oil-contaminated soil purifier of the present invention is extremely useful as a technology that can sufficiently purify high-concentration oil-contaminated soil, which was difficult to be decomposed in a short time by the conventional method. It was proved sufficiently.

Claims (3)

  A cleansing agent for oil-contaminated soil, comprising NG007 strain (NITE P-1233) of the genus Pestalotiopsis.   The oil-contaminated soil purifier according to claim 1, further comprising S133 strain (NITE P-461) belonging to basidiomycetes of the genus Polyporus.   Furthermore, the purification | cleaning agent of the petroleum contaminated soil of Claim 2 which contains D7 strain | stump | stock (NITE P-01703) of Trametes genus.