JP3522669B2 - Contaminated soil purification method - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for remediating a polluted soil by degrading the polluted soil containing an environmental pollutant with an enzyme produced by a wood-rotting fungus.

[0002]

2. Description of the Related Art We human beings have developed various scientific technologies and promoted industrialization since the industrial revolution. However, while it has become possible to live a rich and comfortable social life, environmental destruction has progressed on a global scale, which has become a serious social problem. There are many environmental pollutants produced as by-products of industrial activities on the present earth, and removal of these pollutants and restoration to the original environment have become extremely important issues.

The methods used to remove environmental pollutants are roughly classified into chemical methods, physical methods and biological methods. Among these, the most energy-saving and resource-saving process is a biological method, that is, bioremediation utilizing the purification function of microorganisms. This technology is a technology that skillfully utilizes the metabolic functions of living organisms to decompose environmental pollutants, detoxify them, and finally convert them into carbon dioxide, methane, water, inorganic salts, biomass, etc. .

Purification of contaminated soil by bioremediation does not require excavation of the soil, so that it is possible to purify under a building, and by using microorganisms with high decomposition activity, low-concentration organic compounds can be removed in a short time. It has the features of being able to purify by, and being low in cost and environmental load, and has recently attracted attention as a technology for decomposing and removing environmental pollutants.

Purification methods by bioremediation can be roughly classified into the following three types. In other words, a bioreactor system that uses microorganisms that have the ability to decompose pollutants by immobilizing them, biostimulation that supplies various nutrients to microorganisms that originally live in contaminated soil and groundwater, and enhances their decomposition activity. The method is a bio-augmentation method in which microorganisms having the ability to decompose pollutants are directly sprayed to the pollution site. Currently, the bioremediation technology that has been put into practical use is the above-described method that gives conditions that enhance the degrading activity of degrading bacteria that mainly inhabit the pollution site. However, when the pollutants cannot be decomposed only by the microorganisms existing at the polluted site, it is necessary to apply the above method of introducing and decomposing exogenous degrading bacteria into the polluted environment.

By the way, dioxins are one of the most noticeable environmental pollutants. It is known that these are extremely stable in the natural environment, and that some of them are highly toxic and mutagenic. Currently, the spraying of chemicals that may contain dioxins is strictly regulated, but it has become clear that a large amount of dioxins is released to the natural world when garbage is incinerated, and this has become a serious social problem. Since dioxin pollution spreads at low concentrations over a wide area, the purification method by bioremediation is most suitable, but it is unlikely that microorganisms capable of decomposing dioxins are distributed in general soil. There have been some reports that white rot fungi belonging to wood-rotting fungi decompose dioxins, and bioaugmentation using them is currently being investigated.

Wood-destroying fungi are drawing attention as microorganisms capable of degrading various environmental pollutants. Wood-rot fungi are classified into white-rot fungi and brown-rot fungi depending on their ability to decompose lignin, which is a natural persistent compound. The white-rot fungus produces phenol oxidase outside the cell and can decompose lignin. Brown rot fungus does not have lignin degrading ability because it does not produce phenol oxidase, but it is known to mineralize various phenolic compounds, and it is suggested to have degrading ability to aromatic compounds. .

In particular, with regard to white-rot fungi, in addition to dioxins, the insecticide DDT (1,1,1-trichloro-
2,2-bis (4-chlorophenyl) ethane), a herbicide
It is known to decompose various environmental pollutants such as 2,4-D (2,4-dichlorophenoxyacetic acid), polychlorinated biphenyls, polychlorinated phenols and polycyclic aromatic hydrocarbons. Therefore, it can be said that the white rot fungus is a bacterium suitable for bioaugmentation, which is introduced into a contaminated soil as an exogenous decomposition bacterium and used.

However, when wood-rotting fungi such as white-rotting fungi are introduced into contaminated soil for purification, it is difficult to grow and settle wood-rotting fungi. That is, since wood-rotting fungi have a low ability to grow in contaminated soil, they cannot settle in the contaminated soil for a long period of time. Further, there is a problem that even if the wood-destroying fungus is introduced into the contaminated soil, both the production and the maintenance of the activity of the enzyme relating to the decomposition of the environmental pollutant are low. Therefore, the improvement of contaminated soil using wood-destroying fungi has not yet been put to practical use.

[0010]

Therefore, the present invention has been made in view of the above-mentioned circumstances, and enables wood-rotting fungi to be contaminated in contaminated soil and the contaminated soil can be efficiently purified. It is intended to provide a method for improving contaminated soil.

[0011]

[Means for Solving the Problems] In order to achieve the above-mentioned object, as a result of diligent study by the present inventor, it is possible to improve the fixing ability of wood-destroying fungi by growing turfgrass on contaminated soil. The present invention has been completed based on the finding that the ability to produce enzymes that contribute to the decomposition of pollutants can be improved.

That is, the method for purifying contaminated soil according to the present invention uses the enzyme produced by a wood-rotting fungus to purify contaminated soil containing environmental pollutants, and the above-mentioned method is used in the state where grass grass is planted in the contaminated soil. It is characterized in that the wood-rotting fungus is administered to contaminated soil. Further, in the method for purifying contaminated soil according to the present invention, it is preferable to mow the grass after the grass has grown.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.
The method for purifying contaminated soil according to the present invention is applied when purifying contaminated soil containing environmental pollutants.

Examples of environmental pollutants include organic chlorine compounds such as dioxin, PCB, trichloroethylene, tetrachloroethylene, pentachlorophenol (PCP) and DDT, polycyclic aromatic hydrocarbons such as naphthalene, anthracene and phenanthrene, benzene. , Aromatic hydrocarbons such as ethylbenzene, toluene and xylene, dinitrotoluene, trinitrotoluene (TNT)
And nitro compounds such as nitroglycerin, triphenylmethane dyes such as crystal violet, cresol red, and bromophenol blue.

The contaminated soil is not particularly limited in the method of the present invention, and may be any soil, for example, farmland, incineration site, site of a factory, soil at a crude oil spill accident site, and the like. . In this method, first, turfgrass is grown on the contaminated soil to be improved. Subsequently, wood-destroying fungi are introduced into the contaminated soil where the turfgrass has grown to produce the enzyme. When growing turfgrass, first, the seeds and / or the turfgrass itself is sprayed on the surface of the contaminated soil, and the turfgrass is planted in the contaminated soil. After that, by passing a predetermined period,
Turfgrass can be grown on contaminated soil.

Here, the wood-destroying fungus is Phanerochaete chrysospor.
ium), Phanerochaete Soldida
sordida), Pleurotas ostreatus (Pleurotu)
s ostreatus) (oyster mushroom), Coriolus versicolor (kawaratake), and other white-rot fungi. In addition, Tyromyces palustris (Pleurotus ostreatus) and Gloeophyllum trabtum (Gloeophyllum t
Rabeum) and other brown-rot fungi can also be mentioned. These wood-destroying fungi may be used alone or in combination of two or more.

Examples of the turfgrass include moss grass, koiushiba, and turf grass. In particular, when the contaminated soil is in a warm region, it is preferable to grow the zoysiagrass and koiishiba. When the contaminated soil is in a cold area, it is preferable to grow grass.

[0018] In addition, as compared with Nosiba and Koishibushi, the lawn grass has a faster growth rate and higher nutritional requirements and water requirements, and therefore has a characteristic that it contains a large amount of nutrients such as a carbon source and a nitrogen source and easily retains water. Suitable for contaminated soil with. Soil with high nutrient content and high water retention capacity is suitable for growth of wood-destroying fungi, but overnutrition may not activate the enzyme-producing ability of wood-destroying fungi. Therefore, for soil with high nutrient content and high water retention capacity, it is preferable to administer wood-rot fungus that previously produces an enzyme to the contaminated soil to promote growth and colonization in the soil. Therefore, for contaminated soil having a high nutrient content and a high water retention ability, it is possible to grow the lawn grass and purify the contaminated soil in a short term.

[0019] On the other hand, Noshiba and Koishibushi are not suitable for polluted soil which has a relatively low nutrient content and is difficult to retain water because they have a lower growth rate and lower nutritional requirement and water requirement than that of grass. Contaminated soil that is low in nutrients and difficult to retain water is not suitable for growing wood-destroying fungi,
It is suitable for the production of the above enzymes. Therefore, it is preferable to administer wood-destroying fungi to the contaminated soil to promote growth and establishment in the soil over a certain period of time for the contaminated soil having a low nutrient content and difficult to retain water. For this reason, contaminated soil having a small amount of nutrients and having difficulty in retaining water can be used for long-term purification by growing Noshiba or Korushiba to produce the above enzyme.

Specifically, when growing turfgrass in contaminated soil, it is preferable to use the following method. That is,
After investigating the condition of the contaminated soil, the part including the surface of the contaminated soil is vibrated by a slicing machine or the like to eliminate the solidification of the soil and sprinkle water. In particular, the soil environment is prepared by repeating vibration and watering until a three-phase distribution (solid, liquid, air = 50, 25, 25) is reached. Then, turfgrass is sprayed and grown for 2-3 weeks. The method for growing the turfgrass in the contaminated soil is not limited to the above-mentioned method, and a normal method, that is, a method applied when creating a green space can be appropriately used.

The wood-rotting fungi can be administered to the contaminated soil by the method described below. First, for example,
Using wood or sand containing nutrients necessary for the growth of wood-destroying fungi as a culture bed, the wood-destroying fungi are cured in a temperature-controlled room or in a contaminated soil site. In the case of curing in a thermostatic chamber, as shown in FIG.
About 2 g of sand 2 is put, and 2.5% (wt / wt) of compost 3 is added thereon. Furthermore, a glucose-peptone 5% (wt / vol) solution is added to 25 to 250
After adding mL, sterilize (121 ° C., 60 minutes) to prepare a large number of culture beds. Then, the wood decay fungus 4 is inoculated on the compost 3 and cultivated in a constant temperature room at 25 ° C. in the dark for 2 to 3 weeks. As a result, as shown in FIG.
Wood-rotting fungi 4 can be propagated on top and in the sand 2.

However, if the wood-destroying fungus aged under constant conditions in a temperature-controlled room is directly added to the contaminated soil in which conditions such as nutrition, aeration and moisture retention are not constant, turfgrass is grown to contaminate the contaminated soil. Even after the improvement, the wood-rotting fungi may be difficult to grow. In such a case, it is preferable to cure wood-destroying fungi at the contaminated soil site, as described below. By curing at the contaminated soil site, wood-destroying fungi can be acclimated to the environment.

When curing wood-destroying fungi at the site of contaminated soil, first, as shown in FIG. 3, in order to prevent contamination with various fungi in the soil, a laid iron plate 10 sterilized at the site of contaminated soil is used. Set up. Next, sterilized sand is prepared using the sandblasting machine 11 as shown in FIG. Sand-sanding machine 11
Is composed of a carry-in section 12 for carrying in sand before sterilization, a furnace 13 for baking the carried-in sand at a predetermined temperature for sterilization, and a carry-out section 14 for carrying out the sand sterilized in the furnace 13.

Next, the sand sterilized by the sandblaster 11 was sterilized with a 5% (wt / vol) solution of glucose and peptone with a 0.001% chlorine sodium hypochlorite solution or a chlorine dioxide solution. Disperse so as to be 200 L / m ³ . Thereafter, as shown in FIG. 5, sand was put on the iron plate 10 and the wood-destroying fungus cultured by the above-mentioned method was added in an amount of 0.05 to 0.
After inoculating to 5% (vol / vol), cover with blue sheet 14 and cure with sterilized heavy machinery once a week with stirring for 2-3 weeks (hereinafter, obtained by curing. Is referred to as "culture floor 15 containing wood-destroying fungus").

The iron plate 10 and heavy equipment are 0.001 chlorine.
Sterilize with a% sodium hypochlorite solution or chlorine dioxide solution. Here, the chlorine 0.001% sodium hypochlorite solution or the chlorine dioxide solution does not kill wood-destroying fungi but can kill other bacteria.

On the other hand, in this method, the method for administering the wood-destroying fungus to the contaminated soil in which the lawn grass has grown is not particularly limited, but, for example, first, as shown in FIG. 6, the lawn grass 19 is grown as described above. Depth 5 to 1 from contaminated soil 20
A cylindrical core 21 having a diameter of 0 cm and a diameter of 16 mm is extracted to form a plurality of holes 22. After that, as shown in FIG. 7, the wood-rotting fungus-containing culture bed 15 prepared by the above-described method is sprayed on the contaminated soil 20 including the inside of the hole 22 in a thickness of 3 to 10 mm, and rubbing is performed. After that, the water-destroying fungus can be grown and settled on the contaminated soil 20 by appropriately sprinkling water. Then, in the contaminated soil 20, as the growing area of the turfgrass 19 expands, it is possible to widely spread and fix the wood-destroying fungi.

The wood-destroying fungi administered to the contaminated soil 20 are
As the roots of the turfgrass 19 grow, they can propagate to diffuse into the contaminated soil 20. Generally, in the contaminated soil 20, environmental pollutants are distributed up to a depth of about 10 cm from the surface. Further, it is known that the roots of the turfgrass 19 grow sufficiently at a depth of about 10 cm from the surface. Therefore, the wood-destroying fungus can be sufficiently propagated to the depth where the environmental pollutants are distributed as the roots of the turfgrass 19 grow.

In addition, as the turfgrass 19 grows, the contaminated soil 2
When the carbon source and the nitrogen source in 0 become deficient, the enzyme that decomposes the environmental pollutant is activated as a part of the secondary metabolism expressed in the deficiency of the carbon source and the nitrogen source. In this way, by growing the turfgrass 19 on the contaminated soil 20,
The propagation of wood-destroying fungi and the activation of enzymes can be achieved at the same time.

In other words, by controlling the amounts of carbon source and nitrogen source in the contaminated soil 20 by the growth of the turfgrass 19, the growth of the turfgrass 19 is accelerated, the propagation of wood-destroying fungi is promoted, and the amount of environmental pollutants is increased. It is possible to promote the production of an enzyme that contributes to decomposition and activate the enzyme.

In particular, by pruning the grown turfgrass 19, it is possible to further capture the carbon source and the nitrogen source from the contaminated soil 20 by the turfgrass 19. As a result, in the contaminated soil 20, the production and activation of the enzyme that contributes to the decomposition of the environmental pollutant can be further promoted. Further, according to this method, after the purification of the contaminated soil 20 is completed, the turfgrass 19 can be continuously grown to turn the contaminated soil 20 into a green area.

[0031]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto. Examples In the following examples, bentgrass, which is a kind of grass, was used as turfgrass, and the growth behavior and the enzyme activity of white rot fungi in bentgrass growing soil were evaluated. The bentgrass is characterized by high growth rate and high nutritional requirement and water requirement.

(1) Preparation of culture broth containing white rot fungus White rot fungus [Phanerochiete chrysosporium (Ph
anerochaete chrysosporium) BKMF-1767, ATCC42725]
Was inoculated on a 2% potato dextrose (PDA) agar medium (plate) and cultured at 24 ° C. for 7 days. In a 500 mL Erlenmeyer flask, 25 g of 100 g of river sand, 5 g of Tateyama Yuki, and a 5% (wt / vol) solution of glucose and peptone were added.
mL was injected and sterilized to prepare a culture bed. Inoculate the culture bed with 2 cm x 2 cm of the bacterial cells spread on the plate, and
A culture bed containing white rot fungi was prepared by culturing in the dark at 0 ° C. for 2 weeks.

(2) Preparation of bentgrass growth soil Improved soil in a pot (100 g of river sand, Yuki Tateyama 5
Bentgrass was sprayed onto the surface layer of g, glucose / peptone 5% (wt / vol) solution (25 mL), and water was appropriately sprinkled for curing for 2 weeks to prepare bentgrass growing soil.

(3) Spraying of a culture bed containing white-rot fungi onto bentgrass-grown soil A diameter of 2 cm and a depth of 5 from the bentgrass-grown soil described above.
The cm-shaped tubular core was taken out, and the above-mentioned white-rot fungus-containing culture bed was sprayed to a thickness of 5 mm, and rubbed. The plant was cured with watering as appropriate, and the growth behavior and enzyme activity of white rot fungi in bentgrass-grown soil were evaluated.

Comparative Example 1 Comparative Example 1 was carried out in the same manner as in Example 1 except that the bentgrass was not sprayed in (2) in Example, but the white-rot fungus-containing culture bed was directly sprayed on the improved soil for rubbing. The growth behavior and enzyme activity of white rot fungi were evaluated.

Comparative Example 2 In Comparative Example 2, the filamentous fungus Rhizoctonia solani was used in place of the white rot fungus, except that
The growth behavior and enzyme activity of white rot fungi were evaluated in the same manner as in the examples. Comparative Example 3 In Comparative Example 3, the growth behavior and enzyme activity of white rot fungi were evaluated in the same manner as in Example except that the culture bed containing white rot fungi was not sprayed as a control.

Growth behavior of white rot fungi and enzyme activity evaluation The growth behavior of white rot fungi in Example and Comparative Example 1 was
It evaluated as follows. That is, Example and Comparative Example 1
The soil was digged up at intervals of once a week, and the growth status of the hypha of the white-rot fungus was visually observed to evaluate the growth behavior of the white-rot fungus. In addition, the soils of Example and Comparative Example 1 were sampled and microscopically observed to evaluate the growth behavior of white rot fungi.

As a result, it was found that the growth behavior of white-rot fungi was as in Example >> Comparative Example 1. From these results, it was confirmed that it is possible to prepare an environment that promotes the growth behavior of white-rot fungi by growing grass. The enzyme activity was evaluated by the dye evaluation method described below. For the dye evaluation method, a dye solution and a chromaticity table were prepared.

<Preparation of Dye Solution> 10 g glucose, 0.5 g MgSO ₄ , ₂ g KH ₂ PO ₄ , 1 per liter
A dye solution was prepared by adding 1 mL phenol red solution (2 g / 100 mL solution) to a solution containing 00 mg CaCl ₂ , 10 mL of trace element solution, and 20 mM sodium acetate (pH 4.5) as a buffer. Here, the trace element solution means 3 g of MgSO ₄ and M per 1 L of the solution.
nSO ₄ 0.5 g, NaCl 1.0 g, FeSO ₄
・ 7H ₂ O 0.1 g, CoCl ₂ 0.1 g, ZnS
O ₄ · 7H ₂ O and 0.1 g, the CuSO ₄ 0.1 g, A
10 mg of 1K (SO ₄ ) ₂ · 12H ₂ O, H ₃ BO ₃
Is 10 mg, Na ₂ MoO ₄ .2H ₂ O is 10 mg, and nitrilotriacetate is 1.5 g.

<Preparation of Chromaticity Table> In evaluating the color change of the dye, 14 colors from yellow to crimson were arranged, and a chromaticity table corresponding to numerical values from 1 to 14 was prepared. The color mode RGB is used, red is fixed at 100 and blue is fixed at 20, and green is changed from 0 to 100 in increments of 10, and No. in the chromaticity table is changed. 1 to 11 colors are made, green is fixed at 0 and blue is fixed at 20, red is 90, 8
No. in the chromaticity table is changed to 0 or 70. 12-14
The colors of

(4) Evaluation of soil sample using dye solution After transferring 3 mL of the dye solution to a 15 mL centrifuge tube and adding the soil collected from Examples and Comparative Examples 1 to 3,
Mix by shaking for 30 seconds. When the color change of the dye solution was observed using the chromaticity table, it was found that the soils of Examples had No. It had a color of 9.5. On the other hand, the soil of Comparative Example 1 has N in the chromaticity table.
o. No. 5 in the chromaticity table is shown for the soil of Comparative Example 2.
No. 4 in the chromaticity table is shown for the soil of Comparative Example 3. 2.
5 was shown.

From these results, it was found that the activities of the enzymes contained in the soil were: Example >> Comparative Example 1> Comparative Example 2> Comparative Example 3. From these results, it was confirmed that it is possible to prepare an environment for promoting enzyme production and activation by growing grass. Further, in the examples, white-rot fungi had no effect on the growth of bentgrass, but in Comparative example 2, it was found that filamentous fungi inhibit the growth of bentgrass.

[0043]

As described in detail above, the method for improving contaminated soil according to the present invention grows turfgrass in the contaminated soil, and therefore can promote the propagation of wood-destroying fungi and decompose environmental pollutants. The production and activation of contributing enzymes can be promoted. Therefore, according to the present invention,
It is possible to provide a method for improving contaminated soil which is excellent in the purification efficiency of environmental pollutants.

[Brief description of drawings]

FIG. 1 is a schematic view showing a medium for culturing wood-destroying fungi in a thermostatic chamber.

FIG. 2 is a schematic diagram showing a state in which wood-destroying fungi are cultured in the medium shown in FIG.

FIG. 3 is a perspective view of an essential part showing a laid iron plate used when curing wood decay fungi in contaminated soil.

FIG. 4 is a schematic configuration diagram of a sand blasting machine.

FIG. 5 is a perspective view of a main part of a laid iron plate on which a culture bed containing wood-destroying fungi is placed.

FIG. 6 is a perspective view of a main part of a contaminated soil showing a state where a cylindrical core is formed on the contaminated soil.

FIG. 7 is a perspective view of a main part of a contaminated soil showing a state in which a wood-rot fungus-containing culture bed is sprayed after forming a tubular core on the contaminated soil.

[Explanation of symbols]

1 Erlenmeyer flask, 2 Sand, 3 Compost, 4 Wood decay fungus, 10 Laying iron plate, 11 Sand-blasting machine, 15 Wood decay fungus containing culture bed, 19 Turf grass, 20 Contaminated soil, 21 Cylindrical core, 22 holes

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI C12N 9/00 B09B 3/00 ZABE (56) References JP-A-11-319786 (JP, A) JP-A-2001-252646 (JP , A) JP 2001-276806 (JP, A) Tokuhei HEI 11-512970 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) B09C 1/00-1/10

Claims (2)

(57) [Claims] 1. When purifying a contaminated soil containing an environmental pollutant using an enzyme produced by a wood-rotting fungus, a step of planting a lawn on the contaminated soil and a step of planting the wood on the contaminated soil in which the turf grass is planted. A method for purifying contaminated soil, which comprises a step of administering a rot fungus. 2. The method for purifying contaminated soil according to claim 1, further comprising a step of cutting the grass after the grass has grown.