JP2003334063A - White rot fungus having high ability to degrade aromatic compound and/or halogenated organic compound - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide new microorganisms capable of efficiently degrading aromatic compounds such as bisphenol A and/or halogenated organic compounds such as dioxins even under atmospheric conditions. <P>SOLUTION: The new microorganism is white rot fungus FERM P-18828 having high ability to degrade aromatic compounds such as bisphenol A and/or halogenated organic compounds such as dioxins. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel white-rot fungus FERM P-18828 which is excellent in the ability to decompose aromatic compounds and / or halogenated organic compounds. The microorganism of the present invention is extremely excellent in decomposing aromatic compounds and / or halogenated organic compounds, and can also detoxify harmful compounds such as dioxins. Therefore, the treatment of wastewater containing these compounds, the above compounds are targeted. Starting with bioremediation,
It can be widely used in various technical fields for decomposing / detoxifying the above compounds.

[0002]

2. Description of the Related Art In recent years, environmental pollution due to various harmful chemical substances, adverse effects on human bodies, etc. have been greatly highlighted.

Among them, aromatic compounds such as bisphenol A and organic chlorine compounds such as dioxins are harmful and persistent, and have extremely high accumulation in the environment, human body, etc. Inviting problems. Therefore, much research has been conducted on methods of decomposing these harmful compounds.

At present, the method that uses microorganisms [bioremediation] is in the limelight. When microorganisms with excellent resolution are used, compared with conventional physicochemical methods (high temperature melting method, thermal decomposition method, alkali treatment method, supercritical water decomposition method, catalytic oxidation method, ozone decomposition method, etc.) This is because the decomposing action is obtained, the energy consumption is small, and there is no fear of environmental pollution.

In particular, wood-destroying fungi have attracted attention as microorganisms capable of degrading various environmental pollutants. White-rot fungi, which is a kind of wood-destroying fungi, are caused by a phenol oxidase produced outside the cells. It is known that lignin, which is a naturally persistent substance, is excellent in decomposing ability. Furthermore, it has been reported that dioxins having a chlorine substitution number of 4 or more can be decomposed by a white-rot fungus of the genus Fanerocete [Bumpus et al., Sceience, 228, 1434 (1985);
Takada et al., Appl. Environ. Microbiol, 62, 4323 (199
6)].

However, the decomposing action by the microorganisms of the genus Fanococete is not obtained under normal air conditions, and it is exhibited only after a separate treatment such as increasing the oxygen concentration during the decomposing treatment. Nevertheless, the resolution was low and it was still insufficient for practical use.

[0007]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and an object thereof is to provide an aromatic compound such as bisphenol A or a halogenated organic compound such as dioxins under an air condition. It is to provide a novel microorganism that can be decomposed efficiently.

[0008]

The novel microorganism of the present invention, which has been able to solve the above-mentioned problems, is a white-rot fungus FERM which is excellent in decomposing aromatic compounds such as bisphenol A and / or halogenated organic compounds such as dioxins. P-1882
8

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION In order to achieve the above object, the present inventors have searched for new microorganisms capable of exhibiting a desired action among microorganisms existing widely in nature, and found that microorganisms separated from decaying materials Found that it exerts the desired effect,
The present invention has been completed.

The novel microorganism of the present invention will be described below.

First, the microorganism of the present invention has the following mycological properties.

(1) Growth state in medium Table 1 shows the growth state when grown in each of malt extract agar medium, potato-glucose agar medium and Sabouraud agar medium under the following conditions.

PH of medium: 5.0 Culture conditions: 25 ° C.
7 days

[0014]

[Table 1]

(2) Physiological and ecological properties pH range for growth (potato-glucose agar, 25
Cultivated at 7 ° C. for 7 days) It grows around pH 3 to 9 and does not grow at pH 2 and pH 10. The optimum pH is around 4-6.

Growth temperature range (potato-glucose agar medium, pH 5, 7-day culture) Grows in the vicinity of 10 to 40 ° C., but not in 45 ° C. The optimum temperature is around 25 to 30 ° C.

Phenol oxidase reaction (25 ° C,
Cultivation for 4 days) A clear browning was observed, which is positive.

Characteristics of microflora The agar medium of the types shown in Table 1 is white and felt-like.

Characteristics of mycelium It has a faint connection (clamp connection).

Based on the above-mentioned mycological properties, the strain of the present invention is considered to be a white-rot fungus belonging to Basidiomycetes, but it could not be identified as a known fungus. Moreover, as is clear from the examples described below, the microorganisms of the present invention exhibit remarkably excellent decomposing ability as compared with known white-rot fungi known as decomposing microorganisms such as dioxins (genus Phanelocete). Since such an excellent degrading ability was not found in conventional microorganisms, the strain of the present invention was designated as a new strain and named MAT-1 strain, and the patented organism of National Institute of Advanced Industrial Science and Technology It was deposited at the deposit center (accession number: FERMP-18828, date of notification: April 23, 2002).

The microorganism of the present invention exhibits extremely excellent resolution against aromatic compounds such as bisphenol A and halogenated organic compounds such as organic chlorine compounds including dioxins. Incidentally, the "aromatic compound" in the present invention includes a halogenated aromatic compound, but in the present invention, all halides are included in the "halogenated organic compound", and the "aromatic compound" Shall mean non-halogenated aromatic compounds. Therefore, in the "halogenated organic compound" of the present invention, regardless of whether it is an aromatic compound,
All halogenated organic compounds will be included, while halogenated aromatic compounds are excluded from the "aromatic compounds".

Here, the above-mentioned "aromatic compound" is
Except for halogenated compounds, all compounds having an aromatic ring are included, and the type of homocyclic ring or heterocyclic ring does not matter. Among them, the homocyclic ring includes benzene; benzene having a substituent such as nitrobenzene and fluoranthene; phenol; nitrophenol; alkylphenols such as nonylphenol, octylphenol, and pentylphenol; catechol; dimethyl phthalate, diethyl phthalate, dibutyl phthalate, Phthalates such as diheptyl phthalate and dioctyl phthalate; naphthalene; anthracene; pyrenes such as pyrene, benzopyrene, dibenzopyrene; bisphenol compounds such as bisphenol A; estradiol and the like. Further, as the heterocycle, in addition to carbon, 1 hetero atom such as N, O, S, etc.
An aromatic compound such as pyridine, pyrimidine, furan, thiophene, or pyrrole;
These related compounds are included. Of course, a mixture of a homocyclic ring and a heterocyclic ring is also included in the range of the “aromatic compound”. Further, a polymer raw material having an aromatic ring and its decomposition products (oligomers, partial decomposition products, etc.) are also included.

The "halogenated organic compound" is not particularly limited as long as it is an organic compound having at least one of fluorine, chlorine, bromine and iodine, and a vinyl chloride type or vinylidene chloride type organic chlorine compound; Teflon. (Registered trademark), fluorinated compounds such as freon, and the like.
In addition, among the “halogenated organic compounds”, PCDD
s (polychlorinated dibenzodioxins) and PCDFs
(Polychlorinated dibenzofurans) and the like; dioxins containing bromine instead of chlorine in the dioxins; PC containing coplanar PCB
Bs (polychlorinated biphenyls), CB (chlorobenzene), CP (chlorophenol) and the like are also included.

Next, a method of decomposing an aromatic compound and / or a halogenated organic compound using the above microorganism will be described. Decomposition methods include the case where an aromatic compound and / or a halogenated organic compound (hereinafter sometimes referred to as “aromatic compound etc.”) that is a decomposition target is dissolved / suspended in water etc. and is present in the soil. Solids such as ash and ash (hereinafter,
It may be represented by "soil") and is slightly different from the case where it is adsorbed to "soil". Therefore, each case will be described separately below.

Method of Decomposing Solution / Suspension of Aromatic Compound etc. First, the above microorganism is used as a medium for basidiomycetes [low nitrogen synthesis medium (K
irk's medium), potato-glucose medium, Sabouraud's medium, etc.]) and inoculate. The culturing time varies depending on the type of medium used, but it is recommended to cultivate at 20 to 30 ° C. for 3 to 7 days.

Next, the compound to be decomposed (aromatic compound and / or halogenated organic compound) is added to the medium after culturing and further cultivated. The compound concentration at this time is about 1
It is preferable to adjust the concentration to be 10 ^{−6 to} 100 mg / L. In addition, although the culture conditions vary depending on the type of compound and the like, it is generally recommended to culture at 20 to 30 ° C. for 1 to 30 days.

Method for Decomposing Aromatic Compounds in Soil Aromatic compounds in soil are less likely to be decomposed than aromatic compounds in a solution / suspension, and therefore the decomposition activity (proliferation degree) of microorganisms Need to increase. Therefore, in the present invention, a culture obtained by culturing in a predetermined basidiomycete culture medium is further cultivated in a woody material such as wood flour or sawdust which is a nutrient source of white-rot fungi. The following method is particularly useful as a method for purifying soil contaminated with dioxins.

Specifically, first, the above microorganisms are inoculated into a basidiomycete medium [low nitrogen synthetic medium (Kirk's medium), potato-glucose medium, Sabouraud medium, etc.] and cultured. Cultivation conditions vary depending on the type of wood material used in the subsequent step, etc., but it is recommended to culture at approximately 20 to 30 ° C. for 3 to 14 days.

Then, the obtained culture is inoculated into a woody material such as wood flour or sawdust and cultured. The wood material used is not particularly limited as long as it is usually used for culturing white rot fungi, and general trees such as street trees, garden trees and forests may be used. Specifically, zelkova (Zelkova se
rrata), Yoshino cherry tree (Prunus yedoensis), Weeping willow (Salix babylonica), Osmanthu (Osmanthu)
s fragrans), sasanqua (Camellia sasanqua), black vulgare (Ilex rotunba), coral (Viburunum aw)
abuki), Oomurasaki (Rhododendron pulchrum), Satsuki (Rhododendron indicum), Ginkgo bil
oba), American fuu (Platanus occidentalis), napkin (Sapium sebiferum), camphor tree (Cinnamomun)
camphora), antelope (Pasania edulis), dogfish (Podocarpus macrophylla) and the like. In use, it is recommended that these trees be finely crushed and defatted as necessary to be made into wood powder, wood chips, or the like.

Further, the above wood waste (eg straw, wood waste, sawdust, etc.) can also be used. As with the above wood, these wastes may be finely crushed and degreased.

Here, it is recommended that the mass ratio of the woody material and the culture is generally 100: 1 to 100-50. Further, the mass ratio of the solid matter obtained by summing the above woody material and the culture and the water content in the solid matter is about 1
It is preferably from 00: 200 to 100: 300. Cultivation conditions differ depending on the type of wood material used and the like, but it is generally recommended to culture at 20 to 30 ° C. for 1 to 8 weeks.

The wood material culture thus obtained is
Mix in soil containing aromatic compounds etc., and further culture. Here, the compound concentration in the soil is approximately 1 × 10 ⁻⁸
It is preferable to adjust it to 100 mg / g, and such a soil and the above wood material culture are mass ratio 1: 0.1 to 1.1 (preferably 1: 0.3 to). 0.
With 6), good decomposition activity can be obtained. The culture conditions vary depending on the type of the compound to be decomposed and the like, but it is generally recommended to culture at 20 to 30 ° C. for 1 to 180 days.

The present invention will be described in detail below based on examples. However, the following examples do not limit the present invention, and all modifications and implementations are included in the technical scope of the present invention without departing from the spirit of the above and the following. In the following examples, the microorganism of the present invention is abbreviated as "MAT-1 strain".

[0034]

Examples Example 1: Method for decomposing aromatic compounds in water
Method (1) In this example, the ability to decompose bisphenol A, p-nonylphenol and 17β-estradiol, which are typical aromatic compounds, was evaluated.

First, the liquid medium (15 mL) shown in Table 2 was added to a 50 mL Erlenmeyer flask and sterilized by heating at 120 ° C. for 15 minutes, and then the MAT-1 strain was used as an example of the present invention and a known white color for comparison. Phanerochaete chrysosporium ATCC 3454, a decaying fungus
1 was inoculated respectively and cultured at 30 ° C. for 4 days.

[0036]

[Table 2]

In Table 2, the composition of the trace metal solution is shown in Table 3.
Shown in.

[0038]

[Table 3]

Next, 20 m of each Erlenmeyer flask after culturing
The above three kinds of aromatic compounds adjusted to a concentration of g / L were added, and the mixture was cultured at 30 ° C. with shaking. After culturing for 24 hours, it was separated into a supernatant and cells by centrifugation, and the above compound contained in the cells was extracted with acetonitrile. In the bacterial cell extract thus obtained, and the above compounds contained in the supernatant, quantitative analysis by high performance liquid chromatography (HPLC),
The decomposition rate of each compound was measured. This decomposition rate is the ratio of the compound concentration after treatment with each microorganism to the concentration of each aromatic compound before treatment, expressed as a percentage.

The HPLC measurement conditions are as follows.

Device: Hitachi HPLC System D-7000 Column: Shodex C18-5B (4.6 × 250 mm) Mobile phase: Acetonitrile: Water: Acetic acid = 80: 19: 1
(In the case of bisphenol A and p-nonylphenol) Acetonitrile: water: acetic acid = 70: 29: 1 (17β-
In the case of estradiol) Oven temperature: 25 ° C. Flow rate: 1.0 mL / min Injection amount: 10 μL Detection wavelength: 277 nm (for bisphenol A and p-nonylphenol) 285 nm (for 17β-estradiol) These results are shown in FIG. . From the figure, the microorganism MAT-1 strain of the present invention has an extremely high decomposition rate of 100% against any aromatic compound, which is higher than that of known white-rot fungi.
It can be seen that the resolution is remarkably excellent.

Example 2: A halogenated organic compound in water
Method of Decomposition (Part 2) In this example, the ability to decompose pentachlorophenol, which is a typical organic chlorine compound, was evaluated.

Concretely, the decomposition rate was calculated in the same manner as in Example 1 except that pentachlorophenol was added instead of the above aromatic compound. In addition, the measurement condition of HPLC is that the detection wavelength is 302 nm.
The same as Example 1 except that

The results are shown in FIG. From the figure, it is understood that the microorganism MAT-1 strain of the present invention is extremely excellent in the resolution of pentachlorophenol as compared with the known white-rot fungi.

Example 3: Decomposition of dioxins in soil
Method similar to Example 1 In the same manner as in Example 1, the MAT-1 strain (Example of the present invention) and known Phanerochae chrysosporium (Phanerochae)
techrysosporium) ATCC 34541 (comparative example) was inoculated into the liquid medium shown in Table 2 and cultured at 30 ° C. for 4 days.

6 mL of the culture broth thus obtained and 70 mL of the liquid medium shown in Table 2 were added to and mixed with 30 g of defatted beech wood flour, and cultured at 30 ° C. for 2 weeks.

30 g of the wood flour culture thus obtained
Was added to 100 g of soil containing 470 pg-TEQ / g of dioxins, mixed, and cultured at 30 ° C. for 4 to 6 weeks. For comparison, 30 g of defatted beech wood flour to which the above culture solution was not added was added to soil containing dioxin and mixed, and cultured under the same conditions (control example).

[0048] After completion of the culture, homogenized culture, ¹³
Soxhlet extraction was performed after spiked addition of C-dioxin. The collected organic layer was dehydrated with sodium sulfate, concentrated under reduced pressure, and dissolved in 150 mL of hexane. After this solution is treated with sulfuric acid, washed with water and dehydrated to 1 mL.
It was concentrated under reduced pressure until. Then, after purification with silver nitrate silica gel and activated carbon silica gel, each isomer of dioxins [dibenzoparadioxin (tetrachloride, pentachloride, hexachloride, heptachloride, and octachloride) was analyzed by gas chromatograph mass spectrometry. ); Dibenzofuran (tetrachloride, pentachloride, hexachloride, heptachloride,
And octachloride)] over time, and the dioxin concentration in terms of equivalent toxicity (TEQ) was calculated.

Here, the measurement conditions of the gas chromatograph mass spectrometer (“Autospec” manufactured by Micromass) are as follows.

[Gas Chromatograph Measurement Conditions] HP5890 Series II (1) When the measurement sample is tetra-hexachlorinated dibenzoparadioxin and tetra-hexachlorinated dibenzofuran Separation column: CP-Sil 88 [60 m × 0.25 mm ( ID) 0.10μ
m] Column temperature: 100 ℃ (2min) → (10 ℃ / min) → 190 ℃ (0min)
→ (3 ℃ / min) → 250 ℃ (20min) Carrier gas: Helium (1.10ml / min) Injection temperature: 250 ℃ Injection method: Splitless (1min) (2) The measurement sample is 7 to 8 chlorinated dibenzopara For dioxin and 7-octachlorinated dibenzofuran Separation column: DB17 [30m × 0.25mm (ID) 0.25μm] Column temperature: 100 ℃ (2min) → (30 ℃ / min) → 220 ℃ (0min) →
(10 ℃ / min) → 280 ℃ (15min) Carrier gas: Helium (1.12ml / min) Injection temperature: 270 ℃ Injection method: Splitless (1min) [Mass spectrometer condition] VG AutoSpec-Ultmima Ionization method: EI Ionization energy: 35eV Ionization current: 500μA Ion source temperature: 260 ° C Ion acceleration voltage: 8kV Resolution: 10,000 These results are shown in Figs.

Of these, FIG. 3 shows the decomposition rate of each dioxin isomer at 6 weeks after culturing. From FIG. 3, it can be seen that when the strain MAT-1 of the present invention is used, it has a high decomposition rate of about 50 to 70% with respect to any of the isomers and exhibits extremely excellent resolution.
On the other hand, when a known white-rot fungus was used, all the isomers had an extremely low decomposition rate of 10% or less. When only defatted beech wood flour was used, the resolution was zero for all isomers (hence not shown in the figure).

FIG. 4 shows the time-dependent change in the concentration (total concentration) of dioxins used in this example. From FIG. 4, when the known white-rot fungus is used, almost no decomposition of dioxins is observed, whereas when the strain of the present invention is used, the dioxin concentration is about ½ after 4 weeks of culture. To 6 weeks after culturing,
It can be seen that it is reduced to about ⅓, indicating a good decomposition pattern.

From the above results, the microorganism of the present invention becomes a social problem as a hardly decomposable harmful substance regardless of the type of aromatic compound or halogenated organic compound, as compared with known white-rot fungi. It has been confirmed that even dioxins that are present have excellent resolving power regardless of their existence form (in water, in soil, etc.).

[0054]

EFFECT OF THE INVENTION The microorganism of the present invention exhibits extremely excellent resolution with respect to dioxins, any aromatic compounds and halogenated organic compounds. Especially known white-rot fungi have a low decomposition rate under normal air conditions,
Even if the culture conditions such as increasing the oxygen concentration were controlled, the resolution was still insufficient, whereas according to the present invention,
Regardless of the existence form in water or soil, extremely high resolution can be obtained under normal air conditions (culture conditions),
It is extremely useful in industry.

[Brief description of drawings]

FIG. 1 is a graph showing the decomposition rate of Example 1 (aromatic compound in water).

FIG. 2 is a graph showing the decomposition rate of Example 2 (pentachlorophenol in water).

FIG. 3 is a graph showing the decomposition rate of each isomer of dioxins in Example 3.

FIG. 4 is a graph showing the time course of dioxins concentration in Example 3.

Claims (2)

[Claims] 1. A white-rot fungus FERM P-18 excellent in decomposing aromatic compounds and / or halogenated organic compounds.
828. 2. The white-rot fungus FERM P- according to claim 1, wherein the halogenated organic compound is a dioxin.
18828.