JP4247395B2 - Degradable bacteria of hardly degradable substances and environmental purification method using the same - Google Patents

Description

  The present invention relates to a novel white rot fungus FERM P-20326 strain which is excellent in resolution of persistent substances such as aromatic compounds and / or halogenated organic compounds. The microorganism of the present invention is extremely excellent in the resolution of hardly decomposable substances, and can also detoxify harmful compounds such as dioxins. Moreover, the colored waste water containing dye etc. can be decolored or the dye can be decomposed. For this reason, various treatments for decomposing or detoxifying the hardly decomposable substance including treatment of waste water containing these hardly decomposable substance and bioremediation (biological repair) for the hardly degradable substance are included. It can be widely used in various technical fields.

  In recent years, environmental pollution by various harmful chemical substances has become a problem. In particular, environmental pollution caused by dioxins and endocrine disrupting substances has become a serious problem worldwide because they have a serious impact on the ecosystem. Dioxins are said to be the strongest toxicant ever produced by mankind, and exhibit strong toxicity such as carcinogenicity, liver damage, skin damage, and teratogenicity. In addition to being chemically stable and difficult to decompose, it is highly fat-soluble and easily accumulates in living tissues, so it has a great impact on human beings at the top of the food chain. In addition, endocrine disrupting substances such as nonylphenol affect the reproductive functions of wildlife, and the effects can extend for generations. Furthermore, the treatment of colored wastewater containing dyes and the like has also become a problem. Since dyes and the like are designed in consideration of the stability after dyeing, they are not only chemically stable and difficult to be decomposed, but also have many harmful effects on living organisms. For this reason, various treatment methods such as a physicochemical method and a biological method have been studied as a method for decomposing these hardly decomposable harmful compounds.

  As physicochemical methods, high temperature melting method, thermal decomposition method, alkali treatment method, supercritical water decomposition method, catalytic oxidation method, ozonolysis method, etc. have been studied so far. It has problems such as high cost. Biological treatment methods using microorganisms generally have lower energy and lower cost than these, but have not been put into practical use because of low treatment efficiency. Therefore, it is considered that a practical environmental remediation technique can be provided by using microorganisms with excellent resolution, and wood decay fungi are expected. Wood decay fungi are attracting attention as microorganisms that have the ability to resolve various environmental pollutants, and white decay fungi that are a kind of wood decay fungi are oxidoreductases (lignin peroxidase, manganese peroxidase, It is known that lignin, which is a natural hardly decomposable substance, is decomposed by laccase or the like. Furthermore, it has also been reported that dioxins having a chlorine substitution number of 4 or more can be decomposed by white rot fungi belonging to the genus Funerokete (Non-patent Documents 1 and 2).

For this reason, more than 1,500 kinds of white-rot fungi have been isolated and reported so far, but the application to the treatment of harmful substances is Phanerochaete chrysosporium , Pleurotus ostreatus , Coriolis・ Limited to several types, such as Corsilus versicolor and Bjerkandera adusta . Of these, Funeroquite chrysosporium, which is the most researched, was isolated in the United States and is designated as an import quarantine harmful fungus in Japan. In addition, these white-rot fungi are generally active in the secretion of degrading enzymes in a low nutrient state, and the productivity of secreted enzymes is low in a state of rich nutrition and active growth. For this reason, it is difficult to secrete a large amount of degrading enzymes. Therefore, in order to use white rot fungi for environmental restoration in Japan, those isolated in Japan are required, and microorganisms with high productivity of degrading enzymes in an abundant nutritional state capable of active growth. Separation was desired.
Bumpus et al., Science, 228, 1434 (1985) Takada et al., Appl. Environ. Microbiol, 62, 4323 (1996)

  The present invention has been made in view of the above circumstances, and its purpose is to efficiently decompose hardly decomposable substances that are harmful substances such as aromatic compounds, halogenated organic compounds, and dyes in Japan. The object is to provide a new isolated microorganism. Another object of the present invention is to provide a method for degrading a hardly decomposable substance using the microorganism and purifying the environment.

In order to achieve the above-mentioned problems, the present inventors have searched for new microorganisms that can exert a desired action from microorganisms that exist widely in nature. As a result, microorganisms separated from decayed wood exert their intended actions. As a result, the present invention has been completed. That is, the present invention is as follows.
(1) White rot fungus FERM P-20326 strain.
(2) A method for degrading a hardly decomposable substance using the white rot fungus described in (1) above.
(3) The method according to (2) above, wherein the hardly decomposable substance is at least one selected from the group consisting of an aromatic compound, a halogenated organic compound and a dye.
(4) The method according to (2) or (3) above, wherein Mn ²⁺ and / or organic nitrogen is added to the medium.
(5) The method for cultivating white rot fungus according to (1) above, wherein Mn ²⁺ and / or organic nitrogen is added to the medium.
(6) A hardly decomposable substance decomposing agent comprising the white rot fungus according to (1) and / or a culture solution of the white rot fungus as an active ingredient.

  The microorganism of the present invention exhibits extremely excellent resolution with respect to any hardly decomposable substance such as dioxins, aromatic compounds and halogenated organic compounds. In particular, the known white-rot fungi have a low degradation rate under normal nutrient conditions, and the resolution is still insufficient even when using a low nutrient medium or the like. It is extremely useful industrially in that extremely high resolution can be obtained (under culture conditions), and can be used significantly for environmental purification.

  The present invention is described in detail below.

<Physiological and ecological properties>
First, the microorganism of the present invention has the following mycological properties. The following properties were confirmed using a potato dextrose agar medium or a potato dextrose liquid medium.
(I) Growth pH range (30 ° C, 7 days culture)
It grows at pH 3-8 and does not grow at pH 2 and pH 10. A preferred pH range is pH 4-7, more preferably around pH 5-6.5.
(II) Growth temperature range (pH 6, 7 days culture)
It grows around 10-40 ° C and does not grow at 45 ° C. A preferred temperature range is 20 to 35 ° C, more preferably around 25 to 35 ° C.
(III) Characteristics of the flora White and felt-like in a potato dextrose agar medium.
(IV) Features of mycelia It has a grazing connection (clamp connection).
(V) Secretes bacteria versus exocrine enzyme manganese peroxidase and trace amounts of lignin peroxidase.

  Due to the above bacteriological properties, the strain of the present invention is considered to be a white rot fungus belonging to the basidiomycete, but has not been identified as a known fungus. Furthermore, as will be apparent from the examples described later, the microorganism of the present invention has a particularly excellent ability to decompose dioxins and the like, and such an excellent decomposition ability is not found in conventional microorganisms. Therefore, the strain of the present invention was recognized as a new strain and named L-25 strain, and the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st East, 1st Street, Tsukuba City, Ibaraki Prefecture, 6th) Deposited (date of deposit: December 16, 2004, deposit number: FERM P-20326).

<Resistant material>
The microorganism of the present invention has extremely excellent resolution with respect to various naturally or synthetic hardly degradable substances that are known to be hardly degradable in the environment. Examples of the hardly decomposable substance include aromatic compounds, halogenated organic compounds, and dyes. In the present invention, all halides are included in the “halogenated organic compound”, and “aromatic compound” means an aromatic compound that is not halogenated.

  Here, as the above “aromatic compound”, any compound having an aromatic ring is included, except for halogenated compounds, and any kind of simple ring or heterocyclic ring may be used. Among these, as the monocyclic ring, benzenes having a substituent such as benzene and nitrobenzene; alkylphenols such as phenol, nitrophenol, nonylphenol, octylphenol and pentylphenol; catechol; dimethyl phthalate, diethyl phthalate, dibutyl phthalate, Phthalic acid esters such as diheptyl phthalate and dioctyl phthalate; pyrenes such as naphthalene, anthracene, pyrene, benzopyrene and dipentopyrene; bisphenol compounds such as bisphenol A; and estradiol. In addition to carbon, examples of the heterocyclic ring include rings containing one or more heteroatoms such as N, 0, and S. For example, aromatic compounds such as pyridine, pyrimidine, furan, thiophene, and pyrrole; these related compounds Is included. Mixtures of monocyclic and heterocyclic rings are also included within the scope of the above “aromatic compound”, and polymer raw materials having aromatic rings and decomposition products (oligomers, partial decomposition products, etc.) thereof are also included. .

  The “halogenated organic compound” is not particularly limited as long as it is an organic compound having at least one kind of fluorine, chlorine, bromine, and iodine. Vinyl chloride-based and vinylidene chloride-based organic chlorine compounds; Teflon (registered trademark), Fluorine compounds such as chlorofluorocarbons can be used. Among the “halogenated organic compounds”, dioxins typified by PCDDs (polychlorinated dipenzo-p-dioxins) and PCDFs (polychlorinated dipenzofurans); dioxins containing bromine instead of chlorine in the dioxins PCBs (polychlorinated biphenyls) including coplanar PCB, chlorobenzene, chlorophenol and the like are also included.

The above "dyes" include direct dyes, acid dyes, acid mordant dyes, cationic dyes, vat dyes, disperse dyes, reactive dyes, basic dyes, sulfur dyes, naphthol dyes, oxidation dyes, oil-soluble dyes, metal complex hydrochloric acid properties Dyes and the like, specifically, azo structure, anthraquinone structure,
And organic compounds having a carbonium structure, a quinoneimine structure, a phthalocyanine structure, a stilbene structure, a pyrazolone structure, an acridine structure, an indigoid structure, a methine structure, a thiazole structure, and the like. Examples of the organic compound having an azo structure include orange II and Congo red. Examples of the organic compound having an anthraquinone structure include sren brown R and remazole brilliant blue R.
Etc.

  Examples of the environment containing the persistent substance include, but are not limited to, soil such as farmland, factory ruins, and incinerators, and water environments such as rivers, industrial water, groundwater, and drainage.

<Method of decomposing persistent materials>
The microorganism of the present invention can degrade hardly decomposable substances contained in each of the above environments. Hereinafter, a method for degrading a hardly decomposable substance using the microorganism of the present invention will be described.
In order to degrade the aforementioned hardly decomposable substance, the microorganism of the present invention itself, that is, L-25 itself may be used, or a culture solution containing L-25 may be used.
The temperature when L-25 is precultured is 15 to 40 ° C, preferably 20 to 35 ° C. The pH during culture is in the range of 3-8, preferably 4-7. The culture days are not particularly limited as long as the growth of bacteria and the extracellular enzyme are sufficiently produced, but are usually 1 to 30 days, preferably 1 to 20 days.
In order to promote the production of manganese peroxidase, it is preferable to add a manganese source, an organic nitrogen source, moss and the like to the medium used for the culture. The manganese source to be added is not particularly limited as long as it contains Mn ^2+, and examples thereof include manganese chloride and manganese sulfate. Examples of the organic nitrogen source to be added include polypeptone, bactopeptone, yeast extract, and tryptone.
Furthermore, rice straw and wheat straw are preferable as cocoons to be added, but are not limited thereto.
The amount of manganese source added is such that the Mn ²⁺ concentration in the medium is 0.001 mM to 5.0 mM, preferably 0.005 mM to 2.0 mM. The organic nitrogen source is usually added to the medium in an amount of 0.01 g / L to 50 g / L, preferably 0.1 g / L to 30 g / L. The amount of moss added is 1 g / L to 100 g / L, preferably 5 g / L to 50 g / L in the medium.

  The specific decomposition method is as follows. 1. When dissolved / suspended in water or the like; Since it is slightly different from the case where it is adsorbed on solid matter such as soil and ash (hereinafter, it may be represented by “soil”), it will be described separately for each case.

1. Method of Decomposing Solution / Suspension of Refractory Substance First, L-25 is inoculated into a basidiomycete medium (low nitrogen synthesis medium (Kirk medium), potato / glucose medium, etc.) and cultured. The hardly decomposable substance to be decomposed may be added at the start of the culture, or may be added after culturing for about one week. The concentration of the hardly decomposable substance at this time is preferably adjusted so as to be approximately 1 × 10 ^{−6 to} 200 mg / L. Moreover, although culture | cultivation conditions differ also with the kind etc. of a hardly decomposable substance, generally culturing at 20-30 degreeC for 1 to 30 days is recommended.

2. Method of degrading persistent substances in soil Since persistent substances in soil are harder to decompose than persistent substances in solution / suspension, it is necessary to increase the degrading activity (growth degree) of microorganisms. There is. For this reason, in the present invention, a culture cultured in a predetermined basidiomycete medium is further cultivated with a material such as koji, which is a production promoter for degrading enzymes.

  The following method is particularly useful as a method for purifying soil contaminated with dioxins.

  Specifically, first, L-25 is inoculated into a basidiomycete medium (low nitrogen synthesis medium (Kirk's medium), potato / glucose medium, etc.) and cultured. Although the culture conditions vary depending on the type of sputum material used in the subsequent steps, it is recommended that the culture be performed at 20 to 30 ° C. for 3 to 14 days.

  Next, the obtained culture is inoculated into a material such as cocoon and cultured. The straw to be used is not particularly limited, and common straw such as rice straw or wheat straw may be used. Specifically, in use, it is recommended to chop or crush these ridges to make chips or the like as necessary.

  Here, it is recommended that the mass ratio of the koji material to the culture is generally 100: 1 to 100: 50. Moreover, it is preferable that mass ratio of the solid substance which added the said woody material and culture and the water | moisture content contained in this solid is about 100: 200-100: 300 in general. Although the culture conditions vary depending on the type of wood material used, etc., it is generally recommended to culture at 20-30 ° C. for 1-8 weeks.

The koji culture obtained in this way is mixed in soil containing aromatic compounds and further cultured. Here, the compound concentration in the soil is preferably adjusted so as to be approximately 1 × 10 ^{−8 to} 200 mg / g, and such a soil and the straw culture are 1: If it is 0.1 to 1.1 (preferably 1: 0.3 to 0.6), good decomposition activity can be obtained. Although the culture conditions vary depending on the type of the compound to be decomposed, etc., it is generally recommended to culture at 20 to 30 ° C. for 1 to 180 days.

<Method for producing manganese peroxidase>
Furthermore, this invention includes the method of producing manganese peroxidase using L-25. Specifically, 100 mL of a potato dextrose liquid medium was added to a 500 mL baffled Erlenmeyer flask, inoculated with the L-25 strain, and cultured at 25-35 ° C. at 100-250 rpm for about 3 weeks in the medium. A significant amount of manganese peroxidase is secreted. By adding Mn ²⁺ to the medium in an amount of 0.001 mM to 2.0 mM, preferably 0.005 mM to 1.0 mM, the secretion amount of manganese peroxidase increases dramatically. The manganese source to be added is not particularly limited as long as it contains Mn ²⁺ such as manganese chloride and manganese sulfate. In addition, the amount of manganese peroxidase secreted by adding 0.01 g / L to 50 g / L, preferably 0.1 g / L to 30 g / L, of an organic nitrogen source such as polypeptone, bactopeptone, yeast extract, or tryptone to the medium. Will increase dramatically. The organic nitrogen source to be added is preferably a nitrogen source comprising a protein degradation product, but is not limited thereto. Furthermore, the secretion amount of manganese peroxidase is increased by adding moss to the medium at 1 g / L to 100 g / L, preferably 5 g / L to 50 g / L. As rice cakes, rice straw and wheat straw are preferable, but not limited thereto.

  EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

Example 1: Separation of white rot fungi from decayed wood decayed wood was collected from forests in Fukui, Ishikawa and Hokkaido. The mycelium was cut out from the decayed portion of the collected wood and inoculated on the surface of the separated agar medium shown in Table 1. 360 samples were inoculated and cultured at 30 ° C. for 7 days. A colony having an orange color around the grown colony was isolated as a fungus having lignin-degrading activity. The isolated bacteria were streaked on a separate agar medium, and 62 strains were isolated. The isolated strain was named L-1 to 62 strain.

  Furthermore, the isolated strain was cultured on a separated agar medium at 30 ° C. for 5 days, and the productivity of the degrading enzyme was compared from the area of the discolored region. Coriolus hirsutus IFO 4917 was also cultured for comparison. The L-25 strain, which had the largest color change region among the isolated strains, exhibited a color change region 1.3 times that of the comparative control Coriolus hirsutus IFO 4917. From this result, the L-25 strain was selected as a strain with high productivity of degrading enzymes.

Example 2: Manganese peroxidase production Manganese peroxidase was produced by the shaking culture method using the medium shown in Table 2.

The activity of the obtained manganese peroxidase was measured by the following method.
Manganese peroxidase reaction solution: After dissolving 4.45 g of disodium malonate and 0.25 g of manganese sulfate pentahydrate in 400 mL of distilled water, the pH is adjusted to 4.5 to make the total amount 500 mL.
100 μL of the measurement sample was added to 2.8 mL of the above manganese peroxidase reaction solution, and the mixture was kept at 25 ° C. for 5 minutes, and then 100 μL of 0.1 mM hydrogen peroxide was added to start the reaction. The activity of manganese peroxidase was calculated from the change in absorbance at 270 nm of the solution after the start of the reaction (due to the chelate of Mn ³⁺ and malonic acid).

The result of comparing the maximum activity value of the obtained manganese peroxidase with the previously reported literature is shown in FIG. The activity of manganese peroxidase was measured as follows, and 1 U of enzyme activity was defined as the amount of enzyme that oxidizes 1 μmol of Mn ²⁺ to Mn ³⁺ per minute. Note that the values of the previously reported documents shown in FIG. 1 are corrected in consideration of the difference in measurement method.
As shown in FIG. 1, it was revealed that the manganese peroxidase productivity of the L-25 strain was dramatically higher than that of the previously reported manganese peroxidase-producing bacteria.

Example 3 Decomposition of Aromatic Compound in Water In this example, the resolution of bisphenol A, p-nonylphenol, which is a typical aromatic compound, was evaluated. First, a liquid medium (100 mL) containing bisphenol A shown in Table 3 was added to a 500 mL Erlenmeyer flask, sterilized by heating at 121 ° C. for 15 minutes, and then the L-25 strain was inoculated, followed by shaking culture at 30 ° C. and 150 rpm. For 21 days.

The culture solution was appropriately collected and separated into a supernatant and cells by centrifugation, and the supernatant was quantitatively analyzed by high performance liquid chromatography (HPLC). Moreover, after culturing, all the cultures were centrifuged to separate the cells and supernatant, and then the cells were crushed in a mortar and extracted with ethyl acetate. The ethyl acetate solution after extraction was concentrated under reduced pressure using an evaporator, dissolved in methanol, and bisphenol A contained in the cells was quantified by HPLC. As shown in FIG. 2, the concentration of bisphenol A in the culture broth decreased to below the detection limit. Further, bisphenol A was not detected from the cultured cells.
Next, the resolution of nonylphenol was similarly evaluated using the medium shown in Table 4. As shown in FIG. 3, the nonylphenol concentration decreased below the detection limit. Further, nonylphenol was not detected from the cultured cells.

Here, the measurement conditions of HPLC are as follows.
Column: RP-18GP manufactured by Kanto Chemical (4.6 x 150mm)
Mobile phase: methanol: water = 70:30 (bisphenol A)
Methanol: water = 80:20 (nonylphenol)
Column temperature: 40 ° C
Flow rate: 1.0mL / min
Injection volume: 20μL
Peak detection: UV detector (270nm, bisphenol A)
Fluorescence detector (excitation 225nm, fluorescence 300nm, nonylphenol)
From these results, it can be seen that the microorganism L-25 strain of the present invention has an extremely high degradation rate of 100% with respect to any aromatic compound and exhibits excellent resolution.

Example 4: Decomposition of dyes in water In this example, the resolution of 17 representative dyes used in the dyeing industry was evaluated. Specifically, it was cultured for 2 weeks in the same manner as in Example 1 except that the dye was added after heat sterilization. The dye concentration was calculated from the absorbance at the maximum absorption wavelength of each dye.

  The results are shown in Table 6.

  As shown in Table 6, a degradation rate of 98% or higher was achieved for all the dyes studied. In the visual observation, the culture process is as follows. 1) The inoculated microorganism grows. 2) The grown microorganisms are colored by the dye. 3) The color of the dye disappears from the culture solution. 4) Colors of microorganisms that have been colored disappear. From these observation results, the mechanism of the decolorization of the dye by the L-25 strain is presumed that the dye is first adsorbed to the microorganism or the dye is taken into the microorganism, and then the dye is decomposed by the microorganism. Some microorganisms that decompose only specific dyes have been reported so far, but few microorganisms that can decompose many kinds of dyes such as L-25 strain have been reported. Usually, since many kinds of dyes are mixed in actual dye wastewater, the L-25 strain is considered to be effective for the treatment of actual wastewater.

Example 5: Decomposition of dioxins In the same manner as in Example 1, the L-25 strain was inoculated into a potato dextrose liquid medium and cultured at 30 ° C for 7 days. To 100 mL of this culture solution, about 10,000 ng of 1,3,6,8-tetrachlorodibenzo-p-dioxin (1,3,6,8-TeCDD), about 2000 ng of octachlorodibenzo-p-dioxin (OCDD), 1, About 10000 ng of 2,7,8-tetrachlorodibenzofuran (1,2,7,8-TeCDF) and about 6000 ng of octachlorodibenzofuran (OCDF) were added and cultured with shaking at 30 ° C. and 150 rpm for 1 to 3 weeks.
After completion of the culture, concentrated sulfuric acid was added to the culture and extracted with n-hexane. The collected organic layer was dehydrated and concentrated under reduced pressure. This was spiked with ¹³ C-dioxin, concentrated and cleaned up by various column chromatography. This was analyzed using a gas chromatograph mass spectrometer (GC-MS).
The result of decomposition of dioxins is shown in FIG. FIG. 4 shows the concentration of each dioxin isomer in the first to third week of culture. As shown in FIG. 4, the strain L-25 of the present invention usually exhibits high degradability even with respect to 8-chlorinated products (OCDD, OCDF) which are difficult to decompose, and about 50 to about any isomer. It can be seen that it has a high decomposition rate of 80%. Further, the toxic equivalent amount decreased from 11 ng-TEQ / flask at the start of degradation to 4.2 ng-TEQ / flask after 3 weeks of culture. From these results, it can be seen that the L-25 strain exhibits extremely excellent resolution.

  From the above results, the microorganism of the present invention has significantly higher productivity of manganese peroxidase than known white-rot fungi, regardless of the type of aromatic compound / halogenated organic compound, and a hardly decomposable harmful substance. As a result, it has been confirmed that it exhibits extremely excellent resolution even for dioxins that are socially problematic.

FIG. 1 is a graph comparing the productivity of manganese peroxidase with previously reported microorganisms in Example 2. FIG. 2 is a graph showing the change over time of bisphenol A decomposition in Example 3. FIG. 3 is a graph showing the time course of nonylphenol decomposition in Example 3. 4 is a graph showing the decomposition of each isomer of dioxins in Example 5. FIG.

Claims (5)

White rot fungus FERM P-20326 strain having the following mycological properties (1) to (5) :
(1) grows at pH 3-8 under conditions of 30 ° C. and 7 days culture, and does not grow at pH 2 and pH 10;
(2) grows at 10-40 ° C. under pH 6 for 7 days and does not grow at 45 ° C .;
(3) The flora is white and felt-like in potato dextrose agar;
(4) the mycelium has a grazing connection; and
(5) Secretes manganese peroxidase and lignin peroxidase . A method for decomposing at least one hardly decomposable substance selected from the group consisting of an aromatic compound, a halogenated organic compound and a dye , using the white rot fungus according to claim 1. The method according to claim 2, wherein at least one organic nitrogen source selected from the group consisting of Mn ²⁺ and / or polypeptone, bactopeptone, yeast extract, and tryptone is added to the medium. The culture of white rot fungus according to claim 1, wherein at least one organic nitrogen source selected from the group consisting of Mn ²⁺ and / or polypeptone, bactopeptone, yeast extract and tryptone is added to the medium. Method. Decomposing at least one of hardly decomposable substance white rot fungi and / or aromatic compounds to the white rot active ingredient of the culture solution of bacteria, selected from the group consisting of halogenated organic compounds and dyes described in claim 1 agent for.

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