JP5648838B2 - Novel microorganisms with petroleum resolution - Google Patents

Description

  The present invention relates to a novel microorganism having an oil resolving power and a method for purifying an oil-contaminated environment using the microorganism. The novel microorganism of the present invention is capable of degrading polycyclic aromatic compounds (PAHs), which are hardly decomposable substances in petroleum, under both the marine and soil environments contaminated with petroleum, It is preferably used for decomposing polycyclic aromatic compounds that have flowed out or leaked into the ocean.

  In recent years, damage caused by oil pollution has become serious, marine pollution due to grounding accidents of crude oil tankers, etc .; soils in and around the site of oil refineries, stockpiling bases, coal tar manufacturing plants, coal processing plants, etc. Examples include contamination. Petroleum and crude oil contain components that are easily decomposed naturally, such as low-boiling compounds and compounds with relatively high hydrophilicity, but also include components that are extremely difficult to decompose, such as polycyclic aromatic compounds (PAHs). Yes. Polycyclic aromatic components are problematic because they remain in the environment for a long time and adversely affect living organisms.

  Various chemical or physical treatment methods such as an adsorption method using activated carbon, a thermal decomposition method by heating, an insolubilization treatment method of contaminated soil, and the like as environmental purification methods contaminated with persistent petroleum components There is. However, in recent years, development of a treatment method (bioremediation) using microorganisms that can be carried out at a lower cost under milder conditions and is superior from the viewpoint of energy saving has been actively developed.

  For example, Patent Document 1 discloses TY-8 strains belonging to the order of Polyporales and TY-16 strains belonging to the family Hapalopilaceae as microorganisms capable of efficiently degrading polycyclic aromatic compounds in both liquid and soil environments. Yes. The above document discloses that these microorganisms can degrade polycyclic aromatic compounds in a liquid medium, but have not yet demonstrated that the compounds contained in seawater can be degraded.

  Non-patent documents 1 to 3 disclose a method for decomposing a polycyclic aromatic compound in soil using a microorganism. Among them, Non-Patent Documents 1 and 2 disclose methods for treating an extract of contaminated soil or a suspension of contaminated soil with bacteria such as Arthrobacter spp. Or Acidovorax spp. , A method for purifying petroleum components by a sphingomonas genus or the like is disclosed.

JP-A-2005-46048

H.I. ATAGANA et al., Water, Air, and Soil Pollution, Vol. 141, pp. 233-246 (2002) Mikael Eriksson et al., Applied and Environmental Microbiology, Vol. 69, No. 1, pp. 275-284 (2003) Marc Vinas et al., Applied and Environmental Microbiology, Vol. 71, No. 11, pp. 7008-7018 (2005)

  As described above, several methods for degrading polycyclic aromatic compounds in petroleum by using microorganisms and purifying a petroleum-contaminated environment have been disclosed. However, for example, Non-Patent Documents 1 to 3 are methods for mainly purifying soil contamination, and no mention is made of marine contamination. Non-Patent Documents 1 and 2 are purification technologies based on pretreatment such as extraction of petroleum components and slurrying of contaminated soil, and the range and amount of soil contaminated with petroleum components are enormous. Is not practical enough. Non-Patent Document 3 is a technology that can purify contaminated soil even in a solid state, but has a poor decomposition efficiency, such as having a long time of about 6 months or more until the polycyclic aromatic compound can be sufficiently purified. Yes. On the other hand, Patent Document 1 does not disclose an example in which a polycyclic aromatic compound in seawater is decomposed.

  Moreover, it is not disclosed that any microorganism of the above-mentioned document can grow in seawater.

  The present invention has been made in view of the above circumstances, and an object of the present invention is to efficiently remove the ocean or soil contaminated with petroleum components such as polycyclic aromatic compounds, which are hardly decomposable compounds, in a relatively short time. The object is to provide a purification technique using a novel microorganism that can be purified.

  The novel microorganism of the present invention that has solved the above problems is the F092 strain (NITE P-795) belonging to the genus Fusarium that has petroleum resolving power. The above microorganisms are particularly excellent in the resolution of polycyclic aromatic compounds, and are extremely useful as purifiers for petroleum-contaminated environments.

  In addition, the petroleum decomposing agent composition (petroleum pollution environment purifying agent composition) of the present invention that has solved the above problems has a gist in that it contains the above-described microorganisms and a substrate.

  Moreover, the purification method of the petroleum polluted environment which concerns on this invention which could solve the said subject has a summary in the place which adds said microorganisms or said petroleum decomposer composition to a petroleum polluted environment.

  In a preferred embodiment of the present invention, the contaminated environment is seawater or soil.

  In a preferred embodiment of the present invention, the purification method further comprises adding a nutrient source for the microorganism and / or a surfactant.

  The novel microorganism F092 strain of the present invention purifies the ocean and soil (oil-contaminated environment) contaminated with petroleum components such as polycyclic aromatic compounds in a relatively short time without slurrying or extracting the petroleum components. can do. In addition, since the novel microorganism F092 strain of the present invention can grow in seawater, it is possible to decompose and purify oil components (typically crude oil, heavy oil, etc.) spilled or leaked into seawater on the spot. . Therefore, according to the present invention, it is possible to efficiently purify enormous and vast environmental pollution such as marine pollution due to a grounding accident of a crude oil tanker, soil pollution such as a factory site using oil, It is extremely useful in industry.

  The present inventors have searched for new microorganisms that can efficiently decompose difficult-to-decompose substances in petroleum components typified by polycyclic aromatic compounds and the like to purify petroleum-contaminated environments. As a result, the F092 strain, which belongs to the genus Fusarium and is extremely excellent in the resolution of petroleum components (that is, the ability to purify the petroleum-contaminated environment), was found and the present invention was completed.

  This new microorganism is particularly suitable for the purification of marine environmental pollution, as shown in the examples below, which can grow in seawater and can decompose persistent substances such as polycyclic aromatic compounds in a short time. ing. In addition, if this new microorganism is used, it is not necessary to perform a pretreatment such as slurrying petroleum-extracted soil or extracting pollutants as in the technique described in the above-mentioned non-patent document, Soil can be purified simply by spraying microorganisms.

  First, the novel microorganism strain F092 of the present invention will be described in detail.

The F092 strain is deposited at the depository as follows.
(I) Name and address of depositary institution Name: National Institute of Technology and Evaluation, Patent Microorganism Depositary Center Address: 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan
(Ii) Date of deposit: August 13, 2009 (iii) Receipt number: NITE AP-795
(Iv) Accession number: NITE P-795

The F092 strain was cultured in potato-glucose (potato glucose) agar medium (pH 5.6 before sterilization, sterilized at 121 ° C. for 20 minutes) at 25 ° C. for 5 to 7 days. Is as follows. That is, a white colony is formed, a fiaro-type multicellular, spindle-to-crescent-shaped conidia (short axis × long axis is about 1-2 μm × 20-25 μm) and a single-cell small conidia (The minor axis × diameter is approximately 1-2 μm × 3-4 μm). From the above morphological characteristics, the F092 strain was identified as a bacterium belonging to the genus Fusarium in light of the following references.
Tsuneo Watanabe “Soil filamentous fungi-Searching and morphology of cultured strains”, Soft Science, 26-27, and 221-225 (1993)

  Optimal growth conditions for the F092 strain are pH 5.5 to 8.6, temperature 25 to 30 ° C, and growth range is pH 4 to 9 and temperature 10 to 35 ° C.

  When the F092 strain was cultured in the above potato-glucose liquid medium, 1,2-dioxygenase activity, 2,3-dioxygenase activity, laccase (Lac) activity, and manganese veroxidase (MnP) activity were observed in the medium. It was. Therefore, at least one of these enzymes may be involved in the resolution of petroleum components by the F092 strain.

  When the F092 strain was cultured at 20 ° C., 25 ° C., 30 ° C., and 35 ° C., it grew at any temperature, but the best growth was at 30 ° C. Accordingly, the temperature at which the F092 strain is cultured and the temperature at which the petroleum component is decomposed by the F092 strain are preferably about 20 ° C. or more and 35 ° C. or less.

  The F092 strain of the present invention is excellent in the resolution of petroleum components, but in particular, it has been experimentally confirmed that chrysene, which is a polycyclic aromatic compound that does not have a substituent and is extremely difficult to decompose, can be decomposed efficiently. (See examples below). Moreover, according to the knowledge by the present inventors, the F092 strain actively expresses 1,2-dioxygenase and the like. The F092 strain oxidizes chrysene with these enzymes and cleaves its carbon-carbon bond, leading to a benzene compound having a hydrophilic group such as a hydroxyl group or a carboxyl group, and eventually decomposing into carbon dioxide and water. Is done. Therefore, F092 strain is considered to be capable of degrading polycyclic aromatic compounds similar to chrysene, such as tentalene, indene, naphthalene, azulene, fluorene, phenalene, phenanthrene, anthracene, triphenylene, pyrene, benzopyrene, naphthacene, and perylene. In addition, it is considered that the F092 strain can decompose compounds such as benzene, alkylphenols, halogenated phenols, bisphenols, and dioxins in the same manner.

  In addition, the petroleum component which F092 stock | strain can decompose | disassemble is not limited to the thing derived from a crude oil or a petroleum origin, if it can be contained in a crude oil or petroleum. For example, polycyclic aromatic compounds such as chrysene are also generated by wildfires. On the other hand, such polycyclic aromatic compounds are not easily decomposed naturally. Therefore, if the petroleum component which should be processed by this invention is a component contained in crude oil or petroleum, the origin shall not be ask | required. In general, oil before refining is particularly called crude oil, but in the present invention, oil and crude oil are not particularly distinguished. Crude oil includes petroleum products using crude oil (for example, heavy oil, gasoline, kerosene, light oil, etc.). In the examples described later, C heavy oil was used. C heavy oil is three kinds of heavy oil classified according to kinematic viscosity, and 90% or more is residual oil in addition to light oil.

  Since the F092 strain of the present invention can efficiently decompose polycyclic aromatic compounds in petroleum components, it can be suitably used for purification treatment of petroleum-contaminated environments regardless of whether it is a seawater-contaminated environment or a soil-contaminated environment.

  In particular, the F092 strain of the present invention can grow in seawater and is suitably used for purification of seawater-contaminated environment. According to the present invention, it is possible to efficiently decompose contaminated seawater containing the above-described various petroleum components at high concentrations. The concentration of petroleum-contaminated seawater that can be treated according to the present invention is such that the F092 strain is preferably used in combination with a nutrient source or a surfactant, which will be described later, agitation culture is performed, or the culture period of the strain is increased. The contaminated seawater of about 15,000 to 50,000 ppm can be processed.

  Moreover, the F092 strain of the present invention is also suitably used for purification of soil-contaminated environment. According to the present invention, the F092 strain is added to the oil-contaminated soil and the F092 strain is brought into contact with the oil-contaminated soil without the slurry of the oil-contaminated soil or the extraction of the petroleum components in the contaminated soil as in the prior art. The petroleum component can be decomposed with (see Examples described later).

  And according to this invention, the contaminated soil which contains the various petroleum component mentioned above in high concentration can be decomposed | disassembled efficiently. The concentration of petroleum-contaminated soil that can be treated by the present invention can be increased by using the F092 strain, preferably in combination with nutrient sources or surfactants described later, or by increasing the culture period of the strain formula, Generally, contaminated soil of about 15,000 to 50,000 ppm can be treated.

  A method for bringing the F092 strain into contact with petroleum-contaminated soil may be performed according to a conventional method. For example, when the F092 strain is cultured in a liquid medium, the culture solution may be sprayed on petroleum-contaminated soil.

  The F092 strain of the present invention is preferably cultured using a liquid medium such as a malt extract medium or a potato glucose medium. The compositions of the malt extract medium and potato glucose medium used in the examples are as shown in the examples described later. In addition, the composition of the potato glucose medium used in the examples is slightly different from the commonly used potato glucose medium, and the amount of potato is 2.5 times (200 g / L → 500 g / L) compared to the normal medium. It is different from the normal composition in that yeast extract (5 g / L) is further added. The pH of the liquid medium is preferably adjusted to about 5 to 8.5 according to the optimum pH of the F092 strain.

  As described above, the F092 strain can be used as a petroleum decomposing agent or a petroleum-contaminated environmental purification agent because it can efficiently decompose petroleum components in a high degree and in a short time. Specifically, the F092 strain may be used as it is as a petroleum decomposing agent, or a liquid medium containing the F092 strain may be used as a petroleum decomposing agent. Or a nutrient source, surfactant, etc. (it mentions later) may be included. Or what added the additive (typically preservative etc.) normally added to the processing agent containing microorganisms to F092 strain | stump | stock can also be used as a petroleum decomposition agent.

  The petroleum decomposing agent may be one obtained by sufficiently growing the F092 strain in a liquid medium or the like, or one obtained by refrigerated storage.

  Next, the petroleum decomposer composition of the present invention will be described in detail. The petroleum decomposing agent composition of the present invention contains the F092 strain and a base material. Here, said F092 stock | strain may be contained as a petroleum cracking agent which further contains an additive.

  The base material used in the present invention is not particularly limited, and those usually used for supporting microorganisms and capable of absorbing petroleum components are preferably used. Specifically, for example, paper and fiber products such as paper, fiber, fabric, filter and synthetic fiber; building material products such as wood chips; and materials such as film and plastic. For example, representative examples of the plastic include thermoplastic resins such as polyethylene and polypropylene; thermosetting resins such as phenol resins and melamine resins. Among the above-mentioned substrates, biodegradable substrates such as biodegradable plastics and biodegradable fibers that are decomposed by microorganisms are preferably used. This is because the biodegradable base material not only absorbs petroleum components such as polycyclic aromatic compounds but also decomposes the biodegradable base material itself after purification of the petroleum components. Biodegradable base materials are roughly classified into bio-based base materials derived from biological resources (biomass) and petroleum-based base materials based on raw materials, and any of them can be used in the present invention. Representative examples of bio-base materials include polylactic acid, polycaprolactone, modified polyvinyl alcohol, and casein, and representative examples of petroleum base materials include, for example, PET copolymers.

  The method for obtaining the molded product (product) by supporting the F092 strain of the present invention on the substrate is not particularly limited, and a generally used method may be appropriately employed depending on the type of the substrate. For example, after processing a base material into a product, the method of covering the surface of the said product with the F092 stock containing solution, or immersing in the said product, etc. are mentioned. Or you may employ | adopt methods, such as mixing a base material, F092 stock | strain containing solution, etc. before processing into a product.

  The blending amount of the F092 strain (culture) supported on the substrate may be appropriately controlled according to the type of the substrate used. For example, when a plastic material such as polypropylene or a biodegradable base material such as polylactic acid is used, a culture in which the F092 strain is cultured, for example, a culture in which wood flour is cultured is generally added to 100 parts by weight of the whole base material. It is preferable to mix | blend in the ratio of 5-15 mass parts.

  Next, the method for purifying an oil-contaminated environment according to the present invention will be described in detail. The purification method of the present invention is characterized in that the F092 strain or the petroleum decomposing agent composition described above is added to the oil-contaminated environment of seawater or soil contaminated with petroleum.

  When the F092 strain is added to petroleum-contaminated environments such as seawater and soil contaminated with petroleum, it is preferable to add the nutrient source or surfactant of the F092 strain, or a mixture thereof. Nutrient sources can activate the growth of the F092 strain and thus activate the resolution of petroleum components. In addition, it is considered that the surfactant can be more efficiently purified by facilitating the decomposition by the F092 strain by easily detaching petroleum components having high fat solubility and low hydrophilicity from solid soil or the like. It is done.

  As a nutrient source of the F092 strain, for example, a commercially available nutrient source of the genus Fusarium such as Difco malt extract can be used. Alternatively, a carbon source such as glucose; a nitrogen source such as polypeptone; a trace element source such as a magnesium salt or a manganese salt; a pH adjuster such as citric acid may be appropriately selected and used.

The addition amount of the nutrient source may be appropriately adjusted according to the amount of the F092 strain and the like. In general, the larger the amount of the nutrient source, the better the growth of the F092 strain, and the decomposition efficiency of the petroleum component is improved. . On the other hand, if there are too many nutrient sources such as a carbon source and a nitrogen source, 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 approximately 5 to 15 parts by mass with respect to 100 parts by mass of the oil-contaminated seawater or oil-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 there is little adverse effect on the F092 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.

  In addition to improving the dispersibility of petroleum components in the growth environment of the F092 strain, the surfactant may promote the growth of the F092 strain, for example, as a carbon source. However, too much surfactant may adversely affect the growth of the F092 strain. Therefore, the amount of the surfactant added is preferably 0.05 parts by mass or more and 1.0 part by mass or less, generally 0.1 parts by mass or more, with respect to 100 parts by mass of the petroleum-contaminated environment to be treated. More preferably, it is 0.75 parts by mass or less.

  In the culture, the presence or absence of stirring is not particularly limited, and both stationary culture and stirring culture can be employed. The agitation speed during agitation culture varies depending on the type, concentration, amount, etc. of the crude oil to be treated, but in order to effectively demonstrate the resolution of the F092 strain, it is generally controlled within the range of 50 to 150 rpm. Is preferable.

  Oil-contaminated seawater and oil-contaminated soil to be treated are sampled or mined and then inserted into a container, and F092 strains and nutrient sources are added, and the temperature and humidity are adjusted so that the F092 strain can grow well. May be. However, seawater contamination and soil contamination are often widespread, and if the seawater and soil to be treated are collected or mined one by one, the treatment efficiency may be lowered. Therefore, the F092 strain may be added as it is to oil-contaminated seawater or oil-contaminated soil. Since the F092 strain generally grows sufficiently at 25 ° C., it is considered that the F092 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 in a timely manner.

  What is necessary is just to adjust the addition amount of F092 stock | strain suitably according to the contamination state of contaminated seawater or contaminated soil.

  According to the knowledge by the present inventors, it is more efficient to shield the light during the treatment of oil-contaminated seawater or oil-contaminated soil by the F092 strain. Therefore, after adding the F092 strain to petroleum-contaminated seawater or oil-contaminated soil, it is preferable to suppress the amount of irradiation light by covering with a blue sheet or the like.

  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 may be implemented with appropriate modifications within a range that can meet the purpose described above and below. These are all possible and are within the scope of the present invention.

Example 1: Isolation and screening test of F092 strain First, 10 mL of malt extract (ME) agar medium having the following composition is prepared. In this example, an ME agar medium was prepared using artificial seawater (NaCl concentration 3.5% by mass, pH 8.2) instead of distilled water.
ME agar medium: malt extract (20 g / L), glucose (20 g / L),
Polypeptone (1 g / L), agar (20 g / L)

  Next, 0.1 mL of a chrysene solution (1% chrysene solution of dimethylformamide containing 0.25% Tween 80), which is a kind of PAHs, was added to 10 mL of the above ME agar medium to obtain a screening medium.

  In this screening medium, soil and mushroom 42 samples collected at the Faculty of Agriculture, Ehime University; soil and mushroom 57 samples collected in Matsuyama City; soil and mushroom 12 samples collected in the suburbs of Matsuyama City, a total of 111 samples And statically cultured at 25 ° C. for 7 days in the dark. Among the microorganisms that grew on the medium, the novel microorganism F092 strain of the present invention was selected as the microorganism having the fastest growth rate (the highest chrysene degradation rate). This new microorganism F092 strain was selected from soil collected at the campus of the Faculty of Agriculture, Ehime University. The growth rate (decomposition rate) is determined based on the growth diameter (B) of the bacteria cultured on the chrysene-containing ME medium and the growth diameter (A) of the bacteria cultured on the ME agar medium not containing chrysene as described above. The ratio of B to A (B / A × 100) was calculated, and the growth rate of the F092 strain was about 96%.

  In addition, the high chrysene resolution by the F092 strain is cultured in the same manner as described above using an ME agar medium (pH 4.7) prepared by adding distilled water instead of artificial seawater in the above-mentioned chrysene-containing ME agar medium. And the highest decomposition rate (about 96%) in the sample.

  From the above experimental results, it can be seen that the F092 strain grows at an extremely high growth rate under the conditions of the ME agar medium prepared with seawater and the ME agar medium prepared with distilled water, and can highly degrade chrysene. It was. That is, the F092 strain was proved to be extremely useful as a microorganism capable of highly degrading polycyclic aromatic compounds in seawater.

Example 2
In this example, after the F092 strain was cultured for 15 days and 30 days in a chrysene-containing ME liquid medium having the following composition, chrysene remaining in the culture solution was quantified, and chrysene in the ME liquid medium by the F092 strain was determined. The decomposition rate was measured.

Specifically, first, 20 mL of a malt extract (ME) liquid medium having the following composition is prepared. In this example, an ME liquid medium was prepared using artificial seawater (NaCl concentration 3.5% by mass, pH 8.2) instead of distilled water.
ME liquid medium: malt extract (20 g / L), glucose (20 g / L),
Polypeptone (1g / L)

  Next, 0.4 mL of chrysene solution (dimethylformamide 1.14% chrysene solution containing 0.25% Tween 80) was added to 20 mL of the above ME liquid medium to obtain a chrysene-containing ME liquid medium.

  For comparison, a surfactant-containing chrysene-containing ME liquid medium prepared by adding Tween 80, which is a surfactant, at the ratio (volume ratio) shown in Table 1 to the chrysene-containing ME liquid medium was prepared. Similarly, a polypeptone-containing chrysene-containing ME liquid medium prepared by adding polypeptone, which is a nitrogen nutrient source, at a ratio (volume ratio) shown in Table 1 to the chrysene-containing ME liquid medium was prepared.

  For reference, a chrysene-containing ME liquid medium (pH 4.7) in which distilled water was added instead of artificial seawater was prepared for each of the above ME liquid mediums.

  Subsequently, the F092 strain was inoculated into a total of 6 types of chrysene-containing ME liquid media prepared as described above, and after 15 days or 30 days of stationary culture in the dark at 25 ° C., the method described below was used. The degradation rate of chrysene was measured.

  Furthermore, for comparison of culture conditions, stirring culture (stirring speed 80 rpm) was performed in the chrysene-containing ME liquid medium (without addition of surfactant and polypeptone) prepared as described above, and the method described below for measuring chrysene degradation rate did.

(Measurement of chrysene decomposition rate)
After 15 days and 30 days from the start of culture, the filtrate and the residue (cells) were separated by filtration. The filtrate was extracted with ethyl acetate. The residue was Soxhlet extracted with ethyl acetate for 16 hours. Each extract was concentrated under reduced pressure, merged, and added with 4-chlorobiphenyl as an internal standard, and purified with a silica gel column. After concentrating the fraction eluted with dichloromethane, residual chrysene was quantified by GC-MS. The conditions of GC-MS are as follows.
Column: TC-1 manufactured by GL Sciences Inc. (length 30 m × inner diameter 0.25 mm)
Carrier gas: Helium Temperature rising condition: After holding at 80 ° C. for 2 minutes → 150 ° C. → 20 ° C./min.→300° C.
Temperature rise at 25 ° C./min → Hold at 300 ° C. for 10 minutes Column flow rate: 1.5 mL / min Inlet temperature: 260 °
Ionization voltage: 70 eV

  These results are shown in Table 1 (artificial seawater) and Table 2 (distilled water).

  From these tables, it was found that the F092 strain grew at a high growth rate in any of the ME liquid medium prepared with artificial seawater or distilled water, and was able to highly degrade chrysene. It was also found that the chrysene-degrading action by the F092 strain is further promoted by adding polypeptone or a surfactant, which are nutrients in the ME liquid medium, or by culturing under stirring.

Example 3
In this example, the F092 strain was inoculated into a crude oil-containing potato glucose (PG) agar medium, and the growth diameter (cm) of the mycelium after the culture was measured.

Specifically, first, a PG agar medium having the following composition was prepared using artificial seawater or distilled water.
PG agar medium: Potato (500 g / L), glucose (20 g / L),
Yeast extract (5 g / L), agar (20 g / L)

In each PG agar medium (10 mL), crude oil dissolved in a solvent (hexane 1 mL) was added to a concentration of 5000 to 15000 ppm, and then F092 strain was inoculated and cultured at 25 ° C. in the dark. . Since the F092 strain grows concentrically, the diameter (cm) of the mycelium after the culture was measured every 2 days. The crude oil used in this example is crude oil imported from Indonesia (provided by Taiyo Oil), and its composition is as follows.
62% hydrocarbon (alkane fraction)
Aromatic compounds 6%
Compound containing nitrogen, sulfur and oxygen (NSO) 7%
Asphalt 25%

  These results are shown in Table 3.

  From Table 3, it was found that the F092 strain can grow both in crude oil and in crude oil including seawater, and grow in a wide concentration range of 5000 to 15000 ppm. Therefore, it was found that the F092 strain of the present invention can grow in seawater and is extremely useful as a microorganism capable of highly degrading polycyclic aromatic compounds in seawater contaminated with petroleum.

Example 4
In this example, the following experiment was carried out by simulating the decomposition and purification of petroleum in seawater using the F092 strain and a base material (using a biodegradable polypropylene fiber). In this example, C heavy oil was used as a petroleum component.

  First, 20 mL of the PG agar medium described in Example 3 prepared using artificial seawater (NaCl concentration 3.5% by mass, pH 8.2) was added to an Erlenmeyer flask.

Next, polypropylene fiber (using a polypropylene fiber sheet manufactured by Toray Fine Chemical Co., Ltd.) was cut into a size of 2 cm × 2 cm to absorb C heavy oil. The solvent was removed by leaving it overnight in a clean bench. The concentration of C heavy oil is 1000 ppm. In this example, as C heavy oil, it was C heavy oil corresponding to heavy oil standard (JISK2205) Type 3 No. 1, and C heavy oil manufactured by Taiyo Oil Co., Ltd. was used. The composition of the C heavy oil is as follows.
44% hydrocarbon (alkane fraction)
Aromatic compounds 41%
Compound containing nitrogen, sulfur and oxygen (NSO) 8%
Asphalt 7%

  In this way, the C heavy oil impregnated polylactic acid fiber was floated in 20 mL of the above PG liquid medium, and after standing in the dark at 25 ° C. for 0 to 60 days, the degradation rate of C heavy oil was as follows. It was measured. For comparison, only C heavy oil (without using polypropylene fiber) was added to the above PG liquid medium, culture was performed in the same manner, and the degradation rate of C heavy oil was measured.

  The degradation rate of C heavy oil was determined by the method of Misra et al. 2001)). Here, the amount of C heavy oil [total amount of petroleum hydrocarbon (TPH)] and hydrocarbons constituting C heavy oil: aromatic compounds; compounds containing nitrogen, sulfur and oxygen (NSO); Both asphaltene and asphaltene were measured.

  The outline of the measurement method is as follows. First, cultures were collected after 15 days, 30 days and 60 days from the start of culture (day 0), respectively. Hexane was added to the culture and extracted. The solution after hexane extraction was further extracted sequentially with dichloromethane and chloroform. Each extract was combined and concentrated to obtain an extract. On the other hand, a culture cultured in the same manner without adding the 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. It was. Thus, the weight of each extract obtained was compared and the decomposition rate of the total petroleum hydrocarbon part (TPH) was computed.

Furthermore, hexane was added to each extract, and it was divided 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, and chloroform: methanol (1: 1). The hexane elution part thus obtained is hydrocarbon, the toluene elution part is an aromatic compound, the chloroform: methanol (1: 1) elution part is a compound containing nitrogen, sulfur and oxygen (NSO) and asphalt, Includes each. Among these, the decomposition rate of hydrocarbons and aromatic compounds was calculated by analyzing the hexane elution part and the toluene elution part by gas chromatography (GC), and comparing the peak areas of the eluates before and after the treatment. The GC conditions are as follows. The decomposition ratio of NSO and asphalt was calculated by measuring the weight of a chloroform: methanol (1: 1) soluble part and a hexane insoluble part composed of asphalt and comparing the weights before and after the treatment.
Column: TC-5 manufactured by GL Sciences Inc. (length 30 m × inner diameter 0.25 mm)
Carrier gas: helium Temperature rising condition: 60 ° C. to 280 ° C. at a rate of 10 ° C./min → held at 280 ° C. for 10 minutes Column flow rate: 1.5 mL / min Inlet temperature: 300 ° C.

  The decomposition rates of TPH, hydrocarbons, aromatic compounds, and NSO and asphalt are shown in Table 4 (with base material added) and Table 5 (without base material added), respectively.

  First, focusing on the total petroleum hydrocarbon part (TPH), when F092 strain was used, more than half of 1000 ppm C heavy oil could be decomposed in about 2 months after cultivation regardless of the presence or absence of polypropylene fibers. When Table 4 and Table 5 are compared, the decomposition rate was further promoted when polypropylene fibers were not used, and about 80% of C heavy oil could be decomposed in about two months after the culture (see Table 5).

  The same tendency was recognized also about each component which comprises C heavy oil. That is, as shown in Table 4, when F092 strain and polypropylene fiber were used, more than half of the hydrocarbons and aromatic compounds in C heavy oil could be decomposed in about one month after culturing. In addition, as shown in Table 5, when polypropylene fibers are not used, the decomposition rate is further accelerated, and about 2/3 or more of hydrocarbons and aromatic compounds in C heavy oil can be decomposed in about one month after culturing. In about 2 months after culturing, the resolution was accelerated to about 70-80%.

  From the above results, it was sufficiently demonstrated that the method of the present invention is extremely useful as a technique capable of sufficiently purifying contaminated seawater contaminated with C heavy oil.

Example 5
In this example, the experiment was performed in the same manner as in Example 4 except that in Example 4 described above, crude oil was used instead of C heavy oil. As the crude oil, the crude oil used in Example 3 was used, and the added crude oil concentration was 1000 ppm as in the case of C heavy oil.

  The decomposition rates of TPH, hydrocarbons, aromatic compounds, NSO and asphalt are shown in Table 6 (with base material added) and Table 7 (without base material added), respectively.

  First, focusing on the total petroleum hydrocarbon part (TPH), the F092 strain can decompose about 2/3 of 1000 ppm crude oil in about 2 months after culturing regardless of the presence or absence of polypropylene fibers. It was. Comparing Table 6 and Table 7, when polypropylene fibers were used, the decomposition rate was accelerated, and more than half of crude oil could be decomposed in about one month after culturing (see Table 6).

  Similarly, about each component which comprises crude oil, when F092 stock | strain was used, about 2/3 of 1000 ppm crude oil was able to be decomposed | disassembled in about 2 months after culture | cultivation irrespective of the presence or absence of a polypropylene fiber. Comparing Table 6 and Table 7, when polypropylene fibers were used, the decomposition rate was accelerated, and more than half of the hydrocarbons in the crude oil could be decomposed in about one month after culturing.

  From the above results, it was sufficiently demonstrated that the method of the present invention is extremely useful as a technique capable of sufficiently purifying seawater contaminated with crude oil.

Example 6
In this example, chrysene decomposition rate in chrysene contaminated soil by F092 strain was measured.

  First, chrysene contaminated soil was prepared as follows. Soil (weakly acidic pH 6.2) 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 solution in which 0.01% chrysene was added to dimethylformamide containing 0.25% Tween 80 was prepared. This chrysene solution (1 mL) was added to the sterilized soil (100 g in dry weight). For comparison, the chrysene solution was similarly added to soil that was not sterilized. In any case, the chrysene concentration in the soil is 10 ppm.

  The chrysene contaminated soil prepared as described above was inoculated with the F092 strain. More specifically, the viable cell pellet of the F092 strain was prepared by punching out the F092 cell grown on the above-mentioned ME agar medium together with a cork borer (diameter 5 mm), and the live cell pellet in 100 g of the chrysene contaminated soil. Eight pellets were added. According to this method, the weight (raw weight) of the whole pellet in the whole soil was about 2% by mass, and in this example, this was used as the amount of F092 strain added.

  Furthermore, glucose or polypeptone as a nutrient source is added to the chrysene-contaminated soil at a ratio shown in Table 9 (addition of glucose) or Table 10 (addition of polypeptone), and in the dark at 25 ° C. for 15 days. Or it culture | cultivated for 30 days. For comparison, the same culture was performed in chrysene-added soil to which the above nutrient source was not added.

A portion of the soil was collected after 15 or 30 days from the start of the culture and extracted with dichloromethane for 16 hours. The extract was concentrated under reduced pressure, 4-chlorobiphenyl as an internal standard solution was added, and the mixture was purified with a silica gel column. After concentrating the fraction eluted with dichloromethane, residual chrysene was quantified by GC-MS. The conditions of GC-MS are as follows.
Column: TC-1 manufactured by GL Sciences Inc. (length 30 m × inner diameter 0.25 mm)
Carrier gas: helium Temperature rising condition: 80 ° C. → 150 ° C. up to 20 ° C./min Temperature rising up to 300 ° C. at 25 ° C./min
Temperature rise → Hold at 300 ° C. for 10 minutes Column flow rate: 1.5 mL / min Inlet temperature: 260 ° C.
Ionization voltage: 70 eV

  The decomposition rate was calculated from the amount of chrysene before treatment. The results are shown in Table 8 (control), Table 9 (addition of glucose) and Table 10 (addition of polypeptone), respectively.

  From Tables 8 to 10, it can be seen that if F092 strain is used, chrysene in the soil can be decomposed without adding a nutrient source (see Table 8). The rate of chrysene degradation of F092 strain is further accelerated by the addition of glucose and polypeptone, and after 30 days of culturing, it degrades more than half of chrysene in the glucose added group and about 2/3 of chrysene in the polypeptone added group I was able to.

  From the above experimental results, it was proved that the F092 strain can efficiently decompose polycyclic aromatic compounds in petroleum such as chrysene regardless of seawater and soil.

Example 7
In this example, an experiment was performed in the same manner as in Example 5 except that the concentration of crude oil was changed to 15,000 ppm in Example 5 described above.

  The decomposition rates of TPH, hydrocarbons, aromatic compounds, and NSO and asphalt are shown in Table 11 (with base material added) and Table 12 (without base material added), respectively.

  From these tables, when F092 strain is used, even about 15,000 ppm crude oil, about 40% of hydrocarbons in crude oil can be obtained in about 2 months after culturing regardless of the presence or absence of polypropylene fiber. Could be decomposed. In addition, about the decomposition rate of the hydrocarbon until about one month after culture | cultivation, the tendency not to use a polypropylene fiber showed the tendency for a decomposition rate to be accelerated | stimulated (refer Table 11 and Table 12).

  From the above results, it was demonstrated that the method of the present invention is extremely useful as a technique capable of purifying seawater contaminated with high-concentration crude oil of about 15,000 to 50,000 ppm.

Claims (6)

  F092 strain (NITE P-795) belonging to the genus Fusarium having petroleum resolution.   F092 strain (NITE P-795) belonging to the genus Fusarium having the ability to resolve polycyclic aromatic compounds.   A petroleum decomposing agent composition comprising the microorganism according to claim 1 or 2 and a base material.   A method for purifying an oil-contaminated environment, comprising adding the microorganism according to claim 1 or 2 or the petroleum decomposing agent composition according to claim 3 to an oil-contaminated environment.   The purification method according to claim 4, wherein the petroleum-contaminated environment is seawater or soil.   Furthermore, the purification method of Claim 4 or 5 which adds the nutrient source and / or surfactant of the said microorganisms.