JP3922944B2 - Prediction method for petroleum cracking capacity of environmental samples - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for predicting the ability of decomposing petroleum in polluted environmental samples in the purification of microorganisms (bioremediation) of soil, river water, groundwater and the like contaminated with petroleum.
[0002]
[Prior art]
Environmental pollution caused by petroleum is well known for wide-area marine pollution caused by spills of crude oil or fuel oil due to ship accidents, etc., but also on land due to pipeline breakage, leakage from storage tanks, discharge of contaminated wastewater, etc. There have been many reports of soil destruction, river water, and groundwater contamination. Oil pollution is a major cause of soil and groundwater contamination in other countries, and it is reported that about 80% of soil and groundwater contamination by organic compounds originates from oil in the US Supervised by the Council: Actual conditions and countermeasures for soil and groundwater contamination, published by Ohmsha, p.28, 2000).
[0003]
In recent years, techniques for purifying environmental pollution such as soil, river water, and groundwater caused by various harmful substances have been developed. Although the type and concentration of substances that can be decomposed are limited by the purification method using microorganisms, the target pollutants can be decomposed into harmless compounds under mild conditions at normal temperature and pressure, so other physicochemical purification methods It is attracting attention as a low-cost and environmentally friendly purification method.
[0004]
As a purification method using microorganisms, a method using indigenous microorganisms (biostimulation) existing in the environment to be purified is used, but there is no pollutant-degrading bacteria in the indigenous microorganism group. A method of adding degrading bacteria (bioaugmentation) is used. When applying the purification method using microorganisms, samples such as soil and water are collected from the target contaminated environment, and a decomposition experiment (treatability test) based on biostimulation using indigenous microorganisms is performed. Do. In other words, an appropriate amount of nutrients and, if necessary, a carbon source are added to the collected environmental sample and cultured under environmental conditions close to the actual construction, and the degradability of pollutants by indigenous microorganisms is confirmed. Since petroleum-based substances are hardly decomposable substances, the treatability test took 5 to 6 weeks or more or several months.
[0005]
In addition, petroleum has a wide variety of constituents (Wang, Z., M. Fingas, and L. Ken: J. Chromatogr. Sci., 32, 367, 1994), and its composition varies greatly depending on the contaminated site. It is a feature. In other words, because the petroleum components and concentrations contained in the sample to be investigated are not constant, there may be cases where the petroleum components that can be used by petroleum-degrading bacteria are not sufficiently contained, and the petroleum components can be assimilated in the sample to be purified. Even in the presence of petroleum degrading microorganisms, there were cases where the indigenous microorganisms of the sample were mistaken for low petroleum resolution. In addition, the environmental sample may contain a large amount of easily microbially degradable organic substances other than petroleum hydrocarbons. In such a case, even if the nitrogen source and phosphorus source necessary for the growth of microorganisms are added to the environmental sample to be tested and cultured, microorganisms useful for purification of petroleum contamination cannot always be grown. It was difficult to select the construction method. Furthermore, it goes without saying that when there are no indigenous degrading microorganisms in the sample, it takes a long time to determine the resolution.
[0006]
In recent years, due to the development of molecular biological analysis methods, methods have been developed to grasp the microbial community structure in environmental samples in a short time by examining genes in environmental samples (Amann, RI, Ludwig, W , and Schleifer, KH (1995). Phylogenetic identification and in situ detection of individual microbial cells without cultivation, Microbiol Rev. 59: 143-169). However, as mentioned above, there are a wide variety of petroleum components, so in the petroleum-degrading microbial community, the proportion of degrading microorganisms corresponding to individual petroleum components is small, and there is a high possibility that it will cover many types. However, it was not always possible to detect even by genetic analysis (Kasai, Y., Kishira, H., Syutsubo, K., and Harayama, S., Molecular detection of marine bacterial populations on beaches contaminated by the Nakhodka tanker oil-spill accident .. Environ Microbiol vol.3 (4), 246-255, 2001). In the case of environmental samples such as soil containing a large amount of various organic substances, the number of indigenous bacteria is 10 ⁸ / G to 10 ^Ten / G or so (Bitton, G. and Gerba, CP: Groundwater Pollution Microbiology, John Wiley & Sons, New York, p.23, 1984) In comparison, it is more difficult to detect genes derived from petroleum degrading microorganisms.
[0007]
Attempts have been made to identify microorganisms involved in petroleum degradation by creating a culture in which nitrogen and phosphorus are added to an oil-contaminated sample (soil) and analyzing the genes of the microbial communities present in the sample. (Takahata Yo, Ohba Miho, Suzuki Asaka, Houki Toshihiro, Microbial analysis in petroleum-contaminated soil by DGGE method, Annual Meeting of Japan Society on Water Environment, 1999, p412). However, in the existing technology, the petroleum component contained in the sample (soil) in advance is used as the carbon source of the petroleum decomposing microorganism. It was not always possible to supply petroleum-based hydrocarbons of sufficient quality and quantity to promote. As a result, bacteria that assimilate readily degradable organic substances contained in environmental samples proliferate preferentially, and the abundance of genes derived from petroleum-degrading microorganisms can be detected by relatively molecular biological techniques. It could only be maintained below the limit and it was difficult to confirm the presence of petroleum degrading microorganisms in the sample. In addition, as described above, the constituent components of petroleum are diverse, and the group of microorganisms that can be decomposed differs depending on the components. In the existing technology, the situation of petroleum pollution, in other words, the composition and concentration of petroleum hydrocarbons vary greatly depending on the sample. Therefore, as a result of the microbial community structure analysis, which petroleum constituents are identified by the identified microorganisms will be utilized. It was difficult to judge.
[0008]
In addition, since petroleum hydrocarbons are mostly composed of water-insoluble substances, in order to promote the growth of petroleum-decomposing microorganisms, the contact efficiency between petroleum-decomposing microorganisms and petroleum existing in the sample can be increased. is necessary. In existing technology, a small amount of water (5% (w / w)) is added to the soil, and only manual agitation is performed once a day. Oil and decomposing microorganisms present in the sample (soil) Insufficient contact with the oil, the effect of promoting the growth of petroleum-degrading microorganisms was remarkably low.
[0009]
[Problems to be solved by the invention]
Highly promotes the growth of petroleum-degrading microorganisms present in environmental samples such as soil, river water, and groundwater contaminated by petroleum, and analyzes genes derived from petroleum-degrading microorganisms present in the resulting oil-degrading integrated culture. By detecting it, it is possible to predict in a short time the ability to purify oil pollution by microorganisms indigenous to environmental samples when oil pollution occurs.
The purpose of the present invention is to quickly create a highly petroleum-degrading and enriched culture that originates from microorganisms present in environmental samples, analyze and detect genes derived from petroleum-degrading microorganisms, and improve the ability of petroleum samples to decompose oil. It is to provide a means of prediction.
[0010]
[Means for Solving the Problems]
As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that culture conditions that can rapidly accelerate the cultivation of petroleum-degrading microorganisms in environmental samples, and the oil resolution of samples and the accumulated cultures exist. The relationship with genes derived from petroleum degrading microorganisms was clarified and the present invention was completed.
That is, this invention solved the said subject by the following means.
(1) Environmental samples Add 1 to 100 times the sample weight of sterilized water to the soil sample, Petroleum hydrocarbons that are likely to be assimilated by microorganisms Is added to a concentration of 50 to 5000 g / L, a nitrogen source is used as a substance that promotes the formation of microbial communities, a concentration of 1 to 200 mg-N / L, and a phosphorus source is a concentration of 0.2 to 50 mg-P / P L so that the stirring intensity is maintained at 30 to 300 rpm and stirring culture is performed. An environmental sample oil characterized by forming a microbial community corresponding to the decomposition of petroleum hydrocarbons present in the environmental sample and detecting a gene derived from the petroleum decomposing microorganism in the microbial community formed in the environmental sample. Decomposition capability prediction method.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
According to the method for predicting petroleum decomposing ability of an environmental sample of the present invention, a nitrogen source, phosphorus source or petroleum hydrocarbon is added to the environmental sample to produce an oil decomposing integrated culture, and a gene derived from petroleum decomposing microorganisms is detected in the sample. It is characterized by this. Hereinafter, embodiments of the present invention will be described in detail.
[0012]
To obtain a microbial community formed in response to petroleum hydrocarbon contamination in an environmental sample, simply add water as in the case of unsaturated soil previously contaminated with petroleum. In some cases, it may be sufficient to cultivate, but in general, in order to promote the growth of microorganisms, in addition to water, a nitrogen source and a phosphorus source are added and cultured under aerobic conditions. In addition, it is desirable to add petroleum-based hydrocarbons to samples that are not contaminated with petroleum or have a low concentration of petroleum contamination and samples that contain a large amount of organic substances other than petroleum-based hydrocarbons.
[0013]
In order to promote the growth of petroleum-degrading microorganisms, it is desirable to add an appropriate amount of water when the sample to be investigated is a sample having a low water content such as unsaturated soil. Water is added so that at least the sample is saturated, and it is preferable to add 0.5 to 1000 times the weight of the sample, and add 1 to 100 times the weight of the sample. Is more preferable. If the environmental sample has sufficient water, such as river water or groundwater, there is no need to add water. As the water to be added, it is desirable to use sterilized water so as not to affect the genetic analysis of the sample.
In addition, in order to prevent insufficient oxygen supply and improve the contact efficiency between indigenous microorganisms and petroleum hydrocarbons, in other words, to promote the growth of petroleum-degrading microorganisms, stirring culture, swirling culture, or a method similar to this is used. It is preferable to do so. The strength of stirring is preferably 30 to 300 rpm, and more preferably 100 to 200 rpm.
The culture period is 3 to 30 days, preferably 7 to 14 days. The culture temperature is preferably 10 ° C. to 40 ° C., more preferably 15 ° C. to 35 ° C.
Further, when the pH of the sample is not in the neutral range, it is desirable to adjust the pH to the neutral range when performing the shaking culture.
[0014]
As the nitrogen source to be added, it is desirable to use inorganic nitrogen compounds such as ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium nitrate, ferrous ammonium sulfate, etc., but other inorganic ammonium compounds may be used. . Moreover, you may use the fertilizer for agriculture containing isobutyraldehyde condensed urea, formaldehyde processed urea, etc. Organic nitrogen sources such as compost, peptone, yeast extract, corn steep liquor, meat extract can be used, but it is thought that microorganisms that use organic substances contained in them as a carbon source will grow. It is desirable to use an inorganic nitrogen source. The amount of nitrogen source added depends on the carbon source concentration of the petroleum hydrocarbon as a pollutant, but is preferably added so that the concentration is 0.1 to 1000 mg-N / L, preferably 1 to 200 mg- More preferably, N / L is added.
[0015]
As a phosphorus source to be added, it is desirable to use inorganic phosphorus compounds such as potassium phosphate, magnesium phosphate, ammonium phosphate, calcium phosphate, sodium phosphate, etc., but other inorganic phosphate compounds may be used. good. Moreover, you may use the fertilizer for agriculture containing a dilime phosphate, phosphoric acid bitter soil (phosphate magnesia), etc. Organic phosphorus sources such as compost, peptone, yeast extract, corn steep liquor and meat extract can be used, but it is desirable to use an inorganic phosphorus source in actual use. The addition amount of the phosphorus source is preferably added so that the concentration thereof is 0.1 to 1000 mg-P / L, and more preferably 0.2 to 50 mg-P / L.
[0016]
As the petroleum-based hydrocarbon to be added, it is preferable to use alkane or polycyclic aromatic hydrocarbon which is the main component of petroleum. Alkanes and polycyclic aromatic hydrocarbons may be added alone, or a plurality of compounds may be added simultaneously. In addition, crude oil, heavy oil containing a large amount of a plurality of alkanes and polycyclic aromatic hydrocarbons, crude oil obtained by heating and removing light components, mainly aliphatic hydrocarbons (weathered crude oil, hereinafter referred to as w.oil), and further crude oil Saturated fractions, aromatic fractions, etc. obtained by fractionation of the product by thin layer chromatography or the like can also be used. Moreover, you may add gasoline, light oil, and kerosene containing many alkanes.
[0017]
However, crude oil which is a complex of alkane, polycyclic aromatic hydrocarbon, etc., w. When oil or the like is added to an environmental sample, the growth of microorganisms that degrade easily degradable alkanes is extremely fast, and as a result, the growth of polycyclic aromatic hydrocarbon-degrading bacteria may be suppressed ( Hironori Tsuji, Shigeaki Harayama, Influence of inorganic nutrient concentration on microbial degradation of spilled oil, Annual Meeting of Japan Society on Water Environment, 2000, p.533). In this case, it is desirable to separately create a culture system in which only polycyclic aromatic hydrocarbons are added as a carbon source. The amount of these petroleum hydrocarbons added is preferably 10 to 10,000 mg / L, more preferably 50 to 5,000 mg / L.
[0018]
As the alkane, it is preferable to use a normal alkane having 10 to 36 carbon atoms, but alkanes having other carbon numbers, branched alkanes having side chains, cyclic cycloalkanes, and the like are also used. I can do it. As the polycyclic aromatic hydrocarbon, it is preferable to use a compound that is relatively contained in petroleum from 2 to 5 rings such as naphthalene, fluorene, phenanthrene, anthracene, dibenzothiophene, pyrene, and benzpyrene. May be used.
[0019]
By extracting genes from the enrichment cultures cultured by the above method, analyzing the microbial community structure based on the genes, and identifying and detecting genes derived from petroleum-degrading microorganisms in the environmental samples, It is possible to predict the ability to clean up oil pollution by the resident microorganisms present in Japan.
The conventional method (Marmur, J. (1961) A Procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol. 3: 208-218.) Should be used as a method for extracting genes from environmental samples and cultured samples. Yes, but other methods may be used.
[0020]
In order to detect the presence of petroleum-degrading bacteria by analyzing the structure of microbial communities in environmental samples, first a PCR reaction using a universal primer that can amplify the genes of most microorganisms (polymerase chain reaction, Hiroki Nakayama, Cell Engineering) A separate volume Bio Experiment Illustrated 3, Shujunsha, 1996) is performed to amplify a microbial gene (eg, 16S rDNA gene) extracted from the sample. Denaturing gradient gel electrophoresis (DGGE, Muyzer G., S. Hottentrager, A. Teske, and C. Wawer Denaturing gradient gel electrophoresis) of PCR-amplified 16S rDNA-A new molecular approach to analyze the genetic diversity of mixed microbial communities Molecular Microbial Ecology Manual 3.4.4.:1-23, 1996) Ted 4, Shujunsha, 1996). The sequence of the isolated gene was determined by DNA sequencer, and GenBank (Benson DA. Karsch-Mizrachi I, Lipman DJ, Ostell J. Rapp BA, Wheeler DL., GenBank, Nucleic Acids Res 2000 Jan 1, 28 (1) 15 The presence of petroleum degrading microorganisms can be identified and detected by comparing with genetic information registered in databases such as ~ 8).
[0021]
Design highly specific PCR primers based on the specific gene sequences of microorganisms known to have petroleum degradability and detect the presence of petroleum-degrading microorganisms based on whether PCR products are obtained. You can also. Quantitative PCR methods include competitive PCR methods (Hiroki Nakayama, Cell Engineering Separate Bio Experiment Illustrated 3, Shujunsha, 1996), methods using kinetics (Hiroki Nakayama, Cell Engineering Separate Volume Bio Experiment Illustration) (Rated 3, Shujunsha, 1996), real-time PCR (Taq Man PCR, light cycler, Kazuhiro Kanno, clinical pathology, 45, p218, 1997) can be used, but other methods are used. May be. Examples of genes that can be used to design highly specific PCR primers include ribosomal DNA (rDNA), DNA encoding gyrase β subunit (gyrB DNA), alkanes and polycyclic aromatic hydrocarbons, or their degradation. Examples include functional genes related to degrading enzymes of intermediate metabolites. Examples of functional genes related to petroleum degrading enzymes include alkane monooxygenase (alkA, alkB), naphthalene dioxygenase (nahA), catechol 2,3 dioxygenase (nahA), and other alkanes and polycycles. A gene involved in degradation of aromatic hydrocarbons or degradation metabolites of those substances may be detected.
[0022]
Furthermore, detection of genes relating to petroleum degrading microorganisms can also be performed by a hybridization method. That is, after designing a DNA probe that is highly specific for a specific gene derived from petroleum-degrading microorganisms and labeling this DNA with a radioisotope, a fluorescent dye, etc., the gene is extracted from the environmental sample or the microbial cell in the environmental sample. It is a method to detect the target gene or cell by hybridization (hybrid formation) with the existing gene (Amann, RI, Binder, BJ, Olson, RJ, Chisholm, SW, Devereux, R., and Stahl, DA, Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial population.Appl Environ Microbiol 56: 1919-1925, 1990, Amann, RI, In situ identification of microorganisms by whole cell hybridization with rRNA-targeted nucleic acid acid probes. In Molecular Microbial Ecology Manual, Kluwer academic publishers, 3.3.6: 1-15, 1995). Examples of genes that can be used to design highly specific DNA probes include ribosomal DNA (rDNA), DNA encoding gyrase β subunit (gyrB DNA), alkanes and polycyclic aromatic hydrocarbons, or their degradation. Examples include functional genes related to degrading enzymes of intermediate metabolites.
[0023]
Also according to the T-RFLP method (Terminal Restriction Fragment Patterns, Kitts CL., Terminal restriction fragment patterns: a tool for comparing microbial communities and assessing community dynamics. Curr Issues Intest Microbiol 2001 Mar; 2 (1) .17-25). Detection of genes related to petroleum degrading microorganisms is possible. In this method, first, a fluorescent dye is labeled on either the forward or reverse primer, and PCR reaction using them (Polymerase chain reaction, Hiroki Nakayama, Cell Engineering Separate Volume Bio-Experimental Illustrated 3, Shujunsha, 1996) To amplify a microbial gene (eg, 16S rDNA gene) extracted from the sample. Thereafter, a mixture of genes derived from various microorganisms amplified by PCR is treated with a restriction enzyme that cleaves a specific gene sequence. By this treatment, gene restriction fragments (terminal restriction fragments) of various lengths with a fluorescent dye added on one side can be formed. This restriction amplification-treated PCR amplification product is electrophoresed with a DNA seqencer, etc., and the length of the PCR product to which a fluorescent dye is added is patterned to detect genes derived from petroleum-degrading bacteria with specific sequences. it can.
[0024]
【Example】
Examples of the present invention are shown below, but the embodiments of the present invention are not limited thereto.
[0025]
[Example 1] Diagnosis of petroleum decomposing ability of oil-contaminated soil
In order to investigate whether it is possible to predict petroleum decomposing ability of environmental samples by preparing petroleum degrading accumulation cultures by microbial communities originating from environmental samples and identifying and detecting genes derived from petroleum degrading microorganisms Petroleum-decomposing and accumulating cultures using petroleum-contaminated soils (I, II) with different histories were prepared, the oil resolution of each soil was investigated, and the microbial community structure in the soil was analyzed.
[0026]
For the production of the petroleum degrading integrated culture, a sterilized glass centrifuge tube with a capacity of 50 mL was used, and 2 g of each test soil was introduced into the centrifuge tube. The test soil had a petroleum concentration of about 0.1 to 1.5 mg / g and a water content of 15 to 19%, both of which were insufficient for the growth of petroleum microorganisms. Therefore, in order to promote the growth of indigenous petroleum-degrading bacteria, crude oil rich in 10 mL of sterilized water and n-alkane (crude oil that has been previously heat treated to remove volatile components, hereinafter referred to as “w.oil”), or many C of ring aromatic hydrocarbon ₀ -10 mg (5 mg / g) of phenanthrene (hereinafter referred to as phenanthrene) was added to the above glass centrifuge tube. Furthermore, ammonium chloride (35 mg-N / L) was added as a nitrogen source, and disodium hydrogen phosphate (10 mg-P / L) was added as a phosphorus source, followed by shaking culture (130 rpm) at 28 ° C. for 1 week. It was.
[0027]
Moreover, the series which does not add a carbon source (W.oil, phenanthrene) was also provided as a control system. Genes are extracted from each soil to be tested and from the accumulated culture soil sample at the end of the degradation experiment, and gene amplification by PCR method targeting 16S rDNA gene of bacteria, DGGE method (denaturing agent concentration gradient gel of amplified gene mixture) Separation and patterning by electrophoretic method), determination of the gene sequence of the separated microbial gene (DNA band), and identification of microbial groups present in the microbial community. In addition, in order to investigate the aspects of petroleum decomposition in petroleum decomposition accumulation culture experiments, extraction of petroleum components using an organic solvent from the culture solution was performed at the end of the culture experiment, and analysis by GC-MS (gas chromatograph mass spectrometer) was performed. went.
[0028]
FIG. 1 shows the results of analyzing the structure of the microbial community of the enriched culture obtained under each culture condition by the PCR method and the DGGE method. Lanes 1 to 4 show the results of electrophoresis of PCR amplification products using DNA obtained from soil I and its enriched culture as templates, and lanes 5 to 8 show results of electrophoresis of PCR amplification products of soil II and its enriched cultures. ing. Lanes 1 and 5 are test soils, lanes 2 and 6 are control systems to which no petroleum hydrocarbon is added, and lanes 3 and 7 are w. Oil-enriched cultures, lanes 4 and 8 are the results of electrophoresis of PCR amplification products obtained from the phenanthrene-added cultures. Lanes 9 and 10 are results of electrophoresis of PCR amplification products (hereinafter simply referred to as sample names) obtained from petroleum degrading bacteria isolated from soils I and II. In the DGGE method, the presence of microbial groups in a sample can be shown as a DNA band. From this, it was found that the strong signal bands (microorganisms having a high abundance) obtained in each of the I and II soils showed no commonality, and different microorganisms existed depending on the soil. On the other hand, when the culture conditions such as the carbon source to be added were different in the same soil sample, some strong signal bands (microorganism group) coincided (bands A, B, D). When the gene sequences of these bands were determined, most of them were reported to exist in bacteria (bands A and E) that do not seem to be directly involved in petroleum degradation, or in petroleum-contaminated soil. Although it is unknown, it was closely related to the bacterial group (bands B, C, D) presumed to assimilate its degradation intermediate metabolites.
[0029]
The gene sequences of major bands (microorganisms) detected in the soil itself (lanes 1 and 5) or in the control system (lanes 2 and 6) in which water, nitrogen source, and phosphorus source were added to the soil were determined. The presence of petroleum-degrading microorganisms could not be confirmed. This is probably because the growth of microorganisms that assimilate petroleum was not promoted because the oil contamination concentration of the test soil was low. In contrast, w. In the series to which oil or phenanthrene was added (lanes 3 and 6), the genes of petroleum degrading bacteria could be detected (bands F and G).
Further, FIG. 1 shows the culture solution under each culture condition as hexadecane (C ₁₆ (Alkane) or a migration pattern of petroleum-degrading bacteria (isolated bacteria No. 1 and No. 2) isolated using a selective medium containing phenanthrene as a single substrate was also shown (lanes 9 and 10). Bands corresponding to these oil-degrading bacteria are found in the I soil w. It was also confirmed in the enrichment culture system (lane 3, band F) in which oil was added as a carbon source, and in the system (lane 8, band G) in which phenanthrene was added to II soil. When the gene sequences of these bands were determined, the isolate No. No. 1 is Rhodococcus sp., Isolated strain no. 2 are very close to Burkholderia sp., Each of which decomposes alkanes (saturated fraction) and polycyclic aromatic hydrocarbons (hereinafter referred to as PAHs). It was found that it was a fungus involved.
[0030]
In FIG. Alkanes (C) in an enrichment culture system using oil as a carbon source _Ten ~ C ₃₆ ) And PAHs degradation results. Alkane decomposition was better in soil I than in soil II, and about 90% was degraded in the first week after culturing. On the other hand, with regard to the decomposition of PAHs, II soil was superior, naphthalene, phenanthrene, and fluorene were almost completely decomposed, and about 80% of dibenzothiophene was also decomposed.
From the above results, it was found that I. A gene derived from bacteria (band F) with alkane resolution was detected from the system (lane 3) in which oil was added and cultivated, and the alkane resolution of the soil was high, and II phenanthrene was added to the soil and enriched. From the system (lane 8), the genes derived from PAHs-degrading bacteria were detected, and it was found that the soil has high PAHs resolution. In other words, it is possible to predict the petroleum resolution of environmental samples by preparing such highly petroleum degrading and accumulating cultures, analyzing the genes of microbial communities in the cultures, and detecting genes derived from petroleum degrading bacteria. It was shown that there is.
[0031]
【The invention's effect】
By using the method of the present invention to create an oil-degrading accumulation culture in which petroleum-decomposing microorganisms are highly grown and knowing the existence of genes derived from oil-degrading microorganisms originating from environmental samples, It is possible to provide a means for predicting the oil cracking capacity of the sample. In addition, by obtaining genetic information (gene sequence data) derived from petroleum-degrading bacteria, we can obtain systematic classification of petroleum-degrading bacteria active in the sample and knowledge about petroleum-degrading enzymes possessed by the bacteria, It is possible to know what kind of component the petroleum hydrocarbon can be decomposed into.
That is, according to the present invention, it is possible to predict the purification capacity of each petroleum-contaminated component by microorganisms indigenous to an oil-contaminated environmental sample. It is possible to propose an optimal purification method according to the species.
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows the results of analysis of a test soil and an oil-degrading accumulation culture by PCR-DGGE method.
FIG. 2 w. It is a graph which shows the decomposition | disassembly result of alkane and a polycyclic aromatic hydrocarbon compound in the enrichment culture system which used oil as a carbon source.

Claims (1)

Add 1 to 100 times the sample weight of sterilized water to the soil sample of the environmental sample , add petroleum hydrocarbons that are easily assimilated by microorganisms to a concentration of 50 to 5000 g / L, and form microbial communities Nitrogen source is added as a substance to promote the concentration of 1 to 200 mg-N / L, and phosphorus source is added to a concentration of 0.2 to 50 mg-P / L, and stirring intensity is maintained at 30 to 300 rpm and stirring is performed. Culturing, forming a microbial community according to the decomposition of petroleum hydrocarbons present in the environmental sample, and detecting genes derived from petroleum degrading microorganisms in the microbial community formed in the environmental sample A method for predicting the oil cracking capacity of environmental samples

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