JP4836552B2 - Microorganism and purification method for efficiently purifying actual contaminated soil - Google Patents

Description

  The present invention mainly relates to a soil purification technique using microorganisms. More specifically, the present invention relates to a microorganism and a purification method for efficiently purifying actual contaminated soil.

  As a purification technique for contaminated soil, bioremediation using microbial contaminant resolution has attracted attention. Bioremediation can be processed in-situ and can be processed at a cost of 1/10 compared to conventional physical and chemical purification technologies. For these reasons, bioremediation is particularly emphasized as one of the major soil purification technologies in the future.

  Soil bacteria used in bioremediation work to break down the source of contamination, but at the same time are affected by the toxicity of the source. Therefore, in order to efficiently perform bioremediation, it is important to understand the action of soil bacteria as well as the situation of soil contamination.

  Known methods for detecting soil bacteria include a plate method and a DAPI (4 ′, 6diamino-2-phenylindole dihydrochloride) staining method. However, detection of bacteria by the plate method is complicated and time consuming, and only about 0.1 to 1% of microorganisms actually present can be detected, and the status of specific microorganisms can only be grasped. In addition, detection of bacteria by the DAPI staining method is complicated and time consuming. On the other hand, recently, a method has been reported for succinctly and quickly diagnosing environmental characteristics using as an index the amount of environmental DNA obtained by extracting DNA from a sample collected from the environment such as soil and quantifying the amount of DNA. (See Patent Document 1).

  On the other hand, microorganisms in which the resolution of hydrocarbons and oils has been confirmed in simulated contaminated soil have been reported so far (see Patent Documents 2 to 4).

  However, compared to simulated contaminated soil, bioremediation of actual contaminated soil is different from lab-scale simulated contaminated soil, such as changes in the composition of contaminants, soil microbiota, and large-scale bioremediation purification. Includes very different points.

Therefore, it is desired to develop a bioremediation technique that is more suitable for practical use such as purification of large-scale actual contaminated soil.
JP 2004-337027 A JP 2003-102469 A JP 2004-113197 A JP 2004-121068 A

  The main object of the present invention is to provide a microorganism having soil purification ability suitable for actual contaminated soil, a screening method for the microorganism, and a soil purification method suitable for purification of actual contaminated soil.

  As a result of intensive studies to achieve the above object, the present inventor has determined the soil purification capacity of microorganisms suitable for actual contaminated soil by using as an index the number of soil bacteria obtained based on the amount of DNA per unit weight of the sample. The inventors have found that it is possible to evaluate appropriately and that the soil bacteria count can be used as an index to efficiently purify the actual contaminated soil, and further studies have been made to complete the present invention.

  That is, the present invention includes techniques relating to the following microorganisms, microorganism screening methods, and soil purification methods.

Item 1A: a microorganism belonging to the genus Rhodococcus,
When inoculating 1 x 10 ⁸ microorganisms / g-soil of this microorganism into 50 g of soil with an oil concentration of about 10,000 ppm, the residual oil concentration in the soil after 14 days is 4,500 ppm or less, preferably 4,000 ppm or less, more preferably 3,800 ppm. The number of soil bacteria determined on the basis of the amount of DNA per unit weight of the sample collected from the soil is 3.5 × 10 ⁸ cells / g-soil or more, preferably 4.0 × 10 ⁸ cells / g-soil or more. A soil purification microorganism having a soil purification ability of preferably 5.0 × 10 ⁸ cells / g-soil or more.

  Item 1B: 28 days after inoculation of 1% (v / w) of the microorganism in soil with 1% (w / w) of cycloalkane added to 100 g of sterilized soil (autoclaved at 121 ° C for 15 minutes) Item 1. The microorganism according to Item 1A, which has a decomposition ability of a cycloalkane decomposition rate of 23% or more, particularly 23.3% or more.

  In a preferred embodiment, when 1% (v / w) of the microorganism is inoculated into soil obtained by adding 1% (w / w) of cycloalkane to 100 grams of sterilized soil (121 ° C, 15 minutes autoclaved), 14% Item 1A has the ability to decompose at a cycloalkane decomposition rate of 18% or more, particularly 20% or more, 21% or more after 28 days, and a cycloalkane decomposition rate of 23% or more, particularly 23.3% or more after 28 days. Microorganisms.

Item 1C: The microorganism according to Item 1A or 1B, which has an alk gene containing DNA of the following (a) or (a1):
(A) DNA consisting of the base sequence shown in SEQ ID NO: 1
(A1) A DNA that hybridizes with a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 under a stringent condition and encodes a protein having cycloalkane-degrading activity.

  Item 1D: The microorganism according to any one of Items 1A to C, which has 16S rDNA containing the base sequence represented by SEQ ID NO: 2.

Item 2: Rhodococcus sp. RN1 strain having soil remediation ability (Independent Administrative Institution, National Institute of Advanced Industrial Science and Technology, Patent Biodeposition Center Accession Number FERM P-20708 ).
The embodiment of Item 2 includes a Rhodococcus sp. RN1 strain, which is the microorganism according to any one of Items 1A to 1D.

Item 3A: A microorganism belonging to the genus Gordonia,
When inoculating 1 × 10 ⁸ microorganisms / g-soil of the microorganism into 50 g of soil with an oil concentration of about 10,000 ppm, the residual oil concentration in the soil after 14 days is 4,000 ppm or less, preferably 3,800 ppm or less, more preferably The number of soil bacteria determined on the basis of the amount of DNA per unit weight of sample collected from the soil is 3.5 × 10 ⁸ cells / g-soil or more, preferably 3.6 × 10 ⁸ cells / g-soil or more. More preferably, a soil purification microorganism having a soil purification ability of 3.8 × 10 ⁸ cells / g-soil or more.

  Item 3B: 28 days after inoculation of 1% (v / w) of the microorganism into soil with 1% (w / w) of cycloalkane added to 100 g of sterilized soil (autoclaved at 121 ° C for 15 minutes) Item 3. The microorganism according to Item 3A, which has a decomposition ability of a cycloalkane decomposition rate of 15% or more, particularly 17% or more.

  In a preferred embodiment, when 1% (v / w) of the microorganism is inoculated into soil obtained by adding 1% (w / w) of cycloalkane to 100 grams of sterilized soil (121 ° C, 15 minutes autoclaved), 14% Item 3. The microorganism according to Item 3A, which has a degradation ability of a cycloalkane degradation rate of 2.7% or more, particularly 3% or more, further 4% or more, and a cycloalkane degradation rate after 28 days of 15% or more, particularly 17% or more.

Item 3C: alk1 gene containing DNA of the following (b) or (b1):
(B) DNA consisting of the base sequence shown in SEQ ID NO: 3,
(B1) DNA that hybridizes with a DNA comprising the nucleotide sequence shown in SEQ ID NO: 3 under stringent conditions and encodes a protein having a hydrocarbon decomposing activity
And the alk2 gene containing DNA of the following (c) or (c1):
(C) DNA consisting of the base sequence shown in SEQ ID NO: 4
(C1) DNA that hybridizes with a DNA comprising the nucleotide sequence shown in SEQ ID NO: 4 under a stringent condition and encodes a protein having a hydrocarbon decomposing activity
Item 3. The microorganism according to Item 3A or 3B, comprising:

  Item 3D: The microorganism according to any one of Items 3A to 3C, which has 16S rDNA containing the base sequence represented by SEQ ID NO: 5.

Item 4: Gordonia sp. RN2 strain having soil remediation ability (Independent Administrative Institution National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center Accession Number FERM P-20709 ).
The embodiment of Item 4 includes a Gordonia sp. RN2 strain which is the microorganism according to any one of Items 3A to 3D.

  Item 5: A soil purification method comprising introducing the microorganism according to any one of Items 1A to 1D, Item 2, Item 3A to 3D, or Item 4 into a target soil.

  Item 5A: A soil purifier containing the microorganism according to any one of Items 1A to 1D, Item 2, Items 3A to 3D, or Item 4.

Item 6A: The test microorganism is put into the soil,
A method for screening a soil-purifying microorganism, comprising evaluating a test microorganism using as an index the number of soil bacteria determined based on the amount of DNA per unit weight of a sample collected from the soil into which the microorganism has been introduced.

  Item 6B: The screening method according to Item 6A, wherein the test microorganism is evaluated using the decomposition rate of the contaminant in the soil as an index in addition to the number of soil bacteria.

  Item 7: A soil purification method, wherein a microorganism screened by the method according to Item 6A or 6B is introduced into a target soil.

  Item 8A: Using the number of soil bacteria obtained based on the amount of DNA per unit weight of the sample collected from the target soil as an index, (i) input of an indigenous microorganism activating component into the target soil, and (ii) pollutant resolution A soil purification method comprising performing at least one treatment selected from the introduction of microorganisms.

  Item 8B: When the number of soil bacteria falls below a preset reference value, at least one selected from (i) input of an indigenous microorganism activating component and (ii) input of a microorganism having a pollutant resolving power to the target soil. The soil purification method according to Item 8A, wherein the treatment is performed.

  Item 8C: Using the number of soil bacteria obtained based on the amount of DNA per unit weight of the sample collected from the target soil and the concentration of contaminants in the soil as indicators, (i) input of an indigenous microorganism activating component into the target soil, and (Ii) The purification method according to Item 8A or 8B, wherein at least one treatment selected from the introduction of microorganisms having a contaminant resolution is performed.

  Item 8D: The purification method according to any one of Items 8A to 8C, wherein the contaminant is oil.

  Item 8E: The purification method according to any one of Items 8A to 8D, wherein the microorganism having a contaminant resolution is the soil purification microorganism according to any one of Items 1A to 1D, Item 2, Item 3A to 3D, or Item 4.

Item 8F: having a step of monitoring the time-dependent change of the number of soil bacteria,
Using the number of soil bacteria to be monitored as an index, the target soil is subjected to a process of performing at least one process selected from (i) input of an indigenous microorganism activating component and (ii) input of a microorganism having a pollutant resolution ability. Item 8. The method for purifying soil according to any one of Items 8A to 8E.

In other words, (1) the number of soil bacteria determined based on the amount of DNA per unit weight collected from the target soil, the process of monitoring changes over time in the number of soil bacteria, and the number of soil bacteria monitored as an index Item 8A to 8E, wherein the soil is subjected to at least one treatment selected from (i) input of an indigenous microorganism-activating component and (ii) input of a microorganism having a pollutant resolving power. Soil purification method.

  Item 8G: The purification method according to any one of Items 8A to 8F, further comprising a step of diagnosing the state of soil purification using the number of soil bacteria as an index.

Hereinafter, the present invention will be described in more detail.
Number of soil bacteria In the present invention, the number of soil bacteria refers to the soil determined based on the amount of DNA present per unit weight of sample collected from the target soil (hereinafter also referred to as “environmental DNA amount” or “eDNA amount”). Represents the number of bacteria inside. In the present specification, the number of soil bacteria may be referred to as the number of soil microorganisms.

  When the unit weight is 1 g, the number can be expressed in units of the number per unit weight (cells / g-soil or cells / g-sample) of the target soil (or sample).

  In addition, the amount of DNA here refers to the amount of DNA present per unit weight of the sample collected from the target soil. More specifically, the total amount of DNA present per unit weight of the sample is shown regardless of the origin of the DNA.

  The number of soil bacteria can be determined by converting the amount of DNA present per unit weight of sample collected from the target soil by an appropriate technique.

  For example, by using a measuring means such as a microscope, the correlation between the number of soil bacteria in the soil and the amount of DNA is obtained in advance, and the DNA amount measured from the collected sample is collated with the correlation, Can be sought.

  In an example of a preferred embodiment, the number of soil bacteria is determined by converting the amount of DNA per unit weight of a sample collected from the target soil according to the following (formula 1).

  The total amount of DNA derived from all bacteria present in the sample reflects the overall characteristics and status of the target soil. Therefore, the number of soil bacteria obtained based on the amount of DNA present per unit weight of sample collected from the target soil is an index for grasping the characteristics of the soil and the working conditions of the bacteria in the soil.

For example, the number of soil bacteria present in the soil of the field is about 1.0 × 10 ¹⁰ cells / g-soil, whereas the number of soil bacteria in the contaminated soil is about 1.0 × 10 ⁷ cells / g-soil.

  In soil such as paddy fields and farmland, the larger the number of soil bacteria, the better the growth of crops and the higher the yield. In contaminated soil, the higher the degree of contamination, the smaller the number of soil bacteria.

  Thus, the number of soil bacteria is a suitable index indicating the condition of the soil and the condition of the bacteria in the soil.

  The actual soil is in a complex environment due to changes in the composition of pollutants, microbiota in the soil, and purification of large-scale bioremediation. For this reason, conventionally, it has been difficult to numerically grasp and compare soil characteristics and bacterial conditions. However, by using the number of soil bacteria, it is possible to numerically grasp and evaluate the actual condition of the soil in the field and the condition of the bacteria in the soil.

The type of the sample target soil collected from the target soil is not particularly limited, and is appropriately selected and set from soil that requires purification and contaminated soil. In particular, the present invention can be effectively used for actual contaminated soil, for example, actual petroleum contaminated soil, which is soil in an actual contaminated site. The actual contaminated soil includes, for example, soil in a factory site, factory site, gas station site, incinerator, and the like.

  The type of contaminant contained in the target soil is not particularly limited, but specific substances include, for example, petroleum and oil. Oils include hydrocarbons and hydrocarbon derivatives extracted with n-hexane, carbon tetrachloride and the like. In addition, oils include aliphatic hydrocarbons and alicyclics derived from fuel oils such as crude oil, heavy oil, light oil, kerosene and gasoline, mineral oil such as engine oil and lubricating oil, and animal and vegetable oils of food such as lard and salad oil. Also included are hydrocarbons such as formula hydrocarbons, aromatic hydrocarbons, polycyclic aromatic hydrocarbons (PAHs) and hydrocarbon derivatives. Alicyclic hydrocarbons include cycloalkanes and cycloalkenes.

  The sample collected from the target soil is the soil collected (sampled) from the target soil. The collection method is not particularly limited, and can be appropriately performed according to a known method.

  Although the collection conditions can also be set as appropriate, from the viewpoint of appropriately judging the state of microorganisms in the target soil, it is preferable to collect the sample while avoiding the time when the target soil is not in a normal state due to rain or the like.

DNA amount per unit weight of sample The amount of DNA per unit weight of sample collected from the target soil can be measured by eluting the DNA present in the sample collected from the soil to be diagnosed and quantifying the amount of the DNA. it can.

  It is desirable to measure the amount of DNA in a sample collected from the target soil immediately after obtaining the sample. However, the obtained sample is subjected to low temperature (for example, about −4 to −80 degrees, preferably −20 to −80). It can be stored for about 1 day to 3 weeks.

  The method for eluting DNA from all microorganisms contained in the sample is not particularly limited as long as the DNA is not significantly degraded or sheared and adversely affects its quantification.

  For example, one embodiment of the DNA elution method includes a method of treating the sample with a DNA elution solution.

  Examples of the DNA elution solution used here include solutions generally used for eluting DNA from bacteria.

  Specifically, as the DNA extraction solution, an inhibitor of a DNA degrading enzyme such as EDTA or EGTA, a cationic surfactant, a solution containing an anionic surfactant and / or a buffer containing them, etc. Can be used. The buffer can also contain a proteolytic enzyme such as proteinase K, thermolysin, and subtilisin. The blending ratio of each component can be appropriately set within a range that does not significantly impair DNA extraction.

  In the DNA elution treatment using the DNA elution solution, the elution conditions for DNA are not particularly limited. For example, DNA can be eluted by adding 2 to 20 ml, preferably 5 to 15 ml, more preferably 8 to 12 ml of the above DNA extraction solution to 1 g of soil subjected to elution treatment. .

  Further, the elution temperature can be appropriately set according to the DNA elution solution to be used, the kind of soil used for the elution treatment, and the like.

  The elution time varies depending on the type of DNA extraction solution to be used, the type of soil subjected to elution treatment, the elution temperature, etc., and cannot be defined uniformly, but as an example, 0.1 to 4 hours, preferably 0.2 Up to 2 hours, more preferably 0.3 to 1 hour can be mentioned.

  By quantifying the DNA thus eluted, the amount of DNA present in the target soil can be determined.

  The DNA quantification method is not particularly limited, and for example, the eluted DNA can be purified and collected as necessary, and quantified by a known or conventional DNA quantification method.

  Specifically, as a method for quantifying DNA, after the purified DNA is subjected to agarose gel electrophoresis, the DNA is stained with ethidium bromide and the fluorescence intensity of the DNA band on the gel is measured. The method of doing can be mentioned.

  Moreover, for example, a method of dissolving the DNA recovered by purification in a buffer solution and measuring the absorbance at 260 nm of the solution can also be mentioned.

  The method for purifying DNA is not particularly limited, and can be performed according to a conventional method. For example, as a method for purifying DNA, a step of centrifuging the solution after the elution of DNA as described above and collecting the supernatant; chloroform, chloroform- A step of adding and mixing an impurity removal solution that separates the supernatant from the supernatant such as isoamyl alcohol or phenol; and a step of removing impurities by taking out a layer containing DNA from the mixed solution obtained in the step And a method including a step of adding a DNA precipitating agent such as isopropyl alcohol, ethanol or polyethylene glycol to the DNA-containing layer obtained in the above step to precipitate the DNA and recovering the DNA.

  Since the DNA extraction efficiency may vary depending on the type of target soil, the DNA extraction efficiency in each sample should be measured in advance and corrected for each target sample based on the extraction efficiency. It is desirable to determine the amount of DNA.

  The DNA extraction efficiency here means the ratio of the amount of DNA actually eluted and quantified from the sample to the amount of DNA contained in the sample collected from the target soil.

From the amount of DNA measured as described above, the number of soil bacteria can be determined according to the method described above.

Soil purification microorganism According to the present invention, a microorganism suitable for purification of actual contaminated soil, particularly actual petroleum contaminated soil is provided.

  In order for purification of actual contaminated soil to be performed suitably, it is required that the microorganisms used for purification not only have the resolution of pollutants, but also effectively demonstrate the ability at the actual contamination site. The ability can be appropriately characterized by using, as an index, the number of soil bacteria obtained based on the amount of DNA present per unit weight of the sample collected from the target soil.

(I) Soil-purifying microorganisms belonging to the genus Rhodococcus
A microorganism belonging to the genus Rhodococcus,
When inoculating 1 x 10 ⁸ microorganisms / g-soil of the microorganism to 50 g of soil with an oil concentration of about 10,000 ppm, the residual oil concentration in the soil after 14 days is 4,500 ppm or less, preferably 4,000 ppm or less, more preferably 3,800 ppm. The number of soil bacteria determined on the basis of the amount of DNA per unit weight of the sample collected from the soil is 3.5 × 10 ⁸ cells / g-soil or more, preferably 4.0 × 10 ⁸ cells / g-soil or more. Preferably, soil purification microorganisms having a soil purification ability of 5.0 × 10 ⁸ cells / g-soil or more are included.

  Here, the oil concentration indicates a value obtained by analyzing the soil (sample) by gas chromatography (GC), measuring the peak area, and applying it to the following (Equation 2). Gas chromatographic analysis conditions are as shown in Table 1. Simulated contaminated soil is prepared by adding A heavy oil to silica sand (EX NANIWA, Osaka) to 10,000 ppm in advance. The oil concentration is calculated by measuring and correcting the moisture content of the sample.

  In this specification, the soil purification capacity is not limited to the ability to decompose pollutants, but is an ability that comprehensively evaluates the viability in soil and the resistance to pollutants.

  It is preferable that the microorganism is particularly excellent in the ability to decompose cycloalkane, which is a hardly decomposable hydrocarbon.

Specifically, in a preferred embodiment of the soil purification microorganism, 1% (w / w) of cycloalkane was added to 100 g of sterilized soil (121 ° C., 15 minutes autoclave sterilization). v / w) In the case of inoculation, those having the ability to degrade the cycloalkane degradation rate to 23% or more, particularly 23.3% or more after 28 days are included.

  In particular, when inoculating 1% (v / w) of the microorganism into soil obtained by adding 1% (w / w) of cycloalkane to 100 g of sterilized soil (121 ° C, autoclaved for 15 minutes), 14 days later It is preferable that the cycloalkane decomposition rate is 18% or more, particularly 20% or more, and further 21% or more, and the cycloalkane decomposition rate after 28 days is 23% or more, particularly 23.3% or more.

In the present specification, the cycloalkane decomposition rate is calculated by the following method using a chloroform / methanol extraction method.

  Make a mixture at a ratio of chloroform: methanol = 3: 1, add 30 ml of chloroform-methanol mixture to the sample to be measured, and stir well. Then, it is placed in a 300 ml chloroform-methanol extraction centrifuge tube. Centrifuge at 4,000 × g for 30 minutes at a temperature of 20 ° C. The upper aqueous layer portion is removed, and the intermediate layer and the lower layer are transferred to a 50 ml centrifuge tube and centrifuged at 10,000 × g for 10 minutes. The upper layer and the intermediate layer are removed, and the lower chloroform layer is placed in a petri dish previously weighed and dried at room temperature for 24 hours. Measure the weight of the petri dish after drying and removing the chloroform. For comparison, a sample not inoculated with a strain is used as a control.

  By putting the measured numerical value into the following formula, the decomposition rate is obtained.

  A preferred embodiment of the soil purification microorganism belonging to the genus Rhodococcus includes a microorganism having an alk gene.

Here, the alk gene is DNA of the following (a) or (a1):
(A) DNA consisting of the base sequence shown in SEQ ID NO: 1
(A1) DNA that hybridizes with a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 under a stringent condition and encodes a protein having cycloalkane-degrading activity
It is a gene containing

  Among microorganisms belonging to the genus Rhodococcus, those having the alk gene are particularly excellent in cycloalkane decomposition ability.

  In the present specification, “stringent conditions” refers to conditions under which only specific hybridization occurs and non-specific hybridization does not occur. Such conditions are usually about "1xSSC, 0.1% SDS, 37 ° C", preferably about "0.5xSSC, 0.1% SDS, 42 ° C", more preferably "0.2xSSC, 0.1% SDS, 65 ℃ ”.

  DNA that hybridizes under stringent conditions with DNA consisting of the base sequence shown in SEQ ID NO: 1 usually has high homology with DNA consisting of the base sequence shown in SEQ ID NO: 1.

  In the present specification, high homology refers to 90% or more homology, preferably 95% or more, more preferably 97% or more.

In addition, preferred embodiments of the soil purification microorganism include those having 16SrDNA containing the base sequence shown in SEQ ID NO: 2, in particular, having the alk gene and having 16SrDNA containing the base sequence shown in SEQ ID NO: 2. Microorganisms are preferred because they have a high cycloalkane-degrading ability in real petroleum-contaminated soil.

  A preferred example of the soil purification microorganism of the present invention includes Rhodococcus RN1 strain.

  Rhodococcus RN1 strain has 16SrDNA containing the above-mentioned alk gene and the nucleotide sequence shown in SEQ ID NO: 2, and is superior to known microorganisms belonging to the genus Rhodococcus, and has excellent purification ability in real oil-contaminated soil, and cycloalkane. Excellent resolution.

In more detail, Rhodococcus RN1 strain exhibits excellent soil remediation ability in actual contaminated soil. After inoculating 1 × 10 ⁸ microorganisms / g of the microorganism into 50 g of soil with an oil concentration of about 10,000 ppm, 14 days later The soil has a residual oil concentration of 3,800 ppm or less, and the number of soil bacteria determined based on the amount of DNA per unit weight of the sample collected from the soil is 5.0 × 10 ⁸ cells / g-soil or more. .

  Rhodococcus RN1 strain is inoculated with 1% (w / w) of the microorganism in a soil obtained by adding 1% (v / w) of cycloalkane to 100 grams of sterilized soil (121 ° C, 15 minutes autoclaved). In this case, it has the ability to degrade the cycloalkane degradation rate after 14 days to 21% or more and the cycloalkane degradation rate after 28 days to 23% or more.

This strain has been deposited as Rhodococcus sp. RN1 strain ( Accession No. FERM P-20708 ) at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Deposit Date: November 10, 2005).

(Ii) Microorganisms belonging to the genus Gordonia
A microorganism belonging to the genus Gordonia,
When inoculating 1 × 10 ⁸ microorganisms / g-soil of the microorganism into 50 g of soil with an oil concentration of about 10,000 ppm, the residual oil concentration in the soil after 14 days is 4,000 ppm or less, preferably 3,800 ppm or less, more preferably The number of soil bacteria determined on the basis of the amount of DNA per unit weight of sample collected from the soil is 3.5 × 10 ⁸ cells / g-soil or more, preferably 3.6 × 10 ⁸ cells / g-soil or more. More preferably, a soil purification microorganism having an ability of 3.8 × 10 ⁸ cells / g-soil or more is included.

  Here, the oil concentration is a value obtained by analyzing the soil (sample) by gas chromatography (GC) under the conditions shown in Table 1 and measuring the peak area in the same manner as described above, and applying it to the above (Equation 2). Means.

  In particular, it is preferable that the microorganism is excellent in resolution of cycloalkane, which is a hardly decomposable hydrocarbon.

  In a preferred embodiment, when 1% (v / w) of the microorganism is inoculated in a soil obtained by adding 1% (w / w) of cycloalkane to 100 grams of sterilized soil (121 ° C, 15 minutes autoclaved), Included are microorganisms that have a degradation ability of a cycloalkane degradation rate of 15% or more, particularly 17% or more after 28 days.

  In particular, when inoculating 1% (v / w) of the microorganism into soil obtained by adding 1% (w / w) of cycloalkane to 100 g of sterilized soil (121 ° C, autoclaved for 15 minutes), 14 days later It is preferable that the cycloalkane decomposition rate is 2.7% or more, particularly 3% or more, and further 4% or more, and the cycloalkane decomposition rate after 28 days is 15% or more, particularly 17% or more.

  In addition, preferred embodiments of the soil purification microorganism belonging to the genus Gordonia include those having alk1 gene and alk2 gene.

Here, the alk1 gene is a gene containing the following DNA (b) or (b1).
(B) DNA consisting of the base sequence shown in SEQ ID NO: 3
(B1) DNA that hybridizes with a DNA comprising the nucleotide sequence shown in SEQ ID NO: 3 under stringent conditions and encodes a protein having cycloalkane-degrading activity
The alk2 gene is a gene containing the following DNA (c) or (c1).
(C) DNA consisting of the base sequence shown in SEQ ID NO: 4
(C1) Hybridizes under stringent conditions with the DNA comprising the nucleotide sequence shown in SEQ ID NO: 4
DNA encoding a protein that is soybean and has cycloalkane-degrading activity.

  A DNA that hybridizes under stringent conditions with a DNA consisting of the base sequence shown in SEQ ID NO: 3 usually has high homology with a DNA consisting of the base sequence shown in SEQ ID NO: 3.

  DNA that hybridizes under stringent conditions with DNA consisting of the base sequence shown in SEQ ID NO: 4 usually has high homology with DNA consisting of the base sequence shown in SEQ ID NO: 4.

  Among microorganisms belonging to the genus Gordonia, those having both the alk1 gene and the alk2 gene are excellent in cycloalkane degradation ability.

  In addition, the soil purification microorganism of the present invention belonging to the above genus Gordonia preferably has 16SrDNA containing the base sequence shown in SEQ ID NO: 5.

  In particular, a microorganism having 16SrDNA having the alk1 gene and the alk2 gene and including the base sequence shown in SEQ ID NO: 5 is preferable because it has a high ability to degrade cycloalkane in actual contaminated soil.

  A preferred example of the soil purification microorganism in the present invention includes Gordonia RN2 strain.

  The Gordonia RN2 strain has the alk1 gene and the alk2 gene, and has the 16SrDNA shown in SEQ ID NO: 5, which is superior to the known microorganism belonging to the genus Gordonia, and has an excellent purification ability in real oil-contaminated soil, Excellent cycloalkane decomposition ability.

In more detail, Gordonia RN2 strain shows excellent soil remediation ability in actual contaminated soil. After inoculating 1 × 10 ⁸ microorganisms / g of the microorganism into 50 g of soil with an oil concentration of about 10,000 ppm, 14 days later The soil has a residual oil concentration of 3,500 ppm or less, and the number of soil bacteria determined based on the amount of DNA per unit weight of sample collected from the soil is 3.8 × 10 ⁸ cells / g-soil or more.

  In addition, Gordonia RN2 strain is inoculated with 1% (v / w) of the microorganism in a soil obtained by adding 1% (w / w) of cycloalkane to 100 g of sterilized soil (121 ° C, 15 minutes autoclaved). In this case, the cycloalkane decomposition rate after 14 days is 4% or more, and the cycloalkane decomposition rate after 28 days is 15% or more.

This strain has been deposited as Gordonia sp. RN2 ( Accession No. FERM P-20709 ) at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Deposit date: November 10, 2005).

Soil purification agent The soil purification microorganism of the present invention can be made into a soil purification agent by appropriately formulating it according to a known method.

  The soil purification agent of the present invention contains the above soil purification microorganism as an active ingredient, and is suitably used for purification of actual contaminated soil.

  The soil purification agent may be composed of the soil purification microorganism itself, or may contain other components such as pharmaceutically acceptable carriers and additives. Moreover, what contains only 1 type of purification | cleaning microorganisms may be included, and may further contain the microorganisms which have another soil purification ability, and the microorganisms which have a pollutant resolution | decomposability.

  Examples of other components include a suitable medium, organic compounds, inorganic compounds, and / or fertile soil.

  The blending ratio of these other components can be appropriately set within the range where the effects of the present invention are exhibited.

  The form of the soil purification agent is not particularly limited, and can be used, for example, as a solid preparation, a liquid preparation, or the like.

According to the screening method of the present invention soil remediation microorganisms, by an index number soil bacteria, a method for efficiently screening a suitable microorganism to clean the actual contaminated soil is provided.

  In the screening method of the present invention, a test microorganism is introduced into the soil, and the soil purification of the test microorganism is performed using as an index the number of soil bacteria determined based on the amount of DNA per unit weight of the sample collected from the soil into which the microorganism has been introduced. It is characterized by evaluating ability.

Specific embodiments of the screening method in the present invention include:
Putting the test microorganisms into the target soil;
Measuring the amount of DNA per unit weight of the sample collected from the target soil, and determining the number of soil bacteria based on the amount of DNA;
Monitoring the time-dependent change in the number of soil bacteria, and evaluating the soil purification ability of the test microorganism from its behavior;
A method is included.

The soil purification ability can be evaluated as follows, for example.
(1) In monitoring, when the number of soil bacteria increases, it is evaluated that the soil purification capacity is high, and when the number of soil bacteria decreases, it is evaluated that the soil purification capacity is low.
(2) In monitoring, if the soil bacteria count exceeds a preset reference value, it is evaluated that the soil purification capacity is high, and if the soil bacteria count does not meet the preset reference value, soil purification is performed. Assess that ability is not enough.

  By performing such evaluation and selecting microorganisms that are evaluated to have high soil purification capacity, microorganisms suitable for soil purification can be efficiently screened.

  Since the number of soil bacteria reflects the overall characteristics and conditions of the target soil, the soil purification capacity can be appropriately evaluated.

  For example, even if the microorganism has a high resolution of pollutants, it is difficult to sufficiently increase the number of soil bacteria for microorganisms that cannot sufficiently grow at the site of actual contamination. On the other hand, microorganisms having the ability to increase the number of soil bacteria exhibit sufficient work at the actual contamination site, and can make the soil better.

  Therefore, the screening method of the present invention using the number of soil bacteria as an index makes it possible to efficiently select microorganisms suitable for purification of actual contaminated soil.

  In the screening method of the present invention, microorganisms may be evaluated by adding an appropriate index other than the number of soil bacteria.

  For example, it is possible to monitor the time-dependent change in the concentration of contaminants in the soil together with the number of soil bacteria, and to evaluate the soil purification ability of microorganisms using the degradation rate of the contaminants as an index in addition to the number of soil bacteria.

  At this time, by monitoring the concentration for each contaminant, it is possible to evaluate which substance the microorganism has the ability to selectively decompose.

  Examples of microorganisms to be screened by the above method include Gordonia sp. RN2 strain and Rhodococcus sp. RN1 strain.

  Furthermore, the soil purification method including introducing the microorganisms obtained by such screening into the soil can be suitably used particularly as a purification method for actual contaminated soil.

Soil purification method According to the present invention, the amount of DNA present per unit weight of a sample collected from the target soil is measured, and the number of soil bacteria determined based on the amount of DNA is used as an index to treat soil purification. The soil purification method characterized by performing is provided.

  As described above, the number of soil bacteria reflects the situation of the soil. For this reason, soil purification can be efficiently performed by performing the treatment for soil purification using the number of soil bacteria as an index.

  The procedure and content of the treatment for soil purification may be set in advance as a predetermined pattern, or may be set as appropriate while monitoring the monitoring status.

(1) Content of treatment A preferred embodiment of the soil purification treatment includes performing at least one treatment selected from (i) input of an indigenous microorganism activating component, and (ii) input of a microorganism having a pollutant resolution ability. It is.

  In other words, either the process (i) or (ii) may be performed, and the processes (i) and (ii) may be performed simultaneously.

(I) Input of an indigenous microorganism activating component The indigenous microorganism activating component is not particularly limited as long as it activates microorganisms living in the soil and promotes decomposition of the pollutant or purification of the soil.

  Specifically, the indigenous microorganism activating component includes an organic compound, an inorganic compound, and the like. It also contains moisture and oxygen.

  Among these, an organic compound or a mixture of an organic compound and an inorganic compound is particularly preferably used because the growth of indigenous microorganisms proceeds more effectively.

  The charging method is not particularly limited, and can be appropriately set according to a known method. For example, the activating component may be mixed in advance in a suitable soil and added in the form of fertile soil. Further, it may be added after dissolving in an appropriate solvent.

  In addition, a plurality of activation components may be added to the soil all together in a premixed form, or may be added to the soil in multiple batches.

  Further, the input amount can also be set as appropriate within the range where the effects of the present invention are exhibited.

(Ii) Inputting microorganisms having a contaminant resolution The microorganisms having a contaminant resolution are not particularly limited as long as they decompose the contaminants contained in the target soil and promote the purification of the soil. It can be selected appropriately. Further, the input amount can also be set as appropriate within the range where the effects of the present invention are exhibited.

  However, it is preferable that the type and amount of microorganisms to be input are set within a range in which the influence on the external environment is limited and safety is confirmed.

  Microorganisms having a contaminant resolution include microorganisms having an oil resolution and microorganisms having a hydrocarbon resolution.

  Moreover, in the soil purification method of this invention, the soil purification microorganism of the said this invention and the soil purification microorganism obtained by the screening method of this invention can be used suitably as microorganisms which have a pollutant resolution | decomposability.

  More specifically, the microorganism having a pollutant resolution used in the soil purification method of the present invention is a microorganism belonging to the genus Rhodococcus, a microorganism having the alk gene, 16SrDNA containing the base sequence represented by SEQ ID NO: 2 And microorganisms having Rhodococcus RN1.

  In addition, the microorganism having a pollutant resolution used in the soil purification method of the present invention is a microorganism belonging to the genus Gordonia, a microorganism having the alk1 gene and the alk2 gene, 16SrDNA containing the base sequence represented by SEQ ID NO: 5 And microorganisms having Gordonia RN2.

  The method for introducing microorganisms is not particularly limited, and can be appropriately set according to a known method. For example, there can be mentioned a method in which microorganisms are mixed with an appropriate medium or fertilizer solution or a solution in which the microorganisms are dissolved, and the microorganism-containing solution is seeded on soil. Or the method of inject | pouring and press-fit | injecting this microorganism-containing solution from the pipe embedded and inserted in soil is mentioned. Moreover, the method of processing microorganisms with a suitable binder or support | carrier in the solid form which is easy to use, and spraying this on soil and mixing is mentioned. Water is added to the soil to form a slurry, and a method of adding the above-described bacterial solution or the cells processed into a solid form or the cultured cell culture solution while stirring is employed. Examples of solid forms include pellets, powders, and fibers.

(2) Reference value When performing soil remediation treatment, it is possible to easily grasp the exact timing of soil remediation treatment by setting a reference value as a guide using the number of soil bacteria. Become.

  The reference value can be set as appropriate in consideration of soil characteristics and conditions. For example, a higher reference value can be set if the soil has already been subjected to soil purification treatment, and a lower reference value can be set if the soil is seriously contaminated.

Generally, the number of microorganisms present in the soil of the field is about 1.0 × 10 ¹⁰ cells / g-soil, and the number of microorganisms in the contaminated soil is about 1.0 × 10 ⁷ cells / g-soil. Therefore, it is usually possible to set the reference value within the range of about 1.0 × 10 ^{7 to} 1.0 × 10 ¹⁰ cells / g-soil, especially about 1.0 × 10 ^{8 to} 1.0 × 10 ⁹ cells / g-soil. preferable. An example of the reference value is 1.0 × 10 ⁸ cells / g-soil.

  The reference value can be set in advance in consideration of the characteristics of the target soil, or can be set as appropriate while monitoring and taking into account fluctuations in the number of soil bacteria.

  One or more reference values can be set. For example, several stages of reference values can be set according to the soil purification status. In this case, if the number of soil bacteria falls below the first stage reference value, the first additional process is performed, and if the number of soil bacteria falls below the second stage reference value, the second additional process is performed. It can also be set.

(3) Monitoring The soil purification method of the present invention can be carried out while measuring the number of soil bacteria over time and monitoring its behavior. While soil bacteria act to break down pollutants, they are also affected by the toxicity of the pollutants. Therefore, it is possible to perform efficient bioremediation by monitoring, understanding the progress of microorganisms present in the soil and the progress of purification, and performing treatment in a direction in which the microorganisms function effectively. .

Examples of aspects of soil purification methods for monitoring include methods comprising the following steps;
Measuring the amount of DNA per unit weight of the sample collected from the target soil, and determining the number of soil bacteria based on the amount of DNA;
Monitoring the time course of the soil bacteria count; and
When the monitored number of soil bacteria is lower than a preset reference value, at least one selected from (i) input of an indigenous microorganism activating component and (ii) input of a microorganism having a pollutant resolution to the target soil. Processing steps for processing,
A method for purifying soil.

  The monitoring method is not particularly limited, and can be performed according to a known method as appropriate. For example, the number of soil bacteria may be monitored using a further converted value. Moreover, it can also monitor using display means, such as a suitable graph or a figure.

  Monitoring can also be performed using one or more indices in addition to the number of soil bacteria. For example, in addition to the number of soil bacteria, the change with time in the concentration of contaminants in the soil may be monitored. Contaminant concentration includes oil concentration.

(4) Initial treatment In the soil purification method of the present invention, an appropriate treatment may be applied to the target soil in advance before measuring the amount of DNA for determining the number of soil bacteria.

  For example, the target soil is subjected to at least one treatment selected from (i) the introduction of an indigenous microorganism activating component and (ii) the introduction of a microorganism having a contaminant resolving power as an initial treatment. You can also.

  After performing the initial treatment, it is possible to grasp whether or not the initial treatment was suitable for purification of the target soil by monitoring the number of soil bacteria. In addition, the content of the additional process can be set in consideration of how the soil changes the situation with respect to the initial process.

(5) Additional processing After initial processing, the change in the number of soil bacteria over time is monitored, and when the number of soil bacteria falls below a preset reference value or when it is determined that further processing is necessary, Additional processing can be performed.

  Specifically, as the additional process, at least one process selected from (i) input of an indigenous microorganism activating component and (ii) input of a microorganism having a pollutant resolution can be performed.

  The contents of the additional process may be the same as or different from the contents of the initial process. Further, the additional process may be performed once or a plurality of times.

  For example, in the initial process, a microorganism belonging to the genus Gordonia having a contaminant resolution can be introduced, and in the additional process, a microorganism belonging to the genus Rhodococcus having a contaminant resolution can be introduced.

  In addition, in the initial treatment, fertilized soil is introduced, in the first additional treatment, microorganisms belonging to the genus Gordonia having a contaminant resolution are introduced, and in the second additional treatment, the genus Rhodococcus having a contaminant resolution is introduced. The microorganisms to which it belongs can also be input.

  Moreover, the following aspects can also be mentioned.

An initial treatment is performed in which the target soil is charged with (1-i) an indigenous microorganism-activating component and (1-ii) a microorganism belonging to the genus Gordonia having a pollutant resolving power;
Measuring the amount of DNA per unit weight of the sample collected from the soil subjected to the initial treatment, and determining the number of soil bacteria based on the amount of DNA;
Monitoring the time course of the soil bacteria count;
When the number of soil bacteria is less than or equal to a preset reference value, (2-i) input of an indigenous microorganism activating component and (2-ii) input of a microorganism belonging to the genus Gordonia having a contaminant resolving power Perform additional processing;
Measuring the pollutant concentration of a sample collected from the soil subjected to the first additional treatment and monitoring its change over time;
If the pollutant concentration no longer fluctuates, (3-i) perform the second additional process of charging the indigenous microorganism activating component and (3-ii) charging the microorganism belonging to the genus Rhodococcus having the pollutant resolution ability;
Measuring the amount of DNA per unit weight of the sample collected from the soil subjected to the second additional treatment, and determining the number of soil bacteria based on the amount of DNA;
Monitoring the time course of the soil bacteria count;
When the number of soil bacteria is less than or equal to a preset reference value, (4-i) input of an indigenous microorganism activating component and (4-ii) input of a microorganism belonging to the genus Rhodococcus having a contaminant resolving power A soil purification method comprising performing an additional treatment.

  In addition, the said aspect is an illustration to the last, and does not restrict | limit this invention at all.

(6) Diagnosis In the method of the present invention, the soil purification status can also be diagnosed by using the number of soil bacteria as an index. For example, after the soil purification treatment as described above is performed, if the number of soil bacteria exceeds a certain reference value, it can be diagnosed that the soil has sufficiently recovered. In addition, if the number of soil bacteria does not satisfy a certain reference value, it can be diagnosed that the restoration of the soil is not sufficient. As described above, by using the number of soil bacteria as an index, it is possible to evaluate and diagnose the comparison and comparison of soil purification status, which has been difficult in the past, using a numerical standard.

  As described above, the present invention provides a bioremediation technique suitable for actual contaminated soil by using the number of soil bacteria as an index. In addition, the well-known technique regarding soil purification can be added to this invention as needed within the range with the effect of this invention.

  ADVANTAGE OF THE INVENTION According to this invention, the microorganisms which show the suitable soil purification ability with respect to real-contaminated soil, especially real petroleum-contaminated soil are provided. Furthermore, a microorganism having excellent cycloalkane resolution is provided. By administering the microorganisms to the soil, it becomes possible to efficiently carry out soil purification at the site of actual contamination.

  In addition, according to the present invention, a method for efficiently screening microorganisms suitable for actual contaminated soil is provided. By using the screened microorganisms, it becomes possible to efficiently carry out soil purification at the site of actual contamination.

  Moreover, according to this invention, the soil purification method suitable for purification | cleaning of the actual contaminated soil which uses a soil bacteria number as a parameter | index is provided. By using the number of soil bacteria as an index, the timing and content of treatment for soil purification can be appropriately and easily achieved, and soil purification can be carried out simply and efficiently.

  As described above, the present invention provides a technique that makes it particularly effective and easy to purify actual contaminated soil, and greatly contributes to the practical application and spread of bioremediation.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely using an Example, this invention is not limited to these.

Example 1: Confirmation of taxonomic properties of microorganisms
1-1. Rhodococcus RN1 strain
1-1-1. Phenotype
Biochemical identification of RN1 strain was performed. Various tests are described in Holt, G. et al. , Krieg, N .; R. Sneath, P .; H. A. , Staley, J .; T.A. , And Williams, S .; T.A. (Ed.): Bergey's manual of deterministic bacteria (9th edition) Williams and Wilkins Co. , Baltimore (1994).
The identification results are shown in Table 2 below.

  In the above test, “+” indicates positive and “−” indicates negative.

1-1-2.16 Molecular phylogenetic analysis based on the base sequence of SrDNA This strain was used at 25C using an LB medium (pH 7.0) consisting of 1% peptone, 0.5% yeast extract and 0.5% NaCl. After culturing for 1 day, genome extraction was performed, and a base sequence encoding 16S rDNA was obtained by PCR (Polymerase Chain Reaction) method using a universal cycler (20F, 1510R) with a thermal cycler (Temp / Tronic, manufactured by G Thermoline). Amplified. The obtained PCR product was purified with Q I Aqua ™ PCR Purification Kit (manufactured by GIAGEN) to obtain a template DNA. The template DNA was amplified again by cycle sequence PCR method using Thermo Sequenase pre-mixed cycled sequencing Kit (manufactured by Hitachi Instrument Service).

The reaction solution composition of the cycle sequence method is
ATGC each regent 2μm
Primer 2μm
Template 400-600mg
Sterile distilled water Final 25μl
It was. The reaction conditions were 94 ° C. for 5 minutes, then 94 ° C. for 30 seconds and 60 ° C. for 30 seconds for 25 cycles, then left at 4 ° C.

  A sample obtained by the cycle sequencing method was purified by ethanol precipitation. The base sequence of the purified product was analyzed with a DNA sequencer (SQ 5000E manufactured by Hitachi Instrument Service Co., Ltd.), and the base sequence was determined.

  As a result of analysis by a DNA sequencer, it was confirmed that the gene encoding 16S rDNA of this strain contains the base sequence shown in SEQ ID NO: 2 in the sequence listing.

The nucleotide sequences of 16S rDNA of this strain and other Rhodococcus genus hydrocarbon-degrading bacteria were compared.
Rhodococcus sp. NDKK48 strain (C2 strain described in JP-A-2003-102469) and Rhodococcus sp. ODNM2B strain (Rhodococcus sp. GR-002 strain described in JP-A-2004-113197) and The results of 16S rDNA sequence homology are shown in Table 3 below. The homology is determined by Blast.

  From a comparative study of 16S rDNA sequences with other Rhodococcus genus hydrocarbon-degrading bacteria, it was found that the RN1 strain has high homology with the Rhodococcus sp. Strain. It was identified as Rhodococcus sp. In addition, since there is no strain having 100% homology, the RN1 strain is judged to be a novel strain, and this strain is designated as Rhodococcus sp. RN1 strain.

1-2. Gordonia RN2 stock
1-2-1. Phenotype
Biochemical identification of RN2 strain was performed. Various tests are described in Holt, G. et al. , Krieg, N .; R. Sneath, P .; H. A. , Staley, J .; T.A. , And Williams, S .; T.A. (Ed.): Bergey's manual of deterministic bacteria (9th edition) Williams and Wilkins Co. , Baltimore (1994).
The identification results are shown in Table 4 below.

  In the above test, “+” indicates positive and “−” indicates negative.

1-2.16 Molecular phylogenetic analysis based on the nucleotide sequence of SrDNA The genome of this strain was extracted by the same method as in 1-1-2 above, and the nucleotide sequence encoding 16SrDNA was converted into a PCR (Polymerase Chain). The PCR product was purified by amplification by the Reaction method, and the base sequence of the purified product was analyzed by a DNA sequencer to determine the base sequence.

  As a result of analysis, it was confirmed that the gene encoding 16S rDNA of this strain contains the base sequence shown in SEQ ID NO: 5.

The base sequences in 16S rDNA of this strain and other hydrocarbon-degrading bacteria of Gordonia were compared.
Table 5 below shows the results of homology between the Gordonia sp. NDKK46 strain (Gordonia sp. GR-004 described in JP-A-2004-121068) and the 16S rDNA sequence of this strain. The homology is determined by Blast.

  Comparison with the 16S rDNA sequence of other Gordonia genus hydrocarbon-degrading bacteria shows that the RN2 strain has high homology with the Gordonia sp. Strain. It was identified as Gordonia sp. In addition, since there is no strain having 100% homology with this strain, it was judged as a novel strain and this strain was designated as Gordonia sp. RN2.

Example 2: Evaluation of soil remediation ability of microorganisms
For Rhodococcus sp.RN1 and Gordonia sp.RN2, the following methods and materials were used to compare the soil remediation ability of microorganisms in contaminated soil.

2-1. Materials and analytical methods
2-1-1. As the actual contaminated soil contaminated soil, soil contaminated with heavy fuel oil A carried in from the former factory site was used.

2-1-2: Measurement of Oil Concentration by Gas Chromatography (GC) The oil concentration of the soil was determined by gas chromatographic analysis of the sample and measuring the peak area. Gas chromatographic analysis conditions were as shown in Table 6 below. Simulated contaminated soil was prepared by adding A heavy oil to silica sand (EX NANIWA, Osaka) to 10,000 ppm in advance. Then, the oil peak areas of the sample and the simulated contaminated soil were measured, and the oil concentration was calculated using the following (Formula 2). Each was done in duplicate. Moreover, when calculating the oil concentration, the moisture content of the sample was measured and corrected.

2-1-3. Measurement of soil bacterial count by environmental DNA (eDNA) analysis method The following environmental DNA extraction method was used to extract microbial DNA from 1.0 g of soil and measure the amount of DNA to determine the number of soil bacteria.

  As the soil sample, one sampled from three locations and mixed was used.

  Take 1.0 g of soil sample in a Teflon tube, add 1 ml of 20% (w / v) SDS, 8.0 ml of DNA extraction buffer (see Table 7), and stir (room temperature, 1,500 rpm, 20 min) did. From the stirred solution, 1.5 ml was collected in a 1.5 ml Eppendorf tube and centrifuged (20 ° C., 8,000 rpm, 10 min). 700 μl of the upper layer was taken, an equal volume (700 μl) of chloroform / isoamyl alcohol (24: 1; v / v) was added, and the mixture was centrifuged (20 ° C., 14,000 rpm, 10 min). 500 μl of the upper layer was taken, 0.6 volumes (300 μl) of isopropanol was added, and centrifugation (20 ° C., 14,000 rpm, 20 min) was performed to precipitate the DNA. The supernatant was removed, and 1.0 ml of 70% (v / v) ethanol was added and centrifuged (20 ° C., 14,000 rpm, 5 min) and rinsed. Remove the supernatant, dry for 30 min to remove ethanol, and add TE 10: 1 buffer (Trishydroxymethylaminomethane 1.20 g / l, disodium ethylenediaminetetraacetate 0.37 g / l, pH 8.0) 10 μl was added to obtain a DNA extraction sample.

Agarose HT 1% (w / v), 50 × TAE buffer 2% (v / v) in ion-exchanged water (Trishydroxymethylaminomethane 108.00 g / l, ethylenediaminetetraacetic acid disodium salt 18.60 g / l, CH ₃ COOH 54.32 g / l, pH 8.0) and ethidium bromide 3 mM were added and dissolved by heating to prepare a 1% agarose gel. Mix gel extraction buffer 1.0 μl (bromophenol blue 2.50 g / l, xylene cyanol FF2.50 g / l, ethylenediaminetetraacetic acid disodium salt 3.70 g / l, Ficoll 4001.50 g / l) with 5.0 μl of DNA extraction sample Applied to the gel. Electrophoresis was performed at 100 V for 30 minutes using 1 × TAE buffer as an electrophoresis buffer, and then a DNA band was confirmed with a transilluminator UVP (FUNAKOSHI Co. Ltd, Tokyo). The marker and DNA band were compared, and the fluorescence intensity of DNA was quantified. The marker used was a smart ladder (NIPPON GENE, Toyama).

  The gel after electrophoresis was irradiated with UV, and the fluorescence intensity of the DNA band labeled with ethidium bromide was measured with KODAK 1D Image Analysis software (EASTMAN KODAK, Tokyo). In the analysis, first, a calibration curve of the DNA amount with respect to the fluorescence intensity of the band was created by a smart ladder, and based on this, the DNA amount contained in the band was determined from the fluorescence intensity of the band of each DNA sample. From this result, the amount of DNA per 1.0 g of soil was calculated. Based on the amount of DNA, the number of soil bacteria was determined by the following (formula 1). Duplicates were performed for each sample.

2-2. Experimental method When the concentration of contaminated soil was measured by GC, the oil concentration was 15,900 ppm (saturated hydrocarbon concentration 3,900 ppm, aromatic hydrocarbon concentration 10,500 ppm).

  The contaminated soil and mountain sand were mixed at a ratio of about 2: 1 to prepare soil having an oil concentration of about 10,000 ppm.

50 grams of the prepared soil was put in a 200 ml sample bottle, and each strain was added to 1 × 10 ⁸ pieces / gram-soil. Thereto was added 10 ml of a 4-fold concentrated modified SW medium, which was allowed to stand at room temperature. Samples were stirred with a glass rod every 2 days.

  On the 14th day, the residual oil concentration was analyzed by GC, and the number of soil bacteria was analyzed.

2-3. The results are shown in Table 8 below.

  As a result, it was found that Rhodococcus sp. RN1 strain and Gordonia sp. RN2 strain showed excellent soil remediation ability in actual contaminated soil.

Example 3 Analysis of Cycloalkane Decomposition Ability The cycloalkane decomposition ability of Gordonia sp. RN2 strain and Rhodococcus sp. RN1 strain was examined by the following method.

3-1. analysis method
1% (w / w) of cycloalkane was added to 100 g of sterilized soil (autoclaved at 121 ° C. for 15 minutes). Thereafter, 1% (v / w) of each strain was inoculated, and the cycloalkane degradation rate after 14 and 28 days was determined and evaluated.

  The cycloalkane decomposition rate was calculated by the following chloroform / methanol extraction method.

A mixture of chloroform and methanol was prepared at a ratio of 3: 1, and 30 ml of chloroform-methanol mixture was added to the sample to be measured and stirred well. Then, it put into a 300 ml chloroform-methanol extraction centrifuge tube. Centrifugation was performed at 4,000 × g for 30 minutes at a temperature of 20 ° C. The upper aqueous layer was removed, and the intermediate layer and lower layer were transferred to a 50 ml centrifuge tube and centrifuged at 10,000 × g for 10 minutes. The upper layer and the intermediate layer were removed, and the lower chloroform layer was placed in a petri dish previously weighed and dried at room temperature for 24 hours. The weight of the petri dish after the chloroform was dried and removed was measured. For comparison, a sample not inoculated with the strain was used, and this was used as a control.
The measured numerical value was put into the following formula to determine the decomposition rate.

This operation was performed twice per strain, and the average degradation rate was defined as the cycloalkane degradation rate of the strain.

3-2. The results are shown in Table 9 below.

  As a result, it was found that Rhodococcus sp. RN1 strain and Gordonia sp. RN2 strain have excellent cycloalkane degradation ability.

  As described above, using Gordonia sp. RN2 strain and Rhodococcus sp. RN1 strain, which have been confirmed to have excellent soil remediation ability and excellent cycloalkane decomposition ability in actual contaminated soil, The study of purification was conducted.

Example 4 Comparative Study of Bioremediation in Large Scale Actually Contaminated Soil (480 kg) An experiment was performed on the actual contaminated soil 480 kg by the following method to compare the soil purification ability.

4-1. Experimental materials and methods
4-1-1. Rhodococcus sp. RN1 strain and Gordonia sp. RN2 strain were used as strains used and culture strains. A comparative study was also conducted using Alcaligenessp. ODMI79 strain having cycloalkane degradation ability.

The strain was first cultured at 30 ° C. and 180 rpm using 5 ml of LB medium (polypeptone 10 g / l, dry yeast extract 5.0 g / l, NaCl 5.0 g / l). After confirming that the turbidity OD ₆₆₀ was 1.0, inoculate 1% (v / v) of the culture into 200 ml of LB medium, and cultured at 30 ° C and 120 rpm. .

Similarly, in the bioremediation treatment described below, 1% of a culture solution having a turbidity OD ₆₆₀ of 1.0 was inoculated in a necessary amount of LB medium, and a bacterial solution cultured at 30 ° C. and 120 rpm was used.

  Consortium I refers to a group of hydrocarbon-degrading bacteria that have been activated by culturing for 2 days after stimulating the contaminated soil with B medium.

  Modified SW medium and B medium were used as the medium. The composition of the modified SW medium is shown in Table 10, and the composition of the B medium is shown in Table 11.

  Reagents used in the medium were purchased from Wako Pure Chemical Industries, Ltd. and Nacalai Tesque.

4-1-2. The actual contaminated soil used was the soil provided by Nikko Co., Ltd., and the soil contaminated by the leakage of oil from the heavy oil tank carried in from the factory site.

4-1-3. Measurement of oil concentration by S316 extraction-infrared determination The oil concentration of soil was measured by the following S316 extraction-infrared determination method.

(1) S316 extraction method Soil samples were sampled from three locations per sample and mixed. After stirring the soil sample, 7.0 g of soil was collected, and 1.0 g of sodium sulfate and 2.0 g of silica gel were added for dehydration. To this soil, 25 ml of oil extract S316 (chlorotrifluoroethylene) was added and stirred for about 1 hour. After 1 hour, filtration was performed to separate the soil and the oil extract. The filtrate was diluted moderately so as to be within the measurement range of the oil concentration meter.

(2) Infrared quantification method About 6.5 ml of filtrate was put into an absorption cell and measured using an oil concentration meter (OCMA-350, Horiba, Tokyo). Table 12 shows the measurement conditions. The obtained measured value was converted into the oil concentration by the following (formula 3).

4-1-4. Measurement of the number of soil bacteria by the environmental DNA analysis method The number of soil bacteria was determined by the environmental DNA analysis method in the same manner as in 2-1-3 in Example 2.

4-1-5. Treatment conditions for bioremediation (480 kg scale) of actual contaminated soil In this experiment, seven treatment conditions A-G were prepared in order to compare each bioremediation technology. Each processing condition is shown in Table 13 below. For the 480 kg of actual contaminated soil, 20 l was added in the case of the B medium, and 20 l of the 4-fold concentrated solution in the case of the modified SW medium.

  In the biostimulation system (B and C), 5 l of water was inoculated, and in the bioaugmentation system (D to G system), each bacterial solution was inoculated at 1% (v / w). About 15 liters of water was added every other week to keep the water content in the soil constant, and the soil was cut back to increase aeration efficiency. And 6 weeks of bioremediation was done.

4-1-6. Additional treatment conditions for bioremediation (480 kg scale) of actual contaminated soil
A decrease in the number of microorganisms was predicted during the 6 week treatment period. Therefore, additional processing was performed in the sixth week. Each additional processing condition is shown in Table 14 below.

  For systems (B) and (D) using B medium, 15 l of B medium was supplemented.

On the other hand, in the systems (C), (E), (F) and (G) using the modified SW medium, FeSO ₄ and ZnSO ₄ components contained in the modified SW medium were dissolved in 10 l of water and added. At that time, the final concentration of FeSO ₄ at the time of addition to the soil: 2.78 × 10 ⁻³ g / l, and the final concentration of ZnSO ₄ : 2.01 × 10 ⁻³ g / l were prepared. In addition, Gordonia sp. RN2 strain is added to the Rhodococcus sp. / w) was inoculated.

4-2. result
4-2-1. Change in the number of soil bacteria In order to see the state of soil remediation in bioremediation (480 kg scale), the change was monitored using the number of soil bacteria as an index.

  A summary of changes in the number of soil bacteria in the system treated with B medium is shown in FIG. A summary of changes in the number of soil bacteria in the system treated with the modified SW medium is shown in II of FIG.

The number of soil bacteria before the treatment was below the detection limit (3.0 × 10 ⁷ cells / g-sample), confirming that the number of microorganisms in the contaminated soil was very small.

By the initial treatment, the number of bacteria reached 10 ⁹ cells / g-sample in 2 and 3 weeks in all systems, and then tended to decrease to 10 ⁷ cells / g-sample.

  The additional treatment increased the number of soil bacteria in all systems, confirming the effect of the additional treatment. In particular, the number of soil bacteria increased in the (C) (E) (F) (G) system. From this, it was thought that trace metal was insufficient. In addition, the number of soil bacteria decreased around the 10th week, 3 weeks after the additional treatment, and this was thought to be caused by a lack of nutrients as in the initial 6 weeks.

  Looking at the time-dependent change in the number of soil bacteria in the initial 6 weeks and the time-dependent change in the number of soil bacteria in the 5 weeks after the additional treatment, there was a tendency for the number of soil bacteria to increase once and then greatly decrease.

  Compared with the blank system (A), the other system had a large decrease in the number of soil bacteria, and as a reason, the lack of added inorganic nutrients and enzyme components was considered. It was also possible that the soil environment changed due to the decrease in hydrocarbons and accumulation of intermediate metabolites, and the microbiota changed accordingly.

  In addition, the number of soil bacteria in the bioaugmentation system was greatly reduced compared to the biostimulation system, and it was also thought from the monitoring results that the administered degrading bacteria did not settle in the soil. .

4-2-2. Change of oil concentration In order to grasp the progress of oil decomposition in bioremediation (480 kg scale), the oil concentration of soil was measured using S316 extraction-infrared quantitative method.

  Table 15 below shows the values of the oil concentrations at 0 and 9 weeks. In addition, the total oil residual ratio was calculated from the values of the 0th week and the 9th week, and the result is shown in FIG.

  In the bioremediation-treated system, the oil concentration decreased by about 300 ppm compared to the blank system (A), and a clear difference was confirmed. Compared with the biostimulation system (B), the bioaugmentation system shows an oil concentration lower by 200 ppm or more, and this decrease in oil concentration is attributed to the degradation of these components by microorganisms. it was thought.

  In particular, it was confirmed that the treatment for adding the Gordonia sp. RN2 strain to the initial treatment and the Rhodococcus sp. RN1 strain for the additional treatment had the best oil removal capability.

Moreover, from the above results, the soil bacteria count was monitored, and when the soil bacteria count reached about 1.0 × 10 ⁷ cells / g-sample, the effect of oil decomposition was confirmed by additional treatment. Furthermore, it was confirmed that it was effective to monitor the number of soil bacteria in order to measure the timing of the additional treatment.

Example 5: Comparative study of bioremediation in large-scale actual contaminated soil (750 kg) In Example 4, Rhodococcus sp. RN1 and Gordonia sp. Bioremediation was conducted for 9 weeks.

  Furthermore, paying attention to the soils of the Rhodococcus sp. RN1 strain and Gordonia sp. RN2 strain, which had the best oil decomposition in this treatment, these two kinds of treated soils were recovered. Then, these two types of soil were inoculated so as to combine Rhodococcus sp. RN1 strain and Gordonia sp. RN2 strain, respectively, and a fungus combination experiment was conducted on a contaminated soil 500 g scale. Furthermore, an experiment was conducted on the amount of inoculation and the timing of microbial supplementation.

5-1. Experimental materials and methods
5-1-1. As the strain used and the microorganism used, Rhodococcus sp. RN1 strain and Gordonia sp. RN2 strain were used. The culture conditions were the same as in Example 4. Also, the contaminated soil was stimulated with B medium in advance, and after culturing for 2 days, the activated hydrocarbon-degrading bacteria group was named Consortium II and used. As the medium, a modified SW medium, a B medium, and an organic component medium were used. The composition of the modified SW medium, the composition of the LB medium, and the composition of the B medium are as shown in Example 4. The composition of the organic component medium is shown in Table 16 below.

5-1-2. The actual contaminated soil used was provided by Nikko Co., Ltd., and the actual contaminated soil brought in from the gas station site was used. Contaminants such as gasoline, kerosene, and engine oil can be cited as pollutants that can be considered from the site of a gas station.

5-1-3. Treatment conditions in bioremediation (750 kg) The treatment conditions are shown in Table 17 below. In the initial treatment, 15 l of B medium and 5 l of bacterial solution were added to 750 kg of actual contaminated soil. In the case of the additional treatment, 15 l was added for the B medium, 30 l of the 4-fold concentrated modified SW medium was added for the modified SW medium, and 1% (v / w) of each bacterial solution was added. Additional treatment was performed on week 5.

5-1-4. Measurement of the number of soil bacteria by the environmental DNA analysis method The number of soil bacteria was determined using the environmental DNA analysis method in the same manner as in Example 2, 2-1-3.

5-1-5. Measurement of Oil Concentration by S316 Extraction-Infrared Determination Method The oil concentration of soil was measured by the S316 extraction-infrared determination method in the same manner as in Example 4.

5-2. result
5-2-1. Changes in the number of soil bacteria In order to analyze the effects of bioremediation (750 kg scale) on soil bacteria, the number of soil bacteria was measured as an index. The results are shown in FIG.

  As shown in FIG. 3, no significant increase in the number of soil bacteria was observed, and the same tendency was observed in all systems.

  The number of soil bacteria tended to increase at the first and second weeks and then decreased until the fifth week, showing the same change as in Example 4. However, the biostimulation system (I) with B medium had less increase in the number of soil bacteria than the other bioaugmentation systems. The reason is considered that the soil contains almost no easily decomposed hydrocarbons or aromatic hydrocarbons.

5-2-2. Change in oil concentration To understand the progress of oil removal in bioremediation (750 kg scale), measure the oil concentration in the 0th, 5th and 9th weeks using S316 extraction-infrared quantitative method. did. The oil concentration in bioremediation (750 kg) is shown in Table 18 below.

  In the blank system (H), the oil concentration decreased by about 300 ppm. Like bioremediation (480 kg), this was thought to be due to volatilization or degradation by microorganisms in contaminated soil due to aeration and water addition. However, in the system with bioremediation, the oil concentration decreased by 500 ppm or more, so the effect of bioremediation was confirmed in this experiment. Particularly in the Rhodococcus sp. RN1 strain (K) and Gordoniasp. RN2 strain (L), the oil concentration decreased by more than 700 ppm. From these results, it was considered that the oil concentration decreased due to the decomposition of cycloalkane and polymer hydrocarbons. In addition, the oil concentrations of Rhodococcus sp. RN1 strain (K) and Gordonia sp. RN2 strain (L) at week 5 were both 1,300 ppm. In these systems, B medium was used in the initial treatment, but no significant reduction in oil content could be confirmed, so the combination with B medium was considered ineffective. Therefore, the modified SW medium was used at the 5th week.

FIG. 4 shows the total oil remaining rate at the 9th week.
As shown in FIG. 4, bioaugmentation by Gordoniasp. RN2 strain was the most excellent in oil removal, and the total oil remaining rate was 44%. From this, it was found that bio-augmentation using Gordoniasp. RN2 strain was excellent in oil removal against hydrocarbon contamination of macromolecules.

Example 6 Comparison of additional treatments After completion of a 9-week bioremediation experiment using 750 kg of actual contaminated soil, the system of Rhodococcus sp. RN1 (K) and Gordonia sp. A bioremediation experiment was conducted for the purpose of examining additional processing conditions in augmentation.

6-1. experimental method
6-1-1. Additional processing conditions
The soil of the system (K) inoculated with Rhodococcus sp. RN1 strain was defined as soil KK6, and the system of soil inoculated with the Gordonia sp. RN2 strain (L) was defined as soil 76A.

  The treatment conditions for bioremediation of soil KK6 and the treatment conditions for bioremediation of soil 76A are shown in Table 19 below. Each additional treatment was performed for a treatment period of 5 weeks, and bioremediation was performed.

  The experiment was conducted using a 1 / 5,000a pot (As One Corporation, Osaka). For 500 g of contaminated soil, each strain was collected at 5,000 g for 30 min, suspended in 20% (v / w) of 4-fold concentrated modified SW medium, and sprayed on the soil. The inoculated amount is as shown in Table 19 below.

Water was added in a system in which the bacterial solution and the medium were not added. In the K-6 and L-6 systems, 10% (w / w) of farmland soil was added to the contaminated soil. The pot was placed in the laboratory and allowed to stand at room temperature and covered with aluminum to reduce water volatilization. For inoculation in additional treatment, 500 ml of the bacterial solution cultured until the number of microorganisms reached about 1 × 10 ⁹ cells / ml was collected at 5,000 g for 30 min, and then suspended in 25 ml of organic component medium. .

In (K-4) and (L-4), 1.0 × 10 ⁹ (cells / g-soil) was inoculated every week for 4 weeks regardless of the number of soil bacteria. (K-5) (L-5) was treated for 4 weeks by inoculating 1.0 × 10 ⁹ cells / g-soil only when the number of soil bacteria was below 1.0 × 10 ⁸ cells / g-sample.

6-1-2. Measurement of the number of soil bacteria by the environmental DNA analysis method The number of soil bacteria was determined using the environmental DNA analysis method in the same manner as in Example 2, 2-1-3.

6-1-3. Measurement of Oil Concentration by S316 Extraction-Infrared Determination Method The oil concentration of soil was measured by the S316 extraction-infrared determination method in the same manner as in Example 4.

6-2. result
6-2-1. Change in the number of soil bacteria In order to investigate the state of soil purification under the additional treatment conditions, the change in the number of soil bacteria was examined. FIG. 5 shows time-dependent changes in the number of soil bacteria in the soil KK6 system. Moreover, the time-dependent change of the number of soil bacteria of the soil 76A system is shown in FIG.

  There was no significant difference in the number of soil bacteria in (K-2), (K-3), (L-2), and (L-3) compared to the blank lines (K-1) and (L-1). On the other hand, the number of soil bacteria in (K-4), (K-5), (L-4), and (L-5) increased significantly compared to the blank lines (K-1) and (L-1). In particular, the number of soil bacteria increased 10-fold by the additional treatment in the first week and maintained that number thereafter.

  In addition, in (K-4) and (L-4), microorganisms were added every week, but there was little difference from the number of soil bacteria in the systems (K-5) and (L-5) that were added only in the first week. No additional processing more than once proved to be ineffective.

  In this experiment, it was confirmed that it was very effective to monitor the number of soil bacteria and measure the timing of additional treatment.

The field soil had a high initial soil bacteria count and increased further during the treatment period. However, there was almost no difference between (K-4), (K-5), (L-4), and (L-5) in the decrease in oil concentration, so soil bacteria of 1 × 10 ⁸ cells / g-sample or more It was thought that maintaining the number or additional treatment after one week was effective for oil removal. The number of soil bacteria in the (K-6) and (L-6) systems was maintained without showing a decreasing trend. From this, it is considered that the hardly decomposable hydrocarbons remain in the contaminated soil, but have been reduced to an amount that does not affect the growth of microorganisms. Considering the decrease in toxicity of contaminated soil and the increase in the number of microorganisms in the blank system, hydrocarbon-degrading bacteria growing in the soil can be increased in the treated soil by appropriate water addition and aeration. It was thought that.

6-2-2. Changes in oil concentration In order to evaluate the oil content resolution under each additional treatment condition, the oil content concentration was measured using the S316 extraction-infrared quantitative method. Table 20 and FIG. 7 show the oil concentration after bioremediation for 5 weeks using soil KK6.

  Further, Table 21 and FIG. 8 show the oil concentration after the completion of bioremediation for 5 weeks using soil 76A.

  When the initial oil concentration and the value of the blank system were compared, there was no difference and the oil decomposition did not proceed. From this, it was considered that high-molecular-weight hardly decomposable hydrocarbons remained in the soil.

Initially inoculated with 1 × 10 ⁷ cells / g-soil (K-2) (L-2), 1 × 10 ⁹ cells / g-soil inoculated (K-3) (L-3) There was no difference compared to the blank system.

Every ^{1 × 10 9 cells / g-} soil inoculated system (K-4), and ^{1 × 10 9 cells / g-} soil inoculated when the number of microorganisms is below ^{1 × 10 8 cells / g-} sample The system (K-5) (L-5) showed a decrease in oil concentration from 300 to 500 ppm. From this, it was found that additional treatment to maintain a high soil bacteria count is related to the promotion of oil breakdown. In addition, compared to the blank line (K-1) (L-1), the number of soil bacteria in (K-4) (K-5) (L-4) (L-5) is greatly increased. The number of soil bacteria increased 10-fold by the additional treatment in the first week, and there was a correlation between soil bacteria count and oil degradation.

  Depending on the combination order of Rhodococcus sp. RN1 strain and Gordonia sp. RN2 strain, the oil concentration differed by about 100 ppm. As a result of bioremediation (480 kg scale), the treatment of adding Gordonia sp. RN2 strain in the initial stage and Rhodococcus sp. RN1 strain in the additional treatment was superior in oil removal. The system (K-6) (L-6) mixed with soil from the field also showed a decrease in oil concentration from 300 to 500 ppm. It is thought that there are bacteria in the soil of the field that can decompose various substrates, and oil decomposition is possible by the action of bacteria that can decompose hydrocarbon-degrading bacteria and various intermediate metabolites produced by hydrocarbon decomposition. It was thought that it was prompted.

In addition, the system (K-4) (L-4) to which microorganisms were added every week, and the system to which microorganisms were added when it decreased to 1 × 10 ⁸ cells / g-sample or less (K-5) (L-5) However, there was no difference in the number of soil bacteria and oil degradation. Therefore, excessive addition of microorganisms in (K-4) and (L-4) has no effect on the increase of soil bacteria count and oil degradation, and the addition of (K-5) (L-5) system The treatment was suggested to be effective. In the system of (K-4) and (L-4), the reason why the number of soil bacteria did not increase was that the bacterial density became too high due to excessive addition of microorganisms, and growth inhibition was considered to have occurred.

  In addition, the systems (K-4), (K-5), (L-4), and (L-5) were suspended in an organic component medium and added when adding microorganisms. Therefore, the increase in the number of soil bacteria was thought to be due to nutrients in the medium. However, the system of (K-4) (K-5) (L-5) was accompanied by a decrease in oil content, and it was considered that hydrocarbon-degrading bacteria increased or became activated with the increase of soil bacteria. .

  The system (K-6) (L-6) mixed with soil from the field had the highest number of soil bacteria and the most advanced oil decomposition. It is thought that there are bacteria in the soil of the field that can decompose various substrates, and oil decomposition is possible by the action of bacteria that can decompose hydrocarbon-degrading bacteria and various intermediate metabolites produced by hydrocarbon decomposition. It was thought that it was prompted. It was also thought that Rhodococcus sp. RN1 and Gordonia sp. RN2 strains and indigenous hydrocarbon-degrading bacteria that had already existed in the soil increased the organic matter as a nutrient source and promoted oil decomposition.

  In addition, the system of (K-4) (K-5) (L-5) in which a decrease in oil concentration was observed used an organic component medium when adding microorganisms. Like the field soil, the organic matter in the organic medium was thought to have increased soil bacteria count and oil decomposition by stabilizing the nitrogen cycle of the soil. Therefore, bioremediation suggests that the addition of organic substances may be effective for the growth of microorganisms.

  In the additional processing conditions in the fourth and fifth embodiments and the additional processing examination experiment in the sixth embodiment, the reference of timing for performing the additional processing is different.

Additional treatments for bioremediation (480 kg scale) and bioremediation (750 kg scale) were added when the number of soil bacteria fell below the detection limit (3.0 × 10 ⁷ cells / g-sample). In the experiment for examining the treatment conditions, 1.0 × 10 ⁸ cells / g-sample was used as a reference for the timing of the additional treatment. As a result, it was found that when additional treatment was applied when the number of soil bacteria fell below 1.0 × 10 ⁸ cells / g-sample, the number of soil bacteria continued to increase and the oil decomposition proceeded. The timing of this additional processing was earlier than originally expected. From this, it was confirmed that the soil purification treatment can be effectively performed by monitoring the number of soil bacteria and timing the additional treatment.

It is drawing which showed the result of having measured the change of the number of soil bacteria in the bioremediation (480 kg scale) of Example 4. FIG. 1 shows changes in the number of soil bacteria in the system treated with the B medium, and II in FIG. 1 shows changes in the number of soil bacteria in the system treated with the modified SW medium. It is a figure which shows the total oil residual rate in the process for 9 weeks in the bioremediation (480 kg scale) of Example 4. FIG. It is drawing which showed the result of having measured the change of the number of soil bacteria in the bioremediation (750 kg scale) of Example 5. FIG. FIG. 6 is a drawing showing the oil residual ratio at 9 weeks in bioremediation (750 kg scale) of Example 5. FIG. It is drawing which showed the time-dependent change of the number of soil bacteria of the system of soil KK6 in Example 6. It is drawing which showed the time-dependent change of the number of soil bacteria of the system of soil 76A in Example 6. 6 is a drawing showing the total oil concentration after completion of soil KK6-based bioremediation in Example 6. FIG. It is drawing which showed the total oil concentration after completion | finish of the bioremediation of the soil 76A6 type | system | group in Example 6. FIG.

Claims (3)

Rhodococcus sp. RN1 strain having soil remediation ability (Independent administrative agency, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center accession number FERM P-20708 ). Gordonia sp. RN2 (Gordonia sp. RN2) strain (Independent administrative agency, National Institute of Advanced Industrial Science and Technology, Patent Biodeposition Center Accession No. FERM P-20709 ). A soil purification method, wherein the microorganism according to claim 1 or 2 is introduced into a target soil.

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