JP4876086B2 - Bioremediation method - Google Patents

Description

  The present invention relates to a soil purification method, and more particularly, to a bioremediation method for efficiently purifying soil by applying a microbial growth promoter to oil-contaminated soil contaminated with mineral oil or the like.

  Soil contamination is difficult to experience compared to other pollutions such as air pollution and noise because the pollutants penetrate underground, and it is difficult to identify the source of pollution, the degree of pollution, and the hazards to humans. There is a characteristic. If the pollutant is a toxic substance such as heavy metals, it may spread to groundwater or agricultural land due to soil contamination, which may cause health damage in a form that is difficult for local residents to notice. Environmental standards are set for each component. . One of the soil pollutions is the problem of oil pollution, which causes problems in the preservation of the living environment due to oily odor and oil film due to soil containing mineral oils. However, mineral oils, which are the causative substances, have a variety of types and components, and their properties change in the environment. Therefore, it is difficult to grasp the level of oily odor and oil film uniformly. For this reason, in this case, the whole mineral oil is comprehensively captured based on human senses, and the presence or absence of an oily odor or an oil film is determined, and there are no regulation numerical values as in the case of heavy metals. Mineral oils that cause soil contamination include fuel oils such as gasoline, kerosene, light oil and heavy oil, and lubricating oils such as machine oil and cutting oil.

  On the other hand, due to the increasing social interest in environmental problems in recent years, the problem of oil-contaminated soil has become more important. According to the oil pollution countermeasure guidelines published by the Ministry of the Environment, Obstacles in smell and appearance are defined as obstacles to the preservation of the living environment, and the judgment on oil contamination is becoming increasingly severe. For this reason, there are not a few cases where the land cannot be used without sufficient purification of the oil-contaminated soil in a factory site or the like.

  On the other hand, it is very difficult to purify the soil by removing mineral oils and the like, which are pollutants that have permeated the basement extensively, from the soil. Measures against contaminated soil include replacement with new soil, removal of cover and contaminated soil, and soil purification by chemical treatment, but it is extremely difficult to identify the contaminated area. It is necessary to treat the soil, and in any case, a large amount of treatment and enormous costs are required. In addition, there is a problem that replacement of contaminated soil, covering and removing soil does not solve the fundamental problem.

  Under such circumstances, establishment of a technique capable of purifying oil-contaminated soil in as short a time as possible, efficiently, and inexpensively is desired. Under these circumstances, it can be applied to a wide range of soils in recent years, requires less processing costs than physical or chemical treatments, and has little impact on the environment. Bioremediation technology that uses and purifies the soil is attracting attention.

  For example, Patent Literature 1 discloses a method of activating microorganisms by supplying a nitrogen source, a phosphorus source, vitamins and metals to soil contaminated with oil. In Patent Document 2, the amount of DNA in the soil into which the test microorganism is introduced is measured, and the test microorganism is evaluated using the number of bacteria determined based on the DNA amount as an index. A soil purification method to be used is disclosed.

  As described above, the bioremediation method has various advantages, but it takes a long time to use the biological activity of microorganisms and is unsuitable for short-term treatment. There is a problem that it is very easily affected and it is extremely difficult to stably maintain an efficient processing state. In addition, it is considered that there are various microorganisms having a purification ability for oil-contaminated soil. However, as described in Patent Document 2 described above, the oil purification ability of microorganisms was evaluated in advance and evaluated. In the method of screening and adding microorganisms, since much time and labor are required for evaluation and the like, development of faster and more economical treatment is awaited.

  On the other hand, as described above, mineral oils contain various components. Extremely speaking, there are different microorganisms with excellent purification ability for each component, and it is considered that the agents that can activate the activity are different for each microorganism. However, if there is an economical method for maintaining a soil environment that can perform a rapid purification treatment on all petroleum hydrocarbons including polycyclic aromatic hydrocarbons without introducing special microorganisms, It is very useful in practice.

JP 2007-268451 A JP 2007-135425 A

  Therefore, an object of the present invention is to maintain a soil environment in which a rapid purification treatment can be performed on all petroleum hydrocarbons including polycyclic aromatic hydrocarbons without introducing special microorganisms. An object of the present invention is to provide a bioremediation method that enables oil-contaminated soil to be purified more quickly and more economically.

  The above object is achieved by the present invention described below. That is, the present invention is a bioremediation method for purifying soil by adding a microbial growth promoter to oil-contaminated soil, and monitoring the concentration of polycyclic aromatic hydrocarbon dioxygenase gene in oil-contaminated soil And a step of adding the growth promoter such that the concentration of the gene falls within a specific range.

  In the bioremediation method, the concentration of total petroleum hydrocarbons in the oil-contaminated soil is 100 mg / liter or more, and in the monitoring step, DNA is extracted from the oil-contaminated soil and the extracted It is preferable to quantify the polycyclic aromatic hydrocarbon dioxygenase gene by real-time PCR for DNA.

Further, the growth promoter contains alanine and alginate, phosphate group (PO ₄ ³⁻ ) and ammonium root (NH ₄ ⁺ ), and the content of alanine in the growth promoter is A, When the alginate content is B, the ratio of the two is preferably A: B = 100 parts by mass: 5 to 500 parts by mass, and the concentration of the gene is 10 ¹⁰ per cm ^{3 of the} soil. It is preferable to stop adding the growth promoter when the number of copies is exceeded.

  According to the present invention, it is possible to quickly purify all petroleum hydrocarbons including polycyclic aromatic hydrocarbons (hereinafter abbreviated as “TPH”) without introducing special microorganisms. A bioremediation method is provided that can maintain a soil environment capable of purifying oil-contaminated soil more quickly and more economically.

  Hereinafter, the present invention will be described in detail with reference to preferred embodiments. The present inventors have paid attention to the fact that soil purification proceeds even when oil-contaminated soil is left, although it takes time per year. This fact means that microorganisms that have a purification capacity for soil contaminated with mineral oil always exist in the soil. Therefore, we thought that if the activity of these microorganisms can be activated and the state can be maintained, purification can be promoted simply without selecting a special microorganism. Therefore, first, taking as an example polycyclic aromatic hydrocarbons with inferior degradability among the components of TPH, whether or not microorganisms in contaminated soil can be activated by mixing various compounds into the soil is as follows. And examined.

  The inventors first collected soil in the vicinity where oil contamination was actually confirmed, and added various compounds to the soil to which naphthalene, which is a polycyclic aromatic hydrocarbon, was added. Was measured for the amount of naphthalene remaining in the soil. Specifically, naphthalene is added to the soil to 200 ppm, a predetermined amount of sodium alginate, alanine, potassium dihydrogen phosphate or ammonium sulfate is added to the soil and kept at 30 ° C. for 4 days. The concentration of naphthalene remaining therein was measured.

  The results are shown in FIGS. From these results, by adding each compound, the decomposition of naphthalene in the soil is surely accelerated, and the amount of addition (concentration in the soil at the time of addition) decomposes the naphthalene in the soil. Found that the degree of. Furthermore, as a result of studying various combinations of these compounds, there is not necessarily a synergistic effect when the effective compounds are combined with each other. The knowledge that a ratio exists was obtained.

  These facts indicate that if compounds that can promote the decomposition of naphthalene in the soil, and if multiple types of compounds that can make the effect more remarkable, are mixed in the soil, these compounds will inhabit the soil. It functions as a growth promoting agent for microorganisms and can promote soil remediation. On the other hand, it is important to control the combination of compounds, the ratio of addition, and the timing of mixing in order to enable the compound to function effectively in a smaller amount and to perform soil remediation quickly. I thought. For this purpose, it is necessary to grasp the biological purification ability of the target soil by some method.

  In contrast, the present inventors use the gene concentration of soil pollutant degrading enzyme in soil as an index for evaluating the soil purification ability of the soil when adding a microbial growth promoter to oil-contaminated soil. I came up with that. As a result of detailed research, polycyclic aromatic hydrocarbon (hereinafter abbreviated as “PAH”) dioxygenase gene is excellent as an index for grasping the biological purification ability of the soil to be treated. As a result, the present invention has been completed. That is, the bioremediation method of the present invention comprises the steps of monitoring the concentration of the PAH dioxygenase gene in oil-contaminated soil and adding a growth promoter to the soil so that the concentration of the gene falls within a specific range. It is characterized by having at least. Hereinafter, each process which comprises the bioremediation method of this invention, the growth promoter to be used, etc. are demonstrated in detail.

  The oil-contaminated soil to which the bioremediation method of the present invention is applied has the same meaning as that used in the “Guidelines for Countermeasures against Oil Contamination” by the Central Environmental Council of Japan, and oil refers to mineral oils. means. These mineral oils include fuel oils such as gasoline, kerosene, light oil and heavy oil, and lubricating oils such as machine oil and cutting oil.

  There is no particular limitation on the concentration of oil in the oil-contaminated soil to which the bioremediation method of the present invention is applied. Can be processed. Generally, when the concentration of oil in the soil is determined as TPH, if the TPH concentration in the soil is 100 mg / liter or more, biotreatment is considered possible.

The method for measuring the concentration of the PAH dioxygenase gene required in the step of monitoring the concentration of the PAH dioxygenase gene in the oil-contaminated soil constituting the present invention is not particularly limited, but it is easy to use the real-time PCR method. It is.
The real-time PCR method is a method for monitoring and analyzing PCR amplification products in real time. A commercially available real-time PCR apparatus can be used, and an example is LightCycler (registered trademark; Roche Applied Science).

  In the present invention, when the real-time PCR method is used, DNA can be extracted from oil-contaminated soil, and the PAH dioxygenase gene can be quantified with respect to the extracted DNA by the real-time PCR method. A commercially available kit can be used for extraction of DNA from soil. An example of such a kit is ISOIL (Nippon Gene Co., Ltd.).

  The real-time PCR method includes various methods such as a method using a DNA binding dye, a method using a hybridization probe, and a method using a TaqMan (registered trademark) probe, and any of these methods is suitable in the present invention. Can be used for An example of a DNA binding dye is SYBR ™ Green I. Probes used in the real-time PCR method are prepared based on sequences well known to those skilled in the art (see, for example, Kasuga et al., Biochem. Biophys. Res. Commun., 2001, 283, 195-204). Can do.

In addition, the microorganism growth promoter used in the present invention has an action of promoting the growth of microorganisms, and in particular, any microorganism can be used as long as it promotes the growth of microorganisms involved in the decomposition of mineral oils in oil-contaminated soil. Can also be used. A composition containing a plurality of compounds may synergistically exert the effect of promoting the growth of microorganisms compared to the case where these compounds are used alone, and it is more preferable to use such a composition. As an example of a composition that exhibits such a synergistic growth promoting effect, a composition containing alanine and alginate, which has been developed by the present inventors during the examination process of the present invention, or a phosphate group (PO ₄ ^{3 -} )) And ammonium radicals (NH ₄ ⁺ ).

The alanine is a kind of amino acid, and a commercially available product can be suitably used.
Alginate is a salt of alginic acid, a kind of polysaccharide. Alginic acid is a natural product obtained from the cell wall of brown algae, and is a block copolymer composed of β-D-mannuronic acid and α-L-guluronic acid. Alginates with various degrees of polymerization are commercially available, but the higher the degree of polymerization, the less soluble it is in water, and the increased viscosity when dissolved in water makes it difficult to handle, so it is used in the bioremediation method of the present invention. In this case, the polymerization degree is preferably 5 to 1,000.

  Examples of the alginate include, but are not limited to, alkali metal salts such as sodium alginate and potassium alginate, alkaline earth metal salts such as magnesium alginate and calcium alginate, and ammonium alginate. Alkali metal salts of alginic acid are preferably used in the present invention because of their high water solubility.

  Examples of compounds containing phosphate radicals that can be used as the above-mentioned microorganism growth promoter include trisodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, tripotassium phosphate, dipotassium hydrogen phosphate, and diphosphate phosphate. Examples include potassium hydrogen, tricalcium phosphate, calcium monohydrogen phosphate, calcium dihydrogen phosphate, triammonium phosphate, and ammonium dihydrogen phosphate. Among them, it is preferable to use trisodium phosphate, ammonium dihydrogen phosphate, potassium dihydrogen phosphate, or sodium dihydrogen phosphate because the reagent cost is low.

  On the other hand, examples of the compound containing an ammonium root include ammonium sulfate, triammonium phosphate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate, ammonium carbonate, ammonium hydrogen carbonate, ammonium nitrate, ammonium acetate, and ammonium chloride. it can. Among them, it is preferable to use ammonium sulfate, ammonium chloride, or diammonium hydrogen phosphate because the reagent cost is low.

The growth promoter for microorganisms may contain a compound serving as an oxygen source in addition to the above-described compounds. Examples of such an oxygen source include compounds containing nitrate radicals (NO ₃ ⁻ ), and specific examples include sodium nitrate, potassium nitrate, magnesium nitrate, calcium nitrate, iron nitrate, and ammonium nitrate. Among them, it is preferable to use sodium nitrate, potassium nitrate or ammonium nitrate because the reagent cost is low.

  When the microorganism growth promoter contains alanine and alginic acid, the ratio between the two is A: B = 100 parts by mass: 5 to 500 parts by mass, where A is the alanine content and B is the alginate content. It is preferable that When B is less than 5 parts by mass with respect to A100 parts by mass, the synergistic effect between alanine and alginate is weakened. Similarly, when B is more than 500 parts by mass with respect to 100 parts by mass of A, the synergistic effect of alanine and alginate becomes weak, which is not preferable.

When the growth promoter for microorganisms further contains a compound containing a nitrate radical as an oxygen source, the content C of the nitrate radical (NO ₃ ⁻ ) is 2,000 parts by mass or less with respect to A100 parts by mass. It is preferably 5 to 300 parts by mass. Even if C is more than 2,000 parts by mass with respect to 100 parts by mass of A, no further effect is observed, which is not preferable from the viewpoint of economy.

  When the microbial growth promoter contains all of alanine, alginic acid, nitrate radical, phosphate radical, and ammonium root, a particularly remarkable microbial growth promotion effect can be exhibited by the synergistic effect of these compounds.

  The method of applying the growth promoter to oil-contaminated soil may be sprayed on the soil as a solid powder. However, an aqueous solution is more effective because it can be uniformly applied to the soil.

  When applying the above growth promoter to oil-contaminated soil, it may be sprayed on the surface of the soil with a sprinkler or the like, but oil contamination of the soil penetrates underground and sometimes reaches deep underground. It is preferable to install a well within the application range of the bioremediation method of the present invention and to allow the growth promoter aqueous solution to permeate from the well. The depth of the well needs to be determined according to the degree of soil contamination, the state of oil penetration, the geology of the applied soil, the groundwater level, etc. If the depth of the well becomes deeper, the cost required for drilling and installing the well will be higher, but if it is unnecessarily deeper, the growth promoter will penetrate to the extent that it is not necessary, and the cost for the chemical will increase. May not be possible. For this reason, by measuring the amount of TPH in the sampled soil in advance, the distribution state including the depth direction of the oil in the applied soil is confirmed, and the depth of the well and the position to dig the well based on this are confirmed. It is effective to decide.

  The amount of the growth promoter applied to the soil varies depending on the amount of pollutants contained in the soil, the amount of microorganisms living in the soil, the frequency of application of the growth promoter, etc. The amount which is about 0.5 ppm to 1,000 ppm of the mass of the hit application target soil is preferable. When it is less than 0.5 ppm, there is little effect on microorganisms even if a growth promoter is mixed in the soil, and the concentration of the PAH dioxygenase gene may not be rapidly controlled. On the other hand, even if it is administered in excess of 1,000 ppm, there is little contribution to the effect of improving the activity of microorganisms, the cost effectiveness is reduced, and the economics of the treatment may be impaired. In addition, when using the growth promoter as an aqueous solution, if the concentration is too low, it takes too much time for the whole amount to penetrate into the soil, and if it is too thick, it may be harmful to microorganisms, so it can penetrate quickly, It is not preferable to adjust the concentration of the growth promoter so that it can be uniformly distributed in the soil.

When a growth promoter aqueous solution is applied to soil by a well, the volume (application range) of the soil to which the growth promoter aqueous solution is applied varies depending on the depth of the well, soil geology, saturated layer thickness, etc. Although it is difficult to determine, as a convenient example, it can be assumed that the radius is within 5 m centered on the well in the saturated layer, as calculated from Equation 1 below.
[Formula 1] Application range (m ³ ) = saturation layer thickness (m) × 5 (m) × 5 (m) × π

  In the bioremediation method of the present invention, the growth promoter is added to the soil so that the concentration of the PAH dioxygenase gene measured in the monitoring step falls within a specific range. The main technical feature of the present invention is that it has been found that the concentration of the PAH dioxygenase gene is an index for evaluating the soil purification ability of microorganisms living in the applied soil. It is considered that the numerical value of the concentration range of the PAH dioxygenase gene used in the present invention itself varies depending on the contamination state of the soil to be applied, the type of microorganisms inhabiting, and the like. Therefore, before treatment, examine the amount of TPH in the applied soil, the concentration of the PAH dioxygenase gene, and the tendency of changes in the PAH dioxygenase gene concentration when a growth promoter is added to the applied soil. It is only necessary to determine the optimum range after performing this.

As an upper limit of the concentration range of the PAH dioxygenase gene in the oil-contaminated soil, for example, 10 ¹⁰ copies per 1 cm ^{3 of} soil can be mentioned. In the implementation of the bioremediation method of the present invention, while monitoring the number of PAH dioxygenase genes, when the concentration of the genes becomes 10 ¹⁰ copies or more per 1 cm ³ of soil, Therefore, it is possible to prevent excessive addition of a growth promoter to the soil and economically remediate the soil.

Moreover, addition of a growth promoter can also be controlled so that the PAH dioxygenase gene concentration in oil-contaminated soil is in a range between a specific upper limit value and a lower limit value.
Here, the “upper limit value” refers to the PAH dioxygenase gene concentration in the soil, which is considered to be close to the upper limit to improve the purification ability of contaminants by microorganisms in the soil. Even if a growth promoter is added to increase the concentration of the PAH dioxygenase gene in the oil-contaminated soil above the upper limit, it is difficult to rapidly increase the concentration of the PAH dioxygenase gene. The amount of the accelerator used is increased unnecessarily, and economical treatment cannot be performed. Such an upper limit varies depending on the degree of contamination of oil-contaminated soil, the type of microorganisms inhabiting, the geology, and the like, and examples include 10 ⁹ or 10 ¹⁰ copies per 1 cm ³ of soil.

  On the other hand, the “lower limit value” means that the ability to purify pollutants by microorganisms is low, and if the value remains at that value, even if the treatment can be carried out, it usually takes a yearly period for the treatment. Oxygenase gene concentration. It has been found that if the growth promoter used in the present invention is used, the PAH dioxygenase gene concentration increases, and the same purification treatment can be performed in units of several months. The purpose of the present invention is to efficiently use a growth promoter during this purification treatment and achieve a more rapid treatment. For this purpose, the concentration of the PAH dioxygenase gene in the applied soil is reduced. The amount of increase when a growth promoter is added, and further, how the amount of polycyclic aromatic hydrocarbons to be treated decreases when the growth promoter is increased. The “value” and the “upper limit value” described above may be determined.

In this way, the lower limit of the concentration of the PAH dioxygenase gene in the oil-contaminated soil is determined, and if it is lower than this value, it is determined that the ability to purify the pollutants in the soil has decreased, and the growth of microorganisms in the soil is promoted. If the purification ability is activated by adding an agent, more rapid processing becomes possible. The lower limit value varies depending on the degree of contamination and the geology of oil-contaminated soil, and examples include 10 ⁶ copies or 10 ⁷ copies per 1 cm ^{3 of} soil.

The bioremediation method of the present invention is configured to add a growth promoter to the soil so that the concentration of the PAH dioxygenase gene measured in the monitoring step falls within the specific range determined by the method as described above. can do. Specifically, the concentration of the PAH dioxygenase gene in the soil during the purification treatment is measured at appropriate time intervals, and when the concentration of the gene reaches 10 ¹⁰ copies or more per 1 cm ^{3 of} soil, Try to stop the addition.

As a modification, if the gene concentration is between a specific upper limit value and a lower limit value, it is judged that the soil purification capacity at that time is appropriate, and no growth promoter is added. . However, if the gene concentration shows a rapid decrease (for example, 1/10) compared to the concentration measured before that, a rapid decrease in the purification treatment capacity is expected. Even if it is inside, it is preferable to add a growth promoter to soil. Examples of such a specific concentration range of the PAH dioxygenase gene include 10 ⁶ to 10 ⁹ copies per 1 cm ^{3 of} soil.

  EXAMPLES Hereinafter, although an Example and a comparative example are given and this invention is demonstrated concretely, this invention is not limited to these.

[Example 1]
<Application of microbial growth promoter to soil>
Drilling a 10m deep well in the factory site, filling it with a 50mm nominal diameter 10m long rigid polyvinyl chloride pipe with many small holes in the side wall in the vertical direction, a 10m deep microorganism growth promoter A well for water solution injection was installed.

As a result of the geological survey, a depth of 6 to 10 m was a saturated layer. For this reason, assuming that the permeation range of the growth promoter aqueous solution is within a radius of 5 m from the well in the saturated layer (layer thickness 4 m), the application target soil of the growth promoter is calculated to be about 314 m ³ .
The growth promoter was applied to the soil by injecting a growth promoter aqueous solution having the composition described later into the well four times at intervals of one week.

<Preparation of microbial growth promoter>
The microbial growth promoter aqueous solution was prepared using a tank with a wide mouth of about 500 liters. The composition of the growth promoter aqueous solution used for one injection is as follows.
Oxygen source: Sodium nitrate 675g
Growth promoting substances: 6,675g alanine, 675g sodium alginate
Water: 942 liters The mass of the growth promoter is 8,025 g. Assuming that the density of the application target soil is 1.5 g / cm ³ , the mass of the application target soil is about 4.7 × 10 ⁵ kg. Therefore, the growth promoter is about 17 ppm of the application target soil by mass ratio.
As sodium alginate, Nacalai Tesque Co., Ltd. (product code: 31130-95, polymerization degree: about 450) was used.

<Collecting soil samples>
Any five points in the test zone within a radius of 5 m from the injection well before the first growth promoter aqueous solution injection and 71 days after the first growth promoter aqueous solution injection (50 days after the fourth injection) A soil sample was taken from. With respect to these soil samples, the PAH dioxygenase gene and the total petroleum hydrocarbons were quantified by the following procedure. In the test section, soil contamination was evaluated by smell (oil odor) and vision (oil film).

<Quantification of PAH dioxygenase gene>
Using a soil DNA extraction kit ISOIL (Nippon Gene Co., Ltd.), DNA was extracted from 1 g of a soil sample to obtain a DNA sample. Next, the number of PAH dioxygenase genes contained in the DNA sample was determined by a real-time PCR method. That is, while performing PCR on a known amount of PAH dioxygenase gene, the fluorescence intensity value in the amplification process was measured to obtain a corrected fluorescence intensity value (FIG. 5), and a calibration curve was created based on the data (FIG. 6). . Subsequently, the fluorescence intensity value of the DNA sample was measured in the same manner while performing PCR, and the number of PAH dioxygenase genes contained in the DNA sample was determined from the calibration curve.

In the real-time PCR method, a primer for a sequence specific to the PAH dioxygenase gene was used. As a forward primer, the following sequence SEQ ID NO: 5′-TGYCGGBCAYCGBGGSAWG-3 ′
As a reverse primer,
SEQ ID NO: 5'-CCAGCCGTRRTRSTGCA-3 '
I used something with. The synthesis of the primer DNA strand was outsourced to Sigma-Aldrich Japan.
In the above sequences, Y means C or T, B means G, C or T, S means G or C, W means A or T, and R means G or A. means.

The reaction solution used for the real-time PCR method was prepared by increasing the volume of a reagent having the following composition to 50 μl with distilled water.
^{· SYBR (R) Premix Ex Taq} TM (Perfect Real Time) (2 ×) (TaKaRa Ex Taq HS, dNTP mixture, Mg ²⁺ and SYBR Green including I; Takara Bio Inc.): 25 [mu] l
-Known amount of PAH dioxygenase gene-Forward primer (SEQ ID NO: 1): 20 pmol
Reverse primer (SEQ ID NO: 2): 20 pmol

In addition, as the above reaction solutions, those containing no PAH dioxygenase gene and those containing 10 copies, 10 ² copies, 10 ³ copies, 10 ⁴ copies, 10 ⁵ copies, and 10 ⁶ copies of the PAH dioxygenase gene, respectively. Two were prepared and used for real-time PCR to create a calibration curve.

In the PCR, after the following (1) heat denaturation step, the steps (2) to (4) were performed for 60 cycles, and finally the extension step (5) was performed. The fluorescence intensity was measured using an excitation wavelength of 494 nm and a detection wavelength of 521 nm in the annealing step (3).
(1) Thermal denaturation step: 95 ° C, 120 seconds (2) Thermal denaturation step: 95 ° C, 45 seconds (3) Annealing step: 55 ° C, 60 seconds (4) Extension step: 72 ° C, 45 seconds (5) Extension Process: 72 degreeC, 45 second iCycler (Bio-Rad Laboratories Co., Ltd.) was used for the thermal cycler of PCR reaction.

The corrected fluorescence intensity value was determined by subtracting the fluorescence intensity value of the reaction solution not containing the PAH dioxygenase gene from the fluorescence intensity values obtained for the six reaction solutions containing the PAH dioxygenase gene. That is, the corrected fluorescence intensity value is obtained from the following equation 2.
[Formula 2]
Corrected fluorescence intensity value = fluorescence intensity value of reaction solution containing PAH dioxygenase gene−fluorescence intensity value of reaction solution not containing PAH dioxygenase gene

FIG. 5 is a graph in which the corrected fluorescence intensity value is plotted on a graph with the PCR cycle number as the horizontal axis.
Here, the number of PCR cycles with a corrected fluorescence intensity value of 0.5 was defined as the Ct value (Threshold Cycle) of each reaction solution, and the average value of the Ct values for the above six reaction solutions was determined. The average value of the Ct values was plotted on a semilogarithmic graph with the copy number of the PAH dioxygenase gene in each solution as the vertical axis, and a calibration curve was prepared by the method of least squares. As a result, a calibration curve having a very good correlation with R ² = 0.9991 was obtained. This figure is shown in FIG.

  Next, real-time PCR was performed in the PCR reaction solution using a DNA sample obtained from the soil sample in place of a known amount of PAH dioxygenase gene, and a Ct value was determined. This Ct value was applied to the calibration curve, and the copy number of the PAH dioxygenase gene in the sample was calculated. Table 1 shows the calculated number of copies.

<Quantification of TPH>
The amount of TPH in the soil sample was measured by a gas chromatograph (GC-FID) method with a flame ionization detector. Specifically, 10 g of a soil sample is put into an Erlenmeyer flask, 30 ml of hydrogen disulfide is added to this, and after sufficiently shaking, an extract is obtained, and the extract is measured by the GC-FID method. The TPH concentration in the medium was calculated. The values are shown in Table 1. From the results shown in Table 1, it was found that the TPH concentration in the soil, which was 1,628 mg / kg before the start of the experiment, decreased to 374 mg / kg or less than a quarter after 71 days from the start of the experiment. Understood.

Moreover, it became clear that the presence of the oily odor of the soil and the oil film by the naked eye was improved by the injection of the growth promoter.
From these results, it was revealed that by applying the bioremediation method of the present invention to oil-contaminated soil, the TPH concentration in the soil can be reduced and the degree of soil contamination can be improved.

  According to the present invention, it is possible to maintain a soil environment in which rapid purification treatment can be performed on all petroleum hydrocarbons including polycyclic aromatic hydrocarbons without introducing special microorganisms, and oil-contaminated soil. Thus, a bioremediation method capable of purifying the water more quickly and more economically is provided. By applying the present invention to oil-contaminated soil such as a factory site, more economical and quick soil purification becomes possible.

It is a graph which shows the decomposition amount of naphthalene in the soil which added sodium alginate. It is a graph which shows the decomposition amount of naphthalene in the soil which added alanine. It is a graph which shows the decomposition amount of naphthalene in the soil which added potassium dihydrogen phosphate. It is a graph which shows the decomposition amount of naphthalene in the soil which added ammonium sulfate. In order to prepare a calibration curve, real-time PCR is performed using a known amount of DNA, and the obtained corrected fluorescence intensity value is shown. A calibration curve for quantifying the PAH dioxygenase gene by real-time PCR is shown.

Claims (6)

A bioremediation method for purifying soil by adding a microbial growth promoter to oil-contaminated soil, the step of monitoring the concentration of polycyclic aromatic hydrocarbon dioxygenase gene in the soil, and the monitoring step And adding a growth promoter for microorganisms containing alanine and alginate to the soil so that the concentration of the gene falls within a specific range based on the concentration of the gene measured in (1) above. Mediation method.   The method according to claim 1, wherein the concentration of all petroleum hydrocarbons in the oil-contaminated soil is 100 mg / liter or more.   The method according to claim 1, wherein in the monitoring step, DNA is extracted from the oil-contaminated soil, and the polycyclic aromatic hydrocarbon dioxygenase gene is quantified by real-time PCR for the extracted DNA. The method of claim 1, wherein the growth promoter further comprises a phosphate group (PO ₄ ³⁻ ) and an ammonium group (NH ₄ ⁺ ). The method according to claim 1 , wherein when the alanine content of the growth promoter is A and the alginate content is B, the ratio between the two is A: B = 100 parts by mass: 5 to 500 parts by mass. . The method according to claim 1, wherein the addition of the growth promoter is stopped when the concentration of the gene becomes 10 ¹⁰ copies or more per 1 cm ^{3 of the} soil.

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