JP6999893B2 - Manufacturing method of composition for purification of petroleum-contaminated soil - Google Patents

Description

The present invention relates to a method for producing a composition for purifying petroleum-contaminated soil, a composition for purifying petroleum-contaminated soil, and a method for purifying petroleum-contaminated soil.

"Soil pollution caused by petroleum-based hydrocarbons" caused by accidents during transportation of petroleum and leaks from factories has been a problem for some time, and legislation and measures against leaks are being promoted. As a law for measures against petroleum-based hydrocarbon-contaminated soil, the United States first enacted the "Superfund Law" in 1980. The law requires all parties involved in soil contamination to bear purification costs, etc., and requires that the total petroleum hydrocarbon (TPH) concentration in the soil be 1,000 mg / kg or less.

In Japan, the "Soil Contamination Countermeasures Law" was enacted in 2002, but petroleum was not targeted as a pollutant because the countermeasures against petroleum-based hydrocarbon pollution were not sufficiently prepared. After that, in 2006, "Guidelines for Oil Contamination Countermeasures-Concept of Response by Land Owners to Oil Smell and Oil Film Problems Caused by Soil Containing Mineral Oil-" was announced, and in the purification of petroleum-based hydrocarbon-contaminated soil, " It was necessary to "eliminate oily odors and oil slicks" and "reduce the oil concentration in the soil". Furthermore, from April 2010, the amendment of the "Soil Contamination Countermeasures Law" restricted the transportation of contaminated soil, and it became necessary to purify the contaminated soil in the in-situ as much as possible.

Currently, incineration treatment and heat decomposition treatment using heavy oil are mainly performed for purification of petroleum-based hydrocarbon-contaminated soil. With these methods, the contaminated soil must first be dug up and transported to the treatment plant. However, the amendment of the Soil Contamination Countermeasures Law has restricted the transportation of contaminated soil, making this treatment method unsuitable. Incinerator requires 10 times as much fuel as contaminated oil, and there is a problem that the cost fluctuates greatly depending on the oil price. Furthermore, since the soil after incineration is free of organic matter including microorganisms, it is difficult to reuse the soil.

Therefore, in recent years, research on bioremediation, which purifies pollution by microbial function, has been progressing. Bioremediation is resource-saving compared to incineration and cleaning, and has the advantage of reusing soil. Furthermore, since the soil can be purified in-situ, it is expected to become more widespread with future revisions to the Soil Contamination Countermeasures Law. Bioremediation includes biostimulation, which administers microbial nutrients to activate indigenous microorganisms, and bioaugmentation, in which microorganisms with pollutant resolution are introduced from the outside.

The present inventors have so far isolated petroleum-degrading bacteria capable of decomposing persistent hydrocarbons in order to improve the efficiency of bioremediation of petroleum-contaminated soil (Patent Document 1). It has also been reported that by performing preculture and main culture using a medium having a specific composition, it is possible to obtain a microbial preparation for highly active bioremediation having a highly degrading function (Patent Document 2). .. Furthermore, it was reported that the number of soil microorganisms could be increased and maintained and oil decomposition could be promoted by adjusting the weight and ratio of nutrient components (Total-C, Total-N, Total-P) in the soil to a specific range. (Patent Document 3).

The present inventors have found that there are more petroleum-degrading bacteria in compost than in natural soil, and after adding such compost to petroleum-contaminated soil, the content of nutritional components in petroleum-contaminated soil is adjusted to a specific range. It is reported that petroleum contamination can be efficiently purified by doing so (Patent Document 4).

Japanese Unexamined Patent Publication No. 2007-135425 Japanese Unexamined Patent Publication No. 2008-228623 Japanese Unexamined Patent Publication No. 2012-71255 Japanese Unexamined Patent Publication No. 2014-61489

When petroleum-degrading bacteria are cultivated using a medium as in Patent Document 2, the medium and energy are costly. On the other hand, when biomass (organic material) such as compost is put into contaminated soil as in Patent Document 4, the cost can be reduced as compared with the case where petroleum-degrading bacteria are cultivated using a medium. However, for the purification of petroleum pollution, more excellent biomass is required for the growth of petroleum-degrading bacteria and the maintenance of the number and activity of the bacteria.

It is an object of the present invention to provide a method for producing a composition for purifying petroleum-contaminated soil and a composition for purifying petroleum-contaminated soil, which have high efficiency and low cost for purifying petroleum pollution. Furthermore, an object of the present invention is to provide a method for purifying petroleum-contaminated soil using the composition for purifying petroleum-contaminated soil.

As a result of diligent research to achieve the above object, the present inventor can achieve the above object by using an unfermented material such as soybean residue or peat moss for culturing petroleum-degrading bacteria. I got the finding.

The present invention has been completed by further studies based on these findings, and is the following method for producing a composition for purifying petroleum-contaminated soil, a composition for purifying petroleum-contaminated soil, and purifying petroleum-contaminated soil. It provides a method.

Item 1. A method for producing a composition for purifying petroleum-contaminated soil, which comprises a step of inoculating and culturing petroleum-degrading bacteria in an organic material including an unfermented material.
Item 2. Item 2. The production method according to Item 1, wherein the C / N ratio in the organic material is 10 to 30.
Item 3. Item 2. The production method according to Item 1 or 2, wherein the unfermented material is contained in the organic material in an amount of 60% by mass or more.
Item 4. The unfermented material consists of soybean meal, oil cake, rice husk, fish flour, rice bran, okara, coconut fiber, peat moss, rice straw, water moss, waterweed, rice cake, chips, straw, fallen leaves, cut grass, and bark. Item 6. The production method according to any one of Items 1 to 3, which is at least one selected.
Item 5.1 A composition for purifying petroleum-contaminated soil containing petroleum-degrading bacteria of 1 × 10 ⁶ cells / g or more and 60% by mass or more of unfermented materials.
Item 6. The unfermented material consists of soybean meal, oil cake, rice husk, fish flour, rice bran, okara, coconut fiber, peat moss, rice straw, water moss, aquatic plant, rice cake, chips, straw, fallen leaves, cut grass, and bark. Item 5. The composition according to Item 5, which is at least one selected.
Item 7. A composition for purifying contaminated soil produced by the production method according to any one of Items 1 to 4, or an oil-contaminated soil comprising a step of adding the composition according to Item 5 or 6 to the oil-contaminated soil. Purification method.

According to the present invention, by using an organic material excellent in the growth of petroleum-degrading bacteria and the maintenance of the number and activity of petroleum-degrading bacteria, a composition for purifying petroleum-contaminated soil with high efficiency of petroleum-contaminated purification can be produced at low cost. be able to. Further, by using the composition for purifying petroleum-contaminated soil of the present invention, it becomes possible to efficiently purify petroleum pollution.

Hereinafter, the present invention will be described in detail.

It should be noted that, in the present specification, "comprise" also includes the meaning of "essentially consist of" and the meaning of "consist of".

Definitions of terms used herein are given below.

"Total carbon" (sometimes referred to herein as TC) means the sum of organic and inorganic carbons in soil. Total carbon can be measured with a total organic carbon meter (TOC-V _CPH , Shimadzu Corporation) and a solid sample combustion device (SSM-5000A, Shimadzu Corporation).

"Total nitrogen" (sometimes referred to herein as TN) means the sum of organic and inorganic nitrogen in the soil. Total nitrogen can be measured by the Kjeldahl method and the indophenol blue method.

"C / N ratio" means the ratio of total carbon / total nitrogen (TC / TN).

The following petroleum-degrading bacteria count can be determined by the methods described in Examples, JP-A-2014-60966, JP-A-2014-61489 and the like.

Method for Producing Composition for Purifying Petroleum-Contaminated Soil The method for producing a composition for purifying petroleum -contaminated soil of the present invention is characterized by including a step of inoculating and culturing petroleum-degrading bacteria in an organic material including an unfermented material. And.

The petroleum-contaminated soil in the present invention is not particularly limited as long as it is petroleum-contaminated soil that can be purified by the present invention, and is contaminated with, for example, gasoline, kerosene, crude oil, light oil, heavy oil, lubricating oil, engine oil, or the like. The soil is mentioned. Examples of such soil include soil at a factory site, a factory site, a gas station site, an incineration plant, a pipeline area, an oil pollution accident site, and the like.

Specific examples of hydrocarbons contained in petroleum-contaminated soil include cycloalkanes such as cyclopropane, cyclobutane, cyclopentane, cycloheptane, cyclohexane, cyclooctane, and decalin; benzene, toluene, ethylbenzene, xylene, phenol, cresol, and the like. Monocyclic aromatic hydrocarbons; polycyclic aromatic hydrocarbons such as naphthalene, anthracene, phenanthrene, biphenyl, phenolphthalene, triphenylmethane; 1,1-dichloroethane, chloroform, 1,2-dichloropropane, dibromochloromethane , 1,1,2-Trichloroethane, 2-chloroethylvinyl ether, tetrachloroethane (PCE), chlorobenzene, 1,2-dichloroethane, 1,1,1-trichloroethane, bromodioxide, trans-1,3-dichloropropene, Sis-1,3-dichloropropene, bromoform, chloromethane, bromomethane, vinyl chloride, chloroethane, 1,1-dichloroethane, trans-1,2-dichloroethene, trichloroethene (TCE), dichlorobenzene, cis-1,3- 2-dichloroethane, dibromoethane, 1,4-dichlorobutane, 1,2,3-trichloropropane, bromochloromethane, 2,2-dichloropropane, 1,2-dibromomethane, 1,3-dichloropropane, etc. Examples thereof include halogen-containing hydrocarbons; long-chain linear hydrocarbons; and long-chain cyclic hydrocarbons.

The organic material in the present invention is not particularly limited as long as it contains an unfermented material, and a material containing 60% by mass or more of the unfermented material is preferable, a material containing 70% by mass or more is more preferable, and a material containing 80% by mass or more is preferable. Further preferred, those consisting only of unfermented materials are particularly preferred. Further, the upper limit of the content of the unfermented material is 100% by mass, 95% by mass, 90% by mass, 85% by mass and the like. It is desirable that the content of the unfermented material is high because it is excellent in the growth of petroleum-degrading bacteria and the maintenance of the number and activity of petroleum-degrading bacteria.

The unfermented material is not particularly limited as long as it is an organic material that has not been fermented. Examples include waterweed, rice husk, chips, straw, fallen leaves, cut grass, and bark. The unfermented material can be used alone or in combination of two or more.

The organic material in the present invention may contain materials other than unfermented materials as long as the effects of the present invention can be obtained, and examples thereof include compost and chemical fertilizers.

Examples of compost include plant compost such as bark compost, horse manure compost, chicken manure compost, cow manure compost, pig manure compost and other livestock compost, and seaweed compost. As described in Patent Document 4, compost contains a relatively large amount of petroleum-degrading bacteria.

Chemical fertilizers include urea, ammonium nitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammonium phosphate, sodium nitrate, calcium nitrate, potassium nitrate, potassium chloride, potassium sulfate, lime nitrogen, lime perphosphate, and lime heavy phosphate. , Phosphoric acid, Phosphoric acid, Phosphoric acid, Sulfuric acid, Phosphoric acid potassium, Salt Phosphoric acid, Potassium sulfate, Potassium sulfate, Potassium bicarbonate, Potassium phosphate and the like.

The C / N ratio in the organic material is preferably 10 to 30, more preferably 10 to 25. Organic materials with a C / N ratio in this range are excellent in the growth of petroleum-degrading bacteria and the maintenance of the number and activity of petroleum-degrading bacteria. If one type of organic material does not satisfy the above C / N ratio, the C / N ratio can be set within the above range by using a plurality of organic materials. Such adjustment of the C / N ratio is performed by measuring the total carbon and total nitrogen contents of various organic materials and then mixing the organic materials at a mixing ratio such that the C / N ratio is within the above range. be able to.

It is desirable that the organic material in the present invention has a total carbon content of 150,000 mg / kg or more and a total nitrogen content of 5,000 mg / kg or more.

The petroleum-degrading bacterium in the present invention means a bacterium capable of decomposing petroleum, particularly hydrocarbons contained in petroleum. Examples of such petrolytic bacteria include the genus Gordonia (eg, Gordonia terrae), the genus Rhodococcus (eg, Rhodococcus erythropolis), and the genus Acinetobacter. ), Bacillus, Pseudomonas, Achromobacter, Alcaligenes, Mycobacterium, Sphingomonas, Ralstonia, etc. Bacteria are exemplified. Among these microorganisms, those isolated from soil contaminated with petroleum can be preferably used because of the high resolution of petroleum in general.

The amount of petroleum-degrading bacteria inoculated into the organic material is preferably 1 × 10 ⁴ cells / g or more, more preferably 1 × 10 ⁵ cells / g or more, and further preferably 1 × 10 ⁶ to 1 × 10 ⁷ cells. / g.

Culturing of petroleum-degrading bacteria can be carried out by a known method, for example, by allowing to stand at room temperature. In addition, the culture can also be performed by repeatedly cutting back the deposited organic material. Water may be added as appropriate in order to adjust the water content of the organic material to an amount suitable for culturing. In order to adjust the temperature of the organic material to a temperature suitable for culturing, the organic material may be appropriately heated. The culture period is usually 1 day to 3 months, preferably 5 days to 1 month.

The number of petroleum-degrading bacteria in the purifying composition obtained by the production method of the present invention is preferably 1 × 10 ⁶ cells / g or more, more preferably 1 × 10 ⁷ cells / g or more, and further preferably 1 × 10 ⁸ to 1 × 10 ¹¹ cells / g.

Composition for Purifying Petroleum-Contaminated Soil The composition for purifying petroleum-contaminated soil of the present invention is characterized by containing 1 × 10 ⁶ cells / g or more of petroleum-degrading bacteria and 60% by mass or more of unfermented material. ..

The purification composition can be produced by the method described above. Petroleum-degrading bacteria, unfermented materials, etc. are the same as those described above.

The number of petroleum-degrading bacteria in the purifying composition of the present invention is preferably 1 × 10 ⁷ cells / g or more, and more preferably 1 × 10 ⁸ to 1 × 10 ¹¹ cells / g.

The purification composition in the present invention preferably contains 70% by mass or more of an unfermented material, more preferably 80% by mass or more, and further preferably contains only an unfermented material. Further, the upper limit of the content of the unfermented material is 100% by mass, 95% by mass, 90% by mass, 85% by mass and the like. The content of the unfermented material here means the content of the unfermented material as a raw material before culturing the petroleum-degrading bacteria.

The purifying composition of the present invention may contain materials other than unfermented materials as long as the effects of the present invention can be obtained, and examples thereof include compost and chemical fertilizers.

Method for Purifying Oil-Contaminated Soil The method for purifying oil -contaminated soil of the present invention includes a step of adding a composition for purifying contaminated soil produced by the above-mentioned production method or the above-mentioned purification composition to oil-contaminated soil. It is characterized by.

The amount of the purification composition added to the petroleum-contaminated soil is usually 1 to 10% by volume, preferably 2 to 8% by volume. By setting the amount of the purification composition added so that the total carbon content, C / N ratio, etc. in the oil-contaminated soil are within a specific range (for example, the range disclosed in Patent Document 4), petroleum It is also possible to improve the efficiency of decontamination.

In addition, the number of days required to purify petroleum-contaminated soil is usually 1.5 months or more, preferably 2 to 6 months.

In the method for purifying petroleum-contaminated soil of the present invention, in addition to the addition of the above-mentioned purification composition, additional treatments such as administration of petroleum-degrading bacteria and nutrients of petroleum-degrading bacteria can be performed, if necessary.

The production method of the present invention can produce a composition for purifying petroleum-contaminated soil at a lower cost than in the case of culturing petroleum-degrading bacteria using a medium. In addition, the composition for purifying petroleum-contaminated soil of the present invention is excellent in maintaining the number and activity of petroleum-degrading bacteria, so that petroleum-contaminated purification can be performed efficiently. Moreover, according to the present invention, recovery of vegetation can be expected after purification of petroleum pollution.

Hereinafter, examples will be given to explain the present invention in more detail. However, the present invention is not limited to these examples and the like.

[experimental method]
・ Total number of bacteria
(1) 1.0 g of sample in a 50 ml falcon tube sterilized (121 ° C, 15 minutes), 8.0 ml of DNA extraction buffer (pH 8.0) shown in Table 1 below, and 20% (w / v) sodium dodecyl sulfate solution. 1.0 ml was added.

(2) A falcon tube was set in a stirrer and stirred (1,500 rpm, room temperature, 20 minutes).
(3) 1.5 ml of the stirred solution was separated into a sterilized Eppendorf tube and centrifuged (8,000 rpm, 20 ° C., 10 minutes).
(4) Divide 700 μl of the aqueous layer into a new Eppendorf tube, add 700 ml of chloroform / isoamyl alcohol (24: 1, v / v), stir gently, and centrifuge (14,000 rpm, 20 ° C., 10 minutes). )did.
(5) 500 μl of the aqueous layer was separated into a new Eppendorf tube, 300 μl of 2-propanol was added, the mixture was gently stirred, and the mixture was centrifuged (14,000 rpm, 20 ° C., 20 minutes).
(6) The aqueous layer was removed, 1.0 ml of 70% (v / v) ethanol was added, and the mixture was centrifuged (14,000 rpm, 20 ° C., 5 minutes).
(7) The aqueous layer was removed and dried under reduced pressure (30 minutes with an aspirator).
(8) 50 μl of 10: 1 TE buffer (pH 8.0) shown in Table 2 below was added and dissolved to prepare an eDNA solution.

(9) The eDNA solution (5 μl) was subjected to agarose gel electrophoresis. In addition, smart Ladder (Nippon Gene Co., Ltd.) was used as a marker.
(10) The DNA band of smart Ladder was analyzed using the image analysis software KODAK 1D Image Analysis software (KODAK), a calibration curve of the amount of DNA with respect to the fluorescence intensity was created, and the amount of gel DNA was obtained using this calibration curve. ..
(11) From the obtained gel DNA amount, the eDNA amount was calculated by the following formula.

(12) From the calculated amount of eDNA, the total number of bacteria per 1.0 g of sample was calculated by the following formula.
Total number of bacteria (cells / g-soil) = amount of eDNA (μg / g) x 1.70 x 10 ⁸ [R ² = 0.938]

-Number of petroleum-degrading bacteria An eDNA solution was prepared by the same method as described above. 15 μl of the eDNA solution was subjected to agarose gel electrophoresis. A DNA band was excised from the agarose gel and the eDNA was purified. KAPA SYBR® FAST qPCR Master Mix 10 μl, 10 μM forward primer (5'-AACTAYMTCGARCAYTAYGG-3': SEQ ID NO: 1) and reverse primer (5'-TGRTCKSWRTGNCGYTGVARGTG-3': SEQ ID NO: 2) 1 μl, ROX A 20 μl reaction solution containing 0.4 μl of high and 1 to 5 μl of purified eDNA was added to a 200 μl volume tube and set in the Applied Biosystems 7300 Real Time System (Applied Biosystems) for real-time PCR. The reaction conditions for PCR were heating at 95 ° C. for 5 to 10 minutes, followed by reactions at 95 ° C. for 15 to 30 seconds and 60 ° C. for 30 to 60 seconds for 40 cycles. Of the samples used for real-time PCR, KAPA SYBR and ROX high were used according to the protocol of the KAPA SYBR qPCR kit (Kapa Biosystems). From the obtained Ct value, the number of petroleum-degrading bacteria was calculated using the following formula.
Petroleum-degrading bacteria count (cells / g-sample) = (3 × 10 ¹⁴ ) × e ^{(-0.516 × Ct value)}
By this method, petroleum-degrading bacteria can be specifically quantified with high sensitivity.

[Test Example 1] Analysis of components contained in various organic materials (biomass) Cow dung compost (fermented dried cow dung, Kyoshu Co., Ltd.) and chicken manure compost (fermented chicken manure, Agrienwai) are used as fermented materials, and soybean waste (fermented chicken manure, Agrienwai) is used as unfermented materials. Processed soybeans, J-Oil Mills Co., Ltd.) and Pete Moss (Pete Moss, Green Mail) were selected. The total carbon content (TC) of each biomass in a dry state was measured using a total organic carbon meter (TOC-V CPH, Shimadzu Corporation) and a solid sample combustion device (SSM-5000A, Shimadzu Corporation). In addition, the total nitrogen content (TN) was measured by the Kjeldahl method and the indophenol blue method. The TC, TN and C / N ratios of the dry biomass are shown in Table 3 below.

[Test Example 2] Growth of the number of hydrocarbon-degrading bacteria using fermented materials, fermented materials and unfermented materials (Rhodococcus erythropolis NDKK6)
We analyzed the growth behavior of Rhodococcus erythropolis NDKK6 in mixed biomass using only fermented materials and mixed biomass using fermented and unfermented materials. The culture was carried out by inoculating 300 ml of biomass so as to have 5 × 10 ⁵ cells / g-biomass, and culturing at room temperature and statically. The biomass used was sterilized by autoclave. The number of Rhodococcus erythropolis NDKK6 in 1 g of each mixed biomass was quantified for 4 weeks by real-time PCR using the alkB R2 gene possessed by Rhodococcus erythropolis NDKK6. The results are shown in Table 4 below.

In the mixed biomass of cow dung compost + chicken manure compost, the number of Rhodococcus erythropolis NDKK6 decreased in the first week in the mixed biomass with a high nitrogen source and a C / N ratio of 19, but compared to the 0th week by the second week. It increased up to 1.9 times. After that, it decreased in the 4th week and maintained 1.29 × 10 ⁶ cells / g-biomass in the 4th week. In the mixed biomass with a low nitrogen source and a C / N ratio of 21, the bacterial count reached the maximum at the 1st week and maintained 1.92 × 10 ⁶ cells / g-biomass at the 4th week. No significant change was observed in the growth behavior of Rhodococcus erythropolis NDKK6 in the mixed biomass of two types of cow dung compost + chicken manure compost.

On the other hand, in the mixed biomass of cow dung compost + soybean meal, the mixed biomass with a large nitrogen source and a C / N ratio of 13 increased by about 57 times by the first week compared to the 0th week. The number of bacteria was maintained at the same level until the 3rd week, but decreased to 7.38 × 10 ⁶ cells / g-biomass at the 4th week. In the mixed biomass with a C / N ratio of 16 with a small nitrogen source, it increased by about 56 times by the 1st week compared to the 0th week. After that, the number of bacteria decreased by the 2nd week, and 1.37 × 10 ⁶ cells / g-biomass was maintained by the 4th week. In the mixed biomass of cow dung compost + soybean meal, the mixed biomass with a large nitrogen source and a C / N ratio of 13 maintained and activated Rhodococcus erythropolis NDKK6.

Compared to the mixed biomass of cow dung compost + chicken manure compost, the mixed biomass of cow dung compost + soybean waste using unfermented organic materials increased the number of petrolytic bacteria Rodcoccus erythropolis NDKK6, so cow dung compost + soybean waste Mixed biomass was more suitable for the growth of Rodococcus erythropolis NDKK6. Of the four types of mixed biomass, Rhodococcus erythropolis NDKK6 was the most maintained and activated in the mixed biomass with a C / N ratio of cow dung compost + soybean meal of 13.

Next, the total number of bacteria in each biomass was measured. The results are shown in Table 5. As a result, it showed almost the same behavior as the petroleum-degrading bacterium Rhodococcus erythropolis NDKK6. From this, the bacteria existing in the environment were more suitable for growth when the unfermented material was added, and the growth was better when the nitrogen content was high.

[Test Example 3] Growth of the number of hydrocarbon-degrading bacteria using fermented materials, fermented materials and unfermented materials (Gordonia Terrae NDKY76A)
Similar to Test Example 2, the growth behavior of the petroleum-degrading bacterium Gordonia terae NDKY76A in the mixed biomass using only the fermented material and the mixed biomass using the fermented material and the unfermented material was analyzed. The number of Gordonia terae NDKY76A in 1 g of each mixed biomass was quantified for 4 weeks by real-time PCR using the alkB GT gene possessed by Gordonia terae NDKY76A. The results are shown in Table 6.

In the mixed biomass of cow dung compost + chicken manure compost, the mixed biomass with a large nitrogen source and a C / N ratio of 19 increased steadily and increased to 3.07 × 10 ⁶ cells / g-biomass in the third week. After that, it decreased in the 4th week, but maintained 1.34 × 10 ⁶ cells / g-biomass in the 4th week. In mixed biomass with a low nitrogen source and a C / N ratio of 21, the number of bacteria reached the maximum in the 2nd week (2.78 × 10 ⁶ cells / g-biomass) and decreased slightly in the 4th week, but 2.04 × 10 ⁶ cells. / g-biomass was maintained. In the mixed biomass of two types of cow dung compost + chicken manure compost, there was no significant change in the growth behavior of Gordonia terae NDKY76A as in Rhodococcus erythropolis NDKK6.

On the other hand, in the mixed biomass of cow dung compost + soybean meal, the mixed biomass with a large nitrogen source and a C / N ratio of 13 increased by about 33 times by the first week compared to the 0th week. The increase continued until the 4th week, and decreased to 8.67 × 10 ⁶ cells / g-biomass at the 4th week. In the mixed biomass with a C / N ratio of 16 with a small nitrogen source, it increased about 16 times by the 1st week compared to the 0th week. After that, it started to decrease, and by the 4th week, it became 1.22 × 10 ⁶ cells / g-biomass. In the mixed biomass of cow dung compost + soybean meal, the mixed biomass with a large nitrogen source and a C / N ratio of 13 maintained and activated Gordonia terae NDKY76A NDKK6. This was the same behavior as the petroleum-degrading bacterium Rhodococcus erythropolis.

Compared to the mixed biomass of cow dung compost + chicken manure compost, the mixed biomass of cow dung compost + soybean waste using unfermented organic materials increased the number of petrolytic bacteria Gordonia terae NDKY76A, so the mixed biomass of cow dung compost + soybean waste Biomass was more suitable for the growth of Gordonia Terrae NDKY76A NDKK6. Of the four types of mixed biomass, the mixed biomass with a C / N ratio of cow dung compost + soybean meal was the most maintained and activated. This result was almost the same as that of the petroleum-degrading bacterium Rhodococcus erythropolis NDKK6.

Next, the total number of bacteria in each biomass was measured. The results are shown in Table 7. As a result, it showed almost the same behavior as the petroleum-degrading bacterium Rhodococcus erythropolis NDKK6. From this, the bacteria existing in the environment were more suitable for growth when the unfermented material was added, and the growth was better when the nitrogen content was high.

[Test Example 4] Growth of hydrocarbon-degrading bacteria using unfermented materials (Rhodococcus erythropolis NDKK6)
The growth behavior of Rhodococcus erythropolis NDKK6 in mixed biomass (soybean meal / peat moss) using only unfermented materials was analyzed in the same manner as in Test Example 2. The results are shown in Table 8.

The mixed biomass using soybean meal and peat moss showed a higher bacterial count at 4 weeks than the case where the fermented material and the unfermented material were used in both C / N ratios. This indicates that the unfermented material has better growth of the petroleum-degrading bacterium Rhodococcus erythropolis NDKK6, and it has been clarified that the material is suitable for the growth of the petroleum-degrading bacterium. In addition, all the materials maintain a high number of bacteria even in the 7th week, which is a new method for supplying petroleum-degrading bacteria.

Next, the total number of bacteria in the unfermented mixed material was examined. The results are shown in Table 9. As a result, the total number of bacteria showed the same behavior as the growth of the petroleum-degrading bacterium Rhodococcus erythropolis NDKK6 in all the mixed materials. In addition, most of the total number of bacteria was Rhodococcus erythropolis NDKK6, and it was clarified that these materials are effective Rhodococcus erythropolis NDKK6 growth materials.

[Test Example 5] Growth of Hydrocarbon Degrading Bacteria Using Unfermented Materials (Gordonia Terrae NDKY76A)
The growth behavior of Gordonia terae NDKY76A in mixed biomass (soybean meal / peat moss) using only unfermented materials was analyzed in the same manner as in Test Example 2. The results are shown in Table 10.

The mixed biomass using soybean meal and peat moss showed higher bacterial counts at 3 to 4 weeks than when fermented and unfermented materials were used at any C / N ratio. This indicates that the combination of unfermented materials has better growth of the petroleum-degrading bacterium Gordonia Terrae NDKY76A than the fermented material, and it has been clarified that the material is suitable for the growth of petroleum-degrading bacteria. In addition, all the materials maintain a high number of bacteria even in the 7th week, which is a new method for supplying petroleum-degrading bacteria. These tendencies were similar to those of the petroleum-degrading fungus Rhodococcus erythropolis NDKK6.

Next, the total number of bacteria in the unfermented mixed material was examined. The results are shown in Table 11. As a result, the total number of bacteria showed the same behavior as the growth of the petroleum-degrading bacterium Gordonia terae NDKY76A in all the mixed materials. In addition, most of the total number of bacteria was Gordonia terae NDKY76A, and it was clarified that these materials are effective Gordonia terae NDKY76A growth materials. This result was also similar to Rhodococcus erythropolis NDKK6, and it was clarified that these materials can be applied to any petroleum-degrading bacteria.

[Test Example 6] Bioremediation using biomass material containing Rhodococcus erythropolis NDKK6 When we searched for biomass suitable for growth of Rhodococcus erythropolis NDKK6, we found that C / N was a mixture of unfermented soybean residue and peat moss. It was found that Rhodococcus erythropolis NDKK6 was maintained and activated by biomass with a ratio of 20. Therefore, bioremediation of hydrocarbon-contaminated soil was performed using this biomass.

Similar to Test Example 2, Rhodococcus erythropolis NDKK6 was inoculated into soybean meal + peat moss (C / N: 20) and cultured at room temperature for 3 weeks (microorganism-containing material). Then, 1% (v / v) of the microbial-containing material was added to the contaminated soil to which the base oil (lubricating oil) of 5,000 mg / kg was added. As a control experiment, only soybean meal + peat moss was added to the same contaminated soil at 1% (v / v), and then cultured Rhodococcus erythropolis NDKK6 was added to 1 × 10 ⁷ cells / g-soil. For the oil content analysis, an infrared analysis method, which is an official method, was used.

Table 12 shows the results of Rhodococcus erythropolis NDKK6 counts in hydrocarbon-contaminated soil, Table 13 shows the results of total bacterial counts, and Table 14 shows the results of base oil concentration.

Comparing Rhodococcus erythropolis NDKK6, which is a petroleum-degrading bacterium, and putting it into contaminated soil in a mixed material, and Rhodococcus erythropolis NDKK6, which was cultivated by the same method as before, were put into contaminated soil. The numbers of NDKK6 were almost the same (Table 12). From this, it was clarified that the petroleum-degrading bacterium Rhodococcus erythropolis NDKK6 functions effectively even in a method of growing in a material with no energy cost.

Similarly, when the total number of bacteria in the contaminated soil was measured, the total number of bacteria showed almost the same behavior, and it was clarified that the method of growing in the mixed material was effective (Table 13).

Finally, when the remaining oil content was analyzed, almost the same oil content decomposition was shown (Table 14). These results indicate the effectiveness of a new method for growing petroleum-degrading bacteria in organic materials instead of expensive strain culture.

[Test Example 7] Biomass using biomass material containing Gordonia Terrae NDKY76A Similar to Rhodococcus erythropolis NDKK6, a biomass that is suitable for the growth of the petroleum-degrading bacterium Gordonia terae NDKY76A was searched for unfermented. It was found that Gordonia terae NDKY76A is maintained and activated by biomass with a C / N ratio of 20, which is a mixture of soybean cake and peat moss, which are the materials. This result was similar to that of the petroleum-degrading bacterium Rhodococcus erythropolis NDKK6. Therefore, bioremediation of hydrocarbon-contaminated soil was performed using this biomass.

In the experiment, as in Test Example 6, soybean meal + peat moss (C / N: 20) was inoculated with Gordonia terae NDKY76A and cultured at room temperature for 3 weeks (microorganism-containing material). Then, 1% (v / v) of microbial-containing material was added to the contaminated soil to which 5,000 mg / kg of base oil (lubricating oil) was added. As a control experiment, only soybean meal + peat moss was added to the same contaminated soil at 1% (v / v), and then cultured Gordonia terae NDKY76A was added to 1 × 10 ⁷ cells / g-soil.

Table 15 shows the results of the Gordonia terae NDKY76A number in the hydrocarbon-contaminated soil, Table 16 shows the result of the total number of bacteria, and Table 17 shows the result of the base oil concentration.

Comparing the number of Gordonia terae NDKY76A NDKY76A cultivated by the same method as the conventional method and the NDKY76A NDKY76A cultivated in the contaminated soil. Were almost equivalent (Table 15). From this, it was clarified that the petroleum-degrading bacterium Gordonia Terrae NDKY76A functions effectively even in a method of growing in materials with low energy cost. This result was similar to that of the petroleum-degrading bacterium Rhodococcus erythropolis NDKK6.

Similarly, when the total number of bacteria in the contaminated soil was measured, the total number of bacteria showed almost the same behavior, and it was clarified that the method of growing in the mixed material was effective (Table 16).

Finally, when the remaining oil content was analyzed, almost the same oil content decomposition was shown (Table 17). Based on these facts, the petroleum-degrading bacterium Gordonia Terrae NDKY76A, like the petroleum-degrading bacterium Rhodococcus erythropolis NDKK6, is a new method for growing petroleum-degrading bacteria in organic materials that can significantly reduce costs, instead of culturing expensive strains. The effectiveness of the method was shown.

Claims (4)

A method for producing a composition for purifying petroleum-contaminated soil, which comprises a step of inoculating and culturing a bacterium capable of decomposing petroleum in an organic material including an unfermented material.
The bacterium is a bacterium of the genus Gordonia and / or the genus Rhodococcus.
The method, wherein the unfermented material is at least one selected from the group consisting of soybean meal and peat moss . The production method according to claim 1, wherein the C / N ratio in the organic material is 10 to 30. The production method according to claim 1 or 2, wherein the unfermented material is contained in the organic material in an amount of 60% by mass or more. A method for purifying petroleum-contaminated soil, which comprises a step of adding a composition for purifying contaminated soil produced by the production method according to any one of claims 1 to 3 to petroleum-contaminated soil.