JP2018051431A - Method of purifying contaminated soil - Google Patents
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Abstract
Description
本発明は、油分で汚染された土壌の浄化方法に関する。 The present invention relates to a method for purifying soil contaminated with oil.
油分で汚染された土壌を浄化する方法として、土壌中に存在する微生物の油分解性能を利用したバイオレメディエーションが知られている。 As a method for purifying soil contaminated with oil, bioremediation using the oil decomposing ability of microorganisms present in the soil is known.
バイオレメディエーションを行う際には、微生物自体を活性化させるために、土壌に窒素やリン酸のような栄養塩を添加することが一般的である。 When performing bioremediation, in order to activate microorganisms themselves, it is common to add nutrient salts, such as nitrogen and phosphoric acid, to soil.
また、バイオレメディエーションには、有機汚染物質の分解性能を備えた好気性微生物を活性化させて有機汚染物質を発酵分解させる、通気型の工法もある（特許文献１参照）。 Bioremediation also includes an aeration-type method in which aerobic microorganisms capable of decomposing organic pollutants are activated to fermentatively decompose organic pollutants (see Patent Document 1).
このような通気型のバイオレメディエーションにおいて微生物自体を活性化させるための方法として、特許文献２には人工腐植土と糖類を利用する方法が開示されており、特許文献３には糟糠類を利用する方法が開示されている。また、特許文献４には、糟糠類を含有する発酵助材を添加した汚染土壌の温度を維持するために通気量を制御する方法も開示されている。 As a method for activating microorganisms in such aeration type bioremediation, Patent Document 2 discloses a method using artificial humus and saccharides, and Patent Document 3 uses moss. A method is disclosed. Patent Document 4 also discloses a method for controlling the air flow rate in order to maintain the temperature of contaminated soil to which fermentation aid containing moss is added.
本発明者らの研究により、汚染土壌に特定の物質を添加することにより、油分の分解に寄与する細菌を増殖させ、油分の分解を促進できることが明らかとなった。 The study by the present inventors has revealed that by adding a specific substance to the contaminated soil, bacteria contributing to the degradation of the oil can be grown and the degradation of the oil can be promoted.
本発明は、汚染土壌に含まれる油分の分解を促進することが可能な汚染土壌の浄化方法を提供することを目的とする。 An object of this invention is to provide the purification method of the contaminated soil which can accelerate | stimulate decomposition | disassembly of the oil component contained in a contaminated soil.
上記課題を解決するために、本発明の汚染土壌の浄化方法は、油分で汚染された汚染土壌を油分解細菌により浄化する方法であって、前記汚染土壌に対してジオレイン酸ポリエチレングリコールを添加する。 In order to solve the above problems, a method for purifying contaminated soil according to the present invention is a method for purifying contaminated soil contaminated with oil by oil-decomposing bacteria, and adding polyethylene glycol dioleate to the contaminated soil. .
本発明によれば、汚染土壌に含まれる油分の分解を促進できる。 According to the present invention, decomposition of oil contained in contaminated soil can be promoted.
＝＝実施形態＝＝
本実施形態は、油分で汚染された汚染土壌を油分解細菌により浄化する汚染土壌の浄化方法に関する。本実施形態に係る浄化方法においては、汚染土壌に対して脂肪酸エステル系の薬剤を添加する。
== Embodiment ==
The present embodiment relates to a method for purifying contaminated soil in which contaminated soil contaminated with oil is purified by oil-degrading bacteria. In the purification method according to the present embodiment, a fatty acid ester chemical is added to the contaminated soil.
［油分］
本実施形態に係る浄化方法が対象とする油分は、たとえば、ガソリン、灯油、軽油、重油、機械油、潤滑油、原油等の石油由来の油分や、タールやベンゼン等の石炭由来の油分、石油化学製品由来の油分である。
[Oil]
Oils targeted by the purification method according to the present embodiment include, for example, petroleum-derived oils such as gasoline, kerosene, light oil, heavy oil, machine oil, lubricating oil, and crude oil, coal-derived oils such as tar and benzene, petroleum Oil derived from chemical products.
［油分解細菌］
油分解細菌は、土壌中に存在する一般的な細菌（一般細菌）のうち、上記の油分を分解することができる細菌である。具体的には、油分解細菌は、油分に含まれるアルカンを分解する酵素を有する細菌である。本実施形態に係る浄化方法では、土壌中に元々存在する油分解細菌を利用してもよいし、浄化方法を実施する際に新たな油分解細菌を土壌に添加することでもよい。
[Oil-degrading bacteria]
Oil-decomposing bacteria are bacteria capable of degrading the above-mentioned oil among general bacteria (general bacteria) present in soil. Specifically, the oil-decomposing bacterium is a bacterium having an enzyme that decomposes alkane contained in oil. In the purification method according to the present embodiment, oil-decomposing bacteria originally present in the soil may be used, or new oil-degrading bacteria may be added to the soil when the purification method is performed.
［脂肪酸エステル系の薬剤］
脂肪酸エステル型の薬剤は、オレイン酸系のポリエチレングリコールを用いることができる。本実施形態では、ジオレイン酸ポリエチレングリコールを用いる。ジオレイン酸ポリエチレングリコールは、油分に含まれるアルカンよりも土壌中における分解速度が速い。油分解細菌は、土壌中で分解したジオレイン酸ポリエチレングリコールを栄養源として増殖する。油分解細菌が増殖することにより、土壌中の油分の分解が促進される。
[Fatty acid ester drugs]
As the fatty acid ester type drug, oleic acid polyethylene glycol can be used. In this embodiment, polyethylene glycol dioleate is used. Polyethylene glycol dioleate has a faster decomposition rate in soil than alkanes contained in the oil. Oil-degrading bacteria grow using polyethylene glycol dioleate degraded in soil as a nutrient source. The decomposition of oil in the soil is promoted by the growth of oil-degrading bacteria.
［浄化方法］
本実施形態に係る浄化方法は、油分で汚染された汚染土壌に対してジオレイン酸ポリエチレングリコールを添加することができれば特に限定されない。ジオレイン酸ポリエチレングリコールは、油分量よりも低い濃度となることが好ましく、土壌に対して重量比で０．０１〜１．０％添加することが好ましい。
[Purification method]
The purification method according to the present embodiment is not particularly limited as long as polyethylene glycol dioleate can be added to contaminated soil contaminated with oil. The polyethylene glycol dioleate preferably has a concentration lower than the oil content, and is preferably added in an amount of 0.01 to 1.0% by weight with respect to the soil.
＝＝実施例＝＝
［油分の分解試験］
土壌に含まれる油分の分解について、以下の試験（実施例１、比較例１、及び比較例２）により検証した。
== Example ==
[Decomposition test of oil]
About the decomposition | disassembly of the oil content contained in soil, it verified by the following tests (Example 1, the comparative example 1, and the comparative example 2).
試験用の汚染土として、軽油汚染土を使用した。軽油汚染土は、油分濃度が約２４５０ｍｇ／ｋｇのものを使用した。油分濃度は、ＴＰＨ試験法（油汚染対策ガイドライン（平成１８年３月）資料３ ＧＣ−ＦＩＤ法によるＴＰＨ試験方法参照）により求めた。 Light oil contaminated soil was used as the contaminated soil for the test. As the light oil-contaminated soil, one having an oil concentration of about 2450 mg / kg was used. The oil concentration was determined by the TPH test method (see Oil Pollution Countermeasure Guidelines (March 2006), Document 3, TPH Test Method by GC-FID Method).
試験方法は、以下の通りである。 The test method is as follows.
（実施例１：ジオレイン酸ポリエチレングリコール＋尿素＋過リン酸石灰）
軽油汚染土６ｋｇを容器に分取し、軽油汚染土に対して重量比で尿素肥料を０．１％、過リン酸石灰を０．９％、ジオレイン酸ポリエチレングリコール（原液）を０．９％添加し、撹拌・混合した。その後、軽油汚染土をワグネルポット（硬質樹脂製。容量１７Ｌ）に入れ、２０℃の温度下で保管し、１週間毎に撹拌をおこなった。
(Example 1: Polyethylene glycol dioleate + urea + lime superphosphate)
Dispense 6 kg of light oil contaminated soil into a container, 0.1% urea fertilizer, 0.9% lime superphosphate, 0.9% dioleic acid polyethylene glycol (raw solution) by weight ratio to the light oil contaminated soil Added, stirred and mixed. Thereafter, the light oil-contaminated soil was placed in a Wagner pot (made of hard resin, volume 17 L), stored at a temperature of 20 ° C., and stirred every week.
（比較例１：対象区）
軽油汚染土６ｋｇをワグネルポット（硬質樹脂製。容量１７Ｌ）に入れ、２０℃の温度下で保管し、１週間毎に撹拌をおこなった。
(Comparative Example 1: Target area)
6 kg of light oil contaminated soil was placed in a Wagner pot (made of hard resin, capacity 17 L), stored at a temperature of 20 ° C., and stirred every week.
（比較例２：尿素＋過リン酸石灰）
軽油汚染土６ｋｇを容器に分取し、軽油汚染土に対して重量比で尿素肥料を０．１％、過リン酸石灰を０．９％添加し、撹拌・混合した。その後、軽油汚染土をワグネルポット（硬質樹脂製。容量１７Ｌ）に入れ、２０℃の温度下で保管し、１週間毎に撹拌をおこなった。
(Comparative Example 2: Urea + lime superphosphate)
6 kg of light oil-contaminated soil was dispensed into a container, 0.1% urea fertilizer and 0.9% superphosphate lime were added to the light oil-contaminated soil by weight, and the mixture was stirred and mixed. Thereafter, the light oil-contaminated soil was placed in a Wagner pot (made of hard resin, volume 17 L), stored at a temperature of 20 ° C., and stirred every week.
＜油分濃度の測定＞
実施例１、比較例１及び比較例２それぞれの汚染土について、７日後、５０日後の油分濃度を上記ＴＰＨ試験法により測定した。表１は、油分濃度の変化、及び５０日後の油分の残存率（５０日後の油分濃度／初期の油分濃度）を示したものである。
<Measurement of oil concentration>
About each contaminated soil of Example 1, Comparative Example 1 and Comparative Example 2, the oil concentration after 7 days and 50 days was measured by the above TPH test method. Table 1 shows the change in the oil concentration and the residual ratio of the oil after 50 days (oil concentration after 50 days / initial oil concentration).
＜一般細菌数の測定＞
また、実施例１、比較例１及び比較例２それぞれの汚染土における一般細菌数の変化を希釈平板法（日本土壌肥料学会監修、土壌環境分析法編集委員会編、「土壌環境分析法」、株式会社博友社、ｐ１３８−１４１。培地：アルブミン培地、温度：２８℃）により測定した。図１は、軽油汚染土中の一般細菌数を示すグラフである。縦軸は一般細菌数（個／ｇ・湿土）であり、横軸は試験開始からの経過日数である。
<Measurement of general bacterial count>
In addition, the change in the number of general bacteria in the contaminated soil of each of Example 1, Comparative Example 1 and Comparative Example 2 was determined by the dilution plate method (supervised by the Japan Soil Fertilizer Society, edited by the Soil Environment Analysis Method Editorial Committee, “Soil Environment Analysis Method”, Hirotomo Co., Ltd., p138-141, medium: albumin medium, temperature: 28 ° C.). FIG. 1 is a graph showing the number of general bacteria in light oil contaminated soil. The vertical axis is the number of general bacteria (cells / g · wet earth), and the horizontal axis is the number of days elapsed from the start of the test.
表１から明らかなように、実施例１においては７日経過時点で油分濃度が大きく低下し、５０日後には油分の残存率が０．３０まで減少した。 As is clear from Table 1, in Example 1, the oil concentration was greatly reduced after 7 days, and the residual ratio of oil was reduced to 0.30 after 50 days.
一方、比較例２においては、油分濃度の低下は見られたがその割合は小さく、実施例１のように、油分濃度が大きく減少することは無かった。 On the other hand, in Comparative Example 2, a decrease in the oil concentration was observed, but the ratio was small, and the oil concentration did not decrease significantly as in Example 1.
また、図１から明らかなように、実施例１においては、比較例１及び比較例２に比べて土中の一般細菌が大きく増殖した。また、増殖した一般細菌の数は、５０日経過後でもほとんど変わらないことが明らかとなった。 As is clear from FIG. 1, in Example 1, the general bacteria in the soil grew larger than in Comparative Examples 1 and 2. In addition, it was clarified that the number of general bacteria that proliferated hardly changed after 50 days.
以上の結果から明らかなように、油で汚染された汚染土壌中にジオレイン酸ポリエチレングリコールを添加することにより一般細菌を増殖させることができ、その結果、油分の分解が促進することが明らかとなった。 As is clear from the above results, it is clear that general bacteria can be grown by adding polyethylene glycol dioleate to contaminated soil contaminated with oil, and as a result, decomposition of oil is promoted. It was.
［油分解細菌の増殖試験］
上述の通り、油分の分解試験の結果によりジオレイン酸ポリエチレングリコールを添加することで一般細菌が増殖することが明らかとなった。一方、油分の分解には一般細菌に含まれる油分解細菌が寄与していると考えられる。そこで、一般細菌の増殖及び油分解細菌の増殖に関して、以下の試験（実施例２、比較例３〜比較例５）により検証した。
[Proliferation test of oil-degrading bacteria]
As described above, it was clarified that general bacteria grow by adding polyethylene glycol dioleate, as a result of the oil decomposition test. On the other hand, it is considered that oil-degrading bacteria contained in general bacteria contribute to the degradation of oil. Therefore, the following tests (Example 2, Comparative Example 3 to Comparative Example 5) were verified with respect to the growth of general bacteria and the growth of oil-degrading bacteria.
試験用の汚染土として、実施例１等と同様の軽油汚染土を用いた。 As the contaminated soil for the test, the same light oil-contaminated soil as in Example 1 was used.
また、ＮＨ４ＮＯ３：５ｇ、Ｋ２ＨＰＯ４：２．５ｇ、ＭｇＳＯ４・７Ｈ２Ｏ：１．０ｇ、滅菌水１Ｌを混合・撹拌した後、オートクレーブ滅菌（１２１℃、１５分）を行うことにより作成した無機培地を用いた。 Further, NH 4 NO 3 : 5 g, K 2 HPO 4 : 2.5 g, MgSO 4 .7H 2 O: 1.0 g, and 1 L of sterilized water are mixed and stirred, and then autoclaved (121 ° C., 15 minutes). The inorganic culture medium prepared by this was used.
試験方法は、以下の通りである。 The test method is as follows.
（実施例２：ジオレイン酸ポリエチレングリコール）
汚染土１ｇを滅菌した２００ｍＬ三角フラスコに分取し、無機培地１００ｍＬを添加した。その後、滅菌水で５％に希釈したジオレイン酸ポリエチレングリコール１０ｍＬを添加し、シリコ栓をした後、２５℃で３日間、振とう培養した（回転数：１２１ｒｐｍ）。
(Example 2: Polyethylene glycol dioleate)
1 g of contaminated soil was dispensed into a sterilized 200 mL Erlenmeyer flask, and 100 mL of inorganic medium was added. Thereafter, 10 ml of polyethylene glycol dioleate diluted to 5% with sterilized water was added, and after silico stoppering, shaking culture was performed at 25 ° C. for 3 days (rotation speed: 121 rpm).
（比較例３：対象区）
汚染土１ｇを滅菌した２００ｍＬ三角フラスコに分取し、無機培地１００ｍＬを添加した。シリコ栓をした後、２５℃で３日間、振とう培養した（回転数：１２１ｒｐｍ）。
(Comparative example 3: Target area)
1 g of contaminated soil was dispensed into a sterilized 200 mL Erlenmeyer flask, and 100 mL of inorganic medium was added. After plugging the silico, it was cultured with shaking at 25 ° C. for 3 days (rotation speed: 121 rpm).
（比較例４：グルコース）
汚染土１ｇを滅菌した２００ｍＬ三角フラスコに分取し、無機培地１００ｍＬを添加した。その後、グルコース０．５ｇを添加し、シリコ栓をした後、２５℃で３日間、振とう培養した（回転数：１２１ｒｐｍ）。
(Comparative Example 4: Glucose)
1 g of contaminated soil was dispensed into a sterilized 200 mL Erlenmeyer flask, and 100 mL of inorganic medium was added. Thereafter, 0.5 g of glucose was added, and after silico stoppering, the cells were cultured with shaking at 25 ° C. for 3 days (rotation speed: 121 rpm).
（比較例５：軽油）
汚染土１ｇを滅菌した２００ｍＬ三角フラスコに分取し、無機培地１００ｍＬを添加した。その後、軽油０．５ｍＬを添加し、シリコ栓をした後、２５℃で３日間、振とう培養した（回転数：１２１ｒｐｍ）。
(Comparative Example 5: Light oil)
1 g of contaminated soil was dispensed into a sterilized 200 mL Erlenmeyer flask, and 100 mL of inorganic medium was added. Thereafter, 0.5 mL of light oil was added, and after silico stoppering, shaking culture was performed at 25 ° C. for 3 days (rotation speed: 121 rpm).
＜遺伝子数の測定＞
振とう培養後の懸濁液をスターラーで撹拌しながら、懸濁液の上澄みを土粒子ごと３００μＬ分取した。市販の抽出キッド（ＩＳＯＩＬ ｆｏｒ Ｂｅａｄｓ Ｂｅａｔｉｎｇ、株式会社ニッポンジーン製）を用い、分取した懸濁液に含まれている細菌のＤＮＡを抽出した。
<Measurement of the number of genes>
While stirring the suspension after shaking culture with a stirrer, 300 μL of the supernatant of the suspension was collected together with soil particles. Using a commercially available extraction kid (ISOIL for Beads Beating, manufactured by Nippon Gene Co., Ltd.), bacterial DNA contained in the collected suspension was extracted.
そして、抽出したＤＮＡを適切な濃度に希釈し、表２に示す反応条件により、リアルタイムＰＣＲ装置（Ｒｏｔｏｒ−Ｇｅｎｅ Ｑ、ＱＩＡＧＥＮ製）で細菌の遺伝子数を測定した。油分解細菌用としては、アルカンの分解酵素を特異的に判別するａｌｋＢ検出用プライマー（表３参照。出典：ＤｉｏＧＯ Ｊｕｒｅｌｅｖｉｃｉｕｓ ｅｔ．ａｌ．“Ｔｈｅ ｕｓｅ ｏｆ ａ Ｃｏｍｂｉｎａｔｉｏｎ ｏｆ ａｌｋＢ Ｐｒｉｍｅｒｓ ｔｏ Ｂｅｔｔｅｒ Ｃｈａｒａｃｔｅｒｉｚｅ ｔｈｅ Ｄｉｓｔｒｉｂｕｔｉｏｎ ｏｆ Ａｌｋａｎｅ−Ｄｅｇｒａｄｉｎｇ Ｂａｃｔｅｒｉａ” ＰＬＯＳ ＯＮＥ，Ｖｏｌ．８（６）ｅ６６５６５（２０１３））を用いた。一般細菌用としては、１６ＳｒＤＮＡを用いた。増幅酵素は、ＴＩＴＡＮＩＵＭ（登録商標） Ｔａｑ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（タカラバイオ株式会社製）を用いた。蛍光酵素は、ＳＹＢＲ（登録商標） Ｇｒｅｅｎ Ｉ（タカラバイオ株式会社製）を用いた。 The extracted DNA was diluted to an appropriate concentration, and the number of bacterial genes was measured with a real-time PCR apparatus (Rotor-Gene Q, manufactured by QIAGEN) under the reaction conditions shown in Table 2. For oil-degrading bacteria, a primer for detecting alkB that specifically discriminates alkane-degrading enzymes (see Table 3. Source: DIOGO Jurelevius et al. -Degrading Bacteria "PLOS ONE, Vol. 8 (6) e66565 (2013)) was used. For general bacteria, 16S rDNA was used. As the amplification enzyme, TITANIUM (registered trademark) Taq DNA Polymerase (manufactured by Takara Bio Inc.) was used. As the fluorescent enzyme, SYBR (registered trademark) Green I (manufactured by Takara Bio Inc.) was used.
図２は、一般細菌の遺伝子数（１６Ｓリボゾームの遺伝子数）を示すグラフである。図３は、油分解細菌の遺伝子数（ａｌｋＢの遺伝子数）を示すグラフである。いずれのグラフも縦軸は遺伝子数（個／ｇ−ｗｅｔ）である。 FIG. 2 is a graph showing the number of genes of general bacteria (the number of genes of 16S ribosome). FIG. 3 is a graph showing the number of genes for oil-degrading bacteria (the number of genes for alkB). In any graph, the vertical axis represents the number of genes (pieces / g-wet).
図２及び図３から明らかなように、実施例２では、一般細菌が増殖すると共に、油分解細菌も増殖した。 As apparent from FIGS. 2 and 3, in Example 2, general bacteria grew and oil-degrading bacteria also grew.
一方、一般的に細菌の栄養源として広く知られているグルコースを添加した場合（比較例４）、一般細菌は実施例１の場合と同程度増殖しているが、油分解細菌はほとんど増殖しなかった。 On the other hand, when glucose, which is generally known as a nutrient source for bacteria, is added (Comparative Example 4), general bacteria grow to the same extent as in Example 1, but oil-degrading bacteria almost grow. There wasn't.
また、油分解細菌の栄養源となりうる軽油を添加した場合（比較例５）、一般細菌数及び油分解細菌のいずれも対象区（比較例３）と同程度しか増殖しなかった。 Moreover, when the light oil which can become a nutrient source of oil-decomposing bacteria was added (Comparative Example 5), both the number of general bacteria and the oil-decomposing bacteria grew to the same extent as the target section (Comparative Example 3).
以上の結果から、土壌にジオレイン酸ポリエチレングリコールを添加することにより、一般細菌に含まれる油分解細菌を特に増殖できることが明らかとなった。 From the above results, it was revealed that oil-degrading bacteria contained in general bacteria can be especially grown by adding dioleic acid polyethylene glycol to soil.
上記実施例１〜２及び比較例１〜５の結果を総合すると、ジオレイン酸ポリエチレングリコールを油分で汚染された汚染土壌に添加することにより、油分解細菌の増殖を促し、その結果、土壌中の油分を効率よく分解できることが明らかとなった。 Summarizing the results of Examples 1-2 and Comparative Examples 1-5 above, by adding polyethylene glycol dioleate to contaminated soil contaminated with oil, it promotes the growth of oil-degrading bacteria, and as a result, It became clear that oil can be decomposed efficiently.
上記実施形態等は、例として提示したものであり、発明の範囲を限定するものではない。上記の構成は、適宜組み合わせて実施することが可能であり、発明の要旨を逸脱しない範囲で、種々の省略、置き換え、変更を行うことができる。上記実施形態やその変形は、発明の範囲や要旨に含まれると同様に、特許請求の範囲に記載された発明とその均等の範囲に含まれる。
The said embodiment etc. are shown as an example and do not limit the scope of the invention. The above configurations can be implemented in appropriate combination, and various omissions, replacements, and changes can be made without departing from the scope of the invention. The above-described embodiments and modifications thereof are included in the invention described in the claims and equivalents thereof in the same manner as included in the scope and spirit of the invention.
Claims (1)
前記汚染土壌に対してジオレイン酸ポリエチレングリコールを添加する汚染土壌の浄化方法。 A method for purifying contaminated soil, which purifies contaminated soil contaminated with oil by oil-degrading bacteria,
A method for purifying contaminated soil, comprising adding polyethylene glycol dioleate to the contaminated soil.
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