JP2003112164A - Cleaning method for soil polluted with organochlorine compound - Google Patents
Cleaning method for soil polluted with organochlorine compoundInfo
- Publication number
- JP2003112164A JP2003112164A JP2001308637A JP2001308637A JP2003112164A JP 2003112164 A JP2003112164 A JP 2003112164A JP 2001308637 A JP2001308637 A JP 2001308637A JP 2001308637 A JP2001308637 A JP 2001308637A JP 2003112164 A JP2003112164 A JP 2003112164A
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- Prior art keywords
- soil
- hydrogen peroxide
- contaminated soil
- degrading
- bacteria
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- Processing Of Solid Wastes (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、汚染土壌を特定の
分解微生物を用いて浄化（バイオオーグメンテーショ
ン）する方法に関する。TECHNICAL FIELD The present invention relates to a method for purifying (bioaugmentation) contaminated soil by using a specific degrading microorganism.
【０００２】[0002]
【従来の技術】有機塩素化合物の中でもダイオキシンに
よる土壌汚染が顕在化しつつあり、効率的かつ安全な浄
化方法が望まれている。ダイオキシン汚染土壌の浄化方
法は、完全焼却法、熱分解処理法、アルカリ触媒分解
法、光分解法、超臨界水分解法及び微生物分解法に大別
できるが、光分解法又は微生物分解法以外を利用する場
合には、いずれも高温／高圧による処理が必要である。
また、土壌のダイオキシン汚染は極低濃度で表面的かつ
広範囲に汚染が広がっているため、常温常圧での浄化が
可能な微生物分解法（バイオ・レメディエーション）に
よる手法が注目されている。しかし、土壌中のダイオキ
シン量は極めて低濃度であるため、土壌生物相における
土着分解菌の占有率は極めて低いことが予測される。そ
のため、ダイオキシン汚染土壌のバイオレメデーション
では、土着菌を活性化させ浄化する方法、すなわちバイ
オスティムレーションの適用は困難であり、特定の分解
菌又は分解酵素を外部から土壌に注入し活性化させる手
法、すなわちバイオオーグメンテーションが注目され
る。2. Description of the Related Art Among organic chlorine compounds, soil pollution due to dioxin is becoming apparent, and an efficient and safe purification method is desired. The methods for cleaning dioxin-contaminated soil can be broadly divided into complete incineration method, thermal decomposition method, alkali catalytic decomposition method, photodecomposition method, supercritical water decomposition method and microbial decomposition method, but methods other than photodecomposition method or microbial decomposition method are used. In that case, both require high temperature / high pressure treatment.
In addition, since dioxin contamination of soil spreads over a wide area over a wide area at an extremely low concentration, a method using a microbial decomposition method (bio-remediation) that can be purified at room temperature and atmospheric pressure is drawing attention. However, since the amount of dioxins in soil is extremely low, it is predicted that the occupancy rate of indigenous degrading bacteria in soil biota will be extremely low. Therefore, it is difficult to apply the biostimulation method that activates indigenous bacteria to biodegradation of dioxin-contaminated soil, that is, biostimulation is difficult. , Ie bioaugmentation is of interest.
【０００３】ダイオキシン分解菌は、シュードモナス
（Pshudomonas）属及びバチルス（Bacillus）属等の細
菌類、並びに白色腐朽菌等の担子菌であることが明らか
となっている。特に、Phanerochaete chrysosporiumを
始めとする特定の白色腐朽菌は、リグニン分解に関与す
るリグニンペルオキシダーゼ、マンガンペルオキシダー
ゼ及びラッカーゼの複合酵素群を生成することによっ
て、ダイオキシンを、塩素、酸素及び二酸化炭素まで完
全分解できることが明らかとなっている。さらにこれら
の白色腐朽菌は、ダイオキシン以外にも、DDT（1,1,1-
トリクロロ-2,2-ビス(4-クロロフェニル)エタン）等の
殺虫剤、2,4-D（2,4-ジクロロフェノキシ酢酸）、2,4,5
-T（2,4,5-トリクロロフェノキシ酢酸）等の除草剤、及
びその他の有機塩素化合物の分解に関与することが確認
されており、様々な環境汚染物質の分解が期待される微
生物である。しかしながら、白色腐朽菌の増殖及び分解
酵素の生成には、温度、酸素、土壌中の水分量、pH等の
環境要因が大きく影響するとともに、土着菌との競合に
よって増殖阻害を受け、単に白色腐朽菌を汚染土壌に接
種するだけでは汚染土壌の浄化は不可能である。It has been clarified that dioxin-degrading bacteria are bacteria such as the genus Pshudomonas and the genus Bacillus, and basidiomycetes such as white-rot fungi. In particular, certain white-rot fungi such as Phanerochaete chrysosporium can completely decompose dioxin to chlorine, oxygen and carbon dioxide by producing a complex enzyme group of lignin peroxidase, manganese peroxidase and laccase involved in lignin decomposition. Has become clear. In addition to dioxins, these white-rot fungi are also found in DDT (1,1,1-
Trichloro-2,2-bis (4-chlorophenyl) ethane) and other insecticides, 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5
-T (2,4,5-trichlorophenoxyacetic acid) and other herbicides, and it has been confirmed to be involved in the decomposition of other organochlorine compounds, and is a microorganism expected to decompose various environmental pollutants. . However, the growth of white-rot fungi and the production of degrading enzymes are greatly affected by environmental factors such as temperature, oxygen, water content in soil, and pH, and growth inhibition is caused by competition with indigenous bacteria, and the white-rot fungus is simply destroyed. It is impossible to clean the contaminated soil simply by inoculating the contaminated soil with the fungus.
【０００４】汚染土壌中で分解菌及び分解酵素を十分に
作用させるには、分解菌の競合となる土壌中の微生物を
低減させ、さらにダイオキシン分解酵素（リグニンペル
オキシダーゼ及びマンガンペルオキシダーゼを始めとす
るペルオキシダーゼ）の活性阻害となる各種酵素及び腐
植物質等を低下させる必要がある。しかしながら、オー
トクレーブ滅菌や加熱滅菌では土壌滅菌の施設が必要で
あるだけでなく、莫大なエネルギーを要するため実用的
ではない。また、塩素系の殺菌剤等の使用は環境の二次
汚染につながり、これらの殺菌剤の使用も不可能であ
る。従って、バイオレメディエーションにより汚染土壌
を浄化する方法において、より効率的に分解菌及び分解
酵素を作用させることが望まれていた。In order for the decomposing bacteria and the degrading enzymes to sufficiently act in the contaminated soil, the microorganisms in the soil that compete with the decomposing bacteria are reduced, and further dioxin degrading enzymes (peroxidase such as lignin peroxidase and manganese peroxidase). It is necessary to reduce various enzymes and humic substances that inhibit the activity of the. However, autoclave sterilization and heat sterilization are not practical because not only a facility for soil sterilization is required but also enormous energy is required. Further, the use of chlorine-based bactericides leads to secondary pollution of the environment, and it is impossible to use these bactericides. Therefore, in the method of purifying contaminated soil by bioremediation, it has been desired to make the degrading bacteria and degrading enzymes act more efficiently.
【０００５】[0005]
【発明が解決しようとする課題】本発明は、特定の汚染
物質分解菌及び／又は分解酵素を用いて汚染土壌を浄化
する方法において、より効率的に浄化を行うために前処
理を行う浄化方法に関する。DISCLOSURE OF THE INVENTION The present invention is a method for purifying a contaminated soil by using a specific pollutant degrading bacterium and / or a degrading enzyme, and a preparative method for performing pretreatment for more efficient purification. Regarding
【０００６】[0006]
【課題を解決するための手段】本発明者は、上記課題を
解決すべく鋭意研究を重ねた結果、過酸化水素水溶液を
用いて汚染土壌を前処理することによって、汚染土壌中
の競合土着菌を殺菌し、競合物質の作用を低減できるた
め、この前処理を行うと、汚染物質分解菌及び／又は分
解酵素による汚染土壌の浄化を効率的に行えることを見
出し、本発明を完成した。すなわち、本発明は、汚染物
質による汚染土壌を過酸化水素水溶液を用いて前処理し
た後、該汚染土壌に汚染物質分解菌及び／又は汚染物質
分解酵素を作用させることを特徴とする汚染土壌の浄化
方法である。Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventor has found that by pre-treating contaminated soil with an aqueous hydrogen peroxide solution, competitive indigenous bacteria in the contaminated soil can be obtained. The present invention has been completed by discovering that this pretreatment can effectively purify contaminated soil with pollutant-degrading bacteria and / or degrading enzymes because it can sterilize and reduce the action of competing substances. That is, the present invention is a method of pre-treating a polluted soil polluted with an aqueous hydrogen peroxide solution, and then treating the polluted soil with a pollutant-degrading bacterium and / or a pollutant-degrading enzyme. It is a purification method.
【０００７】ここで、汚染物質としては、有機塩素化合
物が挙げられるが、特にダイオキシンが好ましい。ま
た、汚染物質分解菌としては、例えばシュードモナス属
若しくはバチルス属に属する微生物、又は担子菌類に属
する微生物、あるいはこれらの任意の組み合わせが挙げ
られる。より具体的には、シュードモナス属に属する微
生物としては、Pseudomonas sp. NCIB9816の変異株、Ps
eudomonas sp. HH69株等、バチルス属に属する微生物と
しては、Bacillus midousuji SH2BJ2等、担子菌類に属
する微生物としては、白色腐朽菌（例えばPhanerochaet
e chrysosporium、Phanerochaete sordida、Trametes v
ivsicolor等が挙げられる。本発明の浄化方法において
は、特に、白色腐朽菌を使用することが好ましい。Examples of the pollutants include organic chlorine compounds, and dioxins are particularly preferable. Examples of the contaminant-decomposing bacteria include microorganisms belonging to the genus Pseudomonas or Bacillus, microorganisms belonging to basidiomycetes, or any combination thereof. More specifically, as a microorganism belonging to the genus Pseudomonas, a mutant strain of Pseudomonas sp. NCIB9816, Ps
As microorganisms belonging to the genus Bacillus such as eudomonas sp. HH69 strain, Bacillus midousuji SH2BJ2 and the like, as microorganisms belonging to basidiomycetes, white-rot fungi (for example, Phanerochaet
e chrysosporium, Phanerochaete sordida, Trametes v
ivsicolor and the like. In the purification method of the present invention, it is particularly preferable to use white rot fungi.
【０００８】さらに本発明の浄化方法において、前処理
は、汚染土壌中の競合土着菌を殺菌するため、及び／又
は汚染土壌中の競合物質の作用を低減させるために行わ
れることが好ましい。また前処理は、例えば、過酸化水
素水溶液と汚染土壌とを混合して行ってもよいし、又は
過酸化水素水溶液を汚染土壌に散布して行ってもよい。Further, in the purification method of the present invention, the pretreatment is preferably carried out in order to sterilize the competitive indigenous bacteria in the contaminated soil and / or to reduce the action of the competitive substance in the contaminated soil. The pretreatment may be carried out, for example, by mixing an aqueous solution of hydrogen peroxide and contaminated soil, or by spraying the aqueous solution of hydrogen peroxide on the contaminated soil.
【０００９】[0009]
【発明の実施の形態】以下、本発明を詳細に説明する。
本発明の浄化方法は、汚染物質による汚染土壌に過酸化
水素水溶液を散布又は混合する前処理を行うことを特徴
としており、該過酸化水素によって汚染物質分解菌の競
合となる土着菌（以下、競合土着菌という。）を殺菌
し、そしてさらに汚染物質分解酵素の活性阻害となる酵
素又は腐食物質（以下、競合物質という。）の作用を低
減した後で、該分解菌又は該分解酵素を用いて汚染土壌
の浄化を効率的に行うものである。BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.
The purification method of the present invention is characterized by performing a pretreatment of spraying or mixing an aqueous hydrogen peroxide solution on a soil polluted by a pollutant, and an indigenous bacterium that competes with a pollutant-degrading bacterium by the hydrogen peroxide (hereinafter, Competitive indigenous bacteria) is sterilized, and the action of an enzyme or a corrosive substance (hereinafter, referred to as a competitive substance) that inhibits the activity of the pollutant degrading enzyme is further reduced, and then the degrading bacterium or the degrading enzyme is used. It efficiently purifies contaminated soil.
【００１０】１．過酸化水素水溶液による前処理
本発明の浄化方法では、過酸化水素水溶液を用いて汚染
物質による汚染土壌を前処理する。本発明において「浄
化」とは、汚染土壌中の汚染物質を分解又は無害化し、
環境の改善を図ることを指す。また本発明の方法で浄化
の対象となる汚染物質は、限定するものではないが、有
機塩素化合物が好ましく、例えば、ダイオキシン類とし
て、2,3,7,8-四塩化ダイオキシン（TCDD）等のポリ塩化
ジベンゾ-パラジオキシン（PCDDs）、ポリ塩化ジベンゾ
フラン（PCDF）及びコプラナーPCBなど；殺虫剤成分と
して1,1,1-トリクロロ-2,2-ビス(4-クロロフェニル)エ
タン（DDT）など；除草剤成分として2,4-ジクロロフェ
ノキシ酢酸（2,4-D）、2,4,5-トリクロロフェノキシ酢
酸（2,4,5-T）など；並びにその他の有機塩素化合物
（例えば四塩化炭素、PCB、クロロベンゼン類、トルエ
ン、キシレン、アントラセン、フェナントレン、アゾ色
素）が挙げられる。1. Pretreatment with Hydrogen Peroxide Solution In the purification method of the present invention, the contaminated soil with pollutants is pretreated with the hydrogen peroxide solution. In the present invention, "purification" means decomposing or detoxifying pollutants in contaminated soil,
Refers to improving the environment. Further, the pollutants to be purified by the method of the present invention are not limited, but are preferably organic chlorine compounds, for example, dioxins such as 2,3,7,8-tetrachloride dioxins (TCDD). Polychlorinated dibenzo-paradioxin (PCDDs), polychlorinated dibenzofuran (PCDF) and coplanar PCB, etc .; 1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane (DDT) etc. as an insecticide component; weeding As an agent component, 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), and the like; and other organic chlorine compounds (for example, carbon tetrachloride, PCB, chlorobenzenes, toluene, xylene, anthracene, phenanthrene, azo dyes).
【００１１】過酸化水素は、それ自体で汚染物質浄化能
を有するが、本発明では過酸化水素自体の汚染物質浄化
能を利用するものではなく、過酸化水素が競合土着菌を
殺菌する性質と、競合物質の作用を低減させる性質を利
用するものである。本発明において「競合土着菌」と
は、本発明の浄化方法を実施する前から汚染土壌中に存
在する微生物を指し、本発明の浄化方法により作用させ
る汚染物質分解菌（以下、分解菌という。）の増殖を阻
害するものである。また、「競合物質」とは、汚染物質
分解酵素（以下、分解酵素という。）の活性阻害となる
酵素又は腐食物質などを指し、そのような競合物質とし
ては、例えば、カタラーゼ、プロテアーゼ等の酵素や、
フミン質、フルボ酸等の腐食物質が挙げられる。Hydrogen peroxide has a pollutant-purifying ability by itself, but the present invention does not utilize the pollutant-purifying ability of hydrogen peroxide itself, and it has the property of sterilizing competitive indigenous bacteria. , Utilizing the property of reducing the action of competitors. In the present invention, “competitive indigenous bacteria” refers to microorganisms existing in contaminated soil before the purification method of the present invention is carried out, and pollutant-decomposing bacteria (hereinafter referred to as decomposing bacteria) to be acted by the purification method of the present invention. A) inhibits the growth of. The term “competitive substance” refers to an enzyme or a corrosive substance that inhibits the activity of a pollutant degrading enzyme (hereinafter referred to as a degrading enzyme), and such a competing substance is, for example, an enzyme such as catalase or protease. Or
Examples include corrosive substances such as humic substances and fulvic acid.
【００１２】前処理に使用する過酸化水素水溶液は、濃
度が30mg/L〜30％程度であれば特に制限されず、好まし
くは１g/L〜１％である。また、市販の30％過酸化水素
水溶液を用いてもよい。前処理は、例えば上記のように
調製した過酸化水素水溶液を汚染土壌と混合するか、又
は汚染土壌に散布することにより行う。混合又は散布す
る過酸化水素量は、土壌中の土着菌数及び強熱減量によ
り異なるが、当業者であれば容易に決定しうる。例えば
30％過酸化水素水溶液を用いる場合には、汚染土壌１m3
当たり約0.5〜10トン、好ましくは0.5〜6トン/m3で過酸
化水素水溶液を混合又は散布する。The aqueous hydrogen peroxide solution used for the pretreatment is not particularly limited as long as the concentration is about 30 mg / L to 30%, preferably 1 g / L to 1%. Alternatively, a commercially available 30% hydrogen peroxide aqueous solution may be used. The pretreatment is performed, for example, by mixing the hydrogen peroxide aqueous solution prepared as described above with the contaminated soil or by spraying it on the contaminated soil. The amount of hydrogen peroxide to be mixed or sprinkled varies depending on the number of indigenous bacteria in the soil and the loss on ignition, but can be easily determined by those skilled in the art. For example
1m 3 of contaminated soil when using 30% hydrogen peroxide solution
The aqueous hydrogen peroxide solution is mixed or sprinkled at a rate of about 0.5 to 10 tons, preferably 0.5 to 6 tons / m 3 .
【００１３】前処理を混合により行う場合には、投入し
た汚染土壌と過酸化水素水溶液を機械撹拌などによって
十分に混合することが好ましい。前処理を散布により行
う場合には、過酸化水素水溶液の散布を数回繰り返すこ
とによって、汚染土壌中に過酸化水素水がまんべんなく
行き渡るようにすることが好ましい。散布回数は特に制
限されないが、汚染土壌からの発泡の消滅及び反応熱の
低下を確認した上で決定する。従って、例えば数回散布
して反応熱が上がらなければ過酸化水素による有機物分
解が飽和になったと考えてよい。When the pretreatment is carried out by mixing, it is preferable that the introduced contaminated soil and the hydrogen peroxide aqueous solution are thoroughly mixed by mechanical stirring or the like. When the pretreatment is performed by spraying, it is preferable to spray the hydrogen peroxide aqueous solution several times so that the hydrogen peroxide solution is evenly distributed in the contaminated soil. The number of times of spraying is not particularly limited, but it is determined after confirming disappearance of foaming from the contaminated soil and reduction of reaction heat. Therefore, for example, if the reaction heat does not rise after being sprayed several times, it can be considered that the decomposition of organic substances by hydrogen peroxide is saturated.
【００１４】前処理後は、適切な分離装置、例えば振動
篩、膜、ベルトプレス又は遠心分離装置等を用いて、過
酸化水素水溶液と土壌とを固液分離するか、又は、過酸
化水素水溶液が土壌中を通過して排水管に回収されるよ
うな浄化処理用装置において前処理を行うことにより過
酸化水素を分離しうる。分離された液分は別途中和する
か又はBOD/COD除去等の水処理を行って、下水道、河川
等への放流基準以下にまで処理してから廃棄することが
好ましい。あるいは、前処理完了後に作用させる分解菌
及び／又は分解酵素に対し過酸化水素が有害な影響を及
ぼさない程度の濃度にまで過酸化水素が分解するのに必
要な時間にわたって放置してもよい。After the pretreatment, the hydrogen peroxide aqueous solution and the soil are solid-liquid separated by using a suitable separator such as a vibrating sieve, a membrane, a belt press or a centrifugal separator, or the hydrogen peroxide aqueous solution is used. Hydrogen peroxide can be separated by performing a pretreatment in a purification treatment device in which is passed through soil and collected in a drainage pipe. The separated liquid is preferably neutralized separately or subjected to water treatment such as BOD / COD removal so that it is treated to a level not more than the discharge standard for sewers, rivers, etc., and then discarded. Alternatively, it may be left for a time necessary for the hydrogen peroxide to be decomposed to a concentration at which hydrogen peroxide does not have a detrimental effect on the degrading bacteria and / or the degrading enzyme to act after completion of the pretreatment.
【００１５】上記前処理によって、土壌中の競合土着菌
が過酸化水素から生成されるヒドロキシラジカルによっ
て酸化殺菌されると共に、競合物質であるカタラーゼが
過酸化水素を利用して反応することにより土壌中のカタ
ラーゼ活性、すなわち競合物質の作用が低減する。過酸
化水素は時間経過と共に速やかに酸素と水に分解される
ため、過酸化水素によって土壌が汚染される危険性はな
い。By the above-mentioned pretreatment, competitive indigenous bacteria in the soil are oxidatively sterilized by the hydroxy radicals produced from hydrogen peroxide, and catalase, which is a competitive substance, reacts by utilizing hydrogen peroxide. Catalase activity, ie the action of competitors, is reduced. Since hydrogen peroxide is rapidly decomposed into oxygen and water over time, there is no risk of soil contamination with hydrogen peroxide.
【００１６】２．浄化処理
前処理後、汚染土壌に分解菌及び／又は分解酵素を作用
させ、汚染土壌の浄化を行う。使用しうる分解菌は、汚
染物質、特に有機塩素化合物を分解又は無害化して浄化
する能力を有するものであれば特に限定されず、例え
ば、シュードモナス（Pshudomonas）属、バチルス（Bac
illus）属に属する微生物、又は白色腐朽菌等の担子菌
類が挙げられる。これらの微生物は、それぞれ単独で又
は組み合わせて使用することができる。具体的な菌株と
して例示すると、シュードモナス属に属する微生物とし
ては、例えば、Pseudomonas sp. NCIB9816の変異株、Ps
eudomonassp. HH69株、Pseudomonas pseudoalcaligenes
KF707等が挙げられる。またバチルス属に属する微生物
としては、例えば、Bacillus midousuji SH2BJ等が挙げ
られる。さらに担子菌としては、白色腐朽菌であるPhan
erochaete chrysosporiumIFO31249、Phanerochaete chr
ysosporium DSM1556、Phanerochaete sordida、Tramete
s vivsicolor IFO7043等が挙げられる。本発明の浄化方
法においては、白色腐朽菌を使用することが好ましく、
特にPhanerochaete chrysosporiumが好ましい。2. After the pretreatment of the purification treatment, the decomposing bacteria and / or the degrading enzyme are allowed to act on the contaminated soil to purify the contaminated soil. The decomposing bacterium that can be used is not particularly limited as long as it has the ability to decompose or detoxify pollutants, particularly organic chlorine compounds, and examples thereof include genus Pshudomonas and Bacillus (Bac
illus), or basidiomycetes such as white-rot fungi. These microorganisms can be used alone or in combination. Exemplifying as a specific strain, as a microorganism belonging to the genus Pseudomonas, for example, a mutant strain of Pseudomonas sp. NCIB9816, Ps
eudomonassp. HH69 strain, Pseudomonas pseudoalcaligenes
Examples include KF707. Examples of microorganisms belonging to the genus Bacillus include Bacillus midousuji SH2BJ. Furthermore, as a basidiomycete, Phan which is a white-rot fungus
erochaete chrysosporium IFO31249, Phanerochaete chr
ysosporium DSM1556, Phanerochaete sordida, Tramete
s vivsicolor IFO 7043 and the like. In the purification method of the present invention, it is preferable to use white-rot fungus,
Phanerochaete chrysosporium is particularly preferable.
【００１７】上記分解菌が汚染物質を浄化するように該
分解菌を培養する条件は、通常の培養条件を用いればよ
く、例えば、培地として、グルコース、デンプン、スク
ロース等の炭素源、硝酸カリウム、硝酸アンモニウム、
酵母エキス、ペプトン等の窒素源を含有し、好ましく
は、リン酸カリウム、硝酸マグネシウム、塩化カルシウ
ム等の無機塩類や微量金属類、アミノ酸類、ビタミン類
等の微量成分を含有する培地を用いて、静置培養、振と
う培養、通気培養、通気撹拌培養等の各種培養条件を用
いて培養を行うことができる。ここで、培養の最適条件
に関しては、用いる分解菌により異なるので、上記培地
及び培養方法は用いる分解菌に適するものに適宜選択及
び調製され、また、温度、pH、培養期間等のその他の培
養条件も適宜選択されて培養が行われることが好まし
い。As the conditions for culturing the decomposing bacterium so that the decomposing bacterium purifies pollutants, usual culturing conditions may be used. For example, as a medium, carbon sources such as glucose, starch, sucrose, potassium nitrate, ammonium nitrate may be used. ,
Yeast extract, containing a nitrogen source such as peptone, preferably potassium phosphate, magnesium nitrate, trace metals such as inorganic salts such as calcium chloride, amino acids, using a medium containing trace components such as vitamins, Culture can be performed using various culture conditions such as static culture, shaking culture, aeration culture, aeration and agitation culture. Here, regarding the optimal conditions of the culture, since it depends on the degrading bacterium used, the medium and the culturing method are appropriately selected and prepared to those suitable for the degrading bacterium to be used, and other culturing conditions such as temperature, pH, and culturing period. Also, it is preferable that the culture is carried out by appropriately selecting.
【００１８】例えば、本発明の浄化方法に好ましく用い
られる白色腐朽菌を培養する場合には、好ましい培地と
しては、水道水１m3当たりKH2PO4２kg、MnSO4・H2O 500
g、CaCl 100g、NH4Cl 120g、チアミン１g及びグルコー
ス10kgを含む培地等が挙げられ、好ましい培養条件は、
温度15〜37℃、pH４〜７であり、培養期間はおよそ７〜
20時間程度である。また、白色腐朽菌は固体培養を行っ
てもよいし、液体培養を行ってもよい。For example, in the case of culturing white rot fungi preferably used in the purification method of the present invention, preferable media include KH 2 PO 4 2 kg and MnSO 4 .H 2 O 500 per 1 m 3 of tap water.
g, CaCl 100 g, NH 4 Cl 120 g, thiamine 1 g and glucose 10 kg, and the like.
Temperature is 15 ~ 37 ℃, pH is 4 ~ 7, culture period is about 7 ~
It takes about 20 hours. The white-rot fungus may be subjected to solid culture or liquid culture.
【００１９】上記分解菌は、その培養液のまま使用して
もよいし、培養液を濾過、遠心分離若しくは抽出等の精
製処理を行って使用してもよいし、又は培養液を水等で
希釈して使用してもよい。一方、本発明の浄化方法に使
用する分解酵素は、汚染物質を分解又は無害化して浄化
する作用を有するものであれば特に限定されず、例え
ば、リグニンペルオキシダーゼ、マンガンペルオキシダ
ーゼ、ラッカーゼ等が挙げられる。さらに上記分解酵素
は、上記分解菌が産生したものであってもよい。分解酵
素もまた、そのまま使用してもよいし、又は水等で希釈
して使用することができる。The above-mentioned degrading bacteria may be used as the culture solution as it is, or may be used after being subjected to a purification treatment such as filtration, centrifugation or extraction, or the culture solution may be treated with water or the like. You may dilute and use it. On the other hand, the degrading enzyme used in the purification method of the present invention is not particularly limited as long as it has an action of decomposing or detoxifying pollutants and purifying, and examples thereof include lignin peroxidase, manganese peroxidase, and laccase. Further, the degrading enzyme may be one produced by the degrading bacterium. The degrading enzyme may also be used as it is, or may be diluted with water or the like before use.
【００２０】上記分解菌及び／又は上記分解酵素は、汚
染土壌の表面に散布する方法、注入管から土壌中に注入
する方法などにより汚染土壌に作用させることができ
る。さらにまた、上記分解菌及び／又は上記分解酵素を
不織布等に接種したものを汚染土壌上に静置して作用さ
せてもよい。また、上記分解菌及び／又は分解酵素は、
それぞれを単独で作用させてもよいし、又は適切に組み
合わせて作用させてもよい。The above-mentioned degrading bacteria and / or the above-mentioned degrading enzyme can be made to act on the contaminated soil by a method of spraying on the surface of the contaminated soil, a method of injecting it into the soil through an injection pipe, or the like. Furthermore, the above-mentioned degrading bacterium and / or the above-mentioned degrading enzyme may be inoculated on a non-woven fabric or the like and allowed to stand on contaminated soil to act. Further, the above-mentioned degrading bacteria and / or degrading enzyme,
Each of them may act alone, or may act in an appropriate combination.
【００２１】作用させる上記分解菌及び／又は分解酵素
の量は、分解菌又は分解酵素の種類、汚染土壌の汚染の
程度を考慮して、望ましい浄化の結果が得られるように
決定しうる。さらに、汚染土壌の浄化をさらに効率的に
行うために、上記分解菌及び／又は分解酵素と共にその
他の追加成分を使用してもよく、そのような追加成分と
しては、限定するものではないが、分解菌の増殖を促進
する培養液、グルコース溶液、リノール酸やリノレン酸
等の不飽和脂肪酸、アミノ酸、硫酸マンガン等が挙げら
れる。The amount of the above-mentioned degrading bacterium and / or degrading enzyme to be acted can be determined in consideration of the type of degrading bacterium or degrading enzyme and the degree of contamination of contaminated soil so that a desired purification result can be obtained. Further, in order to more efficiently purify the contaminated soil, other additional components may be used together with the degrading bacteria and / or degrading enzymes, and such additional components include, but are not limited to, Examples thereof include a culture solution that promotes the growth of degrading bacteria, a glucose solution, unsaturated fatty acids such as linoleic acid and linolenic acid, amino acids, and manganese sulfate.
【００２２】分解菌及び／又は分解酵素による汚染土壌
の浄化を行った後は、分解菌及び／又は分解酵素を分離
してから土壌を埋め戻す。分解菌及び／又は分解酵素の
分離は、上述した過酸化水素の分離と同様にして行うこ
とができる。以下の３及び４に、本発明の好ましい浄化
方法の例として、培養液混合法及び分解菌接種法による
浄化方法をそれぞれ記載する。After cleaning the contaminated soil with degrading bacteria and / or degrading enzymes, the degrading bacteria and / or degrading enzymes are separated before the soil is backfilled. Separation of the degrading bacteria and / or degrading enzyme can be performed in the same manner as the above-mentioned separation of hydrogen peroxide. In the following 3 and 4, as examples of preferable purification methods of the present invention, purification methods by a culture solution mixing method and a degrading bacterium inoculation method are described, respectively.
【００２３】３．培養液混合法による浄化方法
図１に基本的な処理フローを示す。
汚染土壌の掘削／運搬
最初に、汚染土壌を掘削回収し、処理施設へ運搬する。
汚染土壌の過酸化水素水溶液による前処理
続いて、掘削／運搬した汚染土壌を、図１に示す前処理
装置(1)に過酸化水素水溶液と共に混入してスラリーと
する。投入した汚染土壌と過酸化水素水溶液を機械撹拌
によって十分に混合する。
スラリーの固液分離
過酸化水素で処理したスラリーを分離装置(2)にて固液
分離を行う。分離された液分は下水道、河川等への放流
基準以下にまで処理を行ってから廃棄する。3. Purification method by culture solution mixing method Fig. 1 shows a basic processing flow. Excavation / transportation of contaminated soil First, contaminated soil is excavated and collected and transported to a treatment facility. Pretreatment of Contaminated Soil with Hydrogen Peroxide Aqueous Solution Subsequently, the excavated / transported contaminated soil is mixed with the hydrogen peroxide aqueous solution in the pretreatment apparatus (1) shown in FIG. 1 to form a slurry. Thoroughly mix the input contaminated soil with the hydrogen peroxide solution by mechanical stirring. Solid-Liquid Separation of Slurry Solid-liquid separation of the slurry treated with hydrogen peroxide is carried out by a separator (2). Separated liquids should be disposed of after being processed to below the standard for discharge into sewers and rivers.
【００２４】土壌の酵素処理
分離した土壌と、別施設にて培養した白色腐朽菌の液体
培養液又は部分精製酵素液とを処理装置(3)で混合す
る。次に、緩やかな撹拌によるスラリー培養を行い、土
壌中の汚染物質と分解酵素の接触効率を向上させる。運
転期間は1日〜7日程度であれば良く、運転温度は20〜37
℃、好ましくは25〜28℃の範囲である。
スラリーの固液分離
再酵素処理したスラリーを分離装置(2)にて固液分離を
行う。分離された液分は下水道、河川等への放流基準以
下にまで処理を行ってから廃棄する。
土壌の埋め戻し
分離した土壌を回収後、掘削現場に搬送し埋め戻しす
る。Enzyme Treatment of Soil The separated soil is mixed with a liquid culture solution of white rot fungus cultivated in another facility or a partially purified enzyme solution in a treatment device (3). Next, slurry culture is performed with gentle stirring to improve the contact efficiency between pollutants in soil and degrading enzymes. The operating period should be 1 to 7 days, and the operating temperature should be 20 to 37.
C., preferably in the range of 25 to 28.degree. Solid-Liquid Separation of Slurry Solid-liquid separation of the re-enzymatically treated slurry is carried out by a separator (2). Separated liquids should be disposed of after being processed to below the standard for discharge into sewers and rivers. Backfilling of soil After collecting the separated soil, it is transported to the excavation site and backfilled.
【００２５】４．分解菌接種法による浄化方法
図２に基本的な処理フローを示す。
処理ヤードの構築
汚染現場に汚染土壌の処理ヤードを建設する。本ヤード
の底版は防水性及び過酸化水素による耐食性を有するも
のであれば特に制限されない。底版側面には、反応液を
処理ヤードから除外するための排水管(4)が設置されて
おり、両出口にはバルブ及びポンプを設けている。排水
管の片側はピット(5)に導入されており、ピット(5)に収
集された反応液はポンプ(6)で揚水された後、反応液散
布管(7)に繋がる。また、処理ヤード底版に砂層(8)を厚
さ0.1〜0.5m程度に敷き詰める。
汚染土壌の掘削と設置
汚染土壌を掘削回収し、処理ヤードヘ厚さ0.1〜0.5m程
度に敷き詰める。また、反応を効率的にするために、敷
き詰めた汚染土壌の転厚は行わないものとする。4. Purification method by decomposing bacterium inoculation method Fig. 2 shows a basic processing flow. Construction of treatment yard Construct a treatment yard for contaminated soil at the contaminated site. The bottom plate of this yard is not particularly limited as long as it has waterproofness and corrosion resistance against hydrogen peroxide. A drain pipe (4) for removing the reaction solution from the processing yard is installed on the side surface of the bottom plate, and a valve and a pump are provided at both outlets. One side of the drainage pipe is introduced into the pit (5), and the reaction liquid collected in the pit (5) is pumped by the pump (6) and then connected to the reaction liquid spray pipe (7). Further, a sand layer (8) is spread over the bottom slab of the processing yard to a thickness of about 0.1 to 0.5 m. Excavation and installation of contaminated soil Excavate and collect contaminated soil, and spread it to a processing yard to a thickness of 0.1 to 0.5 m. In addition, in order to make the reaction efficient, the spread of contaminated soil shall not be changed.
【００２６】汚染土壌の過酸化水素水溶液による前処
理
排水管のバルプAを閉じ、ピット(5)に繋がるバルブBを
開放とする。次に、濃度を調整した過酸化水素水溶液を
汚染土壌中に散布する。汚染土壌及び砂層(8)を透過し
た過酸化水素水溶液が排水管を通じてピット(5)に回収
される。次に、ポンプ(6)により過酸化水素水溶液を揚
水し、反応液散布管(7)より汚染土壌表面に再度散布す
る。なお、ピットに回収された過酸化水素濃度が低けれ
ば高濃度過酸化水素水溶液にて濃度調整することとす
る。散布回数は、汚染土壌からの発泡の消滅及び反応熱
の低下を確認した上で決定する。
分解菌注入
処理後の過酸化水素水溶液を排除した後、土壌表面に不
織布(9)を設置する。さらに、液体培養又は固体培養し
た白色腐朽菌を不織布表面に一様に接種する。
培養液循環
土壌表面に水道水で調製した培養液を散布する。土壌層
／砂層を透過した培養液はピット(5)に導入される。次
に、ポンプ(6)により培養液を揚水し、反応液散布管(7)
より汚染土壌表面に繰り返し散布される。この運転期間
は、特に限定されないが、約7日〜30日間の範囲であ
る。
処理土壌埋め戻し
処理終了後の土壌を埋め戻し浄化終了とする。Pretreatment of contaminated soil with aqueous hydrogen peroxide solution The valve A of the drain pipe is closed and the valve B connected to the pit (5) is opened. Next, the hydrogen peroxide aqueous solution having the adjusted concentration is sprayed on the contaminated soil. The hydrogen peroxide solution that has permeated the contaminated soil and sand layer (8) is collected in the pit (5) through the drainage pipe. Next, the hydrogen peroxide solution is pumped up by the pump (6) and sprayed again on the surface of the contaminated soil through the reaction solution spraying pipe (7). If the concentration of hydrogen peroxide collected in the pit is low, the concentration will be adjusted with a high-concentration hydrogen peroxide solution. The frequency of spraying is determined after confirming the disappearance of foaming from the contaminated soil and the decrease in reaction heat. After removing the hydrogen peroxide aqueous solution after the degrading bacterium injection treatment, the non-woven fabric (9) is placed on the soil surface. Further, the non-woven fabric surface is uniformly inoculated with white-rot fungi that have been liquid-cultured or solid-cultured. Culture solution circulation Spray the culture solution prepared with tap water on the soil surface. The culture solution that has permeated the soil layer / sand layer is introduced into the pit (5). Next, the culture solution was pumped up by the pump (6), and the reaction solution spray tube (7)
Repeatedly sprayed on the contaminated soil surface. The operating period is not particularly limited, but is in the range of about 7 days to 30 days. The soil after the backfilling of the treated soil is finished by backfilling and purification.
【００２７】[0027]
【実施例】以下、実施例により本発明をさらに具体的に
説明する。但し、本発明は下記実施例にその技術的範囲
が限定されるものではない。
〔実施例１〕各種手法による土壌中の競合土着菌に対す
る殺菌効果
（１）実験条件
土壌殺菌性能について以下の各処理条件で試験した。
・供試土壌：黒木土（人参畑の表層土）
・土壌処理方法：
No.1：オートクレーブ滅菌（121℃×２時間）後、滅菌
蒸留水で50g/30mLに調整した。
No.2：加熱処理（85℃×２時間）後、滅菌蒸留水で50g/
30mLに調整した。
No.3：30％過酸化水素水溶液30mLに土壌50gを混合し30
分撹拌した。
No.4：30％過酸化水素水溶液30mLに土壌20gを混合し30
分撹拌した。
No.5：30％過酸化水素水溶液30mLに土壌10gを混合し30
分撹拌した。
No.6：30％過酸化水素水溶液30mLに土壌5gを混合し30分
撹拌した。
No.7：対照（非滅菌）
上記処理条件の内、過酸化水素水溶液を用いたNo.3〜7
は、耐熱バイアル瓶に過酸化水素水溶液を30mL入れ、マ
グネチックスターラーで十分撹拌しながら所定量の土壌
を徐々に添加した。さらに、約30分撹拌を継続して得ら
れたスラリーをサンプルとした。EXAMPLES The present invention will be described in more detail below with reference to examples. However, the technical scope of the present invention is not limited to the following examples. [Example 1] Sterilization effect against competitive indigenous bacteria in soil by various methods (1) Experimental conditions Soil sterilization performance was tested under the following respective treatment conditions.・ Test soil: Kuroki soil (surface soil of ginseng field) ・ Soil treatment method: No. 1: After autoclave sterilization (121 ° C x 2 hours), it was adjusted to 50 g / 30 mL with sterile distilled water. No.2: 50g / in sterile distilled water after heat treatment (85 ℃ × 2 hours)
Adjusted to 30 mL. No.3: 30 mL of 30% hydrogen peroxide solution mixed with 50 g of soil
Stir for minutes. No.4: 30 mL of 30% hydrogen peroxide solution mixed with 20 g of soil
Stir for minutes. No.5: 30 mL of 30% hydrogen peroxide solution mixed with 10 g of soil
Stir for minutes. No. 6: 5 g of soil was mixed with 30 mL of 30% hydrogen peroxide solution and stirred for 30 minutes. No.7: Control (non-sterile) Of the above treatment conditions, No.3-7 using hydrogen peroxide solution
For 30 ml, 30 mL of hydrogen peroxide solution was put in a heat-resistant vial, and a predetermined amount of soil was gradually added while sufficiently stirring with a magnetic stirrer. Furthermore, the slurry obtained by continuously stirring for about 30 minutes was used as a sample.
【００２８】（２）生菌数測定
上記の各処理条件で調製した土壌スラリー50μLをCGY液
体培地（5g/Lカシトン、1g/Lグリセロール及び5g/L酵母
エキス）450μLに添加した。さらに、新しいCGY液体培
地450μLにその混合液を順次添加／混合することによっ
て希釈列を作製した。30℃恒温室内にて7日間の振とう
培養を行い、MPN法（最確法）にて各処理土壌中の生菌
数を決定した。(2) Measurement of viable cell count 50 μL of the soil slurry prepared under each of the above treatment conditions was added to 450 μL of CGY liquid medium (5 g / L Casitone, 1 g / L glycerol and 5 g / L yeast extract). Further, a dilution series was prepared by sequentially adding / mixing the mixed solution to 450 μL of new CGY liquid medium. After shaking culture for 7 days in a constant temperature room at 30 ° C, the viable cell count in each treated soil was determined by the MPN method (most probable method).
【００２９】（３）実験結果
表１に各処理方法で処理した土壌性状を示す。実験に用
いた土壌（黒木土）中の生菌数は、No.7に示すように乾
燥土壌1g当たり1.0×108（cell/g）であり、強熱減量は
8.9（％）であった。一方、121℃×２時間のオートクレ
ーブ処理後の土壌（No.1）からは生菌が検出されず完全
滅菌されていた。しかし、85℃×２時間の加熱処理（N
o.2）では、土壌から1.6×107（cell/g）の生菌が確認
され、この処理条件では殺菌効果が十分に得られないこ
とがわかった。一方、30％過酸化水素水溶液で処理した
土壌（No.3〜6）からは、今回実験した土壌と過酸化水
素の配合比に係わらず121℃×2時間のオートクレーブ滅
菌（No.1）と同様に生菌は検出されなかった。このよう
に、過酸化水素水溶液を用いた場合には競合土着菌に対
し高い殺菌効果があることが確認された。(3) Experimental results Table 1 shows the soil properties treated by each treatment method. The number of viable bacteria in the soil (Kuroki soil) used in the experiment was 1.0 × 10 8 (cell / g) per 1 g of dry soil as shown in No. 7, and the loss on ignition was
It was 8.9 (%). On the other hand, no viable bacteria were detected in the soil (No. 1) after the autoclave treatment at 121 ° C. for 2 hours, and it was completely sterilized. However, heat treatment at 85 ℃ x 2 hours (N
In o.2), 1.6 × 10 7 (cell / g) of live bacteria were confirmed from the soil, and it was found that the bactericidal effect was not sufficiently obtained under this treatment condition. On the other hand, from soil treated with 30% hydrogen peroxide solution (No.3 to 6), autoclave sterilization (No.1) at 121 ° C for 2 hours was performed regardless of the mixture ratio of soil and hydrogen peroxide tested this time. Similarly, no viable bacteria were detected. As described above, it was confirmed that the use of the aqueous hydrogen peroxide solution has a high bactericidal effect against the competitive indigenous bacteria.
【００３０】[0030]
【表１】 [Table 1]
【００３１】〔実施例２〕土壌スラリー混入による白色
腐朽菌の増殖と分解酵素生成への影響
（１）実験方法
グルコースを主炭素源とするBasalII液体培地をフィル
ター滅菌し、500mL三角フラスコ8本に各々50mLずつ投入
した。これに、PDA寒天培地で5日間固体培養した白色腐
朽菌を無菌的に植菌し、28℃の恒温室内で静置培養を行
った。培養1日後に、上記実施例１の各処理方法で得た
土壌スラリー500μLを無菌的に添加し静置培養を継続し
た。なお、コントロールとして土壌スラリーを添加しな
い系も実施した。[Example 2] Effect of mixing of soil slurry on growth of white-rot fungi and production of degrading enzyme (1) Experimental method Basal II liquid medium containing glucose as a main carbon source was filter sterilized into 8 500 mL Erlenmeyer flasks. 50 mL of each was added. White rot fungi that had been solid-cultured on a PDA agar medium for 5 days were aseptically inoculated into this, and static culture was performed in a constant temperature room at 28 ° C. After 1 day of culturing, 500 μL of the soil slurry obtained by each treatment method of Example 1 was aseptically added, and static culture was continued. As a control, a system without addition of soil slurry was also implemented.
【００３２】培養12日目に培養液を採取し、リグニンペ
ルオキシダーゼ（LIP）活性及びマンガンペルオキシダ
ーゼ（MnP）活性を調べた。以下に各酵素活性のアッセ
イ法を示す。なお、本実験で使用した白色腐朽菌は、国
内の森林から分離した野性株であり、LIP及びMnPを生成
するものである。また、この菌株は、4塩素ジオキシン
及び3塩素ジベンゾフランの分解活性を有するダイオキ
シン分解菌でもある。On the 12th day of culture, the culture solution was collected and examined for lignin peroxidase (LIP) activity and manganese peroxidase (MnP) activity. The assay method for each enzyme activity is shown below. The white-rot fungus used in this experiment is a wild strain isolated from domestic forests and produces LIP and MnP. This strain is also a dioxin-degrading bacterium that has the activity of degrading tetrachlorodioxin and trichlorinated dibenzofuran.
【００３３】（２）酵素活性のアッセイ方法
・リグニンペルオキシダーゼ（LIP）アッセイ
アッセイ試薬として、50mM酒石酸ナトリウム(pH4.5)にA
zureBを480mM、H2O2を150μMで混合したものを用いた。
このアッセイ試薬400μLに培養液200μLを混合し、25℃
で20分間651nmにおける吸光度の減少速度を測定した。
この間の最大傾き(Δabs/min)をリグニンペルオキシダ
ーゼ活性とした。なお、対照としては培養液の代わりに
50mM酒石酸ナトリウム(pH4.5)200μLを用いた。
・マンガンペルオキシダーゼ（MnP）アッセイ
アッセイ試薬として、50mMコハク酸ナトリウム(pH4.5)
にフェノールレッドを0.00375％、H2O2を150μMで混合
したものを用いた。このアッセイ試薬400μLに培養液20
0μLを混合し、25℃で5分間431nmにおける吸光度の減少
速度を測定した。この間の最大傾き(Δabs/min)をマン
ガンペルオキシダーゼ活性とした。なお、対照としては
培養液の代わりに50mMコハク酸ナトリウム(pH4.5)200μ
Lを用いた。(2) Method for assaying enzyme activity-Lignin peroxidase (LIP) assay As an assay reagent, 50 mM sodium tartrate (pH 4.5) A
A mixture of 480 mM zureB and 150 μM H 2 O 2 was used.
Mix 200 μL of culture solution with 400 μL of this assay reagent and
The rate of decrease in absorbance at 651 nm was measured for 20 minutes at.
The maximum slope (Δabs / min) during this period was defined as the lignin peroxidase activity. As a control, instead of the culture solution
200 μL of 50 mM sodium tartrate (pH 4.5) was used.・ Manganese peroxidase (MnP) assay 50 mM sodium succinate (pH 4.5) as an assay reagent
Phenol red was mixed at 0.00375% and H 2 O 2 was mixed at 150 μM. Add 400 μL of this assay reagent to 20
0 μL was mixed and the rate of decrease in absorbance at 431 nm was measured at 25 ° C. for 5 minutes. The maximum slope (Δabs / min) during this period was defined as the manganese peroxidase activity. As a control, instead of the culture solution, 50 mM sodium succinate (pH 4.5) 200 μ
L was used.
【００３４】（３）実験結果
表２に実験結果を示す。土壌スラリーを添加しない条件
（No.8）での12日後のLIP活性及びMnP活性はそれぞれ0.
0635abs/min及び0.0542abs/minであった。一方、滅菌し
なかった土壌スラリーを混合した条件（No.7）及び85℃
で2時間加熱処理した土壌スラリーを混合した条件（No.
2）では、LIP活性及びMnP活性ともに確認されなかっ
た。一方、過酸化水素で処理した土壌スラリーを混合し
た条件（No.3〜6）では、121℃で2時間処理した条件と
同様にLIP活性及びMnP活性ともに確認され、土壌スラリ
ーを添加しない系（No.8）とほぼ同様の活性が得られ
た。なお、培養終了時の液体表面での菌の繁殖を見る
と、LIP活性及びMnP活性ともに検出されなかった土壌非
滅菌（No.7）及び85℃加熱処理の系（No.2）では、接種
した白色腐朽菌以外のカビのコロニーが多数発生してい
た。一方、121℃オートクレーブ処理（No.1）及び過酸
化水素処理した土壌の条件（No.3〜6）では、接種した
白色腐朽菌以外のコロニーは確認されなかった。これら
の結果より、土壌を過酸化水素で処理した場合には、競
合土着菌の多くを酸化殺菌できるとともに、接種した白
色腐朽菌の増殖が阻害されず、かつダイオキシン分解酵
素が生成され、その酵素活性が阻害されないことがわか
った。(3) Experimental Results Table 2 shows the experimental results. LIP activity and MnP activity after 12 days under the condition that the soil slurry was not added (No. 8) were 0.
The values were 0635 abs / min and 0.0542 abs / min. On the other hand, the condition (No.7) of mixing unsterilized soil slurry and 85 ℃
The condition (No.
In 2), neither LIP activity nor MnP activity was confirmed. On the other hand, under the condition that the soil slurry treated with hydrogen peroxide was mixed (No.3 to 6), both LIP activity and MnP activity were confirmed similarly to the condition treated for 2 hours at 121 ° C. The activity similar to that of No. 8) was obtained. Looking at the growth of bacteria on the liquid surface at the end of the culture, inoculation was observed in non-sterilized soil (No. 7) and in the system heat-treated at 85 ° C (No. 2) in which neither LIP activity nor MnP activity was detected. A large number of mold colonies other than the white rot fungus were found. On the other hand, no colonies other than the inoculated white-rot fungi were confirmed under the conditions of 121 ° C autoclave treatment (No. 1) and hydrogen peroxide-treated soil (No. 3 to 6). From these results, when the soil was treated with hydrogen peroxide, many of the competing indigenous bacteria could be oxidatively sterilized, the growth of inoculated white rot fungi was not inhibited, and dioxin degrading enzyme was produced, It was found that the activity was not inhibited.
【００３５】[0035]
【表２】 [Table 2]
【００３６】[0036]
【発明の効果】本発明により、分解菌及び／又は分解酵
素をより効率的に汚染土壌に作用させることができる浄
化方法が提供される。EFFECTS OF THE INVENTION According to the present invention, there is provided a purification method capable of causing a degrading bacterium and / or a degrading enzyme to more efficiently act on contaminated soil.
【図１】培養液混合法による汚染土壌の浄化フローであ
る。FIG. 1 is a purification flow of contaminated soil by a culture solution mixing method.
【図２】分解菌接種法による汚染土壌の浄化フローであ
る。FIG. 2 is a purification flow of contaminated soil by a degrading bacterium inoculation method.
【符号の説明】 １ 前処理装置 ２ 分離装置 ３ 処理装置 ４ 排水管 ５ ピット ６ ポンプ ７ 反応液散布管 ８ 砂層 ９ 不織布[Explanation of symbols] 1 Pretreatment device 2 Separation device 3 processing equipment 4 drainage pipe 5 pits 6 pumps 7 Reaction solution spray tube 8 sand layers 9 non-woven fabric
フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ テーマコート゛(参考） Ｃ１２Ｎ 1/20 Ｃ１２Ｒ 1:645 Ｂ０９Ｂ 3/00 Ｅ //(Ｃ１２Ｎ 1/00 ３０４Ｋ Ｃ１２Ｒ 1:38） ＺＡＢ (Ｃ１２Ｎ 1/00 Ｃ１２Ｒ 1:07） (Ｃ１２Ｎ 1/00 Ｃ１２Ｒ 1:645） (Ｃ１２Ｎ 1/20 Ｃ１２Ｒ 1:38） (Ｃ１２Ｎ 1/20 Ｃ１２Ｒ 1:07） (Ｃ１２Ｎ 1/20 Ｃ１２Ｒ 1:645） Ｆターム(参考） 4B065 AA15X AA41X AA71X AC20 BA22 BB40 CA56 4D004 AA41 AB07 BA10 CA17 CA36 CB27 CC07 CC11 Front page continuation (51) Int.Cl. 7 Identification code FI theme code (reference) C12N 1/20 C12R 1: 645 B09B 3/00 E // (C12N 1/00 304K C12R 1:38) ZAB (C12N 1 / 00 C12R 1:07) (C12N 1/00 C12R 1: 645) (C12N 1/20 C12R 1:38) (C12N 1/20 C12R 1:07) (C12N 1/20 C12R 1: 645) F term ( Reference) 4B065 AA15X AA41X AA71X AC20 BA22 BB40 CA56 4D004 AA41 AB07 BA10 CA17 CA36 CB27 CC07 CC11
Claims (7)
溶液を用いて前処理した後、該汚染土壌に汚染物質分解
菌及び／又は汚染物質分解酵素を作用させることを特徴
とする汚染土壌の浄化方法。1. Purification of contaminated soil, which comprises pretreating the polluted soil with a pollutant with an aqueous solution of hydrogen peroxide, and then causing the pollutant-degrading bacteria and / or the pollutant-degrading enzyme to act on the polluted soil. Method.
１記載の方法。2. The method of claim 1, wherein the contaminant is an organochlorine compound.
求項２記載の方法。3. The method according to claim 2, wherein the organic chlorine compound is dioxin.
びバチルス属に属する微生物、並びに担子菌類に属する
微生物からなる群から選ばれる少なくとも１つである、
請求項１〜３のいずれか１項に記載の方法。4. The contaminant decomposing bacterium is at least one selected from the group consisting of microorganisms belonging to the genus Pseudomonas and Bacillus and microorganisms belonging to basidiomycetes.
The method according to claim 1.
ある、請求項４記載の方法。5. The method according to claim 4, wherein the microorganism belonging to Basidiomycetes is a white-rot fungus.
菌するため、及び／又は汚染土壌中の競合物質の作用を
低減させるために行われるものである請求項１〜５のい
ずれか１項に記載の方法。6. The pretreatment is carried out in order to sterilize competitive indigenous bacteria in the contaminated soil and / or to reduce the action of the competitor in the contaminated soil. The method according to item 1.
とを混合し、又は過酸化水素水溶液を汚染土壌に散布す
るものである請求項１〜６のいずれか１項に記載の方
法。7. The method according to claim 1, wherein the pretreatment is mixing an aqueous solution of hydrogen peroxide with contaminated soil or spraying the aqueous solution of hydrogen peroxide on the contaminated soil.
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|---|---|---|---|
| JP2001308637A JP2003112164A (en) | 2001-10-04 | 2001-10-04 | Cleaning method for soil polluted with organochlorine compound |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2001308637A JP2003112164A (en) | 2001-10-04 | 2001-10-04 | Cleaning method for soil polluted with organochlorine compound |
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|---|---|
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007029860A (en) * | 2005-07-27 | 2007-02-08 | Dna Future:Kk | Method for decomposing or concentrating toxic substance in soil |
| CN105728446A (en) * | 2016-05-12 | 2016-07-06 | 温州泓呈祥科技有限公司 | Soil remediation device capable of removing omethoate components in farmland soil |
| CN105728447A (en) * | 2016-05-12 | 2016-07-06 | 温州泓呈祥科技有限公司 | Soil remediation method capable of removing omethoate components in farmland soil |
| JP2016150272A (en) * | 2015-02-16 | 2016-08-22 | 株式会社鴻池組 | Clarifier by bioremediation of contaminated soil, and clarification method using the same |
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2001
- 2001-10-04 JP JP2001308637A patent/JP2003112164A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007029860A (en) * | 2005-07-27 | 2007-02-08 | Dna Future:Kk | Method for decomposing or concentrating toxic substance in soil |
| JP2016150272A (en) * | 2015-02-16 | 2016-08-22 | 株式会社鴻池組 | Clarifier by bioremediation of contaminated soil, and clarification method using the same |
| CN105728446A (en) * | 2016-05-12 | 2016-07-06 | 温州泓呈祥科技有限公司 | Soil remediation device capable of removing omethoate components in farmland soil |
| CN105728447A (en) * | 2016-05-12 | 2016-07-06 | 温州泓呈祥科技有限公司 | Soil remediation method capable of removing omethoate components in farmland soil |
| CN105728447B (en) * | 2016-05-12 | 2018-09-07 | 涂瑞强 | The soil remediation method of flolimat ingredient in a kind of removal agricultural land soil |
| CN105728446B (en) * | 2016-05-12 | 2018-09-07 | 涂瑞强 | The soil restoring device of flolimat ingredient in a kind of removal agricultural land soil |
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