JP6739792B2 - Environmental pollutant removal method and environmental pollutant removal kit - Google Patents
Environmental pollutant removal method and environmental pollutant removal kit Download PDFInfo
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本発明は環境汚染物質の除去方法および環境汚染物質の除去キットに関する。より詳細には、微生物および線虫を用いた環境汚染物質の除去方法および環境汚染物質の除去キットに関する。 The present invention relates to a method for removing environmental pollutants and a kit for removing environmental pollutants. More specifically, it relates to a method for removing environmental pollutants using microorganisms and nematodes, and an environmental pollutant removal kit.
従来、放射性物質、重金属類、環境ホルモンなどによる環境汚染が問題となっている。なかでも、原発事故により漏えいした放射性物質による土壌汚染は深刻な被害をもたらしている。 Conventionally, environmental pollution by radioactive substances, heavy metals, environmental hormones, etc. has been a problem. Above all, soil pollution by radioactive materials leaked from the nuclear accident has caused serious damage.
そこで、放射性物質により汚染された土壌の浄化方法が検討されている。特許文献１〜３には、化学的または物理的に土壌中の放射性物質を除去する方法が記載されているが、大規模な設備が必要であり莫大な費用がかかるという問題、大量の汚染水などが発生するという問題がある。 Therefore, methods for purifying soil contaminated with radioactive substances are being studied. Patent Documents 1 to 3 describe a method of chemically or physically removing radioactive substances in soil, but the problem that large-scale equipment is required and the cost is huge, and a large amount of contaminated water is required. There is a problem that such as occurs.
生物的に土壌中の放射性物質を除去する方法として、ヒマワリや菜種などの放射性物質を吸収するといわれる植物を汚染土壌で栽培する方法（例えば特許文献４など参照）が報告されているが、除去効率が不充分という問題がある。また、環境汚染物質を吸収し貯留する微生物を用いた方法もあるが、汚染物質を吸収した微生物を効率的に回収する方法が確立されておらず、実施困難という状況である。 As a method of biologically removing radioactive substances in soil, a method of cultivating a plant that is said to absorb radioactive substances such as sunflower and rapeseed in contaminated soil (see, for example, Patent Document 4) has been reported. There is a problem of insufficient efficiency. There is also a method using a microorganism that absorbs and stores environmental pollutants, but a method for efficiently collecting the microorganisms that absorbed the pollutant has not been established, and it is difficult to implement.
本発明は、汚染水などを発生させず簡便に、環境汚染物質により汚染された土壌などから環境汚染物質を除去することで、無害化して再利用することができる方法を提供することを目的とする。 An object of the present invention is to provide a method capable of detoxifying and reusing by simply removing environmental pollutants from soil polluted with environmental pollutants without generating contaminated water or the like. To do.
本発明は、
［１］環境汚染物質を吸収する微生物を増殖させる工程Ａ１、前記微生物を線虫に捕食させる工程Ａ２、および前記線虫を回収する工程Ｂを含む環境汚染物質の除去方法、
［２］前記工程Ａ１および工程Ａ２を同時に行う［１］記載の除去方法、
［３］除去する環境汚染物質が放射性物質である［１］または［２］記載の除去方法、および
［４］環境汚染物質を除去する対象が汚染土壌である［１］〜［３］のいずれかに記載の除去方法、
［５］環境汚染物質を吸収する微生物および前記微生物を捕食する線虫を含む環境汚染物質の除去キットに関する。
The present invention is
[1] A method for removing environmental pollutants, which comprises a step A1 of growing microorganisms that absorb environmental pollutants, a step A2 of feeding the microorganisms to nematodes, and a step B of collecting the nematodes,
[2] The removal method according to [1], wherein the step A1 and the step A2 are performed simultaneously.
[3] The removal method according to [1] or [2], wherein the environmental pollutant to be removed is a radioactive substance, and [4] the target to remove the environmental pollutant is contaminated soil [1] to [3]. Removal method described in
[5] A kit for removing an environmental pollutant including a microorganism that absorbs an environmental pollutant and a nematode that preys on the microorganism.
本発明の環境汚染物質の除去方法および環境汚染物質の除去キットによれば、汚染水などを発生させず簡便に、環境汚染物質により汚染された土壌などから環境汚染物質を除去することができる。 According to the method for removing environmental pollutants and the kit for removing environmental pollutants of the present invention, environmental pollutants can be easily removed from soil polluted with environmental pollutants without generating contaminated water.
本発明の環境汚染物質の除去方法は、環境汚染物質を吸収する微生物を増殖させる工程Ａ１、前記微生物を線虫に捕食させる工程Ａ２、および前記線虫を回収する工程Ｂを含むことを特徴とする。すなわち、本発明の除去方法は、除去対象物中に所定の微生物を増殖させることで、対象物中の環境汚染物質を微生物に吸収させ、環境汚染物質を吸収した微生物を線虫に捕食させることで、環境汚染物質を線虫の体内に移行させた後、この線虫を回収することで除去対象物中の環境汚染物質を除去することを特徴とする。 The method for removing environmental pollutants of the present invention is characterized by including a step A1 of growing a microorganism that absorbs the environmental pollutant, a step A2 of feeding the microorganism to a nematode, and a step B of collecting the nematode. To do. That is, the removal method of the present invention, by growing a predetermined microorganisms in the object to be removed, to absorb the environmental pollutants in the object to the microorganisms, to let the nematodes prey on the microorganisms absorbed the environmental pollutants. Then, after the environmental pollutants are transferred to the body of the nematodes, the nematodes are collected to remove the environmental pollutants in the removal object.
本明細書における環境汚染物質としては、微生物が吸収し貯留できる環境汚染物質であれば特に限定されず、放射性セシウム、放射性ストロンチウムなどの放射性物質、カドミウム、鉛、ヒ素などの重金属類、ビスフェノールＡ、フタル酸エステル類などの環境ホルモンなどが挙げられる。 The environmental pollutant in the present specification is not particularly limited as long as it is an environmental pollutant that can be absorbed and stored by microorganisms, radioactive cesium, radioactive substances such as radioactive strontium, cadmium, lead, heavy metals such as arsenic, bisphenol A, Examples include environmental hormones such as phthalates.
本発明により、環境汚染物質を除去する対象物は、汚染土壌、汚染水などが挙げられる。なかでも、本発明の効果がより発揮されることから、山林、水田、畑地、公園、緑地または花壇などの汚染土壌に含まれる環境汚染物質を除去する方法とすることが好ましい。 According to the present invention, examples of objects for removing environmental pollutants include contaminated soil and contaminated water. Among them, the method of removing environmental pollutants contained in contaminated soil such as forests, paddy fields, uplands, parks, green spaces or flower beds is preferable because the effects of the present invention can be more exerted.
工程Ａ１
工程Ａ１は、環境汚染物質を除去する対象物中において、環境汚染物質を吸収する微生物を増殖させる工程である。対象物中で所定の微生物を増殖させることにより、環境汚染物質を微生物の細胞内に吸収させることができる。
Process A1
Step A1 is a step of growing microorganisms that absorb environmental pollutants in an object from which environmental pollutants are removed. By growing a predetermined microorganism in an object, environmental pollutants can be absorbed into the cells of the microorganism.
環境汚染物質を除去する対象物中に、環境汚染物質を吸収する微生物が存在する場合はその微生物を増殖させればよい。一方、対象物中にそのような微生物が存在しない場合は、環境汚染物質を吸収する微生物を添加して増殖させればよい。 When a microorganism that absorbs the environmental pollutant is present in the object from which the environmental pollutant is removed, the microorganism may be grown. On the other hand, when such a microorganism does not exist in the object , a microorganism that absorbs environmental pollutants may be added and grown.
さらに、環境汚染物質を除去する対象物に微生物の生育および増殖を活性化させる基質を添加することが、微生物の増殖が活性化することから好ましい。当該基質としては、糖類、窒素源などが挙げられるが、使用する微生物や除去対象物の状態に応じて適宜調整することができる。 Furthermore, it is preferable to add a substrate that activates the growth and proliferation of microorganisms to an object from which environmental pollutants are removed, because the growth of microorganisms is activated. As the substrate, sugars, although such a nitrogen source and the like, may be appropriately adjusted according to the state of the microorganisms and removal object to be used.
環境汚染物質を吸収する微生物としては、既知の微生物を用いることもできる。既知の微生物としては、例えば、放射性セシウムを吸収するＲｈｏｄｏｃｏｃｃｕｓ ｅｒｙｔｈｒｏｐｏｌｉｓ、放射性セシウム、放射性ストロンチウム、クロム、水銀、銅、鉛、ヒ素を吸収するＲｈｏｄｏｂａｃｔｅｒ ｓｐｈａｅｒｏｉｄｅｓ、フェナントレンを吸収するＭｉｃｒｏｃｙｓｔｉｓ ａｅｒｕｇｉｎｏｓａなどが挙げられる。また、前記微生物としては、活性汚泥や自然界からスクリーニング等により発見された微生物を用いることもできる。なお、本明細書における微生物とは、細菌、酵母、糸状菌、微細藻類、原生動物を指す。 Known microorganisms can also be used as the microorganisms that absorb environmental pollutants. Examples of known microorganisms include Rhodococcus erythropolis that absorbs radioactive cesium, radioactive cesium, Rhodocobacter sphaeroides that absorbs radioactive strontium, chromium, mercury, copper, lead, and arsenic, and Microcystis aerugus that absorbs phenanthrene. Further, as the above-mentioned microorganism, activated sludge or a microorganism found from the natural world by screening or the like can be used. In addition, the microorganism in this specification refers to bacteria, yeast, filamentous fungi, microalgae, and protozoa.
工程Ａ１の温度は微生物が増殖できる温度であれば特に限定されないが、１５〜４０℃が好ましい。また、工程Ａ１は微生物が定常期に入るという理由から、６〜９日間行うことが好ましい。 The temperature of step A1 is not particularly limited as long as it is a temperature at which the microorganism can grow, but 15 to 40° C. is preferable. Further, the step A1 is preferably performed for 6 to 9 days because the microorganism enters the stationary phase.
工程Ａ２
工程Ａ２は、環境汚染物質を除去する対象物中において、前記微生物を線虫に捕食させる工程である。微生物を線虫に捕食させることにより、環境汚染物質を線虫の体内に凝集させることができる。
Process A2
Step A2 is a step in which nematodes prey on the microorganism in an object from which environmental pollutants are removed. By causing the nematodes to prey on the microorganisms, environmental pollutants can be aggregated in the body of the nematodes.
環境汚染物質を除去する対象物中に、微生物を捕食する線虫が存在する場合はその線虫に捕食させればよい。一方、対象物中にそのような線虫が存在しない場合は、微生物を捕食する線虫を添加して捕食させればよい。 If a nematode that preys on microorganisms is present in the object from which environmental pollutants are removed, the nematode may prey on it. On the other hand, when such a nematode does not exist in the target object , a nematode which preys on the microorganism may be added to prey on it.
前記線虫としては、微生物を捕食する線虫であれば特に限定されないが、様々な微生物を捕食するという理由から、Ｃａｅｎｏｒｈａｂｉｔｉｓ ｅｌｅｇａｎｓが好ましい。また、線虫の日齢は、特に限定されないが、捕食効率に優れるという理由から２〜８日が好ましい。 The nematode is not particularly limited as long as it is a nematode that preys on microorganisms, but Caenorhabitis elegans is preferable because it preys on various microorganisms. The age of the nematode is not particularly limited, but is preferably 2 to 8 days because of its excellent predation efficiency.
工程Ａ２の温度は線虫が捕食および増殖できる温度であれば特に限定されないが、１５〜４０℃が好ましい。また、工程Ａ２は線虫が十分に増殖し、かつ死亡個体数が増え始めないという理由から、７〜９日間行うことが好ましい。 The temperature of step A2 is not particularly limited as long as it is a temperature at which nematodes can prey and grow, but 15 to 40°C is preferable. In addition, step A2 is preferably performed for 7 to 9 days because the nematodes grow sufficiently and the number of dead individuals does not start increasing.
前記の環境汚染物質を吸収する微生物を増殖させる工程Ａ１、および前記微生物を線虫により捕食させる工程Ａ２は、独立して行うこともできるが、並行して行うことが好ましい。すなわち、環境汚染物質を除去する対象物に対し、前記の微生物および線虫を同時に添加し、微生物および線虫にとって好ましい環境下で静置することで、工程Ａ１および工程Ａ２を同時に行うことが好ましい。なお、必要に応じて対象物を撹拌等してもよい。 The step A1 of growing the microorganism that absorbs the environmental pollutant and the step A2 of feeding the microorganism with a nematode can be performed independently, but are preferably performed in parallel. That is, it is preferable to simultaneously perform the step A1 and the step A2 by simultaneously adding the above-mentioned microorganisms and nematodes to an object from which environmental pollutants are to be removed, and allowing them to stand in an environment preferable for the microorganisms and nematodes. .. The object may be agitated or the like if necessary.
工程Ｂ
工程Ｂは、環境汚染物質を除去する対象物から環境汚染物質が体内に凝集した線虫を回収する方法である。環境汚染物質を吸収した微生物を土壌などから直接回収することは極めて困難であるが、微生物より大きく、光や熱に対して忌避行動をとる線虫を回収する本発明の方法によれば容易に環境汚染物質を除去することができる。
Process B
Step B is a method of collecting nematodes, which are aggregates of environmental pollutants, from an object from which environmental pollutants are removed. Although it is extremely difficult to directly collect microorganisms that have absorbed environmental pollutants from soil or the like, it is easy to collect the nematodes which are larger than the microorganisms and take repellent action against light and heat according to the method of the present invention. Environmental pollutants can be removed.
線虫の忌避行動としては、光忌避行動、熱忌避行動、アルカリ忌避行動などがあるが、簡便性から、光忌避行動または熱忌避行動を利用した回収方法とすることが好ましい。具体的には、工程Ａ１および工程Ａ２を経た、汚染土壌などの環境汚染物質を除去する対象物をメッシュ状板の上に載せ、上側から光または熱を加えることで、メッシュ状板の穴を抜けて下部に落下してきた線虫を回収する方法などが挙げられるが、この方法に限定されるものではない。 Examples of nematode repellent behaviors include a light repellent behavior, a heat repellent behavior, and an alkali repellent behavior. However, for the sake of simplicity, it is preferable to use a light repellent behavior or a heat repellent behavior. Specifically, an object for removing environmental pollutants such as contaminated soil, which has undergone steps A1 and A2, is placed on the mesh plate, and light or heat is applied from above to open the holes in the mesh plate. Examples include a method of collecting nematodes that have passed through and dropped to the lower part, but are not limited to this method.
環境汚染物質による汚染具合によるが、前記の工程Ａ１、工程Ａ２および工程Ｂを繰り返し行うことが汚染物質の除去率を高めることができることから好ましい。例えば、前記工程Ｂの後、工程Ａ１の微生物および工程Ａ２の線虫を対象物に再度添加してもよいが、微生物および線虫が残存している場合は、そのまま最適な環境下で静置等することで工程Ａ１および工程Ａ２を繰り返し実施することができる。 Although it depends on the degree of pollution by environmental pollutants, it is preferable to repeat the above-mentioned step A1, step A2 and step B because the removal rate of pollutants can be increased. For example, after the step B, the microorganisms of the step A1 and the nematodes of the step A2 may be added again to the object. However, if the microorganisms and the nematodes remain, they are left as they are under the optimum environment. By doing so, step A1 and step A2 can be repeatedly performed.
なお、工程Ｂにより回収された線虫は、その体内に環境汚染物質が凝集されていることから、燃焼、隔離保管などの汚染物質に適した処理を行うことが好ましい。 Since the nematodes collected in the step B have environmental pollutants aggregated in their bodies, it is preferable to carry out a treatment suitable for the pollutants such as burning and storage in isolation.
除去キット
本発明の環境汚染物質を吸収する微生物および前記微生物を捕食する線虫を含む環境汚染物質の除去キットは、前記の工程Ａ１における微生物および工程Ａ２における線虫を含む環境汚染物質の除去キットである。汚染土壌などの環境汚染物質を除去する対象物に、キット内の微生物および線虫を加えて、数日間静置した後、前述の回収方法などにより線虫を回収することで環境汚染物質を除去することができる。
Removal kit An environmental pollutant removal kit containing a microorganism absorbing an environmental pollutant of the present invention and a nematode that preys on the microorganism is a removal kit for an environmental pollutant containing a microorganism in step A1 and a nematode in step A2. Is. To remove environmental pollutants by adding the microorganisms and nematodes in the kit to the object to remove environmental pollutants such as contaminated soil and allowing them to stand for several days, and then collecting the nematodes by the recovery method described above. can do.
本発明を実施例に基づいて説明するが、本発明は実施例のみに限定されるものではない。 The present invention will be described based on examples, but the present invention is not limited to the examples.
以下に実施例および比較例において用いた各種薬品などをまとめて示す。
ＮＧＭ寒天培地（ＮａＣｌ：３ｇ／Ｌ、ペプトン：２．５ｇ／Ｌ、寒天：１８ｇ／Ｌ、５ｍｇ／ｍＬコレステロール溶液：１ｍＬ／Ｌ、１Ｍリン酸カリウム緩衝液：１ｍＬ／Ｌ、１Ｍ ＣａＣｌ2：１ｍＬ／Ｌ、１Ｍ ＭｇＳＯ4：１ｍＬ／Ｌ、塩化セシウム：表１に示す）
塩化セシウム：和光純薬工業株式会社製（放射性セシウムと同様の挙動を示すことから、環境汚染物質のモデルとして使用）
Ｃｓ吸収菌：Ｒｈｏｄｏｃｏｃｃｕｓ ｅｒｙｔｈｒｏｐｏｌｉｓ ＣＳ９８
大腸菌：Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ ＯＰ５０
線虫：Ｃａｅｎｏｒｈａａｂｄｉｔｉｓ ｅｌｅｇａｎｓ野生種Ｎ２
Various chemicals and the like used in Examples and Comparative Examples are summarized below.
NGM agar medium (NaCl: 3 g/L, peptone: 2.5 g/L, agar: 18 g/L, 5 mg/mL cholesterol solution: 1 mL/L, 1M potassium phosphate buffer: 1 mL/L, 1M CaCl 2 :1 mL / L, 1M MgSO 4: 1mL / L, cesium chloride: shown in Table 1)
Cesium chloride: manufactured by Wako Pure Chemical Industries, Ltd. (because it behaves similarly to radioactive cesium, it is used as a model for environmental pollutants)
Cs absorbing bacteria: Rhodococcus erythropolis CS98
E. coli: Escherichia coli OP50
Nematode: Caenorhabditis elegans wild species N2
実施例１〜８
滅菌済みの樹脂製シャーレに、表１に示す量のセシウムを含有するＮＧＭ寒天培地（１５ｍＬ）を作製した。寒天培地表面にＣｓ吸収菌の培養液２００μＬを滴下してスプレッドし、線虫を１匹ずつピッカーで乗せた（線虫の日齢および乗せた数を表１に示す）。その後、各シャーレを２０℃で７日間静置させ、寒天培地上に生育したＣｓ吸収菌および線虫を生理食塩水を用いて回収した。回収液を３０分静置後、上部１０ｍＬをＣｓ吸収菌の懸濁液として回収し、下部５ｍＬに沈降した線虫を孔径３ｍＬのメンブレンフィルターを用いて回収した。回収した線虫は純水で数回洗浄後、遠心分離（１５０００ｒｐｍ、１０分）により分離させて回収した。
Examples 1-8
NGM agar medium (15 mL) containing the amount of cesium shown in Table 1 was prepared in a sterilized resin dish. 200 μL of the Cs-absorbing culture solution was dropped onto the surface of the agar medium and spread, and nematodes were placed one by one with a picker (the age of nematodes and the number of them are shown in Table 1). Then, each petri dish was allowed to stand at 20° C. for 7 days, and Cs-absorbing bacteria and nematodes grown on an agar medium were recovered using physiological saline. After the collected solution was allowed to stand for 30 minutes, 10 mL of the upper part was collected as a suspension of Cs-absorbing bacteria, and the nematodes that had settled in the lower part of 5 mL were collected using a membrane filter having a pore size of 3 mL. The collected nematodes were washed several times with pure water and then separated by centrifugation (15000 rpm, 10 minutes) to be collected.
比較例１
セシウム吸収菌に替えて大腸菌を用いたこと以外は実施例１と同様に行った。
Comparative Example 1
The same procedure as in Example 1 was repeated except that Escherichia coli was used instead of the cesium-absorbing bacterium.
評価
７日間静置後の線虫数、および回収した線虫内の凝集したセシウム量を評価した。
Evaluation The number of nematodes after standing for 7 days and the amount of aggregated cesium in the collected nematodes were evaluated.
線虫数
７日間静置後の寒天培地上の９点をデジタル顕微鏡（ａｉｇｏ社製のＤｉｇｉｔａｌ Ｍｉｃｒｏｓｃｏｐｅ ＤＭＳ０１２、倍率４０倍）により観察し、視野内の線虫数を計測し、下記式により前線虫数（Ｎ）を算出した。結果、全ての線虫数が約１０万匹となっており、線虫は充分数に増殖したことがわかる。
式 Ｎ＝（ＣＳS）／ＳO
式中、Ｃは９点で計測した線虫数の平均値を、ＳSはシャーレの面積（５６７５ｍｍ2）を、ＳOはデジタル顕微鏡の視野面積（１５．９０ｍｍ2）を示す。
Number of nematodes 9 points on the agar medium after standing for 7 days were observed with a digital microscope (Digital Microscope DMS012 manufactured by aigo, magnification 40 times), the number of nematodes in the visual field was measured, and the number of nematodes in the visual field was calculated according to the following formula. The number (N) was calculated. As a result, the number of all nematodes was about 100,000, which means that the nematodes grew to a sufficient number.
Formula N=(CS S )/S O
Wherein, C is the average value of the line number insects measured at nine points, S S is the area of the petri dish (5675mm 2), S O represents a digital microscope field area (15.90mm 2).
線虫内のセシウム量
６０℃で１日乾燥させた線虫に純硝酸２００μＬを加え、９０℃に加熱し、細胞壁を溶かして菌内部のセシウムを溶出させ、そのまま蒸発乾固させた。蒸発乾固後、１Ｍ硝酸を２００μＬ滴下して溶解させ、純水で溶解液を０．１Ｍに希釈したものを偏光ゼーマン原子吸光光度計（株式会社日立ハイテクサイエンス製のＺＡ３３００）でＣｓ濃度を測定し、線虫内のセシウム量を算出した。結果を表１に示す。
Amount of cesium in nematodes 200 μL of pure nitric acid was added to nematodes dried at 60° C. for 1 day, heated to 90° C., cell walls were dissolved to elute the cesium inside the bacteria, and evaporated to dryness. After evaporation to dryness, 200 μL of 1 M nitric acid was added dropwise to dissolve the solution, and the solution was diluted to 0.1 M with pure water, and the Cs concentration was measured with a polarized Zeeman atomic absorption spectrophotometer (ZA3300 manufactured by Hitachi High-Tech Science Co., Ltd.). Then, the amount of cesium in the nematode was calculated. The results are shown in Table 1.
表１の結果より、寒天培地中のセシウムをＣｓ吸収菌が吸収し、このＣｓ吸収菌を線虫が捕食した結果、線虫内にセシウムが凝集していることがわかる。これにより、本発明の環境汚染物質の除去方法および除去キットによれば、汚染水などを発生させず簡便に、環境汚染物質により汚染された土壌などから環境汚染物質を除去することで、無害化して再利用することができることがわかる。 From the results of Table 1, it can be seen that Cs-absorbing bacteria absorb cesium in the agar medium and nematodes prey on the Cs-absorbing bacteria. As a result, cesium is aggregated in the nematodes. Thus, according to the method and kit for removing environmental pollutants of the present invention, by simply removing the environmental pollutants from soil polluted by the environmental pollutants without generating polluted water, etc., it becomes harmless. You can see that it can be reused.
Claims (5)
環境汚染物質を除去する対象物中において、前記微生物を線虫により捕食させる工程Ａ２、および
環境汚染物質を除去する対象物から前記線虫を回収する工程Ｂを含む環境汚染物質の除去方法。 A step A1 of growing a microorganism that absorbs the environmental pollutant in the object from which the environmental pollutant is removed ,
Step A2 of predating the microorganisms by nematodes in an object from which environmental pollutants are removed , and
A method for removing environmental pollutants, which comprises a step B of collecting the nematodes from an object from which environmental pollutants are removed.
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