JP2013184994A - Volatile organic compound decomposer - Google Patents
Volatile organic compound decomposer Download PDFInfo
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- JP2013184994A JP2013184994A JP2012049048A JP2012049048A JP2013184994A JP 2013184994 A JP2013184994 A JP 2013184994A JP 2012049048 A JP2012049048 A JP 2012049048A JP 2012049048 A JP2012049048 A JP 2012049048A JP 2013184994 A JP2013184994 A JP 2013184994A
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- 239000012855 volatile organic compound Substances 0.000 title claims abstract description 44
- 239000010802 sludge Substances 0.000 claims abstract description 34
- 239000002689 soil Substances 0.000 claims abstract description 16
- 239000003673 groundwater Substances 0.000 claims abstract description 15
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 7
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 7
- CYTYCFOTNPOANT-UHFFFAOYSA-N Perchloroethylene Chemical group ClC(Cl)=C(Cl)Cl CYTYCFOTNPOANT-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229950011008 tetrachloroethylene Drugs 0.000 claims abstract description 5
- 238000002360 preparation method Methods 0.000 claims abstract description 4
- 239000000706 filtrate Substances 0.000 claims description 34
- 239000012528 membrane Substances 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 20
- 239000003795 chemical substances by application Substances 0.000 claims description 18
- 230000001143 conditioned effect Effects 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 4
- 238000000108 ultra-filtration Methods 0.000 description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 230000015556 catabolic process Effects 0.000 description 5
- 238000006731 degradation reaction Methods 0.000 description 5
- 239000011521 glass Substances 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 239000011148 porous material Substances 0.000 description 4
- 238000005273 aeration Methods 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000000354 decomposition reaction Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 206010052804 Drug tolerance Diseases 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- FFBHFFJDDLITSX-UHFFFAOYSA-N benzyl N-[2-hydroxy-4-(3-oxomorpholin-4-yl)phenyl]carbamate Chemical compound OC1=C(NC(=O)OCC2=CC=CC=C2)C=CC(=C1)N1CCOCC1=O FFBHFFJDDLITSX-UHFFFAOYSA-N 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 230000026781 habituation Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000588986 Alcaligenes Species 0.000 description 1
- 208000033962 Fontaine progeroid syndrome Diseases 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- XSTXAVWGXDQKEL-UHFFFAOYSA-N Trichloroethylene Chemical group ClC=C(Cl)Cl XSTXAVWGXDQKEL-UHFFFAOYSA-N 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- KFUSEUYYWQURPO-UPHRSURJSA-N cis-1,2-dichloroethene Chemical group Cl\C=C/Cl KFUSEUYYWQURPO-UPHRSURJSA-N 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 229910000388 diammonium phosphate Inorganic materials 0.000 description 1
- 235000019838 diammonium phosphate Nutrition 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000005108 dry cleaning Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000003365 glass fiber Substances 0.000 description 1
- 239000000383 hazardous chemical Substances 0.000 description 1
- 238000003988 headspace gas chromatography Methods 0.000 description 1
- 231100000206 health hazard Toxicity 0.000 description 1
- 239000002440 industrial waste Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 150000004045 organic chlorine compounds Chemical class 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 238000002731 protein assay Methods 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Processing Of Solid Wastes (AREA)
- Separation Using Semi-Permeable Membranes (AREA)
- Removal Of Specific Substances (AREA)
Abstract
Description
本発明は土壌や地下水に残留する揮発性有機化合物を分解するための分解剤に関する。 The present invention relates to a decomposing agent for decomposing volatile organic compounds remaining in soil or groundwater.
これまでに多くの電子基板工場やドライクリーニング店などで揮発性有機化合物（以下、VOC）が使用されてきた。そのVOCの漏出などにより、各地で土壌・地下水の汚染が見つかっている。主な汚染VOCには、テトラクロロエチレン（以下、PCE）、トリクロロエチレン、シス−１，２−ジクロロエチレンなどの揮発性有機塩素化合物があり、いずれも難分解性である。さらに、VOCは人体への変異原性や発がん性を有する恐れがあることから、健康被害も懸念される。 To date, volatile organic compounds (hereinafter referred to as VOCs) have been used in many electronic substrate factories and dry cleaning stores. Due to the leakage of VOC, soil and groundwater contamination has been found in various places. Major polluting VOCs include volatile organochlorine compounds such as tetrachloroethylene (hereinafter PCE), trichlorethylene, and cis-1,2-dichloroethylene, all of which are hardly decomposable. Furthermore, since VOCs may be mutagenic and carcinogenic to the human body, there are concerns about health hazards.
PCE等の揮発性有機化合物で汚染された土壌や地下水を浄化するための一つの方法として、バイオレメディエーションが知られている。バイオレメディエーションは微生物を用いるため、省エネルギーかつ低コストで土壌や地下水の汚染を浄化することが期待できる（特許文献１）。 Bioremediation is known as one method for purifying soil and groundwater contaminated with volatile organic compounds such as PCE. Since bioremediation uses microorganisms, it can be expected to purify contamination of soil and groundwater at low energy and cost (Patent Document 1).
しかしながら、実際の浄化対象の土壌や地下水の温度、ｐＨや酸素濃度等は様々であるので、実験室で発揮されたような微生物の増殖や分解作用が進まないことも多く、結果として浄化に長時間を要することがある。 However, since the temperature, pH, oxygen concentration, etc. of the actual soil and groundwater to be purified vary, the growth and decomposition of microorganisms as demonstrated in the laboratory often does not proceed, resulting in long purification. It may take time.
従って本発明の課題は、実際の浄化対象の土壌や地下水の条件に影響を受けにくい、揮発性有機化合物分解剤を提供することにある。 Accordingly, an object of the present invention is to provide a volatile organic compound decomposing agent that is not easily affected by the conditions of actual soil and groundwater to be purified.
本発明者が鋭意検討した結果、比較的高濃度のPCEを炭素源として好気条件下で馴養して得られた活性汚泥の菌体外成分が、強いPCE分解能力を有することを見出した。かかる知見に基づいて本発明者はさらに検討を重ね、本発明を完成させた。 As a result of intensive studies by the present inventors, it has been found that the extracellular components of activated sludge obtained by acclimatizing under aerobic conditions using a relatively high concentration of PCE as a carbon source has a strong PCE degrading ability. Based on this knowledge, the present inventors have further studied and completed the present invention.
即ち、本発明の要旨は、
＜１＞ 炭素源として０．０００６〜６００ｍＭのテトラクロロエチレンを含有する条件で活性汚泥を馴養する工程〔工程１〕、及び
馴養された活性汚泥から、分子量３０００以下の画分を分取する工程〔工程２〕、
を含む調製方法によって得られた該画分からなる、揮発性有機化合物分解剤；
＜２＞ 工程１で馴養された活性汚泥をメンブレンフィルターを用いてろ過してろ液を得る工程〔工程Ａ〕をさらに含み、馴養された活性汚泥の代わりに該ろ液を工程２に提供する、前記＜１＞に記載の揮発性有機化合物分解剤；
＜３＞ 前記＜１＞又は＜２＞に記載の揮発性有機化合物分解剤を、土壌中の揮発性有機化合物１ｇに対して２〜２０ｇ適用する工程を有する、土壌中の揮発性有機化合物を分解する方法；並びに
＜４＞ 前記＜１＞又は＜２＞に記載の揮発性有機化合物分解剤を、地下水中の揮発性有機化合物１ｇに対して２〜２０ｇ適用する工程を有する、地下水中の揮発性有機化合物を分解する方法；に関するものである。
That is, the gist of the present invention is as follows.
<1> A step of acclimatizing activated sludge under conditions containing 0.0006 to 600 mM tetrachloroethylene as a carbon source [Step 1] and a step of fractionating a fraction having a molecular weight of 3000 or less from the acclimatized activated sludge [Step 2],
A volatile organic compound decomposing agent comprising the fraction obtained by the preparation method comprising:
<2> The method further includes the step of obtaining the filtrate by filtering the activated sludge conditioned in the step 1 using a membrane filter, and providing the filtrate to the step 2 instead of the conditioned activated sludge. The volatile organic compound decomposing agent according to the above <1>;
<3> A volatile organic compound in soil having a step of applying 2 to 20 g of the volatile organic compound decomposing agent according to <1> or <2> to 1 g of volatile organic compound in soil. A method of decomposing; and <4> a step of applying 2 to 20 g of the volatile organic compound decomposing agent according to <1> or <2> to 1 g of volatile organic compound in groundwater. A method for decomposing volatile organic compounds.
本発明の揮発性有機化合物分解剤は比較的低分子の成分からなるものなので、土壌や地下水の諸条件に左右されることなく、揮発性有機化合物を分解することができる、という効果が発揮される。 Since the volatile organic compound decomposing agent of the present invention consists of relatively low-molecular components, the effect of being able to decompose volatile organic compounds without being affected by various conditions of soil and groundwater is exhibited. The
本発明の揮発性有機化合物分解剤は、下記の工程１及び工程２を含む調製方法によって得られた該画分からなるものである。 The volatile organic compound decomposing agent of the present invention comprises the fraction obtained by the preparation method including the following step 1 and step 2.
工程１
工程１は、炭素源として０．０００６〜６００ｍＭのPCEを含有する条件で活性汚泥を馴養する工程である。
Process 1
Step 1 is a step of acclimatizing activated sludge under conditions containing 0.0006 to 600 mM PCE as a carbon source.
本発明に用いられる活性汚泥としては、特に限定されないが、活性汚泥法を利用する廃水処理施設、例えば下水処理場やその他の産業廃棄物処理場等に由来する活性汚泥を利用することができる。馴養の際の活性汚泥の量としては、馴養期間を通して撹拌や曝気が可能な程度であれば特に限定されないが、例えば、１Ｌの水に対して、乾燥重量で５〜１０ｇの範囲が好ましい。 Although it does not specifically limit as activated sludge used for this invention, The activated sludge derived from the wastewater treatment facility using the activated sludge method, for example, a sewage treatment plant, other industrial waste disposal plants, etc. can be utilized. The amount of activated sludge at the time of acclimatization is not particularly limited as long as it can be stirred and aerated throughout the acclimatization period. For example, a range of 5 to 10 g in dry weight with respect to 1 L of water is preferable.
かかる活性汚泥を、炭素源として０．０００６〜６００ｍＭのPCEを含有する条件で馴養することにより、特定の細菌を含む微生物を含有する馴養化活性汚泥が得られる。馴養時のPCEの濃度としては、PCEを分解し得る微生物を効率的に選択する観点から、０．００６〜１８０ｍＭの範囲が好ましく、０．０６〜６０ｍＭの範囲がより好ましい。 By acclimating such activated sludge under conditions containing 0.0006 to 600 mM PCE as a carbon source, a conditioned activated sludge containing microorganisms containing specific bacteria can be obtained. The concentration of PCE at the time of acclimatization is preferably in the range of 0.006 to 180 mM, more preferably in the range of 0.06 to 60 mM, from the viewpoint of efficiently selecting microorganisms that can degrade PCE.
活性汚泥を馴養する期間としては、特に限定されないが、１〜２４週間の期間が好ましい。馴養の際の他の条件、例えば温度、溶存酸素濃度、ｐＨとしては、以下の範囲が好ましい。温度としては、１０〜４０℃の範囲が好ましく、１５〜３０℃の範囲がより好ましい。馴養中の溶存酸素濃度としては特に限定されないが、空気、酸素等を供給しながら馴養を実施する程度でよい。従って、馴養に用いる装置としては、曝気手段を備えたものが好ましい。さらに、馴養の際のｐＨとしては、馴養時の温度において、例えば６．０〜９．５の範囲が好ましく、７．０〜８．５の範囲がより好ましい。 Although it does not specifically limit as a period which acclimatizes activated sludge, The period of 1 to 24 weeks is preferable. As other conditions at the time of acclimatization, such as temperature, dissolved oxygen concentration, and pH, the following ranges are preferable. As temperature, the range of 10-40 degreeC is preferable, and the range of 15-30 degreeC is more preferable. The dissolved oxygen concentration during the acclimatization is not particularly limited, but may be an extent that the acclimatization is performed while supplying air, oxygen and the like. Therefore, a device equipped with aeration means is preferable as a device used for habituation. Furthermore, as pH at the time of acclimatization, in the temperature at the time of acclimatization, the range of 6.0-9.5 is preferable, for example, and the range of 7.0-8.5 is more preferable.
工程２
工程２は、工程１で馴養された活性汚泥から、分子量３０００以下の画分を分取する工程である。
分子量３０００以下の画分を分取するためには、例えば分画分子量３０００の公知の限外ろ過膜を使用すればよい。この時、活性汚泥を分画分子量３０００の限外ろ過膜で処理することもできるが、膜の目詰まりを抑制する観点から、数段階に分けて、例えば、分画分子量１００００程度（例えば、孔径約２０〜３０ÅのＵＦ膜）の膜を用いて活性汚泥からろ液を分取し、次いで分画分子量５０００程度（例えば、孔径約１０〜２０ÅのＵＦ膜）の膜を用いて、先のろ液からさらにろ液を分取し、最後に分画分子量３０００の膜を用いてろ液、即ち本発明の揮発性有機化合物分解剤を得ることができる。本発明の揮発性有機化合物分解剤は、ろ液の状態、即ち溶液又は分散液の状態であってもよく、ろ液を凍結乾燥等の処理により乾燥させて得られる固体の状態であってもよい。
Process 2
Step 2 is a step of fractionating a fraction having a molecular weight of 3000 or less from the activated sludge conditioned in Step 1.
In order to fractionate a fraction having a molecular weight of 3000 or less, for example, a known ultrafiltration membrane having a molecular weight cutoff of 3000 may be used. At this time, the activated sludge can be treated with an ultrafiltration membrane having a fractional molecular weight of 3000, but from the viewpoint of suppressing clogging of the membrane, it is divided into several stages, for example, a fractional molecular weight of about 10,000 (for example, The filtrate is separated from the activated sludge using a membrane of about 20 to 30 mm UF membrane, and then filtered using a membrane with a molecular weight cut off of about 5000 (for example, a UF membrane having a pore diameter of about 10 to 20 mm). The filtrate is further collected from the liquid, and finally, the filtrate, that is, the volatile organic compound decomposing agent of the present invention can be obtained using a membrane having a molecular weight cutoff of 3000. The volatile organic compound decomposing agent of the present invention may be in the state of a filtrate, that is, in the state of a solution or dispersion, or in the state of a solid obtained by drying the filtrate by a treatment such as freeze drying. Good.
工程Ａ
さらに本発明では、限外ろ過膜の目詰まりを防ぎ、効率的に揮発性有機化合物分解剤を調製する観点から、工程Ａを含むことが好ましい。本明細書における工程Ａとは、工程１で馴養された活性汚泥をメンブレンフィルターを用いてろ過してろ液を得る工程であり、工程Ａを適用する場合、工程２においては、馴養された活性汚泥の代わりに、メンブレンフィルターを用いて得られたろ液が限外ろ過に適用されることとなる。
Process A
Furthermore, in this invention, it is preferable to include the process A from a viewpoint of preventing clogging of an ultrafiltration membrane and preparing a volatile organic compound decomposer efficiently. Step A in this specification is a step of obtaining the filtrate by filtering the activated sludge conditioned in step 1 using a membrane filter. When applying step A, in step 2, the conditioned activated sludge is used. Instead, the filtrate obtained using a membrane filter is applied to ultrafiltration.
工程Ａで用いられるメンブレンフィルターとしては、孔径が０．０２５〜０．４５μｍのものが好ましく、０．１０〜０．２２μｍのものがより好ましい。 The membrane filter used in step A preferably has a pore size of 0.025 to 0.45 μm, and more preferably 0.10 to 0.22 μm.
本発明の揮発性有機化合物分解剤を用いて、土壌中又は地下水中の揮発性有機化合物を分解することができる。本発明は、このような揮発性有機化合物を分解する方法を包含する。 The volatile organic compound decomposing agent of the present invention can be used to decompose volatile organic compounds in soil or groundwater. The present invention includes a method for decomposing such volatile organic compounds.
例えば、土壌中の揮発性有機化合物を分解する方法としては、本発明の揮発性有機化合物分解剤を、土壌中の揮発性有機化合物１ｇに対して２〜２０ｇ適用する工程を有する方法が挙げられる。 For example, as a method for decomposing volatile organic compounds in soil, there is a method having a step of applying 2 to 20 g of the volatile organic compound decomposing agent of the present invention to 1 g of volatile organic compounds in soil. .
さらに、地下水中の揮発性有機化合物を分解する方法としては、本発明の揮発性有機化合物分解剤を、地下水中の揮発性有機化合物１ｇに対して２〜２０ｇ適用する工程を有する方法が挙げられる。 Furthermore, as a method of decomposing | disassembling the volatile organic compound in groundwater, the method which has the process of applying 2-20g with respect to 1g of volatile organic compound decomposing agents of this invention with respect to 1g of volatile organic compounds in groundwater is mentioned. .
以下、本発明を実施例に基づいて説明するが、本発明はこれらの実施例により何ら制限されるものではない。 EXAMPLES Hereinafter, although this invention is demonstrated based on an Example, this invention is not restrict | limited at all by these Examples.
実施例１
工程１：活性汚泥とPCEへの馴養
日本国岐阜県にある電子機器の製造工場に設置された、該工場に由来する廃液を活性汚泥法を利用して処理する施設から、活性汚泥を得た。リービッヒ冷却器を上部に、フラスコ内部を曝気するためのポンプを取り付けた丸底フラスコに、得られた活性汚泥（１０００ｍＬ、乾燥重量は１ｇ）を投入した。
Example 1
Process 1: Acclimatization to activated sludge and PCE Activated sludge was obtained from a facility installed at an electronic equipment manufacturing factory in Gifu Prefecture, Japan, that uses the activated sludge method to treat waste liquid derived from the factory. . The obtained activated sludge (1000 mL, dry weight 1 g) was put into a round bottom flask equipped with a Liebig condenser at the top and a pump for aeration of the inside of the flask.
次いで、炭素源としてのPCE（和光純薬工業）のエタノール溶液を、PCEの終濃度が１２ｍＭとなるように、該フラスコに添加した。該エタノール溶液を一週間ごとに追加し、最終的にフラスコ内のPCEを６０ｍＭの濃度とした。リン源及び窒素源としてのリン酸水素二アンモニウムを、終濃度が３７．８８ｍＭとなるように該フラスコに添加した。さらに、ミネラル源及びビタミン源としての少量の酵母エキス（Difco、細胞培養用）も該フラスコに添加した。該フラスコ内の温度を２０℃とし、ｐＨを６．８〜８．０の範囲に維持しながら、ポンプから空気を該フラスコ内に曝気することによって、好気的条件下で馴養を行った。馴養期間は２週間とした。 Next, an ethanol solution of PCE (Wako Pure Chemical Industries) as a carbon source was added to the flask so that the final concentration of PCE was 12 mM. The ethanol solution was added every week, and finally the PCE in the flask was adjusted to a concentration of 60 mM. Diammonium hydrogen phosphate as a phosphorus source and a nitrogen source was added to the flask to a final concentration of 37.88 mM. In addition, a small amount of yeast extract (Difco, for cell culture) as mineral source and vitamin source was also added to the flask. The flask was conditioned under aerobic conditions by aeration of air from the pump into the flask while maintaining the temperature in the flask at 20 ° C. and maintaining the pH in the range of 6.8-8.0. The acclimatization period was 2 weeks.
活性汚泥の細菌叢の分析
馴養後の活性汚泥の細菌叢の分析を、タカラバイオ社が提供するGenome Sequencer FLXシステムを用いて行った。結果を表１に示す。
Analysis of bacterial flora of activated sludge The analysis of bacterial flora of activated sludge after acclimatization was performed using the Genome Sequencer FLX system provided by Takara Bio Inc. The results are shown in Table 1.
表１より、馴養後の活性汚泥の細菌叢では、Alcaligenesに属する微生物が支配的であることが分かった。 From Table 1, it was found that microorganisms belonging to Alcaligenes are dominant in the activated sludge bacterial flora after habituation.
工程Ａ：活性汚泥のろ過
馴養後の活性汚泥をガラスファイバーフィルター（ADVANTEC、GS-25、ポアサイズは約１μｍ）でろ過して、活性汚泥からフロックを除去した。次いで、ポアサイズが０．２μｍの膜フィルター(ADVANTEC)でろ過して、細菌を除去した。得られたろ液をろ液１とした。
Step A: Filtration of activated sludge The activated sludge after acclimatization was filtered with a glass fiber filter (ADVANTEC, GS-25, pore size is about 1 μm) to remove flocs from the activated sludge. Subsequently, it filtered with the membrane filter (ADVANTEC) whose pore size is 0.2 micrometer, and removed bacteria. The obtained filtrate was designated as filtrate 1.
工程２：ろ液の限外ろ過
ろ液１を、最初に分画分子量が１００００の限外ろ過膜を用いて限外ろ過し、ろ液（ろ液２）を得た。次にろ液２を、分画分子量が５０００の限外ろ過膜を用いて限外ろ過し、ろ液（ろ液３）を得た。最後にろ液３を、分画分子量が３０００の限外ろ過膜を用いて限外ろ過し、ろ液（ろ液４）を得た。ここで用いた限外ろ過膜は、GEヘルスケア社の商品名：Vivaspinであった。このろ液４を揮発性有機化合物分解剤とした。
Step 2: Ultrafiltration of filtrate Filtrate 1 was first ultrafiltered using an ultrafiltration membrane having a fractional molecular weight of 10,000 to obtain a filtrate (filtrate 2). Next, the filtrate 2 was ultrafiltered using an ultrafiltration membrane having a fractional molecular weight of 5000 to obtain a filtrate (filtrate 3). Finally, the filtrate 3 was ultrafiltered using an ultrafiltration membrane having a fractional molecular weight of 3000 to obtain a filtrate (filtrate 4). The ultrafiltration membrane used here was Vivaspin, a trade name of GE Healthcare. This filtrate 4 was used as a volatile organic compound decomposer.
PCEの分解能力の定量
上記の各ろ液についてのPCEの分解能力を定量した。
１００ｍＬ容のガラス瓶に、２０ｍＬのサンプルと、少量のエタノールに溶解させた１２μｍｏｌのＰＣＥを添加した。ガラス瓶を密閉し、２０℃で２２±２時間、好気状態となるように振盪を続けてPCEの分解処理を行った。
Quantification of PCE decomposition ability The PCE decomposition ability of each of the above filtrates was quantified.
A 20 mL sample and 12 μmol PCE dissolved in a small amount of ethanol were added to a 100 mL glass bottle. The glass bottle was sealed, and the PCE was decomposed by continuing shaking at 20 ° C. for 22 ± 2 hours so as to be in an aerobic state.
振盪終了後、ガラス瓶を４℃で１時間静置し、それぞれのガラス瓶の液相から１ｍＬをサンプルとして採取した。９ｍＬの純水及び３ｇの塩化ナトリウムを入れた別の２０ｍＬ容のガラス瓶に、採取したサンプルを注入した。密閉した後、ヘッドスペースガスクロマトグラフィー質量分析装置（島津製作所、GCMS-QP2010 Plus、Perkin Elmer、Headspace Sampler Turbo Matrix 40）を用いてPCEの量を定量した。測定されたPCEの量から、２０ｍＬのサンプルあたりのPCEの減少量を算出した。結果を表２に示す。 After completion of the shaking, the glass bottle was allowed to stand at 4 ° C. for 1 hour, and 1 mL was collected as a sample from the liquid phase of each glass bottle. The collected sample was poured into another 20 mL glass bottle containing 9 mL of pure water and 3 g of sodium chloride. After sealing, the amount of PCE was quantified using a headspace gas chromatography mass spectrometer (Shimadzu Corporation, GCMS-QP2010 Plus, Perkin Elmer, Headspace Sampler Turbo Matrix 40). The amount of decrease in PCE per 20 mL sample was calculated from the measured amount of PCE. The results are shown in Table 2.
タンパク質の定量
上記の各ろ液中のタンパク質の量を、Quanti-iTタンパク質アッセイキット(Invitorogen)を用いて定量した。該キットに添付の説明書に記載の方法に従って、定量操作を行った。結果を表２に示す。
Protein quantification The amount of protein in each of the above filtrates was quantified using a Quanti-iT protein assay kit (Invitorogen). The quantitative operation was performed according to the method described in the instructions attached to the kit. The results are shown in Table 2.
表２より、ろ液１、即ち、０．２μｍの膜フィルターのろ液はPCEを大きく減少させることが分かった。このろ液１を位相差顕微鏡で確認したところ、細菌が観察されなかったことから、菌体外の物質がPCEの分解に関与することが示唆された。 From Table 2, it was found that the filtrate 1, that is, the filtrate of the membrane filter of 0.2 μm, greatly reduces PCE. When this filtrate 1 was confirmed with a phase-contrast microscope, no bacteria were observed, suggesting that substances outside the cells are involved in the degradation of PCE.
ろ液１をさらに限外ろ過に供した結果、ろ液３、即ち分画分子量が５０００未満の画分は、ろ液１の１．７３倍、ろ液４、即ち分画分子量が３０００未満の画分は、ろ液１の１．６８倍のPCE分解能力を有することが分かった。分画分子量が３０００未満の画分がこのような高いPCE分解能力を有すること、及び該画分中のタンパク質性成分の量を考慮すると、比較的低分子量のタンパク質性成分、例えばオリゴペプチドがPCEの分解に関与することが示唆された。 The filtrate 1 was further subjected to ultrafiltration. As a result, the filtrate 3, ie, the fraction having a fractional molecular weight of less than 5000, was 1.73 times the filtrate 1, and the filtrate 4, ie, the fractional molecular weight was less than 3000. The fraction was found to have 1.68 times the PCE degradation capacity of filtrate 1. Considering that fractions with a molecular weight cut-off less than 3000 have such a high PCE degradation ability and the amount of proteinaceous components in the fractions, relatively low molecular weight proteinaceous components such as oligopeptides are It was suggested to be involved in the degradation of.
細菌そのものや酵素ではなく、このような、比較的低分子量のタンパク質性成分がPCEの分解に関与することは、当業者にとって予想外であり、低分子量の成分は細菌そのものや酵素に比べて温度やｐＨに対して高い抵抗性を発揮することが期待される。 It is unexpected for a person skilled in the art that such a relatively low molecular weight protein component is involved in the degradation of PCE, not the bacteria themselves or the enzyme, and the low molecular weight components have a temperature higher than that of the bacteria themselves and the enzyme. And high resistance to pH is expected.
本発明は、土壌や地下水に残留する揮発性有機化合物、特にテトラクロロエチレンを分解するための分解剤として利用することができる。 The present invention can be used as a decomposing agent for decomposing volatile organic compounds remaining in soil or groundwater, particularly tetrachloroethylene.
Claims (4)
馴養された活性汚泥から、分子量３０００以下の画分を分取する工程〔工程２〕、
を含む調製方法によって得られた該画分からなる、揮発性有機化合物分解剤。 A step of acclimatizing activated sludge under conditions containing 0.0006 to 600 mM tetrachloroethylene as a carbon source (step 1), and a step of fractionating a fraction having a molecular weight of 3000 or less from the acclimatized activated sludge (step 2);
A volatile organic compound decomposing agent comprising the fraction obtained by the preparation method comprising:
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| JP2016174537A (en) * | 2015-03-18 | 2016-10-06 | イビデン株式会社 | Production method of biosurfactant, and biosurfactant |
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