JP2007189952A - Storage solution for nitrifying bacterium-immobilized membrane - Google Patents
Storage solution for nitrifying bacterium-immobilized membrane Download PDFInfo
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- 230000001546 nitrifying effect Effects 0.000 title claims abstract description 80
- 239000012528 membrane Substances 0.000 title claims abstract description 67
- 241000894006 Bacteria Species 0.000 claims abstract description 45
- KHPXUQMNIQBQEV-UHFFFAOYSA-N oxaloacetic acid Chemical compound OC(=O)CC(=O)C(O)=O KHPXUQMNIQBQEV-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000000758 substrate Substances 0.000 claims abstract description 12
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 claims abstract description 8
- 235000020774 essential nutrients Nutrition 0.000 claims abstract description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims abstract description 5
- 229940005654 nitrite ion Drugs 0.000 claims abstract description 4
- 239000003761 preservation solution Substances 0.000 claims description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 26
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 9
- 241001453382 Nitrosomonadales Species 0.000 claims description 7
- -1 ammonium ions Chemical class 0.000 claims description 7
- 239000000872 buffer Substances 0.000 claims description 7
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 6
- 230000001590 oxidative effect Effects 0.000 claims description 6
- 238000004321 preservation Methods 0.000 claims description 6
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 claims description 6
- 239000010865 sewage Substances 0.000 claims description 4
- AZFNGPAYDKGCRB-XCPIVNJJSA-M [(1s,2s)-2-amino-1,2-diphenylethyl]-(4-methylphenyl)sulfonylazanide;chlororuthenium(1+);1-methyl-4-propan-2-ylbenzene Chemical compound [Ru+]Cl.CC(C)C1=CC=C(C)C=C1.C1=CC(C)=CC=C1S(=O)(=O)[N-][C@@H](C=1C=CC=CC=1)[C@@H](N)C1=CC=CC=C1 AZFNGPAYDKGCRB-XCPIVNJJSA-M 0.000 claims description 3
- 235000019270 ammonium chloride Nutrition 0.000 claims description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 3
- CDMADVZSLOHIFP-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane;decahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.[Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 CDMADVZSLOHIFP-UHFFFAOYSA-N 0.000 claims description 3
- 239000004304 potassium nitrite Substances 0.000 claims description 3
- 235000010289 potassium nitrite Nutrition 0.000 claims description 3
- 235000010288 sodium nitrite Nutrition 0.000 claims description 3
- 235000010339 sodium tetraborate Nutrition 0.000 claims description 3
- 239000011550 stock solution Substances 0.000 claims description 3
- 229940063013 borate ion Drugs 0.000 claims description 2
- 239000002351 wastewater Substances 0.000 claims 1
- 239000001913 cellulose Substances 0.000 abstract description 8
- 229920002678 cellulose Polymers 0.000 abstract description 8
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 abstract description 6
- 239000000661 sodium alginate Substances 0.000 abstract description 6
- 235000010413 sodium alginate Nutrition 0.000 abstract description 6
- 229940005550 sodium alginate Drugs 0.000 abstract description 6
- 239000000126 substance Substances 0.000 description 8
- 235000010443 alginic acid Nutrition 0.000 description 7
- 229920000615 alginic acid Polymers 0.000 description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 238000007444 cell Immobilization Methods 0.000 description 6
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 5
- 229940072056 alginate Drugs 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 231100000614 poison Toxicity 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical group [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 3
- 239000012620 biological material Substances 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 229910001424 calcium ion Inorganic materials 0.000 description 3
- 230000003100 immobilizing effect Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 2
- 241000605121 Nitrosomonas europaea Species 0.000 description 2
- 239000000783 alginic acid Substances 0.000 description 2
- 229960001126 alginic acid Drugs 0.000 description 2
- 150000004781 alginic acids Chemical class 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
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- 238000010586 diagram Methods 0.000 description 2
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- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000002574 poison Substances 0.000 description 2
- 230000029058 respiratory gaseous exchange Effects 0.000 description 2
- 239000003440 toxic substance Substances 0.000 description 2
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 2
- 229920002554 vinyl polymer Polymers 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229910001429 cobalt ion Inorganic materials 0.000 description 1
- XLJKHNWPARRRJB-UHFFFAOYSA-N cobalt(2+) Chemical compound [Co+2] XLJKHNWPARRRJB-UHFFFAOYSA-N 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
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- 235000012489 doughnuts Nutrition 0.000 description 1
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- 230000002708 enhancing effect Effects 0.000 description 1
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- 229920001002 functional polymer Polymers 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 229910001437 manganese ion Inorganic materials 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910052750 molybdenum Inorganic materials 0.000 description 1
- 239000011733 molybdenum Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229920005597 polymer membrane Polymers 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 229910001415 sodium ion Inorganic materials 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
本発明は、上下水道の各処理プロセスの水や河川水、湖沼水などの環境水を対象として、水中の化学成分をモニタリングすることを目的としたバイオセンサ応用水質計測器に用いられる硝化細菌固定化膜の保存液に関する。 The present invention is directed to fixing nitrifying bacteria used in a biosensor-applied water quality measuring instrument for the purpose of monitoring chemical components in water for environmental waters such as water and river water, lake water, and the like in water and sewage treatment processes. The present invention relates to a preservation solution for a chemical film.
バイオセンサは、溶液中の物質を測定対象とする水質計測器に用いられている。そして、測定対象を認識する分子識別素子として、酵素・抗体などの生体機能高分子、又は、微生物・細胞など生体そのものを利用している。バイオセンサは、これらの生体材料を多孔性高分子膜に包括又は共有結合させることにより固定化した膜と、電気化学的検出器などのトランスデューサとを組み合わせて試料液中の化学成分の測定を行う。バイオセンサでは、試料液を、上記生体材料を固定化した膜に接触させ、生化学反応を起こさせる。そして、生成又は消費される物質の濃度変化を、検出器の出力（電流、電圧など）変化に変換して測定する。一方、既知濃度の被測定物質の標準液によって検量線を予め準備する。このようにして、試料液に対し得られたセンサ出力から、試料液中の目的物質の濃度を算出することができる。 Biosensors are used in water quality measuring instruments that measure substances in solutions. And, as a molecular identification element for recognizing a measurement target, a biologically functional polymer such as an enzyme or an antibody, or a living body such as a microorganism or a cell is used. A biosensor measures a chemical component in a sample solution by combining a membrane in which these biomaterials are immobilized or covalently bonded to a porous polymer membrane and a transducer such as an electrochemical detector. . In a biosensor, a sample solution is brought into contact with a membrane on which the biological material is immobilized, thereby causing a biochemical reaction. Then, a change in concentration of a substance to be generated or consumed is converted into a change in detector output (current, voltage, etc.) and measured. On the other hand, a calibration curve is prepared in advance with a standard solution of a substance to be measured having a known concentration. Thus, the concentration of the target substance in the sample liquid can be calculated from the sensor output obtained for the sample liquid.
例えば、特許文献１には、生体材料として有害物質に極めて弱い微生物である硝化細菌をアルギン酸ゲルによってセルロース膜上に包括固定化した硝化細菌固定化膜を用い、トランスデューサとして溶存酸素電極を用いた水中の毒物検出用バイオセンサが開示されている。このバイオセンサでは、硝化細菌の呼吸速度を連続モニタリングして、検水中に毒物が混入したときの硝化細菌の呼吸速度低下率を基に毒物検出を行うことができる。 For example, Patent Literature 1 uses a nitrifying bacteria-immobilized membrane in which nitrifying bacteria, which are microorganisms that are extremely vulnerable to harmful substances as biomaterials, are comprehensively immobilized on a cellulose membrane with an alginate gel, and an underwater solution using a dissolved oxygen electrode as a transducer. A biosensor for detecting poisons is disclosed. With this biosensor, it is possible to continuously monitor the respiration rate of nitrifying bacteria, and to detect toxic substances based on the rate of decrease in the respiration rate of nitrifying bacteria when toxic substances are mixed into the test water.
上記のような硝化細菌を用いたバイオセンサにおいては、センサ出力の安定性を確保するとともに、毒物に対する応答の変動を回避するため、硝化細菌固定化膜を１〜２ヶ月に１回交換する必要がある。そのため、特許文献２に記載されているように、新しい硝化細菌固定化膜は、使用するまでの間、通常硝化細菌固定化膜用の保存液とともにビニールパックの中に封入して５〜１０℃程度の低温で保存している。 In the biosensor using nitrifying bacteria as described above, it is necessary to replace the nitrifying bacteria-immobilized membrane once every 1 to 2 months in order to ensure the stability of the sensor output and to avoid the fluctuation of the response to the poison. There is. Therefore, as described in Patent Document 2, a new nitrifying bacteria-immobilized membrane is usually enclosed in a vinyl pack together with a storage solution for nitrifying bacteria-immobilized membrane until it is used. Store at a low temperature.
硝化細菌固定化膜の供給体制及びセンサ保守頻度の観点から、硝化細菌固定化膜の保存期間は３ヶ月以上であることが望ましい。上記特許文献２のリン酸イオンを含むアンモニア酸化細菌固定化膜の保存期間についても、さらに保存期間を長くするように望まれていた。 From the viewpoint of the supply system of the nitrifying bacteria-immobilized membrane and the frequency of sensor maintenance, it is desirable that the storage period of the nitrifying bacteria-immobilized membrane is 3 months or more. Regarding the storage period of the ammonia-oxidizing bacteria-immobilized membrane containing phosphate ions of Patent Document 2, it has been desired to further increase the storage period.
本発明はこのような問題に鑑みてなされたものであり、硝化細菌固定化膜を、３ヵ月以上の長期にわたって保存することを可能とした、新規な硝化細菌固定化膜の保存液を提供することを目的とする。 The present invention has been made in view of such problems, and provides a novel nitrifying bacteria-immobilized membrane preservation solution that can preserve a nitrifying bacteria-immobilized membrane for a long period of 3 months or more. For the purpose.
本発明者らは鋭意検討を重ねた結果、硝化細菌固定化膜の保存液に含まれるリン酸イオンに問題があることを見出し、本発明の硝化細菌固定化膜の保存液に至った。 As a result of intensive studies, the present inventors have found that there is a problem with phosphate ions contained in the preservation solution for the nitrifying bacteria-immobilized membrane, and have reached the preservation solution for the nitrifying bacteria-immobilized membrane of the present invention.
すなわち、上記目的を達成するために、本発明は、硝化細菌固定化膜の保存液であって、固定化された硝化細菌の基質及び必須栄養素、並びにオキサル酢酸を含有したホウ酸緩衝液から成ることを特徴とする。
上記硝化細菌固定化膜は、一般的にバイオセンサに用いられ、該バイオセンサは、水質計測器に利用される。
That is, in order to achieve the above-mentioned object, the present invention is a preservation solution for a nitrifying bacteria-immobilized membrane, which comprises a borate buffer containing nitrified bacteria substrate and essential nutrients, and oxalacetic acid. It is characterized by that.
The nitrifying bacteria-immobilized membrane is generally used for a biosensor, and the biosensor is used for a water quality measuring instrument.
上記水質計測器の計測対象水は、一般的には、流水であり、水道原水、下水、排水のいずれかである。
上記硝化細菌は、アンモニア酸化細菌及び亜硝酸酸化細菌からなる群から選択される少なくとも一の硝化細菌であることが好ましい。
上記硝化細菌が、アンモニア酸化細菌の場合には、上記基質としてアンモニウムイオンを含むことが好ましい。また、上記硝化細菌が、亜硝酸酸化細菌の場合には、上記基質として亜硝酸イオンを含むことが好ましい。
The water to be measured by the water quality measuring instrument is generally flowing water, and is any of raw water, sewage, and drainage.
The nitrifying bacterium is preferably at least one nitrifying bacterium selected from the group consisting of ammonia oxidizing bacteria and nitrite oxidizing bacteria.
When the nitrifying bacterium is an ammonia oxidizing bacterium, it is preferable that the substrate contains an ammonium ion. Further, when the nitrifying bacterium is a nitrite oxidizing bacterium, it is preferable that the substrate contains nitrite ions.
上記アンモニウムイオン源として、塩化アンモニウム及び硫酸アンモニウムのいずれか少なくとも一を含むことが好適である。上記亜硝酸イオン源として、亜硝酸ナトリウム及び亜硝酸カリウムのいずれか少なくとも一を含むことが好適である。
本発明に係る保存液は、一般的には、四ホウ酸ナトリウム十水和物を含む。
It is preferable that the ammonium ion source contains at least one of ammonium chloride and ammonium sulfate. The nitrite ion source preferably contains at least one of sodium nitrite and potassium nitrite.
The preservation solution according to the present invention generally contains sodium tetraborate decahydrate.
本発明によれば、硝化細菌固定化膜を、３ヵ月以上の長期にわたって保存することを可能とし、バイオセンサ応用水質計の実用性をより高めることのできる硝化細菌固定化膜の保存液が提供される。 ADVANTAGE OF THE INVENTION According to this invention, the preservation | save liquid of the nitrifying bacteria fixed membrane which can preserve | save a nitrifying bacteria fixed membrane over a long period of 3 months or more and can improve the practicality of a biosensor applied water quality meter is provided. Is done.
以下に、本発明に係る硝化細菌固定化膜の保存液を、好適な実施の形態を参照してさらに詳細に説明する。
図１及び図２は、バイオセンサ及び硝化細菌固定化膜の一実施の形態を示す。
Below, the preservation | save liquid of the nitrifying bacteria fixed film | membrane which concerns on this invention is demonstrated still in detail with reference to suitable embodiment.
1 and 2 show one embodiment of a biosensor and a nitrifying bacteria-immobilized membrane.
図1は、バイオセンサの一実施の形態を示す。図に示すように、バイオセンサ１０は、溶存酸素電極１１と、フローセル１２と、硝化細菌固定化膜２０とを備えている。
フローセル１２には試料液の流路１２ａが設けられている。硝化細菌固定化膜２０には、硝化細菌が固定化されている。固定化される硝化細菌としては、アンモニア酸化細菌及び亜硝酸酸化細菌などを挙げることができる。
そして、硝化細菌固定化膜２０の一方の面は、流路１２ａ中の試料液と接し、もう一方の面は溶存酸素電極１１と接するように配置されている。
FIG. 1 shows an embodiment of a biosensor. As shown in the figure, the
The
One surface of the nitrifying bacteria-immobilized
ここで、図２に硝化細菌固定化膜２０の構成について、一実施の形態を示す。図に示すように、硝化細菌固定化膜２０は、円形の多孔質のセルロース膜２１、２２から成る。セルロース膜２１、２２は、両面テープ２３によって貼り合わされている。そして、硝化細菌が固定化された所定の大きさの円形状菌体固定化部２４を、中心部に供える。なお、両面テープ２３は、菌体固定化部２４と重ならないように、中心部が所定の大きさにくりぬかれたドーナツ状をなしている。
菌体固定化部２４の菌体固定化量は、２．５×１０6〜１．０×１０9個/ｍｍ2となるように調製することが好ましく、この範囲内に調整することにより、固定化膜の保存性の均質化を図ることができる
Here, FIG. 2 shows an embodiment of the configuration of the nitrifying bacteria-
The cell immobilization amount of the
本発明に係る硝化細菌固定化膜の保存液は、図1及び図２について説明したような硝化細菌固定化膜を保存するために用いられる。なお、このような実施の形態に係る硝化細菌固定化膜に用途が限定されるものではない。
また、バイオセンサは、水質計測器に用いられ、このような水質計測器は、計測対象水中の化学物質を計測するために用いられる。計測対象水は、一般的には、流水であり、水道原水、下水、排水のいずれかである。
The preservation solution for nitrifying bacteria-immobilized membrane according to the present invention is used for preserving the nitrifying bacteria-immobilized membrane as described with reference to FIGS. The application is not limited to the nitrifying bacteria-immobilized membrane according to such an embodiment.
Biosensors are used for water quality measuring instruments, and such water quality measuring instruments are used for measuring chemical substances in measurement target water. The measurement target water is generally flowing water, and is any of raw water, sewage, and drainage.
そして、本発明に係る硝化細菌固定化膜の保存液は、固定化された硝化細菌の基質及び必須栄養素、並びにオキサル酢酸を含有したホウ酸緩衝液から成る。すなわち、本発明に係る硝化細菌固定化膜の保存液は、リン酸イオンを含有しないホウ酸緩衝液から成る。 And the preservation | save liquid of the nitrifying bacteria fixed film | membrane which concerns on this invention consists of the borate buffer solution containing the fixed substrate and essential nutrient of nitrifying bacteria, and oxalacetic acid. That is, the nitrifying bacteria-immobilized membrane preservation solution according to the present invention is composed of a borate buffer solution that does not contain phosphate ions.
硝化細菌の固定化剤としては、アルギン酸ナトリウム水溶液（ゾル）が用いられている。アルギン酸ナトリウム水溶液（ゾル）は、一価陽イオンであるナトリウムイオンが、多価陽イオンであるカルシウムイオンと置換されて、アルギン酸分子が架橋され、ゲル転移が進行する。しかし、膜保存液中のリン酸イオンとアルギン酸ゲル内のカルシウムとが、化学反応によりリン酸カルシウムを形成して、カルシウムがアルギン酸ゲル外へ流出するため、架橋構造が破壊される。本発明者らは、鋭意検討した結果このようなことを突き止めた。 A sodium alginate aqueous solution (sol) is used as a nitrifying bacteria fixing agent. In the sodium alginate aqueous solution (sol), sodium ions, which are monovalent cations, are replaced with calcium ions, which are polyvalent cations, so that alginic acid molecules are cross-linked and gel transition proceeds. However, the phosphate ions in the membrane preservation solution and the calcium in the alginate gel form calcium phosphate by a chemical reaction, and the calcium flows out of the alginate gel, so that the crosslinked structure is destroyed. As a result of intensive studies, the present inventors have found out such a thing.
このようなことから、本発明に係る硝化細菌固定化膜の保存液は、ホウ酸緩衝液から成り、硝化細菌の基質及び必須栄養素、並びにオキサル酢酸を含有し、リン酸イオンを含有しない。 For this reason, the preservation solution of the nitrifying bacteria-immobilized membrane according to the present invention comprises a borate buffer solution, which contains a substrate and essential nutrients of nitrifying bacteria and oxalacetic acid, and does not contain phosphate ions.
上記基質は、固定化される硝化細菌に対応して選択する。例えば、アンモニア酸化細菌を用いる場合には、アンモニウムイオン、亜硝酸酸化細菌を用いる場合には、亜硝酸イオンを用いる。 The substrate is selected according to the nitrifying bacteria to be immobilized. For example, when ammonia-oxidizing bacteria are used, ammonium ions are used. When nitrite-oxidizing bacteria are used, nitrite ions are used.
アンモニウムイオン源として、塩化アンモニウム、硫酸アンモニウムを含むこと好ましく、亜硝酸イオン源として、亜硝酸ナトリウム、亜硝酸カリウムを含むことが好ましい。 The ammonium ion source preferably includes ammonium chloride and ammonium sulfate, and the nitrite ion source preferably includes sodium nitrite and potassium nitrite.
上記ホウ酸緩衝液は、一般的には、四ホウ酸ナトリウム十水和物を用いて調製する。上記ホウ酸緩衝液のホウ酸イオン濃度は、１．２５ｍＭであることが好ましい。 The borate buffer is generally prepared using sodium tetraborate decahydrate. The borate ion concentration of the borate buffer is preferably 1.25 mM.
必須栄養素とは、基質以外の栄養素であって、鉄イオン、マグネシウムイオン、炭酸イオン、銅イオン、コバルトイオン、マンガンイオン、モリブデンイオン、亜鉛イオン、カルシウムイオン、リン酸イオンを含むが、本発明に係る硝化細菌固定化膜の保存液では、銅イオン以下を添加せずとも良い。 Essential nutrients are nutrients other than substrates, and include iron ions, magnesium ions, carbonate ions, copper ions, cobalt ions, manganese ions, molybdenum ions, zinc ions, calcium ions, and phosphate ions. In such a nitrifying bacteria-immobilized membrane preservation solution, it is not necessary to add copper ions or less.
ホウ酸緩衝液中のオキサル酢酸の濃度は、固定化された硝化細菌の活性及び菌体量に応じて調整する。オキサル酢酸の濃度としては、０．５〜２ｍＭであることが好ましいが、特に１〜２ｍＭとすることが好ましい。オキサル酢酸濃度を０．５〜２ｍＭに調整することにより、アンモニア酸化細菌の培養齢に左右されることのなく安定した高い保存性を得ることができる。 The concentration of oxalacetic acid in the borate buffer is adjusted according to the activity of the immobilized nitrifying bacteria and the amount of cells. The concentration of oxalacetic acid is preferably 0.5 to 2 mM, and particularly preferably 1 to 2 mM. By adjusting the oxalacetic acid concentration to 0.5 to 2 mM, stable and high storage stability can be obtained without being influenced by the culture age of ammonia-oxidizing bacteria.
本発明に係る硝化細菌固定化膜の保存液は、リン酸イオンを含有しないので、硝化細菌固定化膜の固定化剤であるアルギン酸ゲル中のカルシウムイオンと反応して流出することがない。すなわち、硝化細菌の固定化剤であるアルギン酸ゲルを破壊しない。これによって、硝化細菌が菌体固定化部から流出することがなく、硝化細菌固定化膜を３ヶ月以上にわたって保存することが可能としている。 Since the preservation solution of the nitrifying bacteria-immobilized membrane according to the present invention does not contain phosphate ions, it does not flow out by reacting with calcium ions in the alginate gel, which is an immobilizing agent for the nitrifying bacteria-immobilized membrane. That is, the alginate gel that is a fixing agent for nitrifying bacteria is not destroyed. As a result, nitrifying bacteria do not flow out from the cell immobilization section, and the nitrifying bacteria-immobilized membrane can be stored for 3 months or more.
このように、本発明に係る硝化細菌固定化膜の保存液により、硝化細菌固定化膜の保存可能期間を３ヵ月以上とすることが可能となり、バイオセンサ応用水質計の実用性をより高めることができる。 Thus, the preservation solution of the nitrifying bacteria-immobilized membrane according to the present invention makes it possible to increase the storage period of the nitrifying bacteria-immobilized membrane to 3 months or more, thereby further enhancing the practicality of the biosensor applied water quality meter. Can do.
次に、本発明に係る硝化細菌固定化膜の保存液を、実施例にさらに詳細に説明するが、本発明はこれらの実施例に限定されない。 Next, although the preservation | save liquid of the nitrifying bacteria fixed film | membrane which concerns on this invention is demonstrated in detail to an Example, this invention is not limited to these Examples.
アンモニア酸化細菌Nitrosomonas europaea (ＡＴＣＣ２５９７８)を表１に示す高圧蒸気滅菌した培地を用いて培養した。培養条件は、前培養液１０％接種／温度３０℃／振とう機回転数１５０ｒｐｍとし、培養期間は６日間とした。 An ammonia-oxidizing bacterium Nitrosomonas europaea (ATCC 25978) was cultured using the autoclaved medium shown in Table 1. The culture conditions were preculture 10% inoculation / temperature 30 ° C./shaft rotation speed 150 rpm, and the culture period was 6 days.
培養期間を６日経過した後、上記のアンモニア酸化細菌Nitrosomonas europaea (ＡＴＣＣ２５９７８)の培養液を遠心分離して上澄みを除去した。
回収した菌体に１．０％アルギン酸ナトリウム水溶液を適量加えて懸濁し、所定の生菌個数濃度に調整した菌体濃縮液を得た。
そして、図２について説明したようなセルロース膜２２の中心部に、菌体濃縮液を滴下して菌体固定化量を４×１０7個/ｍｍ2となるように調整し、余剰のアルギン酸ナトリウム水溶液を吸引除去した。
さらに、塩化カルシウム水溶液（３０ｇ／Ｌ）を適量滴下して、余剰の塩化カルシウム水溶液を吸引除去し、菌体を固定化した側を内側にして、ドーナツ状の両面テープ２３を介してセルロース膜２１と貼り合わせた。
そして、最後に塩化カルシウム水溶液（３０ｇ／Ｌ）に１５分間浸漬し、アルギン酸ナトリウムを完全にゲル化させて菌体を固定化させた。
After a culturing period of 6 days, the culture solution of the above ammonia oxidizing bacterium Nitrosomonas europaea (ATCC 25978) was centrifuged to remove the supernatant.
An appropriate amount of a 1.0% aqueous sodium alginate solution was added to the collected cells and suspended therein, thereby obtaining a cell concentrate concentrated to a predetermined viable cell count concentration.
Then, a cell concentrate is dropped onto the center of the
Further, an appropriate amount of an aqueous calcium chloride solution (30 g / L) is dropped, and the excess calcium chloride aqueous solution is removed by suction, and the
And finally, it was immersed in calcium chloride aqueous solution (30 g / L) for 15 minutes, sodium alginate was completely gelatinized, and the microbial cell was fixed.
上記のようにして得られたアンモニア酸化細菌固定化膜を、保存液とともにビニールパックの中に封入して５〜１０℃で保存した。 The ammonia-oxidizing bacteria-immobilized membrane obtained as described above was sealed in a vinyl pack together with a storage solution and stored at 5 to 10 ° C.
保存性の良否判定
硝化細菌固定化膜の保存性良否判定では、任意の期間、冷蔵保存後の硝化細菌固定化膜をバイオセンサに装着し、センサ立上り時間（評価開始時点からセンサ出力が０．３５ｍＶ以下になるまでの経過時間）が２４時間以内の場合を「良」とし、２４時間以上の場合を「不良」としていた。
Determination of storable quality Storing quality of the nitrifying bacteria-immobilized membrane is determined by attaching the nitrifying bacteria-immobilized membrane after refrigerated storage to the biosensor for an arbitrary period, and the sensor rise time (the sensor output is 0. The case where the elapsed time until reaching 35 mV or less) was within 24 hours was determined as “good”, and the case where it was 24 hours or longer was determined as “bad”.
しかしながら、センサ保守時間の観点からは、センサ立上り時間による良否判定を４時間以内に行えれば好適である。すなわち、センサ立上り時間の判定基準を、実施例では２４時間から４時間以内、すなわち３時間として判定した。
表２に保存液組成とセンサ立上り時間との関係を示す。
However, from the viewpoint of sensor maintenance time, it is preferable that the quality determination based on the sensor rise time can be performed within 4 hours. That is, the criterion for determining the sensor rise time was determined to be within 24 hours to 4 hours in the example, that is, 3 hours.
Table 2 shows the relationship between the stock solution composition and the sensor rise time.
アンモニア酸化細菌固定化膜の保存液として、リン酸緩衝液を主体とした保存液（表３）と、本発明のホウ酸緩衝液を主体とした保存液（表４）を用いて、バイオセンサ立上り時間を評価した。 A biosensor using a preservation solution mainly composed of a phosphate buffer (Table 3) and a preservation solution mainly composed of a borate buffer of the present invention (Table 4) as a preservation solution for immobilizing ammonia-oxidizing bacteria Rise time was evaluated.
菌体固定化量を４×１０7個/ｍｍ2に調整した硝化細菌固定化膜を冷蔵保存し、２、３、４ヶ月冷蔵保存後に硝化細菌固定化膜をバイオセンサに装着して、センサ立上り時間を評価した。また、ロット間でのデータ再現性を比較検討するため、３ロットについて、上記評価試験を行った。なお、両保存液の基質濃度（６０ｍｇ／Ｌ）及びオキサル酢酸濃度（１ｍＭ）は同一とし、センサ立上り時間の良否判定基準は３時間とした。 Refrigerated storage of nitrifying bacteria-immobilized membrane adjusted to 4 × 10 7 cells / mm 2 , and attached nitrifying bacteria-immobilized membrane to biosensor after refrigerated storage for 2, 3 or 4 months. Rise time was evaluated. In addition, in order to compare and examine the data reproducibility between lots, the above-described evaluation test was performed on three lots. In addition, the substrate concentration (60 mg / L) and the oxalacetic acid concentration (1 mM) of both the storage solutions were the same, and the quality criterion for sensor rise time was 3 hours.
表５に両保存液を用いた場合のセンサ立上り時間比較結果を示す。センサ立上り時間を３時間とした厳しい判定基準下でも、リン酸緩衝液を主体とした保存液を用いた場合の膜保存可能期間は２ヶ月であったのに対し、ホウ酸緩衝液を主体とした保存液を用いた場合のそれは３ヶ月であった。 Table 5 shows the sensor rise time comparison results when both the preservatives are used. Even when the sensor rise time is set to 3 hours, the preservation period of the membrane when the preservation solution mainly composed of phosphate buffer is used is 2 months, whereas the boric acid buffer is mainly used. It was 3 months when the stock solution was used.
本発明の保存液を用いて３ヶ月冷蔵保存し、バイオセンサによる評価試験を行った後の硝化細菌固定化膜の染色状態を調べた。菌体固定化部からの菌体の流出は認められず、固定化剤であるアルギン酸ゲルの破壊が起きなかったことから、保存可能期間が伸長したものと考えられる。 The storage state of the present invention was used for refrigeration for 3 months, and the staining state of the nitrifying bacteria-immobilized membrane after the evaluation test using a biosensor was examined. Since the outflow of the microbial cells from the microbial cell immobilization part was not observed, and the destruction of the alginic acid gel as the immobilizing agent did not occur, it is considered that the storage period was extended.
以上の結果から、本発明の保存液を用いることにより、硝化細菌固定化膜の保存可能期間を、センサ立上り時間を３時間とした厳しい判定基準下でも３ヶ月以上とすることが可能となった。 From the above results, by using the preservation solution of the present invention, the storable period of the nitrifying bacteria-immobilized membrane can be set to 3 months or more even under strict judgment criteria with a sensor rise time of 3 hours. .
１０ バイオセンサ
１１ 溶存酸素電極
１２ フローセル
１２ａ 試料液流路
２０ 硝化細菌固定化膜
２１ セルロース膜
２２ セルロース膜
２３ ドーナツ状両面テープ
２４ 菌体固定化部
DESCRIPTION OF
Claims (10)
The nitrifying bacteria-immobilized membrane preservation solution according to any one of claims 1 to 9, wherein a borate ion concentration is 1.25 mM.
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| JP2016015926A (en) * | 2014-07-09 | 2016-02-01 | 株式会社安藤・間 | Immobilization method for bacteria, immobilization carrier for bacteria, and preservation method for bacteria |
| WO2020088062A1 (en) * | 2018-10-30 | 2020-05-07 | 江南大学 | Method for determining optimal storage temperature aerobic denitrifying bacteria in sewage treatment |
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| JP2003329667A (en) * | 2002-05-15 | 2003-11-19 | Fuji Electric Co Ltd | Preserving solution for nitrifying bacteria immobilized membrane and method of preserving ammonium oxidizing bacteria immobilized membrane |
| JP2004248516A (en) * | 2003-02-18 | 2004-09-09 | Fuji Electric Systems Co Ltd | Method for producing membrane immobilizing ammonia-oxidizing bacterium |
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| WO2020088062A1 (en) * | 2018-10-30 | 2020-05-07 | 江南大学 | Method for determining optimal storage temperature aerobic denitrifying bacteria in sewage treatment |
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