KR100303611B1 - An Electrochemical Method for Enrichment of Microorganism, and a Biosensor for Analyzing Organic Substance and BOD - Google Patents
An Electrochemical Method for Enrichment of Microorganism, and a Biosensor for Analyzing Organic Substance and BOD Download PDFInfo
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- KR100303611B1 KR100303611B1 KR1019990027167A KR19990027167A KR100303611B1 KR 100303611 B1 KR100303611 B1 KR 100303611B1 KR 1019990027167 A KR1019990027167 A KR 1019990027167A KR 19990027167 A KR19990027167 A KR 19990027167A KR 100303611 B1 KR100303611 B1 KR 100303611B1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/18—Water
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/18—Water
- G01N33/186—Water using one or more living organisms, e.g. a fish
- G01N33/1866—Water using one or more living organisms, e.g. a fish using microorganisms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/18—Water
- G01N33/1806—Water biological or chemical oxygen demand (BOD or COD)
Abstract
본 발명에 따라, 혐기적 조건에서 매개체 없는 생물연료 전지를 사용하여 전기화학적인 방법으로 시료 속의 유기물 농도 또는 BOD를 측정할 수 있는 바이오센서가 제공된다. BOD 측정용 바이오센서에 사용되는 생물연료 전지의 전기화학적 활성 세균은 생물연료 전지의 운전 과정에서 농화 배양된 폐수 및 슬러지 속에 포함된 활성 세균이 사용된다. 따라서, 본 발명의 BOD 바이오센서는 인위적인 미생물의 첨가 없이 운전될 수 있으며 폐수에 따라 적절한 세균의 활성이 유지될 수 있다. 바이오센서용 생물 연료 전지는 6개월 이상 안정적으로 작동할 수 있다.According to the present invention, there is provided a biosensor capable of measuring the concentration of organic matter or BOD in a sample by an electrochemical method using a biofuel cell without a medium in anaerobic conditions. As the electrochemically active bacteria of the biofuel cell used in the biosensor for BOD measurement, the active bacteria contained in the concentrated culture wastewater and sludge are used during the operation of the biofuel cell. Therefore, the BOD biosensor of the present invention can be operated without the addition of artificial microorganisms and the proper bacterial activity can be maintained depending on the waste water. Biofuel cells for biosensors can operate stably for more than six months.
Description
본 발명은 유기물 농도 및 BOD 측정용 바이오센서에 관한 것으로, 더욱 구체적으로는 측정이 간단하고 신속하며, 응용성 및 제작, 유지, 보수에 필요한 비용이 저렴한 유기물 농도 및 BOD 측정용 바이오센서에 관한 것이다.The present invention relates to a biosensor for measuring organic substance concentration and BOD, and more particularly to a biosensor for measuring organic substance concentration and BOD which is simple and quick to measure and inexpensive for applicability and manufacturing, maintenance and repair. .
일반적으로, 바이오센서란 측정부의 일부 또는 전부로서 생물체 또는 생물체에서 유래한 물질이 사용되며, 이 측정부가 전기적 장치와 결합된 측정 장치를 의미한다. 바이오센서는 생물 반응이 갖는 고도의 특이성 때문에 측정 물질의 농도 및 성상을 정밀하게 측정할 수 있다는 장점이 있어, 1960년대부터 이에 대한 연구가 지속적으로 이루어져 왔다. 그 결과, 다양한 종류의 바이오센서가 개발되었고,그 측정 물질의 범위도 다양하여졌다. 예를 들면, 글루코스 옥시다제와 산소 전극을 결합하여 제작한 글루코스 농도 측정용 바이오센서 및 항체를 사용한 의학용 바이오센서가 현재 실용화되어 널리 사용되고 있다 [참조. Tuner 등, 1987: Biosensors, Fundamentals and applications, Oxford Science Publications].In general, a biosensor is used as a part or all of a measuring unit, or a living organism or a substance derived from a living organism, and means a measuring device in which the measuring unit is combined with an electrical device. Biosensors have the advantage of being able to precisely measure concentrations and properties of measurement materials because of the high specificity of biological reactions. As a result, various types of biosensors have been developed, and the range of the measurement materials has also been diversified. For example, a glucose biosensor for measuring glucose concentration produced by combining glucose oxidase and an oxygen electrode, and a medical biosensor using an antibody have been put to practical use and are widely used. Tuner et al., 1987: Biosensors, Fundamentals and applications, Oxford Science Publications.
한편, 공장 가동시 발생하는 폐수나 가정에서 발생하는 하수 등의 오염도는 일반적으로 화학적 산소요구량 (Chemical Oxygen Demand, COD) 또는 생화학적 산소요구량 (Biochemical Oxygen Demand, BOD)으로 환산하여 표시되며 이들의 신속한 측정은 환경 및 공해방지 관련 산업에 있어서 대단히 중요한 의미를 갖는다. 그러나, 폐수 중에 존재하는 미생물 자화성 유기물질의 양을 의미하는 BOD를 측정하는 종래의 방법은 시간이 오래걸리고, 여러 가지 복잡한 과정과 장치가 필요하다는 문제점이 있다. 또한, 작업자의 숙련도에 따라 그 값의 편차가 발생할 뿐만 아니라 수질의 오염 상태를 긴급히 확인하고자 할 경우나 또는 폐수처리 공정의 자동화 시설의 설치시 적용하기 어려운 문제점이 있다.On the other hand, pollution levels such as waste water generated from plant operation and domestic sewage are generally expressed in terms of chemical oxygen demand (COD) or biochemical oxygen demand (BOD), and their rapid Measurement is of great importance for the industry concerned with environmental and pollution prevention. However, the conventional method for measuring the BOD, which means the amount of microorganism magnetizable organic substances present in the waste water, takes a long time and has a problem of requiring various complicated processes and devices. In addition, there is a problem that it is difficult to apply not only the deviation of the value according to the skill of the operator, but also to urgently check the pollution state of the water or when installing the automated facility of the wastewater treatment process.
이러한 문제점을 해결하기 위하여 몇 종의 BOD 측정용 바이오센서가 개발되었다 [참조: Hikuma 등, 1979: European Journal of Microbiology and Biotechnology, 8, 289; Riedel 등 1990: Water Research, 24, 883; Hyun 등, 1993: Biotechnology and Bioengineering, 41, 1107]. 상기 언급된 BOD 센서는 일반적으로 용존산소 측정용 전극에 특정 미생물을 고정화시킨 막을 부착한 형태로서, 이때 BOD 센서를 측정하고자 하는 시료와 반응시키면 막에 고정화된 미생물이 시료내의 유기물을 자화하면서 산소를 소비하게 되고, 이때의 용존산소 값을 대조구의 용존산소 값과 비교하여 이를 BOD로 환산함으로써 측정한다. 그러나, 이와같은 BOD 측정용 바이오센서는 아래와 같은 문제점이 있다.Several kinds of biosensors for measuring BOD have been developed to solve this problem. Hikuma et al., 1979: European Journal of Microbiology and Biotechnology, 8, 289; Riedel et al. 1990: Water Research, 24, 883; Hyun et al., 1993: Biotechnology and Bioengineering, 41, 1107. The above-mentioned BOD sensor is generally in the form of a membrane in which a specific microorganism is immobilized to the electrode for measuring dissolved oxygen. When the BOD sensor is reacted with the sample to be measured, the microorganism immobilized on the membrane magnetizes the oxygen while magnetizing the organic matter in the sample. The dissolved oxygen value at this time is measured by comparing it with the dissolved oxygen value of the control and converting it into BOD. However, such a BOD measurement biosensor has the following problems.
첫째, 1종의 미생물을 사용하기 때문에 사용된 미생물의 기질 특이성으로 인하여 폐수 속의 복합 영양 성분에 대한 자화성이 부족하기 때문에 BOD의 전체 값을 나타낼 수 없다.First, because one microorganism is used, the total value of BOD cannot be represented because of the lack of magnetization of the complex nutrients in the waste water due to the substrate specificity of the microorganisms used.
둘째, 다공성 막에 미생물을 고정시켰기 때문에 재현성이 높은 BOD 측정치를 얻기 위해서는 수시로 막을 교환, 수리해야 하는데, 사용되는 미생물 고정막의 가격이 높으므로, 비경제적이고 정비성도 불량하다.Second, since the microorganisms are immobilized on the porous membranes, the membranes need to be replaced and repaired from time to time in order to obtain high reproducible BOD measurements. Since the cost of the microbial fixation membranes used is high, it is uneconomical and poor in maintenance.
셋째, 대조구용 용존산소 전극 또는 대조시료를 사용하여야 하기 때문에 장치가 복잡하고, 장비의 가격 및 고장률이 높다.Third, because the dissolved oxygen electrode or control sample for the control must be used, the apparatus is complicated, and the cost and failure rate of the equipment are high.
넷째, 종래 BOD 측정용 바이오센서에 사용되는 미생물은 미생물의 외부에서 전기화학적 활성을 직접 측정할 수 없으므로 전자전달 매개체 (electrochemical mediator)나 별도의 변환기 (transducer)를 부착하여야 한다.Fourth, the microorganisms used in the conventional BOD measurement biosensors cannot directly measure the electrochemical activity from the outside of the microorganisms, so an electrochemical mediator or a separate transducer must be attached.
한편, 일반적으로 혐기적 환경에서 생장하는 미생물은 산소이외에 다른 전자 수용체를 사용할 수 있으며, 이러한 전자수용체를 사용하여 대사하는 과정을 미생물의 혐기적 호흡이라고 일컫는다. 혐기적으로 호흡하는 미생물이 유기물을 산화할 때 이용할 수 있는 전자 수용체로는 산화제이철, 질산염, 6가 망간, 황산염, 탄산염 등이 있다. 전자 공여체가 동일한 경우, 미생물 전자전달계내의 산화환원 반응에서 발생하는 에너지는 산화제이철이 산화제일철로 환원될 때가 가장 크고, 질산염, 황산염, 탄산염의 차례로 낮아지게 되는데 이는 각각의 전자 수용체가 갖는고유 특성인 산화환원 전위와 관계가 있다 [참조: 김병홍, 1995: 미생물 생리학, 아카데미서적].On the other hand, microorganisms that are generally grown in anaerobic environments may use other electron acceptors in addition to oxygen, and the process of metabolizing using these electron acceptors is called anaerobic respiration of microorganisms. Examples of electron acceptors that can be used by anaerobic microorganisms to oxidize organic materials include ferric oxide, nitrates, hexavalent manganese, sulfates, and carbonates. When the electron donors are the same, the energy generated from the redox reaction in the microbial electron transport system is most likely to be reduced when ferric oxide is reduced to ferric oxide, which in turn is lowered in the order of nitrates, sulfates, and carbonates. It is related to redox potential [Byeonghong Kim, 1995: Microbial Physiology, Academy Books].
이러한 혐기적 호흡을 수행하는 미생물인 금속염 환원세균이 이용하는 전자 수용체 중 산화 제이철 등은 물에 대한 용해도가 극히 낮기 때문에 호기성 미생물의 일반적인 전자 수용체인 산소와는 달리 불용성 전자수용체를 세포 내부로 흡수하여 환원할 수 없다. 따라서, 금속염 환원 세균의 경우 외부에 존재하는 전자 수용체를 환원하기 위해서 특수한 형태의 전자전달 시스템이 존재한다. 예를 들면, 산화제이철을 전자 수용체로 이용하는 금속염 환원 세균의 일종인지오박터 설퍼리듀센스(Geobacter sulfurreducens)와시와넬라 푸트레파시엔스(Shewanella putrefaciens)의 경우 전자전달 단백질인 사이토크롬이 존재하며, 이 사이토크롬을 통하여 미생물 내부에서 산화된 유기물에서 발생한 전자가 외부의 전자 수용체로 전달되고, 이러한 일련의 전자전달 과정을 통하여 발생한 에너지를 이용하여 생장하게 된다 [참조: Myers and Myers, 1992, Journal of Bacteriology, 174, 3429-3438; Seeliger 등 1998, Journal of Bacteriology, 180: 3686-3691]. 따라서, 유사한 성질을 갖는 이들 금속염 환원세균은 유기물의 대사시 발생하는 전자를 외부의 불용성 전자 수용체에 전달하여 전자 수용체를 환원시키므로 유기물의 양은 외부의 전자 수용체의 환원양과 비례하게 되고, 이 전자 수용체를 대체할 수 있는 적당한 전극을 이용하면 미생물 내부에서 발생하는 전자가 전극을 환원시키고 전극에 직접 전달된 전자는 회로를 통하여 외부로 흐를 수 있게 된다. 이러한 미생물의 생리학적 특성을 이용하는 생물 연료전지 등이 대한민국 특허 공개공보 제1998-16777호 (1998년 6월 5일)에 개시되어 있으며, 본원에 참고로 도입된다.Since ferric oxide, among the electron acceptors used by the metal salt reducing bacterium, a microorganism performing anaerobic respiration, has very low solubility in water, unlike oxygen, which is a general electron acceptor of aerobic microorganisms, insoluble electron receptors are absorbed into cells and reduced. Can not. Therefore, in the case of metal salt reducing bacteria, a special type of electron transfer system exists in order to reduce externally present electron acceptors. For example, the electron transfer protein, cytochrome, if any of the ferric oxide in the metal salt form of reducing bacteria used as an electron acceptor Geo bakteo sulfonic Fury dew sense (Geobacter sulfurreducens) and when the Nella Fu tray Pacific Enschede (Shewanella putrefaciens), and Through this cytochrome, electrons generated from oxidized organic matter inside microorganisms are transferred to an external electron acceptor and grown using energy generated through a series of electron transfer processes. [Myers and Myers, 1992, Journal of Bacteriology, 174, 3429-3438; Seeliger et al. 1998, Journal of Bacteriology, 180: 3686-3691]. Therefore, these metal salt reducing bacteria having similar properties reduce electron acceptors by transferring electrons generated during metabolism of organic matter to an externally insoluble electron acceptor, so that the amount of organic matter is proportional to the reduction amount of the external electron acceptor. By using a suitable electrode that can be replaced, electrons generated inside the microorganism reduce the electrode and electrons transferred directly to the electrode can flow through the circuit to the outside. Biofuel cells using the physiological characteristics of such microorganisms are disclosed in Korean Patent Laid-Open Publication No. 1998-16777 (June 5, 1998), which is incorporated herein by reference.
그런데, 상기 금속염 환원 세균을 이용한 생물 연료 전지에 있어서, 전자의 양은 미생물의 농도, 유기물의 양 등에 비례하게 되므로, 발생된 전자의 양을 측정함으로써 시료 중에 존재하는 유기물의 양을 측정할 수 있게 된다.However, in the biofuel cell using the metal salt reducing bacterium, since the amount of electrons is proportional to the concentration of the microorganism, the amount of the organic matter, etc., the amount of the organic matter present in the sample can be measured by measuring the amount of the generated electrons. .
따라서, 본 발명자들은 이러한 생물연료 전지 및 사용된 미생물과 유기물에 대한 연구를 거듭한 결과, 본 발명을 완성하기에 이르렀다.Therefore, the present inventors have conducted studies on such biofuel cells and used microorganisms and organic materials, and have completed the present invention.
본 발명의 목적은 상기한 종래의 BOD 측정용 바이오센서의 문제점을 갖고 있지 않는 개선된 BOD 측정용 바이오센서 및 그를 사용한 BOD 측정 방법을 제공하는 것이다.It is an object of the present invention to provide an improved biosensor for measuring BOD and a method for measuring BOD using the same, which does not have the problems of the conventional biosensor for measuring BOD described above.
상기 본 발명의 목적에 따라, 측정부, 전류검출부 및 검출된 전류변화를 기록하는 기록부를 포함하며, 상기 측정부는 양극 및 음극, 이들 양극 및 음극의 전도매체 및 이들 두 극 사이의 이온 교환막을 포함하는 무매개체 생물 연료 전지로 이루어지고, 상기 음극부에는 전기화학적 활성 세균을 함유하는 시료가 첨가된 것을 특징으로 하는 BOD 측정용 바이오센서가 제공된다.According to the object of the present invention, it comprises a measuring unit, a current detecting unit and a recording unit for recording the detected current change, the measuring unit includes an anode and a cathode, a conductive medium of these anode and cathode and an ion exchange membrane between these two poles A non-mediated biofuel cell is provided, and the cathode portion is provided with a biosensor for measuring BOD, wherein a sample containing electrochemically active bacteria is added.
또한, 상기 BOD 측정용 바이오센서에서, 음극과 양극을 저항을 거쳐 연결한 후, 음극부에는 질소를 공급하여 혐기성 조건으로, 양극부에는 공기를 공급하여 호기성 조건으로 만들어, 시료 중에 존재하는 전기화학적 활성 세균을 상기 음극부에 농화 배양시키고, 상기 농화 배양된 전기화학적 활성 세균을 미생물 촉매로 이용함으로써 발생된 전류를 측정하는 것을 특징으로 하는 시료 중의 BOD 측정 방법이 제공된다.In addition, in the biosensor for measuring BOD, after connecting the negative electrode and the positive electrode through the resistance, and supplied to the anaerobic condition by supplying nitrogen to the negative electrode portion, to the aerobic condition by supplying air to the positive electrode portion, the electrochemical present in the sample There is provided a method for measuring BOD in a sample, wherein the active bacteria are concentrated and cultured on the negative electrode and the current generated by using the concentrated and cultured electrochemically active bacteria as a microbial catalyst is provided.
또한, 본 발명의 또 다른 관점에서, 측정부, 전류 검출부 및 검출된 전류 변화를 기록하는 기록부를 포함하며, 상기 측정부는 양극 및 음극, 이들 양극 및 음극의 전도매체 및 이들 두 극 사이의 이온 교환막을 포함하는 무매개체 생물 연료 전지로 이루어지고, 상기 음극부에 소정의 유기물을 대사하는 단일종의 전기화학적 활성 세균이 포함되어 있는 것을 특징으로 하는 무매개체 생물 연료 전지형 유기물 농도 측정용 바이오센서가 제공된다.Also in another aspect of the present invention, there is provided a measuring unit, a current detecting unit and a recording unit for recording the detected current change, wherein the measuring unit is an anode and a cathode, a conducting medium of these anodes and a cathode, and ion exchange between these two poles. Provided is a medium-free biofuel cell comprising a membrane, and wherein the negative electrode contains a single species of electrochemically active bacteria that metabolize a predetermined organic substance, the biosensor for a medium-free biofuel cell type organic substance concentration measurement is provided. do.
또한, 상기 유기물 농도 측정용 바이오센서에서, 양극부에는 공기를 계속적으로 공급하여 음극부와 전압차를 유지시키면서, 측정하고자 하는 시료를 음극부에 첨가하여 음극부에 포함된 세균이 유기물을 소비하면서 생산되는 전자의 흐름을 측정함으로써 해당 유기물의 농도를 측정하는 것을 특징으로 하는 유기물 농도 측정 방법이 제공된다.In addition, in the biosensor for measuring the organic concentration, while continuously supplying air to the anode portion to maintain a voltage difference with the cathode portion, while adding the sample to be measured to the cathode portion while the bacteria contained in the cathode portion consumes the organic material An organic substance concentration measuring method is provided, wherein the concentration of the organic substance is measured by measuring the flow of electrons to be produced.
본 발명의 또 다른 관점에서, 양극 및 음극, 이들 양극 및 음극의 전도매체 및 이들 두 극 사이의 이온 교환막으로 이루어지는 무매개체 생물 연료 전지에서, 음극부에는 활성 슬러지와 폐수를 첨가하고, 음극과 양극을 저항을 거쳐 연결한 후, 음극부에는 질소를 공급하여 혐기성 조건으로, 양극부에는 공기를 공급하여 호기성 조건으로 만들어 별도의 전자수용체없이 활성 슬러지와 폐수 중에 존재하는 전기화학적 활성 세균을 농화 배양하는 방법이 제공된다.In another aspect of the invention, in a medium-free biofuel cell consisting of a positive electrode and a negative electrode, a conductive medium of these positive and negative electrodes, and an ion exchange membrane between these two poles, active sludge and wastewater are added to the negative electrode, and the negative electrode and the positive electrode After connecting through the resistance, supplying nitrogen to the cathode part in anaerobic condition, supplying air to the anode part to make aerobic conditions to enrich and culture the electrochemically active bacteria present in the activated sludge and waste water without a separate electron acceptor A method is provided.
도 1은 생물 연료전지 형태의 특정 미생물 농화 장치를 이용한 BOD 측정용 바이오센서의 모식도.1 is a schematic diagram of a biosensor for BOD measurement using a specific microbial enrichment device in the form of a biofuel cell.
도 2는 본 발명에 따른 생물연료전지형 바이오센서에 첨가된 시료의 COD와 전류의 관계를 나타내는 그래프.Figure 2 is a graph showing the relationship between the current and the COD of the sample added to the biofuel cell-type biosensor according to the present invention.
도 3은 생물연료전지의 바이오센서에 첨가된 시료의 COD 및 발생한 적산전류량과의 상관 관계를 나타내는 그래프.3 is a graph showing the correlation between the COD and the amount of accumulated current generated in a sample added to a biosensor of a biofuel cell.
도 4은 정전위 전해장치 (potentiostat)을 이용한 특정 미생물의 농화 배양용 장치와 이를 이용한 BOD 측정용 바이오센서의 모식도.Figure 4 is a schematic diagram of a device for culturing specific microorganisms using a potentiostat and a BOD measurement biosensor using the same.
도 5는 실시예 2에 따른 정전위 전해장치를 사용하여 전기화학적 활성 세균이 농화 배양된 생물연료전지형 바이오센서에 첨가된 시료의 COD와 전류의 관계를 나타내는 그래프.Figure 5 is a graph showing the relationship between the current and the COD of the sample added to the biofuel cell-type biosensors electrochemically active bacteria concentrated using the electrostatic potential electrolytic apparatus according to Example 2.
도 6은 본 발명에 따른 생물연료전지형 바이오센서의 작업 전극표면에 농화된 미생물의 주사전자현미경 사진.Figure 6 is a scanning electron micrograph of the microorganisms concentrated on the working electrode surface of the biofuel cell-type biosensor according to the present invention.
도 7은 본 발명에 따른 연료 전지형 젖산 농도 측정 바이오센서의 모식도.7 is a schematic diagram of a fuel cell-type lactic acid concentration measurement biosensor according to the present invention.
도 8은 젖산 농도 측정시 발생하는 전류 기록의 일반적인 형태.8 is a general form of current recording that occurs when measuring lactic acid concentration.
도 9는 젖산 농도 및 전류 발생시 초기 기울기간의 상관 관계를 나타내는 그래프.Figure 9 is a graph showing the correlation between the lactic acid concentration and the initial period of inclination during current generation.
도 10은 본 발명에 따른 BOD 측정용 바이오센서를 사용한 경우 6개월 간의 첨가된 COD 농도에 따른 전류량을 나타내는 그래프.10 is a graph showing the amount of current according to the added COD concentration for 6 months when using a biosensor for measuring BOD according to the present invention.
<도면의 주요 부분에 대한 부호의 설명><Explanation of symbols for the main parts of the drawings>
1, 201: 음극1, 201: cathode
2, 202: 양극2, 202: anode
3, 203: 양이온 교환막3, 203: cation exchange membrane
4, 204: 음극부4, 204: cathode portion
5, 205: 양극부5, 205: anode portion
6: 누수 방지용 실리콘 고무막6: Silicone rubber membrane for preventing water leakage
7, 8: 음극 및 양극 배선 연결부7, 8: cathode and anode wiring connections
9: 시료 및 질소 투입구9: sample and nitrogen inlet
10: 시료 및 질소 방출구10: sample and nitrogen outlet
11: 공기 및 물 또는 인산 완충용액 투입구11: Air and water or phosphate buffer inlet
12: 공기 및 물 또는 인산 완충용액 방출구12: air and water or phosphate buffer outlet
13: 보호대13: guard
14: 고정용 나사14: fixing screw
101: 작업전극101: working electrode
102: 보조전극102: auxiliary electrode
104: 작업전극부104: working electrode
105: 보조전극부105: auxiliary electrode unit
109, 209: 시료 투입 및 채취구109, 209: sample input and collection port
110, 210: 질소 배출구110, 210: nitrogen outlet
111, 211: 질소 투입구111, 211: nitrogen inlet
112: 체크 밸브112: check valve
113: 은/염화은 기준전극113: silver / silver chloride reference electrode
114, 214: 자석 교반기114, 214: magnetic stirrer
본 발명은 전기화학적으로 활성을 갖는 미생물을 사용하여 전자전달 매개체나 변환기 (transducer) 없이 미생물 자체의 유기물 자화력 및 전자전달 능력을 이용하여 폐수속의 미생물 자화성 성분 (BOD)이나 젖산 등의 유기물의 농도를 측정할 수 있는 바이오센서에 관한 것이다.The present invention utilizes microorganisms that are electrochemically active and utilizes microorganism magnetization and electron transfer ability of microorganisms without electron transfer mediators or transducers, and organic matters such as microorganism magnetization components (BOD) and lactic acid in wastewater. It relates to a biosensor capable of measuring the concentration.
본 발명의 일면에서, BOD 측정용 바이오센서는 측정부, 전류 검출부 및 검출된 전류변화를 기록하는 기록부를 포함하며, 상기 측정부는 양극 및 음극, 이들 양극 및 음극의 전도매체 및 이들 두 극 사이의 이온 교환막을 포함하는 무매개체 생물 연료 전지를 이루어지고, 상기 음극부에는 전기화학적 활성 세균을 함유하는 시료가 첨가된다.In one aspect of the present invention, the BOD measurement biosensor includes a measuring unit, a current detecting unit and a recording unit for recording the detected current change, wherein the measuring unit includes a positive electrode and a negative electrode, a conductive medium of these positive and negative electrodes, and a gap between these two poles. An intermediate-free biofuel cell comprising an ion exchange membrane is formed, and a sample containing electrochemically active bacteria is added to the cathode portion.
즉, 특정 시료 내의 유기물과 활성 슬러지를 종균 시료로 하여 전기화학적으로 활성이 있는 전기화학적 활성 세균을 전기화학적인 방법으로 전극 및 전극을 포함하는 전극부에 농화 배양하고, 상기 농화 배양된 전기화학적 활성 세균을 미생물 촉매로 이용함으로써 전력을 생산시키면, 생산된 전력은 측정부로 사용된 생물 연료전지에 첨가된 미생물 자화성 성분인 각종 유기물 농도에 비례하게 됨으로써, 이를 검출 기록함으로써 시료 중의 BOD를 측정할 수 있게 된다.That is, the organic material and the activated sludge in a specific sample as a seed sample is concentrated and cultured electrochemically active electrochemically active bacteria in the electrode unit including the electrode and the electrode by the electrochemical method, the concentrated chemical electrochemical activity When electric power is produced by using bacteria as a microbial catalyst, the generated electric power is proportional to the concentration of various organic substances, which are microorganism magnetizable components added to the biofuel cell used as the measuring unit, thereby detecting and recording the BOD in the sample. Will be.
또한, 바람직하게는 상기 측정부의 음극부에 전기화학적 활성 세균의 농화배양을 촉진하기 위하여, 정전위 전해장치를 사용할 수 있다.In addition, preferably, in order to promote the enrichment culture of electrochemically active bacteria in the cathode portion of the measuring unit, it is possible to use a potential electrostatic device.
본 발명의 목적상 전기화학적 활성 세균은 폐수내의 유기물을 산화하여 발생하는 전자를 세포 외부로 방출하여 직접적으로 전극에 전달하여 전류를 발생시킬 수 있는 세균을 말하며, 대표적인 것으로는 금속염 환원세균을 들 수 있다.Electrochemically active bacteria for the purposes of the present invention refers to a bacterium capable of generating an electric current by releasing electrons generated by oxidizing organic matter in the wastewater to the outside of the cell and directly transferring them to electrodes, and representative examples thereof include metal salt reducing bacteria. have.
본 발명의 또 다른 면에서, 유기물 농도 측정용 바이오센서는 상기 생물 연료전지의 전극 자체 또는 전극부에 특정 유기 물질을 기질로하는 특정한 전기화학적으로 활성인 세균을 포함시키고, 이를 그대로 바이오센서의 측정부로 이용한다. 즉, 특정한 유기물을 대사하는 전기화학적 활성 세균을 음극부에 포함시킴으로써, 생물 연료전지에 의하여 발생한 전력은 시료중에 존재하는 특정 유기물의 대사에 의한 것이되고, 이를 측정함으로써 시료중의 유기물 농도를 측정할 수 있게 된다.In another aspect of the present invention, the biosensor for measuring the organic concentration includes a specific electrochemically active bacteria having a specific organic material as a substrate of the electrode itself or the electrode portion of the biofuel cell, and the measurement of the biosensor as it is We use for wealth. That is, by including the electrochemically active bacteria that metabolize a specific organic matter in the negative electrode portion, the power generated by the biofuel cell is due to the metabolism of the specific organic matter present in the sample, by measuring the concentration of organic matter in the sample It becomes possible.
하기에서는 상기 설명한 바와 같은 BOD 및 유기물 농도를 측정하는 방법에 대해서 구체적으로 설명한다.Hereinafter, a method of measuring the BOD and the organic concentration as described above will be described in detail.
1) 전기화학적 활성 세균의 농화 배양 및 이를 이용한 BOD 측정용 바이오센서1) Concentration culture of electrochemically active bacteria and BOD measurement biosensor using the same
폐수에서 발생하는 활성 슬러지 및 혐기성 슬러지에는 다량의 철환원 세균을 포함한 여러 종류의 금속염 환원세균이 높은 농도로 존재하는 것으로 최근의 연구에서 확인되었다 [참조: Nielsen 등, 1997, Systematic and Applied Microbiology, 20, 645-651; Nielsen 등, 1996, Water Science and Technology, 34, 129-136; Rasmussens 등, 1994, Water Research, 28, 417-425].It has been confirmed in a recent study that active sludge and anaerobic sludge from wastewater are present in high concentrations of various metal salt reducing bacteria, including a large amount of iron-reducing bacteria. Nielsen et al., 1997, Systematic and Applied Microbiology, 20 645-651; Nielsen et al., 1996, Water Science and Technology, 34, 129-136; Rasmussens et al., 1994, Water Research, 28, 417-425].
따라서, 활성 슬러지나 폐수 등 여러 종의 미생물이 혼합되어있는 시료를 종균으로 삼아 전극이 포함된 배양조에서 적당한 배지와 함께 혐기적으로 배양하면 전극을 전자수용체로 사용할 수 있는 미생물만이 최종적으로 생존할 수 있게 되고, 이들 미생물 종은 사이토크롬과 같은 전자전달체를 가지고 있어 전기화학적 활성을 갖는다. 따라서, 이와 같은 방법으로 폐수나 활성 슬러지 등에 존재하는 여러종의 미생물 중 전기화학적 활성을 갖는 균을 선택적으로 농화 배양할 수 있다.Therefore, if a sample containing several kinds of microorganisms such as activated sludge or wastewater is used as a spawn, and anaerobicly cultured with an appropriate medium in a culture tank containing electrodes, only the microorganisms capable of using the electrode as an electron acceptor can finally survive. These microbial species have electron transporters such as cytochromes and thus have electrochemical activity. Therefore, in this way, it is possible to selectively concentrate culture of bacteria having electrochemical activity among various microorganisms present in waste water, activated sludge, and the like.
한편, 폐수나 오염된 하수에는 다양한 유형의 유기물이 포함되어 있으므로,이들 폐수나 하수의 BOD를 일률적인 방법으로, 즉 한 종류의 미생물만으로 측정하는 것은 대단히 힘들며 측정시 오차 또한 크다. 따라서, 서로 다른 유기 폐수 및 활성 슬러지 중의 다양한 전기화학적 활성 세균을 상기 설명한 바처럼 농화 배양하고, 농화배양된 이들 활성 세균들을 측정부의 생물 연료전지의 미생물 촉매로 이용함으로써, 생산된 전력량을 통하여 시료중의 BOD를 측정할 수 있다.On the other hand, since wastewater or polluted sewage contains various types of organic matter, it is very difficult to measure the BOD of these wastewater or sewage in a uniform manner, that is, only one type of microorganism, and the measurement error is also large. Thus, various electrochemically active bacteria in different organic wastewater and activated sludge are enriched and cultured as described above, and the concentrated and cultured active bacteria are used as microbial catalysts in the biofuel cell of the measuring unit, and thus the amount of electricity produced in the sample is increased. The BOD of can be measured.
2) 생물연료전지형 바이오센서를 사용한 유기물 농도 측정2) Organic matter concentration measurement using biofuel cell type biosensor
상기 BOD 측정용 바이오센서에서 설명한 바와 같은 생물연료 전지에서 음극부에는 측정하고자 하는 기질에 따라 적합한 전기화학적으로 활성인 단일종의 미생물을 포함시킨다. 양극부에는 공기를 계속적으로 공급하여 음극부와 전압차를 유지시키고, 측정하고자 하는 시료를 음극부에 첨가하면 음극부에 포함된 미생물이 해당 기질을 소비하면서 생산되는 전자가 직접 음극을 통하여 외부의 회로로 흘러나가게 되고, 이를 측정함으로써 해당 기질의 농도를 측정할 수 있다. 따라서, 이와 같은 원리로 다양한 기질을 소비하는 전기화학적으로 활성이 있는 세균을 사용함으로써 다양한 기질, 즉 상응하는 해당 유기물의 농도를 측정할 수 있다.In the biofuel cell as described in the biosensor for measuring BOD, the negative electrode part includes a single microorganism suitable for electrochemically active according to the substrate to be measured. Air is continuously supplied to the anode to maintain a voltage difference with the cathode, and when the sample to be measured is added to the cathode, electrons produced while the microorganisms contained in the cathode consume the substrate are directly supplied to the outside through the cathode. As it flows out of the circuit, the concentration of the substrate can be determined by measuring it. Thus, by using electrochemically active bacteria consuming a variety of substrates on this principle, it is possible to measure the concentration of various substrates, ie corresponding organic matter.
이제, 첨부된 도면을 참조로 하여, 하기에서 본 발명이 더욱 자세하게 설명된다.Now, with reference to the accompanying drawings, the present invention is described in more detail below.
도 1은 생물 연료전지 형태의 농화 배양 장치를 이용한 BOD 센서의 모식도이다. 양극부 (5)에 산소를 공급하여 양극 (2)와 음극 (1)에 전위차를 부여하였으며, 음극부 (4)에 시료 (예를 들면, 폐수 및 슬러지)를 첨가하고, 양극부에 인산 완충용액 또는 수도물을 첨가한다. 음극부에는 혐기적 상태를 유지하기 위하여 질소를, 양극부에는 공기를 공급하여 단위 전지의 전압차를 유지할 수 있다. 일정 시간이 지난 후 (일반적으로 3주) 음극에는 특정 폐수를 기질로 하여 농화된 전기화학적 활성을 갖는 미생물이 부착되며, 여기서 부착된 미생물이 기질을 산화하여 발생하는 전기를 적절히 측정하면 폐수속의 BOD 증감을 측정할 수 있다. 본 발명에서는 상기 양극과 음극은 동일하게 탄소 부직포로 구성하였으나, 상황에 따라 여러가지 다른 재료의 전극을 사용할 수 있다.1 is a schematic diagram of a BOD sensor using a thickening culture apparatus in the form of a biofuel cell. Oxygen was supplied to the anode portion 5 to impart a potential difference between the anode 2 and the cathode 1, and a sample (e.g., wastewater and sludge) was added to the cathode portion 4, and phosphate buffered at the anode portion. Add solution or tap water. Nitrogen is supplied to the cathode to maintain the anaerobic state, and air is supplied to the anode to maintain the voltage difference between the unit cells. After a certain period of time (usually three weeks), the cathode is attached with a microorganism having a concentrated electrochemical activity with a specific wastewater as a substrate, and when the attached microorganisms properly measure the electricity generated by oxidation of the substrate, The increase and decrease can be measured. In the present invention, the anode and the cathode are composed of the same carbon nonwoven fabric, but it is possible to use electrodes of various different materials depending on the situation.
도 4은 본 발명의 바람직한 구현예인 전기화학적 농화 배양법을 이용한 BOD 센서의 구성도이다. 이 경우 전극의 전압을 일정하게 유지하기 위하여 정전위 전해장치 (potentiostat)를 사용한다. 여기서, 작업전극 (101)은 전자 수용체로서 작용하며 전극에 대한 적용 전압을 변화시킴에 따라 미생물에 대한 전기화학적 작용이 다를 수 있다. 작업전극의 재질은 탄소 부직포이며, 기준 전극 (113)으로는 은/염화은 (Ag/AgCl)이, 보조전극 (102)으로는 백금이 사용된다. 기준전극은 작업전극의 작용전압을 유지, 보정해주는 역할을 하며 보조전극은 작업전극과 전기적 회로를 구성한다. 이 장치는 작업전극에 일정한 전위를 가하고 (염화은 기준전극에 대하여 일반적으로 +0.98 V) 시료 (폐수 및 슬러지)를 공급하여 특정 폐수속의 전기 화학적 활성세균을 일정기간 (일반적으로 2주) 농화 배양하고, 이에 따라 전극에 부착(농화)된 미생물 및 장치를 그대로 BOD 측정용 바이오센서로 사용할 수 있다.Figure 4 is a block diagram of a BOD sensor using an electrochemical thickening culture method which is a preferred embodiment of the present invention. In this case, a potentiostat is used to keep the voltage of the electrode constant. Here, the working electrode 101 acts as an electron acceptor and the electrochemical action on the microorganism may vary according to the change in the applied voltage to the electrode. The working electrode is made of carbon nonwoven fabric, and silver / silver chloride (Ag / AgCl) is used as the reference electrode 113 and platinum is used as the auxiliary electrode 102. The reference electrode serves to maintain and correct the working voltage of the working electrode, and the auxiliary electrode forms an electrical circuit with the working electrode. The device applies a constant potential to the working electrode (typically +0.98 V for the silver chloride reference electrode) and supplies a sample (wastewater and sludge) to enrich and incubate the electrochemically active bacteria in a specific wastewater for a period of time (typically two weeks). Therefore, the microorganism and the device attached (enriched) to the electrode can be used as a biosensor for BOD measurement as it is.
본 발명의 이점 및 목적은 하기 설명되는 실시예를 통하여 더욱 잘 이해될 수 있지만, 이에 제한되는 것은 아니다.Advantages and objects of the present invention can be better understood through the examples described below, but are not limited thereto.
<실시예 1><Example 1>
생물연료전지를 이용한 전기화학 활성을 갖는 미생물 농화와 COD 농도에 따른 생물 연료전지의 전류 변화Current Changes in Biofuel Cells with Electrochemical Activity and COD Concentration with Electrochemical Activity Using Biofuel Cells
특정 폐수중의 유기물을 전자 공여체로 이용하는 전기 화학적으로 활성이 있는 미생물을 농화 배양하기 위하여 도 1에 표시된 것과 같은 장치를 제작하였다.In order to enrich and culture electrochemically active microorganisms using organic matter in a specific wastewater as an electron donor, an apparatus as shown in FIG. 1 was manufactured.
본 실험에서는 전분가공 폐수 (출처: 삼양 제넥스, 대한민국 인천소재)를 이용하였으며, 동일 공장의 폐수처리에서 발생한 활성 슬러지를 접종원으로 사용하였다. 생물 연료전지의 기본 형태는 베네토 등의 문헌 [참조: Bennetto 등, 1985, Biotechnology Letters, 7, 699-704]을 참조하였다. 전극은 양극 (2), 음극 (1) 모두 탄소 부직포 (크기: 5 x 7.5 × 0.6 cm)로 하였으며, 전극의 배선은 백금선을 사용하였다 [본 발명에서는 미생물(또는 미생물의 전자전달물질)이 전극에 의하여 산화되는 장소를 음극부 (4)라 하고, 외부회로를 통하여 이동된 전자가 다시 전극에서 산화제를 환원시키는 부분을 양극부 (5)라 한다]. 음극부와 양극부는 이온 교환막 (3)으로 분리되며 음극 (1)과 양극 (2)은 외부의 회로를 통하여 연결된다. 이때, 외부의 회로에 적당한 저항을 연결하면 음극과 양극 사이의 전류 흐름을 제어할 수 있다. 양극부 (작업 용량: 30 ml)에는 공기를 공급하고 음극부 (작업 용량: 30 ml)에는 폐수와 슬러지 등의 시료를 첨가하였다. 음극부에 폐수 및 슬러지를 첨가한 후 음극과 양극을 저항을 거쳐 연결한 후 음극부에 질소를 공급하여 혐기적 조건으로, 양극부에 공기를 공급하여 호기적 조건으로 만들어 농화 배양을 시작하였다. 약 3주간의 농화배양을 거친 후 배경 전류가 일정하게 유지되었을 때일정한 농도의 BOD를 가지고 있는 폐수를 첨가하여 발생되는 전류의 총량을 적산하였다.In this experiment, starch processing wastewater (source: Samyang Genex, Incheon, Korea) was used, and activated sludge from wastewater treatment in the same plant was used as inoculum. For basic forms of biofuel cells, see Bennet et al., Bennetto et al., 1985, Biotechnology Letters, 7, 699-704. The anode (2) and the cathode (1) were both made of carbon nonwoven fabric (size: 5 x 7.5 x 0.6 cm), and the wiring of the electrode was made of platinum wire. [In the present invention, the microorganism (or electron transfer material of the microorganism) is the electrode. The portion oxidized by the cathode portion 4 is referred to as the cathode portion 4, and the portion where electrons moved through the external circuit reduce the oxidant in the electrode is referred to as the anode portion 5]. The cathode part and the anode part are separated by the ion exchange membrane 3, and the cathode 1 and the anode 2 are connected through an external circuit. At this time, by connecting a suitable resistor to the external circuit it is possible to control the current flow between the cathode and the anode. Air was supplied to the positive electrode (working capacity: 30 ml), and samples such as wastewater and sludge were added to the negative electrode (working capacity: 30 ml). After adding wastewater and sludge to the negative electrode, the negative electrode and the positive electrode were connected through resistance, and then nitrogen was supplied to the negative electrode to anaerobic conditions, and air was supplied to the positive electrode to make aerobic conditions. After about three weeks of incubation, when the background current remained constant, the total amount of current generated by adding wastewater with a constant concentration of BOD was added.
전류가 기본값을 나타낼 때 다른 농도의 COD를 갖는 폐수 (출처: 삼양 제넥스, 대한민국 인천 소재)를 첨가하였다. 도 2에서 보는 바와 같이 발생되는 전류량은 첨가된 폐수의 COD에 비례하여 증가하였다. 또한, 도 3에서 보는 바와 같이 적산 전류값은 첨가된 시료의 COD가 증가함에 따라 비례적으로 증가하였다.Wastewater with different concentrations of COD (source: Samyang Genex, Incheon, Korea) was added when the current indicated the default value. As shown in FIG. 2, the amount of current generated increased in proportion to the COD of the added wastewater. In addition, as shown in FIG. 3, the integrated current value increased proportionally as the COD of the added sample increased.
한편, 상기 제작된 BOD 센서를 6개월 동안 작동시키면서, 1개월 마다 COD 50 ppm과 100 ppm을 각각 첨가하여 발생되는 전류량을 측정하였다. 도 10에 나타낸 것처럼 발생되는 전류량은 거의 변화없이 일정하게 유지되었다. 따라서, 발생되는 전류량은 BOD의 센서의 작동 기간에 관계없이 첨가된 COD의 양에 따라 일정한 값을 유지하며 작동될 수 있다.Meanwhile, while operating the manufactured BOD sensor for 6 months, the amount of current generated by adding 50 ppm and 100 ppm of COD each month was measured. As shown in Fig. 10, the amount of generated current remained constant with almost no change. Therefore, the amount of current generated can be operated while maintaining a constant value depending on the amount of COD added regardless of the operation period of the sensor of the BOD.
<실시예 2><Example 2>
정전위 전해장치가 장착된 생물연료전지를 이용한 전기화학적 활성 미생물의 농화와 COD 농도에 따른 전류변화Current Change by COD Concentration and Concentration of Electrochemically Active Microorganisms Using Biological Fuel Cell with Electrostatic Potential Electrolyzer
전기 화학적으로 활성이 있는 미생물을 효과적으로 농화 배양하기 위하여 도 4에 표시된 것과 같은 장치를 제작하였다. 전기화학 셀 (Electrochemical cell)의 재질은 파이렉스 유리를 사용하였으며 용량은 500 ml로 하였다. 미생물이 농화되는 부분에는 작업 전극 (탄소 부직포) (101)이 정전위 전해장치와 연결되어 부착되어 있으며 전기적 회로를 구성하기 위하여 보조전극 (백금선) (102)을 부착하고 이를 정전위 전해장치와 연결하였다. 미생물이 농화되는 작업전극부와 보조전극부는투석막으로 분리되어있다. 작업전극부 (104) 및 보조 전극부 (105)에는 같은 농도의 폐수를 첨가하였다. 작업전극을 일정전위로 유지하기 위하여 기준전극 (113)을 부착하였으며 작업전극의 전위는 정전위 전해장치에 의하여 조절된다. 시료의 투입 및 채취구 (109)를 장착하고, 필요에 따라 혐기적 조건을 유지하기 위하여 질소가스를 공급하였다. 이 질소 주입구 및 배출구 (110, 111)는 연속적인 시료의 공급의 필요시 질소 대신에 시료의 공급 및 배출구로도 사용할 수 있다. 작업전극과 보조전극간의 전압 및 전류의 변화는 정전위 전해장치를 통하여 증폭되고 이를 컴퓨터를 이용한 기록 장치나 기록지를 이용하는 레코더로 기록하였다. 농화 배양은 작업 전극부에 균원 시료로 활성 슬러지를 첨가한 후 정전위 전해장치를 작동시켜 작업 전극이 고정된 전위를 유지하게 하는 것으로 개시하였다. 본 실험에서 폐수 및 활성 슬러지는 전분가공 폐수 (출처: 삼양 제넥스, 대한민국 인천 소재)를 이용하였다.In order to effectively concentrate and culture electrochemically active microorganisms, a device such as that shown in FIG. 4 was manufactured. The electrochemical cell was made of Pyrex glass and the volume was 500 ml. A working electrode (carbon nonwoven fabric) 101 is attached to the part where the microorganisms are concentrated, and is attached to the electrostatic potential electrolytic device, and an auxiliary electrode (platinum wire) 102 is attached to form an electric circuit and connected to the electrostatic potential electrolytic device. It was. The working electrode part and the auxiliary electrode part where the microorganisms are concentrated are separated by a dialysis membrane. Wastewater having the same concentration was added to the working electrode portion 104 and the auxiliary electrode portion 105. In order to maintain the working electrode at a constant potential, a reference electrode 113 is attached and the potential of the working electrode is controlled by the electrostatic potential electrolytic apparatus. Sample input and collection ports 109 were mounted, and nitrogen gas was supplied as necessary to maintain anaerobic conditions. These nitrogen inlets and outlets 110, 111 can also be used as the supply and outlet of the sample instead of nitrogen when the continuous supply of the sample is required. The change in voltage and current between the working electrode and the auxiliary electrode was amplified by the electropotential electrolytic apparatus and recorded by a recorder using a computer or a recording paper. Thickening culture was started by adding activated sludge as a fungal sample to the working electrode and then operating the electropotential electrolyzer to maintain the working potential at a fixed potential. In this experiment, wastewater and activated sludge were used for starch processing wastewater (Source: Samyang Genex, Incheon, Korea).
농화 배양은 폐수 및 활성 슬러지를 작업전극부에 첨가한 후 작업전극을 +0.98 v로 고정하여 시작하였다. 실험 개시 후 14일간 가동 하였을때 작업 전극과 보조전극간의 전류는 약 50 μA에서 최대 322 μA로 증가하였으며, 작업 18일 경과 후 약 153 μA에서 전류가 안정화되었다. 전류가 안정화되었을 때 농도가 다른 폐수를 시료 투입구를 통하여 투여한 결과 전류의 값이 도 2와 유사한 양상으로 증가하였다. 시료 투입구 (109)와 질소배출구 (110)를 통하여 폐수를 연속적으로 투입-방출하며, 작업 전극과 보조 전극 사이의 전류를 확인하였을 때 투입된 폐수의 농도에 따라 전류의 변화를 확인할 수 있었다 (참조. 도 5). 따라서, 이 장치를 사용하여 BOD를 연속적으로 측정하는 것이 가능하다는 것을 알 수 있으며, 장치를 분해하여 전극을 주사 전자현미경으로 관찰한 결과 다량의 미생물이 전극에 부착되어 있음을 확인할 수 있었다 (도 6). 전극에서 분리한 미생물을 배양하고 순환 전류법으로 조사한 결과 전기화학적으로 활성이 있는 것으로 확인되었다.Thickening culture was started by adding wastewater and activated sludge to the working electrode and then fixing the working electrode to +0.98 v. After 14 days of operation, the current between the working electrode and the auxiliary electrode increased from about 50 μA to 322 μA. After 18 days, the current stabilized at about 153 μA. When the current was stabilized, the wastewater having a different concentration was administered through the sample inlet, and the current value increased in a manner similar to that of FIG. 2. The wastewater was continuously introduced and discharged through the sample inlet 109 and the nitrogen outlet 110, and when the current between the working electrode and the auxiliary electrode was checked, the current was changed according to the concentration of the wastewater introduced (see reference). 5). Therefore, it can be seen that it is possible to continuously measure the BOD using this apparatus, and as a result of disassembling the apparatus and observing the electrode with a scanning electron microscope, it was confirmed that a large amount of microorganisms were attached to the electrode (FIG. 6). ). The microorganisms isolated from the electrodes were cultured and examined by cyclic ammeter to confirm that they were electrochemically active.
<실시예 3><Example 3>
생물연료전지형 BOD 센서의 음극 및 음극부내의 금속염 환원 세균수 변화Change of Metal Salt Reduction Bacteria in Cathode and Cathode of Biofuel Cell BOD Sensor
실시예 2에서 사용된 생물연료전지 형태의 BOD 센서의 농화 배양 과정 및 작동중 음극부에서 음극을 시료로 채취하여 철환원 세균의 균체수를 조사하였다. 배지는 인산염 완충액 기본 배지 (PBBM)를 사용하였으며, 배지성분은 효모 추출물 1 g/L, 염화암모늄 1 g/L, Macro-mineral (II) 25 ml/L (리터당 6g KH2PO4, 12g NaCl, 2.4g MgSO4·7H2O 및 1.6g CaCl2·2H2O 포함), 미량원소 2 ml/L (리터당 12.8g 니트로아세트산, 0.1g FeSO4·7H2O, 0.1g MnCl2·4H2O, 0.17g CoCl2·6H2O, 0.1g CaCl2·2H2O, 0.1g ZnCl2, 0.02g CuCl2·H2O, 0.1g H3BO3, 0.01g 몰리브덴염, 1.0g NaCl, 0.017g Na2SeO3, 0.026g NiSO4·6H2O 포함), 비타민액 0.1ml/L (0.002g 비오틴, 0.002g 엽산, 0.010g B6(피리독신)HCl, 0.005g B1(티아민)HCl, 0.005g B2(리보플라빈), 0.005g 니코틴산(니아신), 0.005g 판토텐산, 0.0001g B12 (시아노콥알라민) 결정, 0.005g PABA, 0.005g 리폰산 (티옥트산), 레사주린 (Resazurin) (0.2) 1 ml/L와 한천 1.8을 첨가하여 평판 배지를 제조하였다. 이때, 전자공여체로 아세트산 20 mM, 젖산 30 mM, 포도당 20 mM을 각각 사용하였으며, 전자수용체로 수용성 철인 페릭 피로포스페이트 (Ferric pyrophosphate) 20 mM을 사용하였다. 1차 시기는 반응 초기 연료전지의 호기성 슬러지와 혐기성 슬러지의 시료를 생리 식염수 (0.85소금물)로 희석하여 CFU (Colony Forming Unit /ml)로 측정하였고, 2차와 3차시기는 반응후 각각 1 개월후 동일한 배지와 방법으로 측정하였다. 결과를 하기 표 1에 나타내었다.During the thickening and culturing process and operation of the biofuel cell-type BOD sensor used in Example 2, the negative electrode was taken as a sample and the cell count of the iron reducing bacteria was examined. The medium used was phosphate buffer basal medium (PBBM), and the medium components were yeast extract 1 g / L, ammonium chloride 1 g / L, Macro-mineral (II) 25 ml / L (6 g KH 2 PO 4 , 12 g NaCl per liter). , 2.4 g MgSO 4 · 7H 2 O and 1.6 g CaCl 2 · 2H 2 O, 2 ml / L of trace elements (12.8 g nitroacetic acid per liter, 0.1 g FeSO 4 · 7H 2 O, 0.1 g MnCl 2 · 4H 2 O, 0.17 g CoCl 2 · 6H 2 O, 0.1 g CaCl 2 · 2H 2 O, 0.1 g ZnCl 2 , 0.02 g CuCl 2 · H 2 O, 0.1 g H 3 BO 3 , 0.01 g molybdenum salt, 1.0 g NaCl, 0.017 g Na 2 SeO 3 , 0.026 g NiSO 4 6H 2 O), vitamin liquid 0.1 ml / L (0.002 g biotin, 0.002 g folic acid, 0.010 g B6 (pyridoxine) HCl, 0.005 g B1 (thiamine) HCl, 0.005 g B2 (riboflavin), 0.005 g nicotinic acid (niacin), 0.005 g pantothenic acid, 0.0001 g B12 (cyanocorpalamine) crystals, 0.005 g PABA, 0.005 g liphonic acid (thioctic acid), resazurin (0.2) 1 A plate medium was prepared by adding ml / L and agar 1.8, where 20 mM acetic acid, 30 mM lactic acid and 20 mM glucose were used as electron donors. Ferric pyrophosphate (20 mM) was used as an electron acceptor, and the first phase was diluted with aerobic and anaerobic sludge samples from the fuel cell at the initial stage of the reaction by diluting CFU (0.85 salt solution). Colony Forming Unit / ml), and the second and third time periods were measured by the same medium and method 1 month after the reaction, respectively.
상기 표 1의 결과에서 알 수 있는 바와 같이, 호기성 슬러지 시료는 연료전지의 음극부위를 혐기 상태로 만들어주므로 통성 혐기성 균주외에는 선별되면서 계속 감소하여 전기화학적 활성을 가진 미생물만 농화 배양되는 것으로 판단되며, 혐기성 슬러지 시료는 2차시기에서 혐기성세균이 증가하다가 3차 시기에는 감소하여 전기화학적 활성을 가진 특정 생물만 농화 배양되었다.As can be seen from the results of Table 1, the aerobic sludge sample is made to anaerobic the negative electrode portion of the fuel cell is determined to be cultured and cultured only the microorganisms with electrochemical activity to continue to decrease while being selected outside the anaerobic strains, Anaerobic sludge samples increased in anaerobic bacteria in the second phase and decreased in the third phase, so that only certain organisms with electrochemical activity were enriched.
<실시예 4><Example 4>
시와넬라 푸트레파시엔스를 이용하는 연료 전지형 바이오센서에 의한 젖산농도 측정Measurement of Lactic Acid Concentration by Fuel Cell Type Biosensor Using Siwanella Putepassience
젖산의 농도를 측정하기 위하여 철 환원 세균의 일종인시와넬라 푸트레파시엔스(Shewanella putrefaciens) IR-1 (수탁번호 KCTC 8753P, 한국과학기술연구원 부설 생명공학 연구소 유전자 은행)을 사용하여 바이오센서를 제작하였다 (참조. 도 7).Use the iron reduction upon the type of bacteria and Nella Fu Trail Pacifico Enschede (Shewanella putrefaciens) IR-1 (accession number KCTC 8753P, Korea Institute of Science and Technology R & D Biotechnology Institute gene bank) to measure the concentration of lactate biosensors (See FIG. 7).
이 세균은 젖산을 아세트산으로 산화하여 발생하는 환원력으로 산화제이철을 환원하는 성질을 갖는다. 양극부 (205)의 용량은 대략 20 ml로 0.1 M의 염화 나트륨이 포함된 0.05 M의 인산염 완충용액을 첨가하였고, 음극부 (204)에는시와넬라 푸트레파시엔스IR-1 (건조 중량: 5 mg)와 0.1 M의 염화 나트륨이 포함된 19 ml의 0.05 M 인산염 완충 용액을 첨가하였다. 음극 (201)은 0.8 X 4 X 0.3 cm 크기의 탄소 부직포를 사용하였으며, 양극 (202)은 다공성 광택 탄소 (Reticulated Vitreous Carbon; 면적: 3 X 3 X 0.3 cm)를 사용하였다. 양극과 음극은 저항 (500Ω)을 통하여 연결되며 이때의 전압의 변화를 측정하여 전극간의 전류를 계산하였다. 전류는 전압계를 거쳐 기록계를 작동시킬 수 있도록 증폭되었으며 전류(전압)의 변화를 기록계를 사용하여 기록하였다. 작업 온도를 25℃로 유지하였고, 측정은 배경전류가 안정화된 후 각각 다른 농도의 젖산이 포함된 1 ml의 시료를 투입구 (209)를 통하여 첨가하고 시간에 따른 전류의 변화를 기록하여 초기 기울기를 구하였다.This bacterium has a property of reducing ferric oxide with a reducing force generated by oxidizing lactic acid with acetic acid. The capacity of the anode portion 205 was approximately 20 ml, with 0.05 M phosphate buffer containing 0.1 M sodium chloride added, and the cathode portion 204 with Siwanella putrfaciens IR-1 (dry weight: 5 mg) and 19 ml of 0.05 M phosphate buffer solution containing 0.1 M sodium chloride were added. The negative electrode 201 used a carbon nonwoven fabric having a size of 0.8 X 4 X 0.3 cm, and the positive electrode 202 used Reticulated Vitreous Carbon (area: 3 X 3 X 0.3 cm). The positive electrode and the negative electrode were connected through a resistor (500Ω) and the current between the electrodes was calculated by measuring the change in voltage at this time. The current was amplified to operate the recorder via a voltmeter and the change of the current (voltage) was recorded using the recorder. The working temperature was maintained at 25 ° C., and the measurement was performed by adding 1 ml of sample containing different concentrations of lactic acid through the inlet 209 after the background current was stabilized, and recording the change in the current over time. Obtained.
일정 농도의 젖산을 바이오센서내로 투입하였을 때 발생하는 전류의 초기 기울기는 젖산의 농도에 비례하였으며 이는 미생물에 의하여 젖산이 산화되어 발생한 전자가 전극을 통하여 이동함을 의미하며, 젖산의 농도는 미생물의 농도가 일정할 때 발생되는 전자의 양에 비례함을 보여주고 있다. 대표적인 전류의 증가예를 도7에 나타내고, 젖산의 농도 변화에 따른 전류 발생시의 초기 기울기를 도 8에 나타내었다. 젖산 농도에 따른 전류 초기 기울기의 상관 계수는 0.84였으며 이는 미생물의 종류, 농도, 전극의 재질 및 넓이를 변화시키거나 저항을 변화시키는 등 바이오센서의 구성을 변화시킴에 따라 개선되었다.The initial slope of the current generated when a certain concentration of lactic acid is introduced into the biosensor is proportional to the concentration of lactic acid, which means that electrons generated by oxidation of lactic acid by the microorganism move through the electrode. It is shown that the concentration is proportional to the amount of electrons generated. A representative example of the increase in current is shown in FIG. 7, and the initial slope at the time of current generation according to the change in the concentration of lactic acid is shown in FIG. The correlation coefficient of the initial slope of the current according to the lactic acid concentration was 0.84, which was improved by changing the composition of the biosensor, such as changing the type of microorganism, concentration, material and width of the electrode, and resistance.
바이오센서에 사용되는 연료전지의 미생물 촉매인 전기화학적 활성 세균으로 BOD 측정을 의한 생물 연료전지의 운전 과정에서 농화배양된 폐수 및 슬러지 속에 포함된 활성 세균이 사용된다. 그러므로, 본 BOD 바이오센서는 인위적인 미생물의 첨가 없이 운전될 수 있으며 폐수에 따라 적절한 세균의 활성이 유지될 수 있고, 폐수의 BOD 값을 연속적으로 측정할 수 있다. 또한, 본 발명의 BOD 측정용 바이오센서에 사용되는 생물 연료 전지는 6개월 이상 안정적으로 작동할 수 있다.As an electrochemically active bacterium that is a microbial catalyst of a fuel cell used in a biosensor, active bacteria contained in concentrated wastewater and sludge are used during the operation of a biofuel cell by measuring BOD. Therefore, the present BOD biosensor can be operated without the addition of artificial microorganisms, the appropriate bacterial activity can be maintained according to the wastewater, and the BOD value of the wastewater can be continuously measured. In addition, the biofuel cell used in the biosensor for BOD measurement of the present invention can operate stably for more than 6 months.
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| KR1019990027167A KR100303611B1 (en) | 1999-07-07 | 1999-07-07 | An Electrochemical Method for Enrichment of Microorganism, and a Biosensor for Analyzing Organic Substance and BOD |
| CNB008099952A CN1211652C (en) | 1999-07-07 | 2000-03-17 | Electrochemical method for enrichment of microorganism, biosensor for analyzing organic substance and BOD |
| AU34607/00A AU3460700A (en) | 1999-07-07 | 2000-03-17 | An electrochemical method for enrichment of microorganism, biosensor for analyzing organic substance and bod |
| CA002378580A CA2378580A1 (en) | 1999-07-07 | 2000-03-17 | An electrochemical method for enrichment of microorganism, a biosensor for analyzing organic substance and bod |
| PCT/KR2000/000230 WO2001004626A1 (en) | 1999-07-07 | 2000-03-17 | An electrochemical method for enrichment of microorganism, a biosensor for analyzing organic substance and bod |
| EP00913115A EP1236043A4 (en) | 1999-07-07 | 2000-03-17 | An electrochemical method for enrichment of microorganism, a biosensor for analyzing organic substance and bod |
| JP2001509985A JP3557528B2 (en) | 1999-07-07 | 2000-03-17 | Method for electrochemically enriching and culturing microorganisms, and biosensor for analyzing organic substances and BOD |
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| JPH0735741A (en) * | 1993-07-20 | 1995-02-07 | Fuji Electric Co Ltd | Bod measuring equipment |
| WO1995006242A1 (en) * | 1993-08-26 | 1995-03-02 | Akebono Brake Industry Co., Ltd. | Bod sensor and bod measuring method |
| KR100224381B1 (en) * | 1996-08-29 | 1999-10-15 | 박호군 | Biofuel cell using metal salt-reducing bacteria |
| KR19980016777U (en) * | 1996-09-19 | 1998-06-25 | 박병재 | Independent suspension control arm structure for automobiles |
| JPH10318965A (en) * | 1997-05-15 | 1998-12-04 | Fuji Electric Co Ltd | Bod biosensor measuring apparatus and standard solution therefor |
| KR100332932B1 (en) * | 1999-07-07 | 2002-04-20 | 박호군 | A Biofuel Cell Using Wastewater and Activated Sludge for Wastewater Treatment |
-
1999
- 1999-07-07 KR KR1019990027167A patent/KR100303611B1/en not_active IP Right Cessation
-
2000
- 2000-03-17 CN CNB008099952A patent/CN1211652C/en not_active Expired - Lifetime
- 2000-03-17 AU AU34607/00A patent/AU3460700A/en not_active Abandoned
- 2000-03-17 CA CA002378580A patent/CA2378580A1/en not_active Abandoned
- 2000-03-17 JP JP2001509985A patent/JP3557528B2/en not_active Expired - Fee Related
- 2000-03-17 WO PCT/KR2000/000230 patent/WO2001004626A1/en not_active Application Discontinuation
- 2000-03-17 EP EP00913115A patent/EP1236043A4/en not_active Withdrawn
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100435817B1 (en) * | 2001-11-10 | 2004-06-12 | 한국과학기술연구원 | Method for Measuring Low BOD Using Fuel Cell-Type Sensor to Measure Low BOD Value Using Electrochemically Active Oligotrophic Anaerobes |
| KR100502885B1 (en) * | 2002-05-15 | 2005-07-25 | 한국과학기술연구원 | Method for Monitoring BOD of Waste Water Continuously Using Microbial Fuel Cell |
| KR20220116668A (en) * | 2021-02-15 | 2022-08-23 | 한양대학교 에리카산학협력단 | Paper-based microbial fuel cell sensor |
| KR102585432B1 (en) | 2021-02-15 | 2023-10-10 | 한양대학교 에리카산학협력단 | Paper-based microbial fuel cell sensor |
Also Published As
| Publication number | Publication date |
|---|---|
| JP3557528B2 (en) | 2004-08-25 |
| JP2003504621A (en) | 2003-02-04 |
| KR20010009030A (en) | 2001-02-05 |
| EP1236043A4 (en) | 2003-01-15 |
| AU3460700A (en) | 2001-01-30 |
| CN1211652C (en) | 2005-07-20 |
| EP1236043A1 (en) | 2002-09-04 |
| CN1360677A (en) | 2002-07-24 |
| CA2378580A1 (en) | 2001-01-18 |
| WO2001004626A1 (en) | 2001-01-18 |
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