Classifications

• • 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
• • 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—Biological oxygen demand [BOD] or chemical oxygen demand [COD]

Definitions

• the present invention relates to a biosensor for the measurement of an organic substance concentration and BOD. More particularly, the present invention relates to a biosensor for measuring an organic substance concentration and BOD. which biosensor enables the performance of a simple and rapid measurement, and is relatively inexpensive in costs required for its. fabrication, application, maintenance, and repair.
• a biosensor means a measuring device in which organisms or substances originated from the organisms are used for at least one part of a measuring unit that is coupled with an electrical device.
• the biosensor has been continuously studied from the 1960 ' s, as it has an advantage in that it enables the precious measurement of concentration and properties of a substance to be measured, by virtue of a high degree of specificity with a biological reaction. As a result, a variety of biosensors were developed, and substances to be measured became varied in their range.
• the pollution level of an industrial wastewater or a domestic sewage is generally represented in terms of Chemical Oxygen Demand (COD) or Biochemical Oxygen Demand (BOD).
• COD Chemical Oxygen Demand
• BOD Biochemical Oxygen Demand
• Their rapid measurements have a significantly important value in environmental and pollution prevention-allied industries.
• the prior method for measuring BOD that shows the amount of microorganism-magnetizable organic substances is problematic in that it requires much time, as well as various complex procedures and devices for the measurement.
• the prior method is disadvantageous in that a variation in measured value occurs depending on a skilled degree of workers.
• this method is difficult to apply to the case where the polluted state needs to be rapidly identified or where an automated facility for wastewater treatment is installed.
• these BOD sensors have a structure in which a membrane, onto which a certain microorganism is immobilized, is attached to a dissolved oxygen-measuring electrode. Where these BOD sensors are reacted with a sample to be measured, the microorganism immobilized onto the membrane magnetizes an organic material contained in the sample while consuming oxygen. The value of dissolved oxygen in the resulting sample is compared to the value of dissolved oxygen in a control sample and converted into BOD.
• these biosensors have the following problems:
• these biosensors employ one microorganism species. Thus, they are short of the magnetic susceptibility to complex nutrient components present in wastewater due to the substrate specificity of the used microorganism, thereby being not capable of indicating the total value of BOD.
• a microorganism is immobilized on a porous membrane. For this reason, the membrane needs to be frequently replaced or repaired in order for BOD to be measured at a high reproducibility.
• the microorganism- immobilizing membrane is expensive, the biosensors are uneconomical, and also poor in maintainability.
• the equipment is complex, and also is high in equipment cost and failure rate.
• a microorganism used in these BOD-measuring biosensors can not transfer electrons directly to its outside. For this reason, the biosensors require the use of an electron transfer mediator or a separate transducer.
• microorganisms growing in an anaerobic environment can commonly utilize electron receptors other than oxygen. The metabolism using these electron receptors is named the anaerobic respiration of microorganisms.
• the electron receptors which can be used in the oxidation of an organic substance by the anaerobically respiratory microorganisms, include ferric oxide, nitrate, hexavalent manganese, sulfate, carbonate and the like.
• the reduction of ferric oxide into ferrous oxide generates the largest level of energy among energy generated from the redox reactions between the respective electron receptors and the electron donor, with the energy level being low in order of nitrate, sulfate and carbonate.
• This energy level is associated with the redox potential which is an inherent characteristic of the respective electron receptors (see, Byoung-Hong, Kim, Microorganism Physiology. Academy Press Co.. Ltd.. Seoul. Korea. 1995).
• Shewanella putrefaciens that are a kind of the metal salt-reducing bacteria using ferric oxide as an electron receptor there is present cytochrome, an electron transfer protein. Through this cytochrome, electrons generated from the oxidation of organic substances within the microorganisms are transferred to the electron receptor outside of the microorganism cell. Using energy generated by this electron transfer procedure, the microorganism grows. [See, Myers and Myers, Journal of Bacteriology, 174, 3429-3438, (1992); and Seeliger et al., Journal of Bacteriology, 180, 3686-3691 , (1998)].
• these metal salt-reducing bacteria having similar characteristics transport electrons generated from a catabolism of organic substances to the external insoluble electron receptor such that the receptor is reduced. For this reason, the amount of the organic substances will be proportional to the amount of the reduced electron receptor. Also, when a suitable electrode is used that can be substituted for the electron receptor, the electrode will be reduced with the electrons generated from the inside of the bacteria, and the electrons transferred directly to the electrode will outwardly flow through a circuit. A biofuel cell using such physiological characteristics of the microorganisms is described in Korean Patent Application Publication No. 1998-
• the quantity of the generated electrons is in proportion to a concentration of the bacteria, the amount of the organic substances and the like.
• the measurement of the quantity of the generated electrons allows the amount of the organic substances present in the sample to be determined.
• a BOD-measuring biosensor comprising a measuring unit, an electric current- detecting unit, and a recording unit serving to record a variation in the detected electric current, the measuring unit being composed of a mediator-less biofuel cell, the biofuel cell including: cathodic and anodic compartments defined therein and contained with a conductive medium, respectively; an anode arranged in the anodic compartment; a cathode arranged in the cathodic compartment: and an ion exchange membrane interposed between the cathodic and anodic compartments and serving to divide the anodic compartment from the cathodic compartment, wherein the anodic compartment is added with a sample containing electrochemically active bacteria.
• a method for measuring BOD of a sample using the BOD-measuring biosensor of the first aspect above comprising: electrically connecting the anode to the cathode via a resistor; introducing nitrogen into the anodic compartment to maintain the anodic compartment in an anaerobic condition, while introducing oxygen into the cathodic compartment to maintain the cathodic compartment in an aerobic condition; densely culturing electrochemically active bacteria present in the sample in the anodic compartment; and measuring electric current being generated while employing the densely cultured, electrochemically active bacteria as a microbial catalyst.
• a mediator-less biofuel cell type biosensor for the measurement of organic substance concentration
• the biosensor comprising a measuring unit, an electric current-detecting unit, and a recording unit serving to record a variation in the detected electric current
• the measuring unit being composed of a mediator-less biofuel cell
• the biofuel cell comprising: cathodic and anodic compartments defined therein and contained with a conductive medium, respectively; an anode arranged in the anodic compartment; a cathode arranged in the cathodic compartment; and an ion exchange membrane interposed between the cathodic and anodic compartments and serving to divide the anodic compartment from the cathodic compartment, wherein the anodic compartment contains a single species of electrochemically active bacterium serving to catabolize the organic substance.
• a method for measuring a concentration of an organic substance using the biosensor comprising: adding a sample to be measured to the anodic compartment while continuing to feed air to the cathodic compartment to maintain the cathodic compartment at a voltage different from the anodic compartment; and measuring an electric current generated from the consumption of an organic substance contained in the sample by the electrochemically active bacterium, whereby the concentration of the organic substance is measured.
• a method for densely culturing electrochemically active bacteria present in active sludge and wastewater comprising: adding an active sludge and a wastewater to the anodic compartment; electrically connecting the anodic compartment to the cathodic compartment via a resistor; introducing nitrogen to the anodic compartment to maintain the anodic compartment in an anaerobic condition, while introducing air to the cathodic compartment to maintain the cathodic compartment in an aerobic condition; whereby a bacterium present in the active sludge and wastewater is densely cultured without a separate electron receptor.
• Fig. 1 is a perspective view schematically showing a biofuel cell used in a BOD-measuring biosensor according to the present invention
• Fig. 2 is a graph showing a correlation of electric current with COD of a sample added to a biofuel cell according to Example 1 of the present invention
• Fig. 3 is a graph showing a correlation of the quantity of the generated electricity with COD of a sample added to a biofuel cell according to Example 1 of the present invention
• Fig. 4 is a schematical view showing a BOD-measuring biosensor according to Example 2 of the present invention, with the biosensor including the use of a microorganism dense-culturing device having a potentiostat;
• Fig. 5 is a graph showing a correlation of electric current with COD of a sample added to a biofuel cell type biosensor in which an electrochemically active bacterium was densely cultured using a potentiostat according to Example 2 of the present invention
• Fig. 6a is a scanning electron micrograph of the surface of a working electrode of the biofuel cell type biosensor according to Example 2 of the present invention, which micrograph was taken before the biosensor is used for densely culturing microorganisms
• Fig. 6a is a scanning electron micrograph of the surface of a working electrode of the biofuel cell type biosensor according to Example 2 of the present invention, which micrograph was taken after the biosensor is used for densely culturing microorganisms
• Fig. 7 is a schemetical view of a biofuel cell type biosensor for measuring a lactic acid concentration according to Example 4 of the present invention
• Fig. 8 shows typical increase in electric current generated during the measurement of a lactic acid concentration
• Fig. 9 is a graph showing a correlation of a lactic acid concentration with an initial slope of the generated electric current, which was obtained according to Example 4 of the present invention: and Fig. 10 is a graph showing the quantity of electricity according to COD of a sample, which electricity was measured for six months using the BOD-measuring biosensor according to Example 1 of the present invention.
• the present invention is directed to a biosensor capable of measuring a concentration of microorganism-magnetizable components (BOD) or organic substances, such as lactic acid, that are present in wastewater.
• BOD microorganism-magnetizable components
• the inventive biosensor employs an organic substance-magnetizing force and electron transfer capacity of electrochemically active microorganisms without an electron transfer mediator or a transducer.
• the BOD-measuring biosensor comprises a measuring unit, an electric current-detecting unit, and a recording unit serving to record a variation in the detected current.
• the measuring unit is composed of a mediator-less biofuel cell.
• a biofuel cell includes cathodic and anodic compartments defined therein and contained with a conductive medium. respectively.
• the biofuel cell includes an anode arranged in the anodic compartment; a cathode arranged in the cathodic compartment; and an ion exchange membrane interposed between the cathodic and anodic compartments and serving to divide the anodic compartment from the cathodic compartment.
• the anodic compartment there is included a sample containing an electrochemically active bacteria.
• the electrochemically active bacteria are electrochemically densely cultured using, as a seed sample, organic substances and active sludge present in a certain sample.
• the densely cultured, electrochemically active bacteria is used as a microbial catalyst to produce electric power.
• the produced electric power is proportional to a concentration of various organic substances, which are magnetizable by microorganisms added to the biofuel cell that serves as the measuring unit.
• the detection and recording of the produced electric power allow BOD of the sample to be determined.
• a potentiostat in order to facilitate the dense culture of the electrochemically active bacteria in the anodic compartment of the measuring unit, there may be preferably used a potentiostat.
• electrochemically active bacteria means bacteria that can discharge electrons generated from the oxidation of an organic substance present in wastewater to the outside of their cells to transfer the electrons to an electrode, thereby generating electric current.
• electrochemically active bacteria typically includes metal salt-reducing bacteria.
• a biosensor for the measurement of an organic substance concentration contains electrochemically active bacteria at its electrode itself or electrode compartment. Such bacteria utilize certain organic substances as a substrate.
• the biosensor containing such bacteria is used by itself as the measuring unit. That is to say, as the anodic compartment contains the electrochemically active bacteria of catabolizing a certain organic substance, electric power generated by the biofuel cell corresponds to that generated by the catabolism of certain organic substances present in a sample.
• the measurement of the generated electric power allows a concentration of the organic substances present in the sample to be determined.
• microorganism species have an electron carrier such as cytochrome. and thus have an electrochemical activity.
• an electron carrier such as cytochrome.
• electrochemical activity As a result, in accordance with such a manner, it is possible to selectively densely culture microbes having an electrochemical activity among various species of microorganisms present in wastewater and active sludge.
• the anodic compartment of the above described biofuel cell in the BOD- measuring biosensor is added with a single species of an electrochemically active microorganism selected depending on the nature of a substrate to be measured.
• the cathodic compartment is continued to feed air such that it is maintained at a voltage level different from the anodic compartment.
• the anodic compartment is added with the sample to be measured.
• the microorganisms contained in the anodic compartment consume the corresponding substrate, while electrons being produced flow out to an external circuit through the anodic compartment.
• the measurement of the produced electric current allows a concentration of the corresponding substrate to be determined. In this way, the use of the electrochemically active bacteria consuming various substrates allows a concentration of the corresponding organic substance to be measured.
• Fig. 1 is a schematical view showing a biofuel cell served as a microorganism dense-culture device in a BOD sensor of the present invention.
• the device includes an anodic compartment 4 and a cathodic compartment 5.
• these electrode compartments 4 and 5 there are defined an anode 1 and a cathode 2, respectively.
• an ion exchange membrane 3 serving to divide these compartments from each other.
• the cathodic compartment 5 is supplied with oxygen such that the cathode 2 is maintained at a potential different from the anode 1.
• the anodic compartment 4 is fed with a sample (such as wastewater and sludge) through a port 9, while the cathodic compartment 5 is fed with a phosphate buffer solution or tap water through a port 1 1. Also, the anodic compartment 4 is supplied with nitrogen through the port 9 such that it is maintained at an anaerobic condition.
• the cathodic compartment is supplied with air through a port 11 such that the electrodes 4 and 5 can be maintained at a potential different from each other.
• the cathode 2 and the anode 1 are preferably made of a carbon felt, but these electrodes may be sometimes made of other materials. Further, a reference numeral 6 in Fig.
• Fig. 4 is a schematical view showing a construction of a BOD sensor for carrying out an electrochemical dense-culture of a microorganism according to a preferred embodiment of the present invention.
• the BOD sensor includes a potentiostat, in order for electrodes of the BOD sensor to be maintained at a constant voltage level.
• a working electrode 101 serves as an electron receptor and can be varied in electrochemical action to microorganisms, depending on a variation in applied voltage to the working electrode 101.
• the working electrode 101 is formed of a carbon felt
• a reference electrode 113 is made of silver/silver chloride (Ag/AgCl).
• an auxiliary electrode 102 is made of platinum.
• the reference electrode 113 serves to maintain and compensate the applied voltage to the working electrode 101.
• the auxiliary electrode 102 serves to constitute an electrical circuit along with the working electrode 101.
• the working electrode 101 is applied with a constant potential (generally. +0.98V with respect to the silver chloride reference electrode 1 13) and a working electrode compartment 104 is supplied with a sample (wastewater and sludge).
• a biofuel cell as shown in Fig. 1 was fabricated.
• a wastewater from the starch processing collected from Samyang Genex, Inchon, Korea
• an active sludge generated from the wastewater treatment in the same factory was used as an inoculum.
• a basic configuration of the biofuel cell used in this example has referred to literature by Bennetto et al. [See. Bennetto et al., Biotechnology Letters, 7, 699-704, (1985)].
• both an anode 1 and a cathode 2 were formed of a carbon felt having a size of 5 x 7.5 x 0.6 cm. respectively. Also, the electrodes 1 and 2 were wired with a platinum wire.
• the term ''anodic compartment' " designated by a reference numeral 4 means a place in which microorganisms or electron carriers of the microorganisms are oxidized by the anode 1.
• cathodic compartment designated by a reference numeral 5 means a portion in which electrons transferred through an external circuit reduce an oxidant in the cathode 2.
• the anodic compartment 4 and the cathodic compartment 5 were divided by an ion exchange membrane 3 from each other, and electrically connected through the external circuit. In this case, the connection of a suitable resistor to the external circuit allows a control of a flow of electric current between the cathode 2 and the anode 1.
• the cathodic compartment 5 (working capacity: 30 ml) was provided with air. while the anodic compartment 4 (working capacity: 30 ml) was added with a sample consisting of wastewater and sludge.
• the cathode 2 and the anode 1 were electrically connected via the resistor. Then, the anodic compartment 5 was supplied with nitrogen such that it was maintained at an anaerobic condition, whereas the cathodic compartment was supplied with air such that it was maintained at an aerobic condition. While maintaining these electrodes at the respective conditions, a dense culture of microorganisms was started. At about three weeks of the dense culture, a background current was maintained at a constant level. At this time, wastewater having a certain BOD value was added to the anodic compartment 4 and the total quantity of electric current being produced was integrated.
• the biosensor includes an electrochemical cell 100 made of a pyrex glass and having a 500 ml capacity.
• a working electrode 101 made of a carbon felt is disposed while being connected to a potentiostat.
• an auxiliary electrode 102 made of a platinum wire to form an electrical circuit.
• a working electrode portion 104 having the working electrode 101 and an auxiliary electrode portion having the auxiliary electrode 102 are divided by a dialyzing diaphragm from each other.
• the working electrode portion 104 and the auxiliary electrode portion 105 were added with wastewaters having the same COD value.
• a reference electrode 113 was also disposed in the electrochemical cell 100.
• a potential of the working electrode 101 was adjusted by the potentiostat.
• a port 109 for the introduction and discharge of the sample was formed on a side of the electrochemical cell 100.
• the electrochemical cell 100 was provided with nitrogen gas so that it was maintained at an anaerobic condition.
• a nitrogen introducing port 110 and a nitrogen discharging port 111 are disposed that also may serve as the sample supplying and discharging ports when the sample needs to be continuously supplied.
• a variation in potential and current between the working electrode 101 and the auxiliary electrode 102 was amplified through the potentiostat and recorded with a recording unit using a computer and a recorder using a recording paper.
• the working electrode portion 104 was added with an active sludge as an inoculum, and the potentiostat was then allowed to operate such that the working electrode 101 was maintained at a fixed potential.
• the dense culture of the microorganism was started.
• a wastewater from a starch processing collected from
• the electric current was stabilized at about 154 ⁇ A.
• the introduction of another wastewater of a different COD value through the sample introducing port 109 resulted in an increase in electric current value, similarly to that in Fig.2.
• electric current between the working electrode 101 and the auxiliary electrode 102 was monitored. From this, it could be confirmed that the electric current was varied depending on COD of wastewater, as shown Fig. 5. Accordingly, it could be found that the use of the biosensor as in shown Fig. 4 permitted the continuous measurement of BOD.
• observation of the electrode by a scanning electron microscope was carried out after the decomposition of the biosensor.
• Figs. 6a and 6b are micrographs of the electrode surface taken before and after the use, respectively.
• the microorganisms isolated from the electrode were cultured and then examined by a cyclic voltammetry. The microorganism was found to be electrochemically active.
• a phosphate buffer solution-based medium (PBBM) was used as a medium.
• the following components were added to the medium to prepare a plate medium: lg/L of an yeast extract. lg/L of ammonium chloride, 25 ml/L of Macro- mineral (II) (including, per IL. 6 g of KH 2 PO 4 . 12 g of NaCl, 2.4 g of MgS0 4
• B6(pyridoxin)HCl 0.005 g of Bl(thiamin)HCl. 0.005 g of B2(riboflavin). 0.005 g of nicotinic acid(niacin). 0.005 g of panthothenic acid, 0.000 lg of B12 (cyanocobalamine) crystal, 0.005 g of PABA. and 0.005 g of lipoic acid (thioctic acid)), lml/L of resazurin (0.2%). and 1.8% of agar agar. As an electron donor, 20 mM of acetic acid.
• Fig. 7 was fabricated using Shewanella putrefaciens IR-1, a kind of an iron-reducing bacterium.
• Such a strain can be available from the Korean Collection for Type Cultures, Korean Research Institute of Bioscience and Biotechnology, under the accession number KCTC 8753P.
• This bacterium has an ability to reduce ferric oxide using a reducing power generated in the oxidation of lactic acid into acetic acid.
• the biosensor includes a cell 200 in which an anodic compartment 204 and a cathodic compartment 205 are defined.
• the anodic compartment 204 and the cathodic compartment 205 are divided by a cation exchange membrane 203 and include an anode 201 and a cathode 202, respectively.
• the cathodic compartment 205 having a 20 ml capacity was charged with 0.05 M of a phosphate buffer solution containing 0.1 M of sodium chloride.
• An anodic compartment 204 was fed with nitrogen through a nitrogen-introducing port 211.
• a reference numeral 210 represents a nitrogen-discharging port.
• the anodic compartment 204 was added with Shewanella putrefaciens IR-1 (dry weight: 5 mg) and 19 ml of a 0.05 M phosphate buffer solution containing 0.01 M sodium chloride.
• the anode 201 was made of a carbon felt having a size of 0.8 cm x 4 cm x 0.3 cm, and a cathode 202 was made of a reticulated vitreous carbon having a size of 3 cm x
• the anode 201 and the cathode 202 were electrically connected with each other via a resistor (500 ⁇ ). In this state, a variation in voltage across the resistor was measured with a voltage-measuring unit, and converted into electric current between the two electrodes. Electric current was amplified through a scanner so that a recording unit could be operated. The recording unit has recorded a variation in electric current (voltage). Working temperature was maintained at 25 °C. After background current was stabilized, 1 ml of the respective samples containing lactic acid at a different concentration were fed into the biofuel cell through a sample-introducing port 209. A variation in electric current according to time was recorded, and an initial slope of electric current was obtained.
• the initial slope of electric current generated when introducing lactic acid of a desired concentration into the biosensor was proportional to a lactic acid concentration. This indicates that electrons generated from the oxidation of lactic acid by the microorganism move toward the electrode and that the lactic acid concentration is proportional to the quantity of electrons generated at a constant microorganism concentration.
• Fig. 8 illustrates the typical increase in electric current according to the lactic acid addition
• Fig. 9 illustrates the initial slope of electric current according to a variation in lactic acid concentration.
• a correlation coefficient of the initial current slope with the lactic acid concentration was 0.84. This improvement in correlation coefficient was obtained by changing the biosensor construction, such as the nature and concentration of the microorganism, the material and size of the electrodes, the resistor and the like.
• the biosensor of the present invention utilizes electrochemically active bacteria that were contained in wastewater and sludge and densely cultured during the operation procedure of the biofuel cell for the BOD measurement, as a microbial catalyst of the biofuel cell used in the biosensor.
• the present biosensor can be operated without the artificial addition of microorganisms, and allows an activity of the bacteria to be maintained at a suitable level depending on the nature of wastewater. Moreover, it enables the continuous measurement for the BOD value of wastewater.
• the biofuel cell used in the BOD-measuring biosensor of the present invention can be operated in a stable manner over six months or more.

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• 1999
  • 1999-07-07 KR KR1019990027167A patent/KR100303611B1/ko not_active IP Right Cessation
• 2000
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