Note: Descriptions are shown in the official language in which they were submitted.
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AN ELECTROCHEMICAL METHOD FOR ENRICHMENT OF
MICROORGANISM, A BIOSENSOR FOR ANALYZING ORGANIC
SUBSTANCE AND BOD
Technical Field
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.
Background Art
In general, 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
2 0 result, a variety of biosensors were developed, and substances to be
measured
became varied in their range. For instance, there are put to practical use and
widely
used a glucose concentration-measuring biosensor fabricated of a glucose
oxidase
coupled to an oxygen electrode, and a medical biosensor containing an antibody
(see, Tuner et al., 1987, Biosensors, Fundamentals and Applications, Oxford
Science
2 5 Publications).
Meanwhile, 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). Their rapid measurements have a
significantly important value in environmental and pollution prevention-allied
3 0 industries. However, 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.
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Moreover, the prior method is disadvantageous in that a variation in measured
value
occurs depending on a skilled degree of workers. In addition, 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.
To solve these problems, there were proposed several kinds of biosensors
for the measurement of BOD (see, Hikuma et al., 1979, European Journal of
Microbiology and Biotechnology, 8, 289; Riedel et al., 1990, Water Research,
24,
883; and Hyun et al., 1993, Biotechnology and Bioengineering, 41, 1107).
Generally, 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. However, these biosensors have the following
problems:
First, 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
2 0 not capable of indicating the total value of BOD.
Second, 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. However, as the microorganism-
immobilizing membrane is expensive, the biosensors are uneconomical, and also
2 5 poor in maintainability.
Third, as there shall be used a dissolved oxygen electrode for a control
sample, the equipment is complex, and also is high in equipment cost and
failure
rate.
Fourth, a microorganism used in these BOD-measuring biosensors can not
3 0 transfer electrons directly to its outside. For this reason, the
biosensors require the
use of an electron transfer mediator or a separate transducer.
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Meanwhile, 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. If the electron donors
are
the same, 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).
Among these electron receptors used by the metal salt-reducing bacteria
that anaerobically respire, ferrous oxide and the like are very low in
solubility in
water. This insoluble electron receptor can not be absorbed into, and reduced
in
microorganism cells, unlike oxygen that is a common electron receptor used by
the
aerobic microorganisms. Thus, in the metal salt-reducing bacteria. there is
present
a specific form of an electron transfer system in order to reduce the electron
receptor
2 0 present in the outside of the cells. For instance, in Geobacter
sulfurreducens and
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
2 5 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)]. As a result. these metal salt-reducing
bacteria having similar characteristics transport electrons generated from a
3 0 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
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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-
16777 (June 5, 1998), the disclosure of which is incorporated herein by
reference.
In the biofuel cell including the use of the metal salt-reducing bacteria, the
quantity of the generated electrons is in proportion to a concentration of the
bacteria,
the amount of the organic substances and the like. Thus, the measurement of
the
quantity of the generated electrons allows the amount of the organic
substances
present in the sample to be determined.
Accordingly, we have continued to study such biofuel cells. and
microorganisms and organic substances that can be used in the biofuel cell. As
a
result of that. we have perfected the present invention.
Disclosure of the Invention
It is therefore an object of the present invention to provide an improved
biosensor for the measurement of BOD, and a method for the measurement of BOD
using the same, which biosensor has no drawbacks with the prior biosensors for
the
2 0 measurement of BOD.
In accordance with a first aspect of the present invention, there is provided
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,
2 5 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
3 0 compartment is added with a sample containing electrochemically active
bacteria.
According to a second aspect of the present invention, there is provided a
method for measuring BOD of a sample using the BOD-measuring biosensor of the
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first aspect above, the method 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
5 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.
In a third aspect of the present invention, there is provided 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
2 0 to catabolize the organic substance.
In a fourth aspect of the present invention, there is provided a method for
measuring a concentration of an organic substance using the biosensor
according to
the third aspect above, the method comprising: adding a sample to be measured
to
the anodic compartment while continuing to feed air to the cathodic
compartment to
2 5 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.
In a fifth aspect of the present invention, there is provided a method for
3 0 densely culturing electrochemically active bacteria present in active
sludge and
wastewater, using the mediator-less biofuel cell included in the biosensor
according
to the third aspect above, the method comprising: adding an active sludge and
a
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wastewater to the anodic compartment; electrically connecting the anodic
compartment to the catholic compartment via a resistor; introducing nitrogen
to the
anodic compartment to maintain the anodic compartment in an anaerobic
condition,
while introducing air to the catholic compartment to maintain the catholic
compartment in an aerobic condition; whereby a bacterium present in the active
sludge and wastewater is densely cultured without a separate electron
receptor.
Brief Description of the Drawings
The above and other objects and aspects of the invention will be apparent
from the following description of embodiments with reference to the
accompanying
drawings, in which:
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
2 0 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
2 5 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,
3 0 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
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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.
Best Mode for Carrying Out the 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. For such a
measurement, the inventive biosensor employs an organic substance-magnetizing
force and electron transfer capacity of electrochemically active
microorganisms
2 0 without an electron transfer mediator or a transducer.
In one embodiment of the present invention, 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. Such a biofuel cell includes
cathodic and
2 5 anodic compartments defined therein and contained with a conductive
medium,
respectively. Further, 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. In the anodic
3 0 compartment, there is included a sample containing an electrochemically
active
bacteria.
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More specifically, in the anodic compartment, 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. Thus, the detection and recording of
the
produced electric power allow BOD of the sample to be determined.
Moreover, 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.
As used herein, the term "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. An example of the
electrochemically
active bacteria typically includes metal salt-reducing bacteria.
In another embodiment of the present invention, a biosensor for the
measurement of an organic substance concentration contains electrochemically
active bacteria at its electrode itself or electrode compartment. Such
bacteria
2 0 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.
2 5 Thus, the measurement of the generated electric power allows a
concentration of the
organic substances present in the sample to be determined.
The method for measuring BOD and an organic substance concentration as
described above will now be described in detail.
(1) Dense Culture of Electrochemically Active Bacterium, and Biosensor for
3 0 Measuring BOD Using the Cultured Bacterium
From recent studies, it was confirmed that active and anaerobic sludges
originated from wastewater have a variety of metal salt-reducing bacteria
including a
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large amount of iron-reducing bacteria in a high concentration [see, Nielsen
et al.,
Systematic and Applied Microbiology, 20, 645-651, (1997); Nielsen et al.,
Water
Science and Technology, 34, 129-136, (1996); and Rasmussens et al., Water
Research, 28, 417-425, (1994)].
Accordingly, if a seed sample, in which various species of microorganisms
are mixed with each other, is anaerobically cultured along with a suitable
culture in a
fermenter including electrodes, only microorganisms that can use the electrode
as an
electron receptor are then finally viable. These microorganism species have an
electron carrier such as cytochrome, and thus have an 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.
Meanwhile, as wastewater and contaminated sewage contain various organic
substances, it is very difficult to measure BOD of wastewater or sewage in a
uniform
manner, i.e., using only one species of microorganism. In addition, this
measurement is high in errors. For these reasons, according to the present
invention, various species of electrochemically active bacteria present in
organic
wastewater and active sludge are densely cultured as described above, and the
densely cultured active bacteria are used as a microbial catalyst of the
biofuel cell of
2 0 the measuring unit, thereby generating electric power. From the quantity
of the
produced electric power, BOD of the sample can be determined.
(2) Measurement of an Organic Substance Concentration Using Biofuel Cell
Type Biosensor
The anodic compartment of the above described biofuel cell in the BOD
2 5 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. Then, the microorganisms contained in
the
3 0 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
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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.
The present invention will now be described in detail with reference to the
5 accompanying drawings.
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.
Referring to Fig. 1, the device includes an anodic compartment 4 and a
cathodic
compartment 5. In these electrode compartments 4 and 5, there are defined an
10 anode 1 and a cathode 2, respectively. Also. between the cathodic
compartment 5
and the anodic compartment 4. there is interposed an ion exchange membrane 3
serving to divide these compartments from each other.
The cathodic compartment ~ 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 11. 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 ~
2 0 can be maintained at a potential different from each other. After a lapse
of a
certain amount of time (generally three weeks), on the anode 1, there is
adhered
electrochemically active microorganisms which were densely cultured using the
wastewater as a substrate. The measurement of electricity generated from the
oxidation of the substrate by the microorganisms allows an increase and
decrease in
2 5 BOD of the wastewater to be determined. In the present invention, 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. 1
represents a leakage-preventing silicon rubber membrane, reference numerals 7
and
8 wirings of connecting the anode and the cathode, a reference numeral 10 a
3 0 discharging port of a sample and nitrogen, the reference numeral 12 a
discharging
port of air and phosphate buffer solution, a reference numeral 13 a protective
element, and a reference numeral 14 a fixing screw.
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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. Referring to Fig. 4, the BOD
sensor includes a potentiostat, in order for electrodes of the BOD sensor to
be
maintained at a constant voltage level. Moreover, in the BOD sensor, 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/AgCI), and 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
113) and a
working electrode compartment 104 is supplied with a sample (wastewater and
sludge). Then, electrochemically active microorganisms are densely cultured
for a
certain time (generally, two weeks). As a result, the device shown in Fig. 4
having
the microorganisms thus adhered (densely cultured) on the working electrode
101
can be used by itself as a BOD-measuring biosensor. Meanwhile, reference
2 0 numerals 114 and 112 in Fig. 4 represent a magnetic stirrer and a check
valve,
respectively. Also, other reference numerals in Fig. 4 that were not described
above will be described in Example 2 below.
The following examples are for further illustration purposes only and in no
way limit the scope of this invention.
Example 1
Dense Culture of Electrochemically Active Microorganisms Using Biofuel
Cell, and the Variation in Electric Current of Biofuel Cell according to COD
For the dense culture of electrochemically active microorganisms that
3 0 utilize organic substances present in a certain wastewater as a substrate,
a biofuel
cell as shown in Fig. 1 was fabricated.
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In this experiment, a wastewater from the starch processing (collected from
Samyang Genex, Inchon, Korea) was used, and 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)].
Referring to Fig. 1, 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. As used herein, 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. The term
"cathodic compartment"' designated by a reference numeral ~ 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 ~ 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. After the sample had
2 0 been added to the anodic compartment 4, 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
2 5 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.
When
the generated electric current was indicated at a basic value, wastewater of
another
3 0 COD value (collected from Samyang Genex, Inchon, Korea) was added to the
biofuel cell. As shown Fig. 2 in which arrows indicate COD values of the
samples,
the quantity of the generated electric current was increased in proportion to
COD of
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the added wastewater. Moreover, as shown in Fig. 3, the quantity of the
generated
electricity was increased in proportion to an increase in COD of the added
sample.
Meanwhile, while operating the fabricated BOD measuring sensor for six
months, a sample having 50 ppm of COD and a sample having 100 ppm of COD
were added to the BOD sensor every one month, respectively, and the quantity
of
electricity being generated was measured. As shown in Fig. 10, the quantity of
the
generated electricity was maintained at a constant level with little or no
change. As
a result, it was confirmed that the BOD sensor could be operated while
maintaining
the quantity of the generated electricity at a constant level depending on the
COD
value of the added sample regardless of operation duration of the sensor.
Example 2
Dense Culture of Electrochemically Active Microorganisms using Biofuel
Cell including Potentiostat, and the Variation in Electric Current according
to COD
For an effective dense culture of an electrochemically active
microorganism, a biosensor as shown in Fig. 4 was fabricated. Referring to
Fig. 4,
the biosensor includes an electrochemical cell 100 made of a pyrex glass and
having
a 500 ml capacity. At a portion of the electrochemical cell 100 in which a
microorganisms will be densely cultured, a working electrode 101 made of a
carbon
2 0 felt is disposed while being connected to a potentiostat. Moreover, at
another
portion of the electrochemical cell 100, there is disposed 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
2 5 working electrode portion 104 and the auxiliary electrode portion 1 OS
were added
with wastewaters having the same COD value. In order for the working electrode
to be maintained at a constant potential, a reference electrode 113 was also
disposed
in the electrochemical cell 100. A potential of the working electrode 101 was
adjusted by the potentiostat. Also, a port 109 for the introduction and
discharge of
3 0 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. For this purpose, a nitrogen introducing port 110 and
a
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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. For
the
dense culture of microorganisms, 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. Thus,
the
dense culture of the microorganism was started. In this experiment, as the
wastewater and active sludge, a wastewater from a starch processing (collected
from
Samyang Genex, Inchon. Korea) was used. The dense culture was started, after
the
wastewater and active sludge were added to the working electrode portion 104
and
the working electrode 101 was fixed at +0.98V. At 14 days of operation after
the
experiment start, electric current between the working electrode 101 and the
auxiliary electrode 102 was increased from about 50 ~A to a maximum of 322 ~A.
At 18 days after the operation start, the electric current was stabilized at
about 154
pA. When the electric current had been stabilized, 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.
While
2 0 continuing to introduce and discharge wastewater through the sample
introducing
port 109 and the nitrogen discharging port 110, 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
2 5 shown Fig. 4 permitted the continuous measurement of BOD. Moreover,
observation of the electrode by a scanning electron microscope was carried out
after
the decomposition of the biosensor. From this observation, it could be
confirmed
that a large amount of microorganisms were adhered on the electrode, as shown
in
Figs. 6a and 6b that are micrographs of the electrode surface taken before and
after
3 0 the use, respectively. In addition, the microorganisms isolated from the
electrode
were cultured and then examined by a cyclic voltammetry. The microorganism
was found to be electrochemically active.
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Example 3
Change in Metal Salt-Reducing Bacteria Count Present in Anode and
Anodic Compartment of Biofuel Cell Type BOD Sensor
A sample was collected from an anode and an anodic compartment during
5 the dense culture and operation of a biofuel cell type BOD sensor used in
Example
2, and was examined for a colony count of iron-reducing bacteria. In this
experiment, 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: 1 g/L of an yeast extract. I g/L of ammonium chloride, 25 ml/L of
Macro
10 mineral (II) (including, per 1L. 6 g of KHZPO~, 12 g of NaCI, 2.4 g of
MgSO~
~7H20. and 1.6g of CaCl~~2H20), 2 ml/L of microelements (including 12.8 g of
nitroacetic acid. 0.1 g of FeS0~~7H,0. 0.1 g of MnCl,~4H20, 0.17 g of
CoC12~6Hz0.
0.1 g of CaCl~~2Ha0. 0.1 g of ZnCl2, 0.02g of CuCI~~H~O. 0.1 g of H;BO;, O.OIg
of
molybdate, 1.0 g of NaCI, 0.017 g of Na2~Se0;. and 0.026 g of NiS0.~~6H~0).
0.1
15 ml/L of a vitamin solution (including 0.002 g of biotin, 0.002 g of
folacin, 0.010 g of
B6(pyridoxin)HCh 0.005 g of B1(thiamin)HCI, 0.005 g of B2(riboflavin), 0.005 g
of
nicotinic acid(niacin), 0.005 g of panthothenic acid, 0.0001 g of B 12
(cyanocobalamine) crystal, 0.005 g of PABA, and 0.005 g of lipoic acid
(thioctic
acid)), 1 ml/L of resazurin (0.2%), and 1.8% of agar agar.
2 0 As an electron donor. 20 mM of acetic acid, 30 mM of lactic acid, and 20
mM of glucose were used, respectively, while 20 mM of ferric pyrophosphate, a
water soluble iron, was used as an electron receptor. In the first
measurement, the
respective samples of the aerobic sludge and the anaerobic sludge of the
biofuel cell
at the early stage of reaction were diluted with a physiological saline
solution
2 5 (0.85% brine) and then measured for Colony Forming Unit per ml of
solution.
Second and third measurements were carried out using the same medium and
method as in the first measurement, at one month and two months after the
reaction, respectively. Results are shown in Table 1 below.
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Table 1: Change
in Colony Count
in Anodic Compartment
of Biofuel
Cell
SampleElectron Electron First Second Third time
time
donor(mM) receptor(mM) time
AerobicAcetic acid(20))FP(20) 2.8x 0.9x S.1 x 10'
10' 10''
sludgeGlucose(20) FP(20) 8.0x10'1.3x10'4.2x10''
Lactic acid(30)FP(30) 6.4x10'1.1x10'4.1x10''
Anaerobic FP(20) 3.6x10'5.4x1061.5x10'
Acetic
acid(20)
sludgeGlucose(20) FP(20) 2.1 8.4x 1.4x 106
x 10' 1 O6
Lactic acid(30)FP(20) 1.7x1051.5x1062.3x10'
FP: Ferric Pyrophosphate
As evident from Table 1 above, in the case of the aerobic sludge sample, it
is believed that. as the anodic compartment of the biofuel cell is maintained
in an
anaerobic condition, strains other than facultative anaerobic strains are
continued to
reduce while being screened, such that only electrochemically active
microorganisms are densely cultured. In the case of the anaerobic sludge
sample,
the anaerobic bacteria were increased at the second measurement, and then
decreased at the third measurement, such that only electrochemically active
microorganisms were densely cultured.
Example 4
Measurement of Lactic Acid Concentration with Fuelcell Type Biosensor
Using Shewanella putrefaciens
For the measurement of lactic acid concentration, a biosensor as shown in
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
2 0 oxide using a reducing power generated in the oxidation of lactic acid
into acetic
acid.
Referring to Fig. 7, the biosensor includes a cell 200 in which an anodic
compartment 204 and a cathodic compartment 20~ are defined. The anodic
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17
compartment 204 and the cathodic compartment 205 are divided by a canon
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. Also, the
anodic
compartment 204 was added with Shen~anella 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
3 cm x 0.3 cm. The anode 201 and the cathode 202 were electrically connected
with each other via a resistor (500 S2). 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
2 0 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
2 5 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, and 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.
3 0 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.
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18
Industrial Applicability
As apparent from the foregoing, 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.
Therefore, 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. In addition, 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.
Although the preferred embodiments of the invention have been disclosed
for illustrative purposes, those skilled in the art will appreciate that
various
modifications, additions and substitutions are possible, without departing
from the
scope and spirit of the invention as disclosed in the accompanying claims.
25
