JPS6282345A - Method and sensor for measuring carbon dioxide - Google Patents

Abstract

PURPOSE:To obtain a carbon dioxide sensor which is so constructed that the deterioration of the life thereof by drying of microorganism bacteria is prevented and that the carbon dioxide past a gas permeable membrane is made hardly dissipatable by providing a buffer soln. reservoir or layer near the microorganism bacteria. CONSTITUTION:The sensor for measuring carbon dioxide has an oxygen electrode body 7, a cathode 5 consisting of platinum and an anode 6 consisting of lead and is provided with an electrolyte 8. The top end of the electrode 7 is coated with the gas permeable membrane 4 in such a manner that the part of the cathode 5 is fully coated. An immobilizing film 2 for the microorganisms 3 disposed to the outside of the membrane 4 is coated with a dialysis membrane 10 made of acetyl cellulose in the form of enclosing the entire part thereof. The immobilized microorganisms 2 and the membrane 10 are immersed into the buffer soln. 13 of a high pH value stored form a spacer 11. The sensor is so constituted that the buffer soln. 13 and the outside contact each other via a gas permeable membrane 12. The membrane 12 is fixed by rubber ring (may be an O-ring) 9. The microorganism bacteria are thus preserved without being dried.

Description

DETAILED DESCRIPTION OF THE INVENTION [1] A carbon dioxide (COZ) sensor using microorganisms is disclosed. The CO2 sensor of the present invention is revolutionary in that it can measure CO2 amperometrically, and is
It can be advantageously used in a number of measurement fields including the environment and fermentation industrial processes.The sensor of the present invention can maintain the lifespan of microorganisms for a long time, and can also reduce measurement errors. I can do it.

[Industrial application field]

The present invention relates to a method and apparatus for measuring the concentration of carbon dioxide in a solution or in air.

More specifically, the present invention relates to a method for measuring carbon dioxide using microbial functions and electrochemical electrodes, and a sensor for measuring carbon dioxide. As described above, the present invention can be advantageously used in many measurement fields including medicine, the environment, and fermentation industrial processes.

[Conventional technology]

It is well known that in the field of measurement, carbon dioxide measurement is considered important. Conventionally, carbon dioxide electrodes based on potentiometry have been developed and actually used as sensors for measuring carbon dioxide, especially for the purpose of measuring carbon dioxide components in body fluids (blood). For details about carbon dioxide electrodes, see, for example, J.W.
house) and 8, F. Bradle.
y), J, A pl, Ph 5iol,, 13゜5
15 (1958)). However, this potentiometric type carbon dioxide electrode has various problems that occur during measurement due to its configuration. For example, this method has problems such as being susceptible to the influence of contaminants and being unable to measure improvements in sensitivity beyond a certain level because the sensitivity depends on the Nernst equation.

As a result of conducting research to solve these problems, the present inventors focused on the fact that the respiratory activity of microorganisms that assimilate carbon dioxide changes depending on changes in the concentration of carbon dioxide near the microorganisms. It was discovered that detecting the 1st phase with an oxygen electrode and performing a fixed star of 4 degrees carbon dioxide would lead to a solution to the problem. The carbon dioxide measuring sensor (carbon dioxide sensor) based on amberometry developed by the present inventors is disclosed in a patent application separately filed on October 3, 1985, for example. A measurement sensor for measuring the concentration of carbon dioxide in a solution such as a body fluid such as blood or in the air, which includes an oxygen electrode and a sensor fixed near the cathode of the oxygen electrode. microorganisms that can selectively assimilate carbon;
covering the entire cathode and each of the microorganisms) W
and a gas permeable membrane. The structure of this carbon dioxide sensor is, for example,
As shown schematically in the accompanying FIG.

7 in the figure is a sensor main body (oxygen electrode), which has a spring cathode 5 and an anode 6. The oxygen electrode 7 also has an electrolyte 8 therein.

The oxygen electrode 7 is covered with a gas permeable membrane 4 made of Teflon (trade name) so that the cathode 5 portion thereof is completely covered. Further, the microorganism 3 immobilization membrane 2 placed outside this membrane 4 is also covered with a gas permeable membrane 1 made of Teflon. 9 is a rubber band (0 ring may be used) for fixing the gas permeable membrane.

[Problem that the invention seeks to solve]

The carbon dioxide sensor recently developed by the inventors is
It allows for higher intensity measurements than conventional potentiometric carbon dioxide electrodes, and also expands the range of applications for the sensor. However, in this sensor structure, the entire microbial cells and the cathode of the oxygen electrode are covered with a gas-permeable membrane, which tends to dry out the area around the microbial cells, which may affect the lifespan of the sensor. It's large. Furthermore, there is a risk that the carbon dioxide that has passed through the gas permeable membrane may be extracted from the gas permeable membrane again before it is assimilated by microorganisms, resulting in new measurement errors. These problems that may occur in the carbon dioxide sensor developed by the present inventors are the problems that the present invention attempts to solve.

[Means for solving problems]

The present inventors conducted research to solve the above-mentioned problems, and found that by providing a buffer reservoir or layer near the microbial cells, it is possible to prevent the lifespan of the microorganisms from being shortened due to drying out, and to It has been discovered that it is possible to provide a carbon dioxide sensor with a structure in which carbon dioxide that has passed through a gas permeable membrane is unlikely to be lost.

According to one aspect of the present invention, there is provided a measuring method for measuring the concentration of carbon dioxide in a solution such as a body fluid or in the air, the method comprising: measuring the concentration of carbon dioxide in the solution or the air through a gas-permeable membrane; The permeable carbon dioxide is converted into carbonic acid and/or carbonate ions in a buffer solution placed in contact with the gas permeable membrane, and the carbonic acid and/or carbonate ions are converted into carbonic acid and/or carbonate ions in the buffer solution. Microorganisms that are in contact with the solution and can selectively assimilate carbon dioxide are assimilated, and the decrease in oxygen concentration in the buffer solution caused at that time is measured as a current value using an oxygen electrode, and the change in this current value is A method for measuring carbon dioxide is characterized in that the concentration of carbon dioxide is measured from the amount of carbon dioxide.

According to another aspect of the present invention, there is provided a measuring sensor for determining the concentration of carbon dioxide in a solution or in the air based on amperometry, comprising: an oxygen electrode; a microorganism that is fixed near the cathode and is capable of selectively assimilating the carbon dioxide;
A sensor for measuring carbon dioxide is characterized in that it comprises a combination of a buffer reservoir disposed surrounding the microorganism and a gas permeable membrane disposed covering the buffer reservoir.

Microorganisms that can be advantageously used in carrying out the present invention are microorganisms that can selectively assimilate carbon dioxide, such as cells belonging to the genus Hydrogenomonas and other genera.

The present inventors discovered that Hydrogenomonas sp.TIT/FJ-00
01 bacteria (Feikoken Bacteria No. 8473).

Unidentified TIT/FJ-0002 bacterium (Microtechnical Research Institute No. 84
No. 74), particularly favorable results were obtained.

The mycological properties of these bacterial cells are as follows.

1,' TIT/FJ-0001 bacterium (National Institute of Microbial Technology, Agency of Industrial Science and Technology, Microbial Accession Number: Tetsukoken Bacterium No. 847
No. 3) - A microorganism that uses carbon oxide as its main carbon source 2 Nitrogen gas as its main nitrogen source, hydrogen gas and oxygen gas as its chemical energy sources, and that can be grown using other commonly used inorganic compounds and water. .

fa) Morphological Properties When cultured on agar medium containing Beefex 0.3% and peptone 0.5% at 30°C for 4 days, 1 x 4-5 micron rods, straight or oily; when cultured for 7 days, the above Although some shapes remain, most of them have a short culm shape with a major axis of 1.9 to 2.1 microns. Most exist singly, but in rare cases two or more may be connected. The same goes for liquid culture, but the time required for the concave shape to change is slightly shorter. However, it shows polymorphism. No motility. Lifeson (Leifs)
No flagella are found by staining using the on) method. No spore formation is observed. Durham negative. Non-acid-fast.

(b) Culture properties (1) Broth agar plate culture: Grows to form yellow, smooth and shiny colonies. Colonies are circular. No dye diffusion is observed.

(2) Meat juice agar slant culture: Same as meat juice agar plate culture.

(3) No growth on the surface of meat juice liquid culture.

The viscosity of the culture solution increases slightly due to mucilage production.

(4) Meat juice gelatin puncture culture: No growth. No liquefaction is observed.

(5) Lidmus milk: No coagulation or liquefaction observed.

When cultured for a long time, it becomes alkaline.

(C) Physiological properties Nitrate reduction: Negative denitrification reaction: Negative MR test: Negative VP test: Negative indole production: Negative hydrogen sulfide production: Positive Starch hydrolysis: Negative citric acid utilization (in Oser medium ): Positive use of citric acid (Christensen medium): Positive use of nitrate: Positive extracellular production of pigment: Negative use of ammonium salt: Positive urease: Positive oxidase: Positive catalase: Positive growth temperature range around 30°C (20 The pH range for good growth is around 7.0 (4.0 to 8.6).The pH range for good growth is around 7.0 (4.0 to 8.6).Attitude towards oxygen is aerobic, but grows slightly in anaerobic conditions.

0-F test (by lugh Leifson method)
:Negative carbon source assimilation, acid and gas production (Assimilation, acid and gas production, non-existence, and ambiguity are indicated by + and -1, respectively)Growth in inorganic medium sex:
When culturing in a solid or liquid medium containing only inorganic compounds, in the presence of hydrogen gas and oxygen gas, carbon dioxide or -
Grows vigorously using fixed carbon oxide and nitrogen gas.

Due to the above properties, especially the remarkable feature of growing in the presence of hydrogen gas, oxygen gas, and carbon dioxide gas in an inorganic medium, it is thought that the native species is clearly classified as a member of the genus Hydrogenomonas. Bergey's Manual
of Determinative Bacteri
8th Edition, The WilHarms &
Wilkins Company/Baltimora
by〕.

However, the genus Hydrogenomonas is generally said to have polar flagella, and the ability to fix nitrogen gas is not known for the genus Hydrogenomonas.For example, Hydrogenomonas nitroba^TCC17707 also does not show the ability to fix nitrogen gas. Since it is different from the previously described genus Hydrogenomonas, it may be appropriate to create a separate genus.

However, on the other hand, it is natural that bacteria with properties similar to those in Japan have been isolated in the past, and that some bacteria have been classified as Hydrogenomonas without knowing their ability to fix nitrogen gas. Therefore, I think it is appropriate to classify it as a genus Hydrogenomonas in its home country for the time being.

2. TIT/FJ-0002 bacterium (National Institute of Microbial Technology, Agency of Industrial Science and Technology, Microbial Accession Number: Microbiological Research Institute Bacteria No. 847)
No. 4) Identification of the country of origin has not been completed. However, it has already been scientifically confirmed that it is a microorganism that assimilates carbon dioxide. In addition, the home country is considered to be thermophilic.

The above-mentioned microorganisms can be immobilized on the oxygen electrode by techniques commonly used in this technical field. Methods for preparing microbial membranes that immobilize microorganisms in membranes while maintaining their respiratory activity include, for example, methods in which microorganisms are chemically bonded to membranes (covalent bonding method), microorganisms are bonded to synthetic polymers or natural There are methods such as entrapping immobilization in a polymer matrine lath (entrapping method) and physically adsorbing it to a membrane (adsorption method). It is particularly advantageous to prepare microbial membranes by adsorbing and immobilizing microorganisms on membranes of porous nitrocellulose.

Examples of the oxygen electrode used to measure the decrease in oxygen concentration include polar mouth type and galvanic type oxygen electrodes. In particular, a galvanic type oxygen electrode does not require the use of a special amplifier, so it can be advantageously used for manufacturing a simple sensor.

In addition, gas permeable membranes used for permeating carbon dioxide and oxygen include silicone rubber membranes, fluorine-containing polymer membranes 9, polytetrafluoroethylene (PTFIE) membranes such as Teflon (trade name), and tetrafluoroethylene (PTFIE) membranes such as Teflon (trade name). Copolymer of ethylene and hexafluoropropylene (FE
Examples include P) films.

The buffer solution in the buffer reservoir to be placed surrounding the microbial cells needs to have a high pH value in order to allow it to absorb carbon dioxide that can pass through the gas-permeable membrane. Carbon dioxide gas dissolves in the buffer solution in the form of carbonic acid and/or carbonate ions, making it more easily taken up by microorganisms. In the present invention, the type of buffer used and pl-1
The activity of microorganisms can be determined by selecting the appropriate value.
Not only can it be kept stable over J periods, but
This has the effect of preventing carbon dioxide from escaping to the outside through the gas-permeable membrane, making stable measurements possible. An example of a suitable buffer is 0.05M KH2PO.

/Na0tl buffer solution can be mentioned.

Furthermore, the buffer reservoir itself contains /! (In order to improve I + constant efficiency and accuracy, it is desirable to arrange the dialysis membrane in such a way as to enclose the entire cathode of the oxygen electrode and the microorganisms. An example of a suitable membrane is an acetyl cellulose film. .

〔Example〕

In this example, a carbon dioxide measuring sensor having the structure shown in FIG. 1 was manufactured. 7 in the figure is an oxygen electrode main body, which has a cathode 5 made of platinum and an anode 6 made of lead. The electrolyte 8 of this electrode is IN
It was KOH. The tip of the oxygen electrode 7 is coated with Teflon (trade name) so that the cathode 5 is completely covered.
It is coated with a gas permeable membrane 4 manufactured by. In addition, this film 4
The microorganism 3 immobilization membrane 2 placed outside is covered entirely with a dialysis membrane 10 made of acetyl cellulose. Further, as shown in the figure, the immobilized microorganism 2 and the dialysis membrane 10 are separated by a spacer 1 made of Teflon (trade name).
The buffer solution 13 is immersed in a high pH buffer solution 13 stored in the buffer solution 13, and the buffer solution 13 is in contact with the outside through a gas permeable membrane 13 made of Teflon (trade name). There is. 9 is a rubber band (an O-ring may be used) for fixing the gas permeable membrane. An example of mounting this sensor is shown below: In a pre-sterilized Sakalo flask, add Beefex 0.3%,
A liquid medium Loom 1 consisting of 0.5% peptone and 99.2% distilled water was added. In this medium, Hydrogenomonas TIT/FJ-0001 bacteria (Feikoken Bacterial Serial No. 8473) were added.
No.) was inoculated and cultured under aerobic conditions at 30°C for 4 days. Next, add 5 ml of the culture medium to the bacteria! 0.05M K11ZPO4/Na
Washed with 0II buffer (pl!5.5).

Next, as shown in FIG. 1, the membrane 2 that has filtered the bacteria as described above is placed with the side with the bacteria 3 facing the cathode 5 of the galvanic type oxygen electrode 7, and then The outside thereof was wrapped with a dialysis membrane 10 made of acetyl cellulose so that the cathode 5 portion of the electrode was completely covered with the bacterial cell immobilization membrane 2 and oxygen 1. Furthermore, these are spacer 1
0.05M K11zPO4/Na accumulated by 1
011 immersed in buffer M (pH 7.5), and contacted with the outside through a gas permeable membrane 12 made of Teflon (trade name). Finally, by fixing with a rubber band 9, the illustrated carbon dioxide sensor was completed.

In this example, as an example of carbon dioxide measurement, the concentration of dissolved carbon dioxide in the buffer solution was measured. Since it was thought that the dissolved oxygen concentration changes due to carbon dioxide, the pH
A carbonate solution was added to a buffer solution maintained at 5 to 8 to convert some of the carbonate ions into dissolved carbon dioxide, which was then measured. The actual measurement is shown below: The carbon dioxide sensor manufactured as described above was
5M K11zPO4/Na011 buffer (30°C,
pH5,5) 50m7! It was immersed in water and left until it stabilized. Then, in order to measure the concentration of dissolved carbon dioxide in the buffer solution, I N K2CO3 was added into the buffer solution and supplied by converting some of the carbonate ions into carbon dioxide.

As a result of the measurement, a stable calibration curve as shown in FIG. 3 was obtained. From this figure, K2CO3? It can be seen that the current value of the sensor decreases in response to a change in M degree (change in dissolved carbon dioxide concentration), and that the dissolved carbon dioxide concentration can be measured from the amount of change in this current value.

〔Effect of the invention〕

According to the present invention, the carbon dioxide sensor can be manufactured amperometrically, which reduces the influence of contaminants and enables more sensitive measurement than conventional potentiometric carbon dioxide electrodes. I can do it, and also.
The application fields of the sensor can also be expanded. Further, according to the present invention, since microbial cells can be stored without drying, it is possible to extend the life of the microbial electrode (sensor) itself. Furthermore, since carbon dioxide that has passed through the gas-permeable membrane can be prevented from escaping through the gas-permeable membrane again, stable measurement characteristics can be obtained.

[Brief explanation of drawings]

FIG. 1 is a structural diagram showing a preferred example of the carbon dioxide sensor of the present invention; FIG. 2 is a structural diagram showing a preferred example of the carbon dioxide sensor developed by the present inventors and for which a patent application has been separately filed; FIG. 3 is an example of a calibration curve obtained using the sensor of the present invention shown in FIG. In the figure, 2 is an immobilized microorganism membrane, 3 is a microorganism, 4 is a gas permeable membrane, 5 is a cathode, 10 is a dialysis membrane, 12 is a gas permeable membrane, and 13 is a buffer solution. Structural diagram of carbon dioxide sensor 1 Fig. 2 Immobilized microorganism membrane 3 Microorganism 4 Gas permeable membrane 5 Cathode 10 Dialysis membrane 12 Gas permeable membrane 13 ...Structure diagram of buffer solution carbon dioxide sensor Figure 2

Claims (1)

[Claims] 1. A measuring method for measuring the concentration of carbon dioxide in a solution or in the air, which comprises selectively permeating the carbon dioxide in the solution or in the air through a gas-permeable membrane. carbon dioxide and/or carbonate ions in the form of carbonic acid and/or carbonate ions in a buffer solution placed in contact with the gas permeable membrane; The reduction in oxygen concentration in the buffer solution caused by selective assimilation by microorganisms is measured as a current value using an oxygen electrode, and the concentration of carbon dioxide is measured from the amount of change in this current value. A carbon dioxide measurement method characterized by: 2. The carbon dioxide measuring method according to claim 1, wherein the gas permeable membrane is a silicone rubber membrane. 3. The carbon dioxide measuring method according to claim 1, wherein the gas permeable membrane is a fluorine-containing polymer membrane. 4. The carbon dioxide measuring method according to any one of claims 1 to 3, wherein the microorganism is a bacterial cell belonging to the genus Hydrogenomonas. 5. The carbon dioxide measuring method according to any one of claims 1 to 4, wherein the oxygen electrode is a galvanic oxygen electrode. 6. The carbon dioxide measuring method according to any one of claims 1 to 5, wherein the buffer has a high pH value. 7. A measuring sensor for measuring the concentration of carbon dioxide in a solution or in the air based on amperometry, which is fixed in the vicinity of an oxygen electrode and a cathode of the oxygen electrode, and is fixed in the vicinity of the oxygen electrode and the cathode of the oxygen electrode. It is characterized by comprising a combination of a microorganism capable of selectively assimilating carbon dioxide, a buffer reservoir disposed surrounding the microorganism, and a gas permeable membrane disposed covering the buffer reservoir. A sensor for measuring carbon dioxide. 8. The sensor for measuring carbon dioxide according to claim 7, wherein a dialysis membrane is disposed within the buffer reservoir and surrounds the entire cathode and the microorganisms. 9. The sensor for measuring carbon dioxide according to claim 7 or 8, wherein the oxygen electrode is a galvanic oxygen electrode. 10. The sensor for measuring carbon dioxide according to claim 7, 8 or 9, wherein the gas permeable membrane is a silicone rubber membrane. 11. The sensor for measuring carbon dioxide according to claim 7, 8, or 9, wherein the gas permeable membrane is a fluorine-containing polymer membrane. 12. The sensor for measuring carbon dioxide according to any one of claims 7 to 11, wherein the microorganism is a bacterial cell belonging to the genus Hydrogenomonas. 13. The sensor for measuring carbon dioxide according to any one of claims 7 to 12, wherein the buffer in the buffer reservoir has a high pH value.