JPH0740009B2 - Biomass measurement electrode - Google Patents

Description

TECHNICAL FIELD The present invention relates to an electrode for on-line measurement of a biomass in a culture device. Therefore, the present invention
Bioindustry, medical, food industry, sewage,
It plays a very important role in fields such as wastewater treatment.

(Prior Art) Bioreactors and culturing devices that produce useful substances using various microorganisms, animal cells, plant cells, etc., control the bioreactor and culturing devices because the amount of living organisms inside them changes moment by moment. It is very important to measure the amount of living organisms in order to carry out or to know the internal condition.

In bioreactors, etc., for various microorganisms with small cell size, in measuring the bacterial cell concentration in the suspension solution,
It is possible to measure the concentration of microorganisms based on various optical properties of the microorganisms in the medium. However, various measurement methods using light have problems such as misidentification of the biomass due to the mixture of SS (suspended solids) other than organisms, increase in error due to color and bubbles of the measurement solution, and complexity of the measurement mechanism. In such a case, at present, cells have to be collected from a reactor or a culture device by a sampling method, and there is a high risk of contamination of various bacteria in the culture system. In many cases, it was necessary to improve the culture efficiency. In addition, it is impossible to reflect information such as biomass on online control of reactors and culture devices, and it has been emphasized to develop a method that can measure biomass online without sampling the organism. .

Recently, the present inventors have invented a method for measuring the amount of living organisms in real time online by mounting electrodes in a culture device and measuring electric conductivity (conductivity) and / or electric capacity (dielectric constant). (See Japanese Patent Application No. 62-224018: Japanese Patent Laid-Open No. 64-67200).

The present invention uses a spray-processed metal as an electrode, and a sintered metal as an electrode, as a biological amount measuring electrode capable of measuring an electric capacity by the above-mentioned measuring method. No such electrode has hitherto been known and is new.

(Problems to be Solved by the Invention) Conventionally, as a biological amount measuring electrode for measuring biological concentration by measuring electric capacity and / or electric conductivity,
Stainless steel, platinum, platinum black, etc. were used. However, it was difficult to measure the amount of organisms in the high ion concentration region for stainless steel and platinum electrodes because the influence of electrode polarization extends to a high frequency band. On the other hand, the platinum black electrode has an excellent property that the effect of electrode polarization is limited to a low frequency band, but it has a drawback that the plated part is peeled off when mechanical stimulus is applied. It was

(Means for Solving Problems) The present invention has been made to solve the above-mentioned drawbacks all at once, in which the surface of a metal used as an electrode is subjected to thermal spraying, and a sintered metal is used as an electrode. Therefore, compared to the case of using stainless steel, platinum, titanium, etc. as the electrode, by reducing the effect of electrode polarization, it is possible to measure the biomass even in a high ion concentration solution, and compared to the platinum black electrode. The present invention has been completed by having excellent strength.

First, we describe what the electrode polarization is and how it affects the capacitance value. Electrode polarization is due to the electric double layer, and the concept of the electric double layer has been established in a very wide range, and when any fixed surface including the electrode comes into contact with the electrolyte solution, the electric double layer is formed at the solid-liquid interface. (Electrochemical assay, Fujishima et al., Gihodo Publishing, P.4)
Four).

The value of the electrode polarization depends on the measured frequency, and exhibits frequency characteristics as shown in FIG. Further, this frequency characteristic is related to the ion concentration in the electrolytic solution, and when the ion concentration is high, it shifts to the high frequency side as shown in FIG. Now, in the frequency band where the measurement frequency is high and the electrode polarization is almost negligible, the capacitance value is not affected by the ions in the solution. Therefore, in such a frequency band, the capacitance of the solution containing cells depends on those existing in the electric field formed by the electrodes (eg, cells, water, gas such as air, substances such as sugar and protein, etc.). It's decided Among them, cells are known to have a very large capacitance value, and it is known that as the number of cells in a solution increases, the capacitance value increases in a constant relationship with the cell concentration (Japanese Patent Laid-Open Publication No. Sho. 64-67200).

If the electrical conductivity does not fluctuate even in the frequency band where the measurement frequency is low and is affected by the electrode polarization, it should be possible to obtain a constant relationship between the capacitance value and the cell concentration. However, in a culture system containing cells, the ion concentration and composition change due to cell metabolism and the like. In addition, the cells themselves become a resistance component in the solution, and the change in the cell concentration itself becomes a factor of fluctuation in the electrical conductivity in the solution. Therefore, it is practically impossible to measure the biomass in the frequency band where the influence of electrode polarization cannot be ignored.

On the other hand, the increase in the capacitance value due to the presence of biological cells is such that the measurement frequency is approximately 10 MHz or less (JP-A-64-67200). Therefore, in order to calculate the cell concentration by measuring the electric capacity, it is necessary to use a frequency of several MHz or less. At this time, the influence of electrode polarization needs to be small. Therefore, the state of the effect of electrode polarization is as small as possible,
In other words, it is desirable to measure with the electrode polarization shifted to the low frequency side as much as possible.

As mentioned above, electrode polarization has a higher frequency band that is affected by higher ion concentration in the solution.
Another factor is the electrode material. In other words, it can be considered that a material having a property that the electrode polarization is shifted to the low frequency side is a better material. Since the distance between electrodes, the electrode area, and the ion concentration in the solution are also related to the electrode polarization as described above, there are cases where the amount of living organisms can be measured even with electrodes of the same material, but a good electrode material By using, it becomes possible to measure under a wider range of conditions.

Therefore, as a result of earnestly examining the target electrode material from various directions, we found that the metal whose surface was spray-processed and sintered metal were suitable, and based on this new knowledge, further research was conducted. The invention was completed.

Hereinafter, the present invention will be described in detail.

First of all, the measuring cell 1 shown in FIG. 2 (for example, 20 mm
X 20mm x 40mm) and prepare its inner wall 2,2 (for example, 20mm x 2)
0 mm surface), stainless steel (a), titanium (b), platinum (c), platinum black (d), stainless steel surface sprayed with stainless steel (e), titanium surface sprayed with titanium (f) The electrodes 3 and 3 each made up of 1 were attached, and the internal space 4 was filled with a 1% NaCl solution, and the frequency characteristic of the electric capacity was measured.

The measurement results are shown in FIG. From these results, it can be seen that in any case, the capacitance value increases as the measurement frequency decreases, which is an effect of electrode polarization. From this result, it was found that there was no difference in the frequency characteristic of the electric capacity between the stainless (a), titanium (b) and platinum (c) electrodes. Further, the sprayed electrodes of stainless steel (e) and titanium (f) are affected by electrode polarization up to a high frequency band as compared with the platinum black (d) electrode, but compared with stainless steel (a) and titanium (b). It was revealed that the effect of polarization was limited to the low frequency band. From this, it was found that the thermal spraying of the metal surface has the effect of reducing the influence of electrode polarization when measuring the electric capacity of the solution.

As the thermal spraying electrode in the present invention, a metal atomized and atomized by using compressed air is sprayed onto the surface of the object to be sprayed, which has been roughened in advance, and mechanically adhered thereto. It includes all electrodes manufactured by the well-known thermal spraying process.

Moreover, it is known that the electrode area of the platinum black electrode is substantially large, and the effect of electrode polarization may be shifted to the lower frequency side by enlarging the electrode area of the electrode. Based on the idea that the influence of electrode polarization can be reduced when measuring the capacitance of a solution), the electrode surface is enlarged by the thermal spraying method as described above, and the electrode area by the sintering method. An electrode having an enlarged size was manufactured.

In the present invention, as the sintered metal electrode, an electrode manufactured by a sintering operation according to a conventional method, in which a powdery metal is pressed and solidified, and this is heated to obtain a briquette or lumpy metal or alloy It is a broad inclusion. It was produced in this way. If the surface of the metal sprayed or sintered metal is used as the electrode surface, the target biological quantity measuring electrode can be freely prepared, and these electrode surfaces have the double structure as described below. By using as
It is possible to accurately and quickly measure the desired amount of biomass.

The electrode having a double structure in the present invention includes all electrodes whose electrode surface has a double structure so that the outer electrode covers the inner electrode, for example, the electrode surface having a double cylindrical shape, Alternatively, an electrode surface having a double shell shape or the like is appropriately used.

FIG. 4 shows an example of the former type,
11 indicates a double cylindrical electrode, 12 indicates an inner electrode, and 13 indicates an outer electrode. 14 is a support made of an insulator. Outer electrode
13 has a cylindrical shape with an open lower surface, and four holes are formed on each of the side surface and the upper surface thereof.

In the double-shell type electrode 11 having such a structure, the outer electrode 13 was made of stainless steel, the inner electrode 12 was made of titanium spraying and stainless sintering, and a biological concentration measurement experiment was performed. In this electrode, the electrode area (inner electrode) is generally smaller and the inter-electrode distance is shorter than the electrode in the measuring cell shown in FIG. In addition, the electric conductivity when measuring a solution with the same ion concentration is much higher than that of the measurement cell (the electric field in the measurement cell can be made up by the cell volume, but the electric field spreads over a wide range in the electrode inserted in the solution ). Under these conditions, the effect of electrode polarization on this electrode shifts to the high frequency side as compared with the measurement cell.

Examples of the present invention will be described below.

Example 1 A concentric cylindrical electrode as shown in FIG. 4 was produced and a yeast measurement experiment was conducted. The outer electrode has a diameter of 40 mm, a height of 40 mm, and a hole with a diameter of 20 mm at four side surfaces and a diameter of 10 mm at four upper surfaces so that the solution can freely flow in and out of the electrode. The underside is open. The electrode material is stainless steel. The inner electrode has a diameter of 10 mm and a height of 2 mm, and the material is sintered stainless steel.

This electrode was mounted in a culture tank (internal volume 5 L), filled with 3 L of culture solution, and the electric capacity was measured. Next, baker's yeast was sequentially put into the solution to measure the electric capacity, 10 ml of the solution was sampled, and the yeast concentration was determined by the dry weight method.

FIG. 5 shows the relationship between the capacitance value and the yeast concentration. The vertical axis represents the difference between the electric capacity of the yeast solution at the measurement frequency of 1 MHz and the culture solution containing no yeast, and the horizontal axis represents the yeast concentration. The ion concentration of the culture solution used here was equivalent to 1% NaCl, and a linear relationship was observed between the two even under such high ion concentration conditions (the inner electrode made of stainless steel has a polarization. The effect of is not measurable.),
It was found that the yeast concentration can be measured using this electrode.

Example 2 A concentric cylindrical electrode as shown in FIG. 4 was produced and a yeast measurement experiment was conducted. The outer electrode has a diameter of 40 mm, a height of 40 mm, and a hole with a diameter of 20 mm at four side surfaces and a diameter of 10 mm at four upper surfaces so that the solution can freely flow in and out of the electrode. The lower surface is an opening. The electrode material is stainless steel. The inner electrode has a diameter of 5 mm and a height of 5 mm, and the material is titanium to titanium with a thickness of 0.1 mm.
It was sprayed.

FIG. 6 shows the relationship between the capacitance value and the yeast concentration. The vertical axis represents the difference between the electric capacity of the yeast solution at the measurement frequency of 1 MHz and the culture solution containing no yeast, and the horizontal axis represents the yeast concentration.
The ion concentration of the culture solution used here was equivalent to 1% NaCl, and a linear relationship was observed between them even under such high ion concentration conditions (the inner electrode made of titanium without thermal spraying was used). However, it was not possible to measure because of the large influence of polarization), and it was found that the yeast concentration can be measured using this electrode.

(Effect of the invention) Due to the electrode using a metal whose surface is sprayed or sintered metal, it is impossible to use it repeatedly in the conventional method. An electrode having excellent properties was obtained.

Therefore, according to the present invention, the amount of microorganisms, animal cells and plant cells can be nondestructively measured, and biotechnology, vaccine production, experiments using microorganisms, animal cells and plant cells, technical fields of research, and others The present invention can be widely used in various fields.

[Brief description of drawings]

FIG. 1 is a diagram showing an example of frequency characteristics of electric capacities in various NaCl solutions. It measured using the measuring cell of cell constant (0.08). The salt concentration of the measurement solution is as follows. a: 0.5% NaCl b: 1% NaCl c: 2% NaCl d: 3% NaCl e: 5% NaCl Figure 2 shows the appearance of the measuring cell. FIG. 3 is a diagram showing the results of measuring the frequency characteristic of the electric capacity of a 1% NaCl solution by mounting various metal electrodes (a to f) on the measuring cell shown in FIG. However, a: stainless steel, b: titanium, c: platinum, d: platinum black, e:
Spraying stainless steel on the surface of stainless steel, f: spraying titanium on the surface of titanium Fig. 4 shows the appearance of the concentric cylindrical electrode. FIG. 5 is a graph showing the measurement results of the yeast concentration using a concentric cylindrical electrode made of sintered stainless steel. FIG. 6 is a graph showing the measurement results of yeast concentration at a concentric cylindrical electrode using sprayed titanium.

Claims (3)

[Claims] 1. An electrode for measuring a biological amount, characterized in that a metal whose surface is sprayed is used as an electrode surface to measure an electric capacity. 2. An electrode for measuring a biological amount, which measures a capacitance by using a sintered metal as an electrode surface. 3. The biomass measuring electrode according to claim 1, wherein the electrode surface has a double structure so that the outer electrode covers the inner electrode.