JPS5836736B2 - Volatile substance measurement method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for selectively measuring volatile substances using microbial electrodes.

Volatile substances such as ethanol, methanol, and acetic acid are used as fermentation raw materials, and gas chromatography and semiconductor detection methods have conventionally been used to measure these volatile substances in fermentation liquids.

However, when a large amount of non-volatile substances are contained, such as in fermentation liquid or waste water, the gas chromatography method has the drawback that the non-volatile substances accumulate in the column, making long-term measurements difficult.

Moreover, since the equipment becomes complicated, online management is not easy.

On the other hand, the semiconductor detection method also has problems with its stability, and it has been pointed out that it lacks reliability when used for online management.

As a result of extensive research into a measurement method that is simple, stable, reliable, and suitable for on-line management, the inventors of the present invention have found that microorganisms that utilize volatile substances and consume oxygen can be detected using the diaphragm of the oxygen electrode. By using a microbial electrode sealed or immobilized between a volatile substance-permeable membrane covering the membrane, we have developed a method for selectively measuring volatile substances suitable for this purpose.

That is, the present invention brings the microbial electrode into contact with a test solution containing a volatile substance, measures the rate of decrease in current of the microbial electrode, and calculates the proportionality between the rate of decrease in current and the concentration of the volatile substance in the test solution. This method uses the relationship to selectively measure the concentration of volatile substances in a test solution.

The method of the present invention is extremely simple, and is a new measuring method that can selectively measure volatile substances stably and with good reproducibility, in an extremely short time of 2 to 10 minutes, without any trial or effort.

The method of the present invention will be explained in detail below.

The microorganisms used in the present invention may be microorganisms that have the ability to assimilate volatile substances and simultaneously consume oxygen, such as yeasts such as Trichosporon brassicae and Satucharomyces cerevisiae, and Plevibacterium flavum A.
Various activated sludges containing bacteria such as TCC14067, 21 475, or a mixture of various microorganisms are used.

Silicone membranes are the best volatile substance permeable membranes.
Polyethylene membranes, polybutadiene membranes, polypropylene membranes, polyester membranes, Teflon (registered trademark) membranes, etc. can also be used.

The point is that it selectively permeates dissolved oxygen and volatile substances, and it is desirable to have a certain degree of permeability and a movement speed of volatile substances that is higher than that of dissolved oxygen.

The oxygen electrode may be either a Galpanic type or a Polaro type, and any commercially available one can be used.

The microbial electrode of the present invention is manufactured as follows.

First, the microorganism is cultured in a conventional nutrient medium using a conventional method, and the microbial cells are separated from the culture solution to form a paste-like suspension of the bacterial cells.

Apply the bacterial cells on the diaphragm of the electrolytic electrode, and if necessary, support it with a nionic wire or the like, cover it with the volatile substance permeable membrane, and securely attach it to the electrode with a rubber band or the like. A microbial electrode as shown in FIG. 1 can be obtained.

Figure 1 shows a microbial electrode using a galpanic type oxygen electrode, in which 1 is a microbial layer, 2 is a support layer, 3 is a volatile substance permeable membrane, 4 is a diaphragm of the oxygen electrode, and 5 is a A platinum cathode, 6 an aluminum anode, 7 a potassium chloride solution, and 8.8' a rubber band.

An immobilized microbial membrane may be used instead of the microbial layer 1 in Figure 1, and the immobilized microbial membrane is obtained by immobilizing microorganisms with collagen, polyacrylamide gel, etc. according to the usual enzyme entrapment immobilization method. All you have to do is make one, cut it to an appropriate size, and enclose it.

However, the effect does not change at all even if an immobilized microbial membrane is used, so there is no particular need to use an immobilized microbial membrane.

The principle of the method of the invention is explained as follows.

The microbial electrode is brought into contact with a test solution containing a non-volatile substance.

Volatile substance permeable membranes such as silicone membranes allow ethanol, methanol, acetic acid, lower ketones, etc. with low boiling points to pass through, but sugars, amino acids, organic acid salts, inorganic salts, etc. do not pass therethrough; Proportional to the vapor pressure of a substance.

Therefore, only oxygen and volatile substances can reach the bacterial cell layer.

Since microorganisms consume (assimilate) volatile substances and at the same time consume oxygen, the dissolved oxygen concentration near the diaphragm decreases in proportion to this, and as a result, the current at the microorganism electrode decreases.

It is known that a proportional relationship exists between the rate of decrease in current in a microbial electrode and the concentration of organic matter consumed and assimilated by the microorganism, but the rate of decrease in current in the microbial electrode of the present invention was also tested. It is well proportional to the concentration of volatile substances in the liquid.

Therefore, by measuring the rate of decrease in this current, the concentration of volatile substances can be determined.

FIG. 2 shows one embodiment in which the method of the invention is carried out continuously.

In Figure 2, 1 is a microbial electrode, 2 is a rubber packing, 3 is a flow cell (content: 2-10 mA!), 4 is a magnetic cooler, 5 is a stirrer, 6 is a recorder, γ is an air inlet, 8 is water ( carrier liquid) injection port, and 9 indicates a sample injection port.

The method of the present invention will be explained along the system shown in FIG.
First, water saturated with oxygen as a carrier liquid is pumped through the measurement cell (flow cell 2), and the sample is placed in the sampler at regular intervals (10 to 2
When the sample is injected from the injection port 12 for 0 minutes), the sample is diluted appropriately with the carrier liquid (0.01-1%) and enters the flow cell, where it comes into contact with the microbial electrode 1. Only the volatile substances and dissolved oxygen of the sample in the flow cell are present. permeates the volatile substance permeable membrane, reaches the microbial layer, is assimilated by the microorganisms, and consumes oxygen.

Therefore, the oxygen concentration decreases, the microbial electrode current decreases, and the peak of the decreased current is recorded on the recorder 9 (see FIG. 4).

By determining the relationship between the concentration of the substance to be measured and the peak height, the concentration can be easily determined from the peak height.

On the other hand, when performing batch measurements using the method of the present invention, the microbial electrode is brought into contact with the sample solution for a certain period of time (0.5 to 5
The amount of decrease in current per minute) or the equilibrium current value may be measured.

When measuring while keeping the dissolved oxygen concentration in the test liquid constant during measurement, the current value becomes constant and reaches an equilibrium state as the measurement time increases.

Since the current value or the current decrease value at equilibrium is also proportional to the volatile substance concentration in the test solution, it can also be determined from the amount of decrease in the equilibrium current.

It is desirable to carry out the measurement under conditions suitable for the activity of the microorganisms used, usually pH 4-8.01 and temperature 15-45.
It is sufficient to perform this under the conditions of ℃.

Since the reaction of microorganisms is fast, a contact time of 0.5 to 10 minutes between the microorganism electrode and the sample is sufficient.

This short contact time with the sample not only shortens the measurement time, but also reduces contamination of the microbial electrode used, making it possible to use it for a long period of time.

When measuring acetic acid by the method of the present invention, it can be easily measured by adjusting the pH at the time of measurement to an acidic pH of 4 or less.

Depending on the type of microorganism used, it is also possible to separate and measure ethanol and methanol, and it is also possible to analyze gas components such as methane and hydrogen.

As described above, the present invention provides an excellent method for selectively measuring volatile substances contained in fermentation liquid or waste water containing a large amount of non-volatile substances.

Example 1 Potato dextrose agar Frant medium (pH 6.0
Trichosporon brassicae (Trichosporonbrassi
cae) CBS 6 3 8 2 microbial cells ←Apply a platinum loop to a Millipore filter with a diameter of 10 mm, paste it on the diaphragm of a galpanic type oxygen electrode, and cover it with a silicone membrane (for DCO2 sensor, si I). icon
e Film, Radiometer, Cop
enha-gen) and fixed to the electrode with a rubber band to prepare a microbial electrode as shown in FIG.

Using this microbial electrode, we assembled the continuous measurement system shown in Figure 2 and conducted the following experiments.

First, water saturated with dissolved oxygen is passed through the system as a carrier liquid (flow rate: 3.OTIL/min), and the electrode current is recorded on a recorder (baseline). After the baseline is stabilized, an ethanol solution with a constant concentration is injected from the sample injection port. were injected sequentially at 30 minute intervals (pulse width 1.5 minutes).

The vertical axis in Figure 3 shows the peak height of the decrease in the obtained electrode current, and the horizontal axis shows the concentration of ethanol in the flow cell. The peak heights are well proportional.

In exactly the same way, ethanol, glucose and ethanol,
The influence of G/I,/course was examined by sequentially injecting aqueous solutions of glucose and glucose, but no influence of glucose was observed.

Example 2 Satucharomyces cerevisiae - CBS 1 1 7 2
was subjected to flask shaking culture at 30° C. for 35 hours using the following medium.

The culture solution was centrifuged to remove bacteria, and ethanol was added to the resulting supernatant to prepare samples with an ethanol concentration of 0.1 to 1.0%.

This was measured in exactly the same manner as in Example 1, and the peak height of the electrode current was plotted against the alcohol concentration in the flow cell measured by gas chromatography.

This is shown as a white circle in Figure 3.

Figure 3 shows that this measurement method is completely unaffected by impurities.

Example 3 Sodium acetate is added from 0.4% to 1.6% glucose aqueous solution
2.0% and the pH was adjusted to 4.0 with phosphoric acid.

This was injected into the flow cell with a pulse width of 10 minutes in exactly the same manner as in Example 1, and the voltage output was measured.

A linear relationship as shown in FIG. 4 was observed between the acetic acid concentration of the sample in the flow cell and the peak of the electrode current.

[Brief explanation of the drawing]

Figure 1 is a schematic diagram of the microbial electrode of the present invention, Figure 2 is a schematic diagram of a volatile substance measurement system using the microbial electrode, Figure 3 is a graph showing the relationship between the ethanol concentration and electrode current in a pure system, and Figure 4 is a graph showing the relationship between acetic acid concentration and electrode current.

Claims (1)

[Claims] 1. A microbial electrode consisting of a microorganism that assimilates volatile substances and consumes oxygen is sealed between the diaphragm of the oxygen electrode and the volatile substance permeable membrane covering it, and the microbial electrode is brought into contact with the test liquid, and the rate of decrease in the electrode current is measured. Or, a method for measuring volatile substances, which comprises measuring equilibrium currents and measuring volatile substances using the rate of decrease of these currents or the proportional relationship between the equilibrium bud current and the concentration of volatile substances.