JPH02285252A - Manipulating method of microbial sensor - Google Patents

Abstract

PURPOSE:To speedily and easily measure the concentration of an object with good reproducibility by a method whereby a microbe is fixed to a porous carrier, passed through a solution containing an object to be measured, bred to be 0.1 mg or more by the dry weight per 1 ml of the porous carrier and used. CONSTITUTION:In using a microbial sensor, first, a microbe is fixedly adhered to a porous carrier and passed through a liquid containing an object to be measured so that said fixed microbe is bred to be 0.1mg or more by the dry weight per 1ml of the porous carrier. The microbial sensor is in such a structure that a part where the porous carrier which is a fixer and carrier of a microbe is filled in a fixed microbe container is separated from an electrochemical device part. In comparison with a film-type carrier, the microbe can move in the porous carrier at much higher speeds. Therefore, the measurement becomes possible even by a small amount of microbes, and the time for breeding the microbes can be shortened.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a novel method of operating microbial sensors.

[Conventional technology and its problems]

Biocatalysts such as enzymes and microorganisms have advanced molecular identification functions that are not found in conventional physical and chemical sensors.
The development of so-called biosensors is actively being carried out in various fields.

For example, in a glucose sensor, an enzyme is immobilized on a membrane-like carrier, and glucose, which is the object to be measured, is reacted with the enzyme within the membrane-like carrier, and the concentration of the reaction product hydrogen peroxide or the enzyme consumed in the reaction is determined. It is being detected and converted into glucose flux. Also, special public Sho 5 B-30
No. 537 discloses that BOD (biochemical oxygen demand) is determined by immobilizing microorganisms on a membrane-like carrier and measuring the concentration of oxygen consumed during the reaction between the immobilized microorganisms and pollutants in wastewater. Disclosed is a BOD sensor that determines the amount of

These glucose sensors and BOD sensors have a structure in which enzymes and microorganisms are immobilized on a membrane-like carrier, and this immobilization membrane and an electrochemical device are in close contact. However, in this case, most of the movement of the substance to be measured is due to diffusion within the membrane-like carrier, resulting in a problem that the rate of substance movement becomes very low and it takes a considerable amount of time for the electrode to show a stable value.

Furthermore, in order to utilize the biocatalysts used in these biosensors as molecular identification elements, it is necessary to immobilize (insolubilize) the biocatalysts without losing their functions. Methods for immobilizing biocatalysts include (1) covalent bonding, (2) crosslinking, (3) entrapping, and (4) adsorption, but the operations are generally complicated and each method is complicated. There is no permanent immobilization method for biocatalysts, and considerable research is required before finding an appropriate immobilization method. Furthermore, with these immobilization methods, it is still inevitable that the biocatalyst itself undergoes chemical or physical denaturation during immobilization, which frequently results in a decrease in its activity. .

On the other hand, if a specific microorganism itself is used as a biocatalyst, it can be immobilized relatively easily without complicated methods. For example, microorganisms such as activated sludge used to purify wastewater release sticky substances outside the cells during their metabolic process.

Therefore, these microorganisms can be easily attached and immobilized on a porous carrier due to the adhesive properties of their sticky substances, and can be used as molecular identification elements in sensors.

However, with such adhesion and immobilization using adhesive substances, the amount of microorganisms that adhere to the porous carrier is small, and the amount of microbial reaction products is reduced. Chemical devices have a problem in that they cannot be used as-is as microbial sensors because they cannot sufficiently detect changes.

[Means to solve the problem]

In this situation, the present inventors conducted intensive research and found that after attaching and immobilizing microorganisms to a porous carrier, a solution containing the analyte to be measured was used as a substrate, and the microorganisms were kept constant on the surface of the porous carrier. They discovered that if the biocatalyst is grown to a larger amount and then used as a microbial sensor, it is possible to easily detect chemical changes caused by the reaction between the biocatalyst and the object to be measured.
The invention has been completed.

That is, the present invention provides a microbial sensor that includes a porous carrier for adhering and immobilizing microorganisms and an electrochemical device for quantifying substances produced or consumed by the action of these microorganisms. The microorganisms are attached and immobilized, and a solution containing the target substance to be measured is passed through the porous carrier, so that the dry microorganism weight is 0 per oli of the porous carrier.
．． The present invention provides a method for operating a microbial sensor, which is characterized in that the microbial sensor is grown until it reaches 1 mg or more before use.

A porous carrier is used as a carrier for immobilizing microorganisms used in the method of the present invention, and as shown in FIG. It has a structure in which the part filled with the porous carrier, which is a carrier, and the electrochemical device part are separated. When a porous carrier is used as in the present invention, the liquid movement speed within the carrier is much higher than that of a membrane carrier, so it is possible to measure even a small amount of microorganisms.
Moreover, the time required for the subsequent proliferation of microorganisms can also be shortened.

The porous material used for the porous carrier has a diameter of 0.00 mm, which allows microorganisms to penetrate into the surface or inside.
The material is not particularly limited as long as it is an inorganic or organic material that has many pores of 1 μm or more. Specifically, for example, porous glass, porous ceramics, activated carbon, porous polymers, cavernous tissue-like materials (porous sponge, etc.), molded bodies of stainless steel wire mesh, etc. may be mentioned.

In the method of the present invention, microorganisms immobilized on a porous carrier are grown before being used as a microbial sensor.

Here, it is necessary to use a solution containing the analyte to be measured as a substrate for the growth of microorganisms.

If a substrate other than the substance to be measured is given, microorganisms that induce enzymes suitable for the given substrate will proliferate, so if measurements are performed after the growth of such microorganisms, the induced enzyme may not be present in the substance to be measured. It becomes unsuitable, and sufficient chemical changes do not occur, resulting in the inability to measure the concentration of the analyte.

Further, the microorganisms are grown until the dry weight of microorganisms becomes 0.1 mg or more, preferably 1 mg or more per ml of the porous carrier. The amount of microorganisms grown is 0 or 1
If the amount is less than mg, when used as a microbial sensor, it will not be possible to cause a sufficient amount of reaction products or chemical changes such as oxygen consumption, making it impossible to detect with an electrochemical device.

Next, regarding the microorganism sensor of the present invention, using FIG. 1 showing one embodiment thereof, a BO using an oxygen electrode as an electrode
This will be explained using D measurement as an example.

In FIG. 1, 1 is a container for selling immobilized microorganisms, and a microorganism immobilization carrier 11 is filled therein. Reference numeral 2 represents an electrode, and the measurement results thereof are recorded on a recorder 7. To measure the BOD value of the measurement sample 8 using this microbial sensor, the following procedure may be performed. That is, first, the switching valve 5.6 is operated so that the microorganism suspension 9 circles around the immobilized microorganism sales container 1 filled with the microorganism immobilization carrier, and the microorganism suspension is sent using the pump 4. Then, the microorganisms are attached and immobilized on the microorganism immobilization carrier 11.

When used as a BOD sensor, the microbial suspension to be passed through may be one containing aerobic microorganisms, and may be a suspension in an activated sludge treatment tank, river water, or the like. The microorganism immobilization operation may be carried out by passing the microorganism suspension into a container filled with resin as described above, but it is also possible to add a microorganism immobilization carrier to the microorganism suspension and shake at specified times. ,
Alternatively, it may be adsorbed by stirring and then taken out and filled into containers for selling immobilized microorganisms.

After the microorganisms are immobilized, the switching valve 6 is switched to allow only the measurement sample 8 to pass through, allowing the microorganisms to grow. The time required for the microorganisms to grow to the required amount has been confirmed in advance in another experiment, and the flow is continued until that time.

Next, operate the switching valve 5.6 to allow the buffer solution to flow through. The liquid is passed through the immobilized microorganism sales container 1 until the dissolved oxygen concentration of the liquid is stabilized and a certain baseline is obtained. In addition, ion exchange water may be used instead of the buffer solution if it does not adversely affect microorganisms.
7. Furthermore, it is preferable that the immobilized microorganism sales container 1, the measurement flow cell 3, the measurement sample 8, and the buffer solution 10 be placed in a thermostatic chamber, and the measurement sample 8 and the buffer solution 10 should be aerated.

Next, the switching valve 6 is switched to pass a certain amount of the sample to be measured, the amount and rate of decrease in the dissolved oxygen concentration is measured, and the BOD value is determined using a calibration curve prepared in advance.

The time required for one measurement depends on the type of microorganisms to be immobilized, the amount of microorganisms immobilized, and the operating conditions such as the amount of microorganism immobilized carrier photon and the liquid passage speed. For example, when microorganisms in an activated sludge treatment tank are When using a 2.5 ml container as a container for selling adsorbed and immobilized microorganisms, the operating conditions are as follows:
Measurement sample injection amount 0.5mj! , liquid passing rate 5mj2/mi
One measurement can be carried out in 5 to 10 minutes at n. In addition, even in high concentration samples with BOD values of 500 or more, 1
It was possible to return to the baseline within 0 minutes and to measure within 10 minutes.

Further, the microbial sensor shown in FIG. 2 is another embodiment of the present invention, and according to this, a certain amount of the measurement sample is injected,
Measurements can be made. That is, first, a certain amount of the measurement sample is filled into the sample loop 13 by the pump 4. At this time, the buffer solution is passed through and a baseline is obtained. Next, by operating the switching valves 14 to 17, the sample to be measured is extruded with a buffer solution and injected into the immobilized microorganism sales container 1, and the change in dissolved oxygen concentration is measured.

Using the method of the present invention, it is possible to combine an appropriate electrochemical device for quantifying not only BOD but also the types of microorganisms attached to the microorganism immobilization carrier and the substances consumed or produced by reactions by these microorganisms. This makes it possible to easily produce various microbial sensors.

For example, Pseudomonas fluorescens (Pseudomonas fluorescens)
If a microorganism that selectively assimilates glucose, such as domonas fluoresens, is adsorbed and immobilized and combined with an oxygen electrode, it can be used as a glucose sensor. At this time, a solution containing glucose may be provided for the growth of microorganisms. In addition, Nitrosomonas (N
1troaoa+onas) and Nitrobacter (Nit
It can be used as an ammonia sensor by adsorbing and immobilizing nitrifying bacteria such as C. robactar and combining it with an oxygen electrode, or as an amino acid sensor by combining Escherichia coli, which has glutamic acid decarboxylase activity, with a carbon dioxide electrode.

These are examples of the microbial sensor of the present invention, and various other microbial sensors suitable for the purpose can be manufactured.

Furthermore, in the example shown in FIG. 1, the immobilized microorganisms are grown after being incorporated into the microbial sensor line, but it is also possible to immobilize and grow them in another line and then incorporate them into the present microbial sensor.

〔Effect of the invention〕

In the method of the present invention, in a microbial sensor that uses microorganisms as molecular identification elements, microorganisms are attached and immobilized on a porous carrier in advance, and then a solution containing the analyte is passed therethrough to induce an enzyme suitable for the analyte. Since the microorganisms that are immobilized are grown, the concentration of the target substance can be measured easily, quickly, and with good reproducibility using an appropriate electrochemical device even when the amount of immobilized microorganisms is small. Furthermore,
This method has the advantage that it does not require complicated immobilization methods or expensive carriers as in the past when using various microorganisms that meet the purpose.

Therefore, according to the microbial sensor of the present invention, it is possible to quickly and easily measure various substances such as sugars, alcohols, organic acids, etc. in addition to BOD in water.

〔Example〕

EXAMPLES Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples in any way.

Example 1 A BoD sensor was manufactured using the system shown in FIG. An oxygen electrode was used as the electrode.

In an immobilized microorganism sales container with a volume of 2.5 ml, -side 3
Fill a polyurethane sponge with a cube size of
Operate in a cycle of 4 → 1 → 5 → 9. Then, the microorganism suspension was pumped to 2.5 ml/a using pump 4.
The microorganisms are adhered and immobilized by passing the liquid for 30 minutes at a flow rate of +in. In this example, activated sludge treated water was used as the microorganism suspension.

Next, liquid was passed from the measurement sample 8 through the switching valve 6 using the pump 4 at 2.5 mA/min for 4 consecutive days to grow the microorganisms.

When a similar liquid passage experiment was conducted using another column, the amount of microorganisms attached was as shown in Table 1 below.

From the results shown in Table 1 in the margin below, it was found that microorganisms could sufficiently proliferate to a usable range by passing the liquid for 4 days, so the time for passing the liquid in this example was set to 4 days.

After passing the solution for 4 days, the buffer solution 10 was added to the immobilized microorganism sales container 1.
A switching valve is operated so that the buffer solution flows through the measurement flow cell 3 until the oxygen concentration is stabilized and a constant baseline is obtained.

A sample solution with a known BOD is prepared, and a calibration curve showing the relationship between the BOD value and the amount of decrease in dissolved oxygen concentration is first created. As for the operation method, first add a buffer solution of 2.5a+1! Flow the liquid at /min. Then, switch the switching valve 6 to 0
．． 2 ml of the sample to be measured is passed through, and the switching valve 6 is switched again to pass the buffer solution. In this example, the measurement flow cell 3, the immobilized microorganism sales container 11, the measurement sample 8, and the buffer solution 10 were all maintained at 20°C.

FIG. 3 shows the relationship between the reciprocal of the dissolved oxygen concentration when measuring known samples with BOD values of 120.240,560 .mu./l and the reciprocal of the BOD values of the known samples.

As will be seen, each reciprocal has a linear relationship, and the BOD value was calculated from the amount of dissolved oxygen reduction in the unknown sample of this example using this graph as a calibration curve.

Table 2 below shows the BOD values measured for several unknown samples using the microbial sensor of this example and the BOD values measured using the BOD measurement method specified in JISKO102.

As is clear from this result, the values measured by the microbial sensor of the present invention and the values measured by the JIS method are in good agreement. Measuring BOD using the JIS method requires a long time of 5 days, and the measurement operation is complicated and requires skill; however, by using the method of the present invention, BOD
can be measured quickly, easily, and with high precision.

Example 2 The system shown in Fig. 1 was used, spherical porous polystyrene beads with an average particle size of 0.5 mm were used as the porous carrier, and microorganisms present in river bottom mud were used as the microorganisms. The BOD value of river water was measured using the same procedure as in Example 1.

For the adhesion, immobilization and proliferation of microorganisms, the river bottom mud and river water are thoroughly stirred and mixed, the bottom mud is allowed to settle, and then the supernatant containing the microorganisms is poured into polystyrene beads in the same manner as in Example 1. This was carried out by passing a liquid through the column and then allowing river water to pass through the column for further 7 days.

The amount of microorganisms immediately after adhesion and immobilization is 0.02 in terms of dry microbial weight.
mg/ml, but the amount of microorganisms after 7 days of growth was 0.6 mg/ml as a dry weight of microorganisms.

Table 3 below shows the BOD values measured for several types of river water using the microbial sensor of this example and the BOD values measured using the BOD measurement method specified in JIS 0102.

As is clear from this result, the values measured by the microbial sensor of the present invention and the values measured by the JIS method are in good agreement.

[Brief explanation of drawings]

FIG. 1 is a diagram showing an embodiment of a microbial sensor in the method of the present invention. FIG. 2 is a drawing showing another embodiment of a microbial sensor in the method of the present invention. Figure 3 shows the reciprocal of the dissolved oxygen concentration measured by the microbial sensor in the method of the present invention shown in Figure 1 and the BOD of the sample.
It is a drawing which shows the relationship of the reciprocal of a value. Above rl engraving Figure 1 Figure 1 Container for immobilized microorganisms 2 Electric oven 3 Measuring flow 7 Lamp 5.6 Switching pulp 7 Recorder 8 Measurement sample 9 Microorganism suspension 10 Buffer solution or ion exchange water 11 Microorganism immobilization carrier 12 Air dunde 1 /BOD (t/m9 0.01 Procedural amendment (method) 6. Drawings subject to amendment 7. Contents of amendment (1) Figure 1 will be corrected as shown in the attached sheet. 1゜Indication of the case 1999 Patent Application No. 106907 2゜Name of the invention Operation method of microbial sensor 3゜Relationship with the person making the amendment Applicant name (091) Kao Corporation 4, Agent address Tokyo 1-3-6, Nihonbashi Ningyocho, Chuo-ku (address:
103) July 25, 1999 (shipped) A;N

Claims (1)

[Claims] 1. When using a microbial sensor equipped with a porous carrier for adhering and immobilizing microorganisms and an electrochemical device for quantifying substances produced or consumed by the action of these microorganisms, microorganisms are attached to the porous carrier. A microbial sensor characterized in that the immobilized microorganisms are grown until the dry microorganism weight becomes 0.1 mg or more per ml of porous carrier by passing a solution containing the target substance to be measured before use. How to operate.