JP3253950B2 - BOD measurement method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for rapidly measuring the biochemical oxygen demand (BOD) by combining a microbial membrane and an oxygen electrode.

[0002]

2. Description of the Related Art At present, the measurement of BOD is carried out in accordance with the method specified in Japanese Industrial Standards (Industrial Wastewater Test Method JIS K-0102-1972), but this method requires 5 days for measurement. Therefore, a sensor combining a microbial membrane and an oxygen electrode has been conventionally developed as an apparatus for measuring BOD. As the microorganisms to be immobilized, Trichosporone ctaneum, activated sludge, and the like are often used (Japanese Patent Publication No. Sho 61-7258; edited by Shuichi Suzuki, biosensors p.135-136, p.140-142 (1989), published by Kodansha) Place).

A method for immobilizing microorganisms on a membrane has also been studied (Japanese Patent Application Laid-Open No. 53-117496). Further, as for the measuring device, there is known a device in which a sensor combining a microbial membrane and an oxygen electrode is mounted on a flow cell and used (Japanese Patent Application Laid-Open Nos. 53-47895 and 3-123851).

[0004]

However, the BOD measuring device which has been developed so far has the following features. (1) Due to its structure, a standard solution (150 mg of glucose and 150 mg of glutamic acid dissolved in water to make 1 L, equivalent to 220 ppm of BOD). thing)
In addition, since the previous standard solution or the sample remains in the liquid introduction path from the inlet of the sample to the flow cell, an error occurs in the measured value when mixing the sample at the next measurement and trying to measure in a short time.

(2) In addition, the membrane in a stored state cannot be used immediately for measurement, and therefore needs to be activated. However, the conventional method requires 48 hours or more for activation. In a site such as a wastewater treatment plant, a demand for immediate measurement cannot be satisfied. (3) Further, when the measurement is not performed, the washing solution and the standard solution must be continuously supplied to the microbial membrane in order to prevent the activity of the microbial membrane from decreasing. Therefore, frequent and large amounts of washing solution, buffer solution and standard solution are required, and much time is required for preparing a solution used for maintaining the apparatus.

(4) In addition, the washing solution, buffer solution, and standard solution are frequently contaminated by microorganisms and easily rotten, so that the frequency of solution exchange is high.
In addition, slime, bacterial mass, and pellicle are clogged in the device path, and maintenance of the device is not easy. Thus, the present inventors
As a result of intensive studies to solve these problems, the present invention has been completed.

[0007]

SUMMARY OF THE INVENTION The present invention relates to a method for measuring BOD quickly by combining a microbial membrane and an oxygen electrode, and reduces the time required for activating the microbial membrane used. Another object of the present invention is to provide an unprecedented and improved method of measuring BOD in which the frequency of preparing a use solution is reduced.

That is, the present invention provides a method for measuring BOD using a microbial membrane on which microorganisms are immobilized and an oxygen electrode, wherein a washing solution or a substrate solution is intermittently applied to the microbial membrane when the microbial membrane is not used for a long time. This is a method for measuring BOD, which comprises supplying a liquid.

The microorganism used in the present invention includes BOD
There is no particular limitation as long as it can be used for the measurement of Klebsiella, for example, microorganisms belonging to the genus Klebsiella . Examples of microorganisms belonging to the genus Klebsiella include Klebsiella oxytoca JCM1665 (Klebsiella ).
ella oxytoca JCM1665), Klebsiella Planty Kola JCM7251 (Klebsiella planticola JCM725
1), Klebsierra Ozaenae JCM1663 ( Kleb
siella ozaenae JCM1663), Klebsiella terrigena
JCM1687 ( Klebsiella terrigena JCM1687) and the like. Furthermore, the present inventors have searched a wide range of microorganisms suitable for BOD measurement from the natural world. As a result, one strain isolated from soil in Kagoshima Prefecture has a wide assimilation property, and BOD measurement can be performed by a short activation treatment. It has been found that it has practically excellent properties such as being able to.

The mycological properties of the strain are described below. A. Morphological properties (1) Cell shape and size: 0.8 to 1.2 μm × 3 to 6 μm in bacilli (2) Cell polyplasia: none (3) Motility: none (4) Spores Presence / absence: None (5) Gram stainability: Negative (6) Acid resistance: None B. Cultural properties (1) Broth agar plate culture: It grows well and forms smooth pale blue-gray colonies.

(2) Gravy agar slant culture: grows well (3) Broth liquid culture: grows well (4) Gelatin puncture culture: not liquefied (5) Litmus milk: acidifies and coagulates C. Physiological properties (1) Reduction of nitrate: + (2) Denitrification reaction: + (3) MR test:-(4) VP test: + (5) Formation of indole: + (6) Formation of hydrogen sulfide:-(7) Starch hydrolysis:-(8) Use of citric acid:-(9) Use of inorganic nitrogen source: + (10) Pigment formation-(11) Urease: + (12) Oxidase:-(13) Catalase: + (14) Growth range: temperature 5-40 ° C pH 4-9.5 (15) Attitude to oxygen: facultative anaerobic (16) OF test: F (fermentation type) (17) Acids and gases from the following saccharides (1) L-arabinose: ++ (2) D-xylose: ++ (3) D-glucose: ++ 4) D-mannose: ++ (5) D-fructose: ++ (6) D-galactose: ++ (7) Maltose: ++ (8) Sucrose: ++ (9) Lactose: ++ (10 ) Trehalose: ++ (11) D-sorbitol: ++ (12) D-mannitol: ++ (13) Inosit: ++ (14) Glycerin: ++ Starch: − − D. Other properties (1) β-galactosidase: + (2) DNase-(3) tryptophan deaminase:-(4) degradation of esculin: + (5) degradation of arginine:-(6) decarboxylation of lysine: + (7) decarboxylation of ornithine Reaction:-(8) Arsenic resistance: Can grow in the presence of 10,000 ppm of arsenic.

Based on the mycological properties described above, the taxonomic status of this strain was determined by the Burgess Manual of Systematic Bacteriology, Vol. 1, pp. 415-416, 461.
~ 465 pages (1984) (Bergey's Manual of Systematic Bac
teriology Volume 1, 415-416, 461-465 (1984)), it was identified as a new strain belonging to Klebsiella oxytoca.
92 ( Klebsiella oxytoca 12092). This strain was sent to the Institute of Microbial Industry and Technology by the National Institute of Advanced Industrial Science and Technology.
No. 616 (FERM BP-3616), deposited on October 22, 1991 and deposited internationally.

The method for culturing the present strain is described below. For culturing this strain, any conventional method for culturing bacteria can be used. The carbon sources include glucose, maltose, sucrose,
Those commonly used such as molasses can be used alone or in combination. As the nitrogen source, various amino acids as organic nitrogen-containing substances, corn steep liquor, malt extract, peptone, yeast extract, meat extract, urea, etc.In addition, as inorganic nitrogen-containing substances, ammonium chloride, ammonium sulfate, ammonium nitrate, etc., alone or in combination Can be used.

In addition, components such as vitamins and minerals can be appropriately added and used. The culture temperature is preferably 20
C. to 40C, more preferably 28C to 37C. Culture pH is preferably 4.5 to 9.0, more preferably 5.5 to 8.0.
The culture method can be any of liquid culture and solid culture, but it is desirable that the cells in the logarithmic growth phase with a strong viability are desirable, and the culture time is preferably 10 when aerobic liquid culture is performed.
Hours to 72 hours, more preferably 12 hours to 48 hours.
When solid culture is performed, it is preferably 12 hours to 96 hours, more preferably 24 hours to 72 hours.

After completion of the culture, the cells can be obtained by a known general method. For example, the cells can be collected by centrifugation. The obtained cells are washed with distilled water or the like and then immobilized.A method commonly used for immobilization can be used, for example, a membrane filter (pore size).
When immobilized between membranes (0.45 micron or less), a stable immobilized microbial membrane with no leakage of cells can be obtained.

The membrane used for immobilizing microorganisms may be made of a hydrophilic material that does not allow the passage of bacterial cells but allows the passage of dissolved components such as oxygen and organic substances in the sample solution. However, an acetylcellulose membrane, nitrocellulose membrane or the like having a diameter of 0.45 μm or less is suitable. Also, when immobilizing microorganisms between two membranes, a stable response is obtained by minimizing the gap between the membranes and maintaining the state in which the microorganisms are encapsulated between the adherent membranes. In addition, since the adhesiveness to the oxygen electrode is improved, the response to a change in oxygen concentration is also improved, which is desirable.

The microbial electrode used in the present invention comprises the above-mentioned immobilized microbial membrane and an oxygen electrode, and the microbial membrane is fixedly attached to the oxygen electrode by a removable cap or the like, so that the membrane can be easily replaced. In addition, this microbial electrode can be used by attaching it to any flow cell that is commonly used.However, Klebsiella oxytoca 12092 has a fast response to the components in the sample solution and has a high detection sensitivity, so it can be used with a small amount of sample. BOD measurement is possible, so that the volume of the flow cell can be made as small as 1 ml or less, more preferably 0.3 ml to 0.6 ml, in order to shorten the measurement time. Does not necessarily need to be completely oxygen-saturated, as long as the oxygen concentration is constant.

Further, by adopting a method in which the flow cell is arranged in a vertical position and the sample solution is passed from below and discharged from above while stirring the inside of the flow cell with a stirrer, there is no adhesion or residue of bubbles or the like to the microbial membrane. Stable measurement is possible.
For example, when a sample solution is passed at 4 ml / min, at 30 ° C., one sample can be measured in about 15 to 20 minutes. Hereinafter, the first invention of the present application will be described.

A feature of the present invention is that, in the BOD measuring apparatus, a drain section is provided before the solution flows into the flow cell. In conventional equipment, washing solution, buffer solution, standard solution,
In the test, the introduction path from each inlet to the flow cell is directly connected. For example, if the BOD of the sample to be measured first is high, the test remains in the liquid introduction path and the BOD of the sample to be measured next is low. In such a case, a method of flowing the next test water until the influence of the previous test water disappears is adopted, but this results in a loss of the advantage of the present apparatus that measurement can be performed quickly. The present inventors have solved this problem by providing a drainage section in the liquid introduction path connected to the inlet of the flow cell.

FIG. 1 shows the mechanism. (1) to (5) are off
Represents a replacement valve and uses a solenoid valve. P is liquid
Represents a pump, which is used for cleaning, standard, or
It has the ability to simultaneously feed with an acid buffer. Washing
Use ordinary tap water, distilled water or deionized water for the cleaning solution.
Can be. The buffer is usually potassium dihydrogen phosphate (KH
_TwoPO_Four) And dipotassium hydrogen phosphate (K_TwoHPO_Four)
Use a phosphate buffer solution at a concentration of 50-300 mM, preferably
Or 130-260 mM. pH depends on the nature of the microorganism used.
PH is usually adjusted to 5.0 to 8.0, preferably pH 6.0 to 7.0.
You. Potassium dihydrogen phosphate and sodium dihydrogen phosphate
(Na _TwoHPO_Four) Can also be used.

As the standard solution, a mixed solution containing 150 mg / L of glucose and 150 mg / L of glutamic acid is adjusted to a BOD value of 220 ppm and, if necessary, diluted to a set BOD value. After passing through the liquid feed pump, the washing solution, the standard solution, and the sample side merge with the phosphate buffer. AP represents an air pump, which is used to eliminate the difference between dissolved oxygen in the standard solution and the sampled water by passing air after passing through the valve of (5) and to remove the influence of supersaturated oxygen. (6) is an oxygen electrode, and the tip (8) is a microbial membrane. (7) is a flow cell having a liquid inlet and a liquid outlet.

Here, the effect of the first invention of the present application will be described.
For example, the time taken for the sample to reach the valve (4) from the sample inlet is X
If the time from valve (4) to valve (5) is Y, after the pre-sample measurement, after installing the sample inlet at the next sample, the valve (5) opens during X or more. At the same time, the valve (4) is also opened. Next, the valve (4) is closed during Y,
The valve (1) is opened only during or longer than Y, and the cleaning liquid flows. By this method, the pre-test water is drained from the valve (5) without flowing into the flow cell, and at the same time the next test water is filled from the test water inlet to the valve (4), and the measurement preparation for the next test water is completed.

At this time, when the valve (5) is opened, only the air from the air pump (AP) is supplied to the flow cell, so that the baseline becomes unstable. Is closed and the cleaning liquid flows to the flow cell, and the baseline is stabilized. Hereinafter, the second invention of the present application will be described.

An object of the present invention is to reduce the time required for activating a microorganism membrane. The microbial membrane used for the measurement of BOD is in a dry state in the storage state in order to maintain the activity for a long period of time, and in order to use it for measurement, it is necessary to change the state from a dry state to a wet state and activate the dry bacteria. . In the conventional method, after immersing the microbial membrane in a buffer solution, it was attached to an oxygen electrode, and it took about two days to use it for measurement in order to continue supplying a standard solution until the output was stabilized.

Therefore, the present inventors have found an activation method which can be performed in a shorter time. In this method, the dried membrane is first immersed in the same buffer solution or water as used for measurement, attached to the oxygen electrode, and then activated at the inlet of one of the standard solutions 1, 2, or 3 or the test sample. A solution containing a nutrient source (hereinafter referred to as “nutrient solution”) is attached. After a certain period of time, the washing solution and the nutrient solution are alternately flowed.

Here, the inflow time of the washing solution and the inflow time of the nutrient solution are both usually between 30 seconds and 30 minutes, preferably around 10 minutes. The composition of the nutrient solution may be a medium that is generally used for culturing microorganisms or a composition suitable for the microorganisms to be used. Monosaccharides such as glucose and fructose, disaccharides such as sucrose and maltose, and nitrogen as a carbon source may be used. As sources, ammonium salts such as ammonium chloride and ammonium sulfate, various amino acids and polypeptones, yeast extracts as vitamin sources, and metal salts such as magnesium sulfate and iron sulfate as metals may be given.

A nutrient solution may be prepared by adding a yeast extract of a vitamin source to a standard solution to be used, and a metal salt may be added as needed. The nutrient solution obtained by adding the yeast extract of the vitamin source to the above-mentioned standard solution is a nutrient solution containing glucose, glutamic acid and yeast extract.In this nutrient solution, the glucose concentration and the glutamic acid concentration are:
Preferably 5-1000 ppm, more preferably 300-600 ppm
The yeast extract concentration is preferably 10 to 10,000 ppm
, More preferably 200 to 400 ppm.

The phosphate important for the growth of the microorganism is supplied from a phosphate buffer. Hereinafter, the third invention of the present application will be described. An object of the present invention is to maintain the activity of a microbial membrane for a long time with a smaller amount of a supply liquid. When the microbial membrane is being measured, it is considered that a nutrient source is provided to assimilate the organic matter in the test water from its principle. But,
When the measurement is not performed, only the cleaning liquid flows,
There is no nutrient supply and activity is reduced. Therefore, in order to maintain the activity of the microbial membrane, it is necessary to continuously supply nutrients necessary for maintaining the activity even when measurement is not performed.
Therefore, as a method of providing a normal nutrient source as a practical method, a method of continuously supplying a washing solution and a standard solution using a method of measuring a standard solution may be considered. In this case, there are a method using all the standard solutions used for the measurement, and a method using only a standard solution of one concentration. However, in any of the above cases, the amounts of the standard solution, the washing solution, and the buffer solution are considerably large because of continuous use. For example, when the apparatus is operated at a flow rate of 5 ml / min for 16 hours, the washing solution, the standard solution, and the buffer solution are consumed in a total amount of nearly 5 L. For this reason, the frequency of preparation of these solutions increases, and time is required.

Therefore, as a result of the research conducted by the present inventors, the microbial membrane activity is maintained even if the nutrient source is not provided continuously as described above but intermittently to such an extent that the microbial membrane activity does not decrease. Was found to be sufficient. In this method, the liquid feed pump is operated for a certain time and then stopped for a certain time. During the further operation, the washing liquid and the substrate solution are alternately applied at regular intervals. The certain period of the pump operation here depends on the nature of the microorganisms in the membrane used, but is usually between 10 seconds and 4 minutes, preferably between 30 seconds and 2 minutes. The time to stop is usually 30 seconds to 3 seconds.
Minutes, preferably between 1 and 2 minutes.

As the substrate solution supplied to the microbial membrane, a nutrient solution containing the above-mentioned nutrient may be used in addition to the standard solution. Furthermore, the interval between the washing solution and the substrate solution is as follows:
As with the start / stop time, depending on the nature of the microorganism used, various combinations may be used, such as alternately applying the washing solution and the substrate solution once each time, or applying the washing solution 10 times and then giving the substrate solution once. Can be.

Hereinafter, the fourth invention of the present application will be described.
As a method of preserving by mixing other reagents in the solution used for measurement, a method of lowering the pH of the preservative by hydrochloric acid or acetic acid is generally known, and other methods such as sodium hypochlorite and A method of adding chloramphenicol at a low pH is known. However, it is preferable that the preservative naturally used when using the microbial membrane does not affect the microorganisms used in the microbial membrane as much as possible.

Thus, the present inventors have studied various preservatives and found that boric acid (H ₃ BO ₃ ), sorbic acid, or a salt thereof provides an excellent effect. Examples of the salts of boric acid or sorbic acid include sodium borate and potassium sorbate. In particular, it is preferable to use boric acid and potassium sorbate. The concentration used depends on the type of preservative used.
0.1-1.0%, preferably 0.3-0.5%, and potassium sorbate usually 0.1-1.0%, preferably 0.25%
~ 0.5%.

Furthermore, if the concentration of the buffer solution is not a problem, a combination of boric acid and low pH is also an effective preservative. pH
The antiseptic effect is further increased by adjusting to a value of 2 to 3 using an inorganic acid such as hydrochloric acid or an organic acid which is not assimilated by the microorganism used in the present invention.

[0034]

EXAMPLES The present invention will be described more specifically with reference to Preparation Examples, Experimental Examples and Examples, but the scope of the present invention is not limited to these. (Preparation Example 1) biofilm Preparation Klebsiella oxytoca twelve thousand and ninety-two (Klebsiella oxytoca
12092) (FERM BP-3616) 1% polypeptone, yeast extract
Liquid medium containing 0.1% and 0.5% sodium chloride (pH 6.5)
A 500 ml Sakaguchi flask containing 100 ml was inoculated and cultured at 30 ° C. for 17 hours under aerobic conditions with shaking. After completion of the culture, the culture was centrifuged at 6000 rpm for 20 minutes to collect the cells. A small amount of sterile distilled water was added to the obtained cells and suspended,
Washing was performed by centrifugation again. This washing was repeated three times, and finally a 20-fold dilution was performed to prepare a bacterial cell concentrate such that the OD ₆₆₀ value was 0.58.

32 μl of this cell concentrate was sterilized to 1.7% κ
-Carrageenan, 0.8% locust bean gum mixture 2
g, and dropped on an acetylcellulose membrane (Millipore membrane filter type HA) having an average pore diameter of 0.8 μm, and aspirated from the bottom of the membrane until all of the microorganism suspension was retained in the membrane. Infiltrated and fixed in the membrane. Excess κ-carrageenan aqueous solution that has passed through the membrane may be removed by dripping from the lower part of the membrane as it is. Next, the membrane holding the microorganism was cooled on ice, and then a 40 mM phosphate buffer solution was added.
It was immersed in 100 ml at room temperature for 5 minutes to solidify κ-carrageenan and locust bean gum to obtain a microbial membrane.

(Experimental Example 1) Comparison between the case with and without the drainage section The BOD was measured using the microbial membrane obtained in Preparation Example 1. For measurement, glucose 150mg / L, glutamic acid 15
A 0 mg / L mixed solution having a BOD value of 220 ppm was diluted to obtain a BOD value of 100 ppm or 5 ppm.

The equipment used was in the path shown in FIG.
The case with and without the drainage part of (5) was used. The air pump was ventilated at a flow rate of 1000 ml / min, and the washing solution and the standard solution were flowed at a flow rate of 4 ml / min and the buffer solution was flowed at a flow rate of 1 ml / min by a liquid feed pump. The measurement was carried out with and without a drainage part. The washing liquid (water) was allowed to flow for 10 minutes to stabilize the baseline before drawing in the standard solution. next,
The standard solution adjusted to 100 ppm as the standard solution was kept flowing. Then, the first point at which the output line of the water sample became constant was obtained as the output value. Next, the washing solution was flowed for 10 minutes, and a 5 ppm standard solution was set at the sample inlet, and was flowed until the output line became constant.

FIG. 2 shows the change in the output of the oxygen electrode as a result. From Fig. 2, it can be seen that the output stabilizes and a peak value is obtained in 15 minutes when the drain is attached, but it takes 30 minutes for the output to stabilize due to the 100 ppm standard solution when the drain is not installed. You can see that it was done. (Experimental example 2) Activation of dried microbial membrane by nutrient solution As nutrient solution, glucose 426 ppm, glutamic acid 426
What contained 300 ppm of yeast extract and 300 ppm of yeast extract was used.

A microbial membrane prepared by the same method as the membrane used in Experimental Example 1 was used. The apparatus used had the drainage part (5) used in Experimental Example 1, and the other conditions were the same as those in Experimental Example 1. The nutrient solution and the buffer were each supplied for 10 minutes. The output state at this time is shown in FIG. The output value in the figure indicates a high value when the dissolved oxygen is high, and a low value when the dissolved oxygen is low.

In FIG. 3, when the nutrient solution is supplied to the microbial membrane, the dissolved oxygen is reduced because the microorganisms in the microbial membrane consume the substrate in the nutrient solution. Therefore, when the nutrient solution is supplied, the output value decreases. Next, when the cleaning liquid is supplied, since the substrate does not exist in the cleaning liquid, the dissolved oxygen is not consumed and the output value becomes high. Next, when the nutrient solution is supplied again, the microorganisms in the microbial membrane are activated or proliferated by the first supply of the nutrient solution, so that the oxygen consumption becomes larger than that in the first supply of the nutrient solution, and the output value is increased. Is even lower. When the supply of the nutrient solution and the washing solution is repeated, as the activity increases, the output value for the nutrient solution gradually decreases, and the return of the output value by washing to this output value becomes slow, and the baseline decreases. come. Here, it is considered that this decrease in the baseline value indicates that the activity of the microbial membrane is increasing, so it is determined that the activity required for the measurement has been reached when the baseline value reaches a certain output value. It is considered possible.

Further, as shown in FIG. 3, the activity of the microbial membrane is increased by the nutrient solution. However, since the baseline is high and cannot be used for measurement as it is, washing is performed again to stabilize the baseline. There is a need. Then, when the output value of the baseline reached the above-mentioned activated constant value, the supply of the nutrient solution was stopped, the washing solution was flown to start the washing, and the baseline was stabilized.

With respect to the output value for judging that the activation has been reached, when the baseline when the nutrient solution is supplied to the microbial membrane reaches 1000, 900, 800, and 700 mV, the cleaning solution is supplied, and the output becomes constant. After that, the measurement was performed. As a result, after the output value reached 800 mV or less, washing was performed to obtain an output similar to the activity of the microbial membrane before drying (Table 1).

[0043]

[Table 1]

The same activity was reached at 700 mV and 800 mV, but the required time was about 17 hours at 700 mV,
In FIG. 3, the cleaning liquid was supplied at the time of reaching 800 mV in order to perform activation in a short time because V was about 11 hours. As a result, the washing was started in about 21 hours, and the washing time was set until the baseline was stabilized.
It has been found that hours or more are preferable. Therefore, all activation was completed in 26 hours until the completion of washing of the microorganism membrane. Table 2 shows the activity of the microorganism membrane activated in this test.

[0045]

[Table 2]

As is evident from the above, the activity of the dried microbial membrane with the nutrient solution was restored to almost the same level as before and activated to the extent that a sufficient output for measurement was obtained. The activation time was also 16 hours. In the conventional method, the activation time is such that the dried microbial membrane is immersed in a buffer solution for one day, and then, after mounting on the device, the standard solution and the washing solution are alternately applied. In the liquid, since the composition is only glucose and glutamic acid, it takes time to activate the microorganisms in the microbial membrane due to lack of vitamins and metal salts, and it is actually necessary for one day to obtain measurable activity, It took a total of 2 days for the microbial membrane to be usable. In contrast, the method of the present invention was able to reduce the time by 32 hours.

(Experimental example 3) Comparison between continuous supply and intermittent supply Condition: Microbial membrane prepared in the same manner as the membrane used in Experimental example 1 was used. After the pump operation time was 30 seconds, the pump stop time was 90 seconds, the cleaning liquid was flowed up to the fifth operation of the pump, and the standard liquid was flowed at the sixth time. After that, the same operation was repeated. The time course is shown in FIG. Conditions: Continuous operation of the pump Cleaning solution 11 minutes Standard solution (50 ppm) 4 minutes Inflow was repeated.

Flow rate: 5 ml / min, operation time: 16 hours The same apparatus as used in Experimental Example 2 was used. Table 3 shows the results. The total of consumed washing solution, standard solution, and buffer solution was about 1.2 L, which was 1/4 of continuous operation. In addition, the activity could maintain the same activity as in continuous operation.

[0049]

[Table 3]

(Experimental Example 4) Influence of preservative (1) The activity (output) when various preservatives were added to the washing solution, buffer solution and standard solution was compared with the case where no preservative was added. As measurement conditions, a standard solution having a BOD of 50 ppm was measured ten times, and the output change at that time was compared with the first output value.

Table 4 shows the results.

[0052]

[Table 4]

Table 4 shows that boric acid and potassium sorbate are excellent. (2) Boric acid was added to the washing solution, buffer solution, and standard solution, and the water discharged from the food factory was measured every day. When not measured, various bacteria on the surface of the microorganism membrane when the activity was maintained under the conditions of Experimental Example 3 The presence or absence of flock was visually inspected. Table 5 shows the results.

[0054]

[Table 5]

* In the case of adding 1.0% boric acid, the measurement was possible, but the microbial membrane was affected, and it took 24 hours or more to activate the microbial membrane as in Experimental Example 2. Table 5 shows that the concentration of boric acid is preferably 0.3 to 0.5%. (3) BOD 50ppm standard solution was applied for 30 days under the conditions shown in Table 6.
The number of viable cells when left at ℃ was measured. pH is 2.
Adjusted to 0.

Table 6 shows the results.

[0057]

[Table 6]

(Example 1) Correlation between BOD ₅ (official method) and this method (microbial membrane method) Conditions: The microbial membrane was prepared by the method of Experimental Example 1. The method of Experimental Example 2 was used to activate the storage film. The washing solution, buffer solution and standard solution were prepared so that boric acid was added at 0.3%, and the pH was adjusted to 2.0 with hydrochloric acid. The activity was maintained by the method under the conditions of Experimental Example 3. Method: Water sampling was performed by sampling the effluent discharged from a food factory once a day, and measured for 5 days by an official method and a sensor method using a microbial membrane, and the correlation between the two was determined. As a result, the correlation between the official method and the sensor method showed a high value of 0.9 or more, and it was found that the method of the present invention had a high correlation with the official method (FIG. 5).

(Example 2) Continuous measurement by the method of the present invention Continuous measurement was carried out under the same conditions as in Example 1 to examine the correlation with the official method and the retention time of the activity of the microorganism membrane. As a result, even in continuous use, the correlation with the official method was high, and the microbial membrane could be used for three months (FIG. 6).

From the above, according to the present invention, the BOD
It can be seen that the BOD in the wastewater can be quickly and easily managed daily without requiring five days as in the official method to measure the BOD.

[0061]

According to the present invention, it is possible to maintain the activity of the microorganism membrane for a long time with a smaller amount of the supply liquid.

[Brief description of the drawings]

FIG. 1 is a diagram showing a route of a BOD measuring device of the present invention.

FIG. 2 is a diagram showing a change in output of an oxygen electrode with and without a drain section.

FIG. 3 is a diagram showing activation of a dried microbial membrane by a nutrient solution.

FIG. 4 is a diagram showing a time course of intermittent liquid supply.

FIG. 5 is a diagram showing a correlation between an official method and a sensor method using a microbial membrane.

FIG. 6 is a diagram showing results of continuous measurement by an official method and a sensor method using a microbial membrane.

[Explanation of symbols]

 1, 2, 3, 4, 5 switching valve 6 oxygen electrode 7 flow cell 8 microbial membrane AP air pump P liquid feed pump

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Yoshiya Kawamura 132, Kowatari, Kochino-cho, Konan-shi, Aichi (56) References Japanese Utility Model Application Sho 61-176448 (JP, U)

Claims (1)

(57) [Claims] 1. A method for measuring a BOD in which a microorganism membrane having microorganisms immobilized thereon and an oxygen electrode are combined, wherein a washing solution or a substrate solution is intermittently supplied to the microorganism membrane when the microorganism membrane is not used for a long time. A method for measuring BOD, characterized in that: