JPH051994A - Measurement method for atp in activated sludge liquid - Google Patents

Abstract

PURPOSE:To enable ATP contained in the microorganism of activated sludge liquid to be easily and accurately measured by restraining hindrance to TCA luminous reaction and a measurement error in diluting the liquid, respectively to a sufficiently low level. CONSTITUTION:Trichloroacetic acid solution as ATP extraction chemical is mixed with activated sludge liquid, and this mixed solution is diluted. In addition, a luminous reagent is added to the diluted mixed solution, and a luminous magnitude in luminous reaction generated due to the addition of the reagent is measured, thereby quantifying ATP in the activated sludge liquid. In this case, a ratio of 1:1 is maintained between the amounts of the activated sludge liquid and trichloroacetic acid solution added thereto, and the concentration of the trichloroacetic acid solution is kept at a value between 5% and 10%. Also, a dilution magnification with dilution liquid is taken at a value between 20 and 100 for measurement.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for extracting adenosine triphosphate (ATP) contained in microorganisms in activated sludge.

[0002]

2. Description of the Related Art It is well known that effective knowledge can be obtained by qualitatively or quantitatively measuring substances existing in cells or cells.

[0003] For example, ATP having a function of phosphoric acid metabolism and energy metabolism of a living body is inevitably present in a living cell, but this ATP disappears promptly when a microorganism is killed. Therefore, ATP exists in a dead cell. No, so AT
It is considered that P can be an index showing the concentration of microorganisms. (1984 version sewage test method P293) In addition, in cells of the same kind, ATP present in one cell
The amount is equal. Therefore, if ATP can be quantified, it is possible to determine the number of cells, determine whether they are living cells or dead cells, and measure the activity of cells.

Therefore, the measurement of ATP in the cells of microorganisms has attracted attention in various fields such as water treatment, food and medicine. Especially in the water treatment field, the activated sludge method is used to decompose and remove organic pollutants such as sewage and factory wastewater.The activated sludge method purifies the microorganisms by ingesting them. Therefore, the concentration of microorganisms involved in purification is a very important operating condition in the activated sludge method.

In order to measure ATP in microorganisms, it is necessary to extract ATP outside the microorganisms. Many extraction methods have been attempted up to the present, and the conditions are that the extraction efficiency of ATP is high, the operability is excellent, and the influence on the measurement is small.

[0006] As an example, the applicant of the present invention has filed Japanese Patent Application No. 1-
Japanese Patent No. 109486 proposes an extraction method using TCA as a reagent for extracting ATP from the inside of the microorganism existing in activated sludge to the outside of the microorganism.

In the method for measuring ATP using the above-mentioned TCA, as shown in FIG. 6, ACA is extracted by adding TCA and a diluent to the sample, and then a luminescent sample such as luciferin and luciferase is added, as shown in FIG. Cause a luminescence reaction,
At this time, luminescence measurement is performed to measure the ATP concentration.

[0008]

However, in the above method of measuring ATP concentration, in order to completely extract ATP from the microbial body in the activated sludge liquid, TC of a relatively high concentration is used.
Although it is necessary to use A, the higher the TCA concentration, the higher the efficiency of ATP extraction from the inside of the microbial body, but on the other hand, the luminescence reaction is inhibited and accurate measurement becomes difficult.

Therefore, it is necessary to dilute the sample solution to lower the TCA concentration during the luminescence reaction, but generally, when the dilution operation is performed, the operation becomes complicated and automation is difficult, and when the dilution ratio becomes high, the measurement time becomes long. In addition, the measurement accuracy is likely to decrease.

If the TCA concentration at the time of ATP extraction is kept low, the extraction of ATP from the inside of the microorganism may be incomplete and the measured ATP value may be smaller than the true value.

Therefore, in order to measure ATP more accurately, the TCA concentration should be kept as low as possible within the range in which the extraction of ATP in the microorganism is completely performed.
It is necessary to suppress the dilution ratio of the solution at the time of the luminescence test to a low value within a range where the influence of CA emission inhibition hardly appears.

However, no quantitative report has yet been made regarding the selection criteria of the above TCA concentration and dilution ratio, and a method for determining the optimum TCA concentration and dilution ratio has not been established.

The present invention has been made under the above-mentioned background, and it is easy and accurate to determine the optimum TCA concentration and the dilution ratio, which are contained in microorganisms in activated sludge.
It is intended to provide a method for measuring TP.

[0014]

In order to solve the above-mentioned problems, according to the present invention, a trichloroacetic acid solution as an ATP extractant is added to an activated sludge liquid to dilute the mixed liquid, and the diluted mixed solution emits light. In the method for measuring ATP in activated sludge liquid, a reagent is added, and the amount of luminescence in the luminescence reaction generated at this time is measured to quantify ATP in the activated sludge liquid.
The concentration of the trichloroacetic acid solution was 5%, with the amount of the activated sludge liquid and the addition amount of the trichloroacetic acid solution being 1: 1.
It is characterized in that it is 10% to 10% and the dilution ratio with the diluent is 20 times to 100 times.

By measuring with the concentration of trichloroacetic acid and the dilution ratio as described above, the inhibition of the luminescence reaction by TCA and the measurement error at the time of dilution can be suppressed to a sufficiently small level. ATP contained in microorganisms can be measured.

[0016]

As a result of the experiment, as shown in Table 1, the extraction efficiency of ATP increases as the TCA concentration increases, and when the TCA solution is added to the activated sludge at a ratio of 1: 1, the TCA concentration in this solution increases. The ATP concentration to be extracted becomes almost constant when the value is 5% or more.

Furthermore, the inhibition of the luminescence reaction is 5% to 10% of T
By diluting the CA solution 20- to 100-fold, the inhibition of the luminescence reaction is reduced and appropriate measurement becomes possible.

Therefore, the above-mentioned TCA brain and the dilution rate enable efficient extraction of ATP contained in the microorganisms in the activated sludge, and the measurement error due to the inhibition of the luminescence reaction can be suppressed sufficiently small. It is possible to determine a good microbial concentration and microbial activity.

[0019]

EXAMPLES The present invention will be described below with reference to examples.

Usually, AT contained in microorganisms in activated sludge
The PCA is extracted by adding luciferin and luciferase as luminescent reagents to a mixed solution of TCA and ATP in TCA used for microorganisms, and determining the ATP concentration by measuring the amount of luminescence during this luminescent reaction. However, this luminescence reaction is hindered by the coexistence of a high concentration of TCA, and the amount of luminescence decreases.

Further, when the ATP concentration is usually measured by the above-mentioned luminescence reaction, a calibration solution is prepared by diluting a mixed solution of TCA and ATP of a predetermined concentration at an appropriate ratio, and using this calibration curve, an experimental sample is prepared. The ATP concentration is determined by converting the light emission amount into the ATP concentration.

Therefore, in this example, the following experiment was conducted in order to investigate the inhibition of the luminescence reaction due to the TCA concentration when preparing the calibration curve. For the sake of convenience, the TCA concentration was 1% = 0.01 g / ml, and the diluent used was a buffer solution consisting of EDTA and NH ₂ C (CH ₂ OH) ₃ [Tris].

First, an ATP standard solution of 10 ^-5 mol / l and a concentration of 0.1
% TCA solution.

Next, 0.5% of the above ATP standard solution was added to the above 0.1%
A mixed solution was prepared by adding 0.5 ml of a TCA solution, and this solution was mixed with 0.1 mol / l NH ₂ C (CH ₂ O) containing 4 mmol / l EDTA.
H) Each sample solution is prepared by diluting 2 times, 5 times, 10 times, 20 times and 50 times with ₃ buffer solution.

Further, 0.5 ml of the luminescent reagent was added to 0.5 ml of each diluted sample solution described above, and the amount of luminescence for 30 seconds from 1 second to 30 seconds was measured to prepare a standard curve.

Next, TCs with concentrations of 0.5%, 1.0% and 5.0%
The solution A and pure water as a comparative example were prepared, and the measurement experiments of the amount of light emission were performed. The result is shown in FIG. In Fig. 2, line A is 0.5 ml of the above-mentioned 10 ^-5 mol ATP standard solution.
0.5ml of pure water is added to each of 2 times, 5 times, 10 times, 20 times,
It is a standard curve obtained by performing a luminescence test after diluting 50 times.

Similarly, B line (overlapping with A line), C line, D line, E
Each line is 0.1% TCA in the above 10 ^-5 mol ATP standard solution.
Solution, 0.5% TCA solution, 1% TCA solution, 5% TCA solution are added respectively, these are diluted to the above-mentioned respective magnifications, and a calibration curve obtained by conducting a luminescence test is shown.

According to this figure, when the 0.1% TCA solution is used, a linear relationship is obtained as in the case of using pure water, and the influence of TCA on luminescence inhibition is hardly recognized.

On the other hand, when a 0.5% to 5% TCA solution is used, luminescence inhibition by TCA is observed, but it is 5% in a 0.5% TCA solution using the above Tris buffer.
Almost no luminescence inhibition was observed by diluting 10 times with 1.0% TCA solution and 50 times with 5% TCA solution.

From the above results, it can be seen that the inhibition of the luminescence reaction by TCA is influenced by both the TCA concentration and the ATP concentration.

Next, an ATP extraction experiment was conducted by TCA.

In the present example, in order to investigate the optimum TCA concentration and the optimum dilution ratio in the ATP measurement, first, 10 ^-5 mol / 0.5 ml of TCA solutions of various concentrations were used.
0.5 ml of an ATP standard solution containing l, 10 ^-6 mol / l and 10 ^-7 mol / l was added, and this solution was diluted to an appropriate ratio.

Then, the ATP concentration and the amount of luminescence at each dilution ratio were measured to prepare a standard curve, and when the ATP concentration was determined from the results of the luminescence test, the ATP concentration was determined based on this standard curve. The details are shown below.

(A) Preparation of standard curve (1) 10 ^-5 mol / l ATP standard solution was diluted with distilled water to prepare 10 ^-6 mol / l, 10 ^-7 mol / l ATP standard solution To do.

(2) 10 ^-5 mol / l, 10 ^-6 mol / l, 10 ^-7 mol /
1 ml of each ATP standard solution is mixed with 0.5 ml of 0.1% TCA solution.

(3) 0.1 mol containing 4 mmol / l EDTA
Dilute the mixed solution of ATP and TCA at the above concentrations 2-fold, 10-fold, and 50-fold with / l Tris buffer.

(4) 0.5 ml of each of the above diluted solutions was added to a luminescent reagent.
Add 0.5 ml to react and measure the amount of luminescence for 30 seconds from 1 second to 30 seconds.

(B) Measurement of ATP concentration in microbial cells present in activated sludge (1) A mixed solution is prepared by adding 0.5 ml of TCA solution to 0.5 ml of activated sludge.

(2) 0.1 mol containing 4 mmol / l EDTA
Dilute the above mixture 2 fold, 5 fold, and 10 fold with / l Tris buffer.

(3) 0.5 ml of each diluted solution is reacted with 0.5 ml of the luminescent reagent, and the amount of luminescence for 30 seconds from 1 second to 30 seconds is measured.

The 2-fold dilution obtained in the above experiment (A),
Standard curves of ATP diluted 10-fold and 50-fold are shown in FIGS. 3, 4 and 5, respectively.

Further, when the ATP concentration is read from the standard curve obtained in the experiment (A) from the luminescence amount obtained in the above experiment (B), it is 5.20 × 10 ⁻⁷ in the 2-fold dilution.
The results were 1.10 × 10 ⁻⁶ mol / l for mol / l and 10-fold dilution, and 1.20 × 10 ⁻⁶ mol / l for 50-fold dilution.

Since the TCA concentration was constant in the above experiment, the amount of ATP extracted from the inside of the microorganism was the same, and therefore these measured values should originally agree with each other. However, the TCA concentration in the luminescence test was 50-fold diluted, 10-fold diluted, 2
The values obtained from the 2-fold diluted solution are the highest in the order of double dilution.
The P concentration is about half of the other values.

Next, the TCA concentration is set to 0.5%, 1.0%, 5.0.
% And 10.0%, a standard curve was prepared in the same manner as in the above experiment, and the ATP concentration in the microbial cells present in the activated sludge was measured. In the above experiment, when the TCA concentration was 0.5%, the dilution ratio was set to 2 times, 5 times, 10 times, 50 times, and 1.0%.
%, 5 times, 10 times, 50 times, and TCA concentration of 5.
At 0% and 10.0%, 2 times, 5 times, 10 times, 20 times,
The measurement was performed at 50-fold and 100-fold dilution ratios.

Table 1 shows the measurement results of the ATP concentration by the above experiment.

[0046]

[Table 1]

From this table, for example, when the dilution ratio is 50 times, the measured value of the ATP concentration at the TCA concentration of 10.0% is twice or more the measured value at the TCA concentration of 0.1%. It can be seen that the extraction efficiency of ATP becomes higher as the value becomes higher.

On the other hand, when the TCA concentration becomes high, the measured value of the ATP concentration becomes small due to the inhibition of luminescence, which sometimes makes the measurement impossible. For example, assuming that the TCA concentration is 10%, ATP cannot be detected at a dilution rate of 10 times or less, and the measured value at a dilution rate of 20 times is about 85% of the measured value at a dilution rate of 100 times. There is.

From the above experimental results, in the case of extracting ATP in the microbial body by TCA to measure the ATP concentration,
TC to increase the efficiency of ATP extraction from the inside of the microorganism
It can be seen that it is necessary to increase the A concentration and also increase the dilution ratio in order to suppress the luminescence inhibition.

On the other hand, when the dilution ratio is increased as described above, the measurement error increases. Therefore, in the present example, a dilution ratio of 50 times was selected as a dilution ratio that suppresses the influence of luminescence inhibition and has a sufficiently small measurement error, and the correlation of the ATP concentration with the TCA concentration was examined.

A correlation diagram between the TCA concentration and the ATP concentration is shown in FIG.

This figure shows that the extracted ATP concentration becomes almost constant when the TCA concentration exceeds 5%.

Therefore, according to the above experimental results and Table 1, highly accurate AT is obtained when the TCA concentration is 5% to 10% and the dilution ratio is about 20 to 100 times in this embodiment.
It can be seen that P can be measured.

[0054]

As described above, according to the present invention, the TCA concentration is kept as low as possible within the range where ATP in microorganisms is completely extracted, and within the range where the effect of luminescence inhibition by TCA hardly appears. Thus, the dilution ratio of the solution in the luminescence test can be suppressed as low as possible.

Therefore, according to the present invention, ATP contained in the microorganisms in the activated sludge can be easily and accurately measured.

[Brief description of drawings]

FIG. 1 is a correlation diagram of TCA concentration and ATP concentration.

FIG. 2 is a correlation diagram of ATP concentration and luminescence amount at each TCA concentration.

FIG. 3 is a correlation diagram of ATP concentration and luminescence amount.

FIG. 4 is a correlation diagram of ATP concentration and luminescence amount.

FIG. 5 is a correlation diagram of ATP concentration and luminescence amount.

FIG. 6 is an ATP extraction process diagram.

FIG. 7 is an explanatory diagram of a luminescence reaction formula of ATP.

Claims (1)

Claims: 1. A trichloroacetic acid solution as an ATP extractant is added to an activated sludge liquid to dilute the mixed liquid, and a luminescent reagent is added to the diluted mixed solution. ATP in activated sludge liquid is measured by measuring the amount of luminescence in the luminescence reaction
In the method for measuring ATP in the activated sludge liquid, the amount of the activated sludge liquid and the addition amount of the trichloroacetic acid solution are set to 1: 1 and the concentration of the trichloroacetic acid solution is set to 5% to 10%. A method for measuring ATP in an activated sludge liquid, characterized in that the dilution ratio with the diluent is 20 to 100 times.