JP2002119300A - Method for rapidly assaying filamentous bacterium and method for controlling operation of activated sludge process tank using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide both a method for rapidly assaying filamentous bacteria, capable of rapidly and accurately assaying the amount of filamentous bacteria in a sludge or only a target specific filamentous bacteria by an image treatment process and a method for controlling an operation of an activated sludge process tank using the same. SOLUTION: Filamentous bacteria in a sludge are dyed by an FISH method, the size and the number of the dyed filamentous bacteria in the sludge are assayed by a flow cytometer to obtain the amount of the filamentous bacteria in the sludge. In this case, preferably the filamentous bacteria are subjected to fluorescent dyeing by using a fluorescent labeled gene probe to be specifically combined with an r-RNA of specific filamentous bacteria to be a detection object. An amount of dissolved oxygen in a tank, an inflow load to the tank, etc., are controlled so as not to make the amount of the filamentous bacteria exceed a fixed value based on the assayed result.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for quickly measuring a filamentous bacterium or a specific filamentous bacterium which is particularly suitable for use in a sewage treatment plant, and a method for controlling the operation of an activated sludge treatment tank using the same. It is.

[0002]

2. Description of the Related Art In an activated sludge treatment tank in a sewage treatment plant, a phenomenon called bulking occurs due to abnormal growth of filamentous bacteria depending on the operating conditions, and sedimentation of flocs is inhibited, so that normal treatment cannot be performed. Sometimes. For this reason, in the sewage treatment plant, activated sludge is periodically sampled, the amount of filamentous bacteria is monitored with a microscope, and operating conditions are controlled so that bulking does not occur.

[0003] The monitoring of such filamentous bacteria is as follows.
Activated sludge is imaged with a microscope, or activated sludge is imaged with a microscope and the image is processed by a computer. This method is a method of distinguishing from other microorganisms and garbage by utilizing the shape peculiar to the filamentous bacterium, but since there are many garbage and the like in a shape confusing with the filamentous bacterium, only the filamentous bacterium is accurately extracted. It is not easy. In addition, recent studies have shown that among filamentous bacteria, filamentous bacteria that do not become bulking-causing filamentous bacteria are found. There was a problem that was large. Further, such a conventional method has a problem that it is difficult to utilize it for automating the operation control of the activated sludge treatment tank because the measurement requires time.

[0004]

DISCLOSURE OF THE INVENTION The present invention solves the above-mentioned conventional problems, and enables the amount of filamentous bacteria in activated sludge or only the specific filamentous bacteria of interest to be detected more quickly and accurately than the image processing method. It is a main object of the present invention to provide a method for rapidly measuring filamentous bacteria which can be measured and a method for controlling the operation of an activated sludge treatment tank using the same. Another object of the present invention is to provide a method for rapidly measuring filamentous bacteria that can accurately measure only filamentous bacteria that cause bulking, and a method for controlling the operation of an activated sludge treatment tank using the same.

[0005]

Means for Solving the Problems To solve the above-mentioned problems, the method of the present invention for rapidly measuring a filamentous bacterium or a specific filamentous bacterium of interest can be performed by ultrasonically treating a filamentous bacterium in sludge, Cut by homogenizer treatment, enzyme treatment, etc.,
After staining by the FISH method (fluorescent staining method), the amount of the filamentous bacteria in the sludge is determined by measuring the size and the number of the filamentous bacteria in the sludge with a flow cytometer. It is characterized by the following.
The cutting of the bacteria is not limited to before the staining by the FISH method, but may be performed after. It is preferable to fluorescently stain the filamentous bacterium using a fluorescently labeled gene probe that specifically binds to the r-RNA of the filamentous bacterium to be measured, such as a filamentous bacterium that causes bulking.

Further, the method for controlling the operation of an activated sludge treatment tank according to the present invention measures the amount of filamentous bacteria in sludge by the above method, and damages the filamentous bacteria so as not to exceed a predetermined value. Of one or more of the following: addition of a chemical that gives the following, control of the dissolved oxygen amount in the tank, control of the inflow load into the tank, adjustment of the dead space, execution of flushing, and adjustment of the nutrient concentration. It is characterized by performing. As the agent, it is preferable to select an agent having an effect such as chlorine, sodium hypochlorite, a polymer, or a polycation, which has a lytic effect of dissolving filamentous bacteria, a bactericidal effect of killing, a flocculating effect of sinking, and a sedimentation effect.

The method for rapid measurement of filamentous bacteria of the present invention
Since only the filamentous bacteria in the sludge were fluorescently stained by the ISH method and the size and the number of filamentous bacteria in the sludge were measured with a flow cytometer, the filamentous filaments in the sludge were used. Only the amount of the bacterium can be measured more quickly and accurately than the image processing method. In particular, if a fluorescent-labeled gene probe that specifically binds to r-RNA of a filamentous bacterium that causes bulking is used, only the filamentous bacterium that causes bulking can be accurately measured.

[0008] The method for controlling the operation of an activated sludge treatment tank according to the present invention comprises the use of an agent capable of damaging the above-described filamentous bacteria in the tank according to the amount of filamentous bacteria rapidly measured by the above method. Addition, control of dissolved oxygen, control of inflow load into the tank, adjustment of dead space, execution of flushing, and adjustment of nutrient concentration. , The occurrence of bulking can be reliably prevented. Hereinafter, the present invention will be described in more detail with preferred embodiments.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In the present invention, first, activated sludge sampled from an activated sludge treatment tank in a sewage treatment plant is biologically fixed with ethanol, paraformamide, or the like. For biological fixation, for example, paraformaldehyde having a concentration of 3% is used as a fixing reagent,
The reaction can be performed at a temperature of 4 ° C. for one hour. Next, a treatment such as an ultrasonic treatment, a homogenizer treatment, and an enzyme treatment is performed to cut the bacteria. Then, the FISH method (Fluoresc
ent In Situ Hybridization method). The bacteria may be cut after fluorescent staining by the FISH method. As is well known, the FISH method is a method in which only a specific microorganism is fluorescently stained using a gene probe having a fluorescent label attached to one end thereof. By using a probe, only filamentous bacteria can be stained.

This fluorescent-labeled gene probe analyzes the genome of a specific microorganism, compares the homology with other microorganisms, selects a specific gene sequence, synthesizes the gene probe with a DNA synthesizer, and labels it with a fluorescent dye. It is created by the procedure of doing. Microorganisms that cause bulking include Sphaerotilus natuns,
There are many species such as the genus Thiothrix,
In Japan, most of bulking was found to be caused by filamentous bacteria classified as type 021N,
A fluorescent-labeled gene probe was prepared using the AP3 strain of this type 021N as a target.

The prepared fluorescently labeled gene probe has a base sequence as shown in Table 1. This base sequence was described as SEQ ID NO: 1 in the sequence listing at the end of the specification.

[Table 1]

Hybridization was carried out as follows. Washing conditions: suspension in PBS, centrifugation twice, temperature: 4
6 ° C., formamide concentration: 35%, reaction time: 1 hour. As a result, the fluorescent-labeled gene probe specifically binds to the ribosome r-RNA of the target filamentous bacterium in the activated sludge and performs fluorescent staining.

Next, in the present invention, the amount of the filamentous bacteria in the sludge is determined by measuring the size and the number of filamentous bacteria in the sludge with a flow cytometer. A flow cytometer is a device that can measure the particle size and the number of particles contained in a droplet by irradiating a laser beam or the like while sludge flows out from a nozzle as a droplet and receiving the image with a photodiode. Capable of measuring thousands of particles. By using, for example, a Mercury xenon lamp as the light source and irradiating light near the excitation wavelength of the fluorescently labeled gene probe, only the fluorescently stained filamentous bacteria can be detected. Using a flow cytometer, one sample can be measured within 4 minutes.

As described above, according to the present invention, only the amount of the target filamentous bacteria in the sludge can be measured quickly and accurately. The overall procedure is shown in FIG. In addition, an example of specific measurement contents is shown in the examples. According to the method of the present invention, the target filamentous bacteria can be measured much more rapidly than the conventional image analysis method, so that the amount of the filamentous bacteria in the sludge does not exceed a predetermined value. Operation control can be performed.

For example, as shown in FIG. 2, based on the measurement results, the bacteriolytic effect of dissolving filamentous bacteria such as chlorine, sodium hypochlorite, polymer, polycation, etc., the bactericidal effect of killing, the flocculating effect of sinking, the sedimentation effect, etc. The activated sludge tank 1, the sedimentation basin 4, or the returned sludge line 5 is supplied with an agent that is effective and damages the filamentous bacteria described above.
, The growth of filamentous bacteria can be suppressed. Also, as shown in FIG. 2, if the amount of dissolved oxygen in the tank is reduced by automatically controlling the amount of air supplied to the diffuser 2 of the activated sludge tank 1 based on the measurement result,
The growth of filamentous bacteria can be suppressed. Also, as shown in FIG. 2, the growth of filamentous bacteria can be suppressed by reducing the inflow load by controlling the amount of sewage flowing into the tank by the flow control device 3. In addition, the growth of filamentous bacteria can be suppressed by adjusting the dead space in the tank, performing flushing in the tank, adjusting the nutrient concentration in the tank, and the like. Here, the adjustment of the dead space means that the growth of the filamentous bacteria is suppressed by adjusting the dead space which is likely to cause the growth. Flushing means washing away sediments in the tank together with water, and has the effect of removing the filamentous bacteria themselves. Adjustment of the nutrient concentration means adjusting the nutrient concentration so that it is not suitable for the growth of filamentous bacteria and is suitable for the growth of normal bacteria in the activated sludge. Good.

[0016]

EXAMPLES Example 1 Measurement Example of Filamentous Bacteria In order to confirm the effect of the present invention, a publicly available strain AP3 (ATCC49788) was used from among bacteria classified into type 021N. , And pure cultured bacteria were measured.

After culturing the bacteria, they are lifted and fixed with a platinum wire (4
C. for 1 hour), washed with PBS (phosphate buffer containing 0.15 M NaCl), treated with a microsonicator to cut the bacteria, and used as a stained sample. This sample was stained with a fluorescent-labeled gene probe having the nucleotide sequence shown in SEQ ID NO: 1. The reaction solution for staining was 0.9 M NaCl, 0.1% S
DS, 20mM Tris / HCl (Ph7.2), 35% Formamide, 5ng / μ
l Fluorescently labeled probe, the reaction was performed at 46 ° C for 1 hour. Thereafter, the supernatant was discarded by centrifugation, a hybridization buffer containing no probe was added in the same amount as the reaction solution, the mixture was allowed to stand at 48 ° C. for 5 minutes, and then dispersed with a syringe.

The samples stained in this manner were measured using a flow cytometer manufactured by Bio-Rad.
The flow rate is 20 μL / min, the light source is a 75 W Mercury Xenon lamp, and when measuring the number of AP3 strains, it is excited at 546 ± 5 nm and simultaneously measures the forward scattered light indicating the particle size and the TRITC fluorescence (Em520-720 nm). did.

The results of measuring the fluorescence of TRITC in this manner are as shown in FIGS. The horizontal axis shows the particle size and the vertical axis shows the fluorescence intensity. FIG. 3 shows the measurement results of the AP3 strain not stained with FISH, and FIG. 4 shows the measurement results of the AP3 strain stained with FISH. From these comparisons, it was confirmed that the fluorescent-labeled gene probe AP3 stained the AP3 strain, and that the fluorescence could be measured with a flow cytometer.

Example 2 Measurement of Filamentous Bacteria in Activated Sludge Next, a sample was prepared by mixing the AP3 strain with activated sludge in a sewage treatment plant. First, 70 cultured AP3 strains were
The cells were fixed with 4% ethanol (4 ° C, 1 hour), washed with PBS, and treated with a microsonicator to cut the bacteria. On the other hand, activated sludge sampled at A sewage treatment plant
The cells were fixed with% ethanol, washed with PBS, and treated with a microsonicator in the same manner as the AP3 strain. Sample is OD1.8
Of the AP3 strain suspension in OD1.4 activated sludge from 0 to 50% by volume
It was made by adding.

Using the probe AP3 described above as a probe, the reaction was carried out in the same reaction solution as in Example 1 at 46 ° C. for 1 hour. Thereafter, centrifugation was performed in the same manner as in Example 1, the supernatant was discarded, a hybridization buffer containing no probe was added, the mixture was allowed to stand at 48 ° C. for 5 minutes, and then dispersed with a syringe. Next, in order to stain all microorganisms in the sample, DAPI as a DNA dye was added to a final concentration of 1 μg / mL, and reacted at room temperature for 5 minutes.

The samples stained in this way were measured using a flow cytometer. Flow rate is 20 μL / min,
The light source was a 75 W Mercury Xenon lamp, and when measuring the number of AP3 strains, excitation was performed at 546 ± 5 nm, and forward scattered light indicating the particle size and TRITC fluorescence (Em520-720 nm) were measured simultaneously. When measuring the total number of microorganisms,
Excitation at 350-425 nm, forward scattered light and DAPI fluorescence (Em
390-490 nm).

From the comparison of the results of measuring the fluorescence of TRITC and the preliminary test using Escherichia coli, it is known that the size of the filamentous bacteria is 1.5 μm or more. L. Also, TRITC fluorescent particles, ie, AP3 stained with probe AP3
Since the number of strains is in the range surrounded by a square frame in FIG. Similarly, the total number of microorganisms in the sample was determined from the results of DAPI fluorescence measurement and was designated as D.

FIG. 5 is a graph showing the ratio of filamentous bacteria to total microorganisms (T / D) in the sample. FIG. 6 is a graph showing the ratio (T / D) of filamentous bacteria to all particles of 1.5 μm or more in the sample.
/ L). Thus, all AP
It was confirmed that there was a high correlation with the contamination rate of the three strains. For T / L, T / D should be used originally to count particles other than filamentous bacteria, but TRITC and DA
The sample needs to be measured twice because the PI excitation wavelengths are far apart. On the other hand, T / L requires only one measurement and does not require staining with DAPI, so it is simple and excellent as a practical method for measuring filamentous bacteria.

[0025]

As described above, according to the method for rapidly measuring filamentous bacteria of the present invention, the amount of filamentous bacteria causing bulking in sludge can be measured more quickly and accurately than by the image processing method. According to the method for controlling the operation of the activated sludge treatment tank of the present invention, the operation control of the activated sludge treatment tank is automatically performed so as not to generate bulking using the measurement result. There are advantages that can be.

[Brief description of the drawings]

FIG. 1 is a flow sheet showing a method for rapid measurement of filamentous bacteria of the present invention.

FIG. 2 is a schematic sectional view showing an operation control method of the activated sludge treatment tank of the present invention.

FIG. 3 is a graph showing the results of measuring the fluorescence of TRITC for a sample of the AP3 strain not stained with FISH.

FIG. 4 is a graph showing the results of measuring the fluorescence of TRITC for a sample of the AP3 strain stained with FISH.

FIG. 5: Ratio of AP3 strain to total microorganisms in the sample
It is a graph which shows (T / D).

FIG. 6: AP for all particles of 1.5 μm or more in a sample
It is a graph which shows the ratio (T / L) of 3 strains.

[Explanation of symbols]

1 activated sludge tank, 2 diffuser, 3 flow controller, 4
Settling tank, 5 Return sludge line

[Sequence List] SEQUENCE LISTING <110> NGK INSURATORS, LTD. 160> 1 <210> 1 <211> 18 <212> RNA <213> Artificial Sequence <400> 1 tccctctcccacattcta 18

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // C12N 1/00 C12N 1/00 S (C12Q 1/68 (C12Q 1/68 A C12R 1: 645) (C12R 1: 645) (72) Inventor Mitsuo Kawase 2-56 Suda-cho, Mizuho-ku, Nagoya-shi, Aichi Japan Inside Nihon Insulators Co., Ltd. (72) Inventor Junko Shimizu 2-56, Suda-cho, Mizuho-ku, Nagoya-shi, Aichi Sun Insulator Co., Ltd. F term (reference) 4B063 QA01 QA20 QQ07 QQ18 QQ54 QR32 QR56 QS11 QS34 QX02 4B065 AA58X BC13 BC14 BC50 BD01 BD50 CA46 CA54 4D028 AA01 CA06 CA07 CC06 CE01

Claims (3)

[Claims] 1. Filamentous bacteria in sludge or specific filamentous bacteria of interest are cut, stained by a FISH method (fluorescent staining method), and then stained particle size and particles in the sludge by a flow cytometer. A method for rapidly measuring filamentous bacteria, comprising determining the amount of filamentous bacteria or a target specific filamentous bacteria in sludge by measuring the number. 2. Filamentous bacteria in sludge or specific filamentous bacteria of interest are stained by a FISH method (fluorescent staining method), cut, and then stained with a flow cytometer. A method for rapid measurement of filamentous bacteria, wherein the number of filamentous bacteria or a target specific filamentous bacteria in sludge is determined by measuring the number. 3. The method according to claim 1, wherein the amount of filamentous bacteria in the sludge is measured, and an agent which damages the filamentous bacteria is added so as not to exceed a predetermined value. Activated sludge treatment tank characterized by performing one or more of controlling dissolved oxygen amount in tank, controlling inflow load into tank, adjusting dead space, performing flushing, and adjusting nutrient concentration. Operation control method.