JPH06153989A - Separation of microorganism from organism immobilizing carrier and evaluation of organism decomposition function - Google Patents

Abstract

PURPOSE:To determine the conditions for separation of a microorganism from an organism-immobilizing carrier by ultrasonic treatment considering the survival ratio of the microorganism with first priority and to evaluate according to a simple method whether the organism-immobilizing carrier performs an organism decomposition and removal function or not. CONSTITUTION:In this method for separation of a microorganism from an organism-immobilizing carrier and the method for evaluation of the organism decomposition function, an ultrasonic wave generator having a duty cycle control function is used and ultrasonic treatment is carried out under conditions of 20 to 80% duty cycle, 15 to 50W ultrasonic wave generation power and 1 to 5min treatment time. Further, the method for evaluation of the organism decomposition function is carried out by sampling the organism-immobilizing carrier with time, extracting ATP from the samples, adding a luminous reagent to the extracts, measuring the intensity of luminescence and continuing the measurement until the concentration of ATP reaches a constant value and used as a standard for judgment of the adsorptivity of the carrier.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for exfoliating microorganisms from a bioimmobilized carrier and a method for evaluating biodegradation function.

[0002]

2. Description of the Related Art In recent years, with the deterioration of water conditions, the number of water purification treatment facilities that must use raw water such as lake water or river water having a high content of organic pollutants is increasing. In these water treatment facilities, in order to secure good quality drinking water, a treatment method in which a biological treatment method is added to an ordinary water treatment facility is being investigated (Shunji Yoshimura et al., Water Pollution Research, 9, 484-489, 198).
See 6 years).

Among them, biological activated carbon
rbon (hereinafter abbreviated as BAC)) is excellent due to the excellent adsorption characteristics of ordinary activated carbon as a bioimmobilization carrier and the regenerating action of adsorption sites by biodegradation which is an adsorbed substance of the activated carbon. It has been clarified that the ability to remove organic matter and its long-term sustainability are possible, and it is expected as an effective method for future advanced water purification treatment (Kurosawa Yoshinori et al., Water Pollution Research, 11, 577-589, 1988).
11: 590-588, 1988, Junzo Suzuki et al., Journal of Japan Society on Water Environment, 15, 45-51, 1992).

The BAC treatment is a method of treating organic matter by a mixed culture system in which various bacteria and microorganisms such as protozoa and micro metazoa are attached and inhabited on a biologically immobilized carrier such as activated carbon.

On the other hand, for the purpose of analyzing the microorganisms in BAC, it has been reported that ultrasonic treatment is applied to the exfoliation of microorganisms attached and inhabiting the BAC (for example, Junzo Suzuki et al., Journal of Japan Society for Water Environment, 15, 45). -51, 1992, Taro Yoshikura et al., Water and Wastewater, 33, 463-470, 1991.
Mitsuho Tojo et al., 25th Annual Conference on Water Pollution Society, 256
257, pp. 1991, Kin Shu Naga et al., 26th Annual Meeting of the Japan Society on Water Environment, pp. 128-129, 1992).

[0006] Although no report has been made on the selection of conditions for removing microorganisms from BAC by such ultrasonic treatment in detail, for example, Suzuki et al. Mentioned above, agar is used as a means for water quality analysis and viable cell count measurement. BA cultured with medium
It was reported that 1 g of wet weight of C was washed with sterilized physiological saline, sterilized physiological saline was added, and the mixture was crushed with a spatula and further sonicated for 15 minutes. Reported that ultrasonic treatment was carried out using UD-200 manufactured by TOMY Seiko Co., Ltd. in order to investigate the decomposition process of 2-MIB by bacteria in a mixed culture system sample. Furthermore, Kin et al. Evaluated the number of bacteria exfoliated from BAC by the acridine orange method, treatment time 3 to 5 minutes, intensity 30W, 40W, 50W, 60.
We are considering ultrasonic treatment of W and 70W and the intensity is 40%.
It is reported that the processing conditions of W and 3 minutes were selected.

On the other hand, as a criterion for deciding whether or not BAC fulfills the function of removing biodegradation, ammoniacal nitrogen (NH 3) which is not removed by adsorption but can be removed by nitrifying bacteria is used.
_3- N), the ability to generate trihalomethane (THMFP), which can be removed by adsorption or microorganisms, diosmin, 2-
A method utilizing the removal property of methylisoborneol (2-MIB) or the like is used (for example, Naoto Akazawa et al., 42nd National Water Works Association Presentation, 635-637, 199).
1 year, Muneyoshi Miyoshi et al., 43rd National Waterworks Association Presentation, 25
0-252, 1992, Teruki Nayuki et al., 43rd National Water Works Association Presentation, 265-267, 1992).

[0008]

However, such conventional ultrasonic treatment is not necessarily the most suitable method from the viewpoint of the survival rate of microorganisms on activated carbon as a biologically immobilized carrier. Yes, there is a current situation where re-examination from a conditional point of view is required.

That is, the above ultrasonic treatment is performed by a grinding method or a Hu method.
Although it is extremely powerful compared to ghes press method, French press method, etc., at the same time, ultrasonic waves have the ability to disrupt many cell structures by cavitation, so it should be examined from the aspect of microbial viability. Problems still remain (Microbial Research Methods Round-table Conference, Microbial Experimental Methods page 269, Kodansha 1
1975). Therefore, it is necessary to search for sonication conditions with high survival rate of microorganisms.

In addition, an ion chromatograph is usually adopted for the measurement of ammonia nitrogen (NH ₃ —N), which is used as a criterion for determining whether or not BAC fulfills the function of removing biodegradation, and also the measurement of trihalomethane. A gas chromatograph with an electron capture detector as the detector is used for the same.
A gas chromatograph / mass spectrometer is required for MIB measurement, but these devices are extremely expensive and require the skill of an operator to use them. Therefore, at present, it is desired to realize a method for easily and quickly evaluating the biodegradation and removal function of BAC. This evaluation is particularly necessary when deciding whether or not a new BAC having a biodegradation removal function should be replenished, for example, at the time of start-up in a bioactive carbon tower.

[0011] Therefore, the present invention, in the selection of conditions for removing microorganisms from BAC by such conventional ultrasonic treatment,
It is an object of the present invention to obtain a treatment method in which the survival rate of the microorganism is primarily considered. Still another object of the present invention is to provide a method capable of evaluating whether or not BAC has a biodegradation-removing function by a simple means.

[0012]

[Means for Solving the Problems] In order to achieve the above-mentioned object, the present invention provides a biologically immobilized carrier having microorganisms attached thereto by subjecting the biologically immobilized carrier to ultrasonic treatment. In the method of separating and analyzing microorganisms from the above, a device having a duty cycle adjusting function is adopted as an ultrasonic wave generator, the duty cycle is 20% to 80%, and the ultrasonic wave generation output is 15 W to 50 W.
Provided is a method for exfoliating a microorganism from a biologically immobilized carrier, wherein ultrasonic treatment is performed under a treatment time of 1 minute to 5 minutes.

Further, according to claim 3, a sample is prepared by sampling the biologically immobilized carrier to which microorganisms are adhered with time, and subjecting the sample to ultrasonic treatment to exfoliate the microorganisms, In the method of evaluating the biodegradation function of the bioimmobilized carrier by continuing the counting until the value becomes a constant value, a device having a duty cycle adjusting function is adopted as the ultrasonic wave generator, and the duty cycle is 20% to 80%, output of ultrasonic wave 15W-5
Provided is a biodegradation function evaluation method in which ultrasonic treatment is performed under the conditions of 0 W and a treatment time of 1 minute to 5 minutes, and according to claim 4, a bioimmobilized carrier to which a microorganism is attached is collected over time. A sample was prepared, ATP of this sample was extracted, a luminescent reagent was added to the extract, the luminescence intensity was measured, and the measurement was performed until the ATP concentration reached a constant value, which was used as a criterion for determining the carrier adsorption capacity. Provided is a biodegradation function evaluation method for a bioimmobilized carrier.

As the bio-immobilized carrier, bio-activated carbon is adopted.

[0015]

According to the method for exfoliating microorganisms from the bioimmobilized carrier, the number of exfoliated microorganisms from the bioimmobilized carrier is maximized for analysis by performing ultrasonic treatment using the above conditions. In addition to the above, it is possible to obtain the result that the survival rate of the microorganism is extremely good. The above conditions are also mild conditions for the survival of microorganisms, and an optimal method can be obtained from the viewpoint of increasing the survival rate of the microorganisms on the immobilized carrier.

Further, according to the method for evaluating the biodegradation function of a bioimmobilized carrier using ultrasonic treatment, direct bioimmobilization is carried out by a simple means of separating and counting the microorganisms from the bioimmobilized carrier under ultrasonic treatment conditions. It is possible to evaluate the biodegradation removal function of the immobilized carrier, further measure ATP in the bioimmobilized carrier, clarify the relationship between the removal properties of ATP and NH ₃ -N, trihalomethane, and determine the emission intensity of ATP. By measuring, the biodegradation removal function of the bioimmobilized carrier can be easily evaluated.

[0017]

EXAMPLES Hereinafter, various examples of the method for exfoliating microorganisms from an immobilized carrier and the method for evaluating biodegradation function according to the present invention will be described.

[Embodiment 1] In this embodiment, Escherichia coli was selected as a microorganism, a device having a DUTY CYCLE adjusting function was selected as an ultrasonic generator, and the survival condition of E. coli was set. The device having the DUTY CYCLE adjusting function can adjust the ultrasonic wave generation time in one second, for example, and is therefore suitable for setting the survival condition of microorganisms. The equipment is ultrasonic generator USP-300 or USP manufactured by Shimadzu Corporation.
-600 was adopted.

1-1. Method (1) Escherichia coli (C-600) 1 platinum loop with 5 ml of broth medium 2
The book is inoculated and shake-cultured at 37 ° C. overnight. (2) The bacterial cells are collected, washed 3 times with physiological saline, and 10 ml of physiological saline is added. (3) 0.3 ml of bacterial cells and 2.7 ml of sterilized physiological saline are added to a test tube, and at the same time, the number of Escherichia coli in the bacterial solution is counted using a 3M Petrifilm aerobic bacteria measurement plate. (4) OUTPUT CONTROL: 1, 2, 3, processing time: 1 minute under the condition of 50% duty cycle using USP-300
2.5 minutes, 5 minutes, 7.5 minutes, 10 minutes, 12.5 minutes, 15
Process with different minutes and parameters. (5) Add 10 treatment liquids
Dilute ^-6 , 10 ^-5 , 10 ^-4 times, and put 1 ml on a Petrifilm aerobic bacteria assay plate. (6) Count E. coli for 48 hours. (7) Determine the sonication time required for the survival of E. coli.

The above-mentioned OUTPUT CONTROL 1 has an ultrasonic output of 15 W, and OUTPUT CONTROL 2 has the same 40-50 W output.
CONTROL3 is the same 70W.

1-2. Results Figures 1 and 2 show the correlation between the ultrasonic treatment time (minutes) in OUTPUT CONTROL 1 and 2 and the survival rate (%) of Escherichia coli, and Fig. 3 shows the ultrasonic treatment time (minutes) in OUTPUT CONTROL 3 and the number of bacteria ( cells / ml). The ultrasonic wave generation conditions in which Escherichia coli has a residual rate of 20% or more are 1 minute, 2.5 minutes, 5 minutes, 7.5 minutes, and 10 minutes in OUTPUT CONTROL 1, and OU
With TPUT CONTROL 2, it takes 1 minute, 2.5 minutes and 5 minutes. Under these conditions, it is possible to obtain good results by exfoliating the microorganisms adsorbed on BAC. It was also found that the OUTPUT CONTROL 3 shown in FIG. 3 killed E. coli due to the intensity of ultrasonic waves being too high.

Example 2 From the results of Example 1, the conditions for exfoliating the microorganisms adsorbed on BAC are determined.

2-1. Method (1) Add 0.5 g of BAC and 3.0 ml of sterile physiological saline to a test tube. (2) DUTY C using USP-300
OU, which is an ultrasonic wave generation condition for E. coli to survive at 50% YCLE
1 minute, 2.5 minutes, 5 minutes, 7.5 minutes, 1 with TPUT CONTROL 1
0 minutes, 1 minute in OUTPUT CONTROL2, 2.5 minutes, BA in 5 minutes
Sonicate C. (3) Treatment liquid is 10 ^-6 , 10 ^-5 ,
Dilute 10 ⁻⁴ , 10 ⁻³ , 10 ⁻² times. 1 ml of this diluent
In a petri dish, and aseptically add about 15 ml of standard agar medium kept at 45 to 50 ° C (n = 2), 3
After culturing at 6 ± 1 ° C. for 48 ± 2 hours, the number of general bacteria exfoliated from BAC is determined (supervised by Ministry of Health, Health and Welfare Bureau, Water Environment Department, water supply test method, Japan Water Works Association, 1985).
(4) Determine optimum ultrasonic conditions for stripping general bacteria adsorbed on BAC.

2-2. Results Figures 4 and 5 show the correlation between the ultrasonication time (minutes) and the bacterial count (cells / ml) when OUTPUT CONTROL was 1 and 2, respectively. With OUTPUT CONTROL 1, the number of peels was maximum under the condition of 5 minutes, and with OUTPUT CONTROL 2, the number of peels was maximum under the condition of 1 minute.

Based on the residual ratio of Escherichia coli of Example 1 and the result of the number of bacteria of Example 2, the ultrasonic conditions for stripping general bacteria adsorbed on BAC were as follows: OUTPUT CONTROL 2 for a treatment time of 1 minute. It turned out to be good. From the results of the experiment, it was found that the duty cycle of the ultrasonic generator was 20% to 80% in a good operable range. At this time, the survival rate of Escherichia coli was 87.5%, which was extremely good.

From the above results, U as an ultrasonic generator
DUTY C using SP-300 or USP-600
Under the conditions of YCLE 20% to 80%, output 15 W to 50 W, and 1 minute to 5 minutes, the number of Escherichia coli detached from BAC was maximized and the survival rate of E. coli was extremely good. This condition is also a very mild condition for the survival of microorganisms.

Example 3 BAC was collected over time and the number of microorganisms was counted by ultrasonic treatment to clarify the relationship between the number of microorganisms adsorbed on BAC and the removal characteristics of NH ₃ —N and trihalomethane. Evaluate the biodegradation and removal function of.

3-1. Method for counting the number of microorganisms in BAC (1) Add 0.5 g BAC collected over time and 3.0 ml of sterile physiological saline to a test tube. (2) USP-600
DUTY CYCLE 50% using the
UT CONTROL2 (output 40W-50W), ultrasonic processing BAC in 1 minute. (3) Treating solution with 10 ^-6 , 10 ^-5 , 10
Dilute ^-4 , 10 ^-3 , 10 ^-2 times. 1 ml of this diluted solution was put in a petri dish, and about 15 ml of standard agar medium kept at 45 to 50 ° C. was aseptically added to each (n = 2), and 36 ±
Incubate at 1 ° C. for 48 ± 2 hours and determine the number of general bacteria detached from BAC.

3-2. Method for measuring NH ₃ —N (1) Samples before and after BAC treatment collected over time are passed through a 0.45 μm membrane filter.
(2) Toa Denpa ICA-3010 pump and ICA-3
NH ₃ − using a 030 electric conductivity meter according to the manual
Measure N.

3-3. Measurement method of THMFP (1) Chlorine treatment of samples before and after BAC treatment collected over time to generate trihalomethane and extract solvent (Ministry of Health and Welfare Bureau Health and Sanitation Bureau Water Environment Department supervision, water supply test method, Japan Waterworks Association, 489, 1985). (2) Trihalomethane is measured using a gas chromatograph GC-14A having an electron capture detector as a detector.

3-4. Results Figure 6 shows the number of microorganisms adsorbed on BAC and NH ₃ -N, THM.
The FP removal characteristic is shown in FIG.
After 40 days, 80% of H ₃ -N was removed and became a constant value.
THMFP was removed 85% after 60 days and became a constant value. Further, the amount of microorganisms per 1 g of wet weight of BAC after 40 days showed a constant value of about 1 × 10 ⁶ cells / ml.

Therefore, USP-60 is used as an ultrasonic wave generator.
The direct method of separating microorganisms from BAC and counting the number of microorganisms by using the ultrasonic wave generation conditions of 0, DUTY CYCLE 50%, and treatment time of 1 minute according to the optimal ultrasonic treatment condition is the biological method of BAC. It was found that the decomposition and removal function can be evaluated.

As a result of continuing the experiment, DUTY CYCLE20
It was found that there is no problem in practice even if the ultrasonic wave generation conditions of% -80% and treatment time 1 minute-5 minutes are used.

[Example 4] BAC was collected over time and ATP in the BAC was measured to clarify the relationship between ATP and NH ₃ -N and trihalomethane removal characteristics, and
The biodegradation and removal function of BAC is evaluated by measuring the luminescence intensity of BAC.

4-1. ATP method (1) Weigh 0.5 g of BAC washed with a spatula in small petri dishes containing 1 ml of distilled water. Next, remove distilled water with a Pasteur pipette, and use a spatula and Pasteur pipette to remove BA into a test tube containing 1 ml of distilled water.
Add C without exception. (2) Next, 1 ml of trichloroacetic acid is added to a test tube containing BAC, and ATP is extracted by vortex for 30 seconds. Then, 10 ml of Tris buffer is added and diluted. At this point, ATP in BAC has been diluted 12-fold. (3) 0, 10 ^-11 , 10 ^-10 ,
10 ^-9 , 10 ^-8 , 10 ^-7 , 10 ^-6 mol / l ATP is prepared and a standard curve is prepared. 0.25 ml of the luminescence reagent of the reagent kit "Lucifer-AS" for Kikkoman sludge activity measuring device (UPD-2000EX) and the ATP
0.25 ml of the extract is added to a polyethylene tube, and the amount of luminescence is measured with a luminometer UPD-8000 manufactured by Meiden Saw. (4) The amount of ATP extracted from BAC is obtained from the ATP standard curve.

4-2. Method for measuring NH ₃ —N (1) Samples before and after BAC treatment collected over time are passed through a 0.45 μm membrane filter.
(2) Toa Denpa ICA-3010 pump and ICA-3
NH ₃ − using a 030 electric conductivity meter according to the manual
Measure N.

4-3. Method for measuring THMFP (1) Chlorine treatment of samples before and after BAC treatment collected over time to generate trihalomethane and solvent extraction.
(2) Trihalomethane is measured using a gas chromatograph GC-14A having an electron capture detector as a detector.

4-4. Results Figure 7, there is shown the amount of ATP and NH ₃ -N of BAC, the removal characteristics of THMFP, according to the figure, NH ₃ -N
After 40 days, 80% was removed and it became a constant value.
After 60 days, P was 85% removed and became a constant value. or,
The amount of ATP per 1 g of wet weight of BAC after 40 days showed a constant value of about 1.2 × 10 ⁻⁸ mol / l. Therefore A
It was found that the TP method can also evaluate the biodegradation removal function of BAC.

As mentioned above, a direct method for separating microorganisms from BAC by ultrasonication and counting the number of microorganisms, and BA
It was found that the biodegradation removal function of BAC can be evaluated by any of the methods of measuring the emission intensity of ATP extracted from C. By such an evaluation method, it is possible to quickly obtain information as to whether or not new BAC should be supplemented at the time of starting up the biological activated carbon tower.

[0040]

As described above in detail, according to the method for exfoliating microorganisms from the bioimmobilized carrier according to the present invention, ultrasonic treatment under prescribed conditions is performed to fix the organism for analysis. It is possible to maximize the number of exfoliated microorganisms from the modified carrier and to keep the survival rate of the microorganisms good.
The defined conditions are mild conditions for the survival of microorganisms, and an optimal method is provided from the viewpoint of increasing the survival rate of microorganisms.

According to the method for evaluating the biodegradation function of a bioimmobilized carrier using ultrasonic treatment, direct bioimmobilization is carried out by a simple means of separating and counting the microorganisms from the bioimmobilized carrier under ultrasonic treatment conditions. The biodegradation removal function of the immobilized carrier can be evaluated, and the biodegradation removal function can be easily evaluated by measuring ATP in the bioimmobilized carrier and measuring the emission intensity of the ATP. The above evaluation method does not require expensive equipment and
It has the advantage that it does not require the skill of an operator, and furthermore, the biodegradation / removal function of the bioimmobilized carrier can be quickly evaluated to determine whether or not new bioactive carbon should be replenished, for example, when starting up in a bioactive carbon tower. You can get the data to judge.

[Brief description of drawings]

FIG. 1 is a graph showing the correlation between the ultrasonic treatment time (minutes) and the residual rate (%) of Escherichia coli in OUTPUT CONTROL 1 for a biologically immobilized carrier.

FIG. 2 is a graph showing the correlation between the ultrasonic treatment time (minutes) and the Escherichia coli residual rate (%) in OUTPUT CONTROL 2 for a biologically immobilized carrier.

[Fig. 3] Ultrasonic treatment time (minutes) and the number of exfoliated bacteria (cells / m) in OUTPUT CONTROL 3 for the biologically immobilized carrier.
The graph which shows the correlation of l).

[Fig. 4] Ultrasonic treatment time (minutes) and the number of exfoliated bacteria (cells / m) in OUTPUT CONTROL 1 for the biologically immobilized carrier
The graph which shows the correlation of l).

[Fig. 5] Ultrasonic treatment time (minutes) and the number of exfoliated bacteria (cells / m) in OUTPUT CONTROL 2 for the biologically immobilized carrier
The graph which shows the correlation of l).

FIG. 6 shows the number of microorganisms adsorbed on the bio-immobilized carrier and NH ₃ −
The graph which shows the removal characteristic of N and THMFP.

FIG. 7: ATP amount of bioimmobilized carrier and NH ₃ —N, TH
6 is a graph showing the removal characteristics of the MFP.

Claims (5)

[Claims] 1. A method for separating a microorganism from a biologically immobilized carrier by subjecting the biologically immobilized carrier to which the microorganism has adhered to ultrasonic treatment and analyzing the microorganism, wherein the ultrasonic generator has a duty cycle adjusting function. The equipment that has it is adopted, and the duty cycle is 2
A method for exfoliating microorganisms from a biologically immobilized carrier, which comprises performing ultrasonic treatment under conditions of 0% to 80%, an ultrasonic wave generation output of 15 W to 50 W, and a treatment time of 1 minute to 5 minutes. 2. The method for separating microorganisms from a bioimmobilized carrier according to claim 1, wherein the bioimmobilized carrier is bioactive carbon. 3. A biologically immobilized carrier having microorganisms attached thereto is collected over time to prepare a sample, and the sample is subjected to ultrasonic treatment to exfoliate the microorganisms so that the number of the microorganisms becomes a constant value. In a method of evaluating the biodegradation function of a bioimmobilized carrier by continuing counting up to, a device having a duty cycle adjusting function is adopted as an ultrasonic generator and the duty cycle is set to 2
A method for evaluating a biodegradation function of a bioimmobilized carrier, which comprises performing ultrasonic treatment under the conditions of 0% to 80%, an ultrasonic wave generation output of 15 W to 50 W, and a treatment time of 1 minute to 5 minutes. 4. An organism-immobilized carrier to which microorganisms adhere is collected over time to prepare a sample, ATP of this sample is extracted, and a luminescent reagent is added to the extract to measure the luminescence intensity.
A method for evaluating a biodegradation function of a bioimmobilized carrier, which is used as a criterion for determining the carrier adsorption capacity by measuring until the P concentration reaches a constant value. 5. The method for evaluating a biodegradation function of a bioimmobilized carrier according to claim 3, wherein the bioimmobilized carrier is bioactive carbon.