JPH0413655B2 - - Google Patents

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a method for measuring the number of methane bacteria or methane production activity in a specimen containing methane bacteria, such as in a methane fermentation tank in a sewage treatment system. The present invention relates to a method for measuring the number of methane bacteria or methane production activity that coexists with a large number of microorganisms and various foreign substances such as digested sludge.

[Prior art] Conventionally, as a method for evaluating the number of bacteria or the activity of microorganisms from the concentration of microorganisms, a certain correlation between the absorbance of visible light of a solution containing microorganisms and the concentration of microorganisms is used to calculate the absorbance. The method used was to measure the number of bacteria, etc. by measurement.

FIG. 2 is a block diagram of a main part of an apparatus system for explaining a conventional measuring method of this type. In the figure, 1 is a subject containing methane bacteria, 2 is a light source, and 3
4 is a power source that applies voltage to this light source 2; 4 is a photomultiplier tube; 5 is a power source that applies voltage to this photomultiplier tube 4; 6 is a detection unit that detects the photocurrent of photomultiplier tube 4. .

The conventional measurement method uses the configuration shown in FIG. 2, in which visible light from a light source 2 is irradiated onto a specimen 1 containing methane bacteria and transmitted, the transmitted light is received by a photomultiplier tube 4, and its intensity is measured by photoelectrons. The photocurrent value of the multiplier tube 4 was measured by the detection unit 6. Then, the microorganism concentration was evaluated based on the known correlation between the absorbance and the microorganism concentration from the measured absorbance, and the related number of bacteria or the activity of the microorganisms was evaluated.

On the other hand, as a method to measure the activity of microorganisms themselves, adenosine triphosphate ATP (ATP) in the energy metabolic cycle of microorganisms is used.
Triphosphate) or nicotinamide adenine dinucleotide phosphate NAD(P)H
(Nicotinamide Adenine Dinucleotide
There was a method to optically measure the amount of biological substances (mainly coenzymes) such as (phosphate) under certain conditions by measuring the absorbance.

[Problem to be Solved by the Invention] The conventional measurement method using the means shown in FIG. In the case of methane bacteria, the total amount is measured as the concentration, so it has been impossible to selectively measure the concentration of methane bacteria alone, the number of bacteria related thereto, or the activity. On the other hand, since ATP and HAD(P)H are biological substances related to the energy metabolism system of living organisms, methods that measure the activity of microorganisms exist in all microorganisms; Measurement was impossible.

This invention was made in order to solve the above-mentioned problem, and no matter what other microorganisms or foreign substances coexist with the methane bacteria to be measured in the sample, the turbidity caused by these coexisting substances can be reduced. It is an object of the present invention to provide a measurement method that can measure the number of methane bacteria or methane production activity without being influenced by methane bacteria.

[Means for Solving the Problems] A method for measuring the number of methane bacteria or methane production activity according to the present invention involves irradiating a diluted or alkali-treated sample containing methane bacteria with excitation light while changing its wavelength. , the radiation fluorescence emitted by the subject is measured, and this radiation fluorescence falls within the wavelength range of the excitation light.
Detect the wavelength of the excitation light that shows the peak intensity in the range of 380 to 440 nm, irradiate the test object with the excitation light of this wavelength, and measure the peak intensity of the emitted fluorescent light in the wavelength range of 450 to 490 nm emitted by the test object. At the same time, the amount of transmitted light that the excitation light transmitted through the specimen is measured, and from this amount of transmitted light, the attenuation effect resulting from the presence of the specimen is corrected for the intensity of the emitted light, and the turbidity due to coexisting substances other than methane bacteria is corrected. The true fluorescence intensity emitted by the methane bacteria is determined by removing the influence of the methane bacteria, and the number of methane bacteria or the methane production activity is calculated from the correlation between the number of methane bacteria and the fluorescence intensity.

[Function] In the present invention, a specimen containing methane bacteria is irradiated with excitation light of varying wavelength, and the radiation fluorescence emitted from the specimen is irradiated with excitation light of a wavelength at which the maximum peak intensity is exhibited. Methanobacteria or methanogenic activity is determined by measuring the peak intensity of the emitted fluorescent light emitted by the specimen, and correcting the attenuation effect on the above peak intensity based on the amount of transmitted excitation light measured at the same time. . Here, the operation based on the measurement principle will be explained as follows.

Methanobacteria have different physiological properties from normal microorganisms, and although the full details of the electron transport system involved in the energy metabolism of methanobacteria are still unknown, it is believed that this is due to coenzymes unique to methanobacteria. It has been known. In other words, in the electron transport chain that exists in the energy metabolism system of methane bacteria, F ₄₂₀
It is known that a substance called a coenzyme (coenzyme) functions as an electron carrier, and this substance is unique to methane bacteria and does not exist in other biological systems. In this way, F ₄₂₀ , a substance (coenzyme) that binds only to methane bacteria and is centrally involved in the electron transport system (oxidation-reduction system), produces methane from various fermentation products produced from organic matter in methane fermentation. It is said to be actively functional. In other words, substances involved in the electron transport system of methane bacteria, mainly F ₄₂₀ (hereinafter referred to as F ₄₂₀ substances), can be measured in the state of living bacteria (living bacteria) and have specific characteristics that are different from other microorganisms or foreign substances. Because it has physical and chemical properties that are both physical and measurable, it is directly related to the methane production function from the physiological function of methane bacteria, and is an effective measurement target for measuring the number of methane bacteria or methane production activity. .

In fact, when methane bacteria are irradiated with excitation light in a specific wavelength range, the fluorescent properties thought to be emitted by the F ₄₂₀ substance of methane bacteria are caused by microorganisms other than methane bacteria and foreign substances such as digested sludge. It has become clear that the behavior of bacteria differs depending on their fluorescent properties and the state of living bacteria. Therefore, it is possible to determine the true number of methane bacteria or methane production activity in the methane fermentation tank by comparing and calibrating the fluorescence properties of a standard sample containing only methane bacteria and the fluorescence properties from the methane fermentation tank to be tested. It is possible to ask for it.

The above is a measurement principle that focuses only on methane bacteria. However, samples containing methane bacteria, such as fermentation liquid in a methane fermentation tank, generally have high turbidity due to contamination with other substances. Even in diluted solutions, the fluorescence intensity is attenuated by turbidity. As a countermeasure against this, the turbidity of the specimen at the excitation light wavelength and fluorescence wavelength is determined from the amount of transmitted light of the excitation light and the above-mentioned attenuation effect is corrected, so that the methane bacteria are not affected by the turbidity of the specimen. The true value of the number or methanogenic activity is determined.

[Embodiment] FIG. 1 is a schematic block diagram of a measuring device showing an embodiment of the measuring method according to the present invention. In the figure, 7 is a methane fermentation tank, 8 is a sampling tube for collecting a sample containing methane bacteria from the methane fermentation tank, and the collected sample is sent to a sample regulator 12 by a pump 9. Reference numeral 10 denotes a liquid reservoir, which stores a solution for diluting or alkali-treating the sample sent to the sample conditioner 12 to make it a suitable specimen for measurement. The solution in the liquid reservoir 10 is measured and sent to the sample adjuster 12 by the pump 11, where it is mixed with the sample.

The mixed solution prepared as a sample is transferred to a sample chamber 13 for measuring fluorescence and turbidity. Next, the subject in the sample chamber 13 is irradiated with excitation light whose wavelength has been measured by passing the light from the light source 15 through the spectrometer 16 . The transmitted light, which is attenuated due to the turbidity of the sample, is
The light is received by a photomultiplier tube 24 through a spectrometer 23,
The photocurrent is converted into a photocurrent and measured by the detector 22. In addition, by irradiating the excitation light through the spectroscope 16, the fluorescence emitted by the substance specific to the methanogen bacteria in the specimen is
The wavelength of the fluorescent light is selected by a spectrometer 20, received by a photomultiplier tube 21, converted into a photocurrent value, and measured by a detector 22. A detection signal is then sent from the detector 22 to the microprocessor 26. The microprocessor 26 has a calculation function that corrects the detection signal indicating the fluorescence value from the detector 22 with the detection signal indicating the turbidity value, and calculates the fluorescence intensity with the turbidity corrected. is obtained. In addition to such calculation functions, the microprocessor 26 also has a function of automatically controlling various functions within the apparatus such as sampling tubes and liquid feeding. Data processed by the microprocessor 26 is output and displayed at an output terminal 27. The output terminal 27 not only outputs and displays processing data and control sequences, but also inputs data to the microprocessor 26 through the output terminal 27. In addition, for the subject whose measurement has been completed,
The liquid is drained from the sample chamber by the pump 18 to the waste liquid reservoir 19.
abandoned.

Next, based on the measurement principle described above, the specific procedure of the measurement method using the measurement apparatus shown in FIG. 1 will be explained.

The measurement method based on the above principle is operated as follows. A sample is collected from the methane fermentation tank 7 using a sampling tube 8, diluted or treated with alkali to obtain a predetermined test sample using a sample conditioner 12, and placed in a container in a sample chamber 13 to be used as a test sample. Next, a spectroscope 16 selects excitation light in a specific wavelength range that causes the F ₄₂₀ substance to emit fluorescence. Light in the wavelength range of 380 to 440 nm is used as the excitation light. This selected excitation light of a specific wavelength is applied to the sample chamber 1.
3, and the fluorescent light generated from the subject is sent to the spectrometer 2.
0 and is received by a photomultiplier tube 21, and the light transmitted through the subject is received by a photomultiplier tube 24 through a spectroscope 24. A photocurrent indicating the intensity of fluorescence and a photocurrent indicating turbidity are measured in time series by a detector 22, and sent to the microprocessor 2 as a detection signal.
6 is input. The microprocessor 26 performs calculations based on the amount of transmitted light to calculate the true intensity of the fluorescent light by correcting the attenuation of the fluorescent light intensity due to the turbidity of the subject. The result is displayed on the output terminal 27.

In addition, in the measuring device shown in FIG. 1, the spectrometer 2
0 is fixed at a specific wavelength (fluorescence wavelength), and the spectrometer 16
By scanning in a specific wavelength range, the excitation spectrum of _F420 material can be obtained. Further, by fixing the spectroscope 16 to a specific wavelength (excitation wavelength) and scanning the spectrometer 20 in a specific wavelength range, a fluorescence spectrum of the F ₄₂₀ substance can be obtained. In this way, in any of these spectra, a peak of spectral intensity due to F ₄₂₀ substances can be obtained, which can be identified as the spectral intensity corresponding to the abundance of F ₄₂₀ substances, that is, the number of methanogens or methanogenic activity. I can do it. In routine measurements, it has been confirmed that it is sufficient to measure either the excitation or fluorescence spectra, as shown in the example shown in the next figure.

FIG. 3 is a characteristic diagram showing the fluorescence characteristics of digested sludge containing methane bacteria collected from a methane fermentation tank before and after turbidity correction. In the figure, the solid line shows the excitation spectrum before correction, and the dashed-dotted line shows the fluorescence spectrum before correction. The vertical axis of the figure is the spectral intensity, and the horizontal axis shows the wavelength λ _E of the excitation light for the excitation (solid line) spectrum, and the wavelength λ _F of the fluorescence for the fluorescence (dotted chain line) spectrum. Note that the regions indicated by A and B indicate the spectral intensities after correction by the excitation spectrum and fluorescence spectrum, respectively.

The solid curve in FIG. 3 indicates the fluorescence wavelength λ _F
It shows an excitation spectrum obtained by fixing it so that only fluorescence of =470 nm passes through and continuously scanning the excitation wavelength λ _E within the range of 350 to 440 nm using the spectrometer 16. In this case, the peak (point a) is at approximately 420 nm.
It shows. Furthermore, the dashed-dotted curve shows the fluorescence spectrum obtained by fixing the spectrometer 16 at the excitation light wavelength λ _E =420 nm and continuously scanning the fluorescence wavelength λ _F in the wavelength range of 380 to 490 nm using the spectrometer 20. It is.
In this case, the peak ( _b
point). The fact that the spectral intensities at point a and point b almost match is a natural result since two spectra were obtained using the maximum values of both spectra as parameters. However, this characteristic requires first setting λ _F arbitrarily to obtain the excitation spectrum, then determining the fluorescence spectrum by keeping λ _E , which indicates the peak of this excitation spectrum, constant; The fluorescence characteristics shown in FIG. 3 can be easily obtained by determining the excitation spectrum with _F constant.

Once the characteristics shown in Figure 3 are obtained, λ _F =
By determining the maximum value of the excitation spectrum at a constant wavelength of 470 nm, or by determining the maximum value of the fluorescence spectrum at a constant λ _E = 420 nm, or both (for confirmation), each spectrum for methanogens can be determined. The maximum value (a, b) before correction can be easily obtained. Note that the maximum value (a, b) of each spectrum shifts to the left or right depending on the concentration of the alkaline solution processed in the sample conditioner 12, so if the wavelength of the maximum value cannot be specified in advance, the maximum value can be determined using this method. It is possible to find the wavelength that shows .

Turbidity correction is performed at the same time as the above spectrum measurement.
The amount of transmitted light is determined in time series by the spectroscope 23 and the photomultiplier tube 24, and the turbidity correction factor is determined from the degree of decrease. By performing the correction, it is possible to obtain the corrected intensities A and B shown in FIG. 3, respectively.
Note that the peaks (points a and b) in FIG. 3 shift depending on the concentration of the alkaline solution treated in the sample conditioner 12, etc., and the wavelength range of the excitation light is 380 to 440 nm, and the wavelength range of the emitted fluorescent light is 380 to 440 nm. The range is
If the wavelength is 450 to 490 nm, it is possible to measure the number of methane bacteria or methane production activity.

Then, from these spectra, methane bacteria and methane production activity are identified. For identification, it is necessary to know in advance the number of methane bacteria or the methane-producing activity corresponding to the intensity peak of the fluorescence spectrum and the change in the intensity of the spectrum. Therefore, we will conduct measurements to determine the relationship between the two using methane bacteria as a standard sample. To do this, methane bacteria are isolated from the digested sludge of the methane fermentation tank. The peak of the intensity of the fluorescence spectrum when the isolated methane bacteria, such as Methanosarcina barkeri, is suspended in a medium containing no other biological substances and the wavelength of the excitation light is changed using the methane bacteria as a standard sample. The point where appears is determined, and the fluorescence intensity after correction is calculated by the turbidity correction described above. On the other hand, the number of methane bacteria can be determined by counting the colonies of methane bacteria growing on this medium, and the amount of methane generated in the standard sample can be determined by measuring the amount of methane generated by these methane bacteria. be able to. As a result, the correlation between the fluorescence spectrum, the number of methanogens, and the methanogenic activity was revealed. Figures 4 and 5 are calibration diagrams showing an example of the correlation obtained in this way, and show the intensity of the fluorescence spectrum when the wavelength of the excitation light is 420 nm, the number of methane bacteria, and the methane generation. This shows the relationship between quantities. The horizontal axis of FIG. 4 is the fluorescence spectrum intensity, and the vertical axis is the number of methane bacteria. The horizontal axis of FIG. 5 is the same as that of FIG. 4, but the vertical axis shows the amount of methane (gas) generated. It is recognized that both calibration curves have good linearity and are fully usable.

The apparatus for carrying out the measurement method of the present invention is shown in FIG. By changing the voltage, the value of the photocurrent flowing through the photomultiplier tubes 20 and 24 is kept within a range suitable for the photomultiplier tubes 21 and 24, and the intensity of the emitted fluorescence or the photocurrent value for each applied voltage is adjusted. The operation of converting into a photocurrent value for a constant fluorescence intensity or a constant applied voltage can also be automatically performed by the system controller. Alternatively, the object of the present invention can be similarly achieved by introducing a detector directly into the methane fermentation tank 7 and detecting fluorescence and turbidity separately.

Although a system in which other microbial groups and foreign substances coexist in a methane fermentation tank such as a sewage treatment system has been described as a test subject, measurements can be made in the same manner even if other microbial groups and foreign substances coexist.

[Effects of the Invention] As explained above, the present invention is capable of measuring the number of methane bacteria or methane production activity without being affected by turbidity, regardless of what other microorganisms and foreign substances coexist with methane bacteria. It becomes possible to do so. Therefore, the methane fermentation tank can operate efficiently,
This has the effect of easily optimizing the operation and management system of a process having a methane fermentation tank, such as a sewage treatment system.

[Brief explanation of drawings]

Fig. 1 is a schematic structural block diagram of a measuring device showing an embodiment of the measuring method of the present invention, Fig. 2 is a block diagram of the main parts including the device system explaining the conventional measuring method, and Fig. 3 is a block diagram of a measuring device according to the present invention. A characteristic diagram showing the fluorescence characteristics before and after turbidity correction of digested sludge containing methane bacteria collected from a methane fermentation tank, and Figure 4 is a calibration diagram showing the relationship between the number of methane bacteria and fluorescence spectrum intensity. FIG. 5 is a calibration diagram showing the relationship between the amount of methane generated and the fluorescence spectrum intensity. In the figure, 7 is a methane fermentation tank, 8 is a sampling tube, 9 is a pump, 10 is a liquid reservoir, 11 is a pump, 12 is a sample conditioner, 13 is a sample chamber, 14 is a power source, 15 is a light source, and 16 is a spectrometer. , 17 is a drain pipe,
18 is a pump, 19 is a waste liquid reservoir, 20 is a spectrometer, 2
1 is a photomultiplier tube, 22 is a detector, 23 is a spectrometer, 24 is a photomultiplier tube, 25 is a power source, 26 is a microprocessor, and 27 is an output terminal.
Note that the same reference numerals in the figures indicate the same or corresponding parts.

Claims (1)

[Claims] 1. (a) irradiating a diluted or alkali-treated specimen containing methane bacteria with excitation light while changing its wavelength, and (b) measuring the emitted fluorescence emitted by the specimen. hand,
The wavelength range of the excitation light is 380 to 440 nm.
(c) Detecting the wavelength of the excitation light exhibiting the peak intensity; (c) Next, irradiating the test subject with the excitation light at the wavelength to obtain a wavelength range of 450 to 450 nm emitted by the test subject.
While measuring the peak intensity of the emitted fluorescent light at 490 nm, (d) measuring the amount of transmitted light through which the excitation light passes through the subject, (e) calculating the peak intensity of the emitted fluorescent light based on the amount of transmitted light. (f) correcting the attenuation effect resulting from the presence of the analyte, and (f) determining the true fluorescence intensity emitted by the methane bacteria.