JPH0440654B2 - - Google Patents

Description

[Detailed Description of the Invention] [Technical Field of the Invention] The present invention relates to a method for measuring the number of methane bacteria or methane production activity in a specimen containing methane bacteria, and particularly in a methane fermentation tank of a sewage treatment system. This invention relates to a method that can also be applied to the measurement of the number or methane production activity of methane bacteria present in a large number of microorganisms and foreign substances such as digested sludge.

[Prior art]

Conventionally, this type of measurement method has been shown in FIG. In the figure, 1 is a specimen containing microorganisms, 2 is a light source, 3 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, and 6 is a detection unit that measures the photocurrent of the photomultiplier tube 4.

Next, the actual measurement method will be explained. Light emitted from the light source 2 is transmitted through the specimen 1 having microorganisms, this transmitted light is received by the photomultiplier tube 4, and its intensity is measured by the detection unit 6 as a photocurrent value of the photomultiplier tube 4. . Since there is a certain relationship between the absorbance obtained in this way using visible light as a light source and the concentration of microorganisms present in the specimen 1, the concentration of microorganisms can be evaluated by measuring the absorbance. As a result, the number of bacteria or the activity of microorganisms can be evaluated.

Another method for measuring the activity of microorganisms is to use ATP (Adenosine) contained in microorganisms.
Triphosphate) or NAD(P)H
(Nicotinamide Adenine Dinucleotide
There was a method to optically measure the amount of biological substances involved in energy metabolism called phosphates.

The conventional method for measuring the number or activity of microorganisms is to measure the absorbance of the specimen 1 as described above. It is effective when there are no microorganisms, but when the specimen 1 is composed of many types of microorganisms and contains foreign substances, it is possible to selectively measure the number or activity of the specific type of microorganism that you want to measure from among them. That was impossible. Furthermore, since ATP and NAD(P)H are biological substances present in all microorganisms, they are not suitable for measuring the number of methanogens alone or the methanogenic activity.

[Summary of the invention]

This invention was made in order to eliminate the drawbacks of the conventional methods as described above, and by making the specimen containing methane bacteria alkaline and irradiating it with excitation light in a specific wavelength range or a wavelength range of 380 nm to 440 nm, Wavelength range emitted by the object: 450nm ~
The purpose is to measure the number of methane bacteria or methane production activity by measuring the intensity of fluorescence at 490 nm, and is particularly useful in mixed microbial systems containing foreign substances such as digested sludge, such as in methane fermentation tanks. However, it is intended to make it possible to measure the number of methane bacteria or methane production activity.

[Embodiments of the invention]

An embodiment of the present invention will be described below with reference to the drawings. In FIG. 2, reference numeral 8 denotes the inside of the methane fermentation tank containing the specimen, 9 an optical fiber for introducing and guiding light into and out of the methane fermentation tank inside 8, 14 a power source,
13 is a light source connected to the power source 14, 12 is light source 1
11 is a selector that adjusts the light intensity of the light source 13; 10 is a condenser that focuses the light emitted from the light source onto the optical fiber 9; 15 is the light emitted from the optical fiber 9. 16 is an optical filter that limits the wavelength of the light on the receiving side; 17 is a photomultiplier tube; 18 is a power source for the photomultiplier tube; 19 is a photomultiplier tube that measures the photocurrent of the photomultiplier tube 17. This is the detection part.

Next, the principle and operation of this invention will be explained. Methanobacteria have different physiological properties from normal microorganisms, and although the full details of the electron transport system involved in the energy metabolism of methanogens are still unknown, it is known that they are unique to methanogens. ing. It is known that a substance called F ₄₂₀ functions as an electron carrier in the electron transport chain that exists in the energy metabolism system of this methane bacteria.
This substance is unique to methane bacteria and does not exist in other biological systems. Therefore, the substances involved in the electron transport chain of methane bacteria, mainly F ₄₂₀ , have specific and measurable physicochemical properties that are different from microorganisms other than methane bacteria and foreign substances in the detected body such as digested sludge. If it has the following properties and can be measured in the state of viable bacteria (living bacteria) in the object to be detected, it can be used as a measurement parameter in measuring the number of methane bacteria or methane production activity. In particular, substances involved in the electron transport system of methanogens, mainly F ₄₂₀ , are directly related to the methane production mechanism in their physiological functions, and therefore can be effective measurement targets for measuring methane production activity.

As a result of intensive research based on the above considerations, we found that the fluorescence characteristics thought to be caused by F ₄₂₀ of methane bacteria behave differently in the viable bacterial state than the fluorescence characteristics caused by microorganisms other than methane bacteria and foreign substances in digested sludge. After discovering this, I created this invention.

FIG. 3 shows the fluorescence excitation spectrum and fluorescence spectrum of E. coli suspended in a nutrient medium (tryptone 10 g/l, sodium chloride 10 g/l, yeast extract 5 g/l). The fluorescence excitation spectrum shows the intensity of fluorescence at a wavelength of 470 nm with respect to changes in the excitation wavelength, and the fluorescence spectrum shows the fluorescence spectrum at an excitation wavelength of 380 nm. Typical biological substances that emit fluorescence include amino acids such as tryptophan, tyrosine, and phenylalanine; however, the test sample used here contained a mixture of these fluorescent substances, including methane. It can be regarded as a model for biological sample systems other than bacteria.

FIG. 4 shows the fluorescence excitation spectrum and fluorescence spectrum of a methanogen (Methanosarcina barkeri here) suspended in a minimal medium (a medium containing no organic carbon source).
For comparison, the fluorescence excitation spectrum and fluorescence spectrum of E. coli suspended in minimal medium are shown. However, the fluorescence excitation spectrum of methane bacteria shows the intensity of fluorescence at a wavelength of 470 nm with respect to changes in the excitation wavelength, and the fluorescence spectrum shows the fluorescence spectrum at an excitation wavelength of 400 nm. In addition, the fluorescence excitation spectrum of E. coli is
This shows the intensity of fluorescence at 470 nm, and the fluorescence spectrum shows the fluorescence spectrum at an excitation wavelength of 400 nm. Since a minimal medium is used here, the fluorescence characteristics shown in FIG. 4 are derived only from microorganisms, and it can be seen that the fluorescence characteristics of Methanobacterium exhibit a behavior that is significantly different from that of Escherichia coli. Also, the third
Comparison with the figure shows that the fluorescence characteristics of methane bacteria behave differently from the fluorescence characteristics of microorganisms other than methane bacteria and model samples of foreign substances.

FIG. 5 shows the fluorescence excitation spectrum and fluorescence spectrum of the digested sludge collected from the methane fermentation tank. However, the fluorescence excitation spectrum shows the intensity of fluorescence at a wavelength of 470 nm with respect to changes in the excitation wavelength, and the fluorescence spectrum shows the fluorescence spectrum at an excitation wavelength of 420 nm. Comparing Figure 4 and Figure 5
Excitation spectrum and wavelength range from 380nm to 440nm
The fluorescence spectra in the wavelength range of 450 nm to 490 nm exhibited well-matched behavior, indicating that the fluorescence characteristics of digested sludge in the wavelength range mentioned above are caused by methane bacteria.

FIG. 6 shows the fluorescence excitation spectra of Methanobacteria and Escherichia coli in the nutrient medium at each pH. However, it shows the intensity of fluorescence at a wavelength of 470 nm with respect to changes in excitation wavelength. From the figure, E. coli has a pH of 7.
The excitation spectrum hardly changes in the range of PH11, but the fluorescence excitation peak wavelength and intensity of methanogens change depending on the PH.At PH11, the peak wavelength shifts about 20 mm to the longer wavelength side compared to PH7, and the peak intensity also increases about 3 times. is increasing. In this way, by adding basic solutions or basic solids such as sodium hydroxide, potassium hydroxide, or ammonium hydroxide, the sample is made alkaline in the range of PH7 to PH14,
The signal intensity of the fluorescence intensity signal originating from methane bacteria is increased, and the fluorescence excitation wavelength range is shifted to the longer wavelength side in the range of 0 to 30 nm, thereby reducing the S/N ratio with respect to the background fluorescence originating from components other than methane bacteria. It is possible to increase In particular, as is clear from Figures 4 to 6, the wavelength range for the excitation light is
Wavelength range of light from 410nm to 430nm as fluorescence
Using light between 460 nm and 480 nm allows measurements near the peaks of the excitation and emission spectra.

In addition, among the solid and liquid components in the specimen, the liquid component may be replaced with a solution, such as water, that does not emit fluorescence in the measurement wavelength range in the excitation wavelength range, using centrifugation or filtration, or The S/N ratio of the fluorescence intensity signal originating from methane bacteria can also be increased by diluting it with a solution that does not emit fluorescence in the measurement wavelength range within the excitation wavelength range, such as water. Furthermore, at this time, if a basic solution such as sodium hydroxide or potassium hydroxide is used as a solution that does not emit fluorescence in the measurement wavelength range within the excitation wavelength range, the above-mentioned alkalinity effect will be added.

Based on the above research results, the following method can be used to measure the number of methane bacteria or methane production activity present in a large number of microorganisms in a methane fermentation tank and in foreign substances such as digested sludge. I understood.

(1) Using light in the wavelength range of 380 nm to 440 nm as fluorescence excitation light, the correlation between the excitation spectrum intensity and the number of methane bacteria or methane production activity is determined.
Identify the number of methanogens or methanogenic activity.

(2) Using light in the wavelength range of 450 nm to 490 nm as fluorescence, the number of methane bacteria or methane production activity is identified by the correlation between the fluorescence spectrum intensity and the number of methane bacteria or methane production activity.

(3) The following three methods are used to increase the S/N ratio of the fluorescence derived from methane bacteria to the background fluorescence derived from components other than methane bacteria.

(b) Make the specimen alkaline (b) Replace the liquid component of the specimen with another solution (c) Season the specimen Correlation between fluorescence excitation spectrum and fluorescence spectrum intensity and bacterial count or methanogenic activity can be determined using methane bacteria isolated from digested sludge of M. barkeri etc. as a standard sample. As an example, FIGS. 7 and 8 show the correlation between the number of bacteria and the excitation spectrum intensity, and between the amount of methane generated and the excitation spectrum intensity, respectively.

According to this measurement method, it is possible to measure the number of methane bacteria or methane production activity in real time during operation of the methane fermentation tank, and therefore, a great effect can be expected on the operation control of the methane fermentation tank.

It is convenient to configure FIG. 2 as follows. That is, when a system controller is provided and wired to the detection unit 19, optical filters 12, 16, etc., the fluorescence excitation intensity or the voltage applied to the photomultiplier tube 17 is changed according to the intensity of light introduced into the photomultiplier tube 17, The value of the photocurrent flowing through the photomultiplier tube 17 is maintained within a range suitable for the photomultiplier tube 17, and the photocurrent value for each fluorescence excitation intensity or each applied voltage is changed to the photocurrent value for a constant fluorescence excitation intensity or a constant applied voltage. The operation of converting into a value can be automatically performed by the system controller.

Furthermore, in the above embodiment, a method was explained in which the optical fiber 9 was directly introduced into the methane fermentation tank interior 8 for measurement, but it is also possible to collect the specimen from the methane fermentation tank and measure it outside the methane fermentation tank. ,
When treating a specimen with an alkali, diluting it, or replacing the liquid component of the specimen with another solution, it is easier to process the specimen if it is collected.

Furthermore, when methane bacteria are immobilized on the immobilization carrier, it is also possible to measure at the position of the immobilized methane bacteria using the optical fiber 9.

In addition, in the previous and previous explanations, we mainly talked about measuring the number of methane bacteria or methane production activity present in the large number of microorganisms in the methane fermentation tank and foreign substances such as digested sludge. It is not limited.

〔Effect of the invention〕

As described above, according to the present invention, the signal intensity of the fluorescence intensity signal derived from methane bacteria is increased by making the subject containing methane bacteria alkaline,
and shifts the fluorescence excitation wavelength range to the longer wavelength side,
The S/N ratio for background fluorescence derived from components other than methane bacteria can be dramatically improved, and the above-mentioned It has the effect of accurately measuring the number of methane bacteria or methane production activity.

[Brief explanation of drawings]

FIG. 1 is a block diagram explaining a conventional method for measuring the number of microorganisms, FIG. 2 is a block diagram explaining a method for measuring the number of methane bacteria or methane production activity according to an embodiment of the present invention, and FIG. A characteristic diagram showing the fluorescence characteristics of a model of components other than methane bacteria in sludge, Figure 4 is a characteristic diagram showing the fluorescence characteristics of methane bacteria,
Figure 5 is a characteristic diagram showing the fluorescence characteristics of digested sludge containing methane bacteria, Figure 6 is a characteristic diagram showing changes in the fluorescence characteristics of methane bacteria and Escherichia coli depending on pH, Figures 7 and 8
The figures are characteristic diagrams showing the correlation between the number of methane bacteria and the fluorescence excitation spectrum intensity, and between the amount of methane generated and the fluorescence excitation spectrum intensity, respectively. In the figure, 1 and 8 are objects to be examined, 2 and 13 are light sources, 3, 5, 14, and 18 are power supplies, 4 and 17 are photomultiplier tubes, 6 and 19 are detection units, 9 is an optical fiber,
10, 15 are condensers, 11 is a selector, 12, 1
6 is an optical filter. Note that the same reference numerals in the figures indicate the same or corresponding parts.

Claims (1)

[Claims] 1. A specimen containing methane bacteria is made alkaline and irradiated with excitation light in a specific wavelength range, and the intensity of fluorescence emitted by the specimen in a wavelength range of 450 nm to 490 nm is measured. A method for measuring the number of methane bacteria or methane production activity, which obtains the number of methane bacteria or methane production activity. 2. A method for measuring the number of methane bacteria or methane production activity according to claim 1, wherein the subject is irradiated with excitation light in a wavelength range of 380 nm to 440 nm. 3. The bacterial count or methane production activity of methane bacteria according to claim 1 or 2, characterized in that the liquid component of the sample is replaced with a solution that does not emit fluorescence within the excitation wavelength range and measurement wavelength range. How to measure. 4. A method for measuring the number of methane bacteria or methane production activity according to claim 1 or 2, which comprises diluting the sample with a solution that does not emit fluorescence in the measurement wavelength range within the excitation wavelength range. .