JPH062077B2 - Method of measuring cell concentration - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention can easily and accurately measure the concentration of methane bacteria and other cells in a biological reaction tank in which fermentation production by a microorganism or biological treatment by a biological reaction is performed. The present invention relates to a method for measuring bacterial cell concentration.

(Prior Art) In the fermentation treatment for producing useful substances by the action of microorganisms and the biological treatment for treating organic pollutants (BOD) in wastewater with aerobic bacteria and anaerobic bacteria, Accurately knowing the bacterial cell concentration in the bioreactor is very important for operation management. So in conventional biological processing,
Introducing the concept of MLSS (total suspended sludge volume) as a representative value of the amount of microorganisms in the treatment tank, this MLSS or MLVS
Operation control is performed by measuring S (total organic suspended solids), but these values come in with the wastewater to be treated because they contain all floating substances in the solution. It is affected by substances other than bacterial cells and should be called an inaccurate value in order to accurately grasp the bacterial cell concentration. Moreover, ML
The sewage test method stipulates that the SS measurement should be carried out by repeating the procedure of centrifugation for 20 minutes, water addition to the precipitate several times, evaporation to dryness, drying for 2 hours, and cooling in a desiccator. , 60 more for MLVSS measurement
Since the ignition loss is required at 0 ° C. for 30 to 40 minutes, there is a drawback that the measurement requires a long time of 4 to 10 hours.

On the other hand, the situation is similar in the field of fermentative production of useful substances using microorganisms. Certainly, when the substrate such as acetic acid fermentation is soluble and the product is also soluble, and the microorganism used is a bacterium that is uniformly dispersed in the fermentation broth, the bacterial cell concentration in the fermentor is
Absorbance (Op
It is possible to measure the bacterial cell concentration with this OD in normal fermentation production. For example, molds (eg Penicillium chrysogenum) used for fermentative production of antibiotics (eg penicillin) or molds used for citric acid fermentation (Aspergillus ni
Germ-like bacteria, such as ger), make it impossible to measure bacterial cell concentration using OD because the bacteria themselves entangle and form flocs. Currently, the dry microbial cell weight is measured by the same method as the above-mentioned MLSS measurement and used as an index of the microbial cell concentration, but as with the above, the measurement takes time and cannot be used as an index for online control. is there.

In soy sauce fermentation production, molds (Aspergil
lus oryzae, Aspergillus sojae, etc.) are involved in the fermentation production, and the cell concentration is represented by the dry cell weight as described above. However, the situation is more complicated in the fermentative production of soy sauce. That is, since a solid material such as soybean is used as a fermentation raw material, a large part of the dried solid matter measured as dry bacterial cells is solid matter derived from this fermentation raw material, and therefore the measurement of the bacterial cell concentration is very inaccurate. It was a thing.

(Problems to be Solved by the Invention) The present invention solves such conventional problems and enables measurement of bacterial cell concentration in a biological reaction tank accurately and in a short time. It was completed for the purpose of the method.

(Means for Solving Problems) The present invention allows the addition of a protein solubilizer to a biological reaction tank liquid extracted from a biological reaction tank such as a fermenter for fermentation production or a biological treatment tank for biological treatment. After performing the solubilization treatment, a solid-liquid separation treatment for removing suspended matter is performed, and the separation liquid is irradiated with an excitation light having a predetermined wavelength to measure the intensity of fluorescence emitted by the protein component in the separation liquid. To do.

In the present invention, first, a solubilization treatment is carried out by adding a protein solubilizing agent to the liquid inside the biological reaction tank drawn from the biological reaction tank. There are many protein solubilizers including triton solubilizers, but SDS (sodium dodecyl sulfate) is most effective and preferably has a concentration of 2 to 20 times the expected concentration of cells. SDS is added.
This is because if the amount added is less than this, the protein may be insufficiently solubilized and a satisfactory correlation between the fluorescence intensity and the amount of cells may not be obtained. As the bacterial cell concentration expected here, a value of about 10 to 50% of MLSS or MLVSS may be adopted, and an appropriate addition amount of SDS can be confirmed by performing preliminary measurement once. In the solubilization treatment, in order to promote solubilization, the biological reaction tank liquid to which SDS or the like has been added is adjusted to 80 to 1
It is preferable to perform heat treatment at 00 ° C. for 5 to 10 minutes, and to make the measurement conditions uniform, it is preferable to boil at 100 ° C. for 10 minutes, for example. However, if the heating time is long, it does not matter if the temperature is lower than this. As a result of such protein solubilization treatment, the protein, which is the main component of the bacterial cells in the liquid in the biological reaction tank, is eluted into the liquid.Next, solid-liquid separation is performed to remove suspended substances from the liquid. Perform processing. For the solid-liquid separation treatment, for example, centrifugation treatment at 3000 rpm × 20 minutes is sufficient, but filtration with filter paper may also be used, whereby inorganic substances such as sand and organic fibers that enter the biological treatment tank with the wastewater are introduced. Separate contaminants such as. In this way, the separated solution containing the protein eluted from the bacterial cells and freed from contaminants is obtained.
A fluorescence spectrophotometer is used to irradiate excitation light of a predetermined wavelength. When the wavelength of the excitation light is 260 to 320 nm, more preferably 280 to 300 nm, it is 34
It has a maximum wavelength of 0 nm and as shown in FIG.
Fluorescence distributed in the wavelength range of 80 nm is generated and is measured. It was confirmed that the measured fluorescence intensity had a first-order correlation with the bacterial cell concentration in the liquid in the biological reaction tank, as will be shown in the following examples, and the bacterial cell concentration should be accurately grasped by measuring this fluorescent intensity. You can It is known that F _420, which is an enzyme involved in the electron transfer system of methane fermentation reaction, is excited by an excitation wavelength of ₄₂₀ nm and emits fluorescence in the vicinity of 470 nm when biological treatment is performed by a methanogen. The fluorescence to be measured in the present invention is clearly different from the fluorescence by F ₄₂₀ in both peak wavelength and fluorescence intensity, and it was confirmed that it was derived from a protein such as tryptophan.

(Example) Example 1 An anaerobic biological treatment tank containing a methanogenic bacterium (Methanobrevibacter arbophilus A2, Methanobrevibacter arboriphilas) kept acclimatized with glucose, acetic acid, formic acid, etc. for 2 to 3 months (digestion temperature 37 ° C) ), The biological treatment solution was drawn out, SDS of 2 to 20 times the expected bacterial cell concentration was added, and the mixture was boiled at 100 ° C. for 10 minutes for solubilization treatment. This was subjected to solid-liquid separation treatment with a centrifuge at 3000 rpm for 20 minutes, and the obtained separated liquid was irradiated with excitation light having a wavelength of 290 nm using a fluorescence spectrophotometer, and the fluorescence intensity was measured at a measurement wavelength of 340 nm. As a result, the second
As indicated by white circles in the figure, it was found that there is a strong correlation between the bacterial cell concentration separately measured as MLSS and the fluorescence intensity. At the same time, the excitation wavelength was 420 nm and the measurement wavelength was 470 n.
When the fluorescence intensity emitted from F ₄₂₀ was also measured as m, the results shown by the black circles in FIG. 2 were obtained, and it was found that the correlation also holds for the concentration of methanogens in the case of F ₄₂₀ . . However, the fluorescence intensity of F ₄₂₀ is relatively weak, and F ₄₂₀ does not exist when cells other than methanogenic bacteria are used, such as in an aerobic biological treatment tank. Accurate measurement is possible.

For comparison, the fluorescence intensity was measured in the same manner after treating the biological treatment solution with acetone, toluene, and ultrasonic waves, but when treated with toluene, almost no fluorescence could be measured as in the untreated case, and Fluorescence was measured in the acetone-treated and sonicated ones, but the fluorescence intensity was much smaller than that obtained by the method of the present invention.

Example 2 Aspergillus niger, which is one of the typical molds used for fermentation production, was cultured and the correlation between the dry cell concentration and the fluorescence intensity according to the present invention was observed.

The results of plotting the concentration of dry cells and the time change of the fluorescence intensity according to the present invention measured by the same method as in Example 1 are shown in Table 3.
Shown in the figure. As can be seen from the graph of FIG. 3, there is a strong correlation between the dry cell concentration shown by the black circles and the fluorescence intensity shown by the white circles, and even in the fermentation production using mold, the method of the present invention It was found that the cell concentration can be easily measured.

(Effect of the invention) As is apparent from the above description, the present invention focuses on the fact that the main component of bacterial cells is a protein, solubilizes this, and then solid-liquid separates to remove impurities, Since the liquid is irradiated with excitation light of a predetermined wavelength to generate fluorescence derived from protein and the intensity thereof is measured, the bacterial cell concentration is known.
Compared with the conventional method for measuring MLSS, only the bacterial cell concentration can be measured accurately, and the time required for the measurement is shortened to 1 to 2 hours, which is ¼ or less of the time required for measuring MLSS. be able to. Therefore, according to the present invention, in any of the anaerobic and aerobic biological treatment methods, and also in the fermentation production, it is possible to easily and accurately measure the bacterial cell concentration in the liquid in the biological reaction tank, and to manage its operation. It is extremely useful for ensuring the appropriateness of.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between fluorescence wavelength and fluorescence intensity measured in the method of the present invention, FIG. 2 is a graph showing the relationship between bacterial cell concentration and fluorescence intensity in the first example, and FIG. 3 is It is a graph which shows the relationship between a dry cell density | concentration and a fluorescence intensity in a 2nd Example.

Claims (4)

[Claims] 1. A solubilizing treatment by adding a protein solubilizing agent to a liquid in a biological reaction tank drawn from a biological reaction tank such as a fermenter for fermentation production or a biological treatment tank for biological treatment, followed by suspension. A method for measuring a bacterial cell concentration, which comprises performing a solid-liquid separation treatment for removing a substance, and irradiating the separated liquid with an excitation light having a predetermined wavelength to measure the intensity of fluorescence emitted by a protein component in the separated liquid. 2. The method for measuring the bacterial cell concentration according to claim 1, wherein SDS (sodium dodecyl sulfate) having a concentration of 2 to 20 times the expected bacterial cell concentration is used as the protein solubilizer. 3. The method for measuring the bacterial cell concentration according to claim 1, wherein in the solubilization treatment, heat treatment is carried out at 80 to 100 ° C. for 5 to 10 minutes in order to promote solubilization. 4. The method for measuring the bacterial cell concentration according to claim 1, wherein the wavelength of the excitation light is 260 to 320 nm and the fluorescence measurement wavelength is 320 to 380 nm.