JP2000079000A - Detection method of assimilable bacterium, and method for measuring latent degradation activity on specific substrate of sample by using the detection method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting assimilable bacteria capable of promptly deciding whether bio-stimulation is performed or whether bio- augmentation is performed, on the assumption of bio-mediation of an oil-polluted soil. SOLUTION: This method for specifically detecting the assimilable bacteria of a specific substance, present in a solid sample comprises detecting the assimilable bacteria by using direct variable counts(DVC). A latent decomposition activity (S) on the specific substrate in the sample after (t) hours is detected by using the method and the equations X=X0eμt and S=YX=YX0eμt (S is the decomposition activity; X is an amount of microbial cells; X0 is an amount of initial microbial cells; Y is a yield of the microbial cells; μ is a specific growth rate; t is a time).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for specifically detecting a specific substance assimilating bacterium present in a sample, for example, a solid sample such as soil, and a detection result obtained by the detection method. A method for predicting the potential degradation activity of the sample based on the information.

[0002]

2. Description of the Related Art (Bioremediation) In bioremediation of contaminated soil and wastewater treatment by the activated sludge method, a method of activating microorganisms already present by adding nutrients (biostimulation method) ) And a method (bioaugmentation method) for introducing useful microorganisms having a strong ability to decompose pollutants.
When a large number of bacteria that decompose pollutants already exist in contaminated soil or the like, the biostimulation method is effective. However, when almost no degrading bacteria are present, the effect cannot be expected even if nutrients are added. Therefore, when almost no degrading bacteria are present, it is necessary to perform a bioaugmentation method. As described above, the presence or absence of degrading bacteria is important information for selecting a means for performing bioremediation, and a method to be adopted is determined based on this information (see FIG. 1).

[0003] Direct activity measurement method (Direct Vari
able Counts, hereinafter also referred to as DVC method) DV
The C method is a method developed as a method for measuring marine microorganisms by Kogure of the Ocean Research Institute of Tsukaumi University. When a microorganism is cultured in the presence of a cell division inhibitor (such as nalidixic acid, pyromidic acid, pipemidic acid, etc.), the cell grows but cannot divide, so that the cells grow to about 10 to 100 times the normal size. The enlarged microorganism can be easily measured with a microscope visual field, and the DVC method is a method of measuring the enlarged microorganism (see FIGS. 2 and 3).

[0004] The DVC method is also a kind of culture method in that it utilizes the growth of microorganisms. However, conventional culture methods such as a short culture time of 20 to 30 hours and the detection of microorganisms having no colony forming ability can be detected. There are many advantages compared to However, since Kogure's DVC method was developed with an eye on adaptation to marine creatures, it could not be applied to soil microorganisms.

[0005] Someya et al. Of Saga University considered that the reason that the DVC method could not be applied to soil-breaking microorganisms was due to the adsorption of cell division inhibitors by contaminants in soil. Therefore, they increased the amount of the cell division inhibitor added and applied the DVC method to soil microorganisms.

In the DVC method for marine microorganisms, when observing the microorganisms, fluorescence observation is performed using acridine orange, which is a stain for nucleic acids. The acridine orange staining method is based on the total bacterial count (Total Direct).
Counts, hereinafter also referred to as TDC method. This method is generally used to obtain non-viable microorganisms and dead cells. However, measurement requires skill, and when this acridine orange staining method is applied to soil microorganisms, soil particles are stained. Good results are not obtained,
There is a problem to say.

Someya et al., Stained soil microorganisms (fluorescent staining).
After examining the method, ethidium bromide staining (hereinafter referred to as EB), which is a typical nucleic acid stain frequently used in genetic engineering,
Also called a dyeing method. ) Is suitable, and DVC
It has been found that a combination of the EB staining method and the EB staining method enables highly efficient measurement of microorganisms in soil.

[0008] A common method for detecting assimilating bacteria is a culture method using a selective medium. Examples of such methods include a method of measuring the number of colonies of assimilating bacteria generated on a plate medium and a method of measuring degrading bacteria growing in a liquid medium as turbidity. However, microorganisms that do not form a colony on a plate medium cannot be detected, and the types of microorganisms that can be detected are limited, and there are problems such as culturing for several days.

[0009]

The first object of the present invention is to provide a method for specifically detecting a metabolic bacterium of a specific substance present in a sample, for example, a solid sample such as soil. Rapid identification and quantity of assimilating bacteria,
And a method for detecting assimilating bacteria characterized by being easily detectable, in particular, on the premise of bioremediation of oil-contaminated soil, detection of petroleum-degrading bacteria in contaminated soil, and furthermore, the number of such bacteria quickly, Another object of the present invention is to provide a method which can be easily detected and can promptly determine whether to perform biostimulation or bioaugmentation in the bioremediation.

[0010] The second aspect of the present invention relates to a method capable of easily estimating a potential decomposition activity of a sample for a specific substrate using the first detection method.

[0011]

A first feature of the present invention is that:
In a method for specifically detecting a decomposed bacterium of a specific substance present in a solid sample, it is to detect a decomposed bacterium, particularly an assimilating bacterium, by using a DVC method. In the present invention, in order to easily observe and measure the hypertrophic microorganisms obtained by the DVC method, a staining method capable of clearly observing only the hypertrophic microorganisms, for example, EB staining is used in combination. Is preferred. In the present invention, detecting a degrading bacterium, particularly an assimilating bacterium, means specifying the ability of the bacterium (eg, the ability to degrade petroleum) and detecting the total number of the bacterium.

The method for detecting a degrading bacterium of the present invention is a petroleum-assimilating bacterium which is a microorganism capable of specifically degrading a sample, particularly a petroleum product present in contaminated soil, and converting it into its own cell. In addition, the present invention is effective for detecting a chemical substance-utilizing bacterium, which is a microorganism capable of specifically decomposing a chemical substance and converting it into its own cell, and for quantitatively measuring the number of the bacterium.

The method for detecting a degrading bacterium of the present invention is basically used mainly for the measurement or detection of a degrading bacterium in which the substance to be measured coincides with the assimilable substrate as described above. Even for degrading bacteria whose substrate and the substance to be measured do not match,
In the case of a degrading bacterium in which the relationship between the assimilable substrate and the substance to be measured is clear, for example, the assimilable substrate is methane, but the methane assimilating bacterium having the resolution of trichloroethylene or the assimilable substrate is phenol, but It can also be used in the case of phenol assimilating bacteria having resolution.

[0014] Among petroleum-decomposing bacteria, there are few types other than petroleum-utilizing bacteria (many decomposed bacteria are considered to be of this type) that grow using petroleum as an energy source or the like. However, there are true degrading bacteria that degrade petroleum but do not grow using petroleum as an energy source. In the present invention, petroleum assimilating bacteria are mainly targeted, but even for the true degrading bacteria as described above, measurement or detection of the bacteria can be performed by clarifying a specific substrate. Can do it.

The method for detecting a degrading bacterium of the present invention is different from the above-described method for detecting assimilating bacterium, and is capable of detecting assimilating bacterium which does not form a colony on a plate medium. Germ can be detected.

The sample that can be employed in the present invention is not particularly limited as long as it can detect assimilating bacteria by the DVC method. The sample may be a solid sample or a liquid sample, and examples thereof include soil, activated sludge, petroleum products, and soil contaminated with chemical substances.

In the present invention, a sample containing many organic substances as a sample (for example, soil contains many organic substances, and these organic substances are energy sources of microorganisms in the soil) is used as it is. When subjected to the DVC method, even if the substance added as a substrate does not have assimilation properties, there may be a problem that microorganisms that assimilate organic substances originally existing in soil and expand are measured as a background. .

Therefore, the present inventors have further studied the means for minimizing the background, and as a result, the background was removed by removing the organic matter as a sample before soil culture. I was able to minimize it.

Furthermore, the present inventors have found that by removing metabolites of microorganisms after culturing, it is possible to clearly observe only hypertrophic microorganisms. As a result, the microorganisms to be detected can be easily detected, and the number thereof can be accurately detected.

As the cell division inhibitor used in the DVC method employed in the present invention, for example, nalidixic acid, pyromidic acid, pipemidic acid, ciprofloxacin and the like can be used.

This is an effective method for judging whether only nutrients need to be added or microorganisms need to be introduced at the time of bioremediation of petroleum-contaminated soil. The present invention is an important invention in commercializing a soil remediation project. The present invention also relates to a method for specifically detecting assimilating bacteria of a specific substance present in a solid sample. This is an effective method for judging whether only a nutrient should be added or a microorganism needs to be introduced at the same time. An important invention in commercializing a soil remediation project

A second feature of the present invention is that a method for specifically detecting a metabolic bacterium of a specific substance present in the first sample is used to detect a potential for a specific substrate of any soil. Another object of the present invention is to provide a method for easily estimating the decomposition activity. That is, the inventor of the present invention, by using the method of the above-mentioned younger student 1, can determine the initial cell mass X ₀ , the volume V _{0 of} one initial cell and the volume V of one cell after t hours, especially the initial cell mass X Focusing on the fact that ₀ can be detected very accurately and easily, the present invention has been achieved. The potential decomposition activity is, for example, an index indicating how fast natural purification proceeds when oil is spilled on a certain soil, and corresponds to the decomposition amount (S) shown in the following equation. Hereinafter, a method for easily estimating the potential decomposition activity will be described using petroleum-utilizing bacteria as an example, but as described above, the present method is not limited to petroleum-utilizing bacteria.

The petroleum assimilating bacterium ingests a petroleum component to produce (grow) cells, and the amount (X) of the cells after a certain time (t) is calculated by the following equation (X) as shown in FIG. It is represented by 1).

X = X ₀ e ^μt (1) (where X ₀ : initial cell mass, μ: specific growth rate, t: time) The initial cell mass (X ₀ ) of the present invention It is necessary to determine by the first method. Μ (specific growth rate)
Can be obtained as follows. Formula X = X
_{Since 0} e ^μt is represented as InX = InX ₀ + μt, for example, when the initial bacterial mass is 1, X ₀ = 1, that is, I
Since nX ₀ = 0, InX = μt.

The above X can be obtained as follows. X is expressed as X = V / V ₀ and is determined by measuring the initial volume V ₀ of one cell and the volume V of one cell after t hours by the first method. The value μ can be determined as follows. It can be obtained by substituting X, X ₀ and t obtained as described above into the equation X = X ₀ e ^μt . The cell yield Y can be determined as follows. The activity of decomposing a specific substance in a sample is detected, and the activity can be determined by using the decomposing activity. For example, when the specific substance is petroleum, petroleum is aerobicly decomposed, and its substrates are oxygen and petroleum.Therefore, the amount of oxygen required to decompose a certain amount of petroleum is detected, and this oxygen consumption and Oxygen consumption and initial bacterial mass X
By utilizing the fact that there is a correlation with ₀ , it can be obtained as a gradient coefficient.

On the other hand, if the cell yield Y is constant,
The amount of degradation (S) is proportional to the amount of cells X, and the amount of cells X is X = X ₀
Since this is ^e.mu.t , this relationship can be expressed by the following equation (2).

S = YX = YX ₀ e ^μt (2) (where, S: decomposition activity, X: cell mass, X ₀ : initial cell mass, Y: cell yield, μ: specific growth rate , T: time) In the above equation (2), at the initial stage, that is, at t → 0, μt → 0, which is close to e ^μt t → 1.
S = YX ₀ can be expressed.

The initial cell mass X ₀ detected or calculated as described above, the volume V _{0 of} one initial cell, the volume V of one cell after t hours, the cell mass X, the specific growth rate μ The potential degradation activity (S) for a specific substrate in a sample can be determined using the cell yield Y and the above formulas (1) and (2).

According to the second aspect of the present invention, when performing bioremediation, a method (biostimulation method) of activating a microorganism that has already existed by adding nutrients may be used. This is very effective as a means for determining whether it is necessary to employ a method for further introducing a microorganism having a substrate decomposing ability (bioaugmentation method).

[0028]

EXAMPLES Hereinafter, the method for detecting assimilating bacteria of the present invention will be described more specifically with reference to examples.

Example 1 Hereinafter, a specific operation method of the culture and staining methods employed in Examples of the present invention will be described. The culturing and staining were performed in the order of the following operation steps shown in FIG. (A) Dispersion treatment Sterile ultrapure water was added to a soil sample collected from a field in Saga University to perform a stirring treatment to obtain a 100-fold diluted soil suspension. (B) Centrifugal washing 1000 μl of the diluted soil suspension (a) was diluted with sterilized water for 3 hours.
The centrifugal washing was performed twice (1200 rpm, 10 minutes). (C) Addition of DVC medium A DVC medium having the composition shown in Table 1 below was added to the diluted soil suspension washed by the centrifugal washing of (b).

[0030]

[Table 1] (D) Incubation The diluted soil suspension to which the DVC medium of (c) was added was prepared as follows.
Place in a 5 ml polypropylene test tube, 600 rpm
The culture was carried out for 24 hours using a rotary shaker of m. (E) Centrifugal washing The metabolite of (d) was centrifugally washed (1200 rpm).
m, 10 minutes). (F) EB staining and fluorescence observation After the centrifugal washing of the above (e), the culture was stained with ethidium bromide (100 μg / ml) for 3 minutes, and observed with a fluorescence microscope (B excitation, objective 100 × oil immersion system). .

Example 2 (1) Soil sample Petroleum-contaminated soil was prepared by adding 1% of light oil to about 9 kg of forest soil on the premises of the Showa Shell Sekiyu KK Central Research Laboratory. The soil was placed in a stainless steel bucket, sealed in a plastic bag, and acclimated at 25 ° C. for about one month. Aeration was performed once a week by stirring and mixing the soil. (2) Detection of petroleum assimilating bacteria from the soil sample The petroleum assimilating bacteria were detected from the soil sample by the method described in Example 1, and the results are shown in FIG.
In FIG. 8, since the sample is double-stained with EB and CFDA (esterase substrate, cells stained with CFDA have esterase activity), petroleum assimilating bacteria glow yellow-green. . Microorganisms of 1 μm or less that glow yellowish green have been observed in the sample before the culture. Culture 2
In the sample after 4 hours, microorganisms (shown by arrows in the figure) which grew to several μm and glowed green green were observed.
As mentioned above, petroleum-utilizing bacteria do not become long even if they enlarge,
It was observed as a nearly spherical or equally deformed cell. When the yeast extract was used, the hypertrophied cells were observed as elongated cells. On the other hand, in the case of petroleum assimilating bacteria, the hypertrophied cells were observed as spherical or remarkably deformed cells without lengthening. This is thought to be because petroleum affects the cell membrane of microorganisms.

Comparative Example 1 The procedure of Example 2 was repeated except that the centrifugal washing was not performed before the culture. FIG. 5A is a fluorescence micrograph of the soil before culturing. In FIG. 5A, microorganisms that glow small and yellow (both have a size of 1 μm or less)
However, it is difficult to determine whether the microorganism is a microorganism because the soil particles are also shining darkly. FIG. 5B shows a fluorescence microscope photograph after the culture. In FIG. 5B, microorganisms (background) that have grown to several μm using organic matter in the soil as an energy source are observed. Further, FIG.
(C) is a fluorescence micrograph after adding yeast extract to soil without centrifugal washing and culturing. Many microorganisms enlarged to several μm are observed. The number of enlarged cells obtained by subtracting the number of enlarged cells observed in FIG. 5B from the number of enlarged cells observed in FIG. 5C corresponds to the number of microorganisms utilizing the newly added yeast extract. It is thought that.

FIG. 6 shows the effect of Example 1 in which centrifugal washing was performed before culturing. All values shown in FIG. 6 indicate the TDC of soil without centrifugal washing.
(1.1 × 10 ⁹ cells / g dry soil) is a relative value with respect to 100. TDC hardly changed after centrifugal washing. In a system cultured without adding yeast extract, the number of bacteria that was about 25% without washing (background)
Was reduced to approximately 0% by centrifugal washing. The number of bacteria in the system to which yeast extract was added was about 70% without washing, and about 50% when centrifugal washing. The difference of about 20% was approximately equivalent to the number of bacteria without washing (background) without addition of yeast extract. did.

Comparative Example 2 The procedure of Example 2 was repeated, except that the centrifugal washing was not performed after the culture. In the sample of this example which was not subjected to centrifugal washing after culturing, soil particles and metabolites of microorganisms were also stained with EB, making it difficult to observe hypertrophic microorganisms (FIG. 7 (a)). In contrast, in Example 2 in which centrifugal washing was performed after culturing, only the hypertrophic microorganisms could be clearly observed [FIG. 7 (b)].

Example 3 Changes in microflora (flora) in soil contaminated with petroleum Changes in flora in soil due to petroleum contamination were examined. As soil contaminated with petroleum, and as soil not contaminated with petroleum, field soil at Saga University was used. The results are shown in FIG. Each value is shown as a relative value with TDC before adding light oil as 100. When light oil was added to 200 ppm and 2000 ppm, the TDC decreased by about 40% in 24 hours. DV
The number of petroleum-utilizing bacteria detected by Method C was 5 to 12%. The above phenomenon is a result showing that oil pollution has a great effect on the flora in soil.

The change in the flora in the soil was examined using soil acclimated with light oil. FIG. 10 shows changes in the number of petroleum assimilating bacteria detected by the TDC and the DVC method in the artificially contaminated soil. Both TDC and petroleum-utilizing bacteria counts decreased over time. In particular, the ratio of the number of petroleum assimilating bacteria to TDC has decreased remarkably,
The percentage of petroleum-utilizing bacteria decreased from 5% to 20% after 5 months. This is probably because light oil, which is the energy source of petroleum assimilating bacteria, was reduced by cracking and utilization. The reason why the total bacterial count decreased more slowly than the number of petroleum-utilizing bacteria is that microorganisms that use metabolites that petroleum-utilizing bacteria decomposed light oil and dead bacteria of petroleum-utilizing bacteria as energy sources. It is thought that it increased. According to the method for measuring assimilating bacteria of the present invention, the state of change of the microflora (flora) in the soil can be measured.

Example 4 The oil-contaminated soil used in this example was prepared by adding 1 ml of light oil to 50 g of soil collected from several places around Atsugi City. This sample soil was put into a culture vessel (a coulometer manufactured by Okura Electric Co., Ltd.), and the culture was started. The cultivation was performed at 30 ° C. for 2 to 3 days, during which the amount of oxygen absorption accompanying the growth of petroleum assimilating bacteria was continuously measured. The number of petroleum assimilating bacteria was determined by the method shown in Example 1 and the amount of oxygen absorption was obtained. The relationship between the number of petroleum assimilating bacteria and oxygen absorption was as shown in FIG. The cell yield Y was determined from the gradient coefficient in FIG. 12, and the potential decomposition activity (S) could be estimated using these results and the above equations (1) and (2).

[0038]

According to the present invention, a method for specifically detecting assimilable bacteria of a specific substance present in a solid sample is characterized in that a wide variety of assimilable bacteria can be quickly and easily detected. A method for detecting a metabolizing bacterium to be used, and a correlation between the total number of the metabolizing bacterium detected by the detection method and the degrading activity of the sample for the specific substrate, the potential degradation of the sample for a specific substrate. A method was provided for easily estimating activity.

[Brief description of the drawings]

FIG. 1 is a diagram illustrating an outline of bioremediation.

FIG. 2 is a diagram illustrating the principle of the DVC method. (A) In the case of normal culture without using a nucleic acid synthesis inhibitor. (B) In the case of the DVC method using a nucleic acid synthesis inhibitor.

FIG. 3 shows morphological changes of soil bacteria before and after incubation in the DVC method. (A) Shows the state before incubation. (B) shows the state after incubation for 24 hours.

FIG. 4 is a diagram showing the culture and staining steps employed in Example 1.

FIG. 5 is a diagram showing a background of DVC in a non-cleaning DVC method. (A) The state of the soil before culturing is shown. (B) shows the state of the soil after culturing the soil that has not been subjected to centrifugal washing. (C) Yeast extract (YE) on soil not subjected to centrifugal washing
Shows the state of the soil after cultivation with the addition of.

FIG. 6 is a diagram showing the effect of the cleaning DVC method.

FIG. 7 is a diagram showing the effect of centrifugal washing on the fluorescence image of the DVC method. (A) The state of the soil before washing is shown. (B) Shows the condition of the soil after washing.

FIG. 8 is a diagram showing effects of detecting petroleum assimilating bacteria by DVC using light oil as an energy source. (A) The state of the soil before culturing is shown. (B) shows the state of the soil after culturing for 24 hours.

FIG. 9 is a diagram showing a decrease in TDC by oil-added DVC method in oil-non-contaminated soil (field soil in Saga University).

FIG. 10: TD in petroleum contaminated soil in a pot (still at room temperature)
It is a figure which shows the time-dependent change of C and DVC.

FIG. 11 is a graph ^{showing a relation between} X = X ₀ e ^μt and μ
It is a figure which shows the execution result of having performed simulation by setting ₀ to 0.01, 0.1, 0.5, 1.0.

FIG. 12 is a view showing the relationship between the number of petroleum assimilating bacteria and the amount of oxygen absorption in Example 4.

[Explanation of symbols]

A Extended / enlarged cells (cells with proliferative ability) B Cells that have not elongated / enlarged and remain small (cells without proliferative ability) ← Elongated / expanded cells (cells with proliferative ability).

Claims (11)

[Claims] 1. A method for specifically detecting a specific substance-utilizing bacterium present in a sample, comprising using a DVC method. 2. The method according to claim 1, wherein the DVC method and the EB staining method are used in combination. 3. The method for detecting assimilating bacteria according to claim 1, wherein organic substances contained in the sample are removed before culturing. 4. The method for detecting assimilating bacteria according to claim 3, wherein the means for removing organic substances is centrifugal washing. 5. The method for detecting assimilating bacteria according to claim 1, wherein the metabolite of the microorganism is removed after the culture. 6. The method according to claim 5, wherein the removal of the metabolite is centrifugal washing. 7. The method according to claim 1, wherein the sample is soil or activated sludge. 8. The method according to claim 1, wherein the specified substance is a petroleum product or a chemical substance. 9. A method for detecting a potential of a specific substrate in a sample after t hours, using the method for detecting assimilating bacteria according to claim 1 and the following formulas (1) and (2). Method for detecting a specific decomposition activity (S). X = X ₀ e ^μt (1) (where, X: amount of cells, X ₀ : initial amount of cells, μ: specific growth rate, t: time) S = YX = YX ₀ e ^μt (2) (where, S: decomposition activity, X: cell mass, X ₀ : initial cell mass, Y: cell yield, μ: specific growth rate, t: time) 10. The method for detecting decomposition activity (S) according to claim 9, wherein the sample is soil or activated sludge. 11. The method according to claim 9, wherein the specific substance is a petroleum product or a chemical substance.