JP3030370B2 - Method for accumulating degrading bacteria that decompose organic pollutants in soil and method for isolating degrading bacteria using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the treatment of soil contaminated with, for example, organic pollutants such as pesticides, and more particularly to the decomposition which can be used to prevent the contamination of groundwater by pesticides in soil. The present invention relates to a technique for accumulating bacteria and a technique for isolating a degrading bacterium using the same, and particularly to a technique capable of greatly reducing the time required for accumulation and isolation.

[0002]

2. Description of the Related Art Agrochemicals are indispensable for maintaining today's agricultural production, and are often used for maintaining flora in golf courses and the like. On the other hand, there is concern that pesticides may become pollutants and cause undesired effects on the environment, especially as a source of groundwater pollution. Therefore, it is desired to develop a technology for effectively preventing the pesticides and the like that have completed their functions from becoming contaminants and remaining or diffused in the environment.

[0003] In the soil, a great variety of microorganisms inhabit in an enormous number on the order of billions / g, and among these microorganisms, there are organic skeletons which are functional skeletons in many pesticides and the like. There are quite a few degrading bacteria that have the ability to degrade compounds to make them harmless or to eliminate them from the environment. Therefore, contaminants such as pesticides can be removed from the environment by utilizing the ability of such decomposing bacteria. However, in the natural state, the density of degrading bacteria capable of decomposing certain organic compounds is low,
It is not usually the case to effectively prevent the contaminants from remaining or diffusing into the environment. Therefore, a method of selectively accumulating and isolating such degrading bacteria from the soil and applying the same to the soil again reduces the possibility that groundwater is contaminated by soil contaminated by organic pollutants such as pesticides. It can be considered as a powerful means to prevent it.

[0004] As a method of accumulating and isolating a specific bacterium from a variety of bacteria living in the soil, there is a soil reflux method. In the soil recirculation method, soil containing degrading bacteria is packed in a column or the like to form a soil layer for accumulation, and an inorganic salt medium containing only contaminants such as pesticides as a carbon and nitrogen source is continuously formed in the soil layer for accumulation. By selectively refluxing, specific degrading bacteria, ie, those that can assimilate the carbon source and nitrogen source contained in the inorganic salt medium or selectively accumulate and isolate those that can be used for conjugation metabolism (co-metabolism) I do. In general, the accumulation and isolation of the degrading bacteria by the conventional soil recirculation method requires one to six months.
In fact, it requires a period of years. The fact that such a long time is required is a major obstacle in making the technology for preventing groundwater contamination by using the above-mentioned degrading bacteria practical.

[0005]

SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances, and an object of the present invention is to realize an accumulation / isolation speed that enables the practical application of a technology for preventing groundwater contamination by using degrading bacteria. An object of the present invention is to provide a method for accumulating degradable bacteria and an isolation method using the same.

[0006]

The present invention is based on the following new findings. In other words, by adding artificial microhabitat with a certain property to the soil layer for accumulation, the above-mentioned soil reflux method can be improved, and the new knowledge that the rate of accumulation and isolation of the degrading bacteria can be greatly improved. is there.

According to the study of the present inventors, a porous material having an infinite number of pores such as a woody carbon material can be easily handled and the effective surface area can be increased.
Fragmentation into a size of about several tens of mm, and mixing this fragmented porous material as an artificial microhabitat in the soil layer for accumulation, depending on the type of pollutant and the type of decomposing bacteria that degrade it, However, it has been found that effective accumulation and isolation can be performed in about three weeks to three months. Such high-speed accumulation and isolation is not only significantly superior to conventional methods that require a period of six months to one year, but also makes practical use of technology such as groundwater pollution prevention technology using degrading bacteria. Can be sufficiently satisfied.

[0008] The mechanism enabling such high-speed integration and isolation is considered as follows. First, regarding the accumulation, the artificial microhabitat made of porous material efficiently and efficiently absorbs the carbon source and nitrogen source, that is, pollutants contained in the inorganic salt medium into the myriad of pores mainly by hydrophobic adsorption. The favorable conditions given by the pores of the habitat make it easy for the degrading bacteria to proliferate and grow in the pores, and that these factors cooperate to efficiently contaminate the pores. The substance is efficiently decomposed by the decomposing bacteria in the pores, and as a result, high-speed accumulation can be realized. That is, by using the artificial microhabitat, the contaminants themselves are efficiently collected in the artificial microhabitat, and the artificial microhabitat is added to the activity promoting action of the degrading bacteria, thereby enabling high-speed accumulation of the degrading bacteria. This effectively works when the artificial microhabitat in which the degrading bacteria are accumulated / isolated is used as a groundwater pollution preventive material in agricultural lands, golf courses, and the like, and enables a more efficient groundwater pollution prevention function.

[0009] Next, regarding the isolation by increasing the accumulation, not only the above mechanism functions, but also it is possible to easily extract the decomposed bacteria accumulated in the soil in the form of an easy-to-handle artificial microhabitat.
In addition, simple inoculation of a degrading bacterium into a new artificial microhabitat using this effectively works in the isolation operation, thereby realizing high-speed isolation.

[0010] The condition for effectively exerting such a mechanism is that the porous material used for the artificial microhabitat has a larger adsorptive power to the organic pollutant than the target soil.

According to a study by the inventors of the present invention, as a more preferable condition, it is preferable that the adsorption constant is several tens times or more and 10,000 times or less than the adsorption constant of the soil used for the accumulation soil layer. Required. Such a condition is that the specific surface area of the porous material is about 50 m ² / g or more and 600 m ² / g.
It is substantially the same as the condition that it is about ² / g or less.

The reason why such a condition is preferable is related to the above mechanism. In other words, if the adsorptive power of the porous material is excessively large, adsorption for the decomposing bacteria to effectively use the contaminants, that is, adsorption that occurs in a form in which the decomposing bacteria can be used in pores of a size easily accessible to the decomposing bacteria. Cannot be efficiently generated. In other words, the adsorption by the pores other than the pores on which the degrading bacteria can be fixed is too large. When the adsorption force is excessive, the use of a porous material on which decomposing bacteria are accumulated as a groundwater pollution control material or the like also leads to a reduction in the efficiency of decomposing pollutants. That is, finding an appropriate adsorption force on the porous material also enhances the functionality when the porous material on which the accumulated and isolated degrading bacteria are established is used as a groundwater pollution control material.

[0013] In addition to these conditions, by giving certain conditions to the pore distribution in the porous material, the mechanism can be more effectively operated. According to the study of the present inventors, such a condition is that the volume ratio of pores having a size capable of forming a colony by the degrading bacteria to the entire pores is 10% or more. That is, for example, in the case of a porous material, for example, a woody carbonized material, a distribution of a wide range of pore diameters can be seen from a nanometer order for a small one to a few hundred micrometer order for a large one. It is. Of these pores, those in which the decomposing bacteria prefer to settle and form more stable colonies are generally pores of about 2 to 50 μm, and particularly pores of about 5 to 20 μm are most suitable. Therefore, a porous material having pores of these sizes at a certain ratio or more has higher suitability as an artificial microhabitat in the present invention. Such conditions have some relationship with the above-mentioned adsorption power or specific surface area. But not equal.

Therefore, the method for accumulating degrading bacteria according to the present invention has an innumerable number of pores and a large number of organic substances for accumulating specific degrading bacteria capable of decomposing organic pollutants contained in soil from the soil. Porous material having a greater adsorption power than the target soil for adsorbing pollutants is fragmented, and the fragmented porous material is mixed with the soil where degrading bacteria inhabit to form a soil layer for accumulation, and the soil for accumulation is formed. It is characterized in that degrading bacteria are accumulated in the crushed porous material by refluxing an inorganic salt medium containing only a target organic pollutant as a carbon source and a nitrogen source in the layer.

Further, the method for accumulating the degrading bacteria according to the present invention comprises the steps of:
The settlement constant is more than the adsorption constant of the soil used for the accumulation soil layer
10 times or more and 10,000 times or less, or a specific surface area of 5
0m ^Two/ G or more and 600m^Two/ G or less
The use of a porous material that is
You.

In the method for accumulating degrading bacteria according to the present invention, it is even more preferable to use a porous material having a volume ratio of 10% or more of the pores having a size capable of fixing the degrading bacteria to the whole pores. Requirements.

The method for isolating a degrading bacterium according to the present invention comprises:
In order to isolate the degrading bacteria using the above-mentioned accumulation method, the degrading bacteria are accumulated only by the fragmented porous material in which the fragmented porous material in which the degrading bacteria have been accumulated is mixed with the new fragmented porous material. A plurality of operations for forming a decomposing bacterium in a new fragmented porous material by forming an inorganic layer medium in which only the target organic pollutant is used as a carbon source and a nitrogen source are formed in the collecting layer. It is characterized in that isolation of the degrading bacteria is performed by increasing the degree of accumulation of the degrading bacteria by repeating the process twice.

Typical examples of the porous material used in the present invention as described above include a woody carbonized material under specific firing conditions. Particularly, for example, 400 to 700 ° C.
A hardwood carbonaceous material fired at moderately low and medium temperature conditions is suitable, and such a woody carbonaceous material satisfies the above-mentioned conditions for the adsorption power, the specific surface area, and the pore distribution. In addition, a porous synthetic apatite or an inorganic foam such as a ceramic foam can be used. All of these porous materials are also excellent in that they do not adversely affect the soil.

[0019]

Embodiments of the present invention will be described below. According to one embodiment of the present invention, as shown in FIG.
An accumulation soil layer 2 is formed in a soil layer tank 1. The accumulation soil layer 2 is formed by using a woody carbonized material crushed to about 5 to 10 mm as an artificial microhabitat and mixing it into soil in which decomposing bacteria live in about 5% by weight. And a target organic pollutant such as simazine (type name; C
AT, whose structural formula is shown in FIG. 2), is returned from the storage tank 4 to the accumulation soil layer 2 from the storage tank 4 using only the carbon source and the nitrogen source. If this reflux is continued for about three weeks, accumulation will proceed sufficiently.

The state of accumulation is monitored by measuring the concentration of simazine in the reflux liquid or the concentration of products resulting from the decomposition of simazine, for example, chloride ions. Perform First, an artificial microhabitat is taken out from the accumulation soil layer 2. Then, the artificial microhabitat having accumulated the degrading bacteria is inoculated or mixed into an artificial microhabitat prepared separately, thereby forming a layer for accumulation using only the artificial microhabitat, and the same reflux is performed on the layer for accumulation. Such an operation is repeated an appropriate number of times, for example, about three times, thereby increasing the degree of accumulation of the degrading bacteria and performing isolation.

[0021]

[Experimental example]

EXPERIMENTAL EXAMPLE 1 An experimental example in which simazine is used as an organic contaminant and the degree of accumulation is compared for a plurality of types of artificial microhabitats having different adsorption powers and different pore distributions will be described. The experimental conditions are as follows. Test pesticide; Simazine standard product Test soil; Upland soil continuously using simazine, sieved to 2 mm or less Reflux; Simazine alone as a carbon and nitrogen source 5 mg /
l Added inorganic salt medium Reflux conditions; 25 ° C, dark place The following A to E were used for artificial microhabitat. The physical properties of each of these artificial microhabitats are as shown in the table of FIG. A: 5 to 10 mm crushed piece of hardwood normally fired woody carbonized material B: 5-1 of chitosan-treated hardwood normally fired woody carbonized material
0 mm debris C: 5 to 10 mm debris of a woody carbonaceous material obtained by further firing a hardwood normally-fired woody carbonized material at 1000 ° C. for 4 hours D: 5 to 5 mm of a coniferous woody carbonized material fired at 1000 ° C. for 8 hours
10 mm crushed pieces E; commercially available granular activated carbon (particle diameter 5 mm)

Here, the chitosan treatment is a treatment in which a woody carbonaceous material is immersed in an acetic acid solution of chitosan and then dried so that a chitosan film is attached to the inner wall of the pores of the woody carbonaceous material. By performing this treatment, it is possible to enhance the habitability of the decomposing bacteria, for example, because the pH in the pores of the woody carbonaceous material that tends to be alkaline is neutralized.

The experimental results under the above conditions are shown in the graphs of FIGS. 4 to 6 and the table of FIG. The graph of FIG. 4 shows simazine, Cl 2 ^- and NO ₃ in the simazine reflux during the experiment.
^-Shows each concentration change. The graph of FIG. 5 shows the difference in the accumulation rate of simazine-decomposing bacteria for each artificial microhabitat with respect to the chloride ion concentration, and the graph of FIG. 6 shows the same for the simazine concentration. The table in FIG. 7 shows the microbial biomass and the viable cell count in each artificial microhabitat 4 weeks after reflux. From these graphs and tables, it can be seen that there is a difference in the accumulation capacity depending on the physical properties of the porous material used for the artificial microhabitat, and that the coniferous wood carbonized material is different from the hardwood carbonized wood whose specific surface area and pore distribution are appropriate. It can be seen that the activated carbon having a very low accumulation capacity and an inappropriate specific surface area and pore distribution cannot provide a practical accumulation ability. This is supported by biomass measurements.

In the case of an artificial microhabitat using a hardwood carbonaceous material, Cl ⁻
And NO ₃ ^- increase of concentration is observed. This means that the accumulation of decomposing bacteria is progressing, and simazine is being actively decomposed in response. It can be seen that sufficient accumulation can be obtained by refluxing for about three weeks.
The viable cell count is not shown for the artificial microhabitats C and E because they were not counted. Here, “replacement of the reflux liquid” in each graph means that the reflux liquid has been replaced with a newly adjusted one at that time.

EXPERIMENTAL EXAMPLE 2 An experiment was conducted by using a soil recirculation method on the assumption that an artificial microhabitat, in which simazine-degrading bacteria had accumulated, was applied as a groundwater contamination preventive material to a golf course soil in which simazine was continuously used. An example in which the groundwater pollution prevention function is compared with an experiment using an unintegrated artificial microhabitat will be described. The respective experimental conditions are as follows.

In the integrated artificial microhabitat,
Test conditions Test pesticide; simazine standard product Test soil; golf course soil continuously using simazine
The artificial microhabitat a; a test artificial microhabitat a; 5 to 10 mm crushed pieces of a chitosan-treated hardwood normally fired woody carbonaceous material in which simazine-degrading bacteria are accumulated under the same conditions as in Experimental Example 1 above. 5 to 10 mm crushed pieces of broad-leaved normal wood-based carbonized wood in which simazine-degrading bacteria are accumulated under the same conditions as in Experimental Example 1 The mixing ratio of artificial microhabitats a and b in the test soil; 2.5% by weight of reflux liquid; Simazine only 5mg /
l Added inorganic salt medium Reflux conditions; 25 ° C, dark place

In the unintegrated artificial microhabitat
Experimental conditions Test pesticide; simazine standard product Test soil; golf course soil continuously using simazine
The artificial artificial microhabitat c: 5 to 10 mm crushed pieces of the non-accumulated chitosan-treated hardwood normally fired woody carbonized material The test artificial microhabitat d; 10 mm debris Contamination rate of artificial microhabitats c and d in test soil; 2.5% by weight reflux liquid; 5 mg / simazine alone as a carbon and nitrogen source
l Added inorganic salt medium Reflux conditions; 25 ° C, dark place

The above experimental conditions substantially realize the conditions in which pesticides are regularly sprayed at intervals of about several days in golf courses and the like. The results of this experiment are shown in the graphs of FIGS. Each of these graphs shows simazine (CA) in the reflux liquid in the experiment for each of the artificial microhabitats a to d.
T), Chloride (Cl ^-) and Nitrate (NO ₃ ^-)
3 shows the change in the concentration.

Looking at the simazine concentration in each of these graphs, it can be seen from the graph of FIG. 9 that the artificial microhabitat a in which simazine-decomposing bacteria have been accumulated shows that simazine is almost completely decomposed in about one week after the start of the experiment. On the other hand, it can be seen from the graph of FIG. 8 that the degradation of the uncollected artificial microhabitat c for comparison is not advanced even after 3 weeks. The same is true for the graph of FIG.
It can be said that the integrated artificial microhabitat b and the non-integrated artificial microhabitat d can be understood from the comparison of the graphs.
From this, it can be understood that groundwater contamination by pesticides can be prevented beforehand by burying artificial microhabitat in which degrading bacteria have been accumulated in soil and expressing its degrading activity.

[0030]

As described above, according to the present invention, it is possible to accumulate and isolate decomposed bacteria living in soil at a high speed, and to prevent the contamination of groundwater by pesticides by utilizing the decomposed bacteria. Can greatly contribute to the development of

[Brief description of the drawings]

FIG. 1 is a schematic view of an apparatus used in an integration method according to an embodiment.

FIG. 2 is a structural formula of simazine.

FIG. 3 is a physical property table of each artificial microhabitat used in Experimental Example 1.

FIG. 4 is a graph showing changes in the concentrations of simazine, Cl ⁻ and NO ₃ ⁻ in the simazine reflux solution in Experimental Example 1.

FIG. 5 is a graph showing the difference in the accumulation rate of simazine-decomposing bacteria for each artificial microhabitat in Experimental Example 1 with respect to chloride ion concentration.

FIG. 6 is a graph showing the difference in the accumulation rate of simazine-decomposed sono with respect to simazine concentration in each artificial microhabitat in Experimental Example 1.

FIG. 7 is a table showing microbial biomass and viable cell count in each artificial microhabitat after 4 weeks of reflux in an experimental example.

FIG. 8 is a graph showing the change in the concentration of simazine, Cl ⁻ and NO ₃ ^{− in} the simazine reflux liquid for artificial microhabitat c that has not been accumulated in Experimental Example 2.

FIG. 9: Simazine, Cl ⁻ in the simazine reflux solution for artificial microhabitat a which has been accumulated in Experimental Example 2.
FIG. 4 is a graph showing a change in concentration of NO ₃ and NO ₃ ⁻ .

FIG. 10 shows simazine and Cl ⁻ in the simazine reflux liquid relating to uncollected artificial microhabitat d in Experimental Example 2.
FIG. 4 is a graph showing a change in concentration of NO ₃ and NO ₃ ⁻ .

FIG. 11 shows simazine and Cl in the simazine reflux solution for the accumulated artificial microhabitat b in Experimental Example 2.
^- and NO ₃ ^- graph showing the change in concentration of.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Soil layer tank 2 Soil layer for accumulation 3 Inorganic salt medium 4 Storage tank

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-7-39369 (JP, A) JP-A-7-213851 (JP, A) JP-A-50-40478 (JP, A) JP-A-6-39369 70753 (JP, A) JP-A-9-276839 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 1/00 WPI (DIALOG) BIOSIS (DIALOG)

Claims (5)

(57) [Claims] 1. A method for accumulating specific decomposing bacteria capable of decomposing an organic pollutant contained in a soil from the soil, comprising a myriad of pores and an adsorption of the organic pollutant that is higher than that of the target soil. The porous material having a large adsorptive power is fragmented, and the fragmented porous material is mixed with the soil in which the decomposing bacteria inhabit to form an accumulation soil layer, and only the target organic pollutants are collected in the accumulation soil layer. A method for accumulating degrading bacteria, wherein degrading bacteria are accumulated on the fragmented porous material by refluxing an inorganic salt medium serving as a carbon source and a nitrogen source. 2. The method according to claim 1, wherein the adsorption constant is several tens times or more and 10,000 times or less than the adsorption constant of the soil used for the accumulation soil layer.
Or a specific surface area and at 50 m ² / g not less than about 600 meters ²
The method according to claim 1, wherein a porous material having a density of about / g or less is used. 3. The method according to claim 1, wherein a porous material having a volume ratio of 10% or more of pores having a size capable of fixing degrading bacteria to the entire pores is used. . 4. The method according to claim 2, wherein a woody carbonaceous material is used as the porous material. 5. A method for isolating a degrading bacterium by using the accumulating method according to any one of claims 1 to 4, wherein a fragmented porous material on which the degrading bacterium has been accumulated is newly obtained. Forming an accumulation layer solely from the fragmented porous material mixed with the non-fragmented porous material, and refluxing the inorganic salt medium containing only the intended organic pollutant as a carbon source and a nitrogen source in the accumulation layer. The method for isolating degrading bacteria by increasing the degree of accumulation of degrading bacteria by repeating the operation of accumulating degrading bacteria on the new fragmented porous material a plurality of times.