JPH1128449A - Method for maintaining contaminated material decomposing activity of microorganism and environment restoring method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To maintain the decomposing activity of a microorganism for a longer time and to much more improve the efficiency of an environment restoration by holding an active microorganism decomposing at least one of an aromatic compound and a halogenated aliphatic hydrocarbon compound at a temp. in which the activity does not appear. SOLUTION: In the case that a contaminated material is absent or low in the concentration in the existing region of decomposing bacteria when the activity of the decomposing bacteria is high, the decomposing bacteria maintains the activity without showing the activity. When the contaminated material exists in high concentration, the activity is restored again. That is, during a decomposing and purifying period, the temp. of the decomposing bacteria is controlled to an activity maintaining temp. at which the decomposing bacteria does not show the activity and maintains the activity and an activity showing temp., at which the contaminated material is substantially decomposed, in accordance with the concentration of the contaminated material in the decomposing bacteria existing region. Then, an efficient restoration and purification are performed.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for maintaining the activity of decomposing microorganisms for pollutants and a method for remediating the environment. In particular, the present invention relates to a biological remediation and purification method effective for remediating an environment contaminated with an aromatic compound or a halogenated aliphatic hydrocarbon compound.

[0002]

2. Description of the Related Art In recent years, environmental pollution by organic chlorine compounds that are harmful to living organisms and are hardly decomposable has become a serious problem. In particular, in the soil of high-tech industrial areas such as IC factories in Japan and overseas, tetrachlorethylene (PC
It is thought that contamination by organic chlorine compounds such as E), trichloroethylene (TCE), and dichloroethylene (DCE) has been spread to a considerable extent, and a number of cases actually detected by environmental surveys and the like have been reported. It is said that those organic chlorine compounds remaining in the soil are dissolved in groundwater by rainwater or the like and spread throughout the surrounding area. Such compounds are suspected of carcinogenicity and reproductive toxicity, and are very stable in the environment. Therefore, in particular, the pollution of groundwater used as a water source for drinking water is regarded as a major social problem.

[0003] For these reasons, the restoration and purification of soil by decomposing and removing organic chlorine compounds is an important issue from the viewpoint of environmental conservation, and technologies necessary for purification have been developed.

[0004] For example, various studies have been made on the decomposition of organic chlorine compounds such as TCE using microorganisms for practical use. This decomposition treatment using microorganisms can decompose organic chlorine compounds into harmless substances by selecting microorganisms to be used, basically, no special chemicals are required, and labor and cost for maintenance can be reduced. There are advantages.

[0005] Examples of isolated microorganisms having the ability to degrade organochlorine compounds include, for example, TCE-degrading bacteria:
Welchia alkenophila sero 5 (USP4877736, ATCC53570),
Welchia alkenophila sero 33 (USP4877736, ATCC5357
1), Methylocystis sp.strain M (Agric. Biol. Chem., 53,
2903 (1989), Biosci. Biotech. Biochem., 56,486 (1992),
56,736 (1992)), Methylosinus trichosprium OB3b (A
m.Chem.Soc.Natl.Meet.Dev.Environ.Microbiol., 29,365
(1989), Appl.Environ.Microbiol., 55, 3155 (1989), App
l.Biochem.Biotechnol., 28,877 (1991), JP-A-02-92274
JP, JP-A-03-292970), Methylomonas sp.M
M2 (Appl.Environ.Microbiol., 57,236 (1991)), Alcalige
nes denitrificans ssp.xylosoxidans JE75 (Arch.micro
biol., 154, 410 (1990)), Alcaligenes eutrophus JMP134
(Appl.Environ.Microbiol., 56.1179 (1990)), Alcaligen
es eutrophus FERM-13761 (JP-A-07-123976), Ps
eudomonas aeruginosa JI104 (Japanese Patent Application Laid-Open No. 07-236895), Mycobacterium vaccae JOB5 (J. Gen. Microbiol., 8
2,163 (1974), Appl.Environ.Microbiol., 54.2960 (198
9), ATCC29678), Pseudomonas putida BH (Sewerage Association Journal,
24, 27 (1987)), Pseudomonassp.strain G4 (Appl.Enviro
n.Microbiol., 52,383 (1986), 53,949 (1987), 54,95
1 (1989), 56,279 (1990), 57,193 (1991), USP492580
2, ATCC53617, which was originally classified as Pseudomonas cepacia, but was changed to Pseudomonas sp. ), Pse
udomonas mendocina KR-1 (Bio / Technol., 7.282 (198
9)), Pseudomonasputida F1 (Appl.Environ.Microbiol.,
54,1703 (1988), 54,2578 (1988)), Pseudomonas fluor
escens PFL12 (Appl.Environ.Microbiol., 54, 2578 (198
8)), Pseudomonas putida KWI-9 (JP-A-06-70753), Pseudomonas cepacia KK01 (JP-A-06-227769), Nitrosomonas europaea (Appl.Environ.Microbi)
ol., 56, 1169 (1990)), Lactobacillus vaginalis sp. nov
(Int. J. Syst. Bacteriol., 39, 368 (1989), ATCC 49540).

[0006]

By the way, in environmental restoration using such microorganisms, ideally, microorganisms having a decomposition activity of a contaminant at a peak or in a state close to the peak are efficiently separated from the contaminant. Good contact is preferred from the viewpoint of improving the efficiency of environmental restoration. However, there are various problems in achieving this ideal situation.

For example, in environmental restoration using microorganisms, it is usually necessary to grow microorganisms having an activity capable of decomposing pollutants to a predetermined number or more. This growth can be performed in the environment to be repaired and can be performed in parallel with the environmental repair. However, it is difficult to accurately control the culture conditions during the growth in the environment. In addition, it is difficult to prevent contamination when growing in an environment.

[0008] In consideration of these factors, a method is conceivable in which the microorganism is introduced into the environment after culturing and multiplying in a situation without contamination. However, when there is a time interval between the growth process of the microorganism and the introduction of the microorganism into the environment to be repaired, the decomposition activity of the contaminants possessed by the microorganism is reduced, and Sometimes the ideal conditions cannot be met.

[0009] Decomposition of contaminants in the environment by microorganisms requires microorganisms having a decomposing activity and contaminants. In an actual environment, contaminants are always present around microorganisms. Not necessarily. For example, after the microorganisms have decomposed the contaminant in the area where the microorganism is present, there may be no inflow of new contaminants from the surrounding area to the area.
Specifically, in the case of a contaminated environment, in the case of contaminated groundwater that is stagnant, even if a carrier in which microorganisms having decomposition activity are immobilized is introduced into the groundwater, there is no circulation of the groundwater. After the pollutants contained in the groundwater around the carrier are decomposed, new contaminants do not move around the carrier, and a situation arises in which no pollutants exist around the microorganisms. And even if there is no contaminant in the surroundings, the activity of decomposing microorganisms decreases with time. From these, it is preferable to improve the frequency of contact between microorganisms and contaminants by adding physical disturbance such as agitation to this environment so that such a situation is resolved for efficient environmental restoration. However, in most situations, such disturbances cannot be applied in an actual environment.

The present invention has been made in view of such problems, and has as its object to maintain the activity of decomposing microorganisms for a longer time. Another object of the present invention is to provide an environmental restoration method that can further improve the efficiency of environmental restoration even in an environment where it is difficult to increase the frequency of contact between microorganisms and contaminants.

[0011]

The above object is achieved by the present invention described below.

[0012] The present invention relates to a microorganism characterized by maintaining a microorganism having an activity capable of decomposing at least one of an aromatic compound and a halogenated aliphatic hydrocarbon compound at a temperature at which the activity is not exhibited. The present invention relates to a method for maintaining pollutant decomposition activity.

[0013] The present invention also provides a method for repairing an environment contaminated with a contaminant, the method comprising the steps of: And controlling the temperature of the environment between a first temperature at which the decomposing activity of the microorganism is expressed and a second temperature at which the degrading activity of the microorganism is not expressed, according to the measurement result. The present invention relates to an environmental restoration method characterized by having.

[0014]

Embodiments of the present invention will be described below in detail.

Decomposition of contaminants by microorganisms requires that the microorganisms have activity as decomposers and that the microorganisms come into contact with the contaminants. When the contaminants are not present in the area where the degrading bacteria exist, for example, when the decomposing bacteria have already decomposed the contaminants in the existing area and the contaminants have not flowed into the area where the degrading bacteria exist, There is a case where it has not flowed into the region where the degrading bacteria exist. Even if the decomposing bacterium has a high activity, the activity is not used for the decomposition in a state where there is no contact with the pollutant, and eventually disappears. In addition, even if a contaminant is present, if the concentration is considerably lower than the decomposing activity of the decomposing bacteria in contact, or is much lower than the contaminating concentration of the entire contaminated area, it does not mean that no decomposition is performed. However, the purification efficiency is very poor.

[0016] As a specific example, for example, when the contaminated medium is groundwater whose movement speed is slow, the region where microorganisms are present (for example, when decomposed bacteria immobilized on a carrier is embedded in a groundwater passage). Due to the slow inflow of groundwater, the amount of contaminants passing through the microbial area may fluctuate significantly. Also, on-site of reactor etc.
In the purification of e, the supply amount of the pollutant may fluctuate. Further, when the contaminated medium is soil and, for example, the pollutant is a volatile substance such as TCE or DCE, the air in the soil is circulated by a pump to transfer these volatile contaminants to the region where microorganisms exist. When supplying, there is a case where the movement of the pollutant by circulation becomes slow due to the influence of soil and the like, and the supply speed to the region where microorganisms are present is low. When the contaminated medium is a gas, for example, when treating the contaminated air extracted from the soil by vacuum in the reactor,
There is a case where the content of the pollutant in the extracted air fluctuates. These examples are merely examples, and given the diversity of contamination sites, there are and are likely to be many more situations.

Therefore, when the activity of the degrading bacterium is high and there is no contaminant in the region where the degrading bacterium exists, or when the concentration is low, the activity of the degrading bacterium is maintained without expressing the activity, It is considered that the purification efficiency is improved by restoring the activity again when the contaminant is present at a high concentration in the region where the degrading bacteria exist.

[0018] One embodiment of the present invention relates to a method in which degrading bacteria are present in a contaminated medium and the contaminants can be degraded and purified (during the period of degrading and purifying), without exhibiting the activity of the degrading bacteria. Temperature (activity maintaining temperature) and the temperature at which activity is developed to actually decompose pollutants (activity manifestation temperature) are determined according to the concentration of contaminants in the region where the degrading bacteria exist. This is a method for efficiently repairing and purifying the contaminated medium by controlling it.

Controlling the environment in which the degrading bacteria are present at the activity maintaining temperature is effective even before the degrading bacteria exist in the contaminated medium. That is, when the degrading bacteria are grown in a culture tank and the degrading bacteria are supplied to the contaminated medium, when there is a time gap between the culture of the degrading bacteria and the supply to the contaminating medium, for example, from the culture tank If it takes time to transport the degrading bacteria to the contamination site, or if it takes time to concentrate the degrading bacteria to a high concentration and supply it to the contaminated medium, the degrading bacteria may become contaminated in the contaminated medium. When contact is made, the activity may have already been reduced. In particular, when the decomposing bacteria are not decomposed in the contaminated medium and the contaminants are decomposed only by the supplied decomposing bacteria, the decrease in activity of the decomposing bacteria at this time greatly hinders purification. In such a case, after the degrading bacteria are propagated in the culture tank to have the degrading activity, the activity can be maintained by controlling the environment in which the degrading bacteria exist, that is, the culture solution at the activity maintaining temperature. If the solution is supplied into the contaminated medium, contact between the degrading bacteria and the contaminant can be realized with almost no decrease in activity from the time of culture.

That is, in another embodiment of the present invention, after the degrading bacteria are cultured to have the degrading activity, and before the degrading bacteria are supplied to the contaminating medium to decompose and purify the contaminants ( Before the decomposition and purification period), the environment in which the degrading bacteria are present is controlled to a temperature at which the activity of the degrading bacteria is maintained without exhibiting the activity (activity maintaining temperature), whereby the contaminated medium is efficiently repaired and purified. Is the way.

The activity maintaining temperature is a temperature at which a normal decomposing bacterium does not exhibit an activity, specifically, in the range of 0 to 10 ° C, more preferably 2 to 4 ° C. In this temperature range, the activity of the microorganism can be maintained for a particularly long period of time as compared with the high temperature range. As an example, in the case of JM1, the activity of about 60% in 3 days and about 40% in 5 days is maintained when stored at 4 ° C.

The decomposition purification temperature (activity manifestation temperature)
The temperature should be a temperature at which normal degrading bacteria exhibit activity, and if it is desired to complete purification promptly, the optimum temperature is used, and if the activity is to be maintained for a relatively long time, the temperature is slightly lower. For example, an optimum temperature may be set according to the contamination state of the contaminated medium, the temperature, the cost, and the like. Specifically, the range of 5 to 40 ° C. is preferable for ordinary decomposing bacteria.

As a method for controlling the temperature of the environment in which the degrading bacteria are present, any method may be employed. For example, when the environment where the degrading bacteria is present is an aqueous medium or a gas, the environment can be controlled relatively easily by introducing a cooler with a thermostat. When the degrading bacteria-containing environment is in the soil, a method of introducing cooling air directly or indirectly through a conduit (in this case, a gas other than air such as ammonia gas may be used in a closed system). ), A method of spraying cooling water, a method of introducing cooling water directly or indirectly such as by passing through a conduit (in this case, a refrigerant or an organic solvent may be used if it is a closed system), and dissolves in water. In such a case, there is a method of introducing a salt or the like that can cause an endothermic reaction.

In order to determine the temperature control timing of the environment in which the degrading bacteria exist, it is optimal to monitor the concentration of contaminants in the area where the degrading bacteria exist. As a sampling method for monitoring, when the contaminated medium is an aqueous medium, contaminated water is collected from a sampling tube. If the polluting medium is soil, take the soil itself, or if it cannot, take the gas if it is a volatile contaminant or the soil water if it is not volatile. If the polluting medium is a gas, collect the gas itself. The concentration of the collected contaminants may be measured using an analyzer or the like.

As the microorganism used in the present invention, any microorganism can be used as long as it can decompose aromatic compounds and / or organochlorine compounds, and more specifically, microorganisms of the genus Pseudomonas and Acinetobact.
er) genus, Alcaligenes genus, Vibrio genus, Nocardia genus, Bacillus genus, Lactobacillus
Genus, Achromobacter, Arthrobacter, Micrococcus
occus), Mycobacterium,
Methylosinus, Methylomonas
ylomonas, Genus Welchia, Genus Methylocystis, Nitrosomona
s) genus, Saccharomyces, Candida, Torulopsis
sis), for example, J1 strain FERM BP-5102, JM1 strain FERM BP-
5352, Pseudomonas species TL1 strain FER
MP-14726, Alkaligenes species TL
Two strains FERM P-14642 and the like can be mentioned.

The medium used for culturing the microorganism used in the present invention may be any medium required for the growth of normal microorganisms, such as LB medium and 2xYT medium, as long as the microorganism can grow. It is also possible to culture with a medium supplemented with a carbon source. As an example of the composition of the M9 medium, Na ₂ HPO ₄ : 6.2 g, KH ₂ P
O ₄ : 3.0 g, NaCl: 0.5 g, NH ₄ Cl: 1.
0 g (in 1 liter of medium, pH 7.0).
Culture can be performed under aerobic conditions, and liquid culture at a culture temperature of about 15 to 30 ° C is desirable.

In the present invention, the decomposition treatment of the aromatic compound and / or the volatile organic chlorine compound is performed by bringing the aromatic compound and / or the organic chlorine compound in the aqueous medium, in the soil, and in the gas phase into contact with the decomposing bacteria. It can be done by doing. The contact between the decomposing bacterium and the aromatic compound and / or the organochlorine compound can be performed by any method as long as the microorganism can exhibit the decomposing activity.
It can be carried out using various methods such as a semi-continuous method and a continuous method. The degrading bacteria can be used in a semi-fixed state or immobilized on a suitable carrier. The object to be treated such as waste liquid, soil, gas phase, etc. may be subjected to various treatments as necessary.

In the present invention, the decomposition treatment of the aromatic compound and / or the organic chlorine compound in the aqueous medium is performed by bringing the aromatic compound and / or the organic chlorine compound present in the aqueous medium into contact with the decomposing bacteria. Can be. The main uses are described below, but without being limited to these forms, the method of the present invention can be used for purification treatment of aromatic compounds and / or organochlorine compounds in any aqueous medium.

For example, the simplest method is to directly introduce a decomposing bacterium into an aqueous medium contaminated with an aromatic compound and / or an organic chlorine compound. In this case, it is desirable to adjust the pH of the aqueous medium, salt concentration, temperature, concentration of contaminants, and the like.

[0030] As another application form, a culture tank is provided to culture the decomposing bacteria, and an aqueous medium contaminated with an aromatic compound and / or an organic chlorine compound is introduced into the culture tank at a predetermined flow rate to decompose. There is a form to make it. The introduction and drainage of the aqueous medium may be performed continuously, but may be performed intermittently or batchwise according to the processing capacity. Such control may be optimized by controlling the system according to the concentration of the aromatic compound and / or the organic chlorine compound.

Further, the decomposing bacteria are adhered to a carrier, for example, soil particles and the like, filled in a reaction layer, and an aqueous medium contaminated with an aromatic compound and / or an organochlorine compound is introduced into the reaction tank to perform a decomposition treatment. There is a form to do. In this case, the carrier to be used is not limited to soil particles, but any carrier can be used. However, a carrier that has excellent ability to retain microorganisms and does not impair air permeability is more desirable. For example, as a material that provides a space for living microorganisms, various microorganism carriers commonly used in bioreactors conventionally used in the pharmaceutical industry, food industry, wastewater treatment systems, and the like can be used. More specifically, porous glass, ceramics, metal oxides, activated carbon, inorganic particulate carriers such as kaolinite, bentonite, zeolite, silica gel, alumina, anthracite, starch, agar, chitin, chitosan, polyvinyl alcohol, alginic acid And gel-like carriers such as polyacrylamide, carrageenan, agarose and gelatin, ion-exchangeable cellulose, ion-exchange resins, cellulose derivatives, glutaraldehyde, polyacrylic acid, polyurethane and polyester. As natural products, cellulosic materials such as cotton, hemp and paper, and lignin-based materials such as wood flour and bark can also be used.

The decomposition treatment of the aromatic compound and / or the organochlorine compound in the soil in the present invention can be performed by bringing the aromatic compound and / or the organochlorine compound present in the soil into contact with the decomposing bacteria. . The main uses are described below, but the present invention is not limited to these forms, and the method of the present invention can be used for purification treatment of aromatic and / or organochlorine compounds in any soil.

For example, the simplest method is a method in which a decomposing bacterium is directly introduced into soil contaminated with an aromatic compound and / or an organic chlorine compound. As a method of introduction, in addition to a method of spraying on the soil surface, in the case of treatment in a relatively deep stratum, there is a method of introduction from a well inserted into the ground. Further, when pressure is applied with air or water, the decomposing bacteria spread over a wide area, which is more effective. In this case, it is necessary to adjust various conditions in the soil so as to be suitable for the degrading bacteria. However, many of the degrading bacteria are advantageously accelerated in the presence of a carrier such as soil particles, which is advantageous. Furthermore, it grows equally well even at about 15 ° C., which is usually the average temperature in soil, and can sufficiently maintain decomposition activity.

Further, the degrading bacteria are attached to a carrier, and the carrier is filled in a reaction tank. The reaction tank is introduced into a soil, which is contaminated with an aromatic compound and / or an organic chlorine compound, mainly into an aquifer. There is a form in which a decomposition process is performed. The form of the reaction tank is desirably one that can cover a wide range in soil, such as a fence or a film. In this case, any carrier can be used, but it has excellent ability to hold fine substances,
Those that do not impair the air permeability are more desirable. For example, as a material that provides a space for living microorganisms, various microorganism carriers commonly used in bioreactors conventionally used in the pharmaceutical industry, food industry, wastewater treatment systems, and the like can be used. More specifically, porous glass, ceramics, metal oxides, activated carbon, inorganic particulate carriers such as kaolinite, bentonite, zeolite, silica gel, alumina, anthracite, starch, agar, chitin, chitosan, polyvinyl alcohol, alginic acid And gel-like carriers such as polyacrylamide, carrageenan, agarose and gelatin, ion-exchangeable cellulose, ion-exchange resins, cellulose derivatives, glutaraldehyde, polyacrylic acid, polyurethane and polyester. As natural products, cellulosic materials such as cotton, hemp and paper, and lignin-based materials such as wood flour and bark can also be used.

The decomposition treatment of the aromatic compound and / or the organic chlorine compound in the gas phase in the present invention is carried out by bringing the aromatic compound and / or the organic chlorine compound present in the gas phase into contact with the decomposing bacteria. Can be. The main uses are described below, but without being limited to these forms, the method of the present invention can be used for purification treatment of any gas phase contamination of aromatic compounds and / or organochlorine compounds in the gas phase. is there.

For example, a culture tank is provided to culture the degrading bacteria,
There is a form in which a gas contaminated with an aromatic compound and / or an organic chlorine compound is introduced into this culture tank at a predetermined flow rate and decomposed. There is no particular limitation on the gas introduction method, but it is more preferable that the introduction of the gas agitates the culture solution and promotes aeration. The introduction and exhaust of gas may be performed continuously, but it is also possible to perform processing intermittently or batchwise according to the processing capacity. Such control may be optimized by controlling the system according to the concentration of the aromatic compound and / or the organic chlorine compound.

As another application form, a decomposing bacterium is attached to a carrier, for example, soil particles and the like, filled in a reaction layer, and an aromatic compound and / or an organic chlorine compound contaminated gas is introduced into the reaction tank. To perform a decomposition process.
In this case, the carrier to be used is not limited to soil particles, and any one can be used.
Those that do not impair the air permeability are more desirable. For example, various materials commonly used in bioreactors conventionally used in the pharmaceutical industry, the food industry, wastewater treatment systems, and the like can be used as a material that provides a habitat for microorganisms. More specifically, porous glass, ceramics, metal oxides, activated carbon, kaolinite,
Inorganic particulate carriers such as bentonite, zeolite, silica gel, alumina, anthracite, starch, agar, chitin, chitosan, polyvinyl alcohol, alginic acid,
Gel-like carriers such as polyacrylamide, carrageenan, agarose, and gelatin, ion-exchangeable cellulose, ion-exchange resins, cellulose derivatives, glutaraldehyde, polyacrylic acid, polyurethane, polyester, and the like. As natural products, cellulosic materials such as cotton, hemp and paper, and lignin-based materials such as wood flour and bark can also be used.

Further, examples of the material which can serve both as the retention of the degrading bacteria and the supply of nutrients include compost used in the agriculture, forestry and fisheries industry. That is, straws of cereals such as wheat straw, dried products derived from plants such as sawdust, rice bran, snowy cabbage, and sugarcane marc, and marine wastes such as crab and shrimp shells can be used.

In the purification treatment of the pollutant gas, a substance to be a carrier may be preliminarily filled and decomposing bacteria may be introduced.
It may be pre-cultured. In order to perform the decomposition reaction more efficiently, the above-described nutrients, water content, oxygen concentration, and the like may be maintained at desired conditions. Further, the ratio of the amount of water to the carrier in the reaction tank may be appropriately selected from the growth of the degrading bacteria and the permeability, and the form of the reaction tank may be appropriately selected depending on the amount and concentration of the gas to be treated. Care should be taken to promote contact between the gas and the degrading bacteria held by the carrier,
For example, columns, tubes, tanks, and boxes can be used. Further, a unit having such a shape may be unitized with an exhaust duct, a filter, or the like, or some units may be continuous according to the capacity.

The contaminant gas may be adsorbed on the carrier material at first, and there are rare cases where the effect of using the decomposing bacteria is not well observed. However, after a certain period of time, the contaminants adhering to the carrier material are decomposed, The adsorbability of the contaminant to the carrier material is regenerated by adsorbing the contaminant again on the surface of the decomposed material. In this way, a constant decomposition can always be expected without saturating the decontamination ability.

As described above, a commonly used culture medium for culturing microorganisms can be used as a growth material of the degrading bacteria in the above purification treatment. For example, a broth medium, an M9 medium, a 2xYT medium, an L medium, or a medium in which polypeptone, yeast extract, and the like are arbitrarily mixed with a carbon source such as a sugar or an organic acid are effective. In addition, any of these media can be used, such as a liquid or a medium prepared by adding agarose to a gel.

The method of the present invention is applicable to both closed and open wastewater treatment, soil treatment and air treatment. The microorganisms may be immobilized on a carrier or the like, or various methods for promoting growth may be used in combination.

[0043]

EXAMPLES The present invention will be further described below with reference to examples, but the present invention is not limited to these examples.

Example 1 Effect of Temperature Control Before Decomposition and Purification Period in TCE Decomposition in Aqueous Medium by JM1 Strain The time was required between the time when the degrading bacteria were cultured and the time when the degrading bacteria were supplied to the contaminated medium. The required case was simulated, and the effect of temperature control before the decomposition and purification period was evaluated.

A colony of the JM1 strain on M9 agar medium containing 1.0% sodium malate was inoculated into 200 ml of M9 medium containing 2.0% of sodium sodium malate in a Sakaguchi flask, followed by shaking culture at 15 ° C. for 70 hours. What was done was stored at 4 ° C. Over time, 10 ml of the solution was transferred to a 27.5 ml vial, sealed with a butyl rubber stopper and an aluminum seal, and then the gaseous TCE was adjusted to 10 ppm with the syringe assuming that TCE had completely dissolved into the solution. And shake at 15 ° C., and after 12 hours,
The concentration was measured by gas chromatography. As a control, all the above-mentioned storage performed at 4 ° C were used at 15 ° C.

Table 1 shows the residual ratio of TCE at each bacterial solution collection time.
Shown in It can be seen that the bacteria stored at 4 ° C. clearly maintain the degrading activity as compared to the bacteria stored at 15 ° C.

[0047]

[Table 1] (Example 2) Effect of temperature control during decomposition and purification period in TCE decomposition in aqueous medium by JM1 strain Concentration of contaminants in groundwater changes when treating contaminants in pumped groundwater with a reactor Was simulated, and the effect of temperature control at that time was evaluated.

A colony of the JM1 strain on M9 agar medium containing 1.0% sodium malate was inoculated into 200 ml of M9 medium containing 2.0% sodium malate in a Sakaguchi flask, followed by shaking culture at 15 ° C. for 70 hours. went.

10 ml of this culture was transferred to a 27.5 ml vial, and (1) after sealing with a butyl rubber stopper and an aluminum seal, a gaseous TCE was added to the syringe so that the TCE was completely dissolved in the liquid and became 10 ppm. Added in. (2) After the vial was shaken at 15 ° C. for 12 hours, the TCE concentration in the gas phase was measured by gas chromatography. (2) Next, the stopper was opened and TCE was volatilized, and then stored at 4 ° C. for 12 hours. Hereinafter, (1)-
The operation of (3) was repeated. As a control, the one stored at 4 ° C. at 15 ° C. was used.

Table 2 shows the residual ratio of TCE each time 12 hours after the addition of TCE. It can be seen that the bacteria stored at 4 ° C. clearly maintain the degrading activity as compared to the bacteria stored at 15 ° C.

[0051]

[Table 2] Example 3 Effect of Temperature Control During Decomposition and Purification Period in DCE Decomposition in Aqueous Medium by JM1 Strain Concentration of contaminants in groundwater changes when treating contaminants in groundwater pumped up in a reactor, etc. Was simulated, and the effect of temperature control at that time was evaluated.

Instead of TCE, cis-1,2-dichloroethylene (cis-1,2-DCE), trans
-1,2-dichloroethylene (trans-1,2-D
CE) at 10 ppm and 1,1-dichloroethylene (1,
DCE concentration was measured in the same manner as in Example 2 except that 1-DCE) was adjusted to 5 ppm. As a control, all the above-mentioned storage performed at 4 ° C were used at 15 ° C.

Table 3 shows the respective residual ratios at the fourth TCE addition. It can be seen that the bacteria stored at 4 ° C. clearly maintain the degrading activity as compared to the bacteria stored at 15 ° C.

[0054]

[Table 3] (Example 4) Effect of temperature control during decomposition and purification period on phenol decomposition in aqueous medium by J1 strain Concentration of contaminants in groundwater changes when treating contaminants in pumped groundwater in a reactor Was simulated, and the effect of temperature control at that time was evaluated.

The colony of the J1 strain on the M9 agar medium containing 0.1% of yeast extract was transferred to the same composition medium 20 in a Sakaguchi flask.
0 ml was inoculated, and shaking culture was performed at 22 ° C. for 24 hours.

(1) Phenol was added to this culture solution at 100 pL.
pm and shaken at 15 ° C. for 12 hours, and then the phenol concentration in the culture solution was measured. (2) Next, the culture solution was centrifuged (8000 rpm, 10 minutes, 4 minutes).
C)), and resuspended in 200 ml of M9 medium to remove residual phenol in the culture solution from the bacteria.
Stored at 12 ° C for 12 hours. Less than. The operations (1) and (2) were repeated.

As a method for determining phenol, 10 g of sand was used.
To the phenol in the supernatant extracted by stirring for 1 minute and using J
Detection method by IS method (JIS K0102-1993,
28.1). As a control, the one stored at 4 ° C. at 15 ° C. was used.

Table 4 shows the residual ratio of phenol in each case 12 hours after the addition of phenol. Compared to bacteria stored at 15 ° C,
It can be seen that the bacteria stored at 4 ° C. clearly maintain the decomposition activity.

[0059]

[Table 4] (Example 5) Effect of temperature control during decomposition and purification period on cresol decomposition in aqueous medium by TL1 strain Concentration of contaminants in groundwater changes when treating contaminants in pumped groundwater with a reactor Was simulated, and the effect of temperature control at that time was evaluated.

The colony of the TL1 strain on M9 agar medium containing 0.1% of yeast extract was transformed into the same medium 2 in Sakaguchi flask.
Inoculation was carried out at 25 ° C. for 24 hours with shaking culture.

As a substance to be decomposed, instead of phenol, o
-Cresol and m-cresol (both 300pp
m), except that the control storage temperature was 20 ° C.
The cresol concentration was measured in the same manner as described above.

The quantification of each cresol was determined by a JIS method using p-hydrazinobenzenesulfonic acid by the same extraction method as that for phenol (JIS K0102-199).
3, 28.2).

Table 5 shows the residual ratio at the third addition of each cresol. It can be seen that the bacteria stored at 4 ° C. clearly maintain the degrading activity as compared to the bacteria stored at 20 ° C.

[0064]

[Table 5] (Example 6) Effect of temperature control before decomposition and purification period in TCE decomposition in soil by JM1 strain Simulating the case where it takes time from cultivation of degrading bacteria to supply of degrading bacteria to contaminated medium To evaluate the effect of temperature control before the decomposition and purification period.

A colony of the JM1 strain on M9 agar medium containing 1.0% sodium malate was inoculated into 200 ml of M9 medium containing 2.0% sodium malate in a Sakaguchi flask, followed by shaking culture at 15 ° C. for 70 hours. went.

The culture was centrifuged (8000 rpm,
The cells were collected for 10 minutes at 4 ° C.) and resuspended in M9 medium to increase the bacterial concentration five-fold. 4 ℃
Saved. Next, 50 g of Sawara sand (powder-sterilized) was placed in a vial of 68 ml capacity, 5 ml of the above bacterial solution was added to the sand over time, sealed with a butyl rubber stopper and an aluminum seal, and gaseous TCE was syringed. Added in. At this time, the TCE concentration in the solution in the vial was adjusted to 50 ppm (the concentration when all TCE was dissolved in water). After standing at 15 ° C for 12 hours, the TCE
The concentration was measured by gas chromatography. A high-concentration bacterial solution similarly prepared and stored at 15 ° C. was used as a control.

Table 6 shows the residual ratio of TCE at each bacterial solution collection time.
Shown in It can be seen that the bacteria stored at 4 ° C. clearly maintain the degrading activity as compared to the bacteria stored at 15 ° C.

[0068]

[Table 6] (Example 7) Effect of temperature control during decomposition and purification period in TCE decomposition in soil by JM1 strain When contaminants in soil are treated in-situ by air circulation, etc. We simulated the change in the amount of contaminants supplied to the area where the degrading bacteria exist due to the concentration change, and evaluated the effect of temperature control at that time.

A JM1 strain colony on an M9 agar medium containing 1.0% sodium malate was inoculated into 200 ml of an M9 medium containing 2.0% sodium malate in a Sakaguchi flask, followed by shaking culture at 15 ° C. for 70 hours. went.

Next, 50 g of Sawara sand (not sterilized) was placed in a vial of 68 ml capacity, and the bacterial solution cultured as above was added to 4.95 ml of M9 medium containing 2.0% sodium citrate.
5 ml of a 1/100 inoculated solution was added to the sand,
Capped with a cotton plug and incubated at 15 ° C. for 70 hours. (1) After sealing with a butyl rubber stopper and an aluminum seal, gaseous TC
E is 5 as the concentration when all TCEs are dissolved in water.
A syringe was added so that the concentration became 0 ppm. (2) After allowing the vial to stand at 15 ° C. for 12 hours, the TCE
The concentration was measured by gas chromatography. (3) Next, the stopper was opened and TCE was volatilized, and then stored at 4 ° C. for 12 hours. Hereinafter, the operations (1) to (3) were repeated.
As a control, all the above-mentioned storage performed at 4 ° C were used at 15 ° C.

Table 7 shows the residual ratio of TCE each time 12 hours after the addition of TCE. It can be seen that the bacteria stored at 4 ° C. clearly maintain the degrading activity as compared to the bacteria stored at 15 ° C.

[0072]

[Table 7] (Example 8) Effect of temperature control during decomposition and purification period in TCE decomposition in soil by J1 strain When contaminants in soil are treated in-situ by air circulation, etc. We simulated the change in the amount of contaminants supplied to the area where the degrading bacteria exist due to the concentration change, and evaluated the effect of temperature control at that time.

The colony of the J1 strain on the M9 agar medium containing 0.1% of yeast extract was transformed into the same composition medium 20 in a Sakaguchi flask.
0 ml was inoculated, and shaking culture was performed at 22 ° C. for 24 hours.

Next, 50 g of Sawara sand (unsterilized) was placed in a vial of 68 ml capacity, and the bacterial solution cultured as described above was mixed with 500 ppm of phenol and 0.2% of yeast extract in M
5 ml of a solution obtained by inoculating 1/100 volume to 4.95 ml of 9 culture media
Was added to the sand in the same manner as in Example 7 except that
The residual ratio was measured.

Table 8 shows the residual ratio of TCE each time 12 hours after the addition of TCE. It can be seen that the bacteria stored at 4 ° C. clearly maintain the degrading activity as compared to the bacteria stored at 15 ° C.

[0076]

[Table 8] Example 9 Effect of Temperature Control During Decomposition and Purification Period on TCE Decomposition in Soil by TL1 Strain When contaminants in soil are treated in-situ by air circulation, etc. We simulated the change in the amount of contaminants supplied to the area where the degrading bacteria exist due to the concentration change, and evaluated the effect of temperature control at that time.

Colonies of the TL1 strain on M9 agar medium containing 0.1% yeast extract were cultivated in Sakaguchi flask using the same medium 2
Inoculation was carried out at 25 ° C. for 24 hours with shaking culture.

Example 7 except that the bacterial solution thus obtained was used.
The TCE residual ratio was measured in the same manner as described above.

Table 9 shows the residual ratio of TCE 12 hours after the addition of TCE. It can be seen that the bacteria stored at 4 ° C. clearly maintain the degrading activity as compared to the bacteria stored at 15 ° C.

[0080]

[Table 9] (Example 10) Effect of temperature control during decomposition and purification period on TCE decomposition in soil by TL2 strain When contaminants in soil are treated in-situ by air circulation, etc. We simulated the change in the amount of contaminants supplied to the area where the degrading bacteria exist due to the concentration change, and evaluated the effect of temperature control at that time.

Colonies of the TL2 strain on M9 agar medium containing 0.1% yeast extract were cultivated in Sakaguchi flasks using the same composition medium 2
Inoculation was carried out at 25 ° C. for 24 hours with shaking culture. The TCE residual ratio was measured in the same manner as in Example 7.

Table 10 shows the residual ratio of TCE each time 12 hours after the addition of TCE. It can be seen that the bacteria stored at 4 ° C. clearly maintain the degrading activity as compared to the bacteria stored at 15 ° C.

[0083]

[Table 10] Example 11 Effect of Temperature Control Before Decomposition and Purification Period in Gas Phase TCE Decomposition by JM1 Strain Simulating the case where it takes time from cultivation of degrading bacteria to supply of degrading bacteria to contaminated medium To evaluate the effect of temperature control before the decomposition and purification period.

A colony of the JM1 strain on M9 agar medium containing 1.0% sodium malate was inoculated into 200 ml of M9 medium containing 2.0% sodium malate in a Sakaguchi flask, followed by shaking culture at 15 ° C. for 70 hours. What was done was stored at 4 ° C.

Of the above culture solution, 30 ml was gradually added to 6 ml over time.
The solution was transferred to an 8 ml vial, and air that had been aerated in a TCE saturated solution was allowed to flow through the solution at a flow rate of 20 ml / min for 30 minutes, and then completely sealed with a butyl rubber stopper and an aluminum seal.
Perform shaking culture at 5 ° C (72 hours in total), and
The decrease in E was measured by gas chromatography. A high-concentration bacterial solution similarly prepared and stored at 15 ° C. was used as a control.

Table 1 shows the residual ratio of TCE at each bacterial solution collection time.
It is shown in FIG. It can be seen that the bacteria stored at 4 ° C. clearly maintain the degrading activity as compared to the bacteria stored at 15 ° C.

[0087]

[Table 11] Example 12 Effect of Temperature Control During Decomposition and Purification Period in Gas Phase TCE Decomposition by JM1 Strain When contaminants in air collected from soil by vacuum extraction are treated in a reactor, the effects of contaminants in air are reduced. We simulated how the concentration changes, and evaluated the effect of temperature control at that time.

A JM1 strain colony on M9 agar medium containing 1.0% sodium malate was inoculated into 200 ml of M9 medium containing 2.0% sodium malate in a Sakaguchi flask, followed by shaking culture at 15 ° C. for 70 hours. went.

30 ml of the above culture solution was transferred to a 68 ml vial, and (1) air aerated in a TCE saturated solution was flowed through the solution at a flow rate of 20 ml / min for 30 minutes, and then sealed with a butyl rubber stopper and an aluminum seal. Then, shaking culture was performed at 15 ° C., and after 12 hours, the TCE concentration in the gas phase was measured by gas chromatography. (2) Next, the stopper was opened and TCE was volatilized and then stored at 4 ° C. for 12 hours.
The following operations (1) and (2) were repeated. As a control, all the above-mentioned storage performed at 4 ° C were used at 15 ° C.

Table 12 shows the residual ratio of TCE each time 12 hours after the addition of TCE. It can be seen that the bacteria stored at 4 ° C. clearly maintain the degrading activity as compared to the bacteria stored at 15 ° C.

[0091]

[Table 12]

[0092]

As is apparent from the above description, according to the present invention, the activity of decomposing microorganisms can be maintained for a longer time. Further, even in an environment where it is difficult to increase the frequency of contact between microorganisms and contaminants, the efficiency of environmental restoration can be further improved.

Continuation of the front page (72) Inventor Yukitoshi Okubo 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon Inc. (72) Inventor Yoshiyuki Higashiya 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon Inc. (72) Inventor Tomoe Mihara 3-30-2 Shimomaruko, Ota-ku, Tokyo Inside Canon Inc.

Claims (22)

[Claims] 1. A microbial contaminant characterized by maintaining a microorganism having an activity capable of decomposing at least one of an aromatic compound and a halogenated aliphatic hydrocarbon compound at a temperature at which the activity is not exhibited. How to maintain decomposition activity. 2. The method according to claim 1, wherein the temperature is in the range of 0 to 10 ° C. 3. The method according to claim 1, wherein the aromatic compound is at least one selected from phenol and cresol. 4. The halogenated aliphatic hydrocarbon compound,
2. The method according to claim 1, wherein the microorganism is at least one of dichloroethylene and trichlorethylene. 5. The microorganism according to claim 1, wherein the microorganism is strain J1 FERM BP-5.
The method for maintaining the activity of decomposing microorganisms for pollutants according to any one of claims 1 to 4. 6. The microorganism according to claim 1, wherein said microorganism is JM1 strain FERM BP-
5. The method according to claim 1, wherein the activity of the microorganism is 5352. 7. The method according to claim 7, wherein the microorganism is TL1 strain FERM P-1.
The method for maintaining the activity of decomposing microorganisms for pollutants according to any one of claims 1 to 4, wherein the activity is 4726. 8. The microorganism according to claim 1, wherein the microorganism is TL2 strain FERM P-1.
The method for maintaining pollutant-decomposing activity of microorganisms according to any one of claims 1 to 4, which is 4642. 9. A method for repairing microorganisms in an environment contaminated with pollutants, the method comprising measuring the amount of pollutants in the environment contaminated with pollutants, including microorganisms having a decomposition activity of the pollutants; A step of controlling the temperature of the environment between a first temperature at which the decomposing activity of the microorganism is expressed and a second temperature at which the decomposing activity of the microorganism is not expressed, according to the measurement result. And environmental restoration method. 10. The method according to claim 9, wherein said first temperature is in the range of 5 to 40 ° C. 11. The environmental restoration method according to claim 9, wherein said second temperature is a temperature in a range of 0 to 10 ° C. 12. The method according to claim 9, wherein the environment is an aqueous medium. 13. The method according to claim 9, wherein the environment is soil. 14. The method according to claim 9, wherein the environment is a gas. 15. The environmental restoration method according to claim 9, wherein the contaminant is an aromatic compound. 16. The aromatic compound is at least one selected from phenol and cresol.
Environmental restoration method described. 17. The method according to claim 9, wherein the contaminant is a halogenated aliphatic hydrocarbon compound. 18. The method according to claim 17, wherein the halogenated aliphatic hydrocarbon compound is at least one of dichloroethylene and trichloroethylene. 19. The method according to claim 19, wherein the microorganism is J1 strain FERM BP-5.
The environmental restoration method according to claim 9, wherein 20. The microorganism according to claim 1, wherein the microorganism is JM1 strain FERM BP-
The environmental restoration method according to claim 9, which is 5352. 21. The microorganism according to claim 1, wherein the microorganism is TL1 strain FERM P-1.
The method of claim 9, wherein the method is 4726. 22. The method according to claim 19, wherein the microorganism is TL2 strain FERM P-1.
The environmental restoration method according to claim 9, which is 4642.