JPH10296220A - Method for decomposition with microbe and method for restoring environment - Google Patents

Abstract

PROBLEM TO BE SOLVED: To mitigate the adverse effect of a produced oxide upon bacteria by decomposing an organic chlorocompound and an aromatic compound with microbes using an antioxidant. SOLUTION: When a medium contaminated in an aromatic compound and an organic chlorine compound is restored and cleaned by bringing it into contact with biodegrading bacteria, the medium contaminated by the aromatic compound and the organic chlorine compound is effectively restored by continuously keeping the proliferation activity and the biodegradation activity of biodegrading bacteria with the help of an antioxidant added. That is, the antioxidant is added to trap an oxide such as epoxide produced and freed in a cell by biodegradation of the aromatic compound and the organic chlorine compound. Thus it is possible to control and mitigate the irreversible metabolism of nucleic acid and protein in the cell.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for decomposing organochlorine compounds and / or aromatic compounds by microorganisms. Further, the present invention relates to an environmental restoration method useful for purifying a contaminated aqueous medium, soil, and air (gas phase).

[0002]

2. Description of the Related Art In recent years, environmental pollution by volatile organic chlorine compounds which are harmful to living organisms and are hardly decomposable has become a serious problem. In particular, the contamination of volatile organic chlorine compounds such as tetrachloroethylene (PCE), trichloroethylene (TCE), and dichloroethylene (DCE) has spread to a considerable extent in the soil of the paper and pulp industry and precision machinery-related industries in Japan and overseas. It has been reported that many cases were actually detected by environmental surveys. It is said that those volatile 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 are very stable in the environment. In particular, the contamination of groundwater used as a drinking water source is regarded as a major social problem.

[0003] Therefore, purification of an aqueous medium such as contaminated groundwater, soil, and the accompanying gas phase by decomposing and removing volatile organic chlorine compounds is an important issue from the viewpoint of environmental conservation. The technology required for purification is being developed.

For example, adsorption treatment with activated carbon, decomposition treatment with light or heat, etc. have been studied, but are not always practical in terms of cost and operability.

On the other hand, volatile organic chlorine compounds such as TCE, which are stable in the environment, have recently been reported to be decomposed by microorganisms, and research for their practical use has begun.
In the biodegradation treatment using this microorganism, it is possible to decompose organic chlorine compounds to harmless substances by selecting the microorganism to be used, basically no special chemicals are required, and labor and cost for maintenance are reduced. There are advantages such as what you can do.

For example, TCE degrading bacteria include Welchia
alkenophila sero 5 (USP4877736, ATCC53570), Welchia
alkenophila sero 33 (USP4877736, ATCC53571), Methylo
cystis sp.strain M (Agric. Biol. Chem., 53, 2903 (198
9), Biosci. Biotech. Biochem., 56,486 (1992), 56,736
(1992)), Methylosinus trichosprium OB3b (Am.Chem.So
c. Natl. Meet. Dev. Environ. Microbiol., 29, 365 (1989), A
ppl.Environ.Microbiol., 55, 3155 (1989), Appl. Bioche
m.Biotechnol., 28,877 (1991), JP-A-02-92274,
JP-A-03-292970), Methylomonas sp.MM2 (Appl.
Environ.Microbiol., 57, 236 (1991)), Alcaligenes deni
trificans ssp.xylosoxidans JE75 (Arch.microbiol., 15
4,410 (1990)), Alcaligenes eutrophus JMP134 (Appl.
viron.Microbiol., 56.1179 (1990)), Alcaligenes eutro
phus FERM-13761 (JP 07-123976 A), Pseudomona
saeruginosa JI104 (Japanese Patent Application Laid-Open No. 07-236895), Mycob
acterium vaccae JOB5 (J. Gen. Microbiol., 82, 163 (197
4), Appl.Environ.Microbiol., 54.2960 (1989), ATCC2967
8), Pseudomonas putida BH (Sewerage Association Journal, 24, 27 (198
7)), Pseudomonas sp.strain G4 (Appl.Environ.Microbi
ol., 52,383 (1986), 53,949 (1987), 54,951 (1989),
56,279 (1990), 57,193 (1991), USP4925802, ATCC536
17. This fungus was initially classified as Pseudomonas cepacia, but was changed to Acinetobactorsp. ), Pseudomona
s mendocina KR-1 (Bio / Technol., 7.282 (1989)), Pseudo
monas putida F1 (Appl.Environ.Microbiol., 54,1703 (19
88), 54,2578 (1988)), Pseudomonas fluorescens PFL
12 (Appl.Environ.Microbiol., 54, 2578 (1988)), Pseudom
onas putida KWI-9 (JP-A-06-70753), Pseudom
onas cepacia KK01 (Japanese Patent Laid-Open No. 06-227769), Nitros
omonas europaea (Appl.Environ.Microbiol., 56,1169 (19
90)), Lactobacillus vaginalis sp.nov (Int.J.Syst.Ba
cteriol., 39, 368 (1989), ATCC 49540).

[0007]

However, when these degrading bacteria are used in actual environmental purification treatment, it is extremely problematic that intracellular cells produced by decomposition intermediate products accompanying the decomposition of volatile organic chlorine compounds such as TCE. Toxic. For example, Methylocystis sp. Strain M (M strain) (Bioscience and Industry, 52,879 (1994)), toluene / phenol degrading bacteria (Appl. Environ. Microbiol., 55, 272)
3 (1989)), ammonia-oxidizing bacteria (Appl.Environ.Microb
iol., 57, 2986 (1991)), and methane assimilating bacteria (Bioscience and Industry, 49, 492 (1991)).

Although a clear conclusion has not been drawn as to the detailed mechanism of these intracellular toxicity, for example, the production of highly reactive oxides causes irreversible intracellular macromolecules, especially nucleic acids and proteins. It has been suggested that the degrading activity and further the life activity itself of the degrading microorganism are lost. The problem of intracellular toxicity caused by these decompositions makes it difficult to maintain continuous expression of degrading activity and maintain degrading bacteria in actual environmental purification using degrading microorganisms. It has become.

As described above, by avoiding the intracellular toxicity caused by the decomposition of the aromatic compound and / or the volatile organic chlorine compound, an effective environment capable of maintaining the resolution and maintaining the degrading bacteria can be obtained. There is a strong demand for a purification method.

Accordingly, an object of the present invention is to provide a medium contaminated with such an aromatic compound and / or an organochlorine compound when the medium is repaired and purified by bringing the medium into contact with a decomposing bacterium. The effect of the medium contaminated with aromatic compounds and / or organochlorine compounds is reduced by reducing damage to the cells by oxides and the like, thereby maintaining and maintaining the growth ability and decomposition activity of the decomposing bacteria. It is to provide an effective repair method.

[0011]

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

The present invention relates to a method for decomposing a microorganism, which comprises decomposing an organic chlorine compound and / or an aromatic compound using an antioxidant together with the microorganism. In particular, the present invention relates to the above decomposition method, wherein the microorganism is a microorganism that expresses an oxygenase, which is an oxidase. The present invention also relates to an environmental restoration method using the decomposition method of the present invention.

[0013]

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

The present invention provides a method for repairing and purifying a medium contaminated with an aromatic compound and / or an organochlorine compound by contacting the medium with a decomposing bacterium, the bacterium being formed by oxides generated by the decomposition of the compound. By adding an antioxidant for the purpose of reducing the damage to the body, the growth ability and decomposition activity of the decomposing bacteria are maintained and maintained, and the medium contaminated with aromatic compounds and / or organic chlorine compounds is effectively repaired. How to

That is, active oxygen species such as oxides such as epoxides, superoxides, peroxides such as hydrogen peroxide, etc., which are generated and released in cells by the decomposition of aromatic compounds and / or organochlorine compounds are removed. By adding an antioxidant to these existing reaction systems and trapping them, irreversible denaturation of intracellular nucleic acids and proteins is suppressed / reduced. It is to realize the maintenance of. Here, the antioxidant refers to a radical inhibitor that suppresses a chain reaction of autoxidation or a peroxide decomposer that deactivates peroxide by non-radical decomposition.

As described above, nucleic acids, proteins, lipids, and the like are mainly damaged by the generated oxide. In proteins, methionine, histidine, cystine, tyrosine, and tryptophan residues are particularly susceptible to damage. Oxidative modification of these amino acid residues not only causes inactivation of the enzyme but also makes them susceptible to degradation by proteases. Further, in a nucleic acid, faithful replication of a template becomes difficult due to alkylation or the like, resulting in mutation and further cell death.

Compounds which can be used to reduce the damage of these formed oxides include, for example, caffeic acid (3,4-dihydroxycinnamic acid), vitamin E (α-tocopherol), vitamin C (ascorbin) Acid), ubiquinol, uric acid, 2-hydroxyestradiol, bilirubin, 3-hydroxyflavone, chlorogenic acid, catechin, protocatechinic acid, pyrogallol,
Antioxidants such as gallic acid; In addition, any antioxidant can be used as long as it is an effective antioxidant for maintaining and maintaining the microbial decomposition of the aromatic compound and / or the organochlorine compound.

The concentration of the antioxidant used in the present invention varies depending on the type of the antioxidant or the type of the medium using the antioxidant, but is preferably in the range of 1 to 1000 ppm, more preferably in the range of 1 to 200 ppm. It is.

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 Acinetobacte.
r) genus, genus Alcaligenes, genus Vibrio, genus Nocardia, genus Bacillus, lactobacillus (Lactobacillus)
Genus, Achromobacter, Arthrobacter, Micrococcus
occus), Mycobacterium,
Methylosinus, Methylomonas
ylomonas, Genus Welchia, Genus Methylocystis, Nitrosomona
s) genus, Saccharomyces, Candida, Torulopsis
and genus Moraxella, for example, strain J1 (FERM BP-5102), JM
1 strain (FERM BP-5352), Pseudomonas cepacia KK
01 strains (Pseudomonas cepacia KK01, FERM P-4235),
Pseudomonas sp. TL1 strain (Pseudomonas sp.
p.TL1, FERM P-14726), Pseudomonas alkaligenes KB2 strain (Pseudomonas alcaligenes KB2, FERM BP)
-5354), Alkagenes species TL2 strain (Alcal
igenes sp. TL2, FERM P-14642), Vibrio sp. KB1 strain (Vibrio sp. KB1, FERM P-14643) and the like.

The inorganic salt medium used for culturing the microorganism used in the present invention may be any medium as long as it is necessary for the growth of normal microorganisms and can grow the microorganisms. It is also possible to culture with a source added. As an example of the composition of the M9 medium,
Na2HPO4: 6.2 g, KH2PO4: 3.0 g, Na
Cl: 0.5 g, NH4 Cl: 1.0 g (in 1 liter of medium, pH 7.0). The culture can be performed under aerobic conditions, and may be a liquid culture or a solid culture. The culture temperature is preferably around 30 ° C.

The decomposition treatment of the aromatic compound and / or the organic chlorine compound in the present invention comprises the step of separating the aromatic compound and / or the organic chlorine compound in the aqueous medium, the soil, and the gas phase with the decomposing microorganism and the antioxidant. This can be done by contact.

The contact between the decomposing microorganism and the antioxidant and the aromatic compound and / or the organochlorine compound can be carried out by any method as long as the microorganism can exhibit the decomposing activity. And various methods such as a continuous method. The microorganism can be used in a semi-fixed state or by being fixed to 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 organochlorine compound in the aqueous medium is performed by bringing the aromatic compound and / or the organochlorine compound present in the aqueous medium into contact with the decomposing microorganism and the antioxidant. It can be done by doing. The main use forms are described below, but the present invention is not limited to these forms, and 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 microorganism and an antioxidant into an aqueous medium contaminated with an aromatic compound and / or an organic chlorine compound. In this case, the pH of the aqueous medium, the salt concentration,
It is desirable to optimize the temperature, the concentration of pollutants, and the like according to the type of the decomposing microorganism.

As another application form, a culture tank is provided to culture degraded microorganisms, an antioxidant is added to the culture tank, and an aqueous medium contaminated with an aromatic compound and / or an organic chlorine compound is subjected to a predetermined method. There is a form that is introduced at a flow rate and decomposed. 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 microorganisms and the antioxidant are attached to a carrier, for example, soil particles, and the like, and the mixture is filled in a reaction tank, and an aqueous medium contaminated with an aromatic compound and / or an organic chlorine compound is introduced into the reaction tank. There is a form in which the disassembly process is performed. 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. Also as a natural product. Cellulosic materials such as cotton, hemp and paper, and lignin materials such as wood flour and bark can also be used.

In the present invention, the decomposition treatment of the aromatic compound and / or the organochlorine compound in the soil is performed by bringing the aromatic compound and / or the organochlorine compound present in the soil into contact with the decomposing microorganism and the antioxidant. Can do it. 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 and / or organochlorine compound contamination in any soil.

For example, the simplest method is to directly introduce a decomposing microorganism and an acid inhibitor 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 by air, water, or the like, the decomposing microorganisms and the antioxidant are spread over a wide range, which is more effective. In this case, various conditions in the soil may be adjusted to be suitable for the degrading microorganism.

Further, the decomposed microorganisms and the antioxidant are attached to a carrier, and the carrier is filled with the carrier. The reactor is mainly used for aquifers of soil contaminated with aromatic compounds and / or organochlorine compounds. There is a form in which it is introduced into the inside to perform a decomposition process. The reaction tank is shaped like a fence or film.
Those that can cover a wide range in soil are desirable. In this case, any carrier can be used, but it is more preferable that the carrier has an excellent ability to retain microorganisms and does not impair air permeability. For example, as a material that provides a habitat for microorganisms, conventionally, pharmaceutical industry, food industry,
Various microorganism carriers commonly used in bioreactors used in 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, paper, wood flour,
Lignin-based materials such as bark are also available.

In the present invention, the decomposition treatment of the aromatic compound and / or the organochlorine compound in the gas phase is performed by bringing the aromatic compound and / or the organochlorine compound present in the gas phase into contact with the decomposing microorganism and the antioxidant. It can be done by doing. The main uses are described below, but without being limited to these forms, the method of the present invention can be used for the purification treatment of any gas phase of aromatic compounds and / or organochlorine compounds in the gas phase. is there.

For example, a decomposing microorganism is cultured in a culture tank, an antioxidant is added to the culture tank, and a gas contaminated with an aromatic compound and / or an organic chlorine compound is introduced at a predetermined flow rate to decompose the microorganism. There is a form. 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 is performed by using an aromatic compound and / or
Alternatively, the system may be controlled and optimized according to the concentration of the organic chlorine compound.

As another application form, a decomposing microorganism and an antioxidant are adhered to a carrier such as soil particles,
There is a form in which this is filled in a reaction layer, and a decomposition treatment is performed by introducing an aromatic compound and / or an organic chlorine compound contaminated gas into the reaction tank. 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, 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,
Inorganic particulate carriers such as kaolinite, bentonite, zeolite, silica gel, alumina, anthracite, starch, agar, chitin, chitosan, polyvinyl alcohol, alginic acid, polyacrylamide, carrageenan,
Gel-like carriers such as agarose and gelatin, ion-exchangeable cellulose, ion-exchange resins, cellulose derivatives, glutaraldehyde, polyacrylic acid, polyurethane, polyester and the like can be mentioned. 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 holding the degrading microorganisms and the antioxidant and supplying nutrients include compost used in the agriculture, forestry and fisheries industry. That is, plant-derived dried products such as straw and sawdust of cereals such as straw, rice bran, snowy greens, sugarcane marc,
Marine waste such as crabs and shrimp shells can also be used.

In the purification treatment of the contaminated gas, a substance to be a carrier is preliminarily filled, and then a decomposing microorganism and an antioxidant may be introduced. In order to perform the decomposition reaction more efficiently, the above-described antioxidants, nutrients, water content, oxygen concentration, and the like are preferably maintained under desired conditions. Further, the ratio of the amount of water to the carrier in the reaction tank may be appropriately selected from the amount and concentration of the gas to be treated, etc. Care should be taken to promote contact with degrading microorganisms. 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 utilizing microorganisms 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 for degrading microorganisms in the above purification treatment. For example, a broth medium, M9 medium, 2XYT medium, 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 gel prepared by adding agarose.
Optimal conditions for the degrading microorganism may be appropriately selected.

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.

[0038]

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 caffeic acid on phenol degradation treatment in soil (brown forest soil) by strain J1 A colony of strain J1 on an agar medium containing 0.2% yeast extract was placed in a 500 ml Sakaguchi flask. Yeast extract 0.0
Inoculate 200 ml of M9 medium containing 2%
Time shaking culture was performed.

To an M9 medium containing 0.02% yeast extract, 600 ppm of phenol and 100 ppm of caffeic acid were added, and 1 ml of the mixture was poured into a 15-ml sterilization tube, and 4 g of brown forest soil collected from Atsugi-shi, Kanagawa was added. After inoculating 10 μl of the bacterial solution of the J1 strain cultured as described above, cotton stoppers were applied, and the culture was allowed to stand at 15 ° C., which is close to the actual temperature in the soil, to measure the phenol concentration.

As a method for quantifying phenol, 5 ml of sterilized water was added to a tube, and phenol in the supernatant extracted by stirring for 1 minute was subjected to JI using aminoantipyrine.
The detection was performed by the S method (JIS K0102-1993, 28.1).

Table 1 shows the residual ratio of phenol on the third day of the culture. Also, as Comparative Example 1, the results when no caffeic acid was added are shown.

Example 2 Effect of caffeic acid on TCE degradation treatment in soil (brown forest soil) by strain J1 15 ml of M9 medium containing 50 ppm of TCE, 300 ppm of phenol as a TCE degradation inducer and 0.02% of yeast extract 4 g of brown forest soil as in Example 1 was poured into a screw vial with a Teflon inner lid.
In addition, caffeic acid was added to a concentration of 100 ppm. Further, 0.1 ml of the bacterial solution of the J1 strain cultured in the same manner as in Example 1 was inoculated, and sealed with a butyl rubber stopper and an aluminum seal. Then, gaseous TCE was added with a syringe, and the mixture was statically cultured at 15 ° C. which was close to the actual temperature in soil.
The TCE amount was determined by measuring the TCE concentration in the gas phase by gas chromatography. As a control, J
The amount of TCE in the system to which one strain was not added was also determined, and the residual ratio relative to the amount of TCE in the control was determined. T on day 3 of culture
Table 1 shows the residual ratio of CE. Also, as Comparative Example 2, the results when no caffeic acid was added are shown.

Examples 3-1 to 3-3 Effect of caffeic acid on DCE decomposition treatment in soil (brown forest soil) by strain J1 Substance to be decomposed cis-1,2-dichloroethylene (cis-1,2) in the medium 2-DCE), trans-1,2-
DCE was prepared in the same manner as in Example 2 except that dichloroethylene (trans-1,2-DCE) or 1,1-dichloroethylene (1,1-DCE) was each 30 ppm.
Was measured. Table 1 shows the residual ratio of each DCE on the third day of culture. The results of Comparative Examples 3-1 to 3-3 when no caffeic acid was added are also shown.

Examples 4-1 to 4-2 Effect of protocatechinic acid on phenol and TCE degradation treatment in soil (brown forest soil) by strain J1 As an antioxidant, 100 ppm protocatechinic acid instead of 100 ppm caffeic acid In the same manner as in Examples 1 and 2, the effect of protocatechinic acid on phenol and TCE degradation treatment in soil by the J1 strain was confirmed. Table 1 shows the residual ratio of phenol and TCE on the third day of culture. Also, the results of Comparative Examples 4-1 and 4-2 when no protocatechinic acid was added are shown.

Examples 5-1 to 5-2 Effect of Vitamin E on Decomposition of Phenol and TCE in Soil (Brown Forest Soil) by J1 Strain As an antioxidant, 100 ppm vitamin E was used instead of 100 ppm caffeic acid. Others were the same as in Examples 1 and 2, and phenol and TC
The effect of vitamin E on the E decomposition treatment was confirmed. Table 1 shows the residual ratios of phenol and TCE on the third day of culture. In addition, the results when no vitamin E was added are shown as Comparative Examples 5-1 and 5-2.

[0047]

[Table 1]

Example 6 Effect of caffeic acid on phenol degradation treatment in soil (brown forest soil) by strain KK01 A colony of strain KK01 on M9 agar medium containing 0.2% yeast extract was placed in a 500 ml Sakaguchi flask. Was inoculated into 200 ml of M9 medium containing 0.2% of sodium glutamate and 0.02% of yeast extract, followed by shaking culture at 30 ° C. for 18 hours.

An M9 medium containing 800 ppm of phenol and 1% sodium glutamate was prepared, and caffeic acid was added to a concentration of 100 ppm. 1 ml of the mixture was poured into a 15-ml sterile tube, and 4 g of brown forest soil collected from Atsugi-shi, Kanagawa was added. After 10 μl of the bacterial solution of the KK01 strain cultured as described above was inoculated thereto, the cotton stopper was inserted, and the culture was allowed to stand at 20 ° C., and the phenol concentration was measured.

As a method for quantifying phenol, 5 ml of sterilized water was added to a tube, and phenol in the supernatant extracted by stirring for 1 minute was subjected to JI using aminoantipyrine.
The detection was performed by the S method (JIS K0102-1993, 28.1).

Table 2 shows the residual ratio of phenol on the second day of the culture. Also, as Comparative Example 6, the results when no caffeic acid was added are shown.

Examples 7-1 to 7-4 Effect of caffeic acid on cresol degradation treatment in soil (brown forest soil) by KK01 strain o-cresol, m-cresol, p-cresol (600 ppm each) ) And toluene (100 pp)
Except for m), the effect of caffeic acid on cresol and toluene degradation treatment in soil under high salt concentration medium conditions by KK01 strain was confirmed in the same manner as in Example 6. In the case of toluene, a Teflon sheet was applied to the inner lid, and the inner lid was sealed to prevent gas leakage.

The quantification of each cresol was determined by the detection method according to the JIS method using p-hydrazinobenzenesulfonic acid (JIS K0
102-1993, 28.2), and the quantitative determination of toluene was performed by a head space gas chromatography method. Culture 2
Table 2 shows the residual ratio of each cresol and toluene on the day. Also, the results when no caffeic acid was added are shown as Comparative Examples 7-1 to 7-4.

Example 8 Effect of Caffeic Acid on TCE Decomposition Treatment in Soil (Brown Forest Soil) by Strain KK01 15 ml of M9 medium containing 50 ppm of TCE, 300 ppm of phenol as a TCE decomposition inducer, and 1% sodium glutamate The mixture was poured into a screw vial with a Teflon inner lid, 4 g of the same brown forest as in Example 1 was added, and caffeic acid was added to a concentration of 100 ppm. To this, KK cultured as in Example 6
0.1 ml of the bacterial solution of the 01 strain was inoculated. After sealing with a butyl rubber stopper and an aluminum seal, gaseous TCE was added with a syringe, and the mixture was allowed to stand at 20 ° C. and cultured. The TCE amount was determined by measuring the TCE concentration in the gas phase by gas chromatography. As a control, the amount of TCE in a similar experimental system without the KK01 strain was also determined, and the residual ratio of the control to the TCE amount was determined. Table 2 shows the residual ratio of TCE on the second day of culture. In addition, as Comparative Example 8, the results when caffeic acid was not added are also shown.

Examples 9-1 to 9-2 Effect of protocatechinic acid on phenol and TCE degradation treatment in soil (brown forest soil) by strain KK01 100 ppm protocatechinic acid instead of 100 ppm caffeic acid as antioxidant In the same manner as in Examples 6 and 8, the effect of protocatechinic acid on the degradation of phenol and TCE in soil by the KK01 strain was confirmed. Table 2 shows the residual ratios of phenol and TCE on the second day of culture. In addition, the results when no protocatechinic acid is added are shown as Comparative Examples 9-1 and 9-2.

Examples 10-1 to 10-2 Effect of Vitamin E on Decomposition of Phenol and TCE in Soil (Brown Forest Soil) by KK01 Strain 100 ppm Vitamin E was used instead of 100 ppm caffeic acid as an antioxidant. Other than that, it carried out similarly to Example 6 and 8, and confirmed the effect of vitamin E to the phenol and TCE decomposition | disassembly process in soil by KK01 strain.
Table 2 shows the residual ratios of phenol and TCE on the second day of culture. In addition, the results when no vitamin E was added are shown as Comparative Examples 10-1 to 10-2.

[0057]

[Table 2]

Examples 11-1 to 11-4 TCE and DCE in soil (fine sand) by JM1 strain
Effect of caffeic acid on degradation treatment J on M9 agar containing 1.0% sodium malate
The M1 strain colony was inoculated into 200 ml of a liquid medium of the same composition in a 500 ml Sakaguchi flask, and cultured with shaking at 22 ° C. for 20 hours.

TCE (50 ppm) as a substance to be decomposed,
1 ml of M9 medium containing cis-1,2-DCE, tmns-1,2-DCE or 1,1-DCE (30 ppm for each) and 2.0% sodium malate is placed in a 15 ml screw vial with a Teflon inner lid. 5 g of Sawara sand was added, and caffeic acid was added to a concentration of 100 ppm. This was inoculated with 0.1 ml of the bacterial solution of the JM1 strain cultured as described above. After sealing with a butyl rubber stopper and an aluminum seal, gaseous TCE was added with a syringe, and the mixture was cultured at 15 ° C. by standing still.

The amount of TCE and DCE is determined by
And DCE concentration were measured by gas chromatography.
As a control, TCE and DCE levels were also determined in the same experimental system without the addition of the JM1 strain, and the residual ratio of the control relative to the TCE and DCE levels was determined.
Table 3 shows the TCE and the residual ratio of each DCE on the third day of culture. Also, the results when no caffeic acid is added are shown as Comparative Examples 11-1 to 11-4.

Examples 12-1 to 12-4 TCE and DCE in soil (fine sand) by JM1 strain
Effect of Protocatechinic Acid on Decomposition Treatment In the same manner as in Example 11 except that 100 ppm of protocatechinic acid was used instead of 100 ppm of caffeic acid as an antioxidant, TCE and DC in soil by JM1 strain were used.
The effect of protocatechinic acid on the E decomposition treatment was confirmed. Table 3 shows the survival rate on the third day of the culture. Comparative Example 1
The results in the case where caffeic acid is not added are shown as 2-1 to 12-4.

Examples 13-1 to 13-4 TCE and DCE in soil (fine sand) by JM1 strain
Effect of Vitamin E on Decomposition Treatment Effect of Vitamin E on decomposition treatment of TCE and DCE in soil by JM1 strain in the same manner as in Example 11 except that 100 ppm vitamin E was used instead of 100 ppm caffeic acid as an antioxidant. It was confirmed. Table 3 shows the survival rate on the third day of the culture. In addition, the results when no caffeic acid was added are shown as Comparative Examples 13-1 to 13-4.

[0063]

[Table 3]

Examples 14 to 17 Effect of caffeic acid on TCE decomposition treatment in soil (loam soil) by other decomposing bacteria As the decomposing bacteria, TL1 strain (which is a TCE degrading bacterium capable of assimilating aromatic compounds) was used. Example 14) Using TL2 strain (Example 15), KB1 strain (Example 16) and KB2 strain (Example 17) as preculture, colonies on M9 agar medium containing 0.2% yeast extract Was inoculated into 200 ml of a liquid medium of the same composition in a 500 ml Sakaguchi flask,
For 36 hours.

Next, TCE (50 pp) was used as the decomposition target.
m) and phenol 200p as a TCE degradation inducer
1 ml of M9 medium containing 0.2% yeast extract containing pm
The mixture was poured into a 5 ml screw vial with a Teflon inner lid, 4 g of loam soil collected from Ibaraki Prefecture was added, and caffeic acid was added to a concentration of 100 ppm. This was inoculated with 0.1 ml of the bacterial solution cultured as described above. After sealing with butyl rubber stopper and aluminum seal, gaseous TC
E was added with a syringe and cultured at 20 ° C. by standing.

The TCE amount was determined by measuring the TCE concentration in the gas phase by gas chromatography. As a control, the quantification of the amount of TCE in the same experimental system to which no strain was added was also performed, and the residual ratio of the control to the amount of TCE was determined. Culture 3
Table 4 shows the survival rate on the day. Also, as a comparative example, the results when no caffeic acid is added are shown.

Examples 18 to 21 Effect of Protocatechinic Acid on TCE Decomposition Treatment in Soil (Loam Soil) by Other Decomposing Bacteria Except that 100 ppm protocatechinic acid was used instead of 100 ppm caffeic acid as an antioxidant. In the same manner as in Example 14, the effect of protocatechinic acid on the TCE degradation treatment in soil under the conditions of a high salt concentration medium by each degrading bacterium was confirmed. Table 4 shows the survival rate on the third day of the culture. In addition, the results when no protocatechinic acid was added are also shown as comparative examples.

Examples 22 to 25 Effect of Vitamin E on TCE Decomposition Treatment in Soil (Loam Soil) by Other Decomposing Bacteria Example 14 except that 100 ppm vitamin E was used instead of 100 ppm caffeic acid as an antioxidant. In the same manner as described above, the effect of vitamin E on the TCE degradation treatment in soil under the conditions of a high salt concentration medium by each degrading bacterium was confirmed. Table 4 shows the survival rate on the third day of the culture. In addition, the results when vitamin E is not added are also shown as comparative examples.

[0069]

[Table 4]

[0070]

As is apparent from the above description, according to the present invention, it is possible to carry out an effective biodegradation treatment of soil contaminated with an aromatic compound and / or an organic chlorine compound.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12N 1/00 C12N 1/20 D 1/20 F B09B 3/00 E // (C12N 1/20 C12R 1:38) (C12N 1/20 C12R 1:05) (C12N 1/20 C12R 1:63)

Claims (37)

[Claims] 1. A method for decomposing microorganisms, comprising decomposing an organic chlorine compound and / or an aromatic compound by using an antioxidant together with the microorganism. 2. The method according to claim 1, wherein the microorganism is a microorganism that expresses an oxygenase that is an oxidase. 3. The microorganism is a genus Pseudomonas, Acinetobacter, Alcaligenes, Vibrio, Nocardia, Bacillus, Lactobacillus, Achromobacter, Arthrobacter, Micrococcus, Mycobacterium, Methylosinus The method according to claim 2, wherein the microorganism is at least one of microorganisms belonging to the genus, Methylomonas, Berchia, Methylostysis, Nitrozomonas, Saccharomyces, Candida, Torulopsis, and Moraxella. 4. The microorganism is J1 strain FERM BP-510.
3. The method for decomposing microorganisms according to claim 2. 5. The method according to claim 5, wherein the microorganism is JM1 strain FERM BP-53.
The method for decomposing microorganisms according to claim 2, which is 52. 6. The method according to claim 2, wherein the microorganism is Pseudomonas sp. 7. The microorganism is Pseudomonas sp.
The method according to claim 2, which is L1 strain FERM P-14726. 8. The method according to claim 2, wherein the microorganism is Pseudomonas alkaligenes. 9. The method according to claim 2, wherein the microorganism is Pseudomonas alkaligenes KB2 strain FERMBP-5354. 10. The method according to claim 2, wherein the microorganism is Pseudomonas cepacia. 11. The method according to claim 11, wherein the microorganism is Judemonas cepacia K.
The method according to claim 2, wherein the strain is K01 strain FERMBP-4235. 12. The method according to claim 2, wherein the microorganism is Alcaligenes species. 13. The method according to claim 2, wherein the microorganism is Alcaligenes sp. TL2 strain FERMP-14642. 14. The method according to claim 2, wherein the microorganism is Vibrio species. 15. The microorganism is Vibrio species KB1.
The method according to claim 2, which is strain FERM P-14643. 16. The antioxidant used with a microorganism,
Coffee acid, vitamin E, vitamin C, ubiquinol,
Uric acid, 2-hydroxyestradiol, bilirubin,
The method according to any one of claims 1 to 15, wherein the compound is at least one compound selected from 3-hydroxyflavone, chlorogenic acid, catechin, protocatechinic acid, pyrogal, and gallic acid. 17. An antioxidant concentration of 1 to 1000 pp
The method for decomposing microorganisms according to any one of claims 1 to 16, which is in the range of m. 18. The organic chlorine compound and / or aromatic compound is decomposed by contacting an antioxidant together with the microorganism to a medium contaminated with the organic chlorine compound and / or aromatic compound. Item 18. The method for decomposing microorganisms according to any one of Items 1 to 17. 19. An environmental restoration method using the method for decomposing microorganisms according to any one of claims 1 to 18. 20. The environment to be repaired is a medium contaminated with organochlorine compounds and / or aromatic compounds.
9. The environmental restoration method according to item 9. 21. An organic chlorine compound and / or aromatic compound is decomposed by bringing an antioxidant together with the microorganism into contact with a medium contaminated with the organic chlorine compound and / or aromatic compound. Item 21. The environmental restoration method according to Item 20, 22. The contaminated medium is an aqueous medium.
Environmental restoration method described. 23. The environmental restoration method according to claim 22, wherein an aqueous medium containing an organic chlorine compound and / or an aromatic compound is brought into contact with a carrier carrying the microorganism and an antioxidant. 24. The contacting is performed by placing a carrier carrying microorganisms and an antioxidant in a container, introducing an aqueous medium containing an organic chlorine compound and / or an aromatic compound from one of the containers, and discharging the aqueous medium from the other. Claim 2.
3. The environmental restoration method according to 3. 25. The environmental restoration method according to claim 21, wherein the contaminated medium is soil. 26. The method according to claim 25, wherein an aqueous medium containing a microorganism and an antioxidant is introduced into the contaminated soil, and the microorganism is grown in the soil by providing nutrients and / or oxygen. Environmental restoration method. 27. The environmental restoration method according to claim 26, wherein the introduction of the aqueous medium containing the microorganisms and the antioxidant into the soil is performed by pressure from an injection well provided in the soil. 28. The method according to claim 25, wherein a soil containing an organochlorine compound and / or an aromatic compound is introduced into an aqueous medium containing a microorganism and an antioxidant. 29. The environmental remediation method according to claim 25, wherein a soil containing an organochlorine compound and / or an aromatic compound is brought into contact with a carrier carrying a microorganism and an antioxidant. 30. The method of claim 21, wherein the contaminated medium is air. 31. The environmental restoration method according to claim 30, wherein air containing an organic chlorine compound and / or an aromatic compound is introduced into an aqueous medium containing a microorganism and an antioxidant. 32. The environmental remediation method according to claim 30, wherein air containing an organic chlorine compound and / or an aromatic compound is brought into contact with the carrier carrying the microorganism and the antioxidant. 33. In the contact, a carrier supporting microorganisms and an antioxidant is contained in a container, air containing an organic chlorine compound and / or an aromatic compound is introduced from one of the containers, and discharged from the other. 33. The environmental restoration method according to claim 32, wherein the method is performed. 34. The organic chlorine compound is at least one of trichloroethylene and dichloroethylene.
19. The method for decomposing microorganisms according to any one of items 18 to 18. 35. The organic chlorine compound is at least one of trichloroethylene and dichloroethylene.
The environmental restoration method according to any one of items 9 to 33. 36. The aromatic compound is at least one of phenol, toluene and cresol.
The method for decomposing microorganisms according to any one of the above. 37. The aromatic compound is at least one of phenol, toluene and cresol.
4. The method for repairing an environment according to any one of items 3 to 3.