JPH04503528A - Methods and means for microbiological improvement of contaminated soil and microorganisms used in this method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Methods and means for microbiological improvement of contaminated soil and microorganisms used in this method The present invention enables soil contaminated with chlorinated phenolic compounds to be treated with chlorinated phenolic compounds. It concerns a method of remediation by microorganisms capable of decomposing chemical compounds. be. The present invention also provides novel microorganisms used in the above method and The present invention relates to the established microorganism.

Chemically contaminated soil and water are becoming increasingly common in industrialized nations. (Chlorinated phenolic compounds and their derivatives are becoming a serious environmental problem.) (hereinafter these substances are abbreviated as "chlorophenols") are dangerously toxic. A pollutant that hardly decomposes in the environment. in finland Soil contaminated with chlorophenols is contaminated with chlorophenols. A problem occurred on the site of the saw wills factory. 5.000 to io, oo for dry soil.

Contains chlorophenols in concentrations up to mg/kg. Also, chlorophyll Nols are found in environmentally hazardous contents in surface and ground waters.

U.S. Patent No. 4,713.340 (Crawford) , surface water and groundwater contaminated with chlorophenols, accession number No. A fluorophore deposited under ATCC 39723 that can decompose chlorophenols. Discloses purification using Rabobacterium bacteria. Also, this US The patent involves leaching chlorophenols from contaminated soil and extracting chlorophenols from the resulting contaminated water. Discloses that contaminated soil can be purified by removing lophenols. Ru.

Purification of contaminated water using microorganisms that decompose chlorophenols is an international patent application No. 111P. cT/FI89100144 (Fin filed on August 8, 1988) (based on RAND Patent Application No. 883,685), which is hereby incorporated by reference. It is listed as a reference. This patent application claims that microorganisms that decompose pollutants are used to treat contaminated water. Discloses a method for microbiologically purifying chlorophenols. The concentration of chlorophenols increases in the solid support where they accumulate. Microorganisms fixed in the body break down accumulated chlorophenols. Poly Urethane is a preferred support and biofilter, removing chlorophyll from water. Captures nols reversibly. The polyurethane is specially designed to immobilize microorganisms. Can be modified separately. In water purification processes, chlorine is immobilized in a support. As a microorganism that decomposes colophenols, it is extremely effective at removing colophenols from water. Chlorophenes of the genus Rhodococcus that can be decomposed It is preferable to use gnole-degrading bacteria.

Other known chlorophenol-degrading microorganisms include, for example, some Arthrobacterium Bacteria and Pseudomonas, and these microorganisms are classified as An internal 0 quarterly (Toxicity As5) esse+5ent: 8n Internal Quarterly), 1st Vol., pp. 501-513 (1986) J. Portier et al. disclosed by.

Removal of chlorophenols from contaminated soil is significantly more difficult than removal from water. . Traditionally, chlorophyll in water or sterile soil samples under favorable conditions in the laboratory. Some chlorophenol-degrading microorganisms that can degrade phenols Are known. However, these known microorganisms cannot be grown in soil under natural conditions. These microorganisms can degrade chlorophenols in the soil depending on the soil conditions. It is known that it is extremely difficult to maintain this condition.

Chlorophenols are toxic to humans, but contaminated soil It contains an abundance of microorganisms that are resistant to species. Some microorganisms like this Some have no effect on chlorophenols, and others, for example, have no effect on chlorophenols. By methylating chlorophenols, human There may even be a significant risk of Resistant to chlorophenols Only some of the microorganisms that can use chlorophenols as a carbon source It is.

Contaminated soil is a naturally occurring compound that can degrade chlorophenols under laboratory conditions. may contain microorganisms present in It is known that it remains in the soil without being decomposed. Natural conditions are chlorophenol It is usually undesirable for degrading microorganisms.

Chlorophenol-degrading microorganisms that naturally exist in contaminated soil are contaminated by contaminated soil. Used to remove substances (such as Valo, etc.) Robiology and Biotechnology 4 (Appl, Microbi ol, Biotechnol, ) + 1986, 25:68-75). Said In the experiment, the temperature of contaminated soil was raised, nutrients were added to the soil, and the pH was brought to near neutrality. This allows naturally occurring microbial populations to function properly.

In parallel experiments in the above-mentioned paper, chlorophyll under laboratory conditions The decomposition of chlorophenols is performed using chlorophenol-degrading microorganisms in sterilized contaminated soil samples. Pure purity of certain Rhodococcus chlorophenols PCP-I DSM 43826 It was found that this was accelerated when cultured bacteria were added. The microorganism is 'Int, J, 5yst.

Bacteriol, (1986) 36: 246-251 J. It is described in detail by Apaja Iahti et al. However, wild In an external scale experiment, 1 x 10' of chlorophenol-degrading bacteria were added to contaminated soil. It was found that even when added at a cell amount of /g, no significant effect was shown.

As mentioned above, some chlorophenol-degrading microorganisms have , unable to compete with other microorganisms that multiply more rapidly. Therefore, such microorganisms Chlorophenols cannot be effectively degraded when added to different environments. It's normal. Moreover, the soil temperature is low, and the biological activity temperature of microorganisms (universal 20-3 5°C).

In the present invention, we have developed a method that is stable in soil even under naturally occurring conditions. Chlorophenol-degrading microorganisms consisting of Bacterium and Mycobacterium strains were added to the soil. It was confirmed that it can be isolated from soil or water. However, the chlorophenol of the microorganism Since the decomposition activity of chlorophylls is low, if no external measures are taken, chlorophylls in contaminated soil will be It was found that the nols content could not be reduced to a significant extent.

In the present invention, we researched the conditions under which the chlorophenol-degrading microorganisms exhibit activity. As a result, the microorganisms can be effectively used to improve contaminated soil. I found a way to do this.

The first object of the present invention is to treat soil contaminated with chlorophenols with chlorophenols. The objective is to provide a method that can be effectively improved by degrading microorganisms. .

A second object of the present invention is to provide a method containing immobilized microorganisms for use in the method. The object of the present invention is to provide a support material that has the following properties.

A third object of the invention is to provide new The aim is to provide standard microorganisms.

More detailed objects of the present invention will become apparent from the following specification and claims. That's it.

In the present invention, chlorinated phenolic compounds and derivatives thereof Microbiological improvement of soil with chlorophenol-degrading microorganisms If the contaminated soil contains either Rhodococcus or Mycobacterium spp. Chlorophenol-degrading microorganisms belonging to one or both of the chlorophenol-degrading microorganisms are Adding a sufficient number of cells to exhibit degradative biological activity and adjusting growth conditions for the microorganism. The aim is to improve the chlorophenol decomposition activity of the microorganisms used. The first objective of the present invention is achieved by the characterized method for microbiological improvement of contaminated soil. Ru.

In order to improve the growth of said microorganisms in the soil from which pollutants are to be removed. , within the scope of the invention, said microorganisms are preferably provided in a solid porous organic support. may be advantageously immobilized in polyurethane specially modified for this purpose. Do you get it.

In addition, in the present invention, physical and/or chemical parameters of soil, e.g. For example, by adjusting temperature, pH1 moisture, redox potential, nutrients, aeration, etc. , it was found to be important to improve the bioactive functions of microorganisms. Regarding this point However, surprisingly, when adding nutrients, carbon is easily utilized by microorganisms. It has been found that it is necessary to avoid adding sources.

The present invention also provides a modified poultice containing immobilized chlorophenol-degrading microorganisms. It relates to a urethane support, in which Rhodococcus spp. and chlorophenols belonging to either or both of the genus Mycobacterium Degrading microorganisms are immobilized and immobilized in the polyurethane resin during the production of this polymer. It is characterized by the presence of

Additionally, the present invention provides a method for treating chlorinated phenols and their derivatives isolated from the environment. The microorganisms are classified under the Budapest Treaty as DSM (Deutsche) Sammlung von Mikroorganismenund Zell Deposited with Kulturen GmbH), accession number DSM 5146 Rhodococcus sp. CG-1, and Based on the Budapest Treaty, on January 10, 1989, the Depositary DSM (Deutsche Sammlung von Mikroorg) Deposited with Zellkulturen GmbH) Mycobacterium sp. acterium sp,) CG-2.

The invention will now be explained by way of example with reference to the drawings.

Figure 1 shows the decomposition of pentachlorophenol PCP versus carbon dioxide release. This shows the influence of the addition of raw materials on the immobilized microbial chlorophyll in the present invention. Corresponds to nols degrading activity.

Figure 2 shows the decomposition of I4C-labeled pentachlorophenol, which was demonstrated by the microorganisms of the present invention. corresponds to the biological activity of

In the method of the present invention, any one of the genus Rhodococcus and the genus Mycobacterium can be used. A sufficient amount of chlorophenol-degrading microorganisms belonging to one or both of the It is added to dry soil in an amount of 1×10b cells/g or more, and at the same time, the above-mentioned microorganisms are by adjusting the biological activity conditions for the microorganisms to obtain effective microbial activity. and biologically remove chlorophenols from contaminated soil.

In the present invention, microorganisms are immobilized in a solid porous organic support and added. was found to be particularly preferable. The support forms a favorable growth environment for microorganisms, Protects microorganisms from attack by predators. The porous support retains chlorine from the liquid phase of the surrounding soil. Accumulates lophenols. Chlorophyte in the growth environment of microorganisms in contact with microorganisms It is more preferable to reduce the concentration of phenols.

This is the genus Rhodococcus and Microbacterium used in the present invention. This is particularly important in the case of microorganisms. The reason is that the above-mentioned Microbial resistance is relatively low, and therefore the support is This is because when the amount is reduced, the activity of the microorganisms is improved. microbial life If the content of chlorophenols decreases due to The species migrate from the support and return to the soil where they are degraded by microorganisms.

Porous materials, especially porous organic materials such as wood chips, bark and polyurethane are suitable as support materials. The reason is that these porous materials are suitable for microorganisms. It was found that it acts well as an adhesion substrate and also accumulates chlorophenols. This is because the.

In the present invention, it is effective as an immobilization substrate for chlorophenol-degrading microorganisms. Particularly suitable as support are polyurethane resins which can be used. use Polyurethane has a large specific surface and porosity. The polyurethane resin used is Modified to immobilize bacteria and its large surface area and contained activity Because of the carbon atoms as well as their surface charge, chlorophenols can accumulate. It can be constructed from a special type of polyurethane that can be used.

In the present invention, wood chips and bark are treated with a solution containing chlorophenols. ``Apajalati'' reversibly accumulates chlorophenols from hti) et al.: Microbiol, Ecol, (1984) 10:359~ 367), and it has been confirmed that it functions extremely well as a microbial immobilization substrate. I chewed it.

Such supports containing immobilized chlorophenol-degrading microorganisms are described above. Such support is disclosed in the international patent application PCT/FI89100144. Supports can also be used in the method of the invention.

Within the scope of the invention, it is further provided that the immobilization of microorganisms in polyurethane supports is It has been found that this can be done during the manufacture of the support material. For example, live cells can be By adding it to a mixture of ethylene glycol and isocyanate compounds, Immobilization takes place during the polymerization of the urethane. During polymerization the whole cell is part of the actual polymer The technique of immobilization of microorganisms by polymerization and polycondensation to form fractions is known per se. ``Klein J., Wagner F. Nearbride Biochemistry and Biochemistry by Wagner F. ``Method for Immobilization of Microbial Cells'' in Volume 4, pp. 25-31. It is revealed in the section that will be used. Microorganisms are encapsulated in polyurethane are particularly well protected for use in the present invention. Moreover, the fruit Polyurethane is modified to meet the requirements of microorganisms and chemicals being treated. can be done.

The biological activity of pollutant-degrading bacteria, especially their rate of degradation, is temperature dependent. outdoor conditions The problem with remediation of contaminated soil is that bacterial activity is low at temperatures below 15°C. and often stops completely at temperatures below 8°C. present invention In one example, for example, composting that generates heat (co+mpostin) g) so that the size and shape of the pits are favorable for thermal energy. make sure that the bits are electrically resistive and/or The required heating is achieved by heating with an external heating means such as.

The amount of composting material added depends on the natural organic matter content of the soil being treated and Depends on the need to increase the temperature. The appropriate amount of digestible organic matter is usually 0.1 ~1%.

Decaying organic materials such as bark, park waste, etc. act as oxygen consumers. In addition to providing heat and porosity. In this way, the biologically active redox potential In the present invention, the chlorophenol decomposition activity of the microorganism is We found that it is better when the oxygen content in the soil is low, e.g. 1% air. does not contain harmful amounts of oxygen, but on the other hand the soil contains more than 50% air. If so, there is an excess amount of air for the microorganisms. Therefore, contaminants It is best to isolate the soil from the environment using plastic film. preferable. Plastic film also prevents rainwater from seeping into the compost. It also prevents chlorophenol-containing water from leaking into the underlying soil layer. dirt The soil from which contamination is to be removed is occasionally mixed to introduce microorganisms into new soil areas. It is preferable to

For biological activity of microorganisms, the pH should be greater than 6, preferably near 7-8. It is preferable to adjust. Contaminated soil can, for example, be treated with ash containing inorganic nutrients at the same time. By adding it, the pH can be adjusted to 4 levels. In the present invention, nitrogen or It was confirmed that it is necessary to add water and phosphorus to the soil.

In the present invention, surprisingly, when adding nutrients, easily metabolized carbon Addition of chlorophenols is preferred for O-methylation of chlorophenols; Easily metabolized carbon sources are added to the soil where CO2 and inorganic chlorides are to be converted. It is extremely important that there is no.

To maintain sufficient gas circulation in the soil, the soil must not be too wet. be. On the other hand, microbial activity decreases in any case in soil that is too dry. The maximum value is not reached. When improving soil according to the present invention, soil moisture is It was found that it is preferable to adjust the water retention function of -HC to a value close to 60%.

Microorganisms useful in the method of the invention include members of the genus Rhodococcus and Microbaterium. A chlorophenol-degrading microorganism, such as the following confirmed microorganisms: Contains strains: Rhodococcus lilorophenolicus PCP-1 (Dinisum 43 826): Apajalahti et al.: Int, J , 5yst.

Bacteriol, (1986) 36:246-251 J. Based on the Budapest Treaty, on January 10, 1989, the Depositary DSM (Deutsche Sammlung von Mikroo Re-deposited at rganismen und Zellkulturen GmbH) and received the accession number Dinism 5128; Rhodococcus sp. CP-2: International patent application PCT/FI89100144 and “H’aggblos+ et al.: Appl, Envir-Micr obil, (1988) 54: 3043-3052 J, Based on the Budapest Treaty, the Depositary It has been deposited with DSM (DSM) and has accession number DSM 4598.

Rhodococcus species CG-1: Dated January 10, 1989 under the Budapest Treaty It has been deposited with the depositary organization DSM (DSM), with accession number DSM 5. and Mycobacterium species CG-2: based on the Budapest Treaty. It was subsequently deposited with the depository organization DSM on January 10, 1989. The accession number was 5129.

The above-mentioned microorganisms were isolated from soil contaminated with chlorophenols. To those skilled in the art , that other corresponding microorganisms can be isolated by corresponding methods, and that other corresponding microorganisms can be It is clear that microorganisms of various types can be used in the method of the invention. Also, the microorganisms mentioned above The chlorophenol-degrading cultured bacteria obtained from the method is suitable for use in the method of the present invention. It is also clear that

Rhodococcus chlorophenolicus PCP-1 strain and Rhodococcus sp. CP-2 The strains are described in the above-mentioned literature, which is hereby incorporated by reference.

Rhodococcus CG-1 strain, which is disclosed as a new microorganism in the present invention, and Mycobacterium CG-2 strain rAppl.

Envir, Microbiol, (1988) 54. :3043-305 It is also described by H. Iggblom et al. deer However, this document does not contain information regarding the deposit of these microorganisms and therefore The identification of microorganisms is based on the literature, which indicates that a person skilled in the art cannot reliably isolate these microorganisms. and thus ensure that these microorganisms can be obtained for use. Therefore, the microorganisms are Disclosed so that the organism can be considered confirmed and reproducible It should be considered a novel microorganism. General characteristics and characteristics of these novel microorganisms Regarding lolophenol decomposition characteristics, please refer to the above-mentioned report.

Pentachlorophyll by the action of Rhodococcus and Mycobacterium spp. The estimated pathway by which phenol is decomposed into carbon dioxide and inorganic chloride is the following reaction. Represented by the formula: Rhodoconox CP-2 bacterium and Rhodococcus chlorophenolicus Although the ability of pcp-i bacteria to degrade chlorophenols is genetically stable, Rodcock Chlorophenol decomposition ability of Mycobacterium CG-1 and Mycobacterium CG-2 is unstable and disappears in the absence of chlorinated phenols on the substrate.

The ability of bacteria to degrade chlorinated phenols was determined by the ability of bacteria to degrade both before and after immobilization. can be induced. Regarding the induction of this resolution, together with the chemicals to be degraded, The bacteria are fed into a nutrient medium to allow enzymes to degrade specific contaminants by the bacteria. produce the element.

Next, the present invention will be described with reference to examples. However, these examples do not limit the invention. It is not determined.

1 cliff ±1 Isolation of chlorophenol-degrading microorganisms Two 500 ml columns each packed with softwood bark chips as a solid support were 10-15 g aliquots of contaminated slurry (sample A) and contaminated soil (sample B) were and then 200 ml of mineral salts medium was passed through these columns. Tetrachloro Guaiacol (TeCG) was added slowly to a concentration of 10-50 μH. 3 After several months, the flowthrough was found to contain a mixed culture. This mixed culture was repeatedly removed and TeCG (10 μM) was added. These percolators? Cultures A and B obtained from Enrichment was performed by adding CG and feeding. Sample A and B was mixed with DSM-65 (glucose 4.0 g, yeast malt extract 4.0g, malt extract 4.0g, Hzo 1000+gl, pH 7,2) Linear inoculation onto agar medium. Monitoring the removal of TeCG from the liquid 130 isolates from each mixed culture were tested for TeCG degradation by Tested. One TeCG-degrading colony was isolated from culture A, and this was -1 and correspondingly one colony from culture B was named CG-2. did.

Similarly, the third chlorophenol decomposition culture (C) was converted into chlorophenol Obtained from contaminated soil. Dilute and dilute cultures in mineral salts medium over a period of 8 months. It was enriched by repeated feeding of PCP and PCP (5 mg/l). This culture Cultured on yeast extract agar containing PCP (20 mg/1), One colony that had degraded was selected and named CP-2.

In addition, other chlorophenol-degrading Rhodococcus bacteria and Mycobacteria were tested using the same method. Cobacterium can be isolated and these bacteria can be produced using the methods of the present invention. It could be used for things.

The pentachlorophenol decomposition ability of the isolated microbial strain was determined using 14C-labeled PCP. and was measured by monitoring the released I4CO2. other krolov Degradation of phenols was also measured.

Strain CG4 (tl) Docox sp. CG-1) is PCP, 2346-TeCP , 2356-TeCP, 235-TCP, 236-TCP, 25- DCP, 34-DCP, TeCG, 346-TCG.

346/356-TCG and TCS were degraded in 48 hours.

Strain CG-2 (?Icobacterium species CG-2) is PCP, 2345-Te CP, 2346-TeCP, 2356-TeCP, 234-TCP, 235-TCP, 236-TCP, 246-TCP, 24-DCP, 26-DCP, TeCG, 345-TCG, 346-TCG, 34 6/356-TCG, 456-TCG, 34-DCG, 35-DCG, 36-DCG, TCS and 35-DCS were completely degraded.

CP-2 (Rhodococcus sp. CP-2) also contains some other phenolics, guaya Coles and syringols in 10μi solution Disassemble. In the above test, PCP, 2345-TeCP.

2346-TeCP, 2356-TeCP, 234-TCP, 235 -TCP, 236-TCP, 245-TCP, 25-DCP, T eCG, 345-TCG, 346-TCG, 356-TCG, 45 6-TCG, 34-DCG, 35-DCG, 36-DCG, TCS and 35 -DCS decomposed in 48 hours.

The abbreviations used above represent the following chlorophenols: pentachlorophenol (PCP), tetrachlorophenol (TeCP), trichlorophenol ( TCP), dichlorophenol (DCP), tetrachloroguaiacol (sugar That is, tetrachloro-2-methoxyphenol) (TeCG), trichloro Guaiacol (TCG) and trichlorosyringol (i.e. trichlorosyringol) -2,6-simethoxyphenol) (Te3).

Factory plate l Immobilization of microorganisms in supports Rhodococcus chlorophenolicus PCP-, a chlorophenol-degrading microorganism 1 (Deenis M5128) and Rodcosk CP-2 (Deenis M4 598) in polyurethane REA manufactured by Bayer AG, West Germany. It was immobilized in a polyurethane resin consisting of 90/16. This polyurethane is a bacterial (polyurethane particle size 10+am) Small; Density 1.14-1.05 kg/1 (20°C) i Weight in suspension (suspension Dry weight in liquid volume 115 kg 10+"; sedimentation rate 94±6 m/h).

A mixed culture of the bacteria was grown with 1% glucose and 1% sorbitol as carbon sources. The culture was carried out in a fermentation device using a mold in the presence of a support. Bacteria grow and secreted a mucilage that appreciably adhered to the surface of the support material. enough amount Cultivation was terminated when biomass of 20% was generated.

Polyurethane containing immobilized Rhodococcus cultures was incubated at -4°C for 2 hours. Stored for 0 days. In tests conducted after storage, the microorganisms It turns out that it is possible to decompose objects.

Chlorophenol-degrading Rhodococcobacteria can be stored for one year at +4°C. Chlorophenols in soil samples Two types of soil samples were used for the decomposition tests. One is organic soil and the other is sand The soil was of good quality. Unsterilized soil samples were intentionally treated with PCP (0.15M NaO). 30 g/l in H) up to a concentration of 665 mg/kg in dry sandy soils. In the case of dry organic soil, it was contaminated to a concentration of 1860 mg/kg.

Rhodococcus chlorophenolicus P as a chlorophenol-degrading bacterium (degrading bacterium) CP-1 strain (DiniSem 512B) was used, and 0-methylated bacteria (methylated Rhodococcus rhodochrouse strain (Dinisum 43241) was used as a bacterium). used. These strains were sterilized using polyurethane PU (West German Leverkusen (16yerkusen) Existence of REA 90/16 manufactured by Bayer The cells were grown in DSM-65 medium. Rhodocotta Skrlov The P. aenorix culture was brought up to a concentration of 50 .mu.- each day with PCP.

0.5 g of polyurethane produced containing immobilized cells when inoculated with microorganisms (dry weight) was used. Add this to the soil, and in the case of sandy soil, add 1 x 108 pieces/g. In the case of soil, the cell concentration was set to 2.5 XIO" cells/g.

Using sterilized PU as a control, the addition of organic matter itself was effective for controlling PCP. We clarified whether or not it affects the solution.

Glucose/yeast extract as an easily metabolized carbon source with 25% glucose by weight and yeast extract in the form of an aqueous solution containing 25% by weight. The exam includes all of the following Nigel course yeast extract using 10 samples of 1) No culture - 2) No culture + 3) Sterilized PU - 4) Sterilized PU + 5) Cultivation with methylated bacteria - 6) Cultivation with methylated bacteria 7) Cultivation with degrading bacteria - 8) Cultivation with degrading bacteria 9) Cultivation with methylating bacteria and degrading bacteria -10) Cultivating with methylating bacteria and degrading bacteria All ten inoculations were cultured with 1% by weight of dry polyurethane, calculated based on moist soil. This was done using letane. Glucose/yeast powder respectively for moist soil Kiss was added to a concentration of 0.5%.

All tests were performed in duplicate using 14c-labeled pcp. In this study, PCP Degradation was monitored based on the percent of 14co□ released. test results It is shown in Figure 1. In Figure 1, for clarity, the tests performed using sterilized PU are shown. Excluding tests conducted by inoculation with methylated bacteria and treating the results as the results of the above tests. The same woody pattern as the uninoculated soil was obtained. In Figure 1, the graph includes the table above. numbered according to the number of In this table, (+) and (-) marks indicate Each indicates the presence or absence of a carbon source.

The results obtained indicate that the microorganisms used can degrade PCP in unsterilized soil. and that addition of easily metabolized carbon sources reduces the mineralization of PCP. clearly shown.

1 hour ■ soil Soil samples collected from two different locations with chlorophenol-contaminated soil (sample A and and B), the same tests as in Example 3 were conducted. Chlorophenol decomposition fines 1. A cell suspension of the organism Rhodococcus CP-2 was added to the sample. I XIO’ pieces /g of dry soil. In order to clarify the degree of decomposition, Inc standard By adding PCP to a soil sample and measuring the amount of 14CO□ produced, its decomposition can be determined. was monitored. Some samples contain glucose/isolate as an easily metabolized carbon source. The yeast extract was added to a concentration of 1%.

The test results are shown in Figure 2. Figure 2 shows that carbon sources that are easily metabolized have a negative impact on the mineralization of PCP. clearly indicate that it will have an impact.

Real ll[[ Field test We lined soil contaminated with chlorophenol with plastic. The soil was then mixed with the following substances: - bark and park to increase the content of digestible organic matter to approximately 0.5% waste; - pH to a value of about 7! ! Ash for cutting; - about 1 kg/m” dissolved in water fertilizer, which contains about 16% N, about 7% P and about 3.3% K; - Water required to adjust the moisture level to approximately 60% of water holding capacity.

In addition, the microorganism Rhodococcus chlorophenolicus P immobilized in polyurethane CP-1 and/or Rhodococcus CG-1 are mixed into the soil and approximately 10"/ The cell concentration was made to be 1 g of dry soil.

After mixing, the soil was covered with plastic film. Wind while comboing The temperature is monitored, and the heath obtained through digestion becomes windloid. If this is not sufficient to raise the temperature to approximately 20-25°C, Heating was performed using an additional heating device attached to the bottom of the chamber. Mix the soil and The chlorophenol content of the samples was measured once a month. Chlorophenols in soil The content has decreased to a predetermined acceptable level. Mouth 2 international search report 1--・Mjl^-+=m+a-w-PCT/FI 9010CIO5S1Al fia+jM61mu,,i,1. ,,N, PCT/Fl 90100055 country international investigation report

Claims (12)

[Claims] 1. soil contaminated with chlorinated phenolic compounds and their derivatives, In microbiological improvement using chlorophenol-degrading microorganisms, The contaminated soil contains either Rhodococcus or Mycobacterium. chlorophenol-degrading microorganisms belonging to the chlorophenol-degrading microorganisms or both Added in a sufficient number of cells to show biological activity, To improve the biological activity function of the microorganisms used by adjusting the growth conditions of the microorganisms. Ru A method for microbiological improvement of contaminated soil, characterized by: 2. The method is characterized in that the microorganism is immobilized in a solid porous support and added. The method according to claim 1. 3. immobilizing the microorganism in polyurethane resin, bark or wood chips; 3. A method according to claim 2, characterized in that: 4. The microorganism is cultured in a medium containing polyurethane, or the microorganism is By producing the polyurethane in a medium containing the organisms, the microorganisms are removed. 4. A method according to claim 3, characterized in that it is immobilized in a polyurethane resin. 5. Selected from the group consisting of temperature, pH, moisture, redox potential, nutrition, aeration, etc. adjustment of soil physical and/or chemical parameters; Claims 1 to 4, characterized in that the biological activity function of the microorganism is improved by The method described in any one of the following sections. 6. It is characterized by avoiding the addition of easily metabolized carbon sources by adjusting nutrition. 6. The method according to claim 5. 7. Add organic matter to the soil, such as tree bark, park waste, etc., which is digested by microorganisms. Adjust the redox potential and/or make the treated soil impermeable by adding A claim characterized in that it is wrapped in a plastic film to reduce ventilation. 5. The method described in 5. 8. Rhodococcus chlorophenolicis, a chlorophenol-degrading microorganism Rhodococcus sp. PCP-1 (DSM 5128), Rhodococcus sp. SM 4598), Rhodococcus sp. CP-2 (DSM 4598), Dococcus sp. CG-1 (DSM 5146) and Mycobacterium sp. Selected from the group consisting of CG-2 (DSM 5129) A method according to any one of claims 1 to 7. 9. Denatured and immobilized chlorophenol-degrading microorganisms to immobilize bacteria In the polyurethane support containing Rhodococcus and Mycobacterium spp. Chlorophenol-degrading microorganisms belonging to one or both of the genuses A polyurethane resin characterized in that it is immobilized in the tan resin during the production of this polymer. Letane support. 10. The microorganism is Rhodococcus chlorophyll, which is a chlorophenol-degrading microorganism. phenolicus PCP-1 (DSM 5128), Rhodococcus sp. CP-2 ( DSM 4598), Rhodococcus species CG-1 (DSM 5146) ) and Mycobacterium sp. CG-2 (DSM 5129) The polyurethane support according to claim 9, characterized in that it is selected from: 11. Microorganisms capable of degrading chlorinated phenolic compounds and their derivatives In a pure culture of Rhodococcus sp. 5146) or a culture obtained therefrom. Culture. 12. Microorganisms capable of degrading chlorinated phenolic compounds and their derivatives In a pure culture, the microorganism is Mycobacterium sp. 5129) or a culture obtained therefrom. pure culture.