PROCESS AND MEANS FOR THE MICROBIOLOGICAL REMEDIATION OF POLLUTED SOIL AND MICROORGANISMS FOR USE IN SAID PROCESS

Abstract: The invention relates to a process for the microbiological remediation of soil polluted by chlorophenols, the soil to be decontaminated being composted with the chlorophenol-degrading microorganisms and the conditions being adjusted so that they favour for the growth of the microorganisms. According to the invention such chlorophenol-degrading microorganisms of the genus Rhodococcus and Mycobacterium are preferably used which are capable of degrading the chlorophenols also in field conditions. According to the invention the microorganisms are preferably immobilized in a solid porous support.

Description: Process and means for the microbiological remediation of polluted soil and microorganisms for use in said process The invention relates to a process for the remediation of soil polluted by chlorinated phenolic compounds with the aid of microorganisms capable of degrading chlorinated phenolic compounds. The invention also relates to- the new micro¬ organisms to be used in said process as well as to said micro¬ organisms immobilized in a solid support. Chemically polluted soil and water is becoming an increasingly serious environmental problem in the industrialized countries. Chlorinated phenolic compounds and their derivatives, which are referred to in the following specification and claims by the term "chlorophenols" for short, are toxic hazardous contaminants which are hardly degradable in the environment. In Finland chlorophenol contaminated soil causes problems at the sites of saw mills where said chlorophenols have been used and where concentrations up to 5000 to 10 000 mg per kg dry soil have been located. Chlorophenols have also been found in surface water and in ground water in environmentally hazardous contents. US Patent 4, 713, 340 (Crawford) describes the purification of chlorophenol contaminated surface and ground water by using bacteria of the genus Flavobacterium deposited under No. ATCC 39723 and capable of degrading chlorophenols. The same Patent also discloses that polluted soil may be purified by leaching the chlorophenols from the soil and removing said chloro¬ phenols from the resulting polluted water. The purification of polluted water with the aid of chloro¬ phenol-degrading microorganisms has been disclosed in the copending international patent application PCT/FI89/00144 (based on the Finnish patent application 883685 filed August 8, 1988) which is incorporated herein by reference. Said application discloses a process for the microbiological purification of polluted water by a contaminant-degrading microorganism, in which process chlorophenols are enriched in a chlorophenol accumulating solid support and microorganisms which are immobilized in said support are made to degrade the accumulated chlorophenols. Polyurethane is the preferred support and biofilter which reversibly captures chlorophenols from the water. Said polyurethane may be specifically modified for the immobilization of microorganisms. As the chlorophenol- degrading microorganisms immobilized in the support the water purification process preferably uses chlorophenol-degrading bacteria of the genus Rhodoσoccus, which are capable of very effectively degrading the chlorophenols in the water. Other known chlorophenol-degrading microorganisms are for instance some Arthrobaσter and Pseudomonas bacteria, which have been disclosed by Portier et al. , in Toxicity Assessment: An International Quarterly, Vol 1, p. 501...513, (1986). The removal of chlorophenols from polluted soil is a much more difficult problem than their removal from water. Several chlorophenol-degrading microorganisms are prior known which are capable of degrading chlorophenols in favourable conditions in water or in a sterilized soil sample in a laboratory. It has, however, been found that it is very difficult to keep these known microorganisms alive in natural conditions in the soil and thus enable them to degrade the chlorophenols in said soil. Although chlorophenols are toxic to humans, contaminated soil includes plenty of microorganisms which tolerate chloro¬ phenols. Some of these do not affect chlorophenols at all, some transform chlorophenols e. g. by methylating them whereby the resulting chloroanisoles may be even more hazardous to humans than the actual chlorophenols. Only a part of the microorganisms which tolerate chlorophenols are ones which are capable of using chlorophenols as their carbon source. Even though contaminated soil may include naturally occurring microorganisms which in laboratory conditions are capable of degrading chlorophenols, it has, however, been found that chlorophenols remain almost undegraded iτi soil. The natural conditions are generally such that they do not favour chloro¬ phenol-degrading microorganisms. Chlorophenol-degrading microorganisms which occur naturally in contaminated soil have been used for decontaminating polluted soil (Valo et al. , Appl. Microbiol. Biotechnol. 1986, 25: 68 75). In said experiment a naturally occurring microbe popu¬ lation was made to function by raising the temperature of the contaminated soil, adding nutrients to the soil and adjusting the pH close to neutral. In a parallel experiment in the above mentioned paper it was noted that the degradation of chlorophenols in laboratory conditions accelerated when a pure culture of the chloro¬ phenol-degrading microorganism Rhodocoσcus chlorophenolicus PCP-1 DSM 43826 was added to a sterilized contaminated soil sample. Said microorganism is described in detail by Apajalahti et al. in Int. J. Syst. Bacteriol. , (1986) 36:246 - 251. In the field scale experiment, however, it was found that the addition of chlorophenol-degrading bacteria in an amount of 1 x 10 s cells/g of contaminated soil did not have any significant effect. As mentioned above several chlorophenol-degrading micro¬ organisms are incapable of competing for the vital function' s with other more quickly multiplying microorganisms. When such microorganisms are added to a foreign environment they are therefore generally incapable of effectively degrading chloro¬ phenols. Moreover, the temperature of the^soil is so low that it does not correspond to the temperature of bioactivity of the microorganisms (generally about 20 to 35 * C). It has now been found that it is possible to isolate from soil or water certain chlorophenol-degrading microorganisms of the strains Rhodocoσcus and Mycobacterium which are stable in soil also in naturally occurring conditions. It has, however, been found that the chlorophenol-degrading activity of said micro¬ organisms is so low that without external means they are incapable of decreasing the chlorophenol content of contami¬ nated soil to any significant degree. In connection with the present invention the conditions for the activity of said chlorophenol-degrading microorganisms were studied and hereby the means were discovered by which the said microorganisms can be made to work effectively so that the contaminated soil can be reclaimed. The object of the present invention is to provide a process, by which chlorophenol contaminated soil can be efficiently reclaimed with the aid of chlorophenol-degrading micro¬ organisms. The object of the invention is also to provide a support material for use in said process which support comprises an immobilized microorganism. The object of the invention is further to provide new micro¬ organisms which are isolated from the environment and which preferably can be used in said process. The more detailed objects of the invention will appear from the following specification as well as from the appended claims. Thus, the object of the invention is a process for the micro¬ biological remediation of contaminated soil from chlorinated phenolic compounds and their derivatives with the aid of chlorophenol-degrading microorganisms, in which process chlorophenol-degrading microorganisms belonging to the genus Rhodocoσcus and/or Mycobacterium are added to the contaminated soil and the facilities for the chlorophenol-degrading activity of the used microorganisms are improved by adjusting the growth conditions of said microorganisms. In order to improve the viability of said microorganisms in the conditions of the soil to be decontaminated it has been discovered within the scope of the invention to be advantageous to immobilize them in a solid porous organic support, preferably in polyurethane which is especially modified for this purpose. According to the invention it has also been found important to improve the facilities for the bioactivity of the micro¬ organisms by adjusting the physical and/or chemical parameters of the soil, such as temperature, pH, moisture, redox potential, nutrients, aeration, etc. In this connection it has surprisingly been discovered that when adding nutrients one should avoid adding a carbon source which is easily utilizable by he microorganisms. The present invention also relates to a modified polyurethane support which contains immobilized chlorophenol-degrading microorganisms, said support being characterized in that chlorophenol-degrading microorganisms of the genus Rhodococcus and/or Mycobacterium are immobilized into the polyurethane resin during the production of the polymer. The invention further relates to microorganisms which are isolated from the environment and which degrade chlorinated phenols and their derivatives, and which microorganisms are: Rhodococcus sp. CG-1, which has been deposited in accordance with the Budapest Treaty on January 10, 1989 in the depository DSM (Deutsche Sam lung von Mikroorganismen und Zellkulturen GmbH) and obtained the number DSM 5146, and Mycobacterium sp. CG-2, which has been deposited in accordance with the Budapest Treaty on January 10, 1989 in the depository DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) and obtained the number DSM 5129. The invention is further illustrated by the following specification and the appended drawings, wherein Fig 1 shows the effect of the addition of a carbon source on the evolution of carbon dioxide indicating the degradation of pentachlorophenol, PCP, responsive to the chlorophenol- degrading activity of a microorganism immobilized in accordance with the present invention. Fig 2 shows the degradation of 1Λ C labeled pentachlorophenol, PCP, responsive to the bioactivity of a microorganism according to the invention. According to the process of the present invention chloro¬ phenols are removed biologically from contaminated soil by adding a sufficient amount, preferably at least 1 x 10 6 cells/g dry soil of chlorophenol-degrading microorganisms of the genus Rhodococcus and/or Mycobacterium and by at the same time adjusting the conditions of bioactivity for said micro¬ organisms so that an effective microbial activity is possible. In the invention it has been found especially preferred to add the microorganisms immobilized in a solid porous organic support. The support forms a favourable living environment for the microorganisms and it protects the microorganisms from the attack of predatory beings. It is further preferred that the porous support is capable of accumulating chlorophenols from the liquid phase of the surrounding soil and thus lowering the chlorophenol concentration in the immediate living environment of the microorganisms. This is especially important when it is a question of the microorganisms of the genus Rhodococcus and Mycobacterium used according to the invention since the tolerance of said microorganisms for chlorophenols is relatively low and their activity is thus improved when the support lowers the chlorophenol content of the water phase of the soil. When the chlorophenol content decreases due to the microbial activity, chlorophenols migrate back from the support into the soil for degradation by the microorganisms. Porous materials, especially porous organic materials such as wood chips and wood bark and polyurethane are. suitable as support materials since they have been found to function well as attachment substrates for the microorganisms and they accumulate chlorophenols. According to the invention polyurethane resins which are capable of effectively acting as immobilization substrates for the degrading microorganisms are especially well suited as supports. The polyurethanes to be used have a large specific area and a large porosity. The utilized polyurethane resin may be of a special polyurethane kind which is modified for the immobilization of bacteria and which is capable of accumulating chlorophenols due to its large surface area and the active carbon that it contains as well as due to its surface charge. Chips of wood and wood bark have been found to reversibly accumulate chlorophenols from chlorophenol containing solutions (see Apajalahti et al. Microbiol. Ecol. (1984) 10: 359...367) and to function very well also as immobilization substrates for microorganisms. Such supports comprising immobilized chlorophenol-degrading microorganisms are disclosed in the previously mentioned international patent application PCT/FI89/00144 and they can be used also in the process of the present invention. In the scope of the present invention it has further been found that the immobilization of the microorganisms into the tr Φ •"3 H- rt rt P> φ tr Hi o μ- CO rt b Hi Φ ι-_ o TJ i Pi 55 M td r Φ rt U 01 φ t P. H Φ O o 0) Φ i μ- CO tr H o o H tr 3 μ- t P» O Φ 3 φ p. Φ p ) li 3 • 3 rt φ TJ Φ ιP μ* o Φ φ 3 o φ rt • iQ TJ Φ 3 rt 3 TJ O TJ Φ H Φ i *<; o b o Φ h Φ φ P. φ μ- o TJ * Φ P P. o b P h tr b 0 ) o a H 01 b o O li H μ- tr μ- H p. Φ Φ μ- I t b Hi rt φ P ) O p. rt ιp rt o p ) Ω J μ- 01 Φ μ- 01 b I-- μ- o P> P rt H rt μ- Φ 01 μ- < a rt O h-> o 3 rt b TJ o μ- b o b μ- ) C H T ft b μ- P 01 b H p ) 01 tr ιθ Φ i N Φ TJ o a H H Φ O rt tr Q P. ) H O P. o i o. J P ) Φ 0 rt ι I- 1 φ b h tr Φ φ b Φ μ- μ- rt I b μ- 01 rt li li H- > • • rt P. φ rt P. 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In accordance with the invention it has been found that the chlorophenol-degrading activity of the said microorganisms is favoured by a low oxygen content in the soil. Thus for instance 1 % air does not contain a harmful amount of oxygen, while on the other hand there is an excessive amount of air for the microorganisms in case the soil contains more than 50 % air. The soil to be decontaminated is therefore preferably isolated from the environment by a plastic film. This also prevents rain water from penetratijig into the compost and chlorophenol containing water from leaking to the lower soil layers. The soil to be decontaminated is preferably mixed at intervals in order to bring the microorganisms into contact with fresh portions of the soil. For the bioactivity of the microorganisms the pH is adjusted to over 6, preferably close to 7 to 8. The contaminated soil may be buffered for instance by the addition of ash which at the same time comprises a mineral nutrient. In accordance with the invention it has been found that nitrogen and phosporus should generally be added to the soil. In the addition of nutrients according to the invention it has surprisingly been found that the addition of an easily meta¬ bolized carbon source favours the o-methylation of the chloro¬ phenols and weakens the mineralization of the chlorophenols to CO_- and inorganic chloride. Thus it is extremely important that no easily metabolized carbon source is added to the soil to be decontaminated. In order to maintain a sufficient gas circulation in the soil said soil must not be too moist. On the other hand the micro¬ organism activity is not either at its maximum in too dry soil. In the soil reclamation according to the invention it has been found preferable to adjust the moisture of the soil to a value which is close to 60 % of the water holding capacity, WHC, of the soil. Microorganisms which are useful in the process of the invention are chlorophenol-degrading microorganisms of the genus Rhodococcus and Mycobacterium which comprise for instance the following identified strains: Rhodococcus chlorophenolicus PCP-1 (DSM 43826), which has been described by Apajalahti et al. in Int. J. Syst. Bacteriol. , (1986) 36: 246...251, and which has been re-deposited according to the Budapest Treaty on January 10, 1989 in the depository DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) and obtained the number DSM 5128; Rhodococcus sp. CP-2, which is disclosed in the international patent application PCT/FI89/00144 as well as in Haggblom et al. Appl. Envir. Microbiol (1988) 54: 3043...3052, and which has been deposited according to the Budapest Treaty on May 13, 1988 in the depository DSM and obtained the number DSM 4598; Rhodococcus sp. CG-1, which has been deposited according to the Budapest Treaty on January 10, 1989 in the depository DSM and obtained the number DSM 5146; and Mycobacterium sp. CG-2, which has been deposited according to the Budapest Treaty on January 10, 1989 in the depository DSM and obtained the number DSM 5129. Said microorganisms were isolated from chlorophenol contami¬ nated soil and water and it is obvious to a person skilled in the art that other corresponding microorganisms may be isolated in a corresponding way and that such may be used in the process of the invention. It is also clear that chloro¬ phenol-degrading cultures derived from said microorganisms are suitable for being utilized in the process of the invention. The characteristics of the strains Rhodococcus chlorophenoli- cus PCP-1 and Rhodocoσcus sp. CP-2 have been described in the above mentioned literature references, which are included herein by reference. The strains Rhodocoσσus CG-1 and Myσobaσterium CG-2 whiσh are disclosed as new miσroorganisms aσσording to the invention have also been desσribed by Haggblom et al. in Appl. Envir. Miσrobiol (1988) 54: 3043...3052. The referenσe does not, however, inσlude information as to the deposition of the miσroorganisms and their identifiσation is therefore not so clear from the said reference that a person skilled in the art σould be sure to isolate them and thus obtain them for use. The said miσroorganisms are therefore to be regarded as being new miσroorganisms whiσh now by the performed deposition have been disclosed in suσh a way that they can be regarded as being identified and reproducible. As regards the general charaσteristiσs and the σhlorophenol-degrading σharacteristics of the new microorganisms reference is made to the above mentioned paper. The probable degradation path of pentachlorophenol to σarbon dioxide and inorganiσ σhloride by the action of bacteria of the genus Rhodocoσcus and Mycobaσterium is shown in the following reaσtion εσheme: The σhlorophenol-degrading σapaσity of the baσteria Rhodococcus CP-2 and Rhodocoσσus σhlorophenoliσus PCP-1 is genetiσally stable while the degrading σapaσity of the bacteria Rhodococcus CG-1 and Mycobacterium CG-2 is instable and vanishes in case the substrate lacks chlorinated phenols. The σapaσity for degrading σhlorinated phenols may be induσed into the bacteria either prior to the immobilization or after the immobilization. In σonneσtion with the induσtion the baσteria are fed with the σhemiσal to be degraded in σonneσtion with a nutrient medium so that the baσteria will produσe the enzymes which degrade the specific contaminant. The following Examples illustrate the invention without, how¬ ever, limiting it in any way. Example 1 The isolation of chlorophenol-degrading microorganisms Two 500 ml columns with softwood bark chips as a solid support were inoculated with 10 to 15 g aliquots of a contaminated slurry (sample A) and soil (sample B) each, and 200 ml of a mineral salts medium was percolated through the columns. Tetrachloroguaijaσol (TeCG) was weekly added up to a ' concen¬ tration of 10 to 50 μM. After 3 months, the percolating fluids were found to contain a mixed culture that repeatedly removed added TeCG (10 μM). The A and B σultures from the perσolators were enriσhed by several dilutions and feeding with 10 to 20 μM TeCG added at 2 to 3 days intervals. Samples of A and B were streaked onto DSM-65 (gluσose -4.0 g, yeast extraσt 4.0 g, malt extraσt 4.0 g, H=0 1000 ml, pH 7.2) agar with 10 μM TeCG. 130 isolates from eaσh mixed culture were tested for TeCG degradation, by monitoring the removal of TeCG from the liquid. From the culture A was isolated one TeCG degrading σolony and it was designated CG-1 and σorrespondingly a σolony isolated from B was designated CG-2. A third σhlorophenol degrading σulture (C) was correspondingly obtained from a chlorophenol contaminated soil. The culture in minerals salts medium was enriσhed for a period of 8 months by repeated dilutions and feeding with PCP (5 mg/1). The σulture was plated on yeast extraσt agar σontaining PCP (20 mg/1), and one σolony that degraded PCP was σhosen and designated CP-2. By σorresponding methods also other σhlorophenol-degrading Rhodoσoσσus and Myσobaσterium baσteria σan be isolated and suσh baσteria may be used in the method and produσt of the present invention. The pentaσhlorophenol-degrading σapaσity of the isolated microbe strains was measured by using 14 C labeled PCP and by monitoring the evolved 14 COa. The degradation of other σhloro¬ phenols was also measured. The strain CG-1 (Rhodoσoσσus sp. CG-1) degraded PCP, 2346-TeCP, 2356-TeCP, 235-TCP, 236-TCP, 246-TCP, 25-DCP, 34-DCP, TeCG, 346-TCG, 346/356 TCG and TCS in 48 hours. The strain CG-2 (Myσobaσterium sp. CG-2) σompletely degraded PCP, 2345-TeCP, 2346-TeCP, 2356-TeCP, 234-TCP, 235-TCP, 236-TCP, 246-TCP, 24-DCP, 26-DCP, TeCG, 345-TCG, 346-TCG, 346/356-TCG, 456-TCG, 34-DCG, 35-DCG, 36-DCG, TCS and 35-DCS. CP-2 (Rhodoσoσσus sp. CP-2) also degraded several other σhlorinated phenols, guajaσols and syringols in 10 μM solutions. In said tests PCP, 2345-TeCP, 2346-TeCP, 2356- TeCP, 234-TCP, 235-TCP, 236-TCP, 245-TCP, 25-DCP, TeCG, 345- TCG, 346-TCG, 356-TCG, 456-TCG, 34-DCG, 35-DCG, 36-DCG, TCS and 35-DCS were degraded in 48 hours. The above used abbreviations refer to the chlorophenols in the following way: pentachlorophenol (PCP), tetrachlorophenol (TeCP), trichlorophenol (TCP), dichlorophenol (DCP), tetra- chloroguajacol (i.e. tetraσhloro-2-methoxyphenol) (TeCG), triσhloroguajaσol (TCG), and triσhlorosyringol (i. e. tri- σhloro-2, 6-dimethoxyphenol ) (TCS). Example 2 Immobilization of the microorganism in the support Pure cultures of the chlorophenol-degrading microorganisms Rhodoσoσcus σhlorophenoliσus PCP-1 (DSM 5128) and Rhodoσoσσus CP-2 (DSM 4598), were immobilized in a polyurethane resin mass of the polyurethane REA 90/16 produced by Bayer Ag, West Germany. Said polyurethane is modified for the immobilization of bacteria (polyurethane particle size below 10 mm; density 1.14 to 1.05 kg/1 at 20 "C; weight in suspension 115 kg dry matter per m 3 of suspension volume; sedimentation rate 94 ± 6 m/h). A mixed culture of the bacteria was incubated in a fermentor using 1% glucose, 1% soritol as a carbon source and in the presence of a support. As they grew the bacteria excreted slime and attached perceptively onto the surface of the support material. When sufficient bio ass had formed the in¬ cubation was ended. The polyurethane mass containing the immobilized Rhodocoσσus σulture was stored for 20 days at +4 "C. In a test performed after said storage the miσrobes were found to be still capable of degrading σhlorophenols. Chlorophenol-degrading Rhodoσoσcus baσteria have been found to degrade σhlorophenols even after having been stored at +4 * C for one year. Example 3 The degradation of σhlorophenols in a soil sample Two soil samples were used in the test, one of an organiσ soil and the other of a sandy soil. The unsterilized soil samples were deliberately σontaminated with PCP (30 g/1 in 0.15 M NaOH) to a σonσentration of 665 mg/kg dry sandy soil and 1860 15 mg/kg dry organiσ soil. As the chlorophenol-degrading baσterium (degrader) the strain Rhodoσoσσus chlorophenoliσus PCP-1 (DSM 5128) was used and as the o-methylating baσterium (methylater) the strain Rhodoσoσσus rhodoσhrous (DSM 43241) was used. The strains were grown in DSM-65 medium for a week in the presenσe of sterile polyurethane, PU, (REA 90/16, Bayer, Leverkusen, West Germany). The Rhodoσoσσus σhlorophenoliσus σultures were induσed with PCP daily up to a σonσentration of 50 μM. In the inoσulation of the miσroorganisms 0.5 g (dry weight) of the resulting polyurethane containing the immobilized cells was used. Added to the soils this gave a concentration of 1 x 10 s cells/g dry weight for the sandy soil and 2.5 x 10 s σells/g dry weight for the organiσ soil. Sterile PU was used as a σontrol to indicate whether the addition of organiσ matter as suσh affects the degradation of PCP. Glucose/yeast extraσt was used as an easily metabolized carbon source in the form of a solution of 25 % by weight of glucose and 25 % by weight of yeast extract in water. The test included a total of the following 10 samples: Gluσose/yeast extraσt 1) No inoσulation 2) No inoσulation 3) Sterile PU 4) Sterile PU 5) Inoc. with methylaters 6) Inoc. with methylaters 7) Inoc. with degraders 8) Inoc. with degraders 9) Inoσ. with meth. and degr. 10) Inoσ. with meth. and degr. All inoσulations were made using 1 % by weight of dry poly¬ urethane σalσulated on the basis of moist soil. Gluσose/yeast extraσt was added to a σonσentration 0.5 % each relative to the moist soil. All tests were duplicated with added a - 4 C-labeled PCP. From said tests the degradation of PCP was monitored on the basis of the percentage of evolved i4 CO_ϊ. The results are shown in Fig. 1, wherein the tests performed with sterile PU and the methylaters have been left out for clarity, as the results of said tests essentially followed the same pattern as the corresponding uninoσulated soils. In the Fig. the graphs are numbered aσσording to the numbering of the above Table whereby the (+) and (-) marks designate the presence and the absence of a σarbon sourσe, respeσtively. The results clearly indicate that the used microorganism is capable of degrading PCP in unsterilized soil and that the addition of an easily metabolized carbon sourσe decreases the mineralization of PCP. Example 4 A test corresponding to Example 3 was performed on soil samples (samples A and B) taken from two different sites of chlorophenol contaminated soil. A cell suspension of the degrading miσroorganism Rhodoσoσcus CP-2 was added to said samples in a concentration of 1.1 x 10" 7 cells/g dry soil. In order to indicate degradation, 14 C-labeled PCP was added to the soil samples and the degradation thereof was monitored by measuring the amount of i4 COa produced. To some of the samples glucose/yeast extract was added in a conσentration of 1 % as an easily metabolized σarbon sourσe. The results are shown in Fig. 2 whiσh σlearly indiσates the negative effeσt of an easily metabolized σarbon sourσe on th mineralization of PCP. Example 5 Field experiment Chlorophenol σontaminated soil is moved to a plastic lined windrow and the following materials are mixed therein wood bark and park refuse in order to raise the σontent of digestible organiσ matter to about 0.5 %; - ash for buffering the pH to a value of about 7; - fertilizer in an amount of about 1 kg/m 3 dissolved in water, said fertilizer containing about 16 % N, about 7 % P and about 3.3 % ; water at need in order to adjust the moisture level to about 60 % of the water holding capacity. Microorganisms Rhodococcus chlorophenolicus PCP-1 and/or Rhodoσoσσus CG-1 immobilized in polyurethane are also mixed into said soil in a σonσentration of about 10 s σells/g dry soil. After mixing the soil is σovered with a plastiσ film. The temperature of the windrow is monitored during the σomposting and in σase the heath obtained from the digestion is not suffiσient to raise the temperature of the windrow to about 20 to 25 " C an additional heating device mounted in the bottom of the windrow is used for heating. The soil is mixed and its chlorophenol content is measured about once a month. When the σhlorophenol σontent of the soil has deσreased to a pre¬ determined aσσeptable level the windrow is broken up and the reclaimed soil is moved away.

Claims: Claims 1. A process for the microbiologiσal remediation of polluted soil from chlorinated phenolic compounds and their derivatives with the aid of chlorophenol-degrading microorganisms, c h a r a c t e r i z e d in that chlorophenol-degrading microorganisms of the genus Rhodococcus and/or Mycobacterium are added to the contaminated soil in σell σounts whiσh are sufficient to provide a chlorophenol-degrading bioactivity and that the facilities for the bioactivity of the used miσro¬ organisms are improved by adjusting the growth conditions for said miσroorganisms. 2. A proσess aσσording to σlai 1, σ h a r a c t e r i z e d in that the miσroorganisms are added immobilized in a solid porous support. 3. A proσess according to claim 2, σ h a r a c t e r i z e d in that the miσroorganisms are immobilized in a polyurethane resin, in wood bark or in wood σhips. 4. A proσess aσσording to claim 3, c h a r a σ t e r i z e d in that the microorganism are immobilized in a polyurethane resin by incubating said microorganisms, in a polyurethane containing medium or by producing said polyurethane from a microorganism containing medium. 5. A process according to one of the preceding claims 1 to 4, c h a r a c t e r i z e d in that the facilities for the bioactivity of said microorganisms are improved by adjusting the physical and/or chemical parameters of the soil, said parameters being chosen from the group consisting of temperature, pH, moisture, redox potential, nutrients, aeration, etc. 6. A proσess aσσording to σlaim 5, σ h a r a σ t e r i z e d in that when adjusting the nutrients the addition of an easily metabolized σarbon sourσe is avoided. 7. A proσess aσcording to claim 5, σ h a r a σ t e r i z e d in that the temperature and the redox potential are adjusted by adding to the soil organiσ matter, such as microbiologiσally digestible wood bark, park refuse or the like and/or that the soil to be treated is enσlosed in a impermeable plastiσ film to deσrease the aeration. 8. A proσess aσσording to any one of the preσeding claims, σ h a r a σ t e r i z e d in that the miσroorganisms are σhosen from the group σonsisting of the chlorophenol-degrading miσroorganisms Rhodoσoσσus σhlorophenoliσus PCP-1 (DSM 5128), Rhodoσoσσus sp. CP-2 (DSM 4598), Rhodoσoσcus sp. CG-1 (DSM 5146) and Myσobaσterium sp. CG-2 (DSM 5129). 9. A polyurethane support modified for the immobilization of baσteria whiσh support σontains immobilized chlorophenol- degrading miσroorganisms, σ h a r a c t e r i z e d in that chlorophenol-degrading microorganisms of the genus Rhodoσoσσus and/or Myσobaσterium are immobilized in said polyurethane resin during the produσtion of the polymer. 10. A polyurethane support aσσording to claim 9, c h a r a σ ¬ t e r i z e d in that the microorganisms are chosen from the group consisting of the chlorophenol-degrading microorganisms Rhodocoσcus chlorophenolicus PCP-1 (DSM 5128), Rhodococcus sp. CP-2 (DSM 4598), Rhodocoσcus sp. CG-1 (DSM 5146) and Myσobaσterium sp. CG-2 (DSM 5129). 11. A pure σulture of a miσroorganism whiσh is σapable of degrading σhlorinated phenoliσ σompounds and their derivatives, σ h a r a σ t e r i z e d in that said miσro¬ organism is Rhodoσoσσus sp. CG-1 (DSM 5146) or a culture derived therefrom. 12. A pure culture of a microorganism which is capable of degrading chlorinated phenolic compounds and their derivatives, c h a r a c t e r i z e d in that said micro¬ organism ' is Mycobacterium sp. CG-2 (DSM 5129) or a σulture derived therefrom.

Inventors: HAEGGBLOM MAX MIKAEL, SALKINOJA-SALONEN MIRJA, VALO RISTO JUHANI, HAEGGBLOM, MAX, MIKAEL, SALKINOJA-SALONEN, MIRJA, VALO, RISTO, JUHANI
Applicants: ALKO LTD, ALKO LTD.
Application year: 1990
Main classification: C02F3
IPC codes: B09C1, C12N11