WO1999033952A1

WO1999033952A1 - Novel coriolus versicolor fungus strain useful for treating polluted materials

Info

Publication number: WO1999033952A1
Application number: PCT/FR1998/002880
Authority: WO
Inventors: Frédéric BAUD-GRASSET; Timothy Vogel
Original assignee: Rhodia Chimie
Priority date: 1997-12-24
Filing date: 1998-12-24
Publication date: 1999-07-08
Also published as: FR2772785A1; AU1883899A; FR2772785B1

Abstract

The invention concerns a Coriolus versicolor fungus strain registered with the CNCM under reference number I-1657, or one of its derivatives, capable of degrading polycyclic aromatic hydrocarbons (PAH) comprising at least five cycles, and its use for treating materials contaminated by resistant polluting compounds.

Description

New strain of fungus Coriolus versicolor useful for the treatment of polluted materials

The present invention relates to a new strain of fungus Coriolus versicolor as well as its use for the treatment of materials contaminated with recalcitrant polluting compounds.

The remediation of polluted sites, that is to say sites on which industrial pollution of the soil or subsoil is likely to generate a nuisance or a lasting risk for people or the environment, is a major current economic and scientific issue.

Pollution control can be done mechanically, by burning the land, chemically or biologically. Bioremediation or bioremediation can be more particularly used. This technique uses microorganisms, such as bacteria or fungi, to degrade recalcitrant polluting compounds, that is to say compounds which resist degradation in the environment. Among these recalcitrant polluting compounds, mention may in particular be made of halobenzoates such as chlorobenzoate and bromobenzoate, and nitro compounds such as 2,4,6-trinitrotoluene (TNT) and other explosives such as PETN (penta-erythritol tetranitrate) and EGDN (ethylene glycol dinitrate). Mention may also be made of organic halides such as in particular DDT [1, 1-bis (4-chlorophenyl) -2,2,2-trichloroethane], 2,3,7,8-tetrachlorodibenzo-p-dioxin, lindane (1,2,3,4,5,6-hexachlorocyclohexane), polyhalogenated biphenyl compounds such as in particular 3,4,3 ', 4'-tetrachlorobiphenyl and 2,4,5,2', 4 ', 5 '-hexachlorobiphenyl. Finally, mention may be made of polycyclic aromatic hydrocarbons

(PAH), some of which are mutagenic and carcinogenic compounds.

Polycyclic aromatic hydrocarbons (PAHs) constitute an important part of the tars resulting from the distillation of coal. They are also found in petroleum distillation tars. PAHs are the systematic pollutants of the soil of old gas factories and coke ovens (steel, carbochemistry). They are also found scattered in other places in relation to the use that has been made of these tars: agglomeration of coal dust into balls, manufacture of wood treatment products called creosotes and wood treatment plants using these products . They have even been used in some cases to manufacture road surfaces, or as sealants (coatings, plugging breaches, building dams).

PAHs are hydrophobic products that strongly adhere to solids. They all have very low solubility, but this solubility is sufficient to displace them and dilute them notably in the soil over time. These are very low volatile products, with the exception of naphthalene, but they nevertheless emit a strong, unpleasant characteristic odor.

The number of PAHs that can be found on a gas site is considerable. The American Environmental Protection Agency (EPA) has published a list of 16 PAHs contaminating the soil, this list making reference in many countries. These

16 PAHs are considered a priority and must be monitored.

The following table 1 presents the formula of these 16 PAHs.

Table 1: Formula of the 16 PAHs

Several fungi forming white rot on wood are known for their degrading action against certain recalcitrant polluting compounds.

These rot fungi, when grown under nutrient-limiting conditions (nitrogen deficiency for example) are capable of degrading lignin as well as many other organic compounds, including in particular PAHs, thanks to a non-enzymatic system specific with strong oxidizing power (ligninases). Certain rot fungi are also capable, when grown under non-limiting conditions, of transforming certain PAHs, such as phenanthrene, into trans-dihydrodiols using monooxygenases according to a mechanism close to that existing in other families. mushrooms.

Some fungi are also capable of degrading PAHs into trans-dihydrodiols or phenols. Thus, for example, the strain Phanerochaete chrysosporium is useful for degrading in particular TNT (WO 94/21394) and PAHs of 2 to 5 cycles such as anthracene, pyrene, benzo (a) pyrene (Hammel et al. ,

1987, INRA colloquia No. 40, INRA Ed., Pp 45-49).

However, no prior art data demonstrates the biodegradation of all PAHs at five and six cycles or the biodegradation of PAHs at more than six cycles.

There was therefore a need to find a strain of microorganism capable of degrading a broad spectrum of recalcitrant polluting compounds, in particular high molecular weight PAHs (with 5 or more aromatic rings).

The Applicant has now discovered a new strain of fungus which meets this need.

The present invention therefore relates to a strain of fungus Coriolus versicolor, characterized in that it is the strain deposited on January 19, 1996 at the CNCM (National Collection of Cultures of

Microorganisms at the Institut Pasteur) under the reference 1-1657, or one of its derivatives, capable of degrading not only polycyclic aromatic hydrocarbons (PAHs) comprising from two to four cycles but also polycyclic aromatic hydrocarbons (PAHs) comprising at least five cycles, including all PAHs comprising five or six cycles, in particular all PAHs five or six cycles from Table 1.

By "derivative" is meant a strain derived from Coriolus versicolor 1-1657 by mutation, spontaneous or induced, and capable of degrading not only polycyclic aromatic hydrocarbons (PAH) comprising two to four cycles, but also polycyclic aromatic hydrocarbons (PAH ) comprising at least five cycles, including all the PAHs comprising five or six cycles, in particular all the PAHs with five or six cycles in Table 1, as well as, in general, the PAHs comprising more than six cycles. Hereinafter, the Coriolus versicolor 1-1657 strain will be designated as well as its derivatives as defined above by the expression "Coriolus versicolor strain according to the invention" or "strain according to the invention".

The present invention also relates to the use of said strain Coriolus versicolor according to the invention for degrading recalcitrant polluting compounds contained in contaminated materials.

More particularly, the subject of the present invention is the use of the strain Coriolus versicolor according to the invention for degrading polycyclic aromatic hydrocarbons (PAHs) which can be chosen in particular from the 16 PAHs from the list of EPA cited above (Table 1 ). The Coriolus versicolor strain according to the invention has proved to be particularly useful for degrading PAHs with at least 6 aromatic cycles such as in particular benzo (g, h, i) perylene and indenol (1, 2,3-cd) pyrene.

The present invention also relates to a method of treating material contaminated with recalcitrant pollutants, in which the fungus of strain Coriolus versicolor according to the invention is brought into contact with the contaminated material under conditions suitable for stimulate the growth of said fungus in the material and guarantee the degrading activity of recalcitrant pollutants.

Said contaminated material can be soil, aquatic sediments, gravel, and other solid materials, as well as water. Preferably, said contaminated material is soil.

The main implementations of soil bioremediation techniques are:

- in situ treatment: the soil is treated without being excavated;

- on-site treatment: the soil is excavated and treated on site; - ex situ treatment: the soil is excavated and treated off site.

By "conditions suitable for stimulating the growth of said fungus in the material and guaranteeing the degrading activity of recalcitrant pollutants", we mean conditions of aeration, temperature and humidity, of the contaminated material, known to those skilled in the art .

When the contaminated material is a soil, its humidity can be, preferably, between 50 and 80% of the useful water content depending on the type of soil. The temperature can be between 10 and 40 ° C, a growth of the fungus being however always possible even at a lower temperature. The nitrogen content in the soil can be such that the carbon / nitrogen / phosphorus ratio is equal to about 100/10/1.

Contaminated material may also advantageously contain nutrients and trace elements.

Aeration can be ensured by forced air circulation, by natural diffusion of air, or by mechanical mixing of the soil. When the contaminated material is soil, an air flow of about 0.1 to 5 m ³ / h / tonne of soil is advantageous for example. The aeration can be made more homogeneous in the soil by ensuring a stripping or a preliminary screening of the soil. The addition of an organic support with an inoculum also improves the structure of the soil, and therefore its aeration. More generally, the homogenization of the soil by stripping / screening / mixing and mixing with the organic support of the inoculum makes it possible to improve the transfers of mass (water, temperature, nutrients, air, enzymes) and therefore the biochemical activity responsible for the degradation of the polluting components.

The strain according to the invention can be brought into contact with the contaminated material either alone or in the presence of an organic support. The strain according to the invention can be brought into contact with such an organic support either at the time of inoculation in the contaminated material, or prior to this inoculation. The latter case is preferred and allows the pre-development of the strain on the organic support before contacting the contaminated material. The inoculum can then be introduced into the contaminated material at a rate of between 1 and 50% by weight, preferably between 5 and 20% by weight relative to the weight of contaminated material. The contacting can be carried out in particular on excavated soil or on unstructured soil.

According to a preferred embodiment of the invention, the material contaminated with recalcitrant pollutants is treated with an inoculum of fungi of the Coriolus versicolor strain according to the invention, said inoculum being prepared by: - treatment of a lignified organic support which can be chosen from wood chips, tree bark, or corn cobs, so as to eliminate microbiological contaminants;

- Inoculation of said lignified organic support thus treated, with said strain of fungus, said strain possibly being in particular in the form of a) a solution of spores and / or fragments of mycelium suspended in a liquid medium, b) of predeveloped mycelium in a liquid medium, or c) of mycelium pre-developed on a solid support, in particular (i) on a solid gelled medium such as for example agar, or (ii) on cereal grains (preculture on cereal grains);

- Then incubation under conditions suitable for the growth of fungi on said lignified organic support. By "conditions suitable for the growth of fungi on said lignified organic support" is meant humidity, temperature and aeration conditions known to those skilled in the art.

The lignified organic support inoculated with the fungus strain can be advantageously placed in a culture chamber saturated with humidity, at a temperature between 15 and 35 ° C, more particularly between 20 and 30 ° C.

Preferably, corn lump or wood chips are chosen as the lignified organic support.

The wood chips can be calibrated or of the "general purpose" type, that is to say uncalibrated.

Wood shavings with a particle size greater than 4.5 mm are preferred, chips with a smaller particle size generally hamper the growth of the fungus due to poor ventilation due to compaction of the chips.

Since fungi are sensitive to microbiological contamination, it is important to minimize the risk of contamination.

To this end, the lignified organic support is treated before inoculation with the fungus strain so as to eliminate microbiological contaminants. This treatment can be carried out by drying between 450 and

550 ° C, for example at 500 ° C for a few seconds or by pasteurization for 1 or 2 cycles each of 8 to 30 hours, preferably

16 hours, each at a temperature between 50 and 100 ° C, preferably between approximately 65 and approximately 85 ° C, or by sterilization (autoclaving) between 110 and 130 ° C, preferably at approximately 120 ° C, for 20 to 240 minutes, preferably for 20 to 60 minutes. The lignified organic support can be moistened preferably to saturation, before the treatment intended to remove microbiological contaminants. Preferably, said treatment is carried out by pasteurization.

The lignified organic support can then be inoculated with the Coriolus versicolor strain according to the invention, which can be: a) in the form of a solution of spores, fragments of mycelium or a mixture of spores and fragments of mycelium, suspended in a liquid medium, which can be prepared, for example, from cultures of fungi on a support solid, in particular on solid gelled medium (in particular agar) in a tube, or on cereal grains; the suspension can thus be obtained in particular by introducing a determined volume of CH culture medium as described in Kirk et al., 1978, Influence of culture parameters on lignin Metabolism by Phanerochaete Chrysosporium. Anch. Microbiol. 117, 277-285, then vigorously shaking the tube to release the spores; b) in the form of pre-developed mycelium in liquid medium: the pre-developed mycelium can be obtained by culturing in CH medium (Kirk et al., 1978) a solution of spores in suspension; in this case, the culture is generally stirred for about a week at 30 ° C; at the end of incubation, the balls of mycelium formed are recovered and then used as an inoculum; c) or in the form of mycelium predeveloped on a solid support (mycelium covering fragments of solid support), in particular (i) on a solid gelled medium, such as for example agar, which can be contained in a petri dish , or (ii) on cereal grains (preculture on cereal grains).

Preferably, the lignified organic support is inoculated with fungi of the Coriolus versicolor strain according to the invention precultured on cereal grains, such as grains of wheat, rye or millet in particular.

Said lignified organic support can be inoculated with cereal grains colonized by the fungus, preferably at a rate of about 1% to about 20%, more preferably from 2 to 10% (grain weight / weight of lignified organic support ).

The cereal grains used are then preferably untreated grains which are sterilized according to the following protocol: soaking in water then cooking at around 100 ° C. for 30 to 60 minutes, preferably 45 minutes and finally autoclaving from 1 10 to 130 ° C, preferably at about 120 ° C for 20 to 240 minutes, preferably 100 to 200 minutes. An abrasive agent facilitating the separation of the grains from one another as well as a source of nutrient can be advantageously added. For example, plaster, calcium carbonate, chalk or talc can act as both a nutrient (source of calcium) and an abrasive agent. Plaster can thus be added to the cereal grains immediately before autoclaving at a concentration of, for example, between approximately 0.5 and approximately 10% (mass / mass). The grains are then inoculated, for example using a) a solution of spores and / or fragments of mycelium suspended in a liquid medium, b) mycelium pre-developed in liquid medium, or c) mycelium pre-developed on a solid support, in particular (i) on a solid gelled medium such as, for example agar, which can be contained in a petri dish or (ii) on cereal grains, then generally placed in sterile jars or in bags sterile with microporous membranes.

The inoculated supports can then be placed in culture bags allowing gas exchange. High capacity solid phase fermenters can also be used in place of bags. According to a preferred embodiment of the invention, as organic support, corn cobs or wood shavings are chosen, which prior to bagging are intimately mixed with the cereal grains colonized by the fungus at a rate of approximately 1 % to 20%, preferably 2 to 10% (weight of grains / weight of chips or stalks). The mixture is then incubated at an optimum temperature for the growth of the fungus strain used (generally 25-30 ° C). This technique makes it possible to obtain a homogeneous and very well developed culture. Successive generations of inoculum can be produced by rediluting either the inoculated grains in a volume of sterile grains, or the mature inoculum in a volume of sterile lignified supports. For example, shavings can be moistened with a mineral solution (including nutritive supplements for the fungus) comprising for one liter of solution:

KH ₂ PO ₄ : 2 g

MgSO ₄ : 0.5 g

CaCI ₂ : 0.1 g

Ammonium tartrate: 0.2 g

Water: in sufficient quantity for 1 I of solution.

The following examples and figures illustrate the invention without limiting it in any way.

LEGEND OF THE FIGURES:

FIG. 1 represents a comparative test of the capacity of four different strains of Coriolus versicolor to degrade four PAHs: BAN (benzo (a) anthracene), BPY (benzo (a) pyrene), BKF

(benzo (k) fluoranthene) and DBA (dibenzo (a, h) anthracene). Biodegradation levels are given semi-quantitatively: 0 = 0-9%, 1 = 10-24%, 2 = 25-49%, 3 = 50-74%, 4 = 75-100%.

FIG. 2 represents the percentages of biodegradation into total PAHs and as a function of the number of cycles after four weeks. The pilot with Coriolus versicolor 1-1657, 20% is compared to the native microflora control.

FIG. 3 represents the percentages of biodegradation into total PAHs and as a function of the number of cycles. The pilot with Coriolus versicolus 1-1657, 10% (after 5 weeks) is compared with the control (native microflora, 8 weeks).

FIG. 4 represents the percentages of biodegradation into total PAHs and as a function of the number of cycles after eight weeks. The pilot with Coriolus versicolor 1-1657, 10% is compared to the native microflora control.

FIG. 5 represents the percentages of biodegradation of some PAHs at five and six cycles in a soil initially containing 7602 mg PAH / kg of soil and after eight weeks of incubation.

EXAMPLE 1:

Degrading activity of the Coriolus versicolor 1-1657 strain towards PAHs.

The following abbreviations were used for each PAH: naphthalene (NA), acenaphthylene (ACY), acenaphthene (ACE), fluorene (FL), phenanthrene (PH), anthracene (AN), fluoranthene (FLH), pyrene (PY), benzo (a) anthracene (BAN), chrysene (CH), benzo (b) fluoranthene (BBF), benzo (k) fluoranthene (BKF), benzo (a) pyrene (BAP), dibenzo (a, h) anthracene (DBA) ), benzo (g, h, i) perylene (BPE) and indenol (1,2,3, cd) ρyrene (INP).

a) Analytical methods

Extraction:

The extraction is carried out on the entire test volume. An organic solvent is added to each bottle at the rate of 40 ml of acetonitrile. Then the bottles are shaken for one hour at 200 rpm before passing through an ultrasonic tank for 5 minutes. The extraction yields have been measured beforehand and are greater than 95%.

Dosages:

The PAHs are determined by GILSON high pressure liquid chromatography / JASCO UV detection, equipped with a Lichrospher 100 C18 column 250 mm long and 4 mm in diameter. The eluent used is isocratic (800 ml acetonitrile + 200 ml water). The wavelength of the detector is 254 nm. This assay method allows the separation and quantification of the peaks corresponding to the 16 PAHs studied.

b) Biodegradation study method: The degradation studies were carried out in 100 ml serum bottles, tightly closed.

Each test bottle contains 20 ml of culture medium allowing the proper development of the fungus. The culture medium used is a medium enriched in nitrogen or nitrogen deficiency and causing stimulation of its extracellular enzymatic activity (secretion of ligninases) depending on the case. Its composition (described by Kirk et al., 1978, Influence of culture parameters on lignin Metabolism by Phanerochaete Chrysosporium. Anch. Microbiol. 1 17, 277-285) is as follows:

• Culture medium: basal medium III medium:

KH ₂ PO ₄ : 20 g / 1

MgSO ₄ : 5 g / 1

CaCI ₂ : 1 g / l

• Composition of culture:

The following products are added per liter of cultures:

Basal medium III (sterilized by filtration), 100 ml

10% glucose (autoclaved), 100 ml

0.1 M 2,2-methylsuccinate, pH 4.2 (autoclaved), 100 ml Thiamine (100 mg / liter of stock solution sterilized by filtration) 10 ml

Ammonium tartrate (8 g / liter of stock solution, autoclaved), 25 ml

Spores (absorbance at 650 nm = 0.5), 100 ml.

The fungi were brought into the test medium as a suspension spore solution, prepared from sloping culture tubes. The tested substances were provided in the form solubilized in an organic solvent at a final concentration of PAHs in the test bottles between 6 and 50 mg / l. However, the concentration tested was generally 6 mg / l in order to limit the toxicity of the substance and the quantities of solvent supplied. In all cases, the total amounts of solvent supplied to the test medium never exceeded 1% and the bottles were, if necessary, left open for the time necessary for the evaporation of the solvent. Oxygen was introduced at the start of the test into the bottles, then, if necessary, regularly over time. The initial amount of pure oxygen added was 50 ml. The test was carried out in static phase and the flasks were incubated for one month in the dark and at 30 ° C. At the end of the test, an extraction with acetonitrile was carried out on the entire volume of test medium after 30 days of incubation. Specific assays were then carried out by HPLC.

c) Results

The parameter measured was the disappearance of the ^" substance studied.

The results obtained for each of the 16 PAHs are presented in Table 2. These results are presented semi-quantitatively: 0 = 0-9%; 1 = 10-24%; 2 = 25-49%; 3 = 50-74%; 4 = 75-100% (% = percentages of biodegradation after one month of incubation).

Table 2:

Conclusion:

The Coriolus versicolor 1-1657 strain is capable of degrading all 16 PAHs. The comparison of the Coriolus versicolor I-1657 strain and three other strains of Coriolus versicolor (Coriolus versicolor ATCC 34578, ATCC 34671, ATCC 32745) highlighted the significant advantage of the Coriolus versicolor 1-1657 strain according to the invention. compared to the other three Coriolus versicolor (see Figure 1) to degrade heavy PAHs. EXAMPLE 2:

Method for the biological treatment of soils contaminated with recalcitrant organic substances

The effectiveness of the Coriolus versicolor strain according to the invention was verified during pilot tests carried out on soils historically polluted by PAHs. Their inoculation in the soil made it possible to significantly improve the biodegradation of compounds known to be recalcitrant. The mushrooms are introduced into the soil on their organic support (wood chips) at a rate of 20% (example 2a) and 10% (examples 2b and 2c).

The organic support was inoculated with pre-developed mycelium on cereal grains (the inoculation of the grains having been carried out initially by solid agar covered with fungi (mycelium pre-developed on solid agar)).

a) Experiments were carried out in 50-liter pilots on soils historically contaminated with PAHs from former gas factory sites. The effectiveness of the fungal strain according to the invention was evaluated and compared to the activity of the native soil microflora in the case of a soil contaminated with PAHs at an initial concentration of total PAHs of 1596 mg / kg. distributed as follows: 431 mg / kg for three-cycle PAHs, 774 mg / kg for four-cycle PAHs, 274 mg / kg for five-cycle PAHs and 117 mg / kg for six-cycle PAHs. The mushrooms were inoculated at a rate of 20% and the environmental parameters (humidity, temperature and, optionally, supply of nutrients) were optimized in order to accelerate the growth of the fungi and their enzymatic activity. Biodegradation percentages of 73% for three-cycle PAHs, 47% for four-cycle PAHs, 15% for five-cycle PAHs and 25% for six-cycle PAHs were obtained after only one month of pilot incubation in the presence of Coriolus versicolor 1-1657 (see Figure 2). Biodegradation of total PAHs was approximately 50% after one month of treatment. At the same time, however, the native microflora is stimulated had only very slightly degraded the PAHs, the final concentration reached not being significantly different from the initial concentration in total PAHs. In this case, only the three- and four-cycle PAHs were degraded, but their percentages of disappearance were only 19 and 16% respectively.

The analytical methods used for all our tests have been validated beforehand and have conventionally consisted, for this example, of an extraction with supercritical CO ₂ followed by an analysis by gas chromatography / mass spectrometry.

b) Tests were carried out in 50 kg pilots on soils historically contaminated with PAHs from former gas factory sites. The effectiveness of fungal strains was evaluated and compared to the activity of the native soil microflora in the case of soil contaminated with PAHs at a concentration of 1092 mg / kg distributed as follows: 122 mg / kg for three-cycle PAHs, 652 mg / kg for four-cycle PAHs, 318 mg / kg for five and six-cycle PAHs. The fungus strain Coriolus versicolor 1-1657 was brought at a rate of 10% (w / w). The environmental parameters have been optimized in order to accelerate the growth of fungi and their enzymatic activity. For this, the temperature was kept around 30 ° C and the soil humidity at 70% of the useful water content of the soil. Biodegradation percentages equal to 75% for the three cycles, 50% for the four cycles, 38% for the five and six cycles including 46% for benzo (a) pyrene, were obtained after only five weeks of incubation in pilot in the presence of the Coriolus versicolor 1-1657 strain (see FIG. 3). Biodegradation in total PAHs reached 49% after only five weeks of incubation. At the same time, the native biostimulated microflora degraded PAHs only very slightly, the percentage of biodegradation reached at the end of the test being barely greater than 10%. After eight weeks of incubation, the results obtained with the native biostimulated microflora had hardly changed, with biodegradation percentages of 18, 21, 6 and 17% for two and three cycles, four cycles, five and six cycles and total PAHs respectively. It is important to note that the native microflora control pilot had also been inoculated with sterile shavings identical to those used for the preparation of the mushroom inoculum and this at a rate of 10% (weight / weight).

The analytical methods used for these tests were validated beforehand and consisted, for this example, of a solvent extraction under pressure using a DIONEX ASE 200 apparatus followed by an analysis by gas chromatography / mass spectrometry or liquid chromatography high pressure / UV detection. The effectiveness of the fungus compared to native microorganisms (bacteria and fungi) is most marked for heavy PAHs with five and six cycles which are the most difficult to biodegrade.

c) Tests have been carried out in 2 kg laboratory pilots on soils historically contaminated with PAHs from former gas factory sites. The effectiveness of fungal strains was evaluated and compared to the activity of the native soil microflora in the case of soil contaminated with PAHs at an initial concentration of total PAHs of 7602 mg / kg distributed as follows : 3903 mg / kg for two and three cycle PAHs, 3233 mg / kg for four cycle PAHs, 466 mg / kg for five and six cycle PAHs. The strain of fungi Coriolus versicolor 1-1657 was brought at a rate of 10% (weight / weight). The environmental parameters have been optimized in order to accelerate the growth of fungi and their enzymatic activity. For this, the temperature was kept around 30 ° C and the soil humidity at 70% of the useful water content of the soil. Biodegradation percentages equal to 49% for the three cycles, 26% for the four cycles, 34% for the five and six cycles including 44% for benzo (a) pyrene, were obtained after only eight weeks of incubation in presence of the Coriolus versicolor 1-1657 strain. Biodegradation in total PAHs reached 38% after eight weeks of incubation. At the same time, the native biostimulated microflora only very slightly degraded the PAH, the percentage of biodegradation reached at the end of the test being equivalent to 25, 9, 0 and 16% for the two and three cycles, the four cycles, the five and six cycles and the total PAHs respectively (see Figure 4).

The analytical methods used for these tests were validated beforehand and consisted, for this example, of a solvent extraction under pressure using a DIONEXASE 200 apparatus followed by an analysis by gas chromatography / mass spectrometry or high liquid chromatography. pressure / UV detection. The effectiveness of the fungus compared to other microorganisms is most marked for heavy PAHs with five and six cycles which are the most difficult to biodegrade (see Figure 5).

Claims

1. Mushroom strain Coriolus versicolor, characterized in that it is the strain deposited on January 19, 1996 at the CNCM under the reference 1-1657, or one of its derivatives, capable of degrading aromatic hydrocarbons polycyclic (PAH) comprising at least five cycles, including all PAH comprising five or six cycles.

2. Coriolus versicolor mushroom strain, deposited on January 19, 1996 at the CNCM under the reference 1-1657.

3. Use of the strain according to any one of claims 1 and 2 for degrading recalcitrant polluting compounds contained in a contaminated material. , _ro

4. Use according to claim 3 in which the recalcitrant polluting compounds are polycyclic aromatic hydrocarbons (PAH).

5. Use according to one of claims 3 and 4, in which the recalcitrant polluting compounds are polycyclic aromatic hydrocarbons chosen from naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo (a) anthracene, chrysene, benzo (b) fluoranthene, benzo (k) fluoranthene, benzo (a) pyrene, dibenzo (a, h) anthracene, benzo (g, h, i) perylene and indenol (1,2,3, cd) pyrene.

6. Use according to one of claims 3 and 4, wherein the recalcitrant polluting compounds are aromatic hydrocarbons with at least five cycles.

7. A method of treating a material contaminated with recalcitrant pollutants in which the strain according to any one of claims 1 and 2 is brought into contact with the contaminated material in appropriate conditions to stimulate the growth of said fungus in contaminated material and guarantee the degrading activity of recalcitrant pollutants.

8. The method of claim 7 comprising the steps of: a) preparing a mushroom inoculum according to any one of claims 1 and 2 on a lignified organic support, said support being previously treated so as to remove the microbiological contaminants, inoculated with said strain of fungus, then incubated under conditions suitable for the growth of the fungus; b) introduce the inoculum into the contaminated material.

9. The method of claim 8, wherein the inoculum is introduced into the contaminated material at a rate between 1 and 50%, preferably between 5 and 20%, by weight.

10. Method according to any one of claims 7 to 9, wherein said contaminated material is a soil.