WO1999033585A1

WO1999033585A1 - Method for treating a material contaminated by resistant polluting compounds

Info

Publication number: WO1999033585A1
Application number: PCT/FR1998/002882
Authority: WO
Inventors: Frédéric BAUD-GRASSET; Dominique Petre
Original assignee: Rhodia Chimie
Priority date: 1997-12-24
Filing date: 1998-12-24
Publication date: 1999-07-08
Also published as: FR2772623A1; AU1971299A; FR2772623B1

Abstract

The invention concerns a method for treating a material contaminated by resistant polluting compounds comprising the following steps: 1) a lignified organic support selected among wood chips, tree bark or corn cob is subjected to a treatment designed to eliminate the microbiological contaminants; 2) said treated support is inoculated with a fungus strain of the polypore family, said strain being in the form (a) of a solution of spores and/or fragment of mycelium suspended in a liquid medium, (b) mycelium previously developed in a liquid medium, or (c) mycelium previously developed on a solid support, in particular (i) on a gelled solid medium, or (ii) on cereal grains; 3) the resulting fungi inoculum is incubated in conditions appropriate for growth of fungi on said support; 4) the fungi inoculum on the organic support obtained from step 3) is introduced in the contaminated material.

Description

Process for treating material contaminated with recalcitrant polluting compounds

The present invention relates to a method for treating material 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 (PAHs), some of which are mutagenic and carcinogenic compounds.

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 PAHs in particular, thanks to a non-specific enzyme system with strong oxidizing power (ligninases). Certain rot fungi are also capable, when cultivated 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 of mushrooms. Some fungi are also capable of degrading PAHs into trans-dihydrodiols or phenols. German patent DE 37 31 816 describes a process for degrading aromatic compounds which are difficult to degrade in contaminated soil, consisting in introducing into these soils certain white rot fungi associated with materials containing hemicellulose, such as straw. These materials containing hemicellulose are subjected beforehand to a treatment at a temperature of 100 to 140 ° C and a pressure of 1 to 3.6 bar, in the presence of water vapor, in order to cause partial autolysis and turgor. of these materials.

The Applicant has now developed a new method for treating materials contaminated with recalcitrant pollutants, by selecting the types of organic supports to be used, their method of treatment and their method of inoculation with the fungus.

The subject of the present invention is therefore a method of treating material contaminated with recalcitrant polluting compounds comprising the following steps:

1) a lignified organic support chosen from wood chips, tree bark or corn cobs undergoes a treatment intended to remove microbiological contaminants; 2) said support thus treated is inoculated with a strain of fungi of the polyporal family, said strain being in the form of 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, or (ii) on cereal grains;

3) the mushroom inoculum obtained is incubated under conditions suitable for the growth of the fungi on said support;

4) the inoculum of mushrooms on an organic support from step 3) is introduced into the contaminated material.

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, for example, be advantageously placed in a culture chamber with controlled humidity, at a temperature between 15 and 35 ° C, more particularly between 20 and 30 ° C, for a period preferably between one and six weeks, for example three weeks.

The highly lignified organic support can be advantageously chosen from hardwood chips, such as poplar or beech or tree bark, such as pine, or even crushed corn cobs, which can be possibly mixed with sawdust. 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 microbioiogical contamination, it is important to ensure that the risks of contamination are very low.

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, by pasteurization for 1 or 2 cycles each from 8 to 30 hours, preferably 8 to 24 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 20 to 60 minutes. The lignified organic support can be humidified, preferably to saturation, before the treatment intended to remove microbiological contaminants. Preferably, this treatment is carried out by pasteurization.

The lignified organic support is then inoculated with the fungus of the polyporal family which is present: a) in the form of a solution of spores, fragments of mycelium or a mixture of spores and fragments of mycelium, in suspension in a liquid medium, which can be prepared for example from mushroom cultures on a solid support, 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, preferably in the form of mycelium pre-developed 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 box of Petri, or (ii) on cereal grains (preculture on cereal grains).

Preferably, the lignified organic support is inoculated with polyporal fungi precultivated 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 fungi, preferably at a rate of about 1% to about 20%, more preferably from 2 to 10% (weight of grains / weight of lignified organic support) .

The cereal grains used are then preferably grains not treated with chemical substances which could inhibit the growth of the fungus, grains which are sterilized according to the following protocol: soaking in water, cooking at around 100 ° C. for 30 to 60 minutes, preferably 45 minutes, then autoclaving from 110 to 130 ° C, preferably at about 120 ° C for 20 to 240 minutes, preferably from 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 to the cereal grains. A pH corrector can also be added advantageously. Plaster, calcium carbonate, chalk or talc can, for example, play the role of a nutrient (source of calcium), an abrasive agent and a pH corrector. 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 or (ii) on cereal grains, then generally placed in sterile jars or in sterile bags equipped with microporous membranes. The incubation is then preferably carried out in a culture chamber with controlled humidity. at a temperature between 15 and 35 ° C, more particularly between 20 and 30 ° C, for a period between one and six weeks, preferably three weeks.

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 an organic support, corn cobs or wood chips are chosen, which, prior to bagging, are intimately mixed with the cereal grains colonized by the fungus, at a rate of '' about 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

MgS0 ₄ : 0.5 g

CaCI ₂ : 0.1 g

Ammonium tartrate: 0.2 g

Water in sufficient quantity for 1 I of solution.

The fungus used in the process of the invention can be any known polyporal. Advantageously, the fungus used can be the strain Coriolus versicolor deposited on January 19, 1996 in the CNCM (National Collection of Cultures of Microorganisms at the Institut Pasteur) under the reference 1-1657 or one of its derivatives, which is capable of degrading not only polycyclic aromatic hydrocarbons (PAH) comprising two to four cycles, but also aromatic hydrocarbons (PAHs) 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.

By "derivative" is meant a strain derived from Coriolus versicolor 1-1657 by spontaneous or induced mutation 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 (namely in particular benzo (b) fluoranthene, benzo (k) floranthene, benzo (a) pyrene, dibenzo (a, h) anthracene, benzo (g, h, i) perylene and indenol (1, 2,3-cd) pyrene), as well as, in general, PAHs comprising more than six cycles .

The present invention also relates to the use of the inoculum prepared according to steps 1) to 3) as defined above for degrading recalcitrant polluting compounds contained in contaminated material. The inoculum is brought into contact with the contaminated material under appropriate conditions to stimulate the growth of fungi in the material and guarantee the degrading activity of recalcitrant pollutants.

Advantageously, the inoculum can be brought into contact with the contaminated material at a rate of between 1 and 50% by weight, preferably between 5 and 20% relative to the weight of the contaminated material. The contacting can be carried out in particular on excavated soil or on unstructured soil.

Said contaminated material can be soils, aquatic sediments, gravel, and other solid materials, and water.

Preferably, said contaminated material is soil.

The main implementations of known techniques for bioremediation of a soil are: - in situ treatment: the soil is left as it is;

- 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% 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 the inoculum according to the invention also makes it possible to improve the structure of the soil, and consequently its aeration. More generally, the homogenization of the soil by demottagc screening / mixing and mixing with the organic support of the inoculum make it possible to improve mass transfers (water, temperature, nutrients, air, enzymes) and consequently the responsible biochemical activity. degradation of polluting components.

The following examples illustrate the invention without limiting it in any way. LEGEND OF THE FIGURES:

Figure 1 shows the percentages of biodegradation in total PAH 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. 2 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).

Figure 3 shows the percentages of biodegradation in total PAH and according to the number of cycles after eight weeks. The pilot with Coriolus versicolor 1-1657, 10% is compared to the native microflora control. FIG. 4 represents the percentages of biodegradation of a few PAHs at five and six cycles in a soil initially containing approximately 7602 mg PAH / kg of soil and after eight weeks of incubation.

EXAMPLE 1:

Choice of organic support

The strains used are:

- Phanerochaete chrysosporium DSM 1547,

- Phanerochaete chrysosporium ATCC 24725,

- Coriolus versicolor CNCM 1-1657. e *.

They were cultivated on a malt agar medium in Petri dishes until a uniform covering of the fungus was obtained on the surface of the dish. Pieces of agar inoculated by the fungi were then used to colonize the organic supports. 9/33585

11

All the supports were previously autoclaved three times at 24 hour intervals for 20 minutes at 120 ° C. They have been moistened to allow optimal development of the fungus.

The supports inoculated with the different strains of fungi were placed in an incubation chamber at a humidity of 100% and at a temperature of 30 ° C until satisfactory development of the fungi was obtained. Their development was assessed visually using a qualitative scale (Table 2):

++: medium development

+++: strong development (dense and uniform)

Table 2:

EXAMPLE 2:

Influence of the particle size of the chips on the growth of fungi

a) Objective:

The objective of this trial was to assess the influence of the size of wood chips on the growth of Coriolus versicolor 1-1657.

b) Methods

The wood chips used come from the company SPS (Société Parisienne de Sciure, Vénissieux). Three different particle sizes in chips were used to assess the influence of the size of the chips on the growth of the fungi: 9-4.5 mm, 4.5-2.5 mm and 2.5-1.8 mm. The shavings originally contain 10% moisture. They were sterilized by autoclaving at 120 ° C for 20 minutes and humidified to 60% with distilled water. They were then inoculated with fragments of solid agar covered with fungus. The chips are then placed in culture chambers saturated with humidity and at a temperature of 30 ° C. The growth of the fungi was assessed regularly over time using a visual method.

c) Results

Slower growth of the fungus was observed on shavings of smaller particle size. This can be explained by poor ventilation due to the compaction of the chips. The fastest growth was obtained for the two largest particle sizes, which correspond to the best aeration. In the latter two cases, the growth of the fungus was very satisfactory.

EXAMPLE 3:

Comparative test for the inoculation of chips by fungi using different sterilization techniques

a) Objective: The objective of this test was to evaluate the effectiveness of different chip sterilization techniques to obtain a quality of microbiologically acceptable and industrially achievable chips. Pasteurization tests have therefore been launched in order to develop an alternative solution to autoclaving.

b) Method:

The wood chips used come from the company SPS (Société Parisienne, Vénissieux). They originally contain 10% moisture. They were humidified to about 60% (weight / weight) using a specific mineral medium, comprising for one liter of solution:

KH ₂ P0 ₄ : 2 g

MgS0 ₄ : 0.5 g CaCI ₂ : 0.1 g

Ammonium tartrate: 0.2 g

Water in sufficient quantity for 1 I of solution.

The shavings were sterilized according to the following techniques: - by autoclaving at 120 ° C for 20 minutes;

- by pasteurization at 65 ° C for 16 hours or 24 hours;

- by pasteurization at 85 ° C for 16 hours;

- by pasteurization at 65 ° C for 2 x 16 hours;

- by pasteurization at 85 ° C for 2 x 16 hours; in the case of double pasteurizations, the two successive cycles of 16 hours each have been separated by a cooling period of 8 hours in order to favor the germination of spores possibly still alive.

They were then inoculated with fragments of solid agar covered with fungus or with pre-developed mycelium in liquid culture, then placed in culture chambers saturated with humidity and at a temperature of 30 ° C. The fungus used is the isolated strain Coriolus versicolor 1-1657. The growth of the fungi was assessed regularly over time using a visual method.

c) Results:

The results obtained by counting the number of contamination points caused by undesirable fungi confirm the effectiveness of pasteurization, in particular pasteurization obtained at a temperature of 85 ° C or for two successive 16-hour pasteurization cycles regardless of the temperature. used (65 or 85 ° C). Indeed, no contamination by a foreign microorganism was observed for the cases mentioned above. Pasteurization therefore represents an effective means of eliminating the risks of contamination of the inoculum. However, it may be necessary to carry out two successive pasteurization cycles or to increase the pasteurization temperature for better efficiency.

EXAMPLE 4:

Biological treatment of soils contaminated with recalcitrant organic substances

The effectiveness of the fungi used was verified during pilot tests conducted 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 fungi are introduced into the soil on their organic support (wood chips) at a rate of 20% (example 4a) and 10% (examples 4b and 4c).

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 efficacy of the strains of fungi 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 nutrient supply) were optimized in order to accelerate the growth of the fungi and their enzymatic activity. Biodegradation percentages equal to 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 1) . Biodegradation of total PAHs was approximately 50% after one month of treatment. At the same time, the native microflora, while being 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 a supercritical CO ₂ extraction 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. 2). 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 respectively the two and three cycles, the four cycles, the five and six cycles and total PAHs. 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 degraded PAHs only very slightly, 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 respectively, the four cycles, five and six cycles and total PAHs (see Figure 3).

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 4).

Claims

1. Method for treating material contaminated with recalcitrant polluting compounds, comprising the following steps:

1) a lignified organic support chosen from wood chips, tree bark or corn cobs undergoes a treatment intended to remove microbiological contaminants;

2) said support thus treated is inoculated with a strain of fungi of the polyporal family, said strain being in the form of 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, or (ii) on cereal grains; 3) the mushroom inoculum obtained is incubated under conditions suitable for the growth of the fungi on said support;

2. The method of claim 1, wherein said lignified organic support is formed of wood chips, preferably of particle size greater than 4.5 mm.

3. The method of claim 1, wherein said lignified organic carrier is formed from corn cobs.

4. Method according to any one of the preceding claims, in which said treatment intended for removing microbiological contaminants is carried out by pasteurization for one or two cycles of 8 to 30 hours, at a temperature between 50 and 100 ° C.

5. Method according to any one of the preceding claims, in which said treatment intended to remove the contaminants microbiological is carried out by pasteurization at a temperature between approximately 65 and approximately 85 ° C, for one or two cycles of 16 hours.

6. Method according to any one of the preceding claims, in which said fungus is pre-inoculated on sterilized cereal grains by soaking in water, cooking at around 100 ° C for 30 to 60 minutes, preferably 45 minutes. , then autoclaving from 110 to 130 ° C, preferably at about 120 ° C for 20 to 240 minutes, preferably from 100 to 200 minutes and is incubated under conditions suitable for the growth of fungi on said grains, prior to inoculation on said lignified organic support.

7. The method of claim 6, wherein an abrasive agent, a nutrient source and / or a pH corrector are added to the cereal grains.

8. The method of claim 7, wherein plaster added immediately before autoclaving at a concentration between about

0.5% and about 10% (mass / mass), acts as an abrasive agent, a source of nutrient and a pH corrector.

9. Method according to any one of the preceding claims, in which said organic support is inoculated with a strain of fungi of the polyporal family, said strain being in the form of mycelium pre-developed on cereal grains, at a rate of about 1% to about 20%, preferably 2 to 10% (grain weight / weight of the organic carrier).

10. Method according to any one of the preceding claims, in which the said fungus is the strain Coriolus versicolor deposited on January 19, 1996 at the CNCM under the reference 1-1657 or one of its derivatives.

11. Method according to any one of the preceding claims, in which the inoculum prepared according to steps 1) to 3) as defined in one of claims 1 to 10, is introduced into said material. contaminated at a rate between 1 and 50%, preferably between 5 and 20%, by weight.

12. Method according to any one of the preceding claims, in which said contaminated material is a soil.

13. Method according to any one of the preceding claims, in which said recalcitrant polluting compounds are polycyclic aromatic hydrocarbons (PAHs).

14. Use of the inoculum prepared according to steps 1) to 3) as defined in one of claims 1 to 10 for degrading recalcitrant polluting compounds contained in contaminated material.

15. Use according to claim 14 in which the inoculum prepared according to steps 1) to 3) as defined in one of claims 1 to 10, is introduced into said contaminated material at a rate of between 1 and 50%, preferably between 5 and 20%, by weight.

16. Use according to one of claims 14 or 15 wherein said contaminated material is a soil.

17. Use according to one of claims 14 to 16, wherein said recalcitrant polluting compounds are polycyclic aromatic hydrocarbons (PAH).