FR2928096A1 - PROCESS FOR THE DEPOLLUTION OF SOILS CONTAMINATED BY HEAVY METALS. - Google Patents

Abstract

The present invention relates to a process for the depollution of soils contaminated with heavy metals.

Description

PROCESS FOR THE DEPOLLUTION OF CONTAMINATED SOILS BY HEAVY METALS

The present invention relates to a process for the depollution of soils contaminated with heavy metals. Heavy metals such as lead, arsenic, cadmium, copper, zinc, nickel and mercury are a major source of soil pollution and are continually introduced into soils due to various activities such as agricultural activities, including the use of agrochemicals or sewage sludge in agricultural soils, as well as industrial activities such as waste incineration, waste disposal or mining (Khan AG, Journal of trace elements in medicine and biology, 18, 355-364, 2005). Soil remediation techniques are usually based on physical or chemical processes and sometimes bacterial (microfilms). These approaches require a relatively large investment and a particular technicality that limits their use, especially in developing countries. In the 1980s, a new concept of remediation of polluted soils was proposed using plants with the particularity to develop on metalliferous environments. This approach called phytoréhabilitation is based on the use of higher plants likely to extract, stabilize or degrade substances polluting the environment. However, these so-called hyperaccumulative plant species are generally little used in phytoextraction because of their slow growth and low biomass production. Mycorrhiza is a mutually beneficial association between a plant and a mycorrhizal fungus. In general, the approach used for phytorehabilitation is a controlled mycorrhizal approach, that is, the selection of an effective fungal strain for its impact on a given parameter, including host plant development or uptake. of metal by the plant. However, the results of this approach are contradictory depending on the fungi or plants used. Thus, the document Arriagada et al. (Journal of Environmental Management, 84 (1), 93-99, 2007) describes the effect of an arbuscular mycorrhizal fungus, Glomus mossae or Glomus deserticola on the growth of Eucalyptus co-cultivated or non-cultured in the presence of soybeans. a mining ground. The Pb and Cd content of the aerial parts of Eucalyptus globulus cocultivated or not in the presence of soybean is not increased in the presence of Glomus mossae by cons, it is more than doubled in the presence of Glomus deserticola. The document Liao et al. (Chemosphere, 50 (6), 847-853, 2003) describes the effect of an arbuscular mycorrhizal fungus on the heavy metal content of maize plants grown with increasing concentrations of these same metals. The biomass weight of aerial parts and roots is higher in the case of non-mycorrhizal plants for Cu concentrations below 3 mg / l or Cd less than 1 mg / l. The Cu content of mycorrhizal plants is significantly higher than in the case of non-mycorrhizal plants with a low Cu concentration. At a high concentration of metals (3 mg / 1 Cu), there is a reverse effect of microorganisms and a lower Cu content in mycorrhizal plants. Therefore, this approach is difficult to generalize to the different heavy metals found in polluted soils and all kinds of plants. One of the objects of the invention is to provide a method, based on a global approach, for the depollution and rehabilitation of large areas of polluted soils, particularly in developing countries. Another object of the invention is to provide a simple process for the depollution of polluted and generalizable soils to the different heavy metals. The present invention relates to the use of at least one endomycorrhizal complex inoculated with fast-growing plants for the depollution of soil spaces contaminated with heavy metals. By endomycorrhizal complex, it is necessary to understand an association of two or more endomycorrhizal fungi. By endomycorrhizal fungus (or arbuscular mycorrhizal fungus) is meant a fungus, the mycelium of which penetrates the root cells and develops arbuscules or vesicles when associated with plant roots. It does not form a fungal mantle around the root (Smith, S.E. & Read, D.J. (1997), Mycorrhizal symbiosis, 2nd edition, UK, Academic Press.). A fast-growing plant refers to a plant for which an increase of at least 10 m3 of biomass per hectare is observed for a plantation under favorable environmental conditions (moisture, soil fertility) and with maintenance provided by adequate silvicultural techniques .

The term "heavy metal contaminated soil spaces" means any land in which a metal or a number of heavy metals is present naturally or accidentally, or has been introduced as a result of various agricultural or industrial activities. In the remainder of the description, the term soil or zone is used and refers strictly to the same thing. Similarly, the expression polluted soil or contaminated soil is used and has exactly the same meaning. Heavy metals refer to natural metallic elements with a density greater than 5g / cm3. In this description, heavy metals can also be referred to as contaminants. By depollution of floor spaces is meant the decontamination of said floor spaces which consists mainly in making the floor and the basement of this space suitable for a new use. A threshold value range may be determined above which a clearance must be considered. The following table I defines these values for the different metals according to two classifications: - Denis Baize (Environmental letter no. 39, February 2000) - The standard AFNOR NF U 44-041. METAL CONTENT (mg / kg) CONTENT (mg / kg) According to D. BAIZE According to AFNOR standard Cd 0.70 2 Co 30 30 Cr 100 150 Cu 35 100 Ni 70 50 Pb 60 100 Zn 150 300 For example, an area whose Cu content in the soil is greater than an interval of 35-100mg / kg will be considered polluted area and a depollution using the method of the invention can then be considered.

In the present invention, it has been found that the use of mycorrhizal complexes allows the depollution of soils contaminated by heavy metals by a phytoréhabilitation mechanism, in particular by phytostabilisation, that is to say by immobilization and reduction of mobility. and the bioavailability of contaminants by the roots of plants and their associated microbes, and / or by phytoextraction, i.e. by removal of metals from the soil and concentration of said metals in the aerial parts of the plant. In an advantageous embodiment, said endomycorrhizal complex consists of at least two endomycorrhizal fungi taken from plants derived from contaminated soil or at the periphery of the zone contaminated with heavy metals. Endomycorrhizal fungi are advantageously taken from the roots of plants The term "plants derived from contaminated soils" denotes plants such as Haloxylon scoparium, Atriplex colerei, Ononis ssp., Hyparrhenia hirta and Cynodon dactylon, but without being limited thereto, capable of develop naturally on soils containing heavy metals, such soils may be the same or different from the contaminated soil spaces identified above, ie they may be from other regions or other contaminated soil spaces. Said plants may also come from the periphery of the contaminated zone in which there is a heavy metal concentration of about 30% (m / m) to about 50% of that of the contaminated zone. For example, in a copper mine in northwestern Marrakesh, the Cu and Pb contents in the different zones are as follows (Table II):

Table II Metals ZNC (mg kg "') PZC (mg kg 1) ZC (mg kg" (uncontaminated area) (Contaminated perimeter of the area (contaminated area)) Cu 104.5 581.8 1085 Pb 23.3 138 , 8 222.9 Metallic limits are, of course, a function of the contaminated soil and its location.

Therefore, the plants come from soils whose content of each metal present in said soils is from at least 30 to more than 100% greater than the threshold value range, i.e. more than 30 times the minimum concentration for Cu and from approximately 1.3 to more than 4 times the concentration allowed for lead according to D. Baize or according to the AFNOR standard NF U 44-041. In another embodiment, said endomycorrhizal complex consists of at least two endomycorrhizal fungi taken from plants derived from soils not contaminated with heavy metals. Said plants come from the uncontaminated zone, that is to say an area in which there is a heavy metal concentration of about 10% (m / m) of the concentration present in the contaminated soils. Consequently, the plants come from soils whose content of each metal present in said soils is less than the threshold value range determined according to D. Baize or according to the AFNOR NF U 44-041 standard. In an advantageous embodiment, said endomycorrhizal complex consists of at least two endomycorrhizal fungi taken from plants derived from a mixture of contaminated soil and soil originating from the periphery of the contaminated zone, or a mixture of soils. from the periphery of the contaminated area and uncontaminated soil, or a mixture of contaminated soil and uncontaminated soil. According to another embodiment, the endomycorrhizal complex defined above comes from soils containing heavy metals selected from Cu, Pb, Zn, Cr, Ni, Cd, W, As. The sol may contain one or more heavy metals chosen from the list above, in identical or different proportions. For example, but not limited thereto, the contaminated soil may contain a mixture of Pb and Cd (respectively 2582 mg / kg and 44.2 mg / kg), Pb, Zn and Cd (respectively 1806 to 2022 mg). / kg, 1025 to 2406 mg / kg and 45.6 to 51.7 mg / kg), Pb, As, Zn (1115 mg / kg, 1605 mg / kg and 1117 mg / kg, respectively) (HM Leung et al. Chemosphere 66 (2007), 905-915). These different mixtures may also contain other heavy metals but in a proportion lower than the threshold value ranges according to D. Baize or according to the AFNOR NF U 44-041 standard. According to an advantageous embodiment, the endomycorrhizal fungi present in the endomycorrhizal complex defined above are in particular chosen from the genus Glomus and preferentially of the genus G. aggregatum and G. fasciculatum.

In an even more advantageous embodiment, the endomycorrhizal complex defined above is inoculated with a fast-growing plant selected from the genus Eucalyptus, Casuarina and Australian acacia, in particular Eucalyptus. These three genera, without limitation, represent the main fast growing tree species used in the rehabilitation of polluted areas. However, other plant species can also be used, such as plants used for biofuels, especially Jatropha curcas. According to another embodiment, the endomycorrhizal complex defined above is multiplied with the aid of a hypermycotrophic plant such as cereals, in particular millet or corn, and legumes. After taking samples from contaminated soil, the periphery of contaminated soil or uncontaminated soil or a mixture of them, the complex is multiplied, by inoculation with a hypermycotropic plant, that is to say a plant capable of developing many mycorrhizae on its roots and therefore to increase the number of endomycorrhizal fungi present on plants from different soils. The multiplication factor thus obtained is about 10, that is to say that there are ten times more spores present on the roots of the hypermycotrophic plant after inoculation. For multiplication, the complex is associated with soil allowing the growth of hypermycotrophic plants. In the case where the propagation is carried out by means of legumes, these are cultivated in a sandy soil previously sterilized for 40 minutes at 120 degrees. According to an even more advantageous embodiment, said multiplied complex defined above is associated with a substrate, in particular consisting of a mixture of contaminated soil and uncontaminated soil, the uncontaminated soil ratio: contaminated soil is comprised of about 10% (v / v) of uncontaminated soil relative to the total of about 100% (v / v) of uncontaminated soil, preferably about 30% (v / v) to about 100% (v / v), more preferably from about 50% (v / v) to about 100% (v / v), more preferably from about 70% (v / v) to about 100% (v / v) and more preferably about 90% (v / v) to about 100% (v / v), especially 100% (v / v) of uncontaminated soil. After multiplication, the complex is therefore associated with a substrate. The substrate is a mixture of contaminated soil and non-contaminated heavy metal soil, which are derived from contaminated soil spaces and uncontaminated soil spaces to begin the growth of the fast growing plant. The increasing proportion of uncontaminated soil present in the substrate promotes the growth of both aboveground biomass and root biomass irrespective of the origin of the mycorrhizal complex, contaminated soil or uncontaminated soil ( Figure 2A and 2B, Figure 3A and 3B, Figure 4A and 4B). In another embodiment, the endomycorrhizal complex defined above is used in a multiplied endomycorrhizal complex ratio: substrate ranging from about 1% (v / v) to about 10% (v / v), preferably about 1% % (v / v) at about 5% (v / v), and more preferably about 1% (v / v). In the case where the ratio is less than 1%, there are not enough endomycorrhizal fungi to allow the depollution. In the case where the ratio is greater than 10%, the amount of endomycorrhizal fungi to be used is too large to be used on a large scale. In an advantageous embodiment, the endomycorrhizal complex defined above is used in soil spaces contaminated by heavy metals such as mining waste rock, urban landfills, industrial waste storage areas. Mineral waste is any product or deposit that results from research and mining or ore processing. These residues may be natural products (sterile, non-exploitable mineral products) or artificial products, resulting from the processing and enrichment phases of the ore (laundered waste) containing any chemical, mineral or organic additives, or generated during a metallurgical melting step (slag). Urban discharge refers to a public or private place where debris and other waste is dumped. Industrial waste storage areas are defined as areas in which industrial waste containing heavy metals is stored prior to disposal. In another aspect, the invention relates to a composition comprising: at least one multiplied endomycorrhizal complex, said complex comprising at least two mycorrhizal fungi, chosen especially from fungi of the genus Glomus and preferably G. aggregatum and G. fasciculatum, a substrate, selected from a mixture of contaminated and uncontaminated soil, the uncontaminated soil ratio: contaminated soil being comprised of about 10% (v / v) of uncontaminated soil relative to the total of about 100% (v / v) of uncontaminated soil, preferably from about 30% (v / v) to about 100% (v / v), more preferably from about 50% (v / v) to about 100% (v / v), still more preferably from about 70% (v / v) to about 100% (v / v) and more preferably from about 90% (v / v) to about 100% (v / v), especially 100% (v / v) ) of uncontaminated soil. In an advantageous embodiment, the multiplied endomycorrhizal complex: substrate ratio of the above defined composition is from about 1% (v / v) to about 10% (v / v), preferably about 1% ( v / v) at about 5% (v, v), and more preferably about 1% (v / v). According to yet another aspect, the invention relates to a method for the depollution of soil spaces contaminated with heavy metals, comprising a step of inoculating fast-growing plants with an endomycorrhizal complex. By inoculation is meant the contact of fast-growing plants with the endomycorrhizal complex to effect a mycorrhizal symbiosis between the plant and the endomycorrhizal fungi. In an advantageous embodiment, the process for the depollution of soil spaces contaminated with heavy metals defined above comprises a prior step of multiplying the endomycorrhizal complex with the aid of a hypermycotrophic plant. According to an advantageous embodiment, the endomycorrhizal complex of the process for the depollution of soil spaces contaminated with heavy metals defined above consists of at least two endomycorrhizal fungi taken from plants derived from soils contaminated with heavy metals. . In another embodiment, the endomycorrhizal complex of the process for the clearance of heavy metal contaminated soil spaces defined above consists of at least two endomycorrhizal fungi taken from plants derived from soils not contaminated with metals. heavy. In an advantageous embodiment, the multiplied endomycorrhizal complex ratio: substrate of the process for the clearance of heavy-metal contaminated soil spaces defined above, is from about 1% (v / v) to about 10% ( v / v), preferably from about 1% (v / v) to about 5% (v / v), and more preferably about 1% (v / v). 9 2928096

According to another embodiment, the substrate of the process for the depollution of soil spaces contaminated with heavy metals, is in particular chosen from a mixture of contaminated and uncontaminated soil, the uncontaminated soil ratio: contaminated soil being comprised of about 10% (v / v) of uncontaminated soil relative to the total of about 100% (v / v) of uncontaminated soil, preferably about 30% (v / v) to about 100% (v / v), more preferably from about 50% (v / v) to about 100% (v / v), more preferably from about 70% (v / v) to about 100% (v / v) and more preferably about 90% (v / v) to about 100% (v / v), especially 100% (v / v) of uncontaminated soil. According to an advantageous embodiment, the heavy metals treated by the process for decontaminating soils contaminated with heavy metals defined above, are chosen from Cu, Pb, Zn, Cr, Ni, Cd, W, As For example, in the case of copper, the copper content in the aerial parts of Eucalyptus camaldulensis plants is significantly increased by treatment with an endomycorrhizal complex from contaminated or uninfected soil compared with the uninoculated control, allowing thus to clean up the soil (FIGS. 5A, 5B and 5C). In an advantageous embodiment, the endomycorrhizal fungi of the process for the depollution of soil spaces contaminated with heavy metals, are in particular chosen from fungi of the genus Glomus and preferentially G. aggregatum and G. fasciculatum. According to an advantageous embodiment, the process for the depollution of soil spaces contaminated with heavy metals defined above comprises a step of introducing a unitary element of culture, in spaces intended for the cultures, containing the endomycorrhizal complex. and the substrate, said unitary crop element being a fast growing plant seed or a fast growing plant. The introduction of a fast-growing plant seed or a fast-growing plant into spaces for culture, containing the endomycorrhizal complex of the invention and the substrate makes it possible to inoculate the seed or the plant thus promoting the growth of above-ground biomass from the seed or the plant. In one advantageous embodiment, the process for the depollution of soil spaces contaminated with heavy metals, comprising a step of transferring high fast growing plants into the endomycorrhizal complex associated with the substrate.

Thus, after three months of growth in the greenhouse, the seeds or plants inoculated with the complex of the invention have a biomass sufficient to be transferred to the spaces to be cleaned.

In an even more advantageous embodiment, the seed or fast-growing plant of the process for the depollution of soil spaces contaminated with heavy metals is chosen from the genus Eucalyptus, Casuarina and Australian acacia trees, in particular Eucalyptus.

The seeds or fast-growing plants are used in the process of the invention so as to allow a rapid development of aboveground biomass and therefore a copper content in the aerial parts increased by a factor of about 2 to about 4 for plants inoculated with a complex from a contaminated area or not, depending on the proportion of uncontaminated soil present in the substrate (Figures 5A, 5B and 5C).

In another embodiment, the method of decontaminating contaminated soil spaces comprises the following steps:

at. removal of at least two endomycorrhizal fungi from plants growing in contaminated soil to obtain an endomycorrhizal complex,

b. multiplication of the endomycorrhizal complex by growth with hypermycotrophic plants to obtain a multiplied endomycorrhizal complex,

vs. mixture of the endomycorrhizal complex multiplied with a substrate chosen in particular from a mixture of contaminated and uncontaminated soil,

the uncontaminated soil ratio: contaminated soil being comprised of about 10% (v / v) of uncontaminated soil relative to the total of about 100% (v / v) of uncontaminated soil, preferably about 30% (v / v); v) at about 100% (v / v), more preferably about 50% (v / v) to about 100% (v / v), more preferably about 70% (v / v) at about 100% (v / v) and more preferably from about 90% (v / v) to about 100% (v / v), in particular 100% (v / v) of uncontaminated soil,

and the multiplied endomycorrhizal complex ratio: substrate is from about 1% (v / v) to about 10% (v / v), preferably from about 1% (v / v) to about 5% (v / v) , and more preferably about 1% (v / v),

d. introducing a unitary crop element consisting of a fast growing plant seed or a fast growing plant into spaces for the cultures containing the mixture for inoculating and growing said seed or plant, e. transfer of fast-growing plants cultivated in said mixture, in a soil space contaminated with heavy metals for the depollution of said soil. The method of the invention used here is carried out with an endomycorrhizal complex derived from contaminated soil. In another advantageous embodiment, the method of decontaminating contaminated soil spaces comprises the following steps: a. removal of at least two endomycorrhizal fungi from plants growing in uncontaminated soil to obtain an endomycorrhizal complex. b. multiplication of the endomycorrhizal complex by growth with hypermycotrophic plants to obtain a multiplied endomycorrhizal complex, c. mixture of the endomycorrhizal complex multiplied with a substrate chosen in particular from a mixture of contaminated and uncontaminated soil, the uncontaminated soil ratio: contaminated soil being comprised of about 10% (v / v) of uncontaminated soil relative to the total of about 100% (v / v) of uncontaminated soil, preferably from about 30% (v / v) to about 100% (v / v), more preferably from about 50% (v / v) to about 100% ( v / v), still more preferably from about 70% (v / v) to about 100% (v / v) and more preferably from about 90% (v / v) to about 100% (v / v), in particular 100% (v / v) of uncontaminated soil, and the multiplied endomycorrhizal complex ratio: substrate is from about 1% (v / v) to about 10% (v / v), preferably about 1% (v / v) at about 5% (v / v), and more preferably about 1% (v / v).

d. introducing a unitary crop element consisting of a fast growing plant seed or a fast growing plant into spaces for the cultures containing the mixture for inoculating and growing said seed or plant. e. transfer of fast-growing plants cultivated in said mixture, in a soil space contaminated with heavy metals for the depollution of said soil. The process of the invention used here is carried out from an endomycorrhizal complex derived from uncontaminated soil. Description of the figures.

Figures 1A, 1B, 1C and 1D represent the effect of the copper soil content on the growth and mineral nutrition of Eucalyptus camaldulensis after 4 months of greenhouse planting: Figure 1A: dry weight of aerial biomass in g of material dry (ordinate) as a function of the Cu content in mg / kg (abscissa).

Figure 1B: dry weight of root biomass in g of dry matter (ordinate) as a function of the Cu content in mg / kg (abscissa).

Figure 1C: Leaf P content in mg / g DM (ordinate) versus Cu content in mg / kg (abscissa), MS = Dry matter.

Figure 1D: Leaf N content in mg / g MS (ordinate) versus Cu content in mg / kg (abscissa), MS = Dry matter. Figures 2A, 2B, 2C and 2D show the effect of inoculation with a mycorrhizal complex from the contaminated (ZC) or uncontaminated (ZNC) zone on the growth of E. coli plants. camaldulensis and their mineral nutrition after 3 months of greenhouse growth on uncontaminated soil.

NI: Not inoculated (control).

Figure 2A: dry weight of aerial biomass in g (ordinate) according to the type of inoculum (abscissa).

Figure 2B: dry weight of root biomass in g (ordinate) according to the type of inoculum (abscissa).

Figure 2C: P content of leaves in mg / g MS (ordinate) according to the type of inoculum (abscissa), MS = dry matter.

Figure 2D: N content of the leaves in mg / g MS (ordinate) according to the type of inoculum (abscissa), MS = dry matter.

For each type of soil and for each measured parameter, the columns indexed by the same letter represent values that are not significantly different from Student's t-test (p <0.05). Figures 3A, 3B, 3C and 3D show the effect of inoculation with a mycorrhizal complex from the contaminated (ZC) or uncontaminated (ZNC) zone on the growth of E. coli plants. camaldulensis and their mineral nutrition after 3 months of growth in a greenhouse on a soil with a contaminated soil / uncontaminated soil ratio = 1/1; V / V.

NI: Not inoculated (control).

Figure 3A: dry weight of aerial biomass in g (ordinate) according to the type of inoculum (abscissa).

Figure 3B: dry weight of root biomass in g (ordinate) according to the type of inoculum (abscissa).

Figure 3C: Leaf P content in mg / g MS (ordinate) according to the type of inoculum (abscissa), MS = dry matter.

Figure 3D: N content of the leaves in mg / g MS (ordinate) according to the type of inoculum (abscissa), MS = dry matter.

For each type of soil and for each measured parameter, the columns indexed by the same letter represent values that are not significantly different from Student's t-test (p <0.05). FIGS. 4A, 4B, 4C and 4D show the effect of inoculation with a mycorrhizal complex originating from the contaminated (ZC) or uncontaminated (ZNC) zone on the growth of E. coli plants. camaldulensis and their mineral nutrition after 3 months of growth in a greenhouse on a soil with a contaminated soil / uncontaminated soil ratio = 2/1; v / v /.

NI: Not inoculated (control).

Figure 4A: dry weight of aerial biomass in g (ordinate) according to the type of inoculum (abscissa).

Figure 4B: dry weight of root biomass in g (ordinate) according to the type of inoculum (abscissa).

Figure 4C: Leaf P content in mg / g MS (ordinate) according to the type of inoculum (abscissa), MS = dry matter.

Figure 4D: N content of the leaves in mg / g MS (ordinate) depending on the type of inoculum (abscissa), MS = dry matter.

For each type of soil and for each measured parameter, the columns indexed by the same letter represent values that are not significantly different from Student's t-test (p <0.05). Figures 5A, 5B and 5C show copper contents (expressed as μg per plant) in the aerial parts of E plants. camaldulensis inoculated or not with a mycorrhizal complex from the contaminated (ZC) or uncontaminated (ZNC) zone in the various soil treatments after 3 months of greenhouse planting on different soils:

NI: Not inoculated (control).

Figure 5A: Cu content (ordinate) according to the type of inoculum (abscissa), obtained on uncontaminated soil.

Figure 5B: Cu content (ordinate) according to the type of inoculum (abscissa), obtained on a soil with a ratio of contaminated soil / uncontaminated soil = 1/1; V / V.

Figure 5C: Cu content (ordinate) as a function of the type of inoculum (abscissa) obtained on a soil with a ratio of contaminated soil / uncontaminated soil = 2/1; v / v /. FIGS. 6A, 6B and 6C show the evolution of height growth of E. camaldulensis plants during the pot experiment as a function of the origin of the mycorrhizal complex originating from the contaminated zone (ZC) or uncontaminated (ZNC) and the rate of soil contamination by copper after different growth times.

NI: Not inoculated (control).

Figure 6A: Height in cm of plants (ordinate) according to the type of inoculum (abscissa), obtained on uncontaminated and uncontaminated soil, after 1 month of growth.

Figure 6B: Height in cm of the plants (ordinate) according to the type of inoculum (abscissa), obtained on uncontaminated and uncontaminated soil, after 2 months of growth.

Figure 6C: Height in cm of the plants (ordinate) according to the type of inoculum (abscissa), obtained on uncontaminated and uncontaminated soil, after 3 months of growth. FIGS. 7A and 7B show the effect of inoculation by the mycorrhizal complexes originating from the contaminated (ZC) or uncontaminated (ZNC) zone on the growth of the aerial and root parts of E. camaldulensis plants after 3 months of growth. on unsterilized contaminated soil and uncontaminated non-contaminated soil.

NI: Not inoculated (control).

Figure 7A: Aerial biomass (g dry weight) of the plants (ordinate) according to the type of inoculum (abscissa), obtained on uncontaminated and uncontaminated soil, after 3 months of growth.

Figure 7B: Root biomass (g dry weight) of the plants (ordinate) according to the type of inoculum (abscissa), obtained on uncontaminated and uncontaminated soil, after 3 months of growth.

EXAMPLES

Example 1: production of endomycorrhizal complexes (fungal inocula) of the contaminated and uncontaminated zone

The work was carried out in an abandoned copper mine located northwest of the city of Marrakech. Plots were materialized in 4 zones with increasing copper contents, ranging from 104.9 mg kg -1 (non-contaminated zone according to AFNOR NF U 44-041) up to 1075.2 mg kg -1.

In each plot, the plant species were recorded and their root systems and soil conditioned by each species were sampled and stored at 4 ° C. The main species identified were: Haloxylon scoparium, Atriplex colerei, Ononis ssp., Hyparrhenia hirta and Cynodon dactylon. The number of spores and the intensity of mycorrhization were determined for each species in each plot. Then the roots taken from the different sites (contaminated sites or not) were collected and inoculated to maize plants in pots filled with unpolluted and disinfected sandy soil (120 ° C, 20 min).

After 4 months of growth in the greenhouse, the corn plants were removed. Their root system was then thoroughly washed and cut into 1 cm fragments. Two types of endomycorrhizal complexes were thus produced:

(i) endomycorrhizal complex (fungal inoculum) from the uncontaminated Cu (ZNC) zone and (ii) endomycorrhizal complex (fungal inoculum) from the Cu (ZC) contaminated zone. EXAMPLE 2 Preparation of Soils and Inoculation of Endomycorrhizal Complexes to Eucalyptus Camaldulensis Plants in a Greenhouse

Both types of soil (contaminated or not) were then collected in the different sites studied and then disinfected (120 ° C, 60 min). The disinfected contaminated soil was then diluted with the uncontaminated soil according to the following reports (contaminated soil / non-contaminated soil): (0/1; v / v) (control), (1/1; v / v) and ( 2/1; v / v /) with Cu contents of 104.9 mg Kg (control), 356 mg Kg and 439 mg Kg, respectively.

The different mixtures were then distributed in 1-liter pots where young seedlings of Eucalyptus camaldulensis, inoculated or not (control), were planted by one or the other fungal inocula, one seedling per pot.

For each soil mixture, there were 3 treatments: NI (not inoculated), ZNC (inoculated with the endomycorrhizal complex from the uncontaminated area) and ZC (inoculated with the endomycorrhizal complex from the contaminated area). Example 3: Transfer of Greenhouse Plants to a Contaminated Natural Environment

Eucalyptus plants were raised in the uncontaminated soil and inoculated or not with fungal inocula from the contaminated area or not were transferred to 20-liter pots filled with soil taken from the contaminated area. The pots were then placed under in situ conditions. Plant height growth was measured monthly.

After 3 months of planting in a greenhouse, the plants were removed and their biomasses (aboveground and root biomass) determined. Results:

1) Effect of copper content on the growth and mineral nutrition of Eucalyptus camaldulensis after 4 months of greenhouse planting.

After 4 months of growth in the greenhouse, the Eucalyptus plants were removed, their biomasses (aerial and root) were determined as well as the P, N and Cu contents of the leaves. Mycorhization rates were also calculated.

During the greenhouse rearing phase (4 months), the increase in copper levels significantly inhibited plant growth in both above-ground and root biomass and mineral nutrition (N, P) (Fig 1). 2) Effect of inoculation with different endomycorrhizal complexes

Inoculation with endomycorrhizal complexes from the contaminated or uninfected area significantly improves plant growth and limits the toxic effect of copper on the development of E. camaldulensis and their mineral nutrition in the different soils (Fig. 2, 3 and 4). It should also be noted that the ZC inoculum is significantly more efficient than the ZNC inoculum in terms of its impact on the growth of aerial parts regardless of the type of soil (Figures 2, 3 and 4). 3) Copper content in the aerial parts of Eucalyptus camaldulensis after 4 months inoculated or not with endomycorrhizal complexes (ZC and ZNC).

Copper contents of aerial parts per plant were also measured in each soil treatment (Fig. 5).

The results show that endomycorrhizal inoculation significantly improves copper uptake by E. camaldulensis with a higher tendency for treatment using the mycorrhizal complex from contaminated soil. 4) Evolution of height growth of E. camaldulensis plants during the pot experiment.

For the experiment to simulate natural planting conditions (transfer of mycorrhizal plants (or not) into 20-liter pots filled with unsterilized and uncontaminated soil, [Cu] = 104.9 mg kg "', or unsterilized and contaminated, [Cu] = 607.6 mg kg l), the results show that inoculation by the fungal complexes ZC and ZNC significantly stimulated the height growth of E. camaldulensis plants in contaminated soil after 3 months of planting (Fig. 6) 5) Effect of inoculation by mycorrhizal complexes from the contaminated (ZC) or uncontaminated (ZNC) zone on the growth of aerial and root parts of E. camaldulensis plants.

For the growth of aerial and root parts in unsterilized and non-copper contaminated soils, the negative effect of heavy metal on the development of E. camaldulensis is found (Fig. 7).

In contaminated soil, inoculation of both complexes significantly increased plant growth after 3 months of culture (Fig. 7). 6) Effect of Inoculation by Mycorrhizal Complexes from the Contaminated (ZC) or Non-Contaminated (ZNC) Zone on Copper Accumulation in Transferred Plants Grown in 20-Liter Greenhouse Pots in Unsterilized and Contaminated Soils or not by copper (simulation of transplantation).

The results are shown in Table 3 and 4 below: Table 3. Copper Accumulation Measures (g per plant) in the Eucalyptus camaldulensis plants inoculated or not with the various endomycorrhizal complexes after 3 months of culture in plants uncontaminated (ZNC) or contaminated soils (CZ). PA: aerial parts; PR: root part; PT: Total dry weight.

Origin of soils Origin of PA PR PT complex ZNC NI 167.4 a (l) 15.7 to 183.2 to ZNC _ _ 261.9 b 234.9 b 26.9 to ZC 510.7 c _ _ 69, 6 b 580.4 c ZC NI 87.8 to 4.8a 92.6 to ZNC 819.3 b 895.3 b 76.1 b ZC 1004.3 c 1135.7 c 131.3 c: for each soil origin, the values followed by the same letter are not significantly different from the Newman & Keuls test (p <0.05)

Table 4: Calculation of Enrichment Coefficient (CE) and Copper Accumulation Factor (FA) in Different Treatments Origin of Soils Origin of EC (IF FA 1 (2) FA 2 (3) ZNC NI 0 complexes, 08 a (4) -ZNC 0.11 ab 1.4 to ZC 0.24 b 2.74 b ZC NI 0.007 a - 1.33 to ZNC 0.06 b 3.8 to 5.07 b 0.08 ZC b 4,6a 6,12 b (I): EC = [CUlpp! [CU] So,; (2): FAI = [CU] Plant mycorrhizal / [Cu] Plant not mycorrhizal (For the same soil origin); (3) FA2 = [Mycorrhizal CUlplant / [CU] Non-Mycorrhizal Plant ZNC According to Zu et al (Zu, YQ, Li, Y., Chen, HY, Qin, L. & Schwartz, C. (2005) Hyperaccumulation of Pb, Zn, and Cd in herbaceous grown on lead-zinc mining in Yunnan, China, Environment International, 31: 755-762.) (4): for each soil origin, the values followed by the same letter are not significantly different from the Newman & Keuls test (p <0.05) Conclusion: The inoculation of arbuscular mycorrhizal fungi complexes of the invention the following advantages: - In sterilized soils and contaminated or not with a heavy metal such as copper o it improves the tolerance of E. camaldulensis at increasing levels of copper, where it allows the plants of E. camaldulensis to have a growth and mineral nutrition substantially equal to those recorded in plants growing in uncontaminated soil, where it allows the plants of E. camaldulensis to fix a greater quantity of copper in its aerial parts, - In unsterilized soils simulating a natural plantation of E. camaldulensis o it improves plant growth, where it allows plants to recover growth identical to that measured in uncontaminated soil, where it improves the enrichment coefficient by a factor of about 10, ie the ratio of the amount of Cu present in the aerial parts of the plant to the amount of Cu present in the soil.

Claims (25)

1. Use of at least one endomycorrhizal complex inoculated with fast growing plants for the depollution of soil spaces contaminated with heavy metals. 2. Use of at least one endomycorrhizal complex according to claim 1, said endomycorrhizal complex consisting of at least two endomycorrhizal fungi taken from plants derived from contaminated soil or at the periphery of the zone contaminated with heavy metals. 3. Use of at least one endomycorrhizal complex according to claim 1, said endomycorrhizal complex consisting of at least two endomycorrhizal fungi taken from plants from soils not contaminated with heavy metals. 4. Use of at least one endomycorrhizal complex according to claim 1 or 2, wherein the heavy metals are selected from Cu, Pb, Zn, Cr, Ni, Cd, W, As. 5. Use of at least one endomycorrhizal complex according to one of claims 1 to 4, wherein the endomycorrhizal fungi are in particular chosen from the genus Glomus and preferentially of the genus G. aggregatum and G. fasciculatum, 6. Use of at least one endomycorrhizal complex according to one of claims 1 to 5, wherein the fast-growing plant is selected from the genus Eucalyptus, Casuarina and Australian acacia, in particular Eucalyptus. 7. Use of at least one endomycorrhizal complex according to one of claims 1 to 6, wherein said complex is multiplied with a hypermycotrophic plant such as cereals, in particular millet or corn, legumes . 8. Use of at least one endomycorrhizal complex according to one of claims 1 to 7, wherein said multiplied complex is associated with a substrate, in particular consisting of a mixture of contaminated soil and uncontaminated soil, the soil ratio not contaminated: Contaminated soil being comprised of about 10% (v / v) of uncontaminated soil relative to the total about 100% (v / v) of uncontaminated soil, preferably about 30% (v / v) to about 100% (v / v), more preferably about 50% (v / v) to about 100% (v / v), more preferably about 70% (v / v) to about 100% (v / v). v) and more preferably from about 90% (v / v) to about 100% (v / v), especially 100% (v / v) of uncontaminated soil. 9. Use of at least one endomycorrhizal complex according to one of claims 1 to 8, wherein the multiplied endomycorrhizal complex ratio: substrate is from about 1% (v / v) to about 10% (v / v) preferably from about 1% (v / v) to about 5% (v / v), and more preferably about 1% (v / v). 10. Use of at least one endomycorrhizal complex according to one of claims 1 to 9, in soil spaces contaminated by heavy metals such as mining waste rock, urban landfills, industrial waste storage areas. 11. Composition comprising: a. at least one multiplied endomycorrhizal complex, said complex comprising at least two mycorrhizal fungi, chosen especially from fungi of the genus Glomus and preferably G. aggregatum and G. fasciculatum, b. a substrate, chosen in particular from a mixture of contaminated and uncontaminated soil, the uncontaminated soil ratio: contaminated soil being comprised of about 10% (v / v) of uncontaminated soil relative to the total of about 100% (v / v); v) uncontaminated soil, preferably from about 30% (v / v) to about 100% (v / v), more preferably from about 50% (v / v) to about 100% (v / v), still more preferably from about 70% (v / v) to about 100% (v / v) and more preferably from about 90% (v / v) to about 100% (v / v), in particular 100% ( v / v) of uncontaminated soil. The composition of claim 11, wherein the multiplied endomycorrhizal complex: substrate ratio is from about 1% (v / v) to about 10% (v / v), preferably about 1% (v / v). at about 5% (v / v), and more preferably about 1% (v / v). 13. Process for the depollution of soil spaces contaminated with heavy metals, comprising a step of inoculating fast-growing plants with an endomycorrhizal complex. 14. Process for the depollution of soil spaces contaminated with heavy metals according to claim 13, comprising a prior step of multiplying the endomycorrhizal complex with a hypermycotrophic plant. 15. The method for the depollution of soil spaces contaminated with heavy metals according to claim 14, wherein the endomycorrhizal complex consists of at least two endomycorrhizal fungi taken from plants derived from soils contaminated with heavy metals. 16. A method for the depollution of soil spaces contaminated with heavy metals according to claim 14, wherein the endomycorrhizal complex consists of at least two endomycorrhizal fungi taken from plants from soils not contaminated with heavy metals. 17. A method for cleaning up soil spaces contaminated with heavy metals according to one of claims 13 to 16, wherein the multiplied endomycorrhizal complex ratio: substrate is from about 1% (v / v) to about 10% (v / v), preferably from about 1% (v / v) to about 5% (v / v), and more preferably about 1% (v / v). 18. A method of depollution of soil spaces contaminated with heavy metals according to one of claims 13 to 17, wherein the substrate is selected from a mixture of contaminated soil and uncontaminated soil ratio uncontaminated soil contaminated ranging from about 10% (v / v) of uncontaminated soil to the total of about 100% (v / v) of uncontaminated soil, preferably from about 30% (v / v) to about 100% ( v / v), more preferably from about 50% (v / v) to about 100% (v / v), more preferably from about 70% (v / v) to about 100% (v / v) and more preferably from about 90% (v / v) to about 100% (v / v), especially 100% (v / v) of uncontaminated soil. 19. A method for the depollution of soil spaces contaminated with heavy metals according to one of claims 13 to 18, wherein the heavy metals are selected from Cu, Pb, Zn, Cr, Ni, Cd, W, As. 20. Process for the depollution of soil spaces contaminated with heavy metals according to one of claims 13 to 19, wherein the endomycorrhizal fungi are in particular chosen from fungi of the genus Glomus and preferentially G. aggregatum and G. fasciculatum, 21. A method for the depollution of soil spaces contaminated with heavy metals according to one of claims 13 to 20, comprising a step of introducing a unitary element of culture, in spaces for cultures, containing the endomycorrhizal complex. and the substrate, said unitary crop element being a fast growing plant seed or a fast growing plant. 22. A method for the depollution of soil spaces contaminated with heavy metals according to one of claims 13 to 21, comprising a step of transferring high fast growing plants in the endomycorrhizal complex associated with the substrate. 23. A method for the depollution of soil spaces contaminated with heavy metals according to one of claims 13 to 22, wherein the seed or the fast-growing plant is selected from the genus Eucalyptus, Casuarina and Australian acacia trees in particular the Eucalyptus. 24. A method for the remediation of contaminated soil spaces according to one of claims 13 to 15 and 17 to 23, comprising the following steps: a. removal of at least two endomycorrhizal fungi from plants growing in contaminated soil to obtain an endomycorrhizal complex, b. multiplication of the endomycorrhizal complex by growth with hypermycotrophic plants to obtain a multiplied endomycorrhizal complex, c. mixture of the multiplied endomycorrhizal complex with a substrate chosen in particular from a mixture of contaminated and uncontaminated soil. the uncontaminated soil ratio: contaminated soil being comprised of about 10% (v / v) of uncontaminated soil relative to the total of about 100% (v / v) of uncontaminated soil, preferably about 30% (v / v); v) at about 100% (v / v), more preferably about 50% (v / v) to about 100% (v / v), more preferably about 70% (v / v) at about 100% (v / v) and more preferably from about 90% (v / v) to about 100% (v / v), especially 100% (v / v) of uncontaminated soil, and the ratio of endomycorrhizal complex multiplied the substrate is from about 1% (v / v) to about 10% (v / v), preferably from about 1% (v / v) to about 5% (v / v), and more preferably from about 1% (v / v), d. introducing a unitary crop element consisting of a fast growing plant seed or a fast growing plant. in spaces for the cultures containing the mixture to inoculate and grow the said seed or plant, e. transfer of fast-growing plants cultivated in said mixture, in a soil space contaminated with heavy metals for the depollution of said soil. 25. A method of remediation of contaminated soil spaces according to one of claims 13 or 14 and 16 to 23, comprising the following steps: a. removal of at least two endomycorrhizal fungi from plants growing in uncontaminated soil to obtain an endomycorrhizal complex, b. multiplication of the endomycorrhizal complex by growth with hypermycotrophic plants to obtain a multiplied endomycorrhizal complex, c. mixture of the endomycorrhizal complex multiplied with a substrate chosen in particular from a mixture of contaminated and uncontaminated soil, the uncontaminated soil ratio: contaminated soil being comprised of about 10% (v / v) of uncontaminated soil relative to the total of about 100% (v / v) of uncontaminated soil, preferably from about 30% (v / v) to about 100% (v / v), more preferably from about 50% (v / v) to about 100% ( v / v), still more preferably from about 70% (v / v) to about 100% (v / v) and more preferably from about 90% (v / v) to about 100% (v / v), in particular 100% (v / v) of uncontaminated soil, and the multiplied endomycorrhizal complex ratio: substrate is from about 1% (v / v) to about 10% (v / v), preferably about 1% (v / v) at about 5% (v / v), and more preferably about 1% (v / v), d. introducing a unitary crop element consisting of a fast growing plant seed or a fast growing plant into spaces for the cultures containing the mixture for inoculating and growing said seed or plant, e. transfer of fast-growing plants cultivated in said mixture, in a soil space contaminated with heavy metals for the depollution of said soil.