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Mining and milling of uranium (U) ores have produced considerable amounts of radioactive waste materials. In local areas, improper waste-storage practices have led to U contamination of the surrounding environment at levels thereafter requiring remedial actions (Abdelouas et al., 1999; Shahandeh et al., 2001). As well as chemical and physical methods suggested for the management and restoration of U contaminated areas (Abdelouas et al., 1999; Vandenhove et al., 2000), the use of plants and associated microbiota to remediate U-contaminated sites, so called phytoremediation, is a method nowadays considered with great interest, because it is not harmful to the environment (Shahandeh et al., 2001). However, the success of any option of phytoremediation, for instance the phytostabilization, is highly dependent on the knowledge and understanding of all parameters and processes involved in U soil–plant transfer.
Several studies with U involving plants with soils (Sheppard et al., 1989; Mortvedt, 1994; Huang et al., 1998) as well as with nutrient solutions (Ebbs et al., 1998) have been conducted, but often without considering the effects of plant symbiotic microflora on U uptake. However, in the field, roots of most plant species are associated with microorganisms that can have either a direct or an indirect effect on U availability. Among the soil microflora, arbuscular mycorrhizal (AM) fungi are root symbionts forming association with 80–90% of all seed plant species (Harrison, 1997). In this association, the extraradical mycelium plays an important role in the absorption of essential nutrients (Smith & Read, 1997; Martin et al., 2001) as well as nonessential heavy metals (Colpaert & Vandenkoornhuyse, 2001; Leyval & Joner, 2001). AM fungi could also influence radionuclides acquisition by plants and hence radionuclides bio-cycling, as was reported for 137Cs (Entry et al., 1999; Berreck & Haselwandter, 2001) and 90Sr (Entry et al., 1999). The contribution of AM fungi to U bio-availability has rarely been reported. Uranium concentration has been shown to be higher in intraradical structures (vesicles) of an undefined species of AM fungi than in the host root tissues (Weiersbye et al., 1999), but information on the role of AM fungi on U uptake process and particularly on U transport by fungal hyphae to plant roots is lacking.
Whatever the radionuclide considered, the difficulty to maintain experimental systems void of undesirable organisms other than the two symbionts and the interferences with soil particles, which may complicate radionuclide availability, has impeded clear identification of the role of AM fungi in uptake and translocation of radionuclides. Root-organ culture systems, associating excised roots with AM fungi on synthetic growth media have been used to study various aspects of the symbiosis (Fortin et al., 2002). It was demonstrated that the spatial separation of mycorrhizal roots and extraradical mycelium ramifying into a root-free compartment (St-Arnaud et al., 1996) was a convenient area to study the uptake and translocation of phosphorus (Joner et al., 2000b; Koide & Kabir, 2000; Maldonado-Mendoza et al., 2001; Nielsen et al., 2002) and could presumably be applied to radionuclides.
The objective of this study was to assess the AM fungal contribution on U uptake and translocation under root-organ culture conditions and to determine unambiguously the role of the extraradical fungal mycelium on U uptake and translocation under various pH conditions.
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- Materials and Methods
The two-compartment in vitro growing system (Joner et al., 2000b) used in the present study was particularly convenient since it avoids interference with soil particles thus allowing to control precisely the form and the concentration of U available to AM hyphae. The results obtained demonstrated, for the first time, that U could be translocated by AM fungal hyphae towards roots, and this translocation was assured only by living hyphae. However, at this stage, we could not determine if U was transferred into the root cells or if it was mainly immobilized in the intraradical fungal structures. Some recent findings suggest a high concentration of U in intraradical AM fungal structures (Weiersbye et al., 1999), probably due to chemical conditions differing between intraradical fungal cells and host root cells. Large differences in P concentration between the intraradical parts of AM fungi (with high concentration) and the host root cells or extraradical hyphae were recently observed (Pfeffer et al., 2001; Solaiman & Saito, 2001). Furthermore, intracellular pH varying between 5.6 and 7.0 was reported for hyphae of G. intraradices (Jolicoeur et al., 1998), and a pH of approx. 6 was reported for the cell wall of rhizodermal cells of roots (Marschner, 1995). Both high P concentration and weakly acidic to neutral pH are factors which may favour the formation of U-phosphate complexes and precipitates in intraradical AM fungal structures, and thus restricting its transfer from the fungus to root cells. A restricted transfer of elements from fungus to root cells due to fungal immobilization was also hypothesized for Cd (Joner & Leyval, 1997), and other metals (Kaldorf et al., 1999), but the mechanisms involved remain unclear and are probably diverse (Colpaert & Vandenkoornhuyse, 2001).
Little information is available in the literature concerning the effect of U speciation on the U bio-availability (Ebbs et al., 1998), although the pH-dependence of U speciation in aqueous systems and in soil has been extensively studied. We did a U speciation calculation using the most up-to-date and coherent U thermodynamic data (Grenthe et al., 1992). Our results support other studies reporting that U speciation is highly pH-dependent (Langmuir, 1978; Mortvedt, 1994; Ebbs et al., 1998). However, considerable uncertainty exists in the value of the formation constant of the UO2(OH)2 (aq) species. Other values for this species may be possible (Silva et al., 1995), and if used may affect the predicted U speciation in the pH range of 6–8. The influence of the U speciation was shown for both U uptake and translocation by the mycorrhizal fungus. It seems that soluble uranyl cations or uranyl-sulfate species that are stable under acidic conditions were translocated to a higher extent through fungal tissues, while phosphate and hydroxyl species dominating under acidic to near neutral conditions or carbonate species dominating under alkaline conditions were rather immobilized by hyphal structures. The effect of pH and U speciation was also reported on U uptake in roots and translocation from roots to the shoots of plants grown in nutrient solution (Ebbs et al., 1998). These authors found highest U content and concentration in shoots at pH 5.0 when U was presumably taken up predominantly as the free uranyl cations, while at this pH, the U content and concentration in roots were the lowest.
The increase of element contents in AM fungi may result from many mechanisms including the metabolism and incorporation in tissues, as demonstrated for essential nutrients such as P and N (Pfeffer et al., 2001), precipitation of nonessential metallic cations in the fungus assumed to occur with PO4 (Turnau et al., 1993) and adsorption on negatively charged constituents of fungal tissues (Joner et al., 2000a). In the present study, most exchange sites of fungal hyphae were probably saturated by H+ at pH 4, and this may result in the low UO22+ adsorption on hyphae. This had probably contributed to the low U content in hyphae at this pH, while its translocation was facilitated. Low rate and extent of bio-sorption of metals at low pH were also reported in another study (Gadd, 1990). By contrast, rising the pH would increase negative charges by deprotonation of constituents of fungal tissues, with enhancement of metallic cation adsorption. However, for U, increasing the pH led to the formation of neutral and even negatively charged species at alkaline conditions. Thus, high pH would impair the bio-adsorption of U. Following this, we could hypothesize that the uptake of U by fungal hyphae in the hyphal compartment was due to its precipitation in fungal structures, especially at pH 5.5 where phosphate species were dominant.
The comparison between data for living and formaldehyde-killed hyphae indicated that the hyphal U concentration was influenced by the metabolic activity of fungal hyphae because killing them resulted in increasing U concentration at low pH and in decreasing U concentration at high pH. The increased U content of the formaldehyde-killed hyphae was exclusively due to passive mechanisms such as the adsorption on exchange sites of hyphae, while for living hyphae, active mechanisms of absorption were also involved. Since the adsorption capacity of hyphae can considerably differ between living and killed hyphae, as it was also reported in another study (Joner et al., 2000a), the respective contribution of absorption and adsorption processes on U content in hyphae could not be determined by simple comparison between data for living and killed hyphae.
The two processes discussed above, that is, U uptake in fungal biomass and translocation by fungal hyphae to roots, are interesting aspects for phytostabilization strategies of U polluted soils. They indicate that AM fungi could contribute to the immobilization of U either in the hyphosphere or in the mycorrhizosphere or in roots, with possible effect in decreasing U dispersion in the environment. Further experiments could quantitatively evaluate the relative importance of this fungal contribution to the U immobilization, for instance by comparing U uptake and translocation by hyphae and by plant roots.
In conclusion, this study has provided, for the first time, fundamental information indicating that the AM fungus G. intraradices can take up and adsorb U, and translocate it to roots. The uptake and adsorption of soluble U by the AM fungus were shown by the amount of U observed in the fungal biomass developing in a U-labelled compartment (HC). The presence of U in a neighbouring compartment initially U-free (RC) demonstrated that U was translocated within the fungal hyphae towards roots, as the contact between the two compartments was mediated only by fungal hyphae. The magnitude of U uptake or translocation by the AM fungus appeared to be influenced by the U speciation which is highly pH-dependent. A next step would be to enhance the sink strength of the mycorrhizal host using entire plants grown in vitro or in vivo, and to determine possible changes in U sequestration by roots or in U transfer to shoots linked to the presence of AM fungus in roots.