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Several plants have been used in combination with chelating agents for enhanced phytoextraction. The ideal plant should be fast growing and produce a large biomass while accumulating high concentrations of polluting metals. It should also be tolerant enough to grow in contaminated soils and be resistant to the chelating agent. It should have known agronomics practice and produce usable fruit or biomass to generate some financial income after harvesting. B. juncea possesses several of these characteristics and is the most commonly used plant species in this remedial approach.

Chelating agents are almost exclusively used as mobilizing agents for enhanced phytoextraction. However, different mineral acids and salts have also been tested. Screening of potential mobilizing agents for radionuclide 137Cs among chelating agents, reducing agents, mineral acids and salts, showed ammonium and potassium salts to be the most effective [30]. In a pot study, Lasat et al. [31] showed that the application of NH4+ (40 - 80 mmol kg-1) increases the accumulation of 137Cs in different plants by 2-12 times. Hammer et al. [32] tested sulphur as a soil amendment. Elemental sulphur is oxidized into sulphuric acid by lithoautotrophic soil microorganisms. This acidifies the soil and possibly mobilizes toxic metals. They used high biomass crops such as willow (Salix viminalis) to extract Cd and Zn from one calcareous and one acidic soil. However, the addition of elemental sulphur to the soil did not yield any additional benefit.

Once a metal is chelated by a chelating agent, the complex has to move from the bulk of the soil to the root's xylem. A threshold concentration of chelating agent is usually required to disrupt the physiological barriers that control metal root uptake under normal conditions [33]. Two pathways are then possible: the solution with complexed metals can enter the symplast by crossing the cell membranes, or move via the apoplast. Further translocation of metal complexes from the roots to the green parts of the plants is driven by plant transpiration. We reported that a single dose addition of 10 mmol EDTA kg-1 to soil contaminated with Pb, Zn and Cd increased their concentration in the aboveground biomass of B. rapa by 104.6, 3.2 and 2.3-times, respectively, while the concentration of the same elements in the roots was 1.7, 3.5 and 3-times lower compared to the corresponding plant tissues from control treatments [34]. These data indicate that metals are probably translocated as a complex with the chelating agent. Indeed, an ultrastructure study using a transmission electron microscope [35] located un-complexed Pb predominantly in the root tissue of Chamaectisus palmensis plants, while HEDTA and EDTA chelated Pb was mainly taken up by the shoots. In B. juncea, measurements of a Pb-EDTA complex in xylem confirmed that the majority of Pb was transported as metal complex in the transpiration stream [33, 36].

Soil contamination is seldom monometallic, and several polluting metals are usually simultaneously present in elevated concentrations. It would therefore be of great practical advantage if the use of a single chelating agent allowed phytoextraction of multi-contaminated soils. Many studies support this. For example, we reported that the addition of 10 mmol kg-1 [S,S]-EDDS to soil contaminated with 1100 mg Pb kg-1, 800 mg Zn kg-1, and 5.5 mg Cd kg-1, enabled the uptake of 1053 ± 125, 211 ± 16 and 5.4 ± 0.8 mg kg-1 of Pb, Zn and Cd, respectively, in the biomass of hemp (Cannabis sativa). This was 105-, 2.3- and 31.7-times higher, respectively, than in the control treatment [37]. However, other authors have reported that the use of chelating agents such as EDTA and DTPA did not enhance, and in some cases even reduced, plant heavy metal uptake [1].

4. Efficiency of chelating agent enhanced phytoextraction

The efficiency of phytoextraction is determined by two key factors: biomass production and the metal bioconcentration factor. The bioconcentration factor is defined as the ratio of metal concentration in the plant shoot to metal concentration in the soil. For phytoextraction to be feasible, the bioconcentration factor of the plant must be greater than 1, regardless of how large the achievable biomass is. Another way to measure the efficiency of phytoextraction is by using the phytoextraction potential. This can be calculated from soil and plant metal concentrations and dry biomass plant yield, as the total amount of metal extracted per ha of soil, in a single phytoextraction cycle, and expressed as kg ha-1. In order to be economically viable, plants for Pb phytoextraction should be able to accumulate at least 10,000 mg Pb kg-1 in their green parts (harvesting the roots is not practical) and achieve a dry biomass of 20 t ha-1 [7].

Shen et al. [38] used 3.0 mmol kg-1 EDTA to treat soil from a mining site in Hong Kong, heavily contaminated with over 10,000 mg kg-1 of Pb. Application of EDTA in three separate doses was the most effective and enabled the Pb concentration in the shoots of B. rapa to exceed 5000 mg kg-1 of dry plant biomass. As explained above, transpiration is believed to be a major force that drives heavy metal accumulation in plant shoots. It has been shown that EDTA soil treatment is toxic for plants and could decrease the plant transpiration rate [33]. Applying EDTA in three separate additions Shen et al. [38], therefore, presumably minimized its adverse effect on the transpiration rate. Barosci et al. [39] applied EDTA in multiple doses to provide time for plants to initiate their adaptation mechanisms and raise their damage threshold against EDTA phytotoxicity. In contrast to these results, we found application of EDTA in multiple doses to be less effective for phytoextraction of Pb, Zn and Cd by B. rapa. [34].

Other authors have reported even higher plant metal concentrations induced by chelating agent application. Blaylock et al. [8] used 3 week-old seedlings and measured more than 15,000 mg Pb kg-1 in the dry weight of shoots of B. juncea after a 10 mmol kg-1 EDTA addition. Huang et al. [36] determined 8960 mg kg-1 and 2410 mg Pb kg-1 in two-week old pea and corn shoots transplanted into a soil substrate pre-treated with 1.5 mmol EDTA kg-1. However, experimental conditions were used in these studies that would be unrealistic in field conditions and full-scale remediation. These include the use of very young plants, a Pb soil fractionation that was favourable for phytoextraction and achieved by "artificial" soil contamination with water-soluble metal salts, and an experimental set up in which no losses of the Pb complex due to leaching occurred.

Since phytoextraction is a long-term technology, it is imperative to keep areas undergoing phytoremediation productive to achieve economically viable and socially acceptable decontamination. Industrial plants, i.e. energy crops or crops for bio-diesel production, are therefore the prime candidates for phytoextraction plants. The use of energy and/or bio-diesel crops for metal phytoextraction would give contaminated soil a productive value and decrease remediation costs. Furthermore, if the phytoextraction cost falls within the margin of interest, or even turns a profit, then the time needed for the operation becomes less important. We evaluated the Pb, Zn and Cd phytoextraction potential of a selection of potential energy plants (C. sativa, Sorghum vulgare, Arundo donax), bio-diesel plants (Brassica napus, Raphanus sativus oleiformis, Sinapis alba) and other plants (Amaranthus spp., Linum usitatissimum, Trifolium pratense, Trifolium repens, Medicago sativa, Zea mays, B. rapa) [37]. Chelating agents EDTA and [S,S]-EDDS were applied when some plants were in the juvenile vegetative and some in the adult vegetative growth phase. The most effective was treatment with 10 mmol kg-1 [S,S]-EDDS and C. sativa, in which the Pb phytoextraction potential reached 26.3 kg ha-1. We used literature data for plant biomass yields in our calculation and potentials were thus probably overestimated. Older, full-grown plants are likely to concentrate less heavy metals than younger ones. With the obtained phytoextraction potential, the percentage of Pb phytoextracted in a single cycle was only approx. 0.6% of the total Pb present in the upper 30 cm of soil. The achieved Pb concentration in C. sativa was clearly far from concentrations required for efficient soil remediation within a reasonable time span. Pb concentrations 10-times higher than actually obtained (exceeding 10,000 mg kg-1 dry biomass) would be required to reduce soil Pb concentrations from an initial 1100 to 300 mg kg-1 Pb (the regulatory limit set by European Union Council, directive 86/278/EEC) in approx. 10-15 years.

There have been very few field demonstrations of chelating agent enhanced phytoextraction. Blaylock [40] showed a significant decrease in soil Pb concentration over two years at two sites in the United States using B. juncea and EDTA. However, the possible leaching of the Pb complex through the soil profile was not determined and it therefore remains uncertain how much Pb the plants really removed. It is well known that EDTA and other chelating agents act as chemical ploughs, redistributing surface contamination from the surface to lower soil horizons, which gives rise to concerns about the environmental safety of enhanced phytoextraction.

5. Concerns relating to the use of chelating agents for enhanced phytoextraction

The ideal chelating agent for enhanced phytoextraction should be specific for targeting metal contaminants, water soluble in its free form to allow easy soil application, and be able to form more lipophilic metal complexes, easily absorbed by plants, and thus decrease the risk of leaching. It should also be non-toxic and inexpensive. EDTA, the most widely tested chelating agent, is not ideal for a number of reasons. EDTA forms highly soluble complexes with Pb and other polluting metals, which can therefore be leached from the soil to the groundwater. EDTA is toxic, especially in its free form [41, 42], and poorly photo-, chemo- and biodegradable in soil [43]. A combined widespread use in fertilizers and slow decomposition has already led to background concentrations of EDTA in European surface waters in the range 10-50 mg L-1 [44].

The increase of soluble metal complexes in pore water following the application of chelating agents raises health, safety and environmental concerns. In a soil column study, we examined the effect of EDTA addition and different watering regimes on the uptake of Pb, Zn and Cd by B. rapa, the leaching of polluting metals and the toxicity effect of EDTA additions on plants and soil microfauna. The most effective was a single dose of 10 mmol EDTA kg-1 soil, whereby 0.06% of soil Pb, 0.03% of Zn and 0.13% of Cd were extracted into shoots, whereas up to 38% of initial total Pb in the soil, 10.5% of Zn and 56% of Cd were leached down the soil profile. EDTA addition had a strong phytotoxic effect on red clover (Trifolium pratense) and inhibited the development of arbuscular mycorrhiza on plants. The results of phospholipid fatty acid analyses indicated a toxic effect of EDTA addition on soil fungi and increased stress of soil microfauna in general, indicated by increased fatty acids trans/cis ratio [34]. Soil microorganisms depend directly on the soil solution for uptake of food, and elevated heavy metal concentrations might be responsible for the toxic effects. Soil microorganisms are largely responsible for important soil processes, such as mineralization and synthesis of soil organic matter, soil micro-aggregate formation, nitrification and denitrification etc., and disturbed microbial activity could thus effect soil functioning. Soil microorganisms are also at the base of the soil food chain. Romkens et al. [45] reported that the number of microbivorous nematodes was greatly reduced during EGTA-enhanced phytoextraction, presumably due to a smaller availability of food. Another hazard is the potential contamination of the food chain if animals graze on the metal contaminated phytoextracting vegetation.

Several other studies investigating phytoextraction enhanced by chelating agents have highlighted the risk of possible mobilization of Pb and other toxic metals from soil to groundwater [9, 46]. Wenzel et al. [47] used canola (B. napus) in a pot and out-door lysimeter experiment and also reported that leaching losses of Cu, Pb and Zn far exceeded the amounts of metal taken up by plants after EDTA was applied. These results indicate that the application of EDTA and other biologically persistent chelating agents may be limited to field conditions in which soil containment and hydrological control of the metal-enriched leachates can be safely achieved, for example to sites where the connection to receiving water has been "broken". In soils that are not hydrologically isolated, control over metal leaching could be possible by maintaining a neutral or negative soil water balance. If a chelating agent is applied and soil irrigation adjusted to natural precipitation in such a way that losses of soil water due to plant transpiration and evaporation are higher or equal to soil water gains, then leaching of metal complexes should in principle not occur. In practice, however, the control of natural factors affecting the soil water balance (precipitation, evapotranspiration) would be very difficult to achieve. To maximize Pb accumulation by plants and reduce the environmental risk of leaching, Epstein et al. [48] proposed that the chelating agent application rate should be selected that maximizes the concentration of complexed Pb, based on the extractability of Pb by the chelating agent. For example; Shen et al. [38] reported that application of EDTA in three separate doses was not just more effective in enhancing the accumulation of Pb in B. rapa, but also reduced mobility and the potential risk of soluble Pb movement into the groundwater.

To extend the time available for plants to accumulate heavy metals and in this way to reduce the leaching of heavy metals from the soil, we tested controlled release as an alternative method of chelating agent application [49]. Controlled release pesticide formulation was first proposed in agriculture to reduce pesticide leaching while maintaining control of pests in soil. We entrapped EDTA in different hydrogel carriers and used it in a column experiment with B. rapa. EDTA in acrylamide, starch, and carrageenan hydrogel granules increased Pb accumulation in a test plant by up to 9.4-times compared to the control. However, the addition of the corresponding amount of EDTA in water solution (5 mmol kg-1 in four separate doses) was more effective and increased the Pb plant concentration by 26.7-times. Controlled release of EDTA was not particularly successful in reducing the Pb leaching, either; 5.4 to 23.6% of initial total Pb was leached through the soil profile. EDTA applied in water solution leached 49.6 % of the total initial Pb.

6. Use of the biodegradable chelating agents for enhanced phytoextraction

The use of biodegradable instead of biologically persistent chelating agents for enhanced phytoextraction could curb off-site migration of their heavy metal complexes. For example, NTA is biodegradable in both aerobic (where it degrades as fast as glucose and citric acid) and anaerobic soil conditions [50]. Kulli et al. [46] used NTA for enhanced phytoextraction of soil contaminated with Cd (2 mg kg-1), Cu (530 mg kg-1) and Zn (700 mg kg-1), with lettuce and Italian ryegrass as test plants. At the highest NTA dose (5.3 mol m-2 soil), the metal concentration in the aboveground plant biomass was 4 to 24 times greater than in the control plants. At this NTA dose, plant growth was almost completely inhibited. Severe visual symptoms indicated metal toxicity as the likely cause. Citric acid used for enhanced U phytoextraction could also represent a useful alternative to more persistent chelating agents. It induces very rapid accumulation of U in plants. Huang et al. [28] reported that shoot U concentrations of B. juncea and Brassica chinensis grown in a U-contaminated soil (750 mg kg-1) increased from less than 5 mg kg-1 to more than 5000 mg kg-1 in citric acid-treated soils. Citric acid complex with U is degraded in soil within a few days. The resulting readsorption of residual U, not extracted by the plants, in the soil reduces the environmental hazard related to potential leaching of U to groundwater.

The [S,S]-isomer of EDDS is a particularly interesting chelating agent because it combines high biodegradability with high chelating strength. [S,S]-EDDS was first isolated as a metabolite of the soil actinomycete Amycolatopsis orientalis [51] and is naturally present in the soil. In addition, the environmental risk of its use in detergent application has already been assessed [52]. The toxicity to fish and daphnia was low (EC50 > 1000 mg/L). The [S,S]-EDDS is readily biodegradable using the criteria stipulated by the Organization for Economic Co-operation and Development (OECD). The OECD criteria state that 60% of the compound must biodegrade within 28 days. For [S,S]-EDDS, the final CO2 yield exceeded 80% after 20 days, assessed by the modified Sturm test [53, 54]. [S,S]-EDDS biodegradation in a concentration of 0.0034 mmol kg-1 occurred rapidly in various environmental compartments. In unacclimated sludge amended soil, the half-life of [S,S]-EDDS was ca. 2.5 days and mineralization was completed in 28 days. [S,S]-EDDS was transformed into benign degradation products, first into N-(2-aminoethyl) aspartic acid, which significantly decreased the chelating capacity [54, 55].

To test the feasibility of using [S,S]-EDDS in enhanced phytoextraction, we compared its efficiency against a benchmark chelating agent EDTA [56]. Applied in a single 10 mmol kg-1 dose, both EDTA and [S,S]-EDDS were almost equally effective in increasing the concentrations of Pb (94.2 and 102.3-fold) and, to a lesser extent, also of Zn and Cd in the leaves of the test plant B. rapa. In separate doses, EDTA was more effective than [S,S]-EDDS, but caused leaching of approx. 22% of Pb, while [S,S]-EDDS leached only 0.8% of initial total Pb concentrations. A biotest with red clover (Trifolium pratense) indicated a greater phytotoxic effect of EDTA than [S,S]-EDDS addition. EDDS was also less toxic to soil fungi, as determined by PLFA analysis, and caused less stress to soil microorganisms, as indicated by the trans/cis PLFA ratio. [S,S]-EDDS and EDTA (5 mmol kg-1, single dose) equally effectively promoted Zn and Cd uptake by oilseed rape (B. napus), amaranth (Amaranthus spp.), Chinese cabbage (B. rapa) and hemp (C. sativa) [37]. Generally, [S,S]-EDDS was also less efficient for Pb plant uptake, except for C. sativa, where the Pb biomass concentration was higher than in the EDTA treatment by 42%. The stability constant for complexes with Pb is substantially higher for EDTA than for [S,S]-EDDS (Table 2). These data therefore illustrate that data on logK does not provide sufficient information on the potency of specific chelating agents for enhanced phytoextraction. The efficiency of chelating agents for phytoextraction seems to be plant-specific, as well as being controlled by the stability constant and soil conditions, as already described above.

To retain the chelating agent solution in the topsoil and thus (i) improve chelating agent availability for Pb mobilisation and plant uptake, and (ii) reduce leaching of Pb-chelate complexes by prolonging the retention time available for [S,S]-EDDS complexes biodegradation in soil, we modified the soil water holding capacity by using synthetic acrylamide hydrogel [57]. Gel-forming soil conditioners are known from agronomy and forestry practice to be effective in increasing the soil water holding capacity, decreasing deep percolation, and minimising losses of water solution through leaching. The addition of 0.2% (w/w) of hydrogel amendment increased soil field water capacity from an initial 24.6% to 31.3%. The use of 5 mmol kg-1 [S,S]-EDDS in hydrogel amended soil increased Pb uptake by B. rapa by 18 times while only 0.2% of total initial Pb was leached through the soil profile. In the control, soil without hydrogel [S,S]-EDDS leached 1.2% of Pb. EDTA was more effective for phytoextraction but caused much higher Pb leaching in all treatments, under any of the soil water sorption condition tested. Using a higher 10 mmol kg-1 [S,S]-EDDS dose in hydrogel amended soil significantly reduced plant Pb uptake and increased Pb leaching to up to 44.2% of initial soil content. This was presumably caused by the toxic effect of high concentrations of [S,S]-EDDS on soil microorganisms (slower biodegradation) and plants (lower Pb uptake). In all treatments in which 10 mmol kg-1 EDTA or [S,S]-EDDS was applied, visual symptoms (necrotic lesions on the leaves of B. rapa) of toxicity were observed. The effect was much less pronounced in treatments with lower amounts of chelating agents.

A serious limitation of [S,S]-EDDS based enhanced phytoextraction is the high price of the chelating agent. The current price for 1 ton of [S,S]-EDDS is approx. 5000 GBP. As [S,S]-EDDS has been substituted for traditional chelating agents in a number of commercial products, e.g. industrial detergents, the price is expected to decrease. In future, the biosynthesis of EDDS by A. orientalis, which produces a chelating agent exclusively in the biodegradable S,S-configuration, instead of the current chemical synthesis, could significantly reduce the production costs [58].

7. Horizontal permeable reactive barriers in enhanced phytoextraction

To further reduce the hazard of heavy metal leaching and off-site migration from treated soil to the environment, we proposed the use of biodegradable chelating agents together with horizontal permeable reactive barriers placed below the contaminated topsoil [59]. A flowsheet of the method is shown in Figure 4. The construction and reactive materials in the barriers are a layer of nutrient enriched substrate with a high water sorption capacity and with extensive surfaces on which microbial films could form, and a layer of absorbent (apatite) for precipitation of metals. The main purpose of the substrate is to enhance the microbial degradation of the metal complexes. Once metal ions are released from the complex they are bound by absorbent, become immobilized and are no longer subject to leaching. After completion of the soil remediation process, the barrier can be excavated and removed.

We borrowed the concept of horizontal permeable reactive barriers from polluted groundwater remediation. Here, a vertical permeable reactive barrier is constructed below ground as a vertical underground wall, filled with reactive materials. The barrier is built by digging a long, narrow trench in the path of the polluted groundwater. Clean groundwater flows out of the other side of the wall. Reactive materials in the barrier trap harmful chemicals or change the chemicals into harmless ones. For example, zero-valent iron can be used for the reduction of toxic Cr6+ to harmless Cr3+, and limestone for Pb precipitation.

Groundwater

Figure 4. Conceptual representation of combined enhanced phytoextraction of heavy metals with biodegradable chelating agent and in situ soil washing using horizontal permeable reactive barriers.

Groundwater

Figure 4. Conceptual representation of combined enhanced phytoextraction of heavy metals with biodegradable chelating agent and in situ soil washing using horizontal permeable reactive barriers.

In our first study, barriers were placed 20 cm deep in soil columns and tested for their ability to prevent leaching of Pb during enhanced phytoextraction using [S,S]-EDDS, EDTA and B. rapa [59]. The reactive materials in the barriers were nutrient enriched vermiculite, peat or agricultural hydrogel, and apatite as absorbent. EDTA and [S,S]-EDDS addition (10 mmol kg-1) increased Pb concentrations in the test plant by 158 and 89 times compared to the control. In EDTA treatments, approx. 25% of total initial soil Pb was leached in a single cycle of chelating agent addition. This was expected, since EDTA is nonbiodegradable, so none of the reactive materials in the barriers were effective. The biodegradability of the chelating agent and metal complexes and binding capacity of adsorptive materials in the barrier for released heavy metals are essential for this type of barrier to function. In [S,S]-EDDS treatments, 20% of the initial Pb was leached from columns with no barrier, while barriers with vermiculite or hydrogel and apatite decreased leaching by more than 60 times, to 0.35%. In total 11.6% of initial Pb was removed from the soil above the barrier with vermiculite and apatite. However, almost all removed Pb was simply washed from the soil and accumulated in the barrier, only a fraction (0.03%) of Pb was phytoextracted. These results pointed toward the possibility of enhanced phytoextraction combined with in situ soil washing. With current plants, however, phytoextraction can be expected to make only a minor contribution.

Most soils are multi-contaminated, so we tested the feasibility of using reactive barriers for remediation of soil contaminated simultaneously with Pb, Zn and Cd. Hemp (C. sativa) was used as the phytoextracting plant [60]. The addition of [S,S]-EDDS (10 mmol kg-1 dry soil) yielded concentrations of 1026 ± 442, 330 ± 114 and 3.8 ± 1.5 mg kg-1 of Pb, Zn and Cd in the dry above-ground plant biomass, respectively. These concentrations were 1926-, 7.5-, and 11- times higher, respectively, compared to treatments with no chelating agent addition. Horizontal permeable barriers, composed of a 3 cm high layer of nutrient enriched sawdust and vermiculite and a 3 cm layer of soil mixed with vermiculite and apatite, were positioned at different soil depths. The barrier placed 30 cm deep reduced leaching of Pb, Zn and Cd by 435-, 4- and 53-times, respectively, compared to columns with no barrier. The lower positioned barrier did not prevent leaching of Zn. In total, 2.5% of initial Pb, 7.3% of Zn and 2.8% of Cd was removed from the contaminated soil in a single [S,S]-EDDS treatment and mostly accumulated in the barrier. The contribution of in situ soil washing prevailed over enhanced phytoextraction, despite a marked increase in Pb plant uptake induced by [S,S]-EDDS. The relative inefficiency of the barrier to prevent leaching of Zn indicates either a lower biodegradability of the Zn-[S,S]-EDDS complex or poor adsorption of released Zn ions. Indeed, Van Devivere et al. (2001) reported that the microbial degradation of Pb-[S,S]-EDDS was much faster than that of Zn-[S,S]-EDDS, and while apatite is an effective absorption material for Pb by conversion into pyromorphite, a poorly soluble Pb phosphate mineral [61], the sorption mechanisms of apatite for metals other than Pb are less clear.

In the follow-up study, the treatment of a vineyard soil with 5 mmol kg-1 [S,S]-EDDS increased the accumulation of Cu in B. rapa by 3.3-times over the control. The reactive barriers composed of nutrient enriched sawdust and apatite were effective and only 0.53+0.32% of the initial Cu was leached, while 36.7% of Cu was washed from the layer of contaminated soil and accumulated in the barriers [29]. Again, these results indicate that with current plants, the use of reactive barriers could be more justified for controlled and environmentally safe in situ soil washing of metals rather than for enhanced phytoextraction.

8. Conclusions

The use of hyperaccumulating plants and continuous phytoextraction is limited to situations in which all of the pollutants present are bioavailable and can be tolerated by plants. At present, it is difficult to judge whether chelating agent assisted phytoextraction can be economically feasible and environmentally sustainable. On the other hand, it is the only phytoextraction option for soils contaminated with highly insoluble metals such as Pb and U. The use of both phytoextraction methods is limited by the climatic and geologic conditions of the site to be cleaned.

The risk of metal leaching is one of the most important limitations of enhanced phytoextraction. We showed that it could be efficiently prevented by the use of a biodegradable chelating agent and reactive barriers. Further research might provide a new environmentally safe mobilizing agent, more effective in enhancing metal accumulation in plants than currently known agents. New methods of chelating agent application, such as alternative formulations allowing the controlled release, could be developed. Another possible option is the use of plants genetically modified to exude strong, metal-selective mobilizing compounds in their rhizosphere. Indeed, environmentally safe methods of enhanced heavy metal phytoextraction must be fully developed and tested before steps towards further commercialization of this remediation technology are attempted.

Since it is the bioavailable fraction and not the total concentration of metals in soil that interact with biological targets and pose an environmental and health threat, phytoextraction research should focus on stripping the bioavailable metal fractions, instead of trying to reduce the concentration of metals in soil below limits set by legislation. Nevertheless, to allow remediation within a reasonable period, the plant heavy metal uptake must be enhanced dramatically. This may be achieved by engineering high-biomass producing plants with as yet unidentified hyper-accumulating genes. One possible strategy to increase heavy metal plant uptake is to increase translocation of chelate complexes through the plant transpiration stream. This could be achieved using high-transpiration plants. For example, Gleba et al. [62] reported that a high-transpiration species of B. juncea phytoextracted 104 % more Pb than the wild-type plant.

Plants exhibiting a high capacity for heavy metal uptake when assisted by chelating agents, i.e. B. juncea, are also generally not very resistant to high levels of Pb and other heavy metals in their foliage. This requires harvesting soon after chelating agent application. There are some indications that the ability to hyperaccumulate heavy metals is the result of high resistance to the metals rather than greater rates of metal uptake [63]. Vacuolar sequestration is likely to be a key component of metal tolerance and hyperaccumulation. To increase resistance to metal by genetic manipulation is therefore another potential approach to improve the efficiency of phytoextraction. Freeman et al. [64] reported that glutathione biosynthesis might play a role in Ni tolerance in hyperaccumulator Thlaspi goesingense. Meagher [65] reported that bacterial genes encoding Hg reductase and organomercurial lyase were successfully transferred into the genome of several plants, including poplar, and that transformed plants showed increased tolerance to Hg.

Although fast progress is being made and studies have demonstrated the power of genetic engineering, more knowledge of the molecular mechanisms responsible for plant metal accumulation, root to shoot transfer and vacuolar sequestration, is necessary before the genetic traits can be transferred into high biomass plants. Practical aspects of enhanced heavy metal phytoextraction also need further research. These might include optimisation of agronomic practices (fertilization, crop protection, harvesting, irrigation) and development of economically feasible techniques for the disposal of metal-enriched plants or, when practical, for metal recovery. Finally, apart from deeper basic research, more pilot-scale outdoor studies and field trials are needed objectively to evaluate the real potential and feasibility of enhanced heavy metal phytoextraction.

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ENZYMES TRANSFERRING BIOMOLECULES TO ORGANIC FOREIGN COMPOUNDS: A ROLE FOR GLUCOSYLTRANSFERASE AND GLUTATHIONE S-TRANSFERASE IN PHYTOREMEDIATION

PETER SCHRÖDER

Institute of Soil Ecology, Department Rhizosphere Biology, GSF National Research Center for Environment and Health, D-85758 Neuherberg, FRG. E-mail: [email protected]

1. Introduction

In the general enzyme list of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB, first published in 1961 and with the last printed edition in 1992) EC 2 is reserved for the enzyme family of transfe-rases. Generally, transferases are enzymes transferring a functional group, for example, methyl- or glycosyl-groups, from one substrate (regarded as donor) to another substrate (regarded as acceptor). Hence, the classification is based on the scheme "donor:acceptor-group transferase". The common names of the enzymes belonging to this group are normally derived from acceptor group-transferase or donor group-transferase. In many cases the donor is a cofactor (coenzyme) carrying the group to be finally transferred.

Whereas most members and subclasses of EC2 are confined to the metabolism of biogenic and natural compounds two subgroups, the glycosyltransferases of EC 2.4 and the aryl/alkyl transferases of EC 2.5, have been recognized as having crucial functions in the metabolism of foreign compounds, xenobiotics, in both animals and plants.

This role is very important, as all organisms are frequently exposed to an array of potentially toxic substances. Organic chemicals are particularly threatening. They may have natural sources e.g. fires, volcano eruptions or processes of biodegradation. They may also be the products of microbial or animal metabolism, or from the secondary metabolism of plants [1]. These organic substances may play a role in defence or in allelopathic reactions. Furthermore, increasing industrialization has provided two novel sources of foreign compounds: (1) through the invention and use of agrichemicals for the protection of crops from pests and weeds, and (2) through the emission of organic xenobiotics in chemical manufacturing processes or the use of synthetic chemicals. The latter compounds of solely anthropogenic origin represent a threat to our environment as these synthetic chemicals are emitted without any control. For plants, the situation is especially difficult as they are rooted in the ground and are dependent on that site for survival. Plants therefore, have to rely on effective detoxification mechanisms.

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