Phytomining

Heavy metals from various sources such as sewage sludge, fertilizers, fossil fuel combustion, mining tailings, and manufacturing waste are significant contributors to environmental pollution. Unlike organic compounds that can be metabolically degraded, remediation of heavy metals requires their removal from the environment or conversion into biologically inert forms. Plants have a remarkable ability to accumulate and concentrate metals. However, the mechanisms involved are complex and still only partially understood. Of particular interest for phytoremediation applications are 'hyperaccumulators' of heavy metals: these are plant species capable of tolerating and storing very high concentrations of metals without deleterious physiological effects. The mechanisms of hyperaccumulation are not fully known and appear to vary significantly for different plant and metal species.

As well as phytoremediation, plants capable of removing heavy metals from the environment are important for the development of phytomining. In phytomining operations, plants growing in high-mineral environments extract and concentrate metals from the soil. The crop is then harvested and dried, and the plant biomass is treated for metal enrichment and recovery. Phytomining is a relatively cheap technology for mineral extraction and has the potential to allow economic exploitation of mineralized soils or low-grade surface ores that are too metal-poor for conventional mining operations.

Hairy root cultures have been used in several studies of heavy metal uptake and tolerance by non-hyperaccumulator and hyperaccumulator plant species. They have also been investigated for the development of appropriate processing strategies to recover metals from plants used in phytomining.

Non-Hyperaccumulators of Heavy Metals

Hairy roots of N. tabacum, Beta vulgaris (sugar beet) and Calystegia sepium (morning glory) were applied as models to evaluate the availability of Cd in anaerobically digested sewage sludge (Metzger et al., 1992). Growth of B. vulgaris roots was found to be highly sensitive to Cd. The level of Cd accumulation in the roots varied with plant species and source of the sludge. A 5-day, non-sterile bioassay procedure was developed to measure Cd accumulation in the root biomass. Cd uptake by tobacco hairy roots was lower than for whole tobacco plantlets; this was attributed to the absence of transpiration in the hairy root cultures.

The effect of experimental conditions on Cd accumulation was investigated using hairy roots of S. nigrum (Macek et al., 1994, 1997). Cd uptake by the roots was biphasic, with an initial phase of rapid uptake due to physical adsorption followed by slower, metabolically-sponsored accumulation. Adsorption equilibria between the root surfaces and media containing 2-50 mg L-1 Cd were reached after 1-3 h of exposure; however, uptake of Cd into the biomass continued by other mechanisms for a further 5-20 h. Cd accumulation was dependent on the solution temperature, composition, pH, and initial Cd concentration. Although root growth and morphology were little affected at the low Cd concentration of 2 mg L"1, growth was almost completely inhibited at 50 mg L"1 Cd and the roots became dark in color and began to callus (Macek et al., 1997).

Hairy roots of Rubia tinctorum (madder) were used to study the response of this plant species to Cd (Maitani et al., 1996). Cd at a concentration of 100 ^M inhibited growth of the roots. The biomass was found to be saturated with Cd after 1 day of exposure; further Cd accumulation occurred as the roots grew. The percentage of biomass-associated Cd recovered in the supernatant fraction of homogenized roots increased from 33% to 77% during the 14-day culture period, indicating a change in the subcellular distribution of Cd. Phytochelatins, which are peptides of general structure (y-Glu-Cys)n-Gly, n = 2-11, involved in the detoxification of heavy metals in plants, were induced in the hairy roots by Cd treatment. Cu as well as Cd was incorporated into the phytochelatins as a metal constituent.

The response of an endangered plant species, Adenophora lobophylla, to Cd was investigated using hairy root cultures (Wu et al., 2001). The properties of A. lobophylla hairy roots were compared with those of a related but non-endangered species, A. potaninii, to determine if the two species differed in their ability to tolerate Cd soil pollution. Growth of the roots was inhibited by 50-400 ^M Cd applied in liquid medium. Cd treatment at concentrations of 10-200 ^M increased root protein contents; A. lobophylla was found to accumulate higher levels of Cd per mg of protein than A. potaninii. Endogenous levels of reduced glutathione (GSH) and cysteine were also higher in A. lobophylla hairy roots than in A. potaninii. Overall, the results suggested that these two species employ different metabolic strategies for Cd detoxification.

Rhizofiltration of U has been studied using hairy roots of B. juncea and Chenopodium amaranticolor (Eapen et al., 2003). Concentrations of uranyl nitrate from 25 to 5000 ^M were tested. Root growth was reduced at U concentrations of 1000-5000 ^M; growth of B. juncea was retarded to a much greater extent than C. amaranticolor. U uptake was 2-4-fold greater using B. juncea hairy roots than with C. amaranticolor.

Hairy roots of A. rusticana were treated with Pb, Ni and Cd to determine the effect on glutathione ^-transferase and peroxidase enzyme activities (Nepovim et al., 2004). The presence of heavy metals decreased peroxidase activity in the roots compared with untreated controls. The expression of constitutive glutathione ^-transferases may have been affected by the toxicity of the heavy metals and the resulting cellular stress response.

Hairy roots of Hyptis capitata (knobweed), Polycarpaea longiflora and Euphorbia hirta (asthma weed or hairy spurge) were studied for their tolerance and accumulation of Cu (Nedelkoska and Doran, 2000a). Whereas the short-term (9-h) Cu uptake capacities of H. capitata and P. longiflora roots were similar, the Cu content of E. hirta roots was lower than for the other species. In longer-term culture experiments (28 days), growth of H. capitata roots was not significantly affected by 20 ppm Cu in the presence of an equimolar concentration of disodium ethylene diaminetetraacetate dihydrate (EDTA). Cu uptake by H. capitata roots was biphasic with initial rapid accumulation followed by a slower increase in Cu content.

Hyperaccumulators of Heavy Metals

Approximately 400 plant species are known to hyperaccumulate heavy metals such as Ni, Cd, Zn, Co, Pb, and Cu (Brooks et al., 1998). Hyperaccumulators are of particular importance for the development of phytoremediation and phytomining technologies, which depend on the ability of plants to accumulate large quantities of metals in their tissues. In order to fully exploit the capacities of hyperaccumulators, or to endow other species with hyperaccumulator traits through genetic engineering, an understanding of the biological mechanisms responsible for hyperaccumulation is required. Efficient transport of heavy metals across the plasma membrane or into the vacuoles of plant cells has been demonstrated to occur in hyperaccumulator species (Lombi et al., 2001; Pence et al., 2000; Persans et al., 2001). In other studies, metal-chelating agents such as organic and amino acids have been implicated in heavy metal transport and tolerance in hyperaccumulator plants (Krämer et al., 1996, 2000; Sagner et al., 1998). To maintain cellular integrity and function in the presence of high external and internal concentrations of heavy metals, hyperaccumulators must possess a means of neutralizing the toxic effects of metal ions as soon as they contact the cells.

Cadmium

Hairy roots of the Cd hyperaccumulator, Thlaspi caerulescens (alpine pennycress), were used to study metal tolerance and hyperaccumulation in this species (Nedelkoska and Doran, 2000b). T. caerulescens hairy roots exhibited superior tolerance to Cd than hairy roots of the non-hyperaccumulator, N. tabacum. Whereas growth of the T. caerulescens roots was essentially unaffected by 20-50 ppm Cd, in contrast, N. tabacum roots turned dark brown after exposure to 20 ppm Cd and growth was severely retarded.

As high concentrations of heavy metals generate stress responses in plants, including oxidative stress, hairy roots of T. caerulescens were applied to test the hypothesis that antioxidative defences play a key role in Cd tolerance in this species (Boominathan and Doran, 2003a). The activities of three antioxidative enzymes, superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), were measured in T. caerulescens and N. tabacum hairy roots as a function of culture time. Figure 3 shows results for maximum enzyme activities measured in the biomass in the absence and presence of 20 ppm (178 ^M) Cd. There were significant differences between T. caerulescens and N. tabacum roots in terms of their antioxidative enzyme activities and responses to Cd. Without Cd, endogenous SOD activities were higher in T. caerulescens than N. tabacum; in addition, Cd treatment resulted in a decrease in SOD activity in N. tabacum (Figure 3 a). The greatest distinction between T. caerulescens and N. tabacum was evident in the results for CAT (Figure 3b). Maximum endogenous CAT activities were more than two orders of magnitude higher in T. caerulescens hairy roots than in N. tabacum. Although CAT activity in N. tabacum increased when Cd was added to the cultures, the induced CAT levels remained substantially lower than in T. caerulescens. APX activities in T. caerulescens were somewhat lower than in N. tabacum and were not significantly altered by Cd treatment in either species (Figure 3c). Overall, the results shown in Figure 3, particularly those for CAT, suggest that T. caerulescens is equipped with superior antioxidative defences compared with the non-hyperaccumulator, N. tabacum.

T. caerulescens N. tabacum

Figure 3. Maximum antioxidative enzyme activities in hairy roots of T. caerulescens (Cd hyperaccumulator) and N. tabacum (non-hyperaccumulator) cultured for 21-28 days with and without 20 ppm Cd. (a) Superoxide dismutase (SOD) activity; (b) catalase (CAT) activity; (c) ascorbate peroxidase (APX) activity. The error bars represent standard errors from triplicate cultures. Data from Boominathan and Doran (2003a).

T. caerulescens N. tabacum

Figure 3. Maximum antioxidative enzyme activities in hairy roots of T. caerulescens (Cd hyperaccumulator) and N. tabacum (non-hyperaccumulator) cultured for 21-28 days with and without 20 ppm Cd. (a) Superoxide dismutase (SOD) activity; (b) catalase (CAT) activity; (c) ascorbate peroxidase (APX) activity. The error bars represent standard errors from triplicate cultures. Data from Boominathan and Doran (2003a).

Sustained build-up of H2O2 is a typical plant cell response to toxic concentrations of Cd (Stroinski and Zielezinska, 1997; Schützendübel et al., 2001). To assess the ability of T. caerulescens and N. tabacum to control the oxidative burst induced by Cd treatment, H2O2 levels were measured in hairy roots of these species (Boominathan and Doran, 2003 a). As shown in Figure 4, maximum H2O2 concentrations in the T. caerulescens roots were not significantly different with and without Cd, suggesting that T. caerulescens exerted tight control over H2O2 accumulation. In contrast, Cd elicited a substantial increase in H2O2 levels in N. tabacum roots, indicating that N. tabacum was not as capable as T. caerulescens of preventing the build-up of reactive oxygen species. The ability of T. caerulescens to keep H2O2 concentrations in check may be an important survival mechanism for this hyperaccumulator species.

Nickel

Hairy roots of three Ni hyperaccumulators, Alyssum bertolonii, A. tenium and A. troodii, were applied in an investigation of Ni tolerance and uptake in plant tissues (Nedelkoska and Doran, 2001). Whereas A. bertolonii hairy roots grew and remained healthy in appearance in the presence of 20-100 ppm Ni, hairy roots of the non-hyperaccumulator, N. tabacum, turned dark brown at 20 ppm Ni and growth was negligible. Enhanced Ni tolerance in A. bertolonii was thus demonstrated independent of the presence of shoots. However, although hairy roots were shown in these studies to be a useful experimental tool, comparisons between A. tenium hairy roots and regenerated plants showed that there were significant differences in Ni uptake capacity and tolerance between these forms of plant culture. Whole A. tenium plants in hydroponic culture were much more tolerant of Ni and capable of accumulating higher Ni concentrations than hairy roots of the same species, suggesting that functional root-shoot translocation plays a significant role in the detoxification of Ni in whole plants.

T. caerulescens N. tabacum

Figure 4. Maximum H2O2 concentrations in hairy roots of T. caerulescens (Cd hyperaccumulator) and N. tabacum (non-hyperaccumulator) cultured for 21-28 days with and without 20 ppm Cd. The error bars represent standard errors from triplicate cultures. Data from Boominathan and Doran (2003a).

T. caerulescens N. tabacum

Figure 4. Maximum H2O2 concentrations in hairy roots of T. caerulescens (Cd hyperaccumulator) and N. tabacum (non-hyperaccumulator) cultured for 21-28 days with and without 20 ppm Cd. The error bars represent standard errors from triplicate cultures. Data from Boominathan and Doran (2003a).

Analysis of Ni uptake, complexation and distribution in A. bertolonii hairy roots showed that 85-95% of the Ni present in the biomass was in the cell symplasm and not associated with the cell walls (Boominathan and Doran, 2003b). About 28% of Ni in the roots was complexed with three organic acids: citric acid, malic acid and malonic acid. Hairy roots of A. bertolonii were used to study the antioxidative responses of this hyperaccumulator species to Ni (Boominathan and Doran, 2002). As shown in Figure 5a, without Ni, the maximum endogenous SOD activity in A. bertolonii hairy roots was significantly greater than in N. tabacum. After treatment with 25 ppm (426 ^M) Ni, SOD activity levels in A. bertolonii roots were about twice those in N. tabacum. Without Ni, maximum endogenous CAT activities in A. bertolonii roots were about 280 times greater than in N. tabacum; CAT activity was not induced by Ni in either species (Figure 5b).

Figure 5. Maximum antioxidative enzyme activities in hairy roots of A. bertolonii (Ni hyperaccumulator) and N. tabacum (non-hyperaccumulator) cultured for 21-28 days with and without 25 ppm Ni. (a) Superoxide dismutase (SOD) activity; (b) catalase (CAT) activity; (c) ascorbate

peroxidase (APX) activity. The error bars represent standard errors from triplicate cultures. Data from Boominathan and Doran (2002).

Without Ni, maximum endogenous APX activities in A. bertolonii roots were lower than in N. tabacum and declined significantly after exposure to Ni (Figure 5c). The maximum APX activity in Ni-treated N. tabacum roots was about three times that in Ni-treated A. bertolonii. Endogenous H2O2 concentrations were similar in the two species (Figure 6); however, A. bertolonii hairy roots were better able to control H2O2 accumulation after Ni treatment than N. tabacum, even though H2O2 levels rose in both cultures. These results indicate that the much higher CAT activities in the hyperaccumulator roots are likely to give A. bertolonii an advantage over N. tabacum for combating Ni-induced oxidative stress (Boominathan and Doran, 2002).

A. bertolonii N. tabacum

Figure 6. Maximum H2O2 concentrations in hairy roots of A. bertolonii (Ni hyperaccumulator) and N. tabacum (non-hyperaccumulator) cultured for 21-28 days with and without 25 ppm Ni. The error bars represent standard errors from triplicate cultures. Data from Boominathan and Doran (2002).

Phytomining

In phytomining, metal-laden plant biomass harvested from mineral-rich soil must be converted into a 'bio-ore' suitable for further processing and metal recovery. Little work has been carried out to investigate methods for generating bio-ores or to examine whether plant-

derived feedstocks are suitable for conventional mineral processing operations such as smelting, flotation or acid leaching.

In initial work in this area, hairy roots of the Ni hyperaccumulator, A. bertolonii, were exposed to NiCl2-6H2O in medium solutions to generate Ni-rich root biomass (Boominathan et al., 2004). The roots were then dried, ground to a powder and placed in a laboratory-scale horizontal-tube furnace operated at 1200°C open to the atmosphere. The weight loss of the samples was about 94% for furnace treatment times of 3 to 17 h and the Ni enrichment factor was 15-18. The resulting furnace residue contained 78-82% Ni by weight, which is a much higher Ni content than the 1-2% Ni typically found in mined ores. The surface morphology of the furnace residue showed the formation of crystalline structures with a dark gray, metallic appearance. Surface energy-dispersive spectroscopy (EDS) indicated that the major elements remaining in the residue other than Ni were Ca, P and Mg. A. bertolonii hairy roots provided a convenient model system in this work for investigating the production of plant-derived Ni-bearing bio-ore.

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