AU775573B2 - Recovering metals from soil - Google Patents

Description

1 RECOVERING METALS FROM SOIL Background of the Invention Field of the Invention This invention pertains to methods for recovering metals, such as nickel and cobalt, from metal-rich soil using phytoextracting or phytomining techniques.

Metals can be selectively extracted from soil by cultivating certain metal hyperaccumulating plants, such as Alyssum plants, on soil treated to adjust the

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15 RelatedArt Industrial practices such as mining, smelting and disposing of manufacturing wastes have increased the concentrations of toxic metals in the environment. For example, at many nickel mining and smelting sites, levels of nickel and cobalt in soil have become so high that few plants survive, resulting S 20 in severe disruption of local ecosystems. Once nickel and cobalt enter soil, their removal is difficult since they are relatively immobile and they do not degrade into less toxic substances. The size of the areas affected by smelter and mine .wastes are usually so large that engineering methods of soil remediation, such as soil removal and replacement, are too expensive to be practical (Cunningham et al., "Phytoremediation of Contaminated Soils," Trends Biotechnol. 13: 393-397 (1995)).

WO 00/28093 PCT/US99/26443 -2- The ability of certain plants to grow in metal-containing or metal-contaminated soil, and to actively accumulate heavy metals in their tissues, has created an interest in using such plants to extract metals from soil. Growing plants, including crops, on contaminated soil to extract contaminants is referred to as phytoextraction. This method is particularly effective in arable contaminated soils because it causes little disruption or dispersal, while preserving soil fertility and landscapes.

Nickel is one of the most widely found, and technologically important metals. It is a natural constituent in all soils, being particularly high in concentration in certain types of soil and geological materials such as serpentine, lateritic serpentine, ultramafic and meteor-derived soils. Cobalt, another valuable metal, has chemical and geological characteristics very similar to nickel and is generally found in the same soils. Other metals that may be found in such soils include those of the platinum and palladium families such as palladium, rhodium, ruthenium, platinum, iridium, osmium and rhenium, and metals such as selenium, zinc and cadmium.

Sites containing serpentine, lateritic serpentine, ultramafic and meteor-derived soils and materials can be conventionally mined or cultivated with metal-accumulating plants. Using such plants to extract metals from mineralized (geogenic) soils is referred to as phytomining.

U.S. Patent No. 5,364,451 to Raskin et al., is directed to a method of remediating polluted soils at a reduced cost. Raskin et al. remove metals from metal-rich soil by growing plants of the family Brassicaceae in the metal-rich soil.

While Raskin et al. generally describe a variety of plants and a large number of metals that may be recovered, the examples mainly describe the recovery of chromium and lead from genetically altered plants. Thus, although promising, Raskin et al. offer little basis for an opportunity to proceed directly with soil phytomining or phytoextraction through plant growth or cultivation.

U.S. Patent No. 5,785,735 to Raskin et al., is also directed to methods of remediating polluted soils. Raskin et al. remove metals from metal-rich soil by growing crop and crop-related members of the plant family Brassicaceae in the metal-rich soil. The methods require the formation of a complex between the metal and a chelating agent added to the soil, the application of an electric field to the soil or a reduction in the pH of the soil. While Raskin et al., generally describe a variety of plants, the specification mainly describes the recovery of metals from genetically altered plants.

Thus, again, Raskin et al., offer little basis for an opportunity to proceed directly with soil phytomining or phytoextraction through plant growth or cultivation.

Scientists recognise that increasing the pH of soil decreases the ability of farm crops to take-up heavy metals. U.S. Patent No. 5,711,784 to Chaney et al., reflects the belief in the art that reducing the pH of the soil "increases the phytoavailability of nickel and cobalt". As disclosed by Chaney et al., a "reduced pH increases solubility, and optimises the release of these metals for absorption by the roots and translocation to the aboveground tissues of the plant." However, reducing the pH of the soil also renders the metals more mobile and may allow for further contamination of the site. Therefore, cultivating plants which are hyperaccumulators of nickel, cobalt and other metals through phytoextraction or phytomining, is a desirable alternative as a means for recovering such metals.

Summary of the Invention Accordingly, this invention relates to improved systems for recovering metals by phytomining or phytoextracting soils rich in metals.

According to a first aspect of the invention there is provided a method for selectively increasing the amount of at least one metal recovered from metal-containing soil, comprising: elevating the pH of the soil; and 25 cultivating at least one metal-hyperaccumulator plant in the soil under conditions sufficient to permit the at least one plant to accumulate the at least one metal from the soil "in above-ground tissue.

According to a second aspect of the invention there is provided a method for selectively increasing the amount of at least one first metal and at least one second metal recovered from metal-containing soil, comprising: elevating the pH of the soil; cultivating at least one metal-hyperaccumulator plant in the soil under conditions sufficient to permit the at least one plant to accumulate the at least one first metal from the soil in above-ground tissue; thereafter lowering the pH of the soil; and [I\DAYLIBLIBH]03621 .doc:UG cultivating the at least one metal-hyperaccumulator plant in the soil under conditions sufficient to permit the at least one plant to accumulate the at least one second metal from the soil in above-ground tissue.

According to a third aspect of the invention there is provided a method for recovering nickel from nickel-containing soil, comprising: elevating the pH of the soil; cultivating at least one nickel-hyperaccumulator plant in the soil under conditions such that at least 0.1% of the above-ground tissue of the at least one plant, on a dry weight basis, is nickel; harvesting the at least one plant; and recovering nickel from the harvested plant.

According to a fourth aspect of the invention there is provided a method for selectively increasing the amount of at least one first metal and at least one second metal recovered from metal-containing soil, comprising: lowering the pH of the soil; cultivating at least one metal-hyperaccumulator plant in the soil under conditions sufficient to permit the at least one plant to accumulate the at least one metal from the soil Sin above-ground tissue; thereafter elevating the pH of the soil; and cultivating the at least one metal-hyperaccumulator plant in the soil under conditions sufficient to permit the at least one plant to accumulate the at least one second metal from the soil in above-ground tissue.

According to a fifth aspect of the invention there is provided a method for selectively increasing the amount of at least one metal recovered from metal-containing 25 soil, comprising: lowering the pH of the soil; and cultivating at least one metal-hyperaccumulator plant in the soil under conditions sufficient to permit the at least one plant to accumulate the at least one metal from the soil in above-ground tissue, wherein the at least one plant is selected from the group consisting of Cyanotis longifolia, Bulbostylis mucronata, Combretum decandrum; Crassula alba, Crassula vaginata, Crassula argyrophylla, Clethra barbinervis, Geissois intermedia, Geissois magnifica, Geissois montana, Geissois trifoliate, Geissois racemosa, Psychotria douarrei, Rinorea bengalensis, Pearsonia metallifera, Dichapetalum gelonioides ssp. tuberculatum and amanicum, Blepharis acuminata, Justicia lanstyakii, Lophostachys villosa, Phidiasia lindavii, Ruellia geminiflora, Adiantum sp., Rhus wildii, [I:\DAYLI B\LIBH]0362 .doc:UG Chromo/aena sp. cf Meyeri, Dicoma niccolifera, Gochnatia crassifolia, Gochnatia recurva, Koanophy/lon grandiceps, Koanophy//on prinodes, Leucanthemopsis a/pina, Pen taca/ia Sen ecio paupercu/us, Shafera p/a typhy/la, So/ida go hispida, Heliotropium sp., Bornmue//era, Cardamine resedifo/ia, Coch/earia aucheri, Goch/earia sempervivium, Peltaria emarginata, Buxus, Campanula scheuchzeri Arenaria, Minuartia laricifo/ia, Minuartia verna, Garcinia bakeriana, Garcinia polyneura, Garcinia revo/ute, Garcinia ruscijfo/ia, Merremia xanthophy//a, Pancheria eng/eriana, Shorea tenuiramu/soa, Argophyl/um grunowii, Argophy//um laxum, Ba/oghia sp., Bonania, Cleidion vie//ardii, Cnidosco/us sp. cf. bahian us, Euphorbia, Gymnanthes recurva, Leucocroton, Phy//anthus, Sapium erythrospermum, Savia, Anthy//is sp., Trifo/ium pa//escens, Casearia si/vana, Xy/osma, Luzu/a /utea, Wa/sura monophy//a, Myristica /auriJo/ia, Mosiera araneosa, Mosiera ekmanii, Mosiera x miraflorensis, Mosiera ophiticola, Psidium araneosum, Psidium havanense, Brackenridgea pa/us tris and ssp. foxworthyi and kje//bergii, Ouratea nitida, Ouratea striata, Chionanthus domingensis, Oncotheca balansae, Trisetum distichophy//um, Ranuncu/us g/acia/is, Ariadne ssp. shaferi and moaensis, Mitracarpus sp., Phy//ome/ia coronata, Psych otria c/em entis, Psychotria costivenia, Psychotria douarrei, Psychotria g/omerata, Psychotria osseana, Psychotria vanhermanii, Ronde/etia, P/anchone//a oxyedra, Saxifraga, Esterhazya sp., Linaria a/pina, Tetra/ix brachyp eta/us, Tetralix crista/ensis, Tetra/ix jaucoensis, Tetra/ix moaensis, Tetra/ix nipensis, Trichospermum kje//bergii, Turnera subnuda, Ve//ozia sp., Agatea deplanchei, Rinorea javanica, Acer pseudop/atanus, Minuartia vernai, Pc/yea rpaea synandrai, Cistus incanus ssp. creticus, Armeria maritime var. ha//en, Agrostis sto/onifera, Agrostis tenuis, Arrhenatherum e/atius, Festuca ovina, Rumex acetosa, Vio/a ca/amimaria, Pandiaka meta//orum, Ce/osia trigyna, Anisopappus chinensis, Anisopappus davyi, Gutenbergia pubescens, Mi//otia myosotidifo/iab, Vernonia petersii, Minuartia verna ssp. hercynicia, Si/ene coba/tico/a, Comme/ina zigzag, Cyanotis longiJo/ia, /pomoea a/pin a, Ascopepis meta//orum, Bu/bosty/is cuprico/a, Bu/bosty/is pseudoperennis, Monadenium cuprico/a, Phy//ant bus wi//iamioides, Crota/aria coba/tico/a, Vigna do/cm itica, G/adiolus gregarious, Aeo//anthus subacau/is var. /inearis, Aeo//anthus homb/ei, Aeo//anthus saxati/is, Aeo//anthus subacau/is var. ericoides and var.

/inearis, Becium grandiflorum var. vanderystii, Haumaniast rum homb/ei, Haumaniastrum robertii, Haumaniastrum rosu/atum, Hibiscus rhodanthus, Abies ba/samea Eragrostis racemosa, Rend/ia a/tera, Sporobo/us con goensis, A ctin iopteris sp., A/ectra sessi/iflora var. senega/ensis, Buchnera henriquesii, Crepidorhopa/on tenuisa, Crepidorhopa/on perennisa, Sopubia mann ii, Sopubia meta//orum, Sopubia neptun ii, (IADAYLI BULBH]0362 I doc.UG Striga hermontheca, Triumfetta dekindtiana, Triumfetta digitata, Triumfetta welwitschii var. descampii, Xerophyta retinervis var equisetoides, Alyxia rubricaulls, Maytenus bureaviania, Maytenus pancheriana, Maytenus sebertiana, Garcinia amplexicaulis, Eugenia clusioides, Beaupreopsis paniculata, Macadamia angustifolia, Macadamia neurophylla, Haplopappus fremontii, Machaeranthera glabriuscula, Machaeranthera ramosa, Machaeranthera venusta, Stanleya pinnata, Stanleya bipinnata, Atriplex confertifolia, Lecythis ollaria, Acacia cana, Astragalus bisulcatus, Astragalus osterhoutii Astragalus pattersonii, Astragalus pectinatus, Astragalus racemosus, Neptunia amplexicaulis, Morinda reticulata and Castilleja chromosa.

According to a sixth aspect of the invention there is provided a method for selectively increasing the amount of at least one metal recovered from metal-containing soil, comprising: lowering the pH of the soil; and cultivating at least one metal-hyperaccumulator plant in the soil under conditions sufficient to permit the at least one plant to accumulate the at least one metal from the soil in above-ground tissue, wherein the at least one plant is selected from the group consisting of Leucocroton sp., Phyllanthus sp. and Psychotria sp.

According to a seventh aspect of the invention there is provided a method for decontaminating or phytomining metal-containing soil, comprising cultivating at least one metal-hyperaccumulator plant in the soil under conditions sufficient to permit the at least one plant to accumulate the at least one metal from the soil in above-ground tissue in an amount exceeding the concentration of the at least one metal in the soil by a factor of at S*least 2, wherein the at least one plant is selected from the group consisting of Cyanotis longifolia, Bulbostylis mucronata, Combretum decandrum; Crassula alba, Crassula 25 vaginata, Crassula argyrophylla, Clethra barbinervis, Geissois intermedia, Geissois magnifica, Geissois montana, Geissois trifoliate, Geissois racemosa, Psychotria douarrei, Rinorea bengalensis, Pearsonia metallifera, Dichapetalum gelonioides ssp. tuberculatum and amanicum, Blepharis acuminata, Justicia lanstyakii, Lophostachys villosa, Phidiasia lindavii, Ruellia geminiflora, Adiantum sp., Rhus wildii, Chromolaena sp. cf Meyeri, Dicoma niccolifera, Gochnatia crassifolia, Gochnatia recurva, Koanophyllon grandiceps, Koanophyllon prinodes, Leucanthemopsis alpina, Pentacalia Senecio pauperculus, Shafera platyphylla, Solidago hispida, Heliotropium sp., Bornmuellera, Cardamine resedifolia, Cochlearia aucheri, Cochlearia sempervivium, Peltaria emarginata, Buxus, Campanula scheuchzeri Arenaria, Minuartia laricifolia, Minuartia verna, Garcinia bakeriana, Garcinia polyneura, Garcinia revolute, Garcinia ruscifolia, Merremia [I;\DAYLIB\LIBH)03621.docLJG xanthophylla, Pancheria engleriana, Shorea ten uiramulsoa, Argophyllum grunowi, Argophyllum laxum, Baloghia sp., Bonania, Cleidion viellardii, Cnidoscolus sp. cf.

bahianus, Euphorbia, Gymnanthes recurva, Leucocroton, Phyllanthus, Sapium erythrospermum, Savia, Anthyllis sp., Trifolium pallescens, Casearia silvana, Xylosma, Luzula lutea, Walsura monophylla, Myristica laurifolia, Mosiera araneosa, Mosiera ekman ii, Mosiera x miraflorensis, Mosiera ophiticola, Psidium araneosum, Psidium havanense, Brackenridgea palustris and ssp. foxworthyi and kellbergii, Ouratea nitida, Ouratea striata, Chionanthus domingensis, Oncotheca balansae, Trisetum distichophyllum, Ranunculus glacialis, Ariadne ssp. shaferi and moaensis, Mitracarpus sp., Phyllomelia coronata, Psychotria clementis, Psychotria costivenia, Psychotria douarrei, Psychotria glomerata, Psychotria osseana, Psychotria vanherman ii, Rondeletia, Planchonella oxyedra, Saxi/raga, Esterhazya sp., Linaria alpina, Tetralix brachypetalus, Tetralix cristalensis, Tetralix jaucoensis, Tetralix moaensis, Tetralix nipensis, Trichospermum kjellbergii, Turnera subnuda, Vellozia sp., Agatea deplanchei, Rinorea lavanica, Acer pseudoplatan us, Minuartia vernai, Polycarpaea synandrai, Cistus incanus ssp. creticus, Armeria maritime var. halleri, Agrostis stolonifera, Agrostis ten uis, Arrhenatherum elatius, Festuca ovina, Rumex acetosa, Viola calaminaria, Pandiaka metallorum, Celosia trigyna, A nisopappus chinensis, A nisopappus davyi, Gutenbergia pubescens, Millotia myosotidifoliab, Vernonia petersii, Minuartia verna ssp.

hercynicia, Silene cobalticola, Commelina zigzag, Cyanotis Iongifolia, lpomoea alpina, Ascopepis metallorum, Bulbostylis cupricola, Bulbostylis pseudoperennis, Monadenium cupricola, Phyllanthus williamicides, Crotalaria cobalticola, Vigna dolomitica, Gladiolus gregarious, Aeollanthus subacaulis var. linearis, Aeollanthus homblei, Aeollanthus saxatilis, Aeollanthus subacaulis var. ericoides and var. linearis, Becium grandiflorum 25 var. vanderystii, Haumaniastrum homblei, Haumaniastrum robe rtii, Haumaniastrum rosulatum, Hibiscus rhodanthus, Abies balsamea Eragrostis racemosa, Rendlia altera, Sporobolus congoensis, Actiniopteris sp., Alectra sessiliflora var. senegalensis, Buchnera henriquesii, Crepidorhopalon tenuisa, Crepidorhopalon perennisa, Sopubia mannii, Sopubia metallorum, Sopubia neptunii, Striga hermontheca, Triumfetta dekindtiana, Triumfetta digit ata, Triumfetta welwitschii var. descampii, Xerophyta retinervis var equisetoides, A lyxia rubricaulls, Maytenus bureaviania, Maytenus pan cheriana, Maytenus sebertiana, Garcinia amplexicaulis, Eugenia clusioides, Beaupreopsis paniculata, Macadamia angustifolia, Macadamia neurophylla, Haplopappus fremontii, Machaeranthera glabriuscula, Machaeranthera ramosa, Machaeranthera venusta, Stanleya pinnata, Stanleya bipinnata, A triplex confertifolia, Lecythis ollaria, Acacia [I \DAYLIB\LIBHj03621.doc:UG cana, Astragalus bisulcatus, Astragalus osterhoutii Astragalus pattersonii, Astragalus pectinatus, Astragalus racemosus, Neptunia amplexicaulis, Morinda reticulata and Castilleja chromosa.

According to an eighth aspect of the invention there is provided a method for decontaminating or phytomining metal-containing soil, comprising cultivating at least one metal-hyperaccumulator plant in the soil under conditions sufficient to permit the at least one plant to accumulate the at least one metal from the soil in above-ground tissue in an amount exceeding the concentration of the at least one metal in the soil by a factor of at least 2, wherein the at least one plant is selected from the group consisting of Leucocroton o1 sp., Phyllanthus sp. and Psychotria sp.

Also disclosed herein are methods for increasing nickel uptake by plants used in phytomining and phytoextraction by elevating the soil pH. Nickel is ultimately recovered from plant tissues at economically acceptable levels without further contaminating the nickel-containing site.

Also disclosed herein are methods for lowering the pH in soils prior or subsequent to nickel recovery to collect, for example, cobalt or any other metal present in the metalladen soil.

~In a particular embodiment disclosed herein, Alyssum plants are cultivated under favourable pH conditions to selectively accumulate certain metals relative to other metals.

20 Also disclosed herein is a method for selectively increasing the amount of at least one metal recovered from metal-containing soil comprising: elevating or lowering the pH of the soil; cultivating at least one metal-hyperaccumulator plant in the soil under conditions sufficient to permit said at least one plant to accumulate at least one metal 25 from the soil in above-ground tissue; elevating the pH of the soil if the pH was lowered in step or lowering the pH of the soil if the pH was elevated in step and cultivating the at least one metal-hyperaccumulator plant in the soil under conditions sufficient to permit said at least one plant to accumulate at least one second metal from the soil in above-ground tissue.

Also disclosed herein is a method for recovering cobalt from cobalt-containing soil comprising: lowering the pH of the soil; [I:\DAYLIB\LIBH]03621doc UG cultivating at least one cobalt-hyperaccumulator plant in the soil under conditions such that at least 0.1% of the above-ground tissue of said at least one plant, on a dry weight basis, is cobalt; harvesting said at least one plant; and recovering cobalt from said harvested plant.

Also disclosed herein is a method for the identification of new hyperaccumulating species of Alyssum whereby collected plants are screened by comparing nickel-uptake by the plants to nickel-uptake by the benchmark nickel-hyperaccumulator A. murale 103.

These new metal-hyperaccumulating species, cultivated on nickel-containing soil, accumulate nickel in above-ground tissue at a concentration of 1.55% or greater by weight based on the gross dry weight of the tissue.

The disclosure further relates to seeds of the Alyssum plant species.

The disclosure further relates to pollen of the Alyssum plant species.

The disclosure further relates to plants that have all the physiological and morphological characteristics of the Alyssum plant species.

The disclosure further relates to propagation material of the Alyssum plant species.

Also disclosed herein is a method for decontaminating metal-containing soil, comprising cultivating at least one hyperaccumulator plant in metal-containing soil, 9* whereby the concentration of metal in the above-ground plant tissue of the at least one hyperaccumulator plant exceeds the concentration of metal in the soil by a factor of at least 2.

Detailed Description of Preferred Embodiments In the present invention, it was discovered that certain metals can be selectively recovered from metal-rich soil using phytoextraction or phytomining techniques 25 employing plants classified as hyperaccumulators of metals. By cultivating selected plants on metal-containing soil, the metals absorbed by the roots can be translocated to above-ground tissues, such as the stems, leaves, flowers, and other leaf and stem tissues.

This feature facilitates the recovery of the metal extracted from the soil. Metal concentrations can be as high as about 5.0% in above-ground plant tissues, when leaves are included, which renders the metal recovery very economical. However, recovering metal in concentrations of less [I:\DAYLI B\LIBH]03621.doc:UG WO 00/28093 PCT/US99/26443 -6than about such as about 1.0% or 0.1% remains useful.

For example, a recovery of about 1.0% or more offers economic return for decontaminating polluted soil and for phytomining. A recovery of about 0.1% to about 1.0% of cobalt is sufficient to decontaminate polluted soil at a low cost, and a recovery of even less than about 0.1% of some metals can still effectively decontaminate polluted soils.

The invention further relates to a method for selectively increasing the amount of at least one metal recovered from metal-containing soil comprising: elevating or lowering the pH of the soil; cultivating at least one metal-hyperaccumulator plant in the soil under conditions sufficient to permit said at least one plant to accumulate at least one metal from the soil in above-ground tissue; elevating the pH of the soil if the pH was lowered in step or lowering the pH of the soil if the pH was elevated in step and cultivating the at least one metal-hyperaccumulator plant in the soil under conditions sufficient to permit said at least one plant to accumulate at least one second metal from the soil in above-ground tissue.

The invention further relates to a method for recovering nickel from nickel-containing soil comprising: elevating the pH of the soil; cultivating at least one nickel-hyperaccumulator plant in the soil under conditions such that at least 0. 1 of the above-ground tissue of said at least one plant, on a dry weight basis, is nickel; harvesting said at least one plant; and recovering nickel from said harvested plant.

The invention further relates to a method for recovering cobalt from cobalt-containing soil comprising: lowering the pH of the soil; WO 00/28093 PCT/US99/26443 -7cultivating at least one cobalt-hyperaccumulator plant in the soil under conditions such that at least 0.1% of the above-ground tissue of said at least one plant, on a dry weight basis, is cobalt; harvesting said at least one plant; and recovering cobalt from said harvested plant.

The invention further relates to the identification of new hyperaccumulating species of Alyssum whereby collected plants are screened by comparing nickel-uptake by the plants to nickel-uptake by the bench-mark nickelhyperaccumulator A. murale 103. These new metal-hyperaccumulating species, cultivated on nickel-containing soil, accumulate nickel in above-ground tissue at a concentration of 1.55% or greater by weight based on the gross dry weight of the tissue.

The invention further relates to seeds of the Alyssum plant species.

The invention further relates to pollen of the Alyssum plant species.

The invention further relates to plants that have all the physiological and morphological characteristics of the Alyssum plant species.

The invention further relates to propagation material of the Alyssum plant species.

The invention further relates to a method for decontaminating metalcontaining soil, comprising cultivating at least one hyperaccumulator plant in metal-containing soil, whereby the concentration of metal in the above-ground plant tissue of the at least one hyperaccumulator plant exceeds the concentration of metal in the soil by a factor of at least 2, preferably by a factor of 2, 3 or 4.

In a preferred aspect of the invention, nickel is selectively accumulated by growing one or more nickel-hyperaccumulating plants in metal-rich, nickelrich, soil and elevating the pH of the soil. The pH of the soil may be elevated before, during or after the plants are cultivated. Preferably, the pH is elevated prior to plant cultivation. Thus, the invention relates to the surprising discovery that raising the pH of the metal-rich soil favors nickel accumulation in plant tissue over other metals. The soil pH can then be lowered to selectively WO 00/28093 PCT/US99/26443 -8accumulate, in the plant tissue, other metals such as cobalt. The preferred pH will depend, inter alia, upon the particular metal and the soil. For example, the preferred pH for nickel extraction ranges between about 6.3 and about 7.0 when the soil is a serpentine soil or when the soil contains high iron oxide levels. The most preferred pH ranges from about 6.3 to about 6.7. However, when the iron oxide level is low, a more alkaline pH may be used.

Cobalt extraction is also affected by the soil chemistry. For example, the most preferred pH for cobalt extraction is about 5.5 when aluminum and/or manganese are present in the soil. For metal extraction in general, the preferred pH ranges between about 5.5 and about Soil pH can be raised and lowered with bases and acids. Such bases and acids may be either naturally occurring or synthetic. To raise the pH, bases such as limestone (calcitic (CaCO 3 or dolomitic (CaMgCO 3 lime (CaO), hydrated lime (Ca(OH) 2 industrial, municipal or agricultural alkaline by-products that contain any of the above bases or a limestone equivalent, or the like can be used.

The phrase "limestone equivalent" is intended to encompass bases that have the same alkalinity as limestone. To lower the pH, acids such as organic and inorganic acids can be used. Examples of such organic and inorganic acids include acetic acid, aqueous hydrogen chloride, aqueous sulfuric acid, sulfur, ammonium, urea-containing fertilizers, nitric acid, sulfide minerals, including, but not limited to, pyrite, and the like.

The amount of base or acid to add depends upon the existing pH of the soil and the soil chemistry. Methods used to determine the amount include, but are not limited to, adding acid or a base, such as CaCO 3 to the soil sample and measuring the resulting pH, then drawing a pH response curve to extrapolate the amount needed to obtain the desired pH.

After cultivation, the hyperaccumulator plant is harvested in a conventional fashion, by cutting the plant at soil level. The harvested materials are then left to dry in the field in the manner in which hay is dried.

Alternatively, the harvested materials are dried in much the same fashion that WO 00/28093 PCT/US99/26443 -9alfalfa is dried, so as to remove most of the water present in the plant tissue by forced heated air drying. After drying, the plant tissue is collected by normal agricultural practices of hay-making, incinerated and reduced to an ash with or without energy recovery. Alternatively, the dried plant material may be hydrolyzed with concentrated acid to produce sugars and the metals recovered according to U.S. Patent Nos. 5,407,817, 5,571,703 and 5,779,164. The sugars may then be fermented to produce ethanol.

The resulting dried plant material may alternatively be further treated by known roasting, sintering or smelting methods which allow the metals in the ash or ore to be recovered according to conventional metal refining methods such as acid dissolution and electrowinning.

Conventional smelting, roasting and sintering temperatures from about 260'C to about 1 000C are sufficient to combust the dried plant material to oxidize and vaporize the organic material present and to prevent dioxin accumulation during incineration. The preferred temperature is sufficient to remove the organic carbon to free the ash. The most preferred temperature is about 1000C. The process leaves a residue of the accumulated metal with few contaminants known to interfere with metal refining. Further, it is expected that the concentration of other components in the ash will be much lower than with conventional mined ore concentrates. For example, serpentine laterite ores generally contain over 10,000 ppm Fe whereas a biomass obtained using phytomining techniques only contains about 100-500 ppm (0.01 0.05%) Fe.

By definition, nickel-hyperaccumulating plants accumulate at least about 1000 mg of nickel per 1 kg dry weight of plant tissue (obtained from a plant grown in soil where the plant naturally occurs). Similarly, cobalthyperaccumulating plants are defined as plants that accumulate at least about 1000 mg of cobalt per 1 kg dry weight of plant tissue (obtained from a plant grown in soil where the plant naturally occurs). However, zinc- and manganesehyperaccumulators are defined as plants that accumulate at least about 10,000 mg of zinc and manganese, respectively, per 1 kg dry weight of plant tissue (obtained WO 00/28093 PCT/US99/26443 from a plant grown in soil where the plant naturally occurs). Finally, cadmiumhyperaccumulators are defined as plants that accumulate at least about 100 mg cadmium per 1 kg dry weight of plant tissue (obtained from a plant grown in soil where the plant naturally occurs).

By screening a wide variety of plants, those of the Alyssum genus (Brassicaceae family) have been identified as hyperaccumulators of nickel. These plants also naturally accumulate cobalt and may accumulate metals such as Zn, Mn and Cd, and metals from the platinum and palladium families including Pd, Rh, Ru, Pt, Ir, Os and Re.

More specifically, plants which naturally concentrate nickel in above-ground tissues and generally exhibit an enhanced uptake of cobalt and other metals include members of the section Odontarrhena of the genus Alyssum.

The metals accumulate in nickel-hyperaccumulatingAlyssum plant species when the plants are grown in contaminated soils. Some 48 taxa within the section Odontarrhena of the genus Alyssum are known to be hyperaccumulators of nickel.

These include the following species: A. akamasicum, A. alpestre, A. anatolicum, A. callichroum, A. cassium, A. chondrogynum, A. cilicicum, A. condensatum, A.

constellatum, A. crenulatum, A. cypricum, A. davisianum, A. discolor, A.

dubertretii, A. eriophyllum, A. euboeum, A. floribundum, A. giosnanum, A. hubermorathii, A. janchenii, A. markgrafii, A. masmenaeum, A. obovatum, A.

oxycarpum, A. penjwinensis, A. pinifolium, A. pterocarpum, A. robertianum, A.

samariferum, A. singarense, A. smolikanum, A. syriacum, A. trapeziforme, A.

troodii, A. virgatum, A. murale, A. pintodasilvae (also known as A. serpyllifolium var. lusitanicum), A. serpyllifolium, A. malacitanum (also known as A.

serpyllifolium var. malacitanum), A. l esbiacum, A. fallacinum, A. argenteum, A.

bertolonii, A. tenium, A. heldreichii, A. corsicum, A. pterocarpum and A. caricum as well as newly discovered species such as A. corsicum G16.A. murale G69 and A. murale G82. These species were deposited on November 6, 1998, under the provisions of the Budapest Treaty at the American Type Culture Collection, WO 00/28093 PCT/US99/26443 -11- 10801 University Blvd., Manassas, VA 20110-2209, and assigned ATCC nos.

203436, 203437 and 203438, respectively.

Species of Alyssum that naturally accumulate nickel in amounts of up to greater than any known Alyssum hyperaccumulator have been isolated.

Species A. murale G49, A. murale G54, A. murale G69 and A. murale G82 isolated in Greece and species A. corsicum G16 isolated in Turkey all accumulate nickel in amounts greater than the known species A. murale 103 which accumulates nickel such that nickel makes up 1.14% by dry weight of a plant shoot from a test field of serpentine soil. The new hyperaccumulators accumulate nickel in amounts such that 1.55-1.60% by dry weight of the shoot is nickel. The results of nickel accumulation of these five new accumulators relative to the benchmark accumulator A. murale 103 is shown in Example 4.

About 250 other plant taxa, including those of tropical origin, have been shown to accumulate quantities of nickel and other metals. However, many of these plants do not exceed about 10,000 mg of metal per kg of plant tissue dry weight. Other metal-accumulating plants includes species of the genus Cyanotis such as Cyanotis longifolia; species of the genus Bulbostylis such as Bulbostylis mucronata; species of the genus Combretum such as Combretum decandrum; species of the genus Crassula such as C. alba, C. vaginata and C. argyrophylla; species of the genus Clethra such as Clethra barbinervis; plants from the Cunoniaceae family such as species of the genus Geissois including G.

intermedia, G. magnifica, G. montana, G. pruinosa, G. trifoliata and G.

racemosa; species of the genus Argophyllum; members of Brassicaceae family such as species of the genus Thlaspi such as Thlaspi caerulescens, Thlaspi montanum var. montanum and Thlaspi montanum var. siskiyouense; species of the genus Serpentine such as Serpentine polygaloides; species of the genus Sebertia such as Sebertia acuminata; species of the genus Hybanthus such as Hybanthus floribundas; species of the genus Psychotria such as Psychotria douarrei; species of the genus Rinorea such as Rinorea bengalensis; species of the genus Pearsonia such as Pearsonia metallifera; species of the genus Sebertia WO 00/28093 PTU9164 PCT/US99/26443 -12such as Sebertia acuminata; and species of the following genera: Homaliwn, Myristica, Trichospermum, Planchonella and Peltaria. Additional plants include, but are not limited to, Streptanthus polygaloides, Berkheya coddii, Phyllanthus palawanensis, Dichapetalum gelonicides ssp. tuberculatum and Stackhousia tryonii.

Additional metal hyperaccumulators are listed below:

ACANTHACEAE

Blepharis acuminata, Justicia lansi'yakii, Lophostachys villosa, Phidiasia lindavii, Ruellia geminiflora

ADIANTACEAE

Adiantum sp.

ANACARDIACEAE

Rhus wild ii

ASTERACEAE

Berkheya coddii, Chromo/aena sp. cf. meyeri, Dicoma niccolifera, Gochnatia crassifolia, G. recurva, Koanophyllon grandiceps, K prinodes, Leucanthemopsis alpina, Pentacalia, Senecio Senecio paupercul us, Shafera platyphylla, Solidago hispida

BORAGINACEAE

Heliotropium sp.

BRASSICACEAE

Bornmuellera, Cardamine resedifolia, Cochlearia aucheri, C sempervivum, Peltaria emarginata, Streptanthus polygalo ides

BUXACEAE

Buxus

CAMPANULACEAE

Campanula scheuchzeri, Arenaria, Minuartia laricifolia, M verna

CLUSIACEAE

Garcinia bakeriana, G. polyneura, Q. revoluta, G. ruscifolia CON VOL VULACEAE Merremia xanthophylla

CUNONIACEAE

WO 00/28093 WO 0028093PCT/US99/26443 -13- Pancheria engleriana

DICHAPETALACEAB

Dichapetalum gelonicides and ssp. tuberculatum and ssp. andamanicum

DIPTEROCARPACEAE

Shorea tenuiramulosa

ESCALLONIACEAE

Argophyllum grunowii, A. laxum

EUPHORBIACEAE

Baloghia sp., Bonania, Cleidion viellardii, Cnidoscolus sp. cf. bahianus, Euphorbia, Gymnanthes recurva, Leucocroton, Phyllanthus, Sapium erythrospermum, Savia

FABACEAE

Anthyllis sp., Pearsonia metallifera, Trifolium pallescens

FLACOURTIACEAE

Casearia silvana, Homalium, Xylosma

JUTNCACEAE

Luzula lutea

MELIACEAE

Walsura monophylla

MYRISTICACEAE

Myristica laurifolia

MYRTACEAE

Mosiera araneosa, M ekmanii, M x miraflorensis, M ophiticola, Psidium araneosum, P. havanense

OCHNACEAF

Brackenridgea pal ust'ris and ssp.foxworthyi and ssp. kjellbergii, Ouratea nitida, 0. striata

OLEACEAE

Chionanthus domingensis

ONCOTHECACEAE

Oncotheca balansae WO 00/28093 PTU9/64 PCT/US99/26443 -14-

POACEAE

Trisetum distichophyllum

RANUNCULACEAB

Ranunculus glacialis

RUBIACEAE

Ariadne shaferi ssp. shaferi and ssp. moaensis, Mitracarpus sp., Phyllomelia coronata, Psychotria clementis, P. costivenia, P. douarrei, P. glomerata, P.

osseana, P. vanhermanfi, Rondeletia

SAPOTACEAE

Planchonella oxyedra, Sebertia acuminata

SAXIFRAGACEAE

Saxifraga

SCROPHULARJACEAE

Eslerhazya sp. and Linaria alpina

STACKHOUSIACEAE

Stackhousia tryonii

TILIACEAE

Tetralix brachypetalus, T cristalensis, T jaucoensis, T moaensis, T nipensis, Trichospermum kellbergii

TURNERACEAB

Turnera subnuda

VELLOZIACEAE

Vellozia sp.

VIOLACEAB

Agalea deplanchei, Hybanthus, Rinorea bengalensis, R. javanica, Rinorea sp.

ACERACEAE

Acer pseudoplai'anus

BRASSICACEAB

Cardaminopsis hal/er, Thiaspi avalanum, T brachypetalum, T caerulescens, T ochroleucum. T rotundifolium subsp. cepaeifolium, T praecox, T stenopterum, T tatrense

CARYOPHYLLACEAB

WO 00/28093 WO 0028093PCT/US99/26443 -Is- Minuaruia verna, Polycarpaea synandra

CISTACEAE

Cistus incanus ssp. creticus

DICHAPETALACEAE

Dichapetalum ge/on joides

PLUMBAGINACEAE

Armeria maritima var. ha//eri

POACEAE

Agrostis stolonifera, A. tenuis, Arrhenatherum elatius, Festuca ovina

POLYGONACEAE

Rumex acetosa

VIOLACEAB

Viola ca/aminaria

AMARANTHACEAE

Pandiaka metallorum, Ce/osia trigyna

ASTERACEAB

Anisopappus chinensis, A davyi, Gutenbergia pubescens, Mi//otia myosotidifo/iab, Vernonia petersii

CARYOPHYLLACEAE

Minuartia verna ssp. hercynica and Silene cobalticola

COMMELINACEAE

Comme/ina zigzag and Cyanotis ion gifolia CON VOL VULACEAE Ipomoea alpina

CRASSULACEAE

Crassula a/ba and C vaginata

CYPERACEAE

Asco/epis meta/lorum, Bulbosty/is cupricola, B. pseudoperennis

EUPHORBIACEAE

Monadenium cupricola and Phy//anthus wi//iamioides WO 00/28093 WO 0028093PCT/US99/26443 -16-

FABACAEAE

Crotalaria cobalticola and Vigna dolomitica

IRIDACEAE

Gladiolus gregarius

LAMIACEAE

Aeollanthus subacaulis var. linearis, A. homblei, A. saxatilis, A. subacaulis var.

ericoides and var. linearis, Becium grandifiorum var. vanderystii, Haumaniastrum homblei, H katangense, H robert ii, H. rosulatum

MALVACEAE

Hibiscus rhodanthus

PINACEAB

Abies balsamea

POACEAE

Era grostis racemosa, Rendlia altera, Sporobolus con goensis

PTERIDACEAE

A ctiniopteris sp.

SCROPHULARIACEAE

Alectra sessiliflora var. senegalensis, Buchnera henriquesii, Crepidorhopalon tenuisa, C perennisa, Sopubia mannii, S. metallorum, S. neptunii, Striga hermontheca

TILIACEAE

Triumfetta dekindtiana, T digita, T' welwitschii var. descampii

VELLOZIACEAE

Xerophyta retinervis var. equisetoides APOC YNACEAE Alyxia rubricaulis

CELASTRACEAE

Maytenus bureaviana, M pancheriana, M sebertiana

CLUSIACEAE

Garcinia amplexicaulis

MYRTACEAE

Eugenia clusioides WO 00/28093 PCT/US99/26443 -17-

PROTEACEAE

Beaupreopsis paniculata, Macadamia angustifolia, M. neurophylla

ASTERACEAE

Haplopappus fremontii, Machaeranthera glabriuscula, M ramosa, M venusta

BRASSICACEAE

Stanleya pinnata, S. bipinnata

CHENOPODIACEAE

Atriplex confertifolia

LECYTHIDACEAE

Lecythis ollaria

LEGUMINOSAE

Acacia cana, Astragalus bisulcatus, A. osterhoutii, A. pattersonii, A. pectinatus, A. racemosus, Neptunia amplexicaulis

RUBIACEAE

Morinda reticulata

SCROPHULARIACEAE

Castilleja chromosa The metals accumulated include nickel, cobalt, barium, gold, beryllium, mercury, molybdenum, copper, arsenic, selenium, antimony, manganese, silver, thallium, tin, lead, rubidium, chromium, cerium, vanadium, cesium, uranium, plutonium, strontium, yttrium, technetium, iridium, ruthenium, palladium, rhodium, platinum, osmium, rhenium, zinc and cadmium.

Metal sequestration can be improved by optimizing soil calcium concentration, using ammonium-containing or ammonium-generating fertilizers rather than other nitrate-containing fertilizers, and by applying chelating agents to the soil in which the hyperaccumulator plants are grown.

Alyssum species which hyperaccumulate metals such as nickel and cobalt evolved in nickel-rich ultramafic and serpentine soils which have low soil calcium and a low Ca:Mg ratio. It is now known that the presence of extremely low and extremely high calcium concentrations in soil inhibits nickel 18 hyperaccumulation by Alyssum. See PCT/US97/15109 (WO 98/08991). Acceptable calcium concentrations in soil range from about 0.128 mM to about 5.0 mM. In terms of percentages, an acceptable calcium concentration in soil ranges from about 2% to about 80% of the exchangeable cations. A preferable range is from about to about 80% of the exchangeable cations. The most preferred range is from about 30% to about 70% of the exchangeable cations. Such ranges can be achieved, if necessary, by adding calcium-containing agents to the soil such as limestone. In addition, gypsum could be added to the soil to raise the exchangeable calcium of the soil to benefit nickel accumulation.

The presence of intermediate concentrations of calcium, between about 0.128 mM and about 5.0 mM, increases nickel uptake whereas calcium values of about 0.128 mM and below, or about 5 mM and above, decrease nickel uptake. Combined with an exchangeable Ca:Mg ratio of between about 0.16 and about 0.40, much lower than recommended, an additional increase in nickel concentration in plant tissues is observed. By "exchangeable Ca:Mg ratio" is intended the ratio of extractable calcium and magnesium in the soil.

Although hyperaccumulators such as Alyssum have developed the ability to hyperaccumulate metals in above-ground tissues, fertilizer supportive of growth, particularly in polluted soil, can be used as an additive to increase 20 hyperaccumulation. Ammonium fertilizers localize acidification adjacent to the root which aids hyperaccumulation of various metals such as Ni, Zn, Cd, Co, etc.

The use of ammonium fertilizersper se is well-known, and acceptable fertilizers and protocols can be readily determined with no more than routine experimentation, by those of ordinary skill in the art. Other additives include, but 0 *0* 25 are not limited to, nutrients such as phosphate which helps to maximize the yield of nickel, for example.

Another possible additive to the contaminated soil is a metal chelating agent. Metal chelates are commonly used in agriculture and occur naturally in living cells. The addition of chelating agents, such as nitrolotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), WO 00/28093 PCT/US99/26443 -19ethyleneglycol-bis-(p-aminoethylether-N, N-tetraacetic acid) or any of a variety of amino-acetic acids known to those of ordinary skill in the art as chelating agents, to the soil to be phytomined or phytoextracted improves the movement of soil metals to root surfaces for uptake and translocation into above-ground tissues. Preferred chelating agents are NTA or EDTA. Typically, chelating agents will be added at a concentration ranging from about 0.5 to about 10 millimoles per kg soil. As with the use of fertilizers, the optimum concentration of chelating agents can be readily determined with no more than routine experimentation.

Chelating compounds which chelate nickel in the presence of high soil levels of Fe, Mg and Ca selectively increase nickel uptake by hyperaccumulator plants.

The following examples are illustrative, but not limiting, of the methods of the present invention. Other suitable modifications and adaptations of the variety of conditions normally encountered which are obvious to those skilled in the art are within the spirit and scope of the present invention.

Examples Example 1 A. murale 103 plants were grown in sets of two for 120 days in 19 pot-sets (4 L) of contaminated or serpentine soils (Mg-nitrate was leached out) without acidification, the first pot in a set, and with acidification, the second pot in a set.

Water was maintained near field capacity by daily watering with deionized water.

The plants were cultivated at a temperature of about 28 0 C during the day and about 20 0 C at night. The soils were acidified using nitric acid and the pH was raised using powdered reagent-grade CaCO 3 The soils included serpentine soils rich in nickel (containing from about 100 to about 5000 ppm nickel) obtained from southwest Oregon (soils 3-19), nickel-refinery contaminated Welland loam from Port Colbome, Ontario (soil 1) and nickel-refinery contaminated Quarry muck from Port Colborne, Ontario (soil Fertilizers containing, inter alia, nickel, potassium, sulfur and phosphorous, were added to optimize plant growth.

WO 00/28093 PCT/US99/26443 Table 1 shows the results of the experiment in contaminated soil.

Table 1 Soil TRT Final Yield Ni Co Mn Zn Cu Fe pH g shoot mg/kg dry matter/ pot S 2 5.16 27.4 9150 119 82.4 117 15.0 58 1 6 4.96 22.7 4220 84.7 145.6 180 19.5 64 2 2 6.04 40.9 4570 5.9 20.9 99.0 4.0 68 2 6 5.40 28.8 2150 7.1 63.0 142 6.5 82 3 2 6.26 21.5 6370 19.9 68.8 61.5 3.5 160 3 6 5.38 19.7 6480 308 680 65.9 5.5 260 4 2 5.61 19.6 12400 56.5 181 88.0 4.0 332 4 6 5.21 15.6 8560 377 140 135 5.0 345 2 5.88 24.0 1860 6.0 53.0 252 3.2 137 6 5.32 21.1 1220 9.8 153 379 3.5 121 6 2 6.03 24.5 4580 14.6 84.2 61.2 5.2 183 6 6 5.42 27.2 5040 58.5 227 70.3 5.5 195 7 2 5.54 23.3 5750 36.3 134 83.7 5.0 250 7 6 5.28 23.2 4870 86.8 272 77.9 5.5 274 8 2 5.77 21.1 9630 28.8 130 52.6 4.0 223 8 6 5.21 17.5 7180 94.0 291 74.9 4.8 221 9 2 6.12 22.1 9770 38.7 122 69.6 4.8 240 9 6 5.62 22.5 9100 196 532 69.7 5.2 273 2 6.25 20.0 12900 31.2 109 79.3 2.5 318 6 5.76 19.3 11500 182 774 93.5 3.2 412 11 2 5.72 32.8 8460 37.3 148 75.5 5.0 266 1 1 6 5.35 24.3 6010 136 339 93.6 4.8 230 12 2 6.54 20.3 8070 29.0 84.4 74.0 3.5 222 12 6 5.78 18.4 8240 86.0 186 66.5 3.2 178 13 2 6.34 18.8 11000 16.2 39.1 51.8 2.2 186 WO 00/28093 PCT/US99/26443 Soil TRT Final Yield Ni Co Mn Zn Cu Fe pH g shoot mg/kg dry matter/ pot 13 6 5.87 19.6 9970 36.0 103 56.6 2.8 181 14 2 5.68 21.3 9150 67.0 331 65.8 4.8 278 14 6 4.84 13.3 5820 313 957 86.0 4.8 567 2 6.04 19.4 7620 30.5 142 69.8 4.8 365 6 5.94 23.7 6110 463 820 88.6 4.8 220 16 2 6.07 21.0 3090 47.4 128 89.1 6.8 172 16 6 5.41 18.2 3560 225 563 105 8.0 267 17 2 6.02 20.6 9080 37.5 124 114 3.8 256 17 6 5.63 23.9 7940 262 973 127 4.2 252 18 2 5.99 19.4 11600 35.3 127 68.5 3.0 440 18 6 5.53 15.4 9500 204 908 116 4.2 548 19 2 5.59 21.8 436 19.1 259 92.4 7.8 190 19 6 5.11 19.5 584 72.4 929 112 8.8 156 "TRT" treatment. In treatment 2, the soil pH was not adjusted. In treatment 6, the soil pH was acidified.

As illustrated in Table 1, the plants grown on soils of less acidic pH generally accumulated far greater amounts of nickel than the plants grown on more acidic soils. In addition, plants taking up larger amounts of nickel on less acidic soils accumulated smaller amounts of other metals such as cobalt, manganese and zinc which are commonly found in lower concentrations in shoots after soil pH is raised.

Example 2 To validate the above example and to obtain optimization, Alyssum plants were grown on nickel-refinery contaminated Welland loam (soil wherein the pH was elevated by applying limestone (Table The plants were also grown on WO 00/28093 PTU9/64 PCT/US99/26443 -22nickel-refinery contaminated Quarry muck (soil 2) and serpentine soils (soils 3- 11) (Table The same cultivation conditions recited in Example I were used in Example 2.

Table 2 Effect of phosphate, pH and Ca:Mg variation on geometric mean shoot yield and micronutrient composition of two Alyssum species grown on nickel-refinery contaminated Welland loam (soil 1) for 120 days.

Soil TRT Yield Ni Co Mn Zn g/kg mg/kg mg/kg mg/kg 1 6.68 b* 7.61 a 127 a 23.7 e 157 fg Phosphate Series: 1 3 7.82 ab 5.94 bc 118ab 72.8 c 209 ab 12 9.78 ab 5.49 cd 109 bcd 59.3 d 170 def 1 4 8.71 ab 6.40 b 114 a-d 66.7 cd 178 c-f 8.03 ab 5.97 bc 98.8 d 60.8 cd 169 def pH Series: 1 6 8.14 ab 3.93 e 132 a 177 a 217 a 1 7 7.46 ab 4.93 d 119 ab 99.8 b 183 b-e 1 2 9.78 ab 5.49 cd 109 bcd 59.3 d 18 10.4 a 8.47 a 101 cd 19.1lf 142 g Ca:Mg Series: 19 9.22 ab 6.10 be 119ab 67.3 cd 168 ef 1 2 9.78 ab 5.49 cd 109 bed 59.3 d 170 def 1 10 7.80 A 5.55 cd 117 abc 64.7 cd 198 abc I1 8.72 ab 5.85 be 120ab 69.8 cd 195 a-d *a-g indicate means followed by the same letter are not significantly different at the P 0.05 level according to the Duncan-Walker K-ratio t-test.

"TRT" treatment I WO 00/28093 PTU9/64 PCT/US99/26443 -23- Table 3 Effect of soil treatments on soil pH and micronutrient composition of Alyssum murale and Alyssum corsicum grown on nickel-refinery contaminated Welland loam (soil nickel-refinery contaminated Quarry muck (soil 2) and serpentine soils (soils 3-1 1) for 120 days.

Soil TRT Final CU Zn CO Ni Mn Fe ___mg/kg mg/kg mg/kg g/kg mg/kg mg/kg 1 5.47 11.0 156 136 8.13 39.2 167.6 Phosphaite Series (hosphate added to the soil in kg/ha b" the addition of Phosphate-containinp fertilizer): 0 3 0 p 5.23 15.0 179 99.1 7.58 56.2 49.6 2 l~o p 5.18 16.0 131 102 7.34 59.7 50.1 4 250 P 5.24 14.5 133 82.2 7.37 56.8 56.4 500 P 5.13 14.5 129 73.8 6.50 53.1 50.8 iHSeries (olwsacidified usingj nitric acid for an MLo vH") 6 Lo pH 4.99 19.2 192 91.0 4.16 129 53.1 7 MLo pH 5.18 16.8 160 104 5.77 81.2 64.0 2 As is pH 5.18 16.0 131 102 7.34 59.7 50.1 8 Limed 5.57 10.1 102 71.1 9.28 19.9 Ca:Mg Ratio Series: D 9 1.0OCa 5.25 17.0 134 108 7.32 65.0 55.0 2 0OCa/Mg 5.18 16.0 131 102 7.34 59.7 50.1 2.5 Mg 5.13 17.4 152 90.4 6.75 48.9 53.0 11 5.0 Mg 5.04 16.2 149 87.6 5.71 54.8 67.1 "TRT" treatment 'MLo pH" medium-low pH The soil designations correspond to the soil designations in Example 1.

WO 00/28093 PCT/US99/26443 -24- The "pH series" experiments demonstrate that the application of limestone increases the uptake of nickel in Alyssum so that plant tissues accumulate an increased concentration of nickel.

Example 3 The results show an increase in the geometric mean of nickel uptake in plant tissue by liming Alyssum plants cultivated on nickel-refinery contaminated Quarry muck (soil 2) (Table 4) and on nickel-refinery contaminated Welloam loam (soil nickel-refinery contaminated Quarry muck (soil 2) and selected serpentine soils (soils 3-11) (Table 5) from Example 1. The cultivation .0 conditions were the same as those for Examples 1 and 2.

WO 00/28093 PTU9164 PCTILJS99/26"3 Table 4 Effects of soil treatments on the mean concentrations of elements in whole shoots and shoot yield of Alyssum murale and Alyssum corsicum grown on nickelrefinery contaminated Quarry muck (soil 2) for 60 days.

Shoot Shoot Shoot Shoot Soil TRT Treatment Yld Ni Co MN glkg mg/kg mg/kg 2 1 None 8.46 d* 7 3.33 abc 8.62 ab 27.9 be Phosphate Series: 2 3 0Op 10.78 a-d 3.24 bc 5.50 b 15.0 be 2 2 100OP 12.09 a 3.23 bc 5.75 ab 14.5Sbc 2 4 250 P 11.53 abc ,3.76 a 5.50 b 18.6 be 2 2 1500P 11. 86 ab _3.3 0abc 6.38 ab 27.7 be pH Series: 2 6 Lo pH 12.01lab 1.48 e 10.25 a 59.8 a 2 7 Med pH 9.44 bcd 2.12 d 6.12 ab 29.0Ob 2 2 As is pH 12.09 a 3.23 bc 5.75 ab 14.5 be 2 8 Limed 11.14 abc 3.2ab 5.88 ab 13.3 c Ca:Mg Series: 2 9 Ca 9.08 ed 3.42 abc 6.38 ab 16.3 bc 2 2 As is Ca 12.09 a 3.23 be 5.75 ab 14.5 be 2 10 Med Mg 11.66 ab 3.03 c 4.62 b 24.9 be 2 111 Hi Mg 9.98 a-d 2.94 c 5.2 5 b 23.3 be indicate means followed by the same letter are not significantly different at the P 0.05 level according to the Duncan-Walker K-ratio t-test.

"TRT" treatment WO 00/28093 PCT/US99/26443 -26- Table Effect of altering nickel-refinery contaminated Welland loam (soil nickelrefinery contaminated Quarry muck (soil 2) and serpentine soils (soils 3-11) by adding phosphate, adjusting the pH or adjusting the Ca:Mg ratio on soil pH, mean yield and micronutrient composition of shoots ofAlyssum species grown for 120 days (GM designates geometric mean).

Soil TRT Final pH GM- GM-Ni GM-Co GM-Mn GM-Zn GM-Fe GM- Yield I I Cu g/pot 9 mg/kg 1 None 6.34 20.2 5460 7.6 11.9 151 61 4.8 Phosphate Treatments (phosphate added to the soil in k2/ha bv the additinn nfnhoxnhate-cnntainino lertilizer 3 0 P 6.09 41.6 4400 5.8 16.5 152 56 4.2 2 100 P 6.05 42.7 4120 5.7 18.6 126 57 4 250 P 6.07 49.9 4120 5.1 21.4 143 57 4.8 500 P 5.98 46.4 3800 5.1 22.9 139 54 4.2 piH Treatments (soil was acidified using nitric acid for "Lo pH" and "Med-pH"): 6 LopH 5.44 32.2 2010 6.8 50.5 153 68 6.4 7 Med- 5.76 36.1 2700 4.5 21.0 143 60 4.8 pH 2 As is 6.05 42.7 4120 5.7 18.6 126 57 pH 8 Limed 6.20 40.5 4520 6.3 15.8 137 55 4.1 Ca:Mg Treatments: 9 0.0 Ca 6.13 38.6 4510 6.3 16.2 135 57 4.8 2 1.0 Ca 6.05 42.7 4120 5.7 18.6 126 56 2.5 Mg 5.98 39.0 4410 5.9 16.2 146 63 4.6 11 5.0 Mg 5.91 44.0 4260 5.8 18.3 158 58 46 "TRT" treatment The soil designations correspond to the soil designations in Example 1.

WO 00/28093 PCT/US99/26443 -27- Example 4 Novel Hyperaccumulators The concentration of elements in the shoots ofAlyssum species grown on a field of serpentine colluvial soil in Josephine County, Oregon, are shown in Table 6 below.

Table 6 Row Species Genotype Block Zn P Cu Co Ni Mn Fe Mg Ca K 139 A. corsicum 16 1 137 5.01 9 14 13400 53 53.8 5.52 20.943.3 483 A. corsicum 16 2 141 4.08 8 16 17500 32 755 5.99 24.244.3 129 A. murale 49 1 99 4.80 7 12 14100 41 397 3.98 32.1 41.4 325 A.murale 49 2 106 4.63 8 16 17100 46 455 5.32 31.741.7 135 A. murale 54 1 119 4.18 5 13 15600 53 927 4.02 25.844.5 143 A. murale 69 1 165 5.78 5 16 16700 53 380 4.52 17.338.8 553 A.murale 69 2 191 4.97 6 15 13400 45 616 5.66 25.46.16 The elements are present in mg/kg amounts.

Whole shoots or side branch samples containing stems and leaves were collected from pots or the field for each genotype, dried in forced air drying ovens and ground with a non-contaminating mill to less than about 0.1 mm. The ground samples were then placed in a borosilicate beaker and ashed at 480 0 C overnight.

Nitric acid was added to dissolve the resultant ash which was then heated until dry on a hot plate. Hydrochloric acid (3.0 M) was added and the beaker was refluxed for two hours to determine recovered nickel concentration.

Concentrations of nickel were measured by an inductively coupled argon plasma emission spectrometer. Low concentrations were measured by atomic absorption spectrometry.

This invention has been described in specific detail with regard to specific plants and methods for increasing metal, such as nickel, uptake via phytomining or phytoextraction. Except where necessary for operability, no limitation to these WO 00/28093 PCT/US99/26443 -28specific materials is intended nor should such a limitation be imposed on the claims appended hereto. From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions without undue experimentation. All patents, patent applications and publications cited herein are incorporated by reference in their entirety.

Claims (22)

1. A method for selectively increasing the amount of at least one metal recovered from metal-containing soil, comprising: elevating the pH- of the soil; and cultivating at least one metal-hyperaccumulator plant in the soil under conditions sufficient to permit the at least one plant to accumulate the at least one metal Irom the soil in above-ground tissue.

2. The method of claim 1, wherein the at least one metal is nickel.

3. The method of claim 1 or claim 2, wherein the pH of the soil is elevated by in adding to the soil at least one agent that results in an increase in the soil pl-H.

4. The method of claim 3, wherein the at least one agent is selected from the o 1 1 't consisting of limestone, dolomitic limestone, lime. hydrated lime. limestone o ciquivalents and mixtures thereof. The method of any one of claims 1-4. wherein the at least one plant is an i' /n'.stm plant.

6. The method of claim 5, wherein the Alyssum plant is selected from the group consisting of A. murale, A. pinlodasilvae, A. serpyllifidium, A. malacilamnm, A. Ie.shiacun, A. fallacinum, A. argen/eum, A. heriolonii, A. Ienium, A. heldreichii, A. cih.-rsicn], A. plerocarpum, A. caricum and mixtures thereof.

7. A method for selectively increasing the amount of at least one first metal and at least one second metal recovered from metal-containing soil. comprising: elevating the pH of the soil; cultivating at least one metal-hyperaccumulator plant in the soil under conditions sufficient to permit the at least one plant to accumulate the at least one first metal from the soil in above-ground tissue; thereafter lowering the pi- of the soil; and cultivating the at least one metal-hyperaccumulator plant in the soil under conditions sufficient to permit the at least one plant to accumulate the at least one second metal from the soil in above-ground tissue.

8. The method of claim 7, wherein the at least one first metal is nickel.

9. The method of claim 7 or claim 8, wherein the pH- of the soil is elevated by adding to the soil at least one agent that results in an increase in the soil pl-H. The method of claim 9, wherein the at least one agent is selected from the group consisting of limestone, dolomitic limestone, lime, hydrated lime, limestone 'lequivalents and mixtures thereof. I RAL 1z]0296.doc:aak I I. [he method of' any one of' claims 7-1 0, wherein the at least one plant is anl Ullvs.Suni plant. 12 The method of 'claimn I I wherein the Alyssum plant is selected f'rom the group coilsistilne of* A. muratle, A. lfinfodaisivaL, A serPYlli/olnun. A. ma7laccitanum, A. le'dhioculm. A. fallacinuni, A. w-genleum, A. 1,erlolonii, A. Ic'nium, A. lwldreichii, A. C01/N/C1111. A. PlerocaIlPin. A. carwculn and mixtures thereo l* I3 The method of any one of claimis 7-1I2, wherein thle at least one second metal selected frlom the OroupI COnlsistin(' of cobalt, palladium, rhodium~l. ruLtheium1.11, platinlum.l rid iLI_ ur.osmium,11 rhieniumI and Mixtures thereol'F 1) 14. The method of any one of'claimis 7-1 3, wherein the pi1I of thle soil is lowered b\ aiddingo to the soil at least one agent that results in a decrease of the soil p1-I. 1.Amethod for recovering nickel fr-om nickel-containing soil, comnprising elevating the p1-1 ol'the soil; Cultivating at least one ickel-hypIer-accumuIlator- plant In thle Soil under SI c0ndi tionIs SuIch that at least 0. of the above-c riounid tissue of theC at least one plant, onl a dix \Vei lht basis. is nickel; harvesting the at least one plant; and recovering nickel from the harvested plant. 1 6. Thie miethod of' claimn 15, wherein the soil has a calclim concentration of' betwveen about 20-80%/. 1 7. The method of' claim 15 or claim 16, wherein the nickel is recovered by irvinic and combuIStII0 the harvested plant to oxidize and v~aporize any organic material :present in thie harvested plant. 1 8. T'he method of any one ol' claimis 1 5-1 7, wherein the at least one plant is anl 13 'IlNl~lplant. 11). The method of' claimn 18, wherein the Al;ysum7 plant is selected From the group consistimne of' A. mulrale, A. I)h/lodaisilive, A serpYlli/oliumn, A. 177/uL'iltmnm, A. Ic yhjuc'iln A. /afll(L'iU~f, A. w-gL'flelm, A. hL'rlwonii, A. Il'nim, A. helireichil, A l PlerWOL'a'un. A. mariumim and Mixtures thereof'. 'The method of claimn 1 9. wherein the Alyssum plant Is selected from the groupI CoInsistinlu of'/I. mn'l-c' G69, A. 11uraclL G82, A. cor-sicum G 16 and mixtures thereof.

21. Tlhe miethod of any one of' claimns 1 5-20, wherein at least about 2.5% of thle above-ground tissue of thle at least one plant, onl a dry weight basis, is nickel.

22.'[he method of claimn 21, wherein at least about 3.0% of the above-ground t.~ISSueC of the at least one plant, onl a dry wveight basis, is nickel. I RALI 13zJ02969.doc:aak 31

23. The method of claim 22, wherein at least about 4.0% of the above-ground tissue of the at least one plant, on a dry weight basis, is nickel.

24. A method for selectively increasing the amount of at least one first metal and at [east one second metal recovered from metal-containing soil, comprising: lowering the pH of the soil; cultivating at least one metal-hyperaccumulator plant in the soil under conditions sufficient to permit the at least one plant to accumulate the at least one metal from the soil in above-ground tissue; thereafter elevating the pl-1 of the soil; and cultivating the at least one metal-hyperaccumulator plant in the soil under conditions sufficient to permit the at least one plant to accumulate the at least one second metal irom the soil in above-ground tissue.

25. The method of claim 24, wherein the at least one second metal is nickel.

26. The method of claim 24 or claim 25, wherein the pl-I of the soil is elevated by o* i; adding to the soil at least one agent that results in an increase in the soil pl-I.

27. The method of claim 26, wherein the at least one agent is selected from the group consisting of limestone, dolomitic limestone, lime, hydrated lime, limestone eq uivalents and mixtures thereof.

28. T he method of any one of claims 24-27, wherein the at least one plant is an A/vs.sv in plant.

29. The method of claim 28, wherein the Alyssum plant is selected from the group o:o consisting of A murale, A. pintodasilvae, A. serpyllifolium, A. malacilainum, A. lesbiacum, /l/a/lcinuim, A argenteum, A. herlolonii, A. lenium, A. hel/h'eichii, A. coirsicum, A. i/)e'ocLurp/ m. A. cariicum and mixtures thereof. 2 S30. The method of any one of claims 24-29, wherein the at least one first metal is selected from the group consisting of cobalt, palladium. rhodium, ruthenium, platinum, iridium. osmium, rhenium and mixtures thereof.

31. The method of any one of claims 24-30, wherein the pl-1 of the soil is lowered by adding to the soil at least one agent that results in a decrease of the soil pH.

32. A method for selectively increasing the amount of at least one metal recovered from metal-containing soil, comprising: lowering the pH of the soil; and cultivating at least one metal-hyperaccumulator plant in the soil under conditions sufficient to permit the at least one plant to accumulate the at least one metal IR:\I.IBzj02969.doc:aak frloml the soil In above-ground tissue,. wherein the at least one plant is selected from thle 2101I P Coils 5St o C vaiolis /lig~fOIlc, Bud/osty/is /uU/cl/ua Comnwi,, dcndui ollm/c uh, vCgilmal, Crassid(ic argyroph (./t'.hrci 1)arhilnervis, Ge 1550/S imc(rmflc' 1. Ge'issoix Inaigniica, Geissvois flilcinac, (iss~o is Irifi/iul', (eiL' /550/ rcenwsa, I .Nvc ho 01/i c0 (IriL' ?iforeci /enigalcns is. Pears oniu meiu//i/era, Dicha/)eta/ufn I..)C017iiC/L'N.VX). /U/117/IUfl?117 and inunicuni, 13/ephuris aumulinla, .Imi~xcia /uanslJyakii, I. vi/hosa, I'hidiasiai lindlavil, IRuellia genini/Ilor,, /1 dianiuni sp.. IRhus wi/dil, Ihromln~t/L'n cf. A4LyL'ri, Diconia niccolifera, Gochnutia cicissi/olia, G'ochnuiia r(cu Ouno,)hpl/on g/uiceps, hounophvllon /)rinoc/ces. Leui /I'no/)sis I 'i niculici imL'iL( ,nuu/)ercu/uts, S5ha/era p/al a v/u So/ic/cno /Uic Ile/io 1ropi n? sp., (.Clwdulnj,1L resedifihci, CoLh/ear/c, acheri, Coch/earia se'nperviviu,, I c/i ecl ma~crgincila, Bivis, ('cinipainc vc'heitchZeri A/1 encrict iAIn,,car',c /criclb/oia, ho/ic.'I 1'roc17(, 6Ciic i keriuna, Gciicinici po/ynerc. (Jcrcinic rL' 1o/ule, Garc/n/a iii.'ltci/olic, MelLr.renuct xanihophy//ai, PuncheracLnglrimma Sh()re Ienuiruni/soa, -phvlm I!(J lnoii, A',guhy//iin l cxuln, Bcdoghic sp., Boncuici, Cleion vie//airdi, 'i /o..co/u.s sp. cf. hahionits Lupholr/a, Ci))11711(f1711ex rL'Lur vc, LL'ucocrolon, I'/v//mlowits. ,Scl/hi/n ei-y//rosperfl ,ii, S civic,/ AihY//ix sp. Irifi4)linl (txccuic si/vanci Xv/osln, L uzu/a /uiiaea. Ii/sura lflnop/y//u. Alyristical I(aurilo/ja, A I )A/Lrie c~rclimvc Ailosiera ekInanjii, A'os.'erci x /nircu//orenA is. M'ox.'erc o/)hil iol/a, i/ill wcm eostiln, I/)5jijj/1 hmavcnLnse, Brackc'nric/geu /)cius frA an ixi fixworlIhyi and ()uraIea niiida, Ouralea stia/a, Chinnhits doliningensis, Oncotheca hc /cwcie T7 /.e/un dislichophy//urn, Rainuincit/us g/cii/s, A riudne ssp shaferi an d ihiOCIicNxixAliI'vraC(urpS sP., Phy//ome/ici coroncila, Psychotria L/leen/is, Psychoiria costi veil/c.. I'x~ycho/ric, doncarrei, PAJychotriai gloin7erala, I's 'Vcholria ossecina. Psychotria Ic/I11i0h170rif'i,/ /?onc/e/ei., Pi~/nchone//a oxyech'c, Scxii-cigc, ElX/LrhazJ' sp., Linariat )/pinc. 7 Circl/ix bra.chYpel'ci/us Teirci/ix cris/ci/ens is, Teovrcix /ac~uoensi, TeIra/ix /MIII 0is 7 enci/ix I/Pens/s, irichos/)ernmin kieI/hergii, 7 innera .%uhniua, Vll/ozia sp., el eplcmnch'i, Iinorea ]c'vcmicc., /1cer pswulO/u/IIcmnUv, Alinucirlia verna', I'oYII'lI)IL'cI yN)cllldr-Cli, (Vis/us inCCanux ssp LrL'//is, Arineric, 177r/ilie var. ha//er, 'I'iis So/onifera, 4,jros/is lenui. Arr/encalherumn '/cuis. Fes/utca ovinc, Runex cc/sa. iola/c(I clcllincirict, Parn/liakci ,ne/ci//orumn, Ce/asic, Irigynci. AnfisopaippUs IlilL'fs/. A 1 isopcppus dc.vyi, Guienbergic' /)ubes ceo'., 'i///otiai myoxolidifi/jab, Ilernonia p/elrx//, Ainucirlc, vernai ssp. hercynicia, Si/ene ccoha//icoci, (.Onl/inc, Zigzcag, Cyanotis /O/M'i/icit, /po.'noea ci/pinai, As copepis rnea//loruIM, BII/hosiy/iA cuprICOla, Bulbosty/is .(2~sii/ocenix Aonadeniuni cupricolai, Phj'//anthus wi/liainioides, Crota/arici I R: I [3z]02969.doc: aak cj( i/ic /olO/ 1 fligim( L(flm/tim, Gladlioluis gr-egarious /1eo//ailhui~.s Att)alaubA var. lineairis, 4 (1/ti ti/hts honh/e, A co/ll/its scixlilis, /leo//an/hus ysitihcauilis var. c'ricoides and v'ar. lilli t rs. BL'um tgrandifloriuu var. vandleriai. Hauinaniaisirutin honib/ci, hiltuunius 1un mbeLr/ ii, liinnaiin rosil/atufl, LHhbscts- r-hodaonhus, A /ies hc t/'.amc'a El i,'o.S.is~ lacLilosa, lRend/ia a//erai, S'por-o/o/us congtoensis, A c/iniop/eris sp., A I(IC it( ANAWiiIorac var. veneg(i/ensis, Buchner-a henriquesyii, Cirepidlorhop~a/on /Cflhis,, tc'pie/rotulon /)erenni.Sa, S'I)INUhi nuann1il, Sopithia me/a/loumn S~opuhiat nepiuni ii, Sl' ltl hIC'ttuon7hL'ca, Triutmklcq dekind/jana, 'Iriuni/a dhp/,ia/a. Triiini/ua iveliischii \iir. c/escolnpii. .VLrphV/ao r-e/mlervis \'ar L'qui se/oicl's, Alv xia( 1i ihlimutlls, Alay/enus 11Circ Plan. AlIyenis paincheriaCi7, MaY/enits sehei iana, (Jarlcifun (ifll/)/exicaulis, Fi tctit c/usioides, Beaupeopsis /)aficii/ct/a, A mcifhl anguis/i/o/ia, Alacadaii I/ h' tt0/)/iv/Io Haplopaipputs fr-emnonii, A4'chaie-n/herat g/a/i-ius Liti, AMachuL'rn/hera m;a.AltfiL' ielwercu ventis/a, San/evat pfinnaa, S/an/eya h ipinna/a., A iriplex Wluo/rtili//1. Leeji his ol/aiait, A cac'io cona, Asua~taius hisulcais, As/ragaus oslei-hou/ij I ii t/ (1/ci/el'SOnmi. As/ cus. pec/hma/its, /1 /rega/i hts raenosts, INep.lunict itti/)/exicuottis, Alor-inda r-e/lic/ait and Co:slille/a chromosci. 3.3. The method of claimn 32. wherein the at least one metal is selected from the "I-r1,1p conSIStine of cobalt, palladium, rhodiUml, ruthenium,. platinum, iridiumn, osmiumn, 111c1111,urn and mixtures thereof 3,4. The method of clm 32 or claim 33. wherein the pH- of the soil is lowered by 1LdInC11u to thle soil at least one agent that results in a decrease of the soil Ill1. 0*35. A\ method for selectively increasing thle amoun.1t of' at least one metal rCcovered fi-r mnetal-containing soil, comprising: lowering the pl-I of thle soil; and Cultivating at least one metal-hlyperaccumul1,1ator plant in the soil under c0ndition1s sLHIcient1 to permit the at least one plant to accumlllate the at least one metal iom the soil in above-(,round tissue. wherein thle at least one plant is selected From the ,11'0,11 consisting of Leiuo/on sp.. Phyllan/hus sp. and Ps )chol/ria sp.

36. A method 66r decontamninatig or- phytomining mectal-containing soil, U C0onpiIsi ngCultivating at least one mletal-hIyperaLccumulLlator- plant in the Soil uinder coniditionS Suff'icienlt to permit the at least one plant to accumulate the at least one metal frlomn thle soil in above-ground tissue in anl amount exceeding the concentration of the at cast one metal in thle soil by a factor of' at least 2. wherein the at least one plant is Icc eted from11 thle group consisting of C'yano/is Ion gi/olia, Bulhoswlis mucronaila, W1,rtuii ion ecaindriun: C.'raisY u/a ct/ha, CraSS11 u/a Vgina/. Irasmtaagy(h/a R\Li Bzj02969.doc:ank I 4 34 cwa/)i/) ifl'Ii, (ic!issois in/ernedCLiai, Ge lAA()is iaii'fiica. (JLiSSO.%i MOWC/(ifl, (IL'issois ii ifi)/ialL' C'eiss~ois /YiL'flosa, PsYc/o/riadiLoihiirei, Ri17O/'ca /)ngl'/1ti/L s i. Peroi miaii/Li. Dichapeta/un ge/aniide% s.sp. luhercultaum? and amanI(iCUi. B/ephairisv (aC11inulaa. *IiiA/icia lon.siyakii, Lop/uosacLyvs vilsta, IPhidiaisict linclavil, Rite//ia ,eiliflora. Adianium sp., Rhus ivii/dii, ('hromo/aena LIf. Aileyeri, Dicoina niccolifera, (;uchnaliah Lvassl/a/ja, Gochiia/ia /LLiiicur, Kaniophy/(), grandic'ps, KoanophY//on /1/il usIL'. LL'uC(an/hL'mopsis alpina, Pl~nacca/ia SenL'Lja /)aii/)Lcu/iis, .S'/a/eia plalyphy/la, io h.id~ca. 1khoL'i/i'/)iif sp., Borninue//era, Caircainine iL'AL'(i V)ia. COL'h/L'Cria uI1IChIu'i. a('Jija sL'ifpL'/viviuin, PeL'/aritt ecir-incila, Buxit. ('aipanulat scheuichze-i I urc'ia. Alinutaria lavrici/olia, Alimiar/ia vL'vna, Garciniat /akL'r tna. Gaicinia /)01)/7CI'I ti. Garciniai irevi'ofe, Garcinia ruscifo/ja, MevvL'inia xandiaph v/la, Pancheria hlerioi/Ci .ShorL't iitiraliilsoa, A rgaphy//uin gruna wii, A ';iop/ivlin laxuln, Ba/aghia S sp.V. BU/1(iia, C/c'iain vl'/ardii, C'nidascolus sp. cf. hahiainus, Euphor-hia Qvinnanihes .'uiui'a.LL'ucocvi)/on, Phv//an/hiA, Sapim erpyhrospwv.'nuin. avia. A n/hid/is sp., :006,, -i/oliiiini pc iIcsc'ens, (.'asLaria si/vanic. L11-1ula//LaI'asuaiifl)y/, .800:Al yr/A/Lac Iaurifi)/ia, Adasiera aranLosa. Alosiera c'kiiia11ii AlosiL'ra x 17iialorens is, Alosiea (phi/ico/a, IPsidiuni acineasitin, IPsidiuln havanense, Brackenridg'a /)a/ius/ris got.*: Lnd ss p. fimoxworhyi and k/e//bergii, Oura/ca ni/ida, ()nvalea s/via/a, C'hionanihus do/iinL4L'nsis, Onco/heca balansa, Tris c/ur /ivsiichaphyv//ui, Runcuuts glacia/is, 2) /1 v IiL'n ssp. .sia/i and nivuens is, AliacarIT"s sp., Phy//oine/ia coronaa, Psycho/nia 000*9 c/Lcn/ lis. Psycholria Los/ivenia, Psychollrio douairrci, P.ycho/via (1017l'tlat, Psycho/via oisSUI If' iii./c',L/o/ia vanhei nni, Ronde/fia IPanchac/ ovda, Sai'Cia, P vAeh-a sp.. Linaria alpina, 7'eIra/ix hivachypL/aus 7'e/rciix L'vis/a/ensis, Teira/ix A ucoc.' iis, Teira/ix inoaensis, ic/ralix nipensis, frichos/wrinun kje///,ergii, Turnera 2 A/sIIa. l/c//(ojIa Agatect dep/anchei, R inorca ]avanica, A cer psL'udop/a/anus, !A'inuaiv/ia i'evnai, PO/YcL'aPaea synandrai, ('is/us incanus ssp. L'i'LiL1i, /1 iU1Lia mlari/imle rif. ha/levi. A gras/is .Wo/ani/evau, A gras/is /L'nuis. A rrhenahL'um? eL'/aius, I'csi uc'a olina, Riincx ace/a.ci, Via/a ca/aininavia, Pandiaka lnc/a//lovum, l/sict Ivigyna, A1nisapappus cIiiiai'/A.Slapa/yusI dai'yi, (Ju/cn/cv ia /)ubescL's, A'i//a/ia in vaolii/fi/ial, IVernonia p/)c'sii. A'iniicii'/ia verne, ssp. hecL'nicia, .Si/L'necob~''a//iCa/e. ('annuc/ina 7 zi 71, CyIanotiS h mii dia. Ipoinaca alpinal, A scopL'/is mne/a//arum, Biilhos/yis cuii/'iL/, Biui/hasty/is /s )A itso Io/)Levil is, Alonaden iutn L'upri ola, Phy//an/hits wi//iai ia ides, C'roaa/ria coho// Wa/a, fl/igna do/am i/ica, Gladio/us gregaious, A ca//an/hus su/,acau/is var. /ineanis, A ca//an/hus hamh/ei, A co//an/hus saxa/i/is, Aeal/an/hus suhacaulis var. erica ides and var. 11incaviS. Beim grandif/arum var. v'anderys/ii, Haiunanica/vum hamblci, 1-aumianiats/rum '<A~r I R :\II fzj02969.doc:aak robel-Iii. HC111111(inia.Y11.11177 rosidalum, Hibiscus rhoclowhus, Abies bals-amea Eragrovis 1*41CC111()NU. Rendlia allera, Sl)orobohts congoensi.v. Actiniol)1cri.v sp.. .41cara sessiliflora 111'. SCIM"(f(I sis. Bitchwra henriquesii, Crel)idorholndon 1clinisa. CrelfidorhOJW11017 1)(TC1717i.S0. Sopubict Inannii, Sopubia ineiallornI17, SOI)uhia nclnuniii. Slr gct hermontheca, 'I'l-illinlella tIckindliana, TriuInkila tl i ilala, Triumlefla wehvilschii vai-. descutnj)ii, X(,1*o1)h.via relinervi.y vai, equiseloi(Jes, AlYxia rubricaulls. Allu.wenus hureaviania, :14a.vietnts pancheriuna, Maylenus seherlitina, Garcinia ainplexicaulis, Eugenia chtsioidc. Beauln-eol).vis 1)(iniculaia, Mucadamia angusl lolia. Macculalnia 1141plopappity fi-enlowii, 1111achaerawhera (4alwhiscula. Alhtchaerawhera ramosa. 11achoerawhera veni/sta, Sitinle-va pinnaia. Slonleju hil)i1,117ala. PI.i1)1Cx coqffi.f lblia, ollaria, Acacia cana, Asirc gahts bisulcums. AsIragalits osterholl1ii Asllvgalus pollersollii. I)eClinalits, Astragalus racemosus, Aleplittv (I an lplexica I dis, 0 Soo lJorindo rclicithact and Ccislilleia chrolnoso.

37. The method of' claim 35 oi- claini 36. whei-ein the Lit least one plant ICCLIIIRIIMCS the Lit least one nietal fi-oni the soil In above-1-01.1nd tISSUe in an aniount 0 see 0: :\cccclfflg the concenti*ation ol'the at least one metal In the soil bv a Cactoi- ol'at least 3.

38. The method of' claim 37. whei-ein the at least one 111allt aCCLII11LIIates the at k:ast one nietal 1rom the soil In above-grOLInd IISSLIC in an L111101.111t exceeding the Colicelill'ation of'tlic at least one metal in theLsoll by a I'actol- ol'at least 4. A method 661- decontaminatino oi- phytonlinino nietal-containing soil, C0111I)HS11111 CLIltivating at least one iiietal-li),pei-acCLIIIIUI ItOl- plant III the Soil under CollclitI011S SLIFficient to permit the at least one plant to accumulate the at least one metal I'l-oni the soil in above-gi-ound tiSSLIC 111 all 11110I.Int exceeding the concentration of the at Icist one iiictal in the soil by a I'actoi- of' Lit least 2. whei-ein [lie Lit least one plant is I'l-0111 the 1-1-01.11) C011SISting ol'Lezicocrolon sp.. PhvIlawhits sp. and Psycholria sp. Dated 8 November, 2001 United States Department of Agriculture University of Maryland University of Sheffield Patent Attorneys for the Applicants/Nominated Persons SPRUSON FERGUSON JR:\LII3zJ02969.doc:aak