AU1346100A - Recovering metals from soil - Google Patents

Description

WO 00/28093 PCTIUS99/26443 Recovering Metals from Soil Background of the Invention Statement as to Rights to Inventions Made Under 5 Federally-Sponsored Research and Development Part of the work performed during development of this invention utilized U.S. Government funds. The U.S. Government has certain rights in this invention. Field of the Invention 0 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 pH. 5 Related Art 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 0 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 engine-ering methods of soil remediation, such as soil removal and replacement, are too expensive to be practical (Cunningham et 5 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 5 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 0 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 ofthe platinum and palladium families such as palladium, rhodium, 5 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 0 (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 5 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 0 remediating polluted soils. Raskin et al. remove metals from metal-rich soil by WO 00/28093 PCT/US99/26443 -3 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 5 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 recognize that increasing the pH of soil decreases the ability of 10 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 optimizes the release of these metals for absorption by the roots and translocation to the above-ground tissues of the plant." However, 15 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 !0 Accordingly, this invention relates to improved systems for recovering metals by phytomining or phytoextracting soils rich in metals. The invention further relates to 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 5 contaminating the nickel-containing site. The invention further relates to lowering the pH in soils prior or subsequent to nickel recovery to collect, for example, cobalt or any other metal present in the metal-laden soil.

WO 00/28093 PCT/US99/26443 -4 In a particular aspect of the invention, Alyssum plants are cultivated under favorable pH conditions to selectively accumulate certain metals relative to other metals. The invention further relates to a method for selectively increasing the 5 amount of at least one metal recovered from metal-containing soil comprising: (a) elevating or lowering the pH of the soil; (b) 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; 10 (c) elevating the pH of the soil if the pH was lowered in step (a) or lowering the pH of the soil if the pH was elevated in step (a); and (d) 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. .5 The invention further relates to a method for recovering nickel from nickel-containing soil comprising: (a) elevating the pH of the soil; (b) 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 0 one plant, on a dry weight basis, is nickel; (c) harvesting said at least one plant; and (d) recovering nickel from said harvested plant. The invention further relates to a method for recovering cobalt from cobalt-containing soil comprising: 5 (a) lowering the pH of the soil; (b) 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; (c) harvesting said at least one plant; and 0 (d) recovering cobalt from said harvested plant.

WO 00/28093 PCTIUS99/26443 -5 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 nickel hyperaccumulator A. murale 103. These new metal-hyperaccumulating species, 5 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. 10 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 metal 15 containing 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. Detailed Description of the Preferred Embodiments 20 In the present invention, it was discovered that certain metals can be selectively recovered from metal-rich soil using phytoextraction or phytomining techniques 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, 5 flowers and other leaf and stem tissues. This feature facilitates 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 WO 00/28093 PCT/US99/26443 -6 than about 5.0%, such as about 4.0%, 3.0%, 2.5%, 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, 5 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: (a) elevating or lowering the pH of the soil; 10 (b) 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; (c) elevating the pH of the soil if the pH was lowered in step (a) or lowering the pH of the soil if the pH was elevated in step (a); and 15 (d) 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: (a) elevating the pH of the soil; (b) 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; (c) harvesting said at least one plant; and 5 (d) recovering nickel from said harvested plant. The invention further relates to a method for recovering cobalt from cobalt-containing soil comprising: (a) lowering the pH of the soil; WO 00/28093 PCT/US99/26443 -7 (b) 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; (c) harvesting said at least one plant; and 5 (d) 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 nickel hyperaccumulator A. murale 103. These new metal-hyperaccumulating species, 0 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. 5 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 metal 0 containing 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 5 growing one or more nickel-hyperaccumulating plants in metal-rich, e.g., nickel rich, 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 0 tissue over other metals. The soil pH can then be lowered to selectively WO 00/28093 PCTIUS99/26443 -8 accumulate, 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 5 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 10 pH ranges between about 5.5 and about 7.0. 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 15 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, i0 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 CaC03, to the soil sample and 5 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, i.e., 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. 0 Alternatively, the harvested materials are dried in much the same fashion that WO 00/28093 PCT/US99/26443 -9 alfalfa 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 5 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 10 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 1 000 0 C are sufficient to combust the dried plant material to oxidize and vaporize the organic material present and to prevent dioxin 15 accumulation during incineration. The preferred temperature is sufficient to remove the organic carbon to free the ash. The most preferred temperature is about 1000 C. 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 M0 conventional mined ore concentrates. For example, serpentine laterite ores generally contain over 10,000 ppm (1%) 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 5 grown in soil where the plant naturally occurs). Similarly, cobalt hyperaccumulating 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 manganese hyperaccumulators are defined as plants that accumulate at least about 10,000 mg 0 of zinc and manganese, respectively, per 1 kg dry weight of plant tissue (obtained WO 00/28093 PCT/US99/26443 -10 from a plant grown in soil where the plant naturally occurs). Finally, cadmium hyperaccumulators 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). 5 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. 10 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-hyperaccumulating Alyssum plant species when the plants are grown in contaminated soils. Some 48 taxa within the section 15 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. huber ?.0 morathii, 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. !5 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 Gi 6, 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 PCTIUS99/26443 -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 20% greater than any known Alyssum hyperaccumulator have been isolated. 5 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 10 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 15 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; 20 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. trifbliata and G. racemosa; species of the genus Argophyllum; members of Brassicaceae family such as species of the genus Thlaspi such as Thlaspi caerulescens, Thlaspi 5 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 0 the genus Pearsonia such as Pearsonia metallifera; species of the genus Sebertia WO 00/28093 PCT/US99/26443 -12 such as Sebertia acuminata; and species of the following genera: Homalium, Myristica, Trichospermum, Planchonella and Peltaria. Additional plants include, but are not limited to, Streptanthus polygaloides, Berkheya coddii, Phyllanthus palawanensis, Dichapetalum gelonioides ssp. tuberculatum and Stackhousia 5 tryonii. Additional metal hyperaccumulators are listed below: ACANTHACEAE Blepharis acuminata, Justicia lanstyakii, Lophostachys villosa, Phidiasia lindavii, Ruellia geminflora 10 ADIANTACEAE Adiantum sp. ANACARDIACEAE Rhus wildii ASTERACEAE 15 Berkheya coddii, Chromolaena sp. cf. meyeri, Dicoma niccolifera, Gochnatia crassifolia, G. recurva, Koanophyllon grandiceps, K prinodes, Leucanthemopsis alpina, Pentacalia, Senecio Senecio pauperculus, Shafera platyphylla, Solidago hispida BORAGINACEAE Z0 Heliotropium sp. BRASSICACEAE Bornmuellera, Cardamine resedifolia, Cochlearia aucheri, C. sempervivum, Peltaria emarginata, Streptanthus polygaloides Z5 BUXACEAE Buxus CAMPANULACEAE Campanula scheuchzeri, Arenaria, Minuartia laricifolia, M verna CLUSIACEAE W0 Garcinia bakeriana, G. polyneura, G. revoluta, G. ruscifolia CONVOLVULACEAE Merremia xanthophylla

CUNONIACEAE

WO 00/28093 PCT/US99/26443 -13 Pancheria engleriana DICHAPETALACEAE Dichapetalum gelonioides and ssp. tuberculatum and ssp. andamanicum DIPTEROCARPACEAE 5 Shorea tenuiramulosa ESCALLONIACEAE Argophyllum grunowii, A. laxum EUPHORBIACEAE Baloghia sp., Bonania, Cleidion viellardii, Cnidoscolus sp. cf. bahianus, 10 Euphorbia, Gymnanthes recurva, Leucocroton, Phyllanthus, Sapium erythrospermum, Savia FABACEAE Anthyllis sp., Pearsonia metallifera, Trifolium pallescens FLACOURTIACEAE 15 Casearia silvana, Homalium, Xylosma JUNCACEAE Luzula lutea MELIACEAE Walsura monophylla 20 MYRISTICACEAE Myristica laurifolia MYRTACEAE Mosiera araneosa, M ekmanii, M x miraflorensis, M ophiticola, Psidium araneosum, P. havanense 25 OCHNACEAE Brackenridgeapalustris and ssp.foxworthyi and ssp. kjellbergii, Ouratea nitida, 0. striata OLEACEAE Chionanthus domingensis 30 ONCOTHECACEAE Oncotheca balansae WO 00/28093 PCTIUS99/26443 -14 POACEAE Trisetum distichophyllum RANUNCULACEAE Ranunculus glacialis 5 RUBIACEAE Ariadne shaferi ssp. shaferi and ssp. moaensis, Mitracarpus sp., Phyllomelia coronata, Psychotria clementis, P. costivenia, P. douarrei, P. glomerata, P. osseana, P. vanhermanii, Rondeletia SAPOTACEAE 10 Planchonella oxyedra, Sebertia acuminata SAXIFRAGACEAE Saxifraga SCROPHULARIACEAE Esterhazya sp. and Linaria alpina 15 STACKHOUSIACEAE Stackhousia tryonii TILIACEAE Tetralix brachypetalus, T cristalensis, T jaucoensis, T moaensis, T nipensis, Trichospermum kjellbergii 20 TURNERACEAE Turnera subnuda VELLOZIACEAE Vellozia sp. VIOLACEAE 15 Agatea deplanchei, Hybanthus, Rinorea bengalensis, R. javanica, Rinorea sp. ACERACEAE Acer pseudoplatanus BRASSICACEAE 0 Cardaminopsis halleri, Thlaspi avalanum, T brachypetalum, T caerulescens, T. ochroleucum, T rotundifolium subsp. cepaeifolium, T. praecox, T stenopterum, T. tatrense

CARYOPHYLLACEAE

WO 00/28093 PCT/US99/26443 -15 Minuartia verna, Polycarpaea synandra CISTACEAE Cistus incanus ssp. creticus DICHAPETALACEAE 5 Dichapetalum gelonioides PLUMBAGINACEAE Armeria maritima var. halleri POACEAE Agrostis stolonfera, A. tenuis, Arrhenatherum elatius, Festuca ovina to POLYGONACEAE Rumex acetosa VIOLACEAE Viola calaminaria AMARANTHACEAE 5 Pandiaka metallorum, Celosia trigyna ASTERACEAE Anisopappus chinensis, A. davyi, Gutenbergia pubescens, Millotia myosotidifoliab, Vernonia petersii 0 CARYOPHYLLACEAE Minuartia verna ssp. hercynica and Silene cobalticola COMMELINACEAE Commelina zigzag and Cyanotis longifolia CONVOLVULACEAE 5 Ipomoea alpina CRASSULACEAE Crassula alba and C. vaginata CYPERACEAE Ascolepis metallorum, Bulbostylis cupricola, B. pseudoperennis 0 EUPHORBIACEAE Monadenium cupricola and Phyllanthus williamioides WO 00/28093 PCT/US99/26443 -16 FABACAEAE Crotalaria cobalticola and Vigna dolomitica IRIDACEAE Gladiolus gregarius 5 LAMIACEAE Aeollanthus subacaulis var. linearis, A. homblei, A. saxatilis, A. subacaulis var. ericoides and var. linearis, Becium grandiflorum var. vanderystii, Haumaniastrum homblei, H. katangense, H robertii, H. rosulatum 10 MALVACEAE Hibiscus rhodanthus PINACEAE Abies balsamea POACEAE 15 Eragrostis racemosa, Rendlia altera, Sporobolus congoensis PTERIDACEAE Actiniopteris sp. SCROPHULARIACEAE Alectra sessiliflora var. senegalensis, Buchnera henriquesii, Crepidorhopalon 20 tenuisa, C. perennisa, Sopubia mannii, S. metallorum, S. neptunii, Striga hermontheca TILIACEAE Triumfetta dekindtiana, T. digitata, T welwitschii var. descampii VELLOZIACEAE 25 Xerophyta retinervis var. equisetoides APOCYNACEAE Alyxia rubricaulis CELASTRACEAE Maytenus bureaviana, M pancheriana, M sebertiana 30 CLUSIACEAE Garcinia amplexicaulis MYRTACEAE Eugenia clusioides WO 00/28093 PCTIUS99/26443 -17 PROTEACEAE Beaupreopsis paniculata, Macadamia angustifolia, M neurophylla ASTERACEAE Haplopappusfremontii, Machaeranthera glabriuscula, M. ramosa, M venusta 5 BRASSICACEAE Stanleya pinnata, S. bipinnata CHENOPODIACEAE Atriplex confertifolia 10 LECYTHIDACEAE Lecythis ollaria LEGUMINOSAE Acacia cana, Astragalus bisulcatus, A. osterhoutii, A. pattersonii, A. pectinatus, A. racemosus, Neptunia amplexicaulis 15 RUBIACEAE Morinda reticulata SCROPHULARIACEAE Castilleja chromosa 0O 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. 5 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 0 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 WO 00/28093 PCT/US99/26443 -18 hyperaccumulation by Alyssum. See PCT/US97/15109. 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 10% 5 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. to The presence of intermediate concentrations of calcium, i.e., 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 .5 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 0 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 fertilizers per 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 5 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), 0 ethylenediaminetetraacetic acid (EDTA), WO 00/28093 PCT/US99/26443 -19 ethyleneglycol-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 5 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 10 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. 15 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. 20 Water was maintained near field capacity by daily watering with deionized water. The plants were cultivated at a temperature of about 28'C during the day and about 20'C at night. The soils were acidified using nitric acid and the pH was raised using powdered reagent-grade CaCO,. The soils included serpentine soils rich in nickel (containing from about 100 to about 5000 ppm nickel) obtained 25 from southwest Oregon (soils 3-19), nickel-refinery contaminated Welland loam from Port Colborne, Ontario (soil 1) and nickel-refinery contaminated Quarry muck from Port Colborne, Ontario (soil 2). Fertilizers containing, inter alia, nickel, potassium, sulfur and phosphorous, were added to optimize plant growth.

WO 00/28093 PCT/US99/26443 -20 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 1 2 5.16 27.4 9150 119 82.4 117 15.0 58 5 ] 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 10 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 5 2 5.88 24.0 1860 6.0 53.0 252 3.2 137 5 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 .5 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 0 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 10 2 6.25 20.0 12900 31.2 109 79.3 2.5 318 10 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 5 11 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 PCTIUS99/26443 -21 Soil TRT Final Yield Ni Co Mn Zn 1 Cu Fe pH I I 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 15 2 6.04 19.4 7620 30.5 142 69.8 4.8 365 5 15 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 .0 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, 5 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, 0 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 1), wherein the 5 pH was elevated by applying limestone (Table 2). The plants were also grown on WO 00/28093 PCT/US99/26443 -22 nickel-refinery contaminated Quarry muck (soil 2) and serpentine soils (soils 3 11) (Table 3). The same cultivation conditions recited in Example I were used in Example 2. Table 2 5 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/pot g/kg mg/kg mg/kg mg/kg 1 1 6.68 b* 7.61 a 127 a 23.7 e 157 fg 0 Phosphate Series: 1 3 7.82 ab 5.94 bc 118 ab 72.8 c 209 ab 1 2 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 1 5 8.03 ab 5.97 bc 98.8 d 60.8 cd 169 def 5 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 170 deg 1 8 10.4 a 8.47 a 101 cd 19.1 f 142 g 0 Ca:Mg Series: 1 9 9.22 ab 6.10 bc 119 ab 67.3 cd 168 ef 1 2 9.78 ab 5.49 cd 109 bcd 59.3 d 170 def 1 10 7.80 ab 5.55 cd 117 abc 64.7 cd 198 abc 1 11 8.72 ab 5.85 bc 120 ab 69.8 cd 195 a-d 5 *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 WO 00/28093 PCTIUS99/26443 -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 1), nickel-refinery contaminated Quarry muck (soil 2) and serpentine 5 soils (soils 3-11) for 120 days. Soil TRT Final Cu Zn Co Ni Mn Fe pH mg/kg mg/kg mg/kg g/kg mg/kg mg/kg 5.47 11.0 156 136 8.13 39.2 67.6 Phosphate Series (phosphate added to the soil in kg/ha by the addition of phosphate-containing fertilizer): 0 3 0 P 5.23 15.0 179 99.1 7.58 56.2 49.6 2 100 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 5 500 P 5.13 14.5 129 73.8 6.50 53.1 50.8 pH Series (soil was acidified using nitric acid for "Lo p1H" and "MLo pH"): 5 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 57.6 Ca:Mg Ratio Series: 0 9 1.0 Ca 5.25 17.0 134 108 7.32 65.0 55.0 2 0 Ca/Mg 5.18 16.0 131 102 7.34 59.7 50.1 10 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 5 "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 5 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 1), 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 PCT/US99/26443 -25 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 nickel refinery contaminated Quarry muck (soil 2) for 60 days. Shoot Shoot Shoot Shoot 5 Soil TRT Treatment Yld Ni Co MN g/pot g/kg mg/kg mg/kg 2 1 None 8.46 d* 3.33 abc 8.62 ab 27.9 bc Phosphate Series: 2 3 0 P 10.78 a-d 3.24 bc 5.50 b 15.0 bc 2 2 100 P 12.09 a 3.23 bc 5.75 ab 14.5 bc 0 2 4 250 P 11.53 abc 3.76 a 5.50 b 18.6 bc 2 2 500 P 11.86 ab 3.30 abc 6.38 ab 27.7 bc pH Series: 2 6 Lo pH 12.01 ab 1.48 e 10.25 a 59.8 a 2 7 Med pH 9.44 bcd 2.12 d 6.12 ab 29.0 b 5 2 2 As is pH 12.09 a 3.23 bc 5.75 ab 14.5 bc 2 8 Limed 11.14 abc 3.72 ab 5.88 ab 13.3 c Ca:Mz Series: 2 9 Ca 9.08 cd 3.42 abc 6.38 ab 16.3 bc 2 2 As is Ca 12.09 a 3.23 bc 5.75 ab 14.5 bc 0 2 10 Med Mg 11.66 ab 3.03 c 4.62 b 24.9 bc 2 11 Hi Mg 9.98 a-d 2.94 c 5.25 b 23.3 bc *a-e 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 5 Effect of altering nickel-refinery contaminated Welland loam (soil 1), nickel refinery 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 5 yield and micronutrient composition of shoots of Alyssum 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 Cu g/pot mg/kg I None 6.34 20.2 5460 7.6 11.9 151 61 4.8 Phosphate Treatments (phosphate added to the soil in kg/ha by the addition of phosphate-containing 0 fertilizer): 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 4.5 4 250 P 6.07 49.9 4120 5.1 21.4 143 57 4.8 5 500 P 5.98 46.4 3800 5.1 22.9 139 54 4.2 5 pH Treatments (soil was acidified usinR nitric acid for "Lo pH" and "Med-pH"): 6 Lo pH 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 4.5 pH 8 Limed 6.20 40.5 4520 6.3 15.8 137 55 4.1 0 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 4.5 10 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 4.6 5 "TRT" = treatment The soil designations correspond to the soil designations in Example 1.

WO 00/28093 PCTIUS99/26443 -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. 5 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 20.9 43.3 483 A. corsicum 16 2 141 4.08 8 16 17500 32 755 5.99 24.2 44.3 129 A. murale 49 1 99 4.80 7 12 14100 41 397 3.98 32.1 41.4 [0 325 A. murale 49 2 106 4.63 8 16 17100 46 455 5.32 31.7 41.7 135 A. murale 54 1 119 4.18 5 13 15600 53 927 4.02 25.8 44.5 143 A. murale 69 1 165 5.78 5 16 16700 53 380 4.52 17.3 38.8 553 A. murale 69 2 191 4.97 6 15 13400 45 616 5.66 25.4 6.16 The elements are present in mg/kg amounts. 5 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'C overnight. Nitric acid was added to dissolve the resultant ash which was then heated until 0 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. 5 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 -28 specific 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 5 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.

WO 00/28093 PCT/US99/26443 -29 What Is Claimed Is: 1. A method for selectively increasing the amount of at least one metal recovered from metal-containing soil comprising: (a) elevating the pH of the soil; and 5 (b) cultivating at least one metal-hyperaccumulator plant in the soil under conditions sufficient to permit said at least one plant to accumulate said at least one metal from the soil in above-ground tissue. 2. The method of claim 1, wherein said at least one metal is nickel. 3. The method of claim 1, wherein the pH of the soil is elevated by adding 0 to the soil at least one agent that results in an increase in the soil pH. 4. The method of claim 3, wherein the at least one agent that results in an increase in the soil pH is selected from the group consisting of limestone, dolomitic limestone, lime, hydrated lime, limestone equivalents and mixtures thereof. 5 5. The method of claim 1, further comprising after said cultivating: (c) lowering the pH of the soil; and (d) 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. 0 6. The method of claim 5, wherein the at least one second metal is selected from the group consisting of: cobalt, palladium, rhodium, ruthenium, platinum, iridium, osmium, rhenium and mixtures thereof.

WO 00/28093 PCT/US99/26443 -30 7. The method of claim 5, wherein the pH of the soil is lowered by adding to the soil at least one agent that results in a decrease of the soil pH. 8. The method of claim 1, wherein said at least one plant is an Alyssum plant. 5 9. The method of claim 8, wherein said Alyssum plant is selected from the group consisting of: A. murale, A. pintodasilvae, A. serpyllifolium, A. malacitanum, A. lesbiacum, A. fallacinum, A. argenteum, A. bertolonii, A. tenium, A. heldreichii, A. corsicum, A. pterocarpum, A. caricum and mixtures thereof. 10 10. A method for recovering nickel from nickel-containing soil comprising: (a) elevating the pH of the soil; (b) 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; l5 (c) harvesting said at least one plant; and (d) recovering nickel from said harvested plant. 11. The method of claim 10, wherein the soil has a calcium concentration of between about 20-80%. 12. The method of claim 10, wherein the nickel is recovered by drying and 0 combusting the harvested plant to oxidize and vaporize organic material present. 13. The method of claim 10, wherein said at least one plant is an Alyssum plant.

WO 00/28093 PCT/US99/26443 -31 14. The method of claim 13, wherein said Alyssum plant is selected from the group consisting of: A. murale, A. pintodasilvae, A. serpyllifolium, A. malacitanum, A. lesbiacum, A. fallacinum, A. argenteum, A. bertolonii, A. tenium, A. heldreichii, A. corsicum, A. pterocarpum, A. caricum and 5 mixtures thereof. 15. The method of claim 14, wherein said Alyssum plant is selected from the group consisting of: A. corsicum Gl 6, A. murale G69, A. murale G82 and mixtures thereof. 16. The method of claim 10, wherein at least about 2.5% of the above-ground 0 tissue of said at least one plant, on a dry weight basis, is nickel. 17. The method of claim 16, wherein at least about 3.0% of the above-ground tissue of said at least one plant, on a dry weight basis, is nickel. 18. The method of claim 17, wherein about 4.0% of the above-ground tissue of said at least one plant, on a dry weight basis, is nickel. 5 19. A method for recovering cobalt from cobalt-containing soil comprising: (a) lowering the pH of the soil; (b) 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; 0 (c) harvesting said at least one plant; and (d) recovering cobalt from said harvested plant. 20. The method of claim 19, wherein the soil has a calcium concentration of between about 20-80%.

Claims (23)

1. 21. The method of claim 20, wherein said at least one plant is an Alyssum plant.
2. 22. The method of claim 21, wherein said Alyssum plant is selected from the group consisting of: A. murale, A. pintodasilvae, A. serpyllifolium, A. 5 malacitanum, A. lesbiacum, A. fallacinum, A. argenteum, A. bertolonii, A. tenium, A. heldreichii, A. corsicum, A. pterocarpum, A. caricum and mixtures thereof.
3. 23. An isolated Alyssum plant species that accumulates nickel in quantities greater than that accumulated by A. murale 103. 0 24. An isolated Alyssum plant species cultivated on nickel-containing soil that accumulates nickel in above-ground tissue at a concentration of 1.55% or greater by weight based on the gross dry weight of said tissue.
4. 25. The isolated Alyssum plant species of claim 23 or 24, wherein the species is A. corsicum G]6. 5 26. The isolated Alyssum plant species of claim 23 or 24, wherein the species is A. murale G69.
5. 27. The isolated Alyssum plant species of claim 23 or 24, wherein the species is A. murale G82.
6. 28. Pollen of the plant of claim 23 or 24. D 29. A plant having all the physiological and morphological characteristics of the plant of claim 23 or 24. WO 00/28093 PCT/US99/26443 -33
7. 30. Propagation material of the plant of claim 23 or 24.
8. 31. An Alyssum corsicum G16 seed as deposited with the American Type Culture Collection under accession number 203436.
9. 32. An Alyssum murale G69 seed as deposited with the American Type 5 Culture Collection under accession number 203437.
10. 33. An Alyssum murale G82 seed as deposited with the American Type Culture Collection under accession number 203438.
11. 34. A plant produced by growing the seed of any one of claims 31-33.
12. 35. Pollen of the plant of claim 34. 0 36. A plant having all the physiological and morphological characteristics of the plant of claim 34.
13. 37. Propagation material of the plant of claim 34.
14. 38. A method for decontaminating metal-containing soil, comprising cultivating at least one hyperaccumulator plant in metal-containing soil, 5 whereby the concentration of metal in the above-ground plant tissue of said at least one hyperaccumulator plant exceeds the concentration of metal in said soil by a factor of at least 2.
15. 39. The method of claim 38, wherein the at least one hyperaccumulator plant exceeds the concentration of metal in said soil by a factor of 3. WO 00/28093 PCTIUS99/26443 -34
16. 40. The method of claim 39, wherein the at least one hyperaccumulator plant exceeds the concentration of metal in said soil by a factor of 4.
17. 41. A method for selectively increasing the amount of at least one metal recovered from metal-containing soil comprising: 5 (a) lowering the pH of the soil; and (b) cultivating at least one metal-hyperaccumulator plant in the soil under conditions sufficient to permit said at least one plant to accumulate said at least one metal from the soil in above-ground tissue.
18. 42. The method of claim 41, wherein said at least one metal is selected from o the group consisting of: cobalt, palladium, rhodium, ruthenium, platinum, iridium, osmium, rhenium and mixtures thereof.
19. 43. The method of claim 41, wherein the pH of the soil is lowered by adding to the soil at least one agent that results in a decrease of the soil pH.
20. 44. The method of claim 41, further comprising after said cultivating: 5 (c) raising the pH of the soil; and (d) 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.
21. 45. The method of claim 44, wherein the pH of the soil is elevated by adding 0 to the soil at least one agent that results in an increase in the soil pH.
22. 46. The method of claim 45, wherein the at least one agent that results in an increase in the soil pH is selected from the group consisting of limestone, dolomitic limestone, lime, hydrated lime, limestone equivalents and mixtures thereof. WO 00/28093 PCT/US99/26443 -35
23. 47. The method of claim 44, wherein the at least one second metal is nickel. PCTIUS99/26443 Applicant's or agent's file International applicatif be SignNi I reference number 1797.009PC05 INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL (PCT Rule 13bis) A. The indications made below relate to the microorganism referred to in the description on page _11_, line 2 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet 0 Name of depositary institution American Type Culture Collection (ATCC) Address of depositary institution (including postal code and country) 10801 University Boulevard Manassas, Virginia 20110-2209 United States of America Date of deposit November 6, 1998 Accession Number ATTC 203436 C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet 0 Alyssum corsicum G16 D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (ifthe indications are notfor all designated States) E. SEPARATE FURNISHING OF INDICATIONS (leave blank ifno applicable) The indications listed below will be submitted to the international Bureau later (specify the general nature of the indications, e.g., "Accession Number of Deposit") For receiving Office use only For International Bureau use only O This sheet was received with the international application 0 This sheet was received by the International Bureau on: Authorized officer Authorized officer Form PCT/RO/134 (July 1998) 009pc05.forml34(1) PCT/US99/26443 Applicant's or agent's file ntraialppctit . bki reference number 1797.009PC05 INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL (PCT Rule 13bis) A. The indications made below relate to the microorganism referred to in the description on page _11-, line 2 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet E Name of depositary institution American Type Culture Collection (ATCC) Address of depositary institution (including postal code and country) 10801 University Boulevard Manassas, Virginia 20110-2209 United States of America Date of deposit November 6, 1998 Accession Number ATTC 203437 C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet 0 Alyssum murale G69 D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are notfor all designated States) E. SEPARATE FURNISHING OF INDICATIONS (leave blank ifnot applicable) The indications listed below will be submitted to the international Bureau later (specify the general nature of the indications, e.g., 'Accession Number of Deposit") For receiving Office use only For International Bureau use only D This sheet was received with the international application -0 This sheet was received by the International Bureau on: Authorized officer Authorized officer Form PCT/RO/134 (July 1998) 009pc05.forml34(2) PCT/US99/26443 Applicant's or agent's file International application No. be aSSigneu reference number 1797.009PC05 INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL (PCT Rule 13bis) A. The indications made below relate to the microorganism referred to in the description on page _11_, line 2 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet 0 Name of depositary institution American Type Culture Collection (ATCC) Address of depositary institution (including postal code and country) 10801 University Boulevard Manassas, Virginia 20110-2209 United States of America Date of deposit November 6, 1998 Accession Number ATTC 203438 C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet 0 Alyssum murale G82 D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are notfor all designated States) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed below will be submitted to the international Bureau later (specify the general nature of the indications, e.g., "Accession Number of Deposit") For receiving Office use only For International Bureau use only o This sheet was received with the international application 13 This sheet was received by the International Bureau on: Authorized officer Authorized officer Form PCT/RO/t34 (July 1998) 009pc05.form1 34(3)