Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
  • B09C—RECLAMATION OF CONTAMINATED SOIL
  • B09C1/00—Reclamation of contaminated soil
  • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
  • B09C1/105—Reclamation of contaminated soil microbiologically, biologically or by using enzymes using fungi or plants
• • C—CHEMISTRY; METALLURGY
  • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
  • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
  • C02F3/00—Biological treatment of water, waste water, or sewage
  • C02F3/32—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae
  • C02F3/327—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae characterised by animals and plants
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
  • Y02W10/00—Technologies for wastewater treatment
  • Y02W10/10—Biological treatment of water, waste water, or sewage

Definitions

• the present invention provides a method for inducing plants to hyperaccumulate metals into their shoots.
• the invention therefore provides a novel and highly advantageous method for phytoremediation of metal-contaminated sites, as plant shoots can readily be harvested and removed from the site.
• the present invention concentrates metals in a readily disposable biomass to levels higher than the concentration of metal in the soil and thereby greatly reduces the weight of contaminated material that must be disposed.
• a cultivar is planted in a metal contaminated environment, the environment is manipulated so that availability of the metal in the environment to the plant is increased, the plant is allowed to take metal up into its roots, and the plant is then exposed to an inducing agent under conditions and for a time sufficient for the plant to hyperaccumulate metal in its shoots.
• Preferred plants for use in the present invention include members ofthe family Brassicaceae, and
• Preferred inducing agents include chelating agents, organic acids, soil acidifiers, herbicides, and high concentrations of heavy metals.
• the present invention provides an improved method for removing metal from an environment by cultivating a plant therein, in which the improvement comprises exposing the plant to an inducing agent under conditions and for a time sufficient for the plant to hyperaccumulate metal into its shoots to a levels higher than it would if it were not exposed to the inducing agent.
• the invention also provides a method for identifying agents that act to induce hyperaccumulation of metal into plant shoots.
• a plant is grown in a metal-contaminated environment, is exposed to a potential inducing agent, such as a chemical or physical stress, and is analyzed to determine the level of metal it accumulated into its shoots.
• a potential inducing agent such as a chemical or physical stress
• Desirable inducing agents according to the present invention are those that stimulate a plant to accumulate more metal after exposure to the agent than it does without such exposure.
• the plant is induce to accumulate at least twice as much metal in its shoots after exposure to the agent than it does without such exposure.
• Figure 1 is a bar graph showing the effects of EDTA on lead accumulation in roots and shoots of a Brassica juncea cultivar.
• Figure 2 is a bar graph showing the effects of acidification on lead accumulation in roots and shoots of a Brassica juncea cultivar after acidification to pH 3.5.
• Figure 3 is a bar graph showing the combined effects of EDTA and acidification on lead accumulation in roots and shoots of a Brassica juncea cultivar.
• Figure 4 is a bar graph showing the combined effects of EDTA, acidification, and an herbicide on lead accumulation in roots and shoots of a Brassica juncea cultivar.
• Figure 5 is a bar graph showing the combined effects of EDTA and an herbicide on metal accumulation in roots and shoots of a Brassica juncea cultivar; the data demonstrate hyperaccumulation of cadmium, copper, nickel, lead, and zinc.
• Figure 6 shows the induction of lead hyperaccumulation into shoots of a
• Brassica juncea cultivar after exposure to high levels of a heavy metal Brassica juncea cultivar after exposure to high levels of a heavy metal.
• Figure 7 is a bar graph showing the effects of certain soil amendments on uranium solubility in contaminated soil.
• Figure 8 is a bar graph showing the effects of certain soil amendments on soil uranium deso ⁇ tion and uranium hyperaccumulation in Brassica juncea shoots.
• Figure 9 is a bar graph showing the ability of citric acid to induce uranium hyperaccumulation into shoots of various B. juncea cultivars.
• Figure 10 shows the ability of citric acid to induce uranium hyperaccumulation in a variety of different plant species.
• Figure 1 1 shows the time-dependent kinetics of uranium accumulation by a B. jun after application of citric acid to uranium-contaminated soil.
• Figure 12 shows the synergistic effect of acidification and herbicide application in inducing uranium hyperaccumulation into plant shoots.
• Metal hyperaccumulation according to the present invention occurs when plants are induced by application of an "inducing agent" to accumulate high levels of metals in their shoots. As noted above, the prior art teaches that plants do not typically transport significant levels of metals into their shoots (see, for example, Cunningham et al.
• the present invention provides novel methods for increasing metal transport into plant shoots.
• the present invention identifies a variety of useful inducing agents that stimulate hyperaccumulation of metals in plant shoots.
• the present invention teaches that phytotoxic substances are useful inducing agents.
• phytotoxic substances induce metal hyperaccumulation by disrupting the plant metabolism in a way that overrides natural safety mechanisms that would otherwise operate to block transport of metal into shoots.
• our theory does not suggest that the induction of metal transport described herein is exclusive of continued uptake of metal into plant roots. That is, metal uptake into plant roots probably continues, and may even be enhanced, during the induction period.
• the negative effect on plant growth can be largely or almost totally avoided by delaying the application ofthe inducing agent until the plants have accumulated a desirable amount of biomass. Then, because once the stimulus is applied, transport of metal into shoots is quite rapid, the metal -containing shoots can be harvested without delay.
• selected plants are cultivated in an environment, typically soil, that is contaminated with metal. After a period of plant growth, plants are induced by exposure to one or more inducing agents to hyperaccumulate metals into their shoots.
• An "inducing agent”, according to the present invention, is any treatment that, when applied to a plant or the soil, induces the plant to accumulate more metal in its shoots than it would accumulate in the absence ofthe treatment. Preferably, the plant is induced to accumulate at least about twice as much metal in its shoots as it would in the absence ofthe treatment.
• a plant is considered to have "hyperaccumulated" a metal in its shoots when, in response to an inducing agent, it has i) achieved a metal concentration in its shoots ( ⁇ g metal/g dry weight shoot mass) that is higher than the concentration of metal in the soil (mg kg soil or mg/L solution); and/or ii) accumulated at least about 1000 ⁇ g of metal per gram dry weight of shoot mass.
• the plant has achieved a metal concentration that is at least about two-fold higher than the concentration in the soil, and/or has accumulated at least about 3000 ⁇ g of metal per gram dry weight of shoot mass. It will be appreciated that the goal is to induce plants to take up sufficient metal to reduce the metal concentration in the soil.
• lead has proven to be a particularly difficult metal for plants to transport into their shoots (see, for example,
• lead is preferably accumulated to at least about 3000 ⁇ g/g d.w. shoot mass, more preferably to at least about 4000 ⁇ g/g d.w. shoot mass, and most preferably to at least about 6000 ⁇ g/g d.w. shoot mass (see Examples).
• the present invention also demonstrates that combinations of inducing agents, applied simultaneously or with intervening time periods, often have synergistic effects on metal accumulation.
• plants are exposed to a first manipulation that increases metal availability (e.g., by employing a first inducing agent that itself increases metal availability and/or by taking additional steps to enhance availability, as is discussed below), and then to a second manipulation
• an inducing agent that stimulates metal transport to the shoots.
• an inducing agent that stimulates metal transport to the shoots.
• Plant members that can be used in accordance with the present invention include any plant that is capable of being induced to hyperaccumulate heavy metals by the methods described herein. Specifically, any plant that can be induced to hyperaccumulate into its shoots a metal to a concentration greater than the corresponding concentration of metal in the growth media (soil) to be treated is useful in the practice ofthe invention. Of course, not all plants can be induced to accumulate high levels of heavy metals in their shoots according to the present invention. In fact, even within a given plant species, not all cultivars will show the desired hyperaccumulation activity.
• any plant that, when cultivated in a metal-contaminated soil and exposed to an inducing agent as described herein, hyperaccumulates metal in its shoots to a greater extent than it would in the absence ofthe inducing agent is desirable.
• the plant is capable of accumulating metal in its shoots to a concentration above that ofthe metal in the soil in response to the inducing agent.
• Preferred plant members for use in the present invention in addition to being capable of hyperaccumulating metal in their shoots to a concentration higher than that in the soil, have one or more ofthe following characteristics:
• Plants that respond to conventional agricultural practices are preferred for the present invention inasmuch as they can be easily cultivated and stimulated to produce vigorous root and shoot growth under intensive agricultural practices (i.e., mechanical tillage, irrigation, fertilization, high plant populations).
• intensive agricultural practices i.e., mechanical tillage, irrigation, fertilization, high plant populations.
• Plants amenable to genetic manipulation may be used to provide material for genetic transformations to inco ⁇ orate into other plants one or more characteristics desired for the practice ofthe present invention.
• plants amenable to genetic manipulation may act as receptors of genetic transformations to develop or improve desired characteristics, thereby becoming useful (or more useful) in the present
• Crop members are those plants that are grown primarily as either vegetative sources (e.g. as vegetables, forage, fodder, and/or condiments), or oilseeds. Crop members are preferred in the practice of the present invention primarily because they tend to produce large amounts of biomass.
• crop-related members are able to exchange genetic material with crop members, thereby permitting breeders and biotechnologists to perform interspecific (i.e., from one species to another) and intergeneric (i.e., from one genus to another) gene transfer, according to known techniques (see, for example, Goodman et al. Science 236:48, 1987, inco ⁇ orated herein by reference).
• Particularly preferred plants for use in the practice ofthe present invention are members ofthe Brassicaceae family, preferably crop and or crop-related members.
• Preferred members ofthe Brassicaceae family include, but are not limited to plants of the genera Brassica, Sinapis, Thlaspi, Alyssum, and Eruca. Particularly preferred are Brassica species B. juncea, B. nigra, B. campestris, B. carinata, B. napus, B. oleracea, and cultivars thereof. An especially useful B. juncea cultivar is number 426308.
• desirable plants for use in the present invention include those that have been mutagenized and/or genetically engineered (e.g., interspecific and/or intergeneric hybrids).
• Methods for mutagenizing plants are well known in the art (see, for example, Konzak et al., Intemational Atomic Energy Agency,
• Plants for use in the present invention can be genetically manipulated using known transformation techniques or using sexual and/or asexual (i.e., somatic) hybridization techniques.
• Hybridization techniques are well-known in the art, and have been employed, for example, to transfer agronomically important traits from related species to crop Brassicas (see, for example,
• metal refers to metals (both stable and radioactive, both ionic and non-ionic forms), mixtures of metals, and combinations of metals with organic pollutants.
• Metals that can be accumulated according to the present invention include antimony, arsenic, barium, beryllium, cadmium, cerium, cesium, chromium, cobalt, copper, gold, indium, lead, manganese, mercury, molybdenum, nickel, palladium, plutonium, rubidium, ruthenium, selenium, silver, strontium, technetium, thallium, thorium, tin, vanadium, uranium, yttrium, zinc, and combinations thereof.
• Common organic pollutants relevant to the present invention include benzene or other aromatics, alkyl benzyl sulfonates (detergents), polycyclic hydrocarbons, polychlorinated biphenyls (PCB's) and/or halogenated hydrocarbons (e.g. trichloroethylene).
• detergents alkyl benzyl sulfonates
• PCB's polychlorinated biphenyls
• halogenated hydrocarbons e.g. trichloroethylene
• the present invention provides a novel method for the accumulation by plants of metals that are not essential to, and/or are detrimental to, plant growth.
• the present invention is particularly useful, and fills a void in existing techniques, because soils to be remediated are typically contaminated with phytotoxic metals.
• metal contaminants that are the primary toxic components of contaminated sites are: lead, chromium, arsenic, zinc, copper, cadmium, and nickel.
• Radioactive metals e.g., uranium, plutonium, etc.
• lead is preferably accumulated to at least about 3000 ⁇ g/g d.w.
• shoot mass more preferably to at least about 4000 ⁇ g/g d.w. shoot mass, and most preferably to at least about 6000 ⁇ g/g d.w. shoot mass; zinc is preferably accumulated to at least about 1000 ⁇ g/g d.w. shoot mass, and more preferably to at least about 2000 ⁇ g/g d.w. shoot mass; copper is preferably accumulated to at least about 1000 ⁇ g/g d.w. shoot mass, and more preferably to at least about 2500 ⁇ g/g d.w. shoot mass; cadmium is preferably accumulated to at least about 500 ⁇ g/g d.w. shoot mass, and more preferably to at least about 1000 ⁇ g/g d.w.
• nickel is preferably accumulated to at least about 200 ⁇ g/g d.w. shoot mass, and more preferably to at least about 500 ⁇ g/g d.w. shoot mass (see Example 7).
• Uranium is preferably accumulated to at least about 10 ⁇ g/g d.w. shoot mass, more preferably to at least about 1000 ⁇ g/g d.w. shoot mass, and most preferably to at least about 4000-6000
• the metal-containing environment in which plants are induced to hyperaccumulate is not intended to limit the scope ofthe present invention. That is, as long as the environment can sustain growth ofthe selected plants, it is suitable for the pu ⁇ oses ofthe present invention.
• Metal-containing environments can range from purely aquatic environments with varying degrees of water saturation, organic matter content, mineral content, etc. to well-drained soils.
• soil as used herein, includes a wide variety of physical types and chemical compositions.
• Plants can be grown in soil, or altematively can be grown hydroponically (see, for example, U.S. Patent No. 5,364,451 ; U.S. Patent No. 5,393,426; U.S.S.N. 08/252,234; U.S.S.N. 08/359,811; U.S.S.N. 08/423,827; and U.S.S.N. 08/443,154, each of which is inco ⁇ orated herein by reference).
• the goal of cultivation in an ordinary crop plant for typical agricultural use is to maximize the crop yield
• the goal when practicing this invention is to increase in an undifferentiated fashion the amount of above-ground biomass prior to induction. That is, the biomass of importance to the effectiveness ofthe invention is the und: entiated amount of biomass produced, in contrast to, for example, in corn, the more typical desire to achieve the maximum yield of edible material. It also should be recognized, as elaborated above, that maximum crop yield per se should not be the sole selection criteria, because it must be balanced with the concentration of metal in shoots upon accumulation.
• the optimal amount of time that a plant should be cultivated before application ofthe inducing stimulus according to the present invention will vary depending on the type of plant, the metal being accumulated, and the character ofthe environment in
• Brassica juncea is being utilized to accumulate lead, it is generally desirable to cultivate the plants for at least three weeks, and preferably four to six weeks, after emergence ofthe plants before applying the induction stimulus (see, for example, Examples 2 and 5)
• the overall goal is to have the largest possible amount of metal taken up into the plant roots and
• Manipulations that can increase the availability of metal to plants include, for example, (i) addition of chelators to the soil; (ii) tilling of soil to bring metals containing soil into contact with the plant root zone; (iii) decreasing pH ofthe metal-containing environment, for example by adding an effective amount of an organic or inorganic acid (such as, for example, nitric acid, acetic acid, and citric acid), or by adding to the environment a compound, such as ammonium sulfate, that will be metabolized by the plant roots (and/or by associated bacteria or other component(s) ofthe rhizosphere) in a manner that produces protons and thereby reduces the soil pH (see, for example, U.S.S.N. 08/252,234, inco ⁇ orated herein
• Desirable inducing agents used either alone or in combination, include metal chelators, organic and inorganic acids, herbicides, plant growth regulators, and other phytotoxic compounds. Chelators
• EDTA ethylenediaminetetraacetic acid
• chelators such as EDTA improve metal solubility in the soil, and thereby increase availability ofthe soil metals to the plant. This increase in metal solubility presumably increases the amount of metal accumulated into the plant.
• EDTA induces hyperaccumulation of lead into plant shoots by stimulating transport of root-accumulated material.
• chelators are also useful to induce metal hyperaccumulation into plant shoots if applied in the manner described.
• metal chelating or complexing agents such as, for example, ethylene glycol-bis( ⁇ -aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), trans- 1 ,2-diaminocyclohexane- N,N,N',N;-tetraacetic acid (CDTA), N-hydroxyethylethylene-diaminetriacetic acid (HEDTA), nitrilotriacetic acid (NTA), citric acid, salicylic acid, and malic acid, can desirably be used in accordance with the present invention, and can follow the teachings ofthe present specification to screen and identify particular chelators and conditions that may be preferred for
• Example 3 reports our finding that exposure of B. juncea plants to pH 3.5 in solution culture induces hyperaccumulation of lead into plant shoots.
• Example 4 demonstrates that the sequential administration of an acid and EDTA induces higher levels of lead accumulation into B. juncea shoots than are
• Example 5 demonstrates that a combination of acid and EDTA induces metal transport into shoots effectively in a field environment. This finding is particularly significant because large- scale acidification of soil to pH 3.5 may well be impractical in soil sites.
• Example 5 demonstrate that such large-scale acidification is not required. Some level of acidification (we note that the quantities of acid used in Example 5 only slightly reduce the soil pH) is still valuable due to its synergistic effects when combined with another inducing agent such as a chelator.
• Example 11 presents our findings that reduction of soil pH dramatically enhances accumulation of uranium into shoots of a wide variety of different plants. As shown in Example 11 , we found citric acid to be a particularly effective inducing agent, probably because of its dual abilities to i) acidify the soil; and ii) chelate the uranium.
• a soil pH greater than about 5.5 is desirable in the initial cultivation stage during which most ofthe biomass is accumulated. This initial cultivation stage is followed by a reduction in pH to induce metal accumulation.
• soil pH is preferably reduced to about pH 3.5, though less dramatic pH reductions are also desirable, especially when an additional inducing agent is employed.
• any acidification (either localized or general) ofthe soil-root system is expected to be beneficial to the induction mechanism when used in combination with other inducing agents, regardless of its ability to stimulate induction in the absence of other inducing agents.
• Alternate acidifying agents such as, for example, acetic acid, ammonium acetate, ammonium sulfate, ferrous sulfate, ferrous sulfide, elemental sulfur, sulfuric
• acid, citric acid, ascorbic acid can be used to reduce the soil pH (see, for example, Example 12). Also, soil pH can be reduced by addition of a metabolite that is processed by the roots or other element ofthe rhizosphere in a manner that produces protons (see above).
• Preferred acidifying agents are those that chemically or biologically degrade within days or weeks without leaving residual salts that may either result in an undesirable buildup of salinity (i.e., ammonium, chloride or sodium) or create a potential environmental hazard from leaching ofthe associated anions (i.e., nitrate from nitric acid). Particularly preferred acidifying agents include, but are not limited to, acetic acid, citric acid, or ascorbic acid.
• Examples 5-7 and 12 demonstrate that several different commercially available herbicides can be used in accordance with the present invention to induce hyperaccumulation of metals into plant shoots;
• Example 12 shows that herbicides can act synergistically with acidification to induce metal hyperaccumulation. It is worth noting that, under the conditions of Examples 5-7, herbicides did not effectively induce hyperaccumulation in soil environments in the absence of an agent (e.g., acid or chelator) that increased metal availability to the plants (see Figure 4).
• an agent e.g., acid or chelator
• metals first accumulate in the plant roots, and that the induction stimulus induces transport to the plant shoots.
• the induction stimulus induces transport to the plant shoots.
• herbicides are applied as inducing agents only after the plants have first been exposed to an agent that increases metals availability (e.g., acid and/or chelator). Furthermore, in many cases, a delay (e.g., 24 hours) is desirably imposed between the application ofthe treatment that increases metal availability and the application ofthe herbicide. The idea is to allow metals to accumulate in the roots during application ofthe treatment that increases metal availability, and then to induce transport of root-accumulated metal into the shoots by application ofthe herbicide.
• One particularly preferred embodiment of the present invention involves sequential application of EDTA and herbicide (e.g., Roundup ® ), with a delay in between.
• herbicides other than those specifically presented in the Examples are useful inducing agents in accordance with the present invention.
• Preferred herbicide compounds have little or no residual soil activity and decompose quickly in the environment.
• Such preferred compounds include commercially available formulations containing, for example, glyphosate, 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4- chlorophenoxyacetic acid (MCP A), or maleic hydrazide.
• any of a variety of other agents can be used as inducing agents in the practice ofthe present invention.
• any agent that disrupts plant metabolism in a way that overrides the natural protective mechanisms that block root-to-shoot transport of phytotoxic materials will be effective in inducing metal hyperaccumulation in plant shoots.
• high levels of heavy metals can also function as inducing agents according to the present invention (see Examples 8- 9).
• hyperaccumulation is only induced above a threshold level of metal.
• the present invention therefore teaches that exposing plants to a physiological stress or phytotoxic substance (e.g. phytotoxic levels of metals or nutrients, low pH, osmotic stress, herbicide, etc.) or combination of such substances, disrupts the plant's natural safety mechanisms normally involved in preventing uptake and/or transport of toxic substances into plant shoots and stimulates metal translocation from the roots to the shoots.
• a physiological stress or phytotoxic substance e.g. phytotoxic levels of metals or nutrients, low pH, osmotic stress, herbicide, etc.
• any agent with phytotoxic activity can be screened to test its ability to induce metal hyperaccumulation in plant shoots according to the procedures described herein.
• any or all chelating agents e.g., EDTA, EGTA, DTPA, CDTA, citric acid, salicylic acid, malic acid
• soil acidifiers e.g.
• acetic acid ammonium acetate, ammonium sulphate, ferrous sulfate, ferrous sulfide, elemental a sulfur, sulfuric acid, citric acid, ascorbic acid), phytotoxic levels of plant nutrients and trace elements (Fe, Mn, Na, Al, etc.) and commercially available herbicides (containing e.g., glyphosate, MCP A, maleic hydrazide) alone or in combination with one another, can be tested for their inducing capabilities, as can other chemical agents such as other toxins, detergents, enzymes, and plant hormones, or physical factors such as drought, extreme heat, ultraviolet radiation, and x-radiation. Also, any of these agents can be tested under conditions of nutritional starvation, but starvation alone is not sufficient to induce metal hyperaccumulation into plant shoots.
• Desirable agents are those that stimulate a plant to accumulate metal in its shoots to a level higher than the plant would accumulate in the absence ofthe agent.
• the agent stimulates the plant to accumulate at least about two-fold more metal in its shoots than the plant would do if not exposed to the agent, more preferably, the agent induces the plant to accumulate at least about 10-fold more metal in its shoots, still more preferably at least about 100-fold more, and most preferably at least about 1000-fold more.
• Plant shoots into which metals have been hyperaccumulated in accordance with the present invention are harvested by any of a variety of standard techniques, such as
• B. campestris, B. juncea, and B. nabus in particular is routine (see, for example, Canola Growers Manual; Canola Council of Canada, 1984, inco ⁇ orated herein by reference).
• EXAMPLE 1 Inducing Hyperaccumulation of Lead by Addition of EDTA.
• a floating styrofoam platform were placed in an 18L tray containing 15L of solution. Experiments were done in an environmentally controlled growth chamber at 25°C, 75% relative humidity, and a 16 hour photoperiod was provided by a combination of incandescent and cool-white fluorescent lights.
• Plant roots and shoots were harvested separately, dried for 48 hours at 70°C in a forced air oven, weighed, ground, and wet digested with nitric and perchloric acids. At least 4 replicates were used for each treatment.
• the metal content ofthe extracted acid was determined with a Fisons Direct Current Plasma Spectrometer, model SS-7.
• soluble lead in the solution is accumulated into plant roots, but is not transported to the shoots in appreciable amounts.
• addition of EDTA results in high levels of accumulation in plant shoots, and a reduced amount of lead remains in the plant roots.
• the chelator may also act to bind lcium at the root surface, thereby reducing metal precipitation, and/or to increase membrane permeability, thereby allowing less restricted movement of metal into the root.
• EXAMPLE 2 Addition of EDTA to Soil Materials and Methods:
• a Sassafras Ap silt loam soil was collected from the Rutgers University Horticultural Farm and amended with lead carbonate.
• the soil was limed to pH 5.1 or 7.5, and was fertilized with urea (150 mg N/kg), potassium chloride (100 mg K 2 O/kg), and gypsum (70 mg CaSO 4 /kg).
• the soil was allowed to equilibrate for two weeks in the greenhouse at saturation, air dried, and remixed before planting.
• the soil was placed in 8.75 cm diameter pots (350 g soil/pot) and planted with Brassica juncea (426308) seeds.
• Phosphate fertilizer was added as a spot placement 1 cm below the seeds at planting at the rate of 100 mg P 2 O 5 /kg. After seedling emergence, the pots were thinned to two plants per pot.
• Plants were grown for three weeks in a growth chamber with a 16 hour photoperiod and were given weekly fertilization treatments of 16 and 7 mg/kg N and K, respectively.
• chelate (EDTA as a K salt) solutions were applied to the soil surface.
• the pots were placed in individual trays to prevent loss of amendments from leaching.
• the soil was irrigated to field capacity on a daily basis.
• the plants were harvested one week after the chelate treatment by cutting the stem 1 cm above the soil surface.
• the plant tissue was dried and analyzed for metal content by ICP as described previously in
• EXAMPLE 3 Inducing Hyperaccumulation of Lead by Altering pH. Materials and Methods:
• Seeds of Brassica juncea cultivar 426308 were obtained from the USDA ARS Plant Introduction Station of Iowa State University.
• Seedlings were cultivated hydroponically in open-ended 1.7 mL microcentrifuge tubes packed with 1 cm 3 of vermiculite, with roots extending into an aerated nutrient solution [1 g/L HydrosolTM supplemented with 0.6 g/L Ca(NO 3 ) 2 ]. During cultivation of the seedlings, six tubes supported by a floating styrofoam platform were placed in an aerated nutrient solution [1 g/L HydrosolTM supplemented with 0.6 g/L Ca(NO 3 ) 2 ]. During cultivation of the seedlings, six tubes supported by a floating styrofoam platform were placed in an
• Roots and shoots were harvested separately, dried for 48 hours at 70° C in a forced air oven, weighed, ground, and wet digested with nitric and perchloric acids. At least 4 replicates were used for each treatment
• the metal content ofthe acid extract was determined with a Fisons Direct Current Plasma Spectrometer, model SS-7.
• Results and Discussion Results are presented in Figure 2. As can be seen, reducing the pH ofthe com inated solution from 5.5 to 3.5 dramatically changed the amount of lead taken up by B. juncea shoots. Plants exposed to 50 mg/L lead solution at a pH of 3.5 accumulated 6 mg/g lead, some 100 times the amount taken up at a pH of 5.5. This phenomenon cannot be explained by increased lead solubility, since the soluble lead remained at 50 mg/L during the entire experimental period at either pH level.
• EXAMPLE 4 Synergistic Induction of Lead Hyperaccumulation by Exposure to a Sequence of Altered pH and EDTA
• pH ofthe solutions was adjusted using a 1.0 N HNO 3 solution.
• EDTA was added after pH adjustment using 0.5 molar stock solution. At least 4 replicates were used for each treatment.
• present invention is practical for phytoremediation of contaminated sites.
• Soil was prepared and plants were grown as described in Example 2. After three weeks of growth, EDTA was applied to the soil solution at the rate of 2.5 mmol/kg. Twenty-four hours after the chelate solutions were applied, herbicide solutions of Paraquat, Roundup ® (glyphosate), or Rockland ® were applied in various concentrations to wet the foliage. Plants were maintained as described in Example 2, and were harvested 7 days after the chelate application. Results and Discussion:
• EXAMPLE 7 Effect of EDTA and Herbicide Applications on Induction of Hyperaccumulation of Various Metals from Contaminated Soil. Materials and Methods:
• the Sassafras Ap soil was amended with oxide and carbonate forms of Cd, Cu, Ni, Pb, and Zn and prepared as in Example 2. Chelate solutions were applied with an herbicide application of 2,4-D as described in Example 5.
• juncea and B. napus were obtained from the Crucifer Genetics Cooperative, Madison, Wisconsin. Seeds of other plants were purchased from local seed markets.
• Seedlings were grown in a greenhouse equipped with supplementary lighting (16 h photoperiod; 24-28°C; see Kumar et al. Environ. Sci. Technol. 29:1232-1238, 1995, inco ⁇ orated herein by reference). Seedlings were grown for 10 days in acid-washed coarse sand and fertilized every two days either with full-strength Hoagland's solution or with 1 g/L HydrosolTM supplemented with 0.6 g/L Ca(NO 3 ) 2 .
• Ten-day-old seedlings were transplanted (in sets of two) into 150 g dry weight (DW) of an acid-washed 1 : 1 (v/v) mixture of coarse sand and coarse Perlite placed in 3.5 inch round plastic pots.
• the pots contained two different levels of lead- 62.5 mg/kg or 625 mg/kg dry weight sand/Perlite.
• Each pot contained two seedlings. At least four replicates for each metal concentration were used.
• Plants were grown for 14-20 days. Plants of metal -treated and control plants were harvested and washed thoroughly with running tap water. Plant tissue was cut into small pieces with scissors, dried for 2 days at 80°C and ashed in a muffle furnace at 500°C for 6 h. The ash was dissolved in a mixture of 2M HCl and 1 M HNO 3 . The metal content ofthe acid extract was determined with a Fisons Direct Current Plasma Spectrometer, model SS-7.
• Table 3 below compared the accumulation of Pb in shoots ofthe 12 species tested at two different levels of Pb.
• concentrations of lead in shoots do not exceed 50 ⁇ g/g DW in plants exposed to 62.5 mg
• high levels of one type of metal can induce plants to hyperaccumulate other types of metal that are not present in such high concentrations in the environment.
• the present invention teaches that high levels of heavy metals can act as an inducing agent to stimulate metal
• EXAMPLE 9 Induction of Hyperaccumulation by Varying Lead Levels Materials and Methods: B. juncea cultivar 182921 was employed in experiments in which plants were grown hydroponically in a manner similar to that described above in Example 1. Roots of 17-day-old seedlings were exposed to 400 mL of aqueous solution containing varying amounts of lead (0, 6, 22, 47, 94 or 188 mg Pb/L). After an additional 14 days, plants were harvested. Metal content of plant parts was analyzed using the procedures detailed in Example 8.
• Results are presented in Figure 6. As can be seen, the concentration of lead accumulated in B. juncea roots increased with increasing solution concentration, though some decline in rate was observed when lead was present in the solution at concentrations above about 50 mg/L. By contrast, the concentration of lead accumulated in B. juncea shoots did not increase significantly until the concentration of lead in the solution approached 100 mg/L. At the highest concentration of lead tested (188 mg/L), lead levels in shoots reached about 1.6%.
• EXAMPLE 10 Manipulations ofthe Environment that Increase Metal Availability
• a variety of different techniques can be used to increase metal availability in soils in accordance with the present invention. These treatments can be applied individually of separately.
• an "effective amount" of a metal chelator is an amount sufficient to increase metal mobility but not sufficient to significantly alter plant growth and development. Desirable "effective amounts" of particular chelators are readily determined through measurements metal mobility effects.
• the concentration of soluble metals in soils can be measured according to the technique described by Mench et al. (J. Environ. Qual. 23:58, 1994, inco ⁇ orated herein by reference). Briefly, metals are extracted from 5g of soil by equilibration with about 25 ml of 0.01 M calcium nitrate (to maintain ionic strength) for about 2 hours on a mechanical shaker. After the equilibration period, the suspension is centrifuged (between 3000-5000 x g) for about 15 minutes to separate the solution from the soil. The supernatant solution is then analyzed for the desired water-soluble metal concentration. Measured metal concentration is correlated with the amount and type of chelator added, so that optimal conditions for maximizing metal availability are determined.
• metal chelators increase metal availability by forming soluble complexes with metals, thereby increasing metal solubility in the soil solutions.
• exemplary solubilizing chelators include ammonium pu ⁇ urate (murexide), 2,3-butane-dione dioxime (dimethylglyoxime), 3,6 disulfo-1 ,8-dihydroxynaphthalene (chromotroic acid), thiourea, alpha-benzoin oxime (cupron), trans- 1,2-diaminocyclohexanetetraacetic acid
• CDTA diethylene-triaminopentaacetic acid
• DTPA diethylene-triaminopentaacetic acid
• NTA nitrilotriacetic acid
• substituted 1,10-phenanthrolines e.g., 5-nitro-l,10 phenanthroline
• sodium diethyldithiocarbamate cupral
• 2-phenoyl- 2-furoylmethane phenoyl-trifluoroacetone
• triethylenetetramine EDTA, citric acid, EGTA, HEDTA, salicylic acid, and malic acid
• Chelating agents are preferably applied to soil by conventional irrigation pipes or other ground level irrigation systems. Chelating agents may alternately be applied through commercially available fertilizer and chemical application equipment, including large volume sprayers. Chelating agents may be applied through broadcast methods for large areas or banding methods for the root zone. Chelating agents are preferably applied at concentrations from 0.1-10 mmol/kg soil. Acidification
• pH ofthe metal-contaminated is reduced to about pH 4.5-5.5 by acidifying soil with an effective amount of organic or inorganic acids (such as nitric acid, hydrochloric acid, sulfuric acid, acetic acid and citric acid).
• Acids are preferably applied to the soil by conventional irrigation pipes or other ground level irrigation systems. Acids may alternately be applied through other commercially available fertilizer and chemical application equipment, including large volume sprayers. Acids are preferably applied at concentrations from O.lmM to 1.0 M at volumes ranging from about 5 to 200 tons per acre or at levels sufficient to drop soil pH in the plant rhizosphere (down to about 40 cm) to between 4.5 and 5.5 pH units.
• Acidification ofthe plant environment may alternately be accomplished by addition to the environment of compounds that depress soil pH because of biological activity of roots and microorganisms.
• these compounds include urea or ammonium sulfate.
• This so-called “biological acidification” occurs because the positively charged ammonium ions that are inco ⁇ orated into the roots and/or microorganisms are replaced with positively charged protons exuded or otherwise released from the rhizosphere into the soil, thus lowering the soil pH.
• the ammonium- containing compounds are applied at 0.5 to about 2.0 tons per acre.
• Metal availability can be enhanced by using electrical fields to increase metal mobility (see, for example, Probstein et al., Science 260:498, 1993, inco ⁇ orated herein by reference).
• a direct current electric field is applied across electrode pairs placed in the ground. The electric field induces motion of liquids and dissolved ions.
• Metal availability to plant roots can be increased by tilling soil to depths greater than 2 cm and as far down as 50 cm.
• Conventional implements may be employed for this pu ⁇ ose, provided that they are suitable for tilling down to the depths required by the sent methods.
• These implements include moldboard plows, chisel plows, tandem and offset disc plows, and various harrowers known to those having ordinary skill in the art. The exact implement used will depend on factors understood in the art, such as soil moisture, soil texture, weed cover and the like.
• EXAMPLE 1 1 Inducing Hyperaccumulation of Uranium Materials and Methods: SOIL CHARACTERIZATION: Uranium-contaminated soils were collected from an
• the soil was screened to pass through a 1.0 cm sieve, and was thoroughly mixed before use.
• the soil had a clay loam texture and an organic matter content of 4.1%.
• the soil pH (1 :1 soil/water) was within the range of 5.5-7.0.
• Uranium was present in the soil at 200-800 mg/kg (total); the solution concentration of uranium in the soil was 2-15 mg/L.
• the soil solution was extracted by a centrifugation method described by Elkhatib et al. (Soil Sci. Soc. Am. J. 51 :578, 1987, inco ⁇ orated herein by reference). Briefly, the soil was watered to field capacity and kept at room temperature for 24 hr before the soil solution was extracted.
• the soil solution was
• SOIL AMENDMENT APPLICATION A variety of different soil amendments were applied to the soil and those that most effectively i) solubilized the uranium (i.e., enhanced deso ⁇ tion ofthe uranium from the soil into the soil solution); and ii) induced uranium hyperaccumulation into plant shoots were identified. Specifically, selected plant species were grown on uranium-contaminated soils in a growth chamber for 4-6 weeks. Subsequently, selected soil amendments were applied to the root-zone ofthe
• a stock solution was prepared for each soil amendment (0.5 M for organic or inorgamc acids; 0.1 M for sodium bicarbonate, potassium bicarbonate, or sodium acetate). The appropriate amount of stock solution was then delivered to the root-zone ofthe plants. Plants were harvested one week after application ofthe soil amendments. Plant samples were digested in a mixture of concentrated HNO 3 /HClO 4 , and the digested samples were analyzed for uranium content by ICP.
• Figure 7 shows the results of a study analyzing the effects of several different soil amendments on uranium availability in the soil. As shown, the solution concentration of uranium in the soil was increased approximately 2-200 fold, depending on the amendment applied. Citric acid was most effective at solubilizing the metal, increasing soil solution concentration from 1.2 mg/L to 240 mg/L. Addition of citric acid to the contaminated soil transiently reduced soil pH by 0.5 to 1.0 pH unit. Because application of nitric acid had a similar effect on soil pH but a less dramatic effect on uranium solubility, we conclude that at least part ofthe solubilizing effect of citric acid is due to its ability to chelate the metal.
• Figure 8 shows the effects of several acidifying agents on the induction of uranium hyperaccumulation into B. juncea shoots. All ofthe agents induced at least about two-fold more accumulation than occurs in the absence of any amendment. Citric acid was the most effective inducing agent, stimulating accumulation at least about 100- fold. We therefore applied citric acid to several individual B. juncea cultivars, and found that it was able to induce from 250 to 300-fold increases in uranium shoot accumulation (Figure 9). We also applied citric acid to a variety of different plant species and found that it was an effective inducing agent for all plants tested (Figure 10). Citric acid application increased metal accumulation at least about 2-fold in all species, and as much as about 1000-fold in some.
• EXAMPLE 12 Synergistic Induction of Uranium Hyperaccumulation by Exposure to both Altered pH and Herbicide Materials and Methods:
• SOIL CHARACTERIZATION Uranium-contaminated soils were collected and analyzed as described in Example 11.
• SOIL AMENDMENT APPLICATION A variety of different soil amendments were applied to the soil and those that most effectively i) solubilized the uranium (i.e., enhanced deso ⁇ tion ofthe uranium from the soil into the soil solution); and ii) induced uranium hyperaccumulation into plant shoots were identified. Specifically, selected plant species were grown on uranium-contaminated soils in a growth chamber for 4-6 weeks. Subsequently, both citric acid and foliar spray ROUNDUP ® solution (containing

Applications Claiming Priority (5)

Publications (1)

Family

ID=26702098

Family Applications (1)

Country Status (4)

Families Citing this family (16)

Family Cites Families (2)

• 1997
  • 1997-03-19 WO PCT/US1997/004956 patent/WO1997034714A1/en not_active Application Discontinuation
  • 1997-03-19 AU AU24242/97A patent/AU725833B2/en not_active Ceased
  • 1997-03-19 EP EP19970919929 patent/EP0888197A1/de not_active Withdrawn
  • 1997-03-19 IL IL12631297A patent/IL126312A0/xx unknown

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events