CA2163666A1 - Method for removing soluble metals from an aqueous phase - Google Patents

Abstract

A process for the removal of metal ions from solution and means for effecting such removal are described. The process is based on the hydroponic growth of sunflowers, terrestrial turfgrasses and/or members of the family Brassicaceae in solutions containing one or more metal ions. Metal ions can be efficiently removed from solutions by passing these solutions through the root biomass of these terrestrial plants. Columns containing the root biomass are also part of the invention.

Description

Wo 94/29226 2 1 6 3 6 6 6 PCT/llS94/06169 METHOD FOR REMOVING SOLUBLE METALS
~ROM AN AQUE~US PHASE

Back~round of the Invention Deposition of metal-rich mine tailings, metal smelting, leather tanning, electroplating, emissions from gas exhausts, energy and fuel production, downwash from powerlines, intensive agriculture and sludge dumping are the most 10 important human activities which contaminate aqueous systems with large amounts of toxic metals. The list of sites cont~min ~t~d with toxic metals grows larger every year, presenting a serious health problem and a formidable danger to the environment. Although water treatment procedures have been developed to remove metals from aqueous environments, water treatment plants, for example, 15 do a relatively poor job of removing toxic metals from residential and industrial aqueous waste, contributing to the overall problem.

Summarv of the Invention The present invention is a method for reducing an amount of metal in a 20 metal-cont~ining solution utili7ing plant roots to absorb, concentrate and precipitate metal from the aqueous solution.
The method includes cont~cting the solution with a root biomass of a terrestrial plant under conditions sufficient for the root biomass to remove themetal from the solution, and then separating the root biomass from the solution.25 In preferred methods of the invention, a terrestrial plant is grown hydroponically in the presence of the soluble metal and the roots, either excised or still attached to the plants, are allowed to remain in contact with the solution cont~ining themetal for a time sufficient for the roots to accumulate the metal and/or for theroots to initiate precipitation of the metal from solution (i.e. to convert the metal 30 from a soluble form to an insoluble form). The roots are then harvested and/or the precipitated metal is separated from the solution.
Preferably, a hydroponic treatment bed is provided for holding a solution, the bed having one and another ends. A solution cont~ining a soluble metal is introduced into one of the ends and allowed to contact the roots of the terrestrial 35 plant that is m~int~ined hydroponically within the bed. The plants will accumulate the metal in the roots and/or will initiate precipitation of the metal out of the Wo 94/29226 2 1 6 3 6 6 6 PCT/US94/06169 the metal in the roots and/or will initiate precipitation of the metal out of the solution. The solution is then removed from the hydroponic treatment bed. The flow of solution through the hydroponic treatment bed can either be continuous or can be intermittent in which the flow stops for a period of time to allow the roots 5 to accumulate the metal from solution and/or precipitate the metal from solution.
The solution is then removed from the hydroponic treatment bed and the process is repeated. Alternately, a column filled with excised roots can be used to achievesimilar results. The method further includes harvesting the roots after allowingaccumulation and/or precipitation of the metal.
The metals that are capable of being accumulated and precipitated by the plants are any one of a variety of heavy metals or radioactive metals selected from the following elements: lead, chromium, mercury, cadmium, cobalt, nickel, molybdenum, copper, arsenic, selenium, zinc, antimony, beryllium, gold, barium, m~ng~nese, silver, th~llium, tin, rubidium, ~IrUnliU~ v~n~linm, yttrium, 15 technecium, ruthenium, palladium, indium, cesium, uranium, plutonium, and cerlum.
The p~fellcd terrestrial plants used in the method of the invention are the sunflower plant (Helianthus annuus L.) and plants selected from the turfgrasses and members of the family Brassicaceae. The most preferred plants are 20 Helianthus and members of the Brassiceae tribe including Brassica juncea and others.
The invention also is a system for reducing an amount of metal in a metal-cont~ining solution. The system includes a means for holding a metal-cont~ining solution; a root portion of a tellesllial plant in contact with the solution; and a 25 means for moving the solution within the holding means. Preferably, the holding means is a column having opposed ends and the moving means is a pump in fluid communication with at least one end of the column. The preferred terrestrial plants are sunflowers, turfgrasses and members of the Brassicaceae, all of whichcan accumulate soluble metal within their roots and/or precipitate the soluble metal 30 out of solution.

Wo 94/29226 2 1 6 3 6 6 6 PCT/US94/06169 Brief Description of the Drawin~
Figure 1 is a schematic, perspective view of a hydroponic column of the present invention;
Figure 2 is a schematic, cross-sectional illustration of another embodiment 5 of the hydroponic column of the invention;
Figure 3 is a schematic, cross-sectional illustration of a flow-through embodiment of the invention;
Figure 4 is a schematic, cross-sectional illustration of an intermittent flow embodiment of the invention;
Figure 5 is a graph illustrating the time course of lead removal by two replicate whole sunflower plants (O, O) and two replicate excised sunflower roots (--, );
Figure 6 is a graph illustrating the positive linear correlation between lead removal and sunflower root biomass; y= 194325.3x + 20845.3 (r=0.91);
Figure 7 is a graph illustrating the time course of lead removal by B.
juncea excised roots (O) and B. iuncea whole plants (~).

Detailed Des~ tion of the Invention This invention is based in part, on exploitation of the ability of certain 20 plants to concentrate metals from solution. This concentration can occur by one, or both of the following mech~ni~m~:
(a) Soluble metals can be accumulated by the plant and transferred into plant biomass, in particular, the root and/or shoot biomass. Preferred methods of the present invention utilize plants that will preferably accumulate metals in the 25 root biomass.
(b) Certain plants can precipitate soluble metal out of a metal-cont~ining solution. This phenomenon is documented for the first time as part of this invention and is believed caused by exudation of inorganic and organic materialsfrom the roots that can act as, for example, chelators to precipitate soluble metals 30 out of solution. That is, in a chemical engineering sense, roots can act as both ion exchange resins and as chemical precipitators.
The present method for reducing an amount of metal in a metal-cont~ining solution is used to remove primarily metal ions from the solution by either wo 94/29226 2 1 6 3 6 6 6 PCT/US94/06169 allowing for uptake of the metal ions into the plant biomass or by conversion ofthe soluble metal ions into an insoluble form. The term "soluble metal ions"
means metal cations or metal-cont~ining anionic species. Soluble metal ions may be present, either alone or bound with anions or chelating agents, that are soluble 5 in the solution at environmentally relevant temperatures (e.g. greater than 0 degrees C to less than about 45 degrees C). The term "insoluble" refers to metalions that are subst~nti~lly insoluble in the solution at environmentally relevant temperatures. The term "insoluble" is also intended to include non-ionic, elemental forms of the metal.
The method is used for the removal of metals that are selected from the commonly known heavy metals and Mdioactive metals such as, for example, lead, chromium, mercury, cadmium, cobalt, nickel, molybdenum, copper, arsenic, selenium, zinc, antimony, beryllium, gold, barium, m~ng~nese, silver, thallium, tin, rubidium, ~llullliuln, vanadium, yttrium, technecium, ruthenium, palladium,15 indium, cesium, uranium, plutonium, and cerium. The term "metal" is also meant to include mixtures of metals and common organic pollutants, for example, lead or chromium in combination with nitrophenol, benzene and/or aLkylbenzyl sulfonates (d~;lelgenls). The method may also be capable of removing more than one metal from an aqueous solution. Literature reports suggest that certain plants may 20 concentrate several different metals in their roots, implying that the mechanism of metal uptake is not always metal-specific.
The term "solution" refers to any metal cont~min~ted liquid such as industrial and residential waste streams, water treatment-plant effluents, ground and surface water, diluted sludge and other aqueous streams cont~ining radioactive 25 and non-radioactive metals.
The plants used in the ~l~relled methods are terrestrial plants. The term "terrestrial" refers to photosynthetic plants that normally grow in soils or sediments. The soils or sediments can include a variety of soil types having wide ranges of water content and organic matter content. The terrestrial plants can 30 therefore include crop-related plants and/or plants associated with environments such as wetlands. The term is also meant to include portions of terrestrial plants (i.e. excised shoots and/or roots). The term "terrestrial" is not, however, meant to refer to strictly aquatic plants that spend their entire life cycle completely

2 1 6 3 6 6 6 PCT/US94/06169 s floating on, or submerged in, an aqueous solution. These aquatic plants also include floating ferns, (e.g., Azolla), duckweed (Lemna), and water hyacinth (Eichhornia). Moreover~ the term "terrestrial" is not intended to include isolated plant cells or cell suspensions capable of metal uptake.
Although the hydroponically-grown terrestrial plants selected for use in the present method can also accumulate metals in their shoot portions (i.e., those portions above the aqueous solution), it is plcrellcd that the terrestrial plants used in the present method do not accumulate significant amounts of metal in their shoots. This is because shoots that do not accumulate metals may be discarded with no special precautions or may be allowed to regenerate new roots. Perennialgrasses offer specific advantages in this regard. Their roots can be continuously harvested and new roots will grow from the rem~ining shoots. Once the new roots are grown, the hydroponic metal uptake methods are repeated. Preferred tel.csl,ial plants are therefore those having roots that absorb and precipitate metals and can be harvested in bulk in the shortest practical period of time.
The terrestrial plants most suitable for the present invention are a variety of turfgrasses and members of the family Brassicaceae as well as the common sunflower, Helianthus annuus L. Exemplary turf grasses include Colonial bentgrass, Kentucky bluegrass, perennial ryegrass, creeping bentgrass, a variety of fescues and lovegrasses, Bermudagrass, Buffalograss, centipedegrass, switch grass, Japanese lawngrass and coastal panicgrass. Members of the Brassicaceae include Brassica juncea and B. oleracea. Other plants also suitable for the present method include spinach, sorghum, tobacco, and corn.
The terrestrial plants may also include those plants that are selectively bred and/or genetically engineered for an enhanced ability to accumulate metals in a hydroponic environment.
A preferred procedure is to grow selected plants from seeds (i.e. Brassica or Helianthus) or grass sod (e.g. turfgrasses) in a hydroponic environment with roots immersed in a nutrient solution. After a period of time, the nutrient solution is replaced with a metal-ion conlail~ing solution. Metal accumulating in the plant tissue is measured by, for example, atomic absorption spectrometry or plasma spectrometry. Metals are extracted with strong acids according to established protocols. See Blincoe et ak, Comm. Soil. Plant Anal., 18: 687 (1987); Baker and wo 94/29226 2 1 6 3 6 6 6 PCT/USg4l06169 Suhr, "Atomic Absorption Spectrometry", pp. 13-27 in Methods of Soil Analysis, part 2, Am. Soc. Agron., Madison, Wisc., (1982). Metal rem~ining in the solution is measured by, for example, atomic absorption or plasma spectrometry.
See, Soltanpour _ ah, "Optical emission spectrometry", pp. 29-65 in Methods of Soil Analysis, part 2, Am. Soc. Agron., Madison, Wisc., (1982). The difference between the decrease in metal in solution and metal concentration in the plant is the amount of metal precipitated out of the solution (see Example 1). Plants exhibiting the best metal uptake and/or precipitation properties may be further tested by obtaining seeds from various germ plasm collecting centers and laboratories. The cultures grown from these seeds are re-tested with the screening assay described above. It will be appreciated that this screening assay can be performed on plants other than those members described herein and using metals other than those specified herein.
Alternatively, or in addition, the plants may be mutagenized using well-known chemical mutagens. For example, ethylmethylsulfonate (EMS) is a potent mutagen which increases genetic variability by increasing the frequency of genomic mutations. See, for example, Redei, G. P. "Genetic Manipulations of Higher Plants", L. Ledoux (ed), Plenum Press, N.Y., (1975).
Ethylmethylsulfonate has been used in selection programs to produce heritable changes in plant biochemistry and physiology, particularly in Arabidopsis th~ n~, a member of the Brassicaceae.
The hydroponic screening system described above is used to identify terrestrial plant species with the highest metal accumulating and preci~ilaling potential. The seeds of these lines are then subjected to EMS mutagenesis using,for example, the methods of Estell et al, "The mutants of Arabidopsis", p. 89 inTrends in Genetics. Elsevier Science Publishers, B.V., Amsterdam, 1986.
Briefly, mutagenesis is accomplished by soaking seeds in EMS solution to induce heterozygous mutations in those cells which will produce the reproductivestructures. The Ml geperation of plants is allowed to self-fertilize and at least 50,000 see lling~ of the M2 progeny are screened for metal tolerance in artificial aqueous solutions cont~ining various metal concentrations. The most tolerant M2 plants, those growing most vigorously, are analyzed for accumulation of metals;
see Example 2.

W094/29226 2 1 6 3 6 6 6 PCT~S94/06169 Furthermore, the terrestrial plants used in the hydroponic methods of the present invention can be genetically manipulated using well-established techniques for gene transfer. It is well-known that a variety of non-photosynthetic org~ni~m~
respond to metals by production of metallothioneins (MT's), low molecular weight 5 proteins encoded by structural genes. See, for example Maroni, G., "Animal Metallothioneins", pp. 215-232 in Heavy Metal Tolerance in Plants; Evolutionary Aspects, (ed. A.J. Shaw), CRC Press Inc., Florida (1990). The present invention contemplates increasing root uptake of metals by heterologous expression of MT'Sin transgenic plants.
A m~mm~ n MT cDNA (e.g. monkey) can be obtained commercially or from an established source and a restriction enzyme fragment cloned into, for example, an Agrobacterium-based plant transformation/expression vector such as pJB90, a derivative of pGSFR780A. See, DeBlock et ah, Physiol Plant., 91: 694-701 (1989).
Seedling segments of terrestrial plants used in the present method are then incubated in the presence of a suspension of bacterial cells (e.g. Agrobacteriumtumefaciens) carrying the expression vector. After several days, the regenerating se~ling segments are transferred to the ap~ plidte selection medium and further incub~ted This results in transformants co,~l~inil-g the m~mm~ n MT gene (see Example 6).
The transformants are analyzed for the presence of MT DNA by Southern and Northern hybridization using m~mm~ n MT as the probe. The transformants are also analyzed for expression of MT protein by immunoblot analysis with antisera against m~mm~ n MT. See established protocols of, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY (1989).
The method for reducing the amount of metal in a metal-cont~ining solution includes contacting the aqueous solution with a root biomass of a terrestrial plant under conditions sufficient for the root biomass to convert the metal from a soluble form to an insoluble form. As discussed above, metal-ion removal can be easily determined by measuring the concentration of metal in the solution and in the plant biomass using a variety of well-known and well-characterized metal detection assays.

The methods of the invention rely on growth of terrestrial plants in a hydroponic system so that the root biomass will have maximum contact with the solution. The term "hydroponic" has a well-recognized meaning to those of ordinary skill in the art and generally refers to the science of growing plants in solutions cont~ining necessary growth promoting materials, instead of in soil. The exact method of growing the plants hydroponically is not intended to limit the scope of the invention.
For example, the methods can include several hydroponic systems currently available. One system is a hydroponic method in which plants are grown in a receptacle for holding aerated solutions in which the plant roots are in contact with the solution cont~ining the metals. Another hydroponic system is a solid supportin which commercially available inert supports, such as for example, rock wool, are saturated with a metal-cont~ining solution. The metal cont~ining solution isperiodically moved through the inert support that anchors the plants. Another hydroponic method involves growing plants in a column through which solution moves at regular intervals so that the roots never have a chance to dry out. This technique of moving the metal-co~ inil-g solution over the roots will be referred to as a "flow-through" system. A further method involves incubating the roots inthe metal-col~ -il-g solution, which is continuously drained and refilled. This method provides for good root aeration and will be referred to hereinafter as an"intermittent" flow method. Another method is the so-called "aeroponic" method which involves contacting the developing roots with the metal-cont~ining solution using ultrasound or compressed air to provide an aqueous mist or aerosol as the solution. Production of a mist or aerosol using ultrasound or compressed air areboth well-known procedures exemplified by U.S. Patent No. 5,017,351 (Rafson).
The plcfcllcd method includes contacting a metal-cont~ining aqueous solution with the root biomass of a tcllc~llial plant by moving the solution through a column co"~ g the root biomass. The column is preferably rendered opaque so that the roots are in darkness. The column can, however, be in a variety of configurations, not intended to limit the scope of the invention. For example, the column can be subst~nti~lly horizontal so that the depth of the solution in the column is substantially less than the length of the column. This typically includes construction of elongated troughs which contain the root biomass. Alternately, the Wo 94/29226 2 1 6 3 6 6 6 PCT/US94/06169 column can be oriented in a vertical position in which the depth of the solution is equal to or greater than the column width. In this configuration, the solution can flow vertically within the column. So long as the aqueous solution is in contactwith the root biomass for a time sufficient for the roots to convert the metal from 5 a soluble form into an insoluble form, the exact configuration of the column is of little significance. After uptake and/or precipitation is completed, the roots are then separated from the solution.
One configuration of a column 10 is shown in Fig. 1. The column 10 includes hydroponic treatment bed 12 for holding one or more plants in solution 10 14. The column 10 is preferably of an inert material such as polyvinylchloride, polytetrafluoroethylene (i.e. Teflon), or glass. The solution 14 has an air/aqueous interface 16. Disposed adjacent to this interface 16 is a mesh material 18 whichcan be made of an inert substance such as stainless steel, plastic or Teflon. In the embodiment illustrated, mesh 18 rests on a lip 19 formed on an inner surface 21 of column 10. On top of mesh 18 is a porous material 20 such as cheesecloth or thin, porous foam pad. On top of porous material 20 is a layer of soil 22 suitable for growing a particular terrestrial plant 24. In the embodiment illustrated, the soil 22 is a layer of a medium for supporting plant growth such as, for example,magnesium-alumin~te-iron silicate (Vermiculite) in which the terrestrial plant seeds can be gerrnin~ted. Alternately, if whole plants are to be used directly, the shoot 26 of the whole plant 24 is anchored by way of the mesh 18, as illustrated.
The plants may also be supported by a wire cage (not shown). The metal-cont~ining solution 14 may be aerated using, for example, an airstone 28 of an aquarium pump (not shown) or any other aeration system. The metal-cont~ining solution 14 can be periodically removed from the column 10. Solution samples may be removed and the total solution volume adjusted through the sampling tube 30. The column can itself be supported by a support element 32. In Figure 1, an outer surface 34 of the column 10 is engaged with an inner surface 36 of supportelement 32.
In the embodiment illustrated, the column is about 11.4 centimeters in diameter and about 9 centimeters deep. The depth of the metal-cont~ining solution is about 4-5 centimeters. The thickness of the metal mesh can be up to several millimeters, covered by an approximately 2 centimeter deep layer of Vermiculite Wo 94/29226 2 1 6 3 6 6 6 PCTIUSg4/06169 in which the seeds are already germin~ted, or the seedlings are transplanted, as in Fig. 1. The height of the support element is approximately the same as the solution depth, about 4-5 centimeters and the diameter (D) of support element 32is slightly larger than the diameter (D') of column 10. In Fig. 1, the element is 5 about 12 centimeters in diameter. The exact dimensions of the column and hydroponic treatment bed described herein are not intended to limit the scope ofthe invention since those having an ordinary skill in the art can easily devise other constructions that can accommodate more plants and a greater liquid volumes.
One example of such an exp~nded system is illustrated in Fig. 2. The 10 hydroponic column 10 is a subst~nti~lly elongated rectangular channel 40 having the length of about 85 centimeters, and a depth and width of about 7 centimeters.
The total internal volume of metal-cont~ining solution 14 in the column is about 7 liters. The column has two opposed ends 42, 44. Each end 42, 44 is connected by way of a conduit 46 to a reservoir 48 conf~ining the metal-cont~ining solution 14. The total volume of reservoir 48 is greater than 7 liters. In Fig. 2, the reservoir volume is 10 liters.
A recirculation pump 50 is disposed within the solution 14 of reservoir 48 and is in fluid communication with one of the conduits 46. The roots 52 of the tellc;sllial plants 24 are m~int~ined hydroponically within the column using, for example, the mesh and Vermiculite treatment bed system 12 described previously with reference to Fig. 1. In use, the solution is m~int~in~d in a constant flow over the roots 52. Alternately, the flow can be stopped and the roots allowed to takeup and/or precipitate the metal under static (i.e., no flow) conditions. The roots are then separated from the solution.
It will be appreciated that a variety of other hydroponic systems can be employed using the columns of the present invention. For example, a series of columns cont~ining hydroponic beds can be aligned in series or they can be aligned in a parallel. Moreover, the solution can flow continuously through the columns cont~ining the hydroponic treatment beds. Further, the flow can be intermittent so that the solution within a given bed is static. Regardless of the type of flow or column configuration, after a certain time sufficient for the plants to convert the soluble metal to insoluble metal, the solution can be removed and the roots separated thererlull~. Other methods, besides pumps, may be used to move wo 94/29226 2 1 6 3 6 6 6 PCT/USg4/06169 the solution through the column(s). Solution can be, for example, gravity-fed tothe column(s).
A schematic illustration of a flow-through system 54 is illustrated in Fig. 3.
A plurality of columns 10 are connected in series and the solution 14 is pumped through the column by one or more pumps 60. A conduit 62 is in fluid communication with a solution inlet conduit 44 and with a solution exit conduit 66.
Conduit 62 serves as a recirculation loop for solution flow. A schematic illustration of an inte,lllillent flow system 56 is illustrated in Fig. 4. A plurality of columns 10 is arranged in series to a solution inlet conduit 64 and to a common exit conduit 68. The flow can conveniently be stopped to provide static conditions with the columns. After uptake and/or precipitation, flow is again started.
Illustrated in Figs. 3 and 4 is a filtration block 58 placed at a downstream end 44 of the flow-through column and placed at the common exit conduit of the intermittent flow system. Filtration block 58 can contain one or more filters (not shown) for separating the precipitated, insoluble metal from the solution 14. The type of membrane used to separate the precipitated metal can be one of a varietyof commercially available filters such as, for example, those manufactured by Millipore Company, Bedford, Massachusetts, and Whatman International Ltd., Maidstone, F.ngl~nd.
There are several ways to prepare plants or their roots for removal of metals using the present methods. The simplest method is to grow plants hydroponically within the columns described herein, the columns being filled with nutrient solution. Growth is continued until the roots reach the appropliate size.
At this point, the roots are exposed to the solution cont~ining metal.
Alternatively, the plants can be grown separately in a "nursery" (either hyd~vpollically or in solid growth medium) and then transferred to the hydroponic treatment beds when the roots reach the apprupliate size.
Applicants have discovered that excised roots of sunflowers and some Brassicaceae members may be even more effective than whole plants in removing metals from solutions. To perform the methods using excised roots, roots are simply cut off from hydroponically cultivated plants and immersed in a metal-cont~ining solution. That is, a column such as those illustrated herein can be filled with excised roots and the metal-cont~ining solution allowed to contact the Wo 94/29226 2 1 6 3 6 6 6 PCT/US94/06169 roots, in a manner similar to that described previously. This particular use of excised roots has been shown to be extremely effective (see Example 4).
The invention will now be illustrated by the following Examples.
Example 1: Screenin~ of terrestrial plants for accumulation and precipitation of metals This Example illustrates removal of lead from a solution by the roots of various cool and warm season turfgrasses and other plants selected for the use in 10 the present method. Seed-grown plants or grass sod are cultivated hydroponically with roots growing in nutrient solution complemented with 0.6 g/L Ca(NO3)2.
The nutrient solution is prefeMbly 1 g/L HydrosolTM . This solution consists of the following components:

Step 1: Dissolve 0.97 g of this material in 1 liter to obtain the following concentrations:
Elemental Composition Total Nitrogen (all Nitrate) NO3- 50.0 Phosphorus P 48.0 Potassium K 210.0 Magnesium Mg 30.0 Sulfate S04 117.0 Iron Fe 3.0 Manganese Mn 0.50 Zinc Zn 0.15 Copper Cu 0.15 Boron B
0.50 Molybdenum Mo 0.10 wo 94129226 2 1 6 3 6 6 6 PCT/US94/06169 Step 2:
Add 0.644 g/L of calcium nitrate to the solution. Total nutrient concentration will be:
Nitrogen as N: 150 ppm N
5 Calcium as Ca: 129 ppm Ca Hydroponic cultivation is performed in a system similar to that shown in Fig. 2 except that a relatively small amount of root tissue is used. After 2 to 4 weeks, the nutrient solution is substituted with a continuously aerated solution of Pb(NO3)2 cont~ining 275 mg/L of lead as lead ion. The total volume of the solution is kept at 400 ml by the addition of distilled water to compensate for water lost through plant transpiration and evaporation. Lead accumulated in the plant tissue and lead rem~ining in the solution is measured after 3 days. The dirre~ ce between lead decrease in the solution and lead uptake by roots 15 represents the amount of lead precipitated by root exudate. (See Table 1, below.) Filter paper controls (thin strips of filter paper, 0.4 g DW (dry weight), immersed in the aerated lead solution) are used to demonstrate that lead uptake and precipitation is root specific. Similar results were obtained in a larger (7 L total volume) flow-through system shown in Fig. 2.

Species, 'Cultivar' Season Cultivation Dis~ a.dllce Pb in roots (Scientific Name) method from the (mg/gm DW
solution (mg root +SE) Pb/gm DW
roots +SE) Colonial bentgrass, 'Exeter' Cool Seed 675 + 200 169 + 11 (A~rostis tenuis Sibth.) Kentucky bluegrass, 'Liberty' Cool Seed 545 + 12 165 + 16 (Poa pratensis L.) Perennial ryegrass, 'Brazil Il' Cool Seed 543 + 34 134 + 3 (Lolium perenne L.) 0 Creeping bentgrass, 'Putter' Cool Seed 485 + 99 146 + 30 (A~rostis palustris Huds.) Chewing Fescue, 'Jamestown' Cool Sod 388 + 277 27 + 9 (Festuca rubra var. c~ uLdta Gaud . ) Sheep fescue, 'Bighorn' Cool Seed 352 + 59 111 + 11 (Festuca ovina L.) Weeping lovegrass CoolSeed 289 + 82142 + 12 (Era~rostis curvula (Schrad.)) Nees Hard fescue, 'Reliant' Cool Sod 258 ~ 37 102 + 9 (Festuca ovina L. var. duriscula (L-) Koch.) Tall fescue, 'Rebel' CoolSeed 243 + 7285 + 3 (Festuca d.~ r~ Schreb.) Kentucky bluegrass, 'Baron' Cool Sod238 + 31 69 + 7 Hard fescue, 'Crystal' Cool Seed 231 + 10 125 + 7 Creeping red fescue, 'Pennlawn' Cool Seed214 + 23 86 + 4 (Festuca rubra L. var. ~enuina) Perennial ryegrass 'Cosmos' Cool Sod157 + 35 80 + 9 B~ dagrass 'Sahara' WarmSeed 507 + 117 90 + 7 (Cvnodon dactvlon (L.) Pers.) Buffalograss, 'Texoka' Warm Seed 393 + 133 56 + 4 (Buchloe dactyloides (Nutt.) Engelm.) C~llLi~,cdegla,s WarmSeed 385 + 82 124 + 13 (Eremochloe ophiuroides (Munro) Hack) Switchgrass 'Blackwell' Warm Seed 342 + 109 116 + 5 (Panicum vir~atum L.) Japanese lawngrass (JM-107) Warm Seed 162 + 25 56 + 2 (Zovsia.iaponica Steud.) Coastal panicgrass 'Atlantic' Warm Seed148 + 27 109 + 9 (Panicum amarum var.
S amoralum (Hitch & Chase)) Wild cabbage (Brassica oleracea) Seed 659 + 205 134 + 15 Spinach (Spinacia oleracea L.) Seed 626 + 309 95 + 25 Sunflower (Helianthus annuus Seed 478 +87 140 + 5 L.) Sorghum (Sor~hum bicolor (L.) Seed 234 +88 88 + 7 Moench) Tobacco (Nicotiana tabacum L.)Seed 214 +41 132 + 6 Indian mustard, (Brassica iunceaSeed 177 +38 103 + 7 (L. ) Czern . ) Corn (~ mavs L.) Seed90 + 2575 + 13 Filter paper control N/A N/A 15 + 4 2 + 0 Example 2 EMS Mutagenesis This example illustrates a protocol for use in mutagenizing plant members of the family Brassicaceae.
1. Dry seeds are placed in about 100 ml of a 0.3% (v/v) solution of EMS (obtained from Sigma chemicals, St. Louis, MO). There may be some variation from batch to batch of EMS so it may be necessary to adjust this concentration somewhat. Between 20,000 to 250,000 seeds are mutagenized at a time. Ethyl methane sulfonate (EMS) is a volatile mutagen. It should be handled only in a fume hood and all solutions and materials which it contacts should be properly disposed of.
2. Seeds are mixed occasionally or stirred on a stir plate and left at room temperature for 16-20 hours. The rate of mutagensis mav be te,-,peldture-dependent so using a magnetic stir plate may alter the results by warming the solution.

3. Seeds are washed with distilled water 10 to 15 times over the course of 2 to 3 hours by dec~nting the solution, adding fresh water, mixing, allowing the Wo 94/29226 2 1 6 3 6 6 6 PCT/US94/06169 seeds to settle, and dec~nting again. After about 8 washes the seeds are transferred to a new container and the original is disposed of.

4. After washing, the seeds are immediately sown at about 3 seeds per square cm (3000 seeds in 50 ml of 0.1 % agar per 35 x 28 x 9 cm flat).

5. After several weeks it is useful to estimate the number of seeds which have gerrnin~tecl in order to know the size of the M1 generation. About 75% of the mutagenized seeds usually germinate. Ideally, the M1 estimate is the number of plants which produce M2 seed, but this is much more difficult to measure.

6. Plants are grown until they begin to die naturally and are then allowed to dry completely before harvesting. Complete drying improves the yield and simplifies harvesting.

Example 3: Kinetics of Metal Removal To determine the kinetics of lead removal, the roots of 7-week-old intact sunflower plants are incubated in a mini~turized system similar to that shown inFig. 1 (total volume 400 ml). Within 4.5 hours, roots of the intact sunflower plants removed more than 90% of 275 mg Pb+2/L initially present in the solution (Fig. 5). Total fresh root mass of plant 1 was 33.2 g (2.1 g dry weight) and of plant 2 was 30.7 g (2.3 g dry weight). Furthermore, the rate of lead removal is significantly correlated (r=0.83) with the root dry biomass used in the experiment (Fig. 6).
Other metal ions (e.g. Ca+2 , Co +2, Cu+2, and K+l ) did not significantly interfere with the ability of rhizofiltrating plants to accumulate lead ion. This suggests that the present methods can be used to remove lead from complex aqueous mixtures cont~ining difre,~lll ions.

Example 4: Metal Accumulation By Excised Roots Roots are excised from hydroponically cultivated t~ llial plants and the excised roots immersed in a column cont~ining a solution with lead ions ( 400 mltotal volume with pb+2 concentration between 275-285 mg /L). The time course of lead removal by the roots connected to the plant is also compared with uptake by excised roots alone. Figure 5 also illustrates the comparison of kinetics of lead _ uptake between the roots of the intact sunflower plants and excised sunflower roots (total fresh root biomass for excised roots was 12.1-15.7 g (0.8-1.0 g dry weight).
Figure 7 shows kinetics of lead uptake for whole B. juncea plants (O) and 5 excised B. juncea root (o). The average fresh root mass for the whole plant was 3.3 + 0.3 g (0.3 + O.lg dry weight; n = 4). The average fresh root mass for the excised roots alone was 3.7 + 1.0 g (0.3 + 0.1 g dry weight; n = 4).

Example 5: Chemical Analysis of Precipitate Lead-treated roots of bermudagrass, B. iuncea and sunflower were subjected to sc~nning electron microscopy with a Jeol 35C SEM operating at 15 kV
acceleration. ~m~ging was done in both secondary electron mode and back scattered electron mode to visualize the sites of lead deposition in the root tissue.
The initial results suggest that most of the lead ion accumulates in the extracellular 15 spaces of the epidermal layer in the form of a mixture of lead carbonate, some lead phosphate and, possibly, lead oxide.
Lead removal in the present method is caused by both metal accumulation in plant roots and by the ability of roots of living plants to precipitate lead ions from the solution. The precipitated lead gives a distinctly miLky appearance to the 20 solution surrounding the roots. Chemical analysis of the root precipitate collected from the system was performed by infrared spectroscopy and direct current plasmaspectrometry and the precipitate was identified as lead phosphate Pb3(PO4)2. Thechemical structure of the precipitate did not vary when different species were used in the system. These results may indicate that phosphate exudation functions as a 25 defense mechanism elicited in grass roots by lead and possibly other metals. The function of this defense mechanism may be to precipitate lead before it has a chance to come in contact with living root tissues.

Example 6: Vector construction and transformation of B. juncea with MT genes 30 A. Vector Construction Monkey MT cDNAs (MTl & MT2) are obtained form Dr. Dean H. Hamer, National In~titlltes of Health, Bethesda, Maryland. A 341 bp Hind m/Bam HI
fragment Co~ g the entire MTl coding sequence including the initiator WO 94/29226 2 1 6 3 6 6 6 PCT/llS94/06169 methionine codon is cloned into the Hind m/Bgl II site of pJB90 to give plasmid pNKl. pJB90 (obtained form Dr. Deepak Pental, Tata Energy Research Institute, New Delhi, India) is an Agrobacterium-based binary, plant transformation/expression, vector. This plasmid contains a plant selectable hpt 5 (hygromycin phosphotransferase) gene and a multiple cloning site for the insertion of foreign DNA, between the T-DNA border repeats. The plasmid also contains a gene for spectinomycin resistance, functional in bacterial cells. pNKl propagated in E. coli DhS was used to transform Agrobacterium tumefaciens strain pGV2260 (Deblaere et ah, Nucl. Acids. Res., 13: 4777, 1985) by the freeze-thaw method (Ebert et al., PNAS, U.S.A., 84: 5745, 1987).
B. Transformation of B. juncea Agrobacterium tumefaciens strain pGV2260 carrying pNKl is grown overnight (t220 rpm, 28C in dark) in 5 mL of liquid YEB [ beef extract-0.5%; yeast extract-0.1 %; peptone-0.5 %; sucrose-0.5 %; MgSO4 7H2O-0.005 %] cont~ining 15 100 mg/L each of spectinomycin and rifampicin. One mL of this suspension is used to inoculate 50 mL of the YEB with the same concentrations of antibiotics and allowed to grow overnight. On the third day, the bacteria are harvested by centrifugation (5500 rpm) and resuspended in filter sterilized liquid MS (see Murashige, T., and Skoog, F., Physiol. Plant. 15: 473-497, 1962) modified 20 merlillm (MS salts & vitamins with 10 g/L each of sucrose, glucose and mannitol) supplemented with 200 micromolar acetosyringone and 100 mg/L each of spectinomycin and rifampicin at pH 5.6 The optical density of the bacterial suspension is adjusted to about A600=l.0 and the bacteria grown for 6 hours, harvested as before are resuspended in the same medium. Freshly cut hypocotyl 25 explants are incubated in the bacterial suspension for lh and co-cultivated on MS
modified medium supplemented with 2 mg/L BAP (6-benzylaminopurine) and 0.1 mg/L NAA (naphthaleneacetic acid). After 2 days the explants are transferred to MS medium supplemented with 2 mg/L BAP, 0.1 mg/L 2,4-D (2-4 dichlorophenoxyacetic acid), 200 mg/L Cefotaxime and 30 micromolar Ag(NO3)2 30 and 10 mg/L Hygromycin B. After 10 days incubation on this medium, the explants are shifted to MS supplemented with 2 mg/L BAP, 0.1 mg/L NAA, 200 mg/L Cefotaxime, 10 mg/L Hygromycin B and 10% coconut milk. Shoots developed in 15-20 days are grown further and rooted in the presence of 20 mg/L

_ hygromycin. We have obtained transformants with the line 173874 at a frequency of about 2 % .
C. Characterization of MT gene expression in transgenic plant lines About 15 independent transgenic plants are generated for the B. juncea line 5 mentioned above. The putative transformants are analyzed for the presence of MT1 DNA by Southern and Northern hybridization analysis using MT1 cDNA as a probe. The putative transformants are analyzed for expression of MT1 protein by immunoblot analysis with antisera against monkey MT.
Transgenic lines expressing high MT levels are selected and tested for lead 10 and chromium accumulation and metal tolerance in greenhouse trials described above. The transgenic lines are evaluated in large scale greenhouse trials whichwill utilize lead and chromium cont~min~ted soil collected from the polluted sites.

Conclusions Roots of the best plants identified in our screens contain about 15 % by weight of lead in dry biomass (Table 1), which is the equivalent of 65% lead by weight in ash. This concentration makes reclaiming metals from ash a viable alternativeto ash burial. By combining uptake and precipitation, the roots of the most efficient plants (i.e. sunflower) remove amounts of lead equal to 60% of their total 20 dry weight. This is far beyond the capacity of all known ion exchange columns, which may be considered as an alternative to the present methods. In addition, our estim~tes suggest that the methods of the invention are an order of m~gnitllde cheaper than ion exchange-based purification schemes.

Equivalents Those skilled in the art will recognize, or will be able to ascertain using no more than routine experimentation, numerous equivalents to the specific productsand processes described herein. Such equivalents are considered to be within the5 scope of the invention and are intended to be covered by the following claims.

Claims (49)

Claims

1. A method for reducing an amount of metal in a metal-containing solution, comprising:
contacting the solution with a terrestrial plant having a root biomass, said contacting under conditions sufficient for the root biomass of the terrestrial plant to remove the metal from the solution;
separating the root biomass of the terrestrial plant from remaining parts of theterrestrial plant; and removing the root biomass from the metal-containing solution.

2. The method of claim 1, wherein the step of contacting comprises contacting the solution with the root biomass by passing the solution through a column containing the root biomass.

3. The method of claim 1, wherein the step of contacting comprises contacting a plant under conditions sufficient to convert the metal from a soluble form to an insoluble form.

4. A method of removing metal from a solution containing said metal, comprising:growing a terrestrial plant derived from a mutagenized progenitor hydroponicallyin the presence of the metal;
allowing roots of the plant to remain in contact with the solution for a time and under conditions sufficient for the roots to accumulate metal therein.

5. The system of claim 20 wherein the root portion consists essentially of excised roots.

6. The method of claims 4 or 5, wherein the step of allowing roots of the plant to remain in contact with the solution comprises allowing roots to promote precipitation of the metal from the solution.

7. The method of claim 6, further comprising separating the precipitated metal from the solution.

8. A method of removing a soluble metal from a solution, comprising:
providing a column for holding at least one plant in a solution the column having one and another ends;
introducing a solution containing the soluble metal into one end of the column;
maintaining a terrestrial plant derived from a mutagenized progenitor hydroponically within the column;
allowing the plant to accumulate the metal in roots of the plant; and removing the solution from at least one of said ends of the column.

9. The method of claim 8, further comprising harvesting the roots after allowingaccumulation of the metal therein.

10. The method of claim 9, wherein the step of harvesting occurs after the step of removing the solution.

11. The method of claim 8, wherein the step of maintaining a plant comprises maintaining a terrestrial plant that accumulates more metal in root biomass than in shoot biomass.

12. The method of claims 8 or 11, further comprising allowing roots of the plant to precipitate metal out of the solution.

13. The method of claim 12, further comprising separating the precipitated metal from the solution at one end of the treatment bed.

14. The method of claims 1, 4 or 8 wherein the metal is selected from the group consisting of lead, chromium, mercury, cadmium, cobalt, nickel, molybdenum, copper, arsenic, selenium, zinc, antimony, beryllium, gold, barium, manganese, silver, thallium, tin, rubidium, strontium, vanadium, yttrium, technecium, ruthenium, palladium, indium, cesium, uranium, plutonium, and cerium.

15. The method of claim 1, wherein the step of contacting the solution comprisescontacting with a terrestrial plant that is selected from the group consisting of sunflower, turfgrasses and members of the Family Brassicaceae.

16. The method of claim 1, wherein the step of contacting the solution with the root biomass comprises contacting under substantially constant flow conditions.

17. The method of claim 1, wherein the step of contacting the solution with the root biomass comprises contacting under intermittent flow conditions.

18. The methnd of claim 15, wherein the turfgrasses are selected from the group consisting of Colonial bentgrass, Kentucky bluegrass, perennial ryegrass, creeping bentgrass, fescues, lovegrass, Bermudagrass, Buffalograss, centipedgrass, switch grass, lawngrass and coastal panicgrass.

19. The method of claim 15, wherein the members of the Brassicaceae are selectedfrom the group consisting of Brassica juncea and B. oleracea.

20. A system for removing a soluble metal from a solution, comprising:
a receptacle for holding a solution containing a soluble metal;
a root portion of at least one terrestrial plant in contact with said solution in said receptacle, the receptacle being arranged and constructed to allow access to said root portion so that, after said root portion has been contacted with said solution and has accumulated metal, said root portion can be removed fmm said receptacle.

21. The system of claim 20, wherein the receptacle includes a column having one and another ends.

22. The system of claim 21, wherein said means for moving includes a pump in fluid communication with at one end of said column.

23. The system of claim 20, wherein said at least one terrestrial plant is capable of accumulating a metal into at least said root portion thereof.

24. The system of claim 20, wherein said at least one terrestrial plant is a turfgrass.

25. The system of claim 20, wherein said at least one terrestrial plant is a member of the Family Brassicaceae.

26. The system of claim 20, wherein said at least one terrestrial plant is a sunflower.

27. A system for removing a soluble metal from a metal-containing solution, comprising:
a column having one and another ends; a metal-containing solution in fluid communication with the column;
an isolated root portion of at least one terrestrial plant in contact with the solution, the root portion capable of accumulating the soluble metal.

28. The system of claim 27, wherein the root portion is capable of precipitating said soluble metal from said solution.

29. The system of claim 27, wherein the root portion is capable of converting the soluble metal into an insoluble form.

30. The system of claim 27, wherein said at least one terrestrial plant is a sunflower.

31. The system of claim 27, wherein said at least one terrestrial plant is a turfgrass or a member of the Family Brassicaceae.

32. The system of claim 31, wherein said turfgrass is selected from the group consisting of Colonial bentgrass, Kentucky bluegrass, perennial ryegrass, creeping bentgrass, fescues, lovegrass, Bermudagrass, Buffalograss, centipedegrass, switch grass, lawngrass and coastal panicgrass.

33. The system of claim 31, wherein said member of the Brassicaceae is selected from the group consisting of Brassica juncea and B. oleracea.

34. A method for reducing an amount of metal in a metal-containing solution, comprising contacting the solution with a root biomass of a plant derived from amutagenized progenitor, the contacting being performed under conditions sufficient for the root biomass to remove metal from the solution.

35. The method of claim 34 wherein the contacting comprises contacting the solution with an isolated root biomass of the plant.

36. The method of claim 34, wherein the terrestrial plant is derived from a progenitor mutagenized with EMS.

37. A method for reducing an amount of metal in a metal-containing solution comprising:
contacting the solution with a root biomass of a terrestrial plant that is a transformant expressing a heterologous gene under conditions sufficient for the root biomass to remove metal from the solution; and separating the root biomass from the solution.

38. The method of claim 37, wherein the heterologous gene comprises a metallothionein structural gene.

39. A method for removing metal from a metal-containing solution comprising the steps of:

growing a terrestrial plant in a solid support;
contacting the solid support with a metal-containing solution under conditions sufficient for roots of the plant to remove metal from the metal-containing solution;
separating the roots of the plant from other parts of the plant; and removing the roots from the metal-containing solution.
allowing accumulation of the metal in roots of the terrestrial plant.

40. The method of claim 39, wherein the step of contacting comprises contacting an aqueous mist or aerosol form of the solution with a root biomass of a terrestrial plant under conditions sufficient for the root biomass to remove the metal from the solution.

41. The method of claim 8 wherein the column is opaque.

42. The method of claim 1, further comprising, allowing the remaining parts of the terrestrial plant to regrow new root biomassunder conditions sufficient for the regrown root biomass to remove the metal from the metal-containing solution; and contacting the metal-containing solution with the terrestrial plant having the regrown root biomass under conditions sufficient for the regrown root biomass to remove the metal from the metal-containing solution.

43. A method of removing metal from a solution containing said metal, comprising:
hydroponically growing a terrestrial plant in the presence of the metal, the terrestrial plant being a transformant expressing a heterologous gene;
allowing roots of the plant to remain in contact with the solution for a time and under conditions sufficient for the roots to accumulate metal therein.

44. The method of claim 43, wherein the step of growing a transformant comprisesgrowing a transformant containing a heterologous gene that comprises a metallothionein structural gene.

45. A method of removing a metal from a solution, comprising:
providing a column for holding at least one plant in a solution, the column having one and another ends;
introducing a solution containing the metal into one end of the column;
hydroponically maintaining within the column a terrestrial plant that is a transformant expressing a heterologous gene;
allowing the plant to accumulate the metal in roots of the plant; and removing the solution from at least one of said ends of the column.

46. The method of claim 45, wherein the step of maintaining a transformant comprises maintaining a transformant containing a heterologous gene that comprises a metallothionein structural gene.

47. A method for reducing an amount of metal in a metal-containing solution, comprising:
contacting excised roots of a terrestrial plant with a metal-containing solutionunder conditions sufficient for the excised roots to remove the metal from the solution;
and separating the excised roots from the solution.

48. The method of claim 47, further comprising hydroponically growing the terrestrial plant and excising the roots thereof prior to contacting the excised roots with the metal-containing solution.

49. A method for reducing an amount of metal in a metal-containing solution, comprising:
selecting a terrestrial plant that accumulates more metal in root biomass than in shoot biomass;
contacting the solution with the root biomass of the terrestrial plant under conditions sufficient for the root biomass of the terrestrial plant to remove the metal from the solution; and separating the root biomass of the terrestrial plant from remaining parts of the terrestrial plant.