¹ Environmental Chemistry Lab, US Department of
Agriculture-Agricultural Research Service, Bldg. 007, BARC-West, Beltsville, MD
² Dept. Natural Resources and Landscape Architecture, University of Maryland, College Park, MD 20742.
³ Dept. Animal and Plant Sciences, University of Sheffield, Sheffield, S1O 2UO, United Kingdom.
Phytoremediation of metal contaminated soils offers a lower cost method for soil remediation and some extracted metals may be recycled for value. Both the phytoextraction of metals and phytovolatilization of Se or Hg by plants offer great promise of commercial development. Natural metal hyperaccumulator phenotype is much more important than high yield ability when using plants to remove metals from contaminated soils. The hypertolerance of metals is the key plant characteristic required for hyperaccumulation; vacuolar compartmentalization appears to be the source of hypertolerance of natural hyperaccumulator plants. Alternatively, soil Pb and Cr6+ may be inactivated in the soil by plants and soil amendments (phytostabilization). Little molecular understanding of plant activities critical to phytoremediation has been achieved, but recent progress in characterizing Fe, Cd, and Zn uptake by Arabidopsis and yeast mutants indicates strategies for developing transgenic improved phytoremediation cultivars for commercial use.
Because the costs of growing a crop are minimal compared to those of soil removal and replacement, the use of plants to remediate hazardous soils is seen as having great promise; other recent reviews on many aspects of soil metal phytoremediation are available [1,2**,3*,4**,5*,6*,7**]. Phytoremediation is the use of plants to make soil contaminants non-toxic and is often also referred to as bioremediation, botanical-bioremediation, and Green Remediation. The idea of using rare plants which hyperaccumulate metals to selectively remove and recycle excessive soil metals was introduced in 1983 , gained public exposure in 1990 , and has increasingly been examined as a potential practical and more cost effective technology than soil replacement, solidification, or washing strategies presently used [2**,3*,7**]. Categories of phytoremediation include phytoextraction (the use of plants to remove contaminants from soils), phytovolatilization (the use of plants to make volatile chemical species of soil elements), rhizofiltration (the use of plant roots to remove contaminants from flowing water) and phytostabilization (the use of plants to transform soil metals to less toxic forms, but not remove the metal from the soil). The use of plants and associated rhizosphere organisms or bioengineered plants to metabolize toxic organic compounds also appears promising (recently reviewed by Cunningham et al. [10**]).
Phytostabilization appears to have strong promise for two toxic elements, chromium and lead. Reduction of Cr6+, which poses an environmental risk, to Cr3+ which is highly insoluble and not demonstrated to pose an environmental risk ), by deep rooted plants can be very effective. Chemical species of Pb in soil are usually somewhat bioavailable if the soil is ingested by children, livestock, or wildlife , while a Pb phosphate mineral, chloropyromorphite is both extremely insoluble and non-bioavailable [13,14**,15,16,17**] but it is formed slowly apparently because the reactants have low solubility. Roots of Agrostis capillaris growing in highly contaminated Pb/Zn mine wastes caused the formation of pyromorphite from soil Pb and phosphate, but the mechanism remains unknown [17**]. Although it was believed that Thlaspi rotundifolium hyperaccumulated Pb, Zea mays accumulated higher Pb levels in controlled tests if soil pH and P were low [18*]. Addition of chelating agents (e.g. HEDTA, EDTA) to such soils increased Pb solubility and mobility within plants; shoot Pb reached 1%, allowing removal of enough Pb to encourage further evaluation of this approach [18*,19**]. Methods to prevent leaching of Pb-chelates down the soil profile would be required to permit such additions in the field in regions where net infiltration occurs. Inactivating soil Pb by use of soil amendments and revegetation to prevent erosion is increasingly seen as the promising soil Pb remediation technology [12,20].
Different views of the potential for use of phytoremediation to clean up contaminated soils have developed among researchers. Some have examined the naturally occurring metal hyperaccumulators, plants which can accumulate 10--500 times higher levels of elements than crops; Reeves  suggested a widely accepted definition of Ni-hyperaccumulators: 'a plant in which a nickel concentration of at least 1000 g g-1 has been recorded in the dry matter of any above-ground tissue in at least one specimen growing in its natural habitat.' This definition can be adapted to other elements. Most plant species suffer significant yield reduction when shoots reach 50-100 mg Ni kg-1 dry weight while Ni-hyperaccumulators tolerate at least 10-20 times the normal maximum tolerable levels; and among the smaller group of plants which can tolerate at least 1% Ni in shoots, a few can reach 5% Ni, or 500 times the shoot Ni tolerated by crop plants. Species which accumulate over 1% Ni have been called 'hypernickelophores' by Jaffré . This term seems appropriate for the plant species which accumulate over 1% of several elements (hypernickelophore, hyperzincophore, etc.) because this ability is qualitatively different than the hyperaccumulators as defined by Reeves . Crop plants tolerate higher shoot Zn and Mn levels than Ni (about 300-500 mg Zn kg-1), so 'hyperaccumulators' contain > 1% shoot Zn or Mn . Shoot Cd levels are usually <1 mg kg-1, so 'hyperaccumulators' must accumulate and tolerate 100 mg Cd kg-1; some hypertolerate > 1% Cd .
How do hyperaccumulators achieve this remarkable bio-accumulation of soil metals? Research has identified several characteristics that are important:
1) The plant must be able to tolerate high levels of the element in root and shoot cells; hypertolerance is the key property which makes hyperaccumulation possible. Such hypertolerance is believed to result from vacuolar compartmentalization and chelation [24,25**]. The most direct demonstration used isolated vacuoles from protoplasts of tobacco cells which had accumulated high levels of Cd and Zn . Whether hypertolerance in the known hyperaccumulators is due to an enhancement of these mechanisms is not yet known. However, electron microprobe analysis  supports vacuolar compartmentation for Zn in leaves of the hyperaccumulator Thlaspi caerulescens.
2) A plant must have the ability to translocate an element from roots to shoots at high rates. Normally root Zn, Cd or Ni concentrations are 10 or more times higher than shoot concentrations, but in hyperaccumulators, shoot metal concentrations can exceed root levels [27,28**,29**]. Krämer et al. [29**] recently found that although the chemical forms of Ni found in extracts of leaves of Alyssum hyperaccumulators are the chelates with malate and citrate, in the xylem exudate histidine chelates about 40% of the total Ni present; nearly all of the histidine in exudate is chelated with Ni. Whether Ni(histidine)2, Ni2+ or a mixed chelate such as Ni(histidine, malate) is pumped into the xylem by a membrane transporter remains unknown. Additions of histidine to nutrient solution increased Ni tolerance and transport to shoots by Alyssum montanum, a nonhyperaccumulator species.
3) There must be a rapid uptake rate for the element at levels which occur in soil solution. Here quite different patterns have been observed in different groups of hyperaccumulators. Brown et al.  found that T. caerulescens accumulated Zn and Cd from nutrient solution only about as well as tomato and Silene vulgaris did, but tomato was severely injured at 30 µM Zn, S. vulgaris at 320 µM Zn, and T. caerulescens only at 10,000 µM Zn. Because this species can keep tolerating and accumulating Zn and Cd at high soil solution levels, it is found in nature with 1--4% Zn while surrounding plants are << 0.05% Zn (Zn excluders). Further, studies have shown that Zn hypertolerant genotypes of T. caerulescens require much higher solution Zn2+ (104-fold) and leaf Zn concentrations (100-300 mg kg-1 vs. 10-12 mg kg-1 in normal plants) to grow normally than do related non hyperaccumulator species [28**]. By implication, the highly effective compartmentalization to reduce the toxicity of Zn and Cd appears to require the plant to accumulate much more Zn to have adequate supply. In contrast, the Ni-hyperaccumulator Alyssum species accumulate remarkably higher shoot Ni levels compared to other species grown at the same Ni2+ activity in solution [29**,30]. And the Se-hyperaccumulating species similarly accumulate higher shoot Se levels and many can volatilize Se at high rates growing beside plants with more normal levels and slow volatilization [31,32].
What evolutionary advantage does metal hyperaccumulation give these species? Boyd et al. [33,34] have demonstrated that high (but not low) Ni levels in leaves of hyperaccumulators can reduce herbivory by chewing insects and reduce the incidence of bacterial and fungal diseases. Similar results were found for Zn in Thlaspi caerulescens (A.J. Pollard and A.J.M. Baker, unpublished data).
For effective development of phytoremediation, each element must be considered separately because of its unique soil and plant chemistry. Both agronomic management practices and plant genetic abilities need to be optimized to develop commercially useful practices. Some elements can be accumulated by plant roots and converted to a volatile species such as dimethylselenide  or Hg0 [35**]. Although many plants can volatilize dimethylselenide (or dimethyldiselenide in the case of the Se hyperaccumulators) , co-contaminating sulfate and salinity in Se contaminated soils commonly inhibit this process) [36,37]; very high B or salinity can kill most plants. So growing species in normal crop rotations which can phytovolatilize soil Se, or accumulate Se into the forage biomass for sale as a Se supplement for livestock feeds are alternative approaches to treating irrigation drainage waters which are much higher in B and sulfate than the water used for irrigation [36,38].
Whether metal hyperaccumulation in shoots or high shoot biomass is more important in phytoremediation of soil metals has been debated [2**,3*,7**]. A quantitative example may provide clarity: presume that a high-biomass crop plant is grown on a contaminated soil and the soil pH is lowered to increase Zn uptake to attain Zn phytotoxicity with 50% yield reduction; Zea mays and Brassica juncea are examples of such annual crops. Under favorable conditions, these plants can reach 20 t dry biomass ha-1. In the case of the usual Zn and Cd co-contamination at 100 mg Zn:1 mg Cd, crop plants suffer significant yield reduction when the shoots have about 500 mg Zn kg-1 at harvest. Because Cd is not 100-times more toxic than Zn, soil Zn phytotoxicity is the factor controlling plant yield. At 50% yield reduction (10 t ha-1), dry biomass contains 500 mg kg-1 (500 g Zn t-1); one removes only 5 kg of Zn ha-1 year-1. T. caerulescens, which can remove both soil Zn and Cd, has a low yield compared to the above species, but can hypertolerate up to 25,000 mg Zn kg-1 (25 kg t-1)  without yield reduction. Even with a low yield of 5 t ha-1 at the point of incipient yield reduction, Zn removal would be 125 kg ha-1. We conclude that the ability to hyperaccumulate and hypertolerate the metals to be phytoremediated is of greater importance than high biomass. Some authors have suggested that yield of a crop would be two orders of magnitude higher than that for hyperaccumulators such as T. caerulescens, but pot and field studies show that such perennial species grown as a crop can attain as high as 5 t ha-1 before breeding to increase the combination of yield and shoot metal concentration [27,28**]. Further, recycling of shoot metals in commerce may provide value for the ash from metal hyperaccumulators such that there is need to pay for safe disposal. Continuing the above model, biomass ash contains 20-40% Zn for T. caerulescens, but only 0.5% for Zea mays; the former is a rich ore, while the latter is a phytotoxic waste requiring disposal. Increasing the yield of a crop could give a linear increase in phytoremediation capacity with increasing yield. But increasing from 'normal' tolerance to 'hypertolerance' and hyperaccumulation increases the potential annual removal of the soil contaminant 25--400 fold. Even for elements which have little value in the biomass, the higher the concentration, the less expensive the disposal of the phytoremediation crop residue or ash (e.g., 137Cs; As; U) will be. Thus, we have emphasized the importance of domestication of metal hyperaccumulator plants and breeding of improved cultivars [7**,28**], the characterization of mechanisms used by hyperaccumulators to accumulate, translocate, and hypertolerate metals, and, eventually, the cloning and use of these genes to convert high biomass agronomic plants into special phytoremediation cultivars if this is required for some elements (7**).
Remediation of other elements (e.g. As, Cu, Cs, Sr, U) from soils by hyperaccumulator crops has not been demonstrated, but is expected to be possible if creative research is applied [2**,3*,7**,35**]. In some cases, the phytoremediation of an element may require soil amendments such as chelating agents because soil or plant chemistry reduces element uptake or translocation to shoots [18*,19**].
Biotechnology approaches to develop phytoremediation plants have been examined. Traditional plant breeding can only use available genetic diversity within a species to combine the characteristics needed for successful phytoremediation. Researchers expected that increasing the concentrations of metal binding proteins or peptides in plant cells would increase metal binding capacity and tolerance. Although plant cell cultures expressing mammalian metallothioneins (MTs)  or phytochelatins (PCs) [41**] are more tolerant of acute Cd toxicity, the transfer of mammalian metallothionein genes to higher plants appear to provide no benefit for phytoremediation. Further, when natural metal hypertolerant plants were examined, the concentration of PCs showed no difference, suggesting that hypertolerance to Cd and Zn in these plants were not due to the hyperaccumulation of PC peptides [42,43]. The evidence for the role of PCs is that their presence does correlate with normal levels of metal tolerance, since mutations that abolished PC production in Arabidopsis and fission yeast resulted in hypersensitivity to Cd [41**,44*,45*]. Cd-sensitive (hypotolerant) single gene mutants cad1 [44*] and cad2 [45*] of Arabidopsis thaliana have been identified and studied (blocked in glutathione synthesis or PC synthesis). For a plant species with normal tolerance ( A. thaliana), PCs were essential for the normal level of tolerance.
Interestingly, when these researchers tested genotypes without and with effective phytochelatin biosynthesis, the outcome was a surprise in that the sensitive mutants (low PCs) had a significantly lower degree of transport of Cd to shoots than the wildtype [45*]. A similar result was observed in corn inbreds which differed substantially in shoot Cd; higher levels of PCs were associated with higher shoot Cd .
Although these studies have allowed cloning of genes involved in acute Cd tolerance, and characterization or confirmation of metabolic pathways, the environmental relevance of findings from such acute Cd exposure has not been established. An alternative view of Cd-catalyzed PC biosynthesis is that chelation of PCs with Cd alleviates the feedback inhibition of the PC-synthase; as long as Cd activity in the cytoplasm is high, an enzyme supports more transfer to form more PCs and longer PCs. Because the level of Zn present in nearly all environments is 100 times higher than that of Cd, if an acutely toxic Cd dose is provided, the plants would be killed by Zn. Even the formation of the sulfide-stabilized high molecular weight Cd-PC complex in vacuoles [4**,25**,41**] may result from the acutely toxic Cd supply without Zn. Further, the finding that the hmt1 vacuolar membrane pump protein (which restored Cd hypertolerance to mutant fission yeast) transported both Cd-PCs and PCs without Cd, raises questions about how the pump works to induce Cd hypertolerance in vivo. Cd phytotoxicity in soil is a recent anthropogenic effect, whereas Zn phytotoxicity and co-accumulation of trace levels of Cd which are normal biogeochemical phenomena. We believe that scientists should place less emphasis on 'Cd hypertolerance' in plants. It seems increasingly likely that the Cd hypertolerance mechanisms are incidental biochemical phenomena. Although Cd-PCs can be found at low levels in plants in the environment, they account for only a small fraction of the tissue Cd [47,48,49*].
Another goal of developing transgenic plants with increased metal binding capacity was to use these metal-binding factors to keep Cd in plant roots, thus reducing Cd movement to the food chain or into tobacco [50,51,52,53]. Vacuolar compartmentation of Cd only in roots may reduce Cd translocation to shoots; expression in plants of the hmt1 vacuolar pump for Cd-PCs from fission yeast [25**] has not yet been successful, and modification of gene sequences may be required before its effectiveness can be tested (similar to the mercury reductase gene sequence changes [35**]). The expression of MT as the whole protein, the Cd binding '-domain' part of the protein, or a fusion protein with -glucuronidase, under several promoters [50,51,52,53] increased Cd tolerance of tobacco and other plants, but had little effect on Cd transport to shoots. Recently use of the improved 35S2 promoter may have increased the ability of MT to keep Cd in roots ; tests have not yet progressed to soil studies which must be the important measure of success. Many of the studies noted here have used acutely toxic levels of Cd, such that the study results do not model metal-contaminated soils in the environment. Rauser and Meuwly  used non toxic levels of Cd (3 M, 30-times the level generally found in soil solution) to study PC physiology in Zea mays, and found that in the short term PCs bound only a small fraction of cell Cd, but, over time, over 90% of root Cd was bound to PCs. McKenna and Chaney  used chelator-buffered [55**] Cd to grow lettuce at Cd levels relevant to food chain safety and found no evidence of Cd-PCs in lettuce leaves.
Because Poaceae species secrete mugineic acid family phytosiderophores (chelating agents) to solubilize soil Fe, and accumulate the intact chelate into root cells [56**], Raskin [5*] suggested that transgenic plants could be developed to secrete metal selective ligands into the rhizosphere which could specifically solubilize elements of phytoremediation interest. Although this approach holds promise, phytosiderophores obtain their specificity not by chelation specifically only of Fe in soils, but from their uptake of nearly only Fe-phytosiderophores by a membrane carrier [55**,56**,57*]. Finding other simple biosynthetic molecules with selective chelation ability, that plants can make and secrete into the rhizosphere at adequate concentrations, and simultaneously creating a selective transport protein for the metal chelate seems difficult, but worth examination to develop unique phytoremediation tools. Regulatory control of phytosiderophore secretion in barley was induced by Fe-deficiency, but not Mn, Zn, or Cu deficiency [58*] in contrast with other reports which indicated that Zn deficiency also induced the biosynthesis and secretion of phytosiderophores.
Lastly, extensive progress has recently been achieved in identifying genes and proteins involved in uptake of Fe by yeast and plants [59**,60**,61*,62**]; high affinity Zn  and Cu membrane transporters have also been found in yeast. A fundamental understanding of both uptake and translocation processes in normal plants and metal hyperaccumulators, regulatory control of these activities, and the use of tissue specific promoters offers great promise that use of molecular biology tools can give scientists the ability to develop effective and economic phytoremediation plants for soil metals.
Extensive progress has been made in characterizing soil chemistry management needed for phytoremediation, and physiology of plants which hyperaccumulate and hypertolerate metals. It is increasingly clear that hypertolerance is fundamental to hyperaccumulation, and high rates of uptake and translocation are observed in hyperaccumulator plants. Fundamental characterization of mechanisms, and cloning of genes required for phytoremediation has begun with the mercuric ion reductase [35**], and hmt1  expression in higher plants is expected soon. Improved hyperaccumulator plants and agronomic technology to improve the annual rate of phytoextraction and to allow recycling of soil toxic metals accumulated in plant biomass is very likely to support commercial environmental remediation which society can afford in contrast with present practices. Although most phytoremediation systems are still in development, or in plant breeding to improve the cultivars for field use, application for Se phytovolatilization has already begun. Many opportunities have been identified for research and development to improve the efficiency of phytoremediation. Progress had been hindered by limited funds for research and development for 12 years since the first report of the model for phytoremediation . New commercial firms are moving into this field and phytoremediation technologies will be increasingly applied commercially in the near term.
Papers of particular interest, published within the annual period of review, have been highlighted as:
* of special interest
** of outstanding interest
This article originally appeared as:
Chaney, R.L, M. Malik, Y.M. Li, S.L. Brown, J.S. Angle and A.J.M. Baker. 1997. Phytoremediation of soil metals. Current Opinions in Biotechnology 8:279-284.
The authors' manuscript has been made available by Dr. Rufus Chaney and has been transcribed to HTML and posted on the server of the Dept. of Soil Science, Univ. of Wisconsin-Madison, by Dr. Phillip Barak for instructional purposes and as a service to the web-browsing public.
--9 Jul 1997, Madison, WI