The Use of Plants for the Removal of Toxic Metals
from Contaminated Soil
Mitch M. Lasat
American Association for the Advancement of Science
Environmental Science and Engineering Fellow
Ni Cd
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NOTICE
This publication was developed under Grant No. CX 824823 a\varded by the U.S.
Environmental Protection Agency. It has not been formally reviewed by EPA. The views
expressed in this-document are solely those of Mitch Lasat-and EPA -doesnoF&idtfrse any
products or commercial services'n,~.,itionedfril'fhis publication."
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54O2OO11
Abstract
Phytoremediation is an emerging technology that employs the use of higher plants for the
cleanup of contaminated environments. Fundamental and applied research have unequivocally
demonstrated that selected plant species possess the genetic potential to remove, degrade,
metabolize, or immobilize a wide range of contaminants. Despite this tremendous potential,
phytoremediation is yet to become a commercial technology. Progress in the field is precluded by
limited knowledge of basic plant remedial mechanisms. In addition, the effect of agronomic
practices on these mechanisms is poorly understood. Another limitation lies within the very
biological nature of this novel approach. For example, potential for phytoremediation depends
upon the interaction among soil, contaminants, microbes, and plants. This complex interaction,
affected by a variety of factors, such as climatic conditions, soil properties, and site hydro-
geology, argues against generalization, and in favor of site-specific phytoremediating practices.
Thus, an understanding of the basic plant mechanisms, and the effect of agronomic practices on
plant/soil/contaminant interaction would allow practitioners to optimize phytoremediation by
cuctomizing the process to site specific conditions.-1.''i-'--
.^.emedistion cf metal cnntaminHted. soil facesta-particular challenge. Unlike organic
contaminants, metals cannot be degraded. .Commonly, dec.vtaminetion of metal-contaminated
soils requires the removal of toxic metals. Recently, phytoextraction, the use of plants to extract
toxic metals from contaminated soils, has emerged as a cost-effective, environment-friendly
cleanup alternative. In this paper, we review the processes and mechanisms that allow plants to
remove metals from contaminated soils and discuss the effects of agronomic practices on these
processes.
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TABLE OF CONTENTS
I. INTRODUCTION
Background
Advantages and disadvantages of phytoremediation
Markets for phytoremediation
H. TOXIC METALS IN SOIL
Sources of contamination
Risk assessment
Total and bioavailable soil fractions
Effect of soil properties on metal bioavailability
III. PHYTOREMEDIATESG PLANTS
Why do plants take up toxic metals?
What is a hyperaccumulator species?
How do plants tolerate high metal concentration in soil?
Mechanisms of metals uptake into root and translocation to shoot
Plant mechanisms for metal detoxification
Plant limitations
Improving phytoremediating plants
IV. PLANT-METAL INTERACTION IN THE RHIZOSPHERE
Metal bioavailability for uptake into roots
Effect of soil microorganisms on metal uptake
Effect of root exudates on metal uptake
V. OPTIMIZATION OF METAL PHYTOEXTRACTION WITH AGRONOMIC
PRACTICES
Plant selection
Soil fertilization and conditioning
Enhancing metal bioavailability with synthetic chelators
Sowing
Crop rotation
Crop maintenance: pest control and irrigation
Handling and disposal of contaminated waste
Cost and time projections
Research needs
VI. LITERATURE CITED
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I. INTRODUCTION
Background
The concept of using plants to clean up contaminated environments is not new. About
300 years ago, plants were proposed for use in the treatment of wastewater (Hartman,1975). At
the end of the 19th century, Thlaspi caerulescens and Viola calaminaria were the first plant
species documented to accumulate high levels of metals in leaves (Baumann,1885). In 1935,
Byers reported that plants of the genus Astragalus were capable of accumulating up to 0.6 %
selenium in dry shoot biomass. One decade later, Minguzzi and Vergnano (1948) identified
plants able to accumulate up to 1% Ni in shoots. More recently, Rascio, (1977) reported
tolerance and high Zn accumulation in shoots of Thlaspi caerulescens. Despite subsequent
reports claiming identification of Co, Cu, and Mn hyperaccumulators, the existence of plants
hyperaccumulating metals other than Cd, Ni, Se and Zn has been questioned and requires
additional confirmation (Salt et al., 1995). The idea of using plants to extract metals from
contaminated soil was reintroduced and developed by Utsunamyia (1980) and Chaney (1983),
and the first field trial on Zn and Cd phytoextraction was conducted in 1991 (Baker et al.). In the
last decade, extensive research has been conducted to investigate the biology of metal
phytoextraction. Despite significant success, our understanding of the plant mechanisms that
aliow metal extraction is still emerging. In addition, relevant applied aspects, such as the effect
of agronomic practices on metal removal by plants are largely unknown. It is conceivable that
maturation of phytoextraction into a commercial technology will ultimately depend on the
elucidation of plant mechanisms and application of adequate agronomic practices. Natural
occurrence of plant species capable of accumulating extraordinarily high metal levels makes the
investigation of this process particularly interesting.
Advantages and disadvantages of phytoremediation
Metal-contaminated soils are notoriously hard to remediate. Current technologies resort to
soil excavation and either landfilling or soil washing followed by physical or chemical separation
of the contaminants. The cost of soil remediation is highly variable and depends on the
contaminants of concern, soil properties, and site conditions. Cost estimates associated with the
use of several technologies for the cleanup of metal-contaminated soil are shown in Table 1.
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Table 1. Cost of soil treatment (Glass, 1999a)
Treatment
Vitrification
Landfilling
Chemical treatment
Electrokinetics
Phvtoextraction
Cost CS/ton)
75-425
100-500
100-500
20-200
5-40
Additional factors/expenses
Long-term monitoring
Transport/excavation/monitoring
Recycling of contaminants
Monitoring
Monitoring
Cleaning of metal-contaminated soils via conventional engineering methods can be
prohibitively expensive (Salt et al., 1995). The costs estimated for the remediation of sites
contaminated with heavy metals, and heavy metals mixed with organic compounds are shown in
Table 2.
Table 2. Projected five-year costs for remediation of sites contaminated with toxic metals
only, and mixtures of toxic metals and organics (U.S. EPA, 1993).
Sector
Superfund1
RCRA2
DOD'
DOE4
State'
Private6
Total
Metals Onlv
2;400
3,000
400
900
200
200
7.100
Metais and Orsanics
—--S- million .-
10,400
12,800
2,400
6,500
800
2,500
35.4CO
1 Sites ranked on the National Priorities List
2 Sites requiring corrective action under the provisions of Resource Conservation and
Recovery Act RCRA
3 Department of Defense
4 Department of Energy
5 State-funded contaminated sites
6 Private-funded contaminated sites
Because of the high cost, there is a need for less-expensive cleanup technologies.
Phytoremediation is emerging as a cost-effective alternative. Several analyses have demonstrated
that the cost of metal phytoextraction is only a fraction of that associated with conventional
engineering technologies (Table 1). In addition, because it remediates the soil in situ,
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phytoremediation avoids dramatic landscape disruption, and preserves the ecosystem. Despite
these advantages, several disadvantages and constraints restrict the applicability of
phytoextraction (Table 3).
Table 3. Major factors limiting the success and applicability of phytoextraction
Plant-based biological limitation Regulatory limitations Other limitations
1) Low plant tolerance 1) Lack of cost and performance 1) Contaminant beneath root
2) Lack of contaminant data zone
translocation from root to 2) Regulators unfamiliarity with 2) Lengthy process
shoot the technology 3) Contaminant in biologically
3) Small size of remediating 3) Disposal of contaminated unavailable form
plants plant waste 4) Lack of remediating plant
4) Risk of fowl chain specie^
contamination
Markets for phytoremediation
A comprehensive analysis of phytoremediation markets was published by Glass (1999a;
1999b). The author indicated that the estimated 1999 phytoremediation markets was two fold
greater than 1998 estimates. This growth has been attributed to an increased number of
companies offering services, particularly companies in the consulting engineering sector, and to
growing acceptance of the technology. An estimate of 1999 U.S. phytoremediation markets
related to a variety of contaminated media and contaminants of concern is shown in Table 4.
Table 4. Estimated 1999 U.S. phytoremediation markets (Glass, 1999b)
Organics in groundwater S 7-12 million
Landfill leachate S 5-8 million
Organics in soil S 5-7 million
Metals in soil S 4.5-6 million
Inorganics in wastewater S 2-4 million
Inorganics in groundwater S 2-3 million
Organics in wastewater S 1-2 million
Metals in groundwater S 1-2 million
Radionuclides S 0.5-1 million
Metals in wastewater S 0.1-0.2 million
Other S 1.9-3.8 million
Total S 30 -49 million
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Current estimates of 1999, and 2000 revenues were slightly lower than what had been
previously projected, largely due to slower commercialization of the technology for the cleanup
of metal- and radionuclide-contaminated sites (Glass, 1999b).
The second largest market for phytoremediation was identified in Europe, although
European market was estimated to be 10-fold-smaller, than the U.S. market (Glass 1999b).
II. TOXIC METALS IN SOIL
Sources of contamination
Heavy metals are conventionally defined as elements with metallic properties (ductility,
conductivity, stability as cations, ligand specificity, etc.) and atomic.number >20. The most
common heavy metal contaminants are: Cd, Cr, Cu, Hg,:Pb, and Zn. Metals are natural
components in soil. Contamination, however, has resulted from industrial activities, such as
mining and smelting of metalliferous ores, electroplating, gas exhaust, energy and fuel
production, fertilizer and pesticide application, and generation of municipal waste (Kabata-
Pendias and Pendias,! 989). Soil concentration range and regulatory limits for several major.
metal contaminants are shown in Table 5.
Table 5. Soil concentration ranges and regulator}':guidelines for some toxic metals
Soil concentration range2 Regulatory limitsb
Metal (mg kg1) (mg kg1)
Pb
Cd
Cr
Hg
Zn
1.00-6,900
0.10-345
0.05-3,950
O.01-UOO
150.00-5,000
600
100
100
270
1,500
/a/., 1992
b) Nonresidential direct contact soil cleanup criteria (NJDEP, 1996)
High ieveis--of metals in soil can be phytotoxic. Poor plant growth and soil cover caused
by metal toxicity can lead to metal mobilization in runoff water and subsequent deposition into
nearby bodies of water. Furthermore, bare soil is more susceptible to .wind erosion and spreading
of contamination by airborne dust. In such situations, the immediate goal of remediation is to
reclaim the site by establishing a vegetative cover to minimize soil erosion and pollution spread.
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Risk assessment
Soil remediation is needed to eliminate risk to humans or the environment from toxic
metals. Human disease has resulted from Cd (Nogawa et al., 1987; Kobayashi 1978; Cai et al,
1990), Se (Yang et al., 1983), and Pb in soil (Chancy et al., 1999). Livestock and wildlife have
suffered from Se poisoning (Rosenfeld and Beath, 1964; Ohlendorf et al., 1986). In addition, soil
contamination with Zn, Ni and Cu caused by mine wastes and smelters is known to be phytotoxic
to sensitive plants (Chaney et al., 1999). One of the greatest concerns for human health is caused
by Pb contamination. Exposure to Pb can occur through multiple pathways, including inhalation
of air and ingestion of Pb in food, water, soil or dust. Excessive Pb exposure can cause seizures,
mental retardation and behavioral disorders. The danger of Pb is aggravated by low
environmental mobility even under high precipitations.
Total and bioavailable soil fractions
In soil, metals are associated with several fractions: (1) in soil solution, as free metal ions
and soluble metal complexes, (2) adsorbed to inorganic soil constituents at ion exchange sites,
(3) bound to soil organic matter, (4) precipitated such as oxides, hydroxides, carbonates, and (5)
embedded in structure of the silicate minerals. Soil sequential extractions are employed to isolate
and quantify metals associated with different fractions (Tessier et al., 1979).
For phytoextraction to occur, contaminants must be bioavailable (ready to be absorbed by
roots). Bioavailability depends on metal solubility in soil solution. Only metals associated with
fractions 1 and 2 (above) are readily available for plant uptake. Some metals, such as Zn and Cd,
occur primarily in exchangeable, readily bioavailable form. Others, such as Pb, occur as soil
precipitate, a significantly less bioavailable form.
Effect of soil properties on metal bioavailability
The chemistry of metal interaction with soil matrix is central to the phytoremediation
concept. In general, sorption to soil particles reduces the activity of metals in the system. Thus,
the higher the cation exchange capacity (CEC) of the soil, the greater the sorption and
immobilization of the metals. In acidic soils, metal desorption from soil binding sites into
solution is stimulated due to FTcornpetition for binding sites. Soil pH affects not only metal
bioavailabilty, but also the very process of metal uptake into roots. This effect appears to be
metal specific. For example, jn T. caerulescens, Zn uptake in'roots showed a small pH
dependence, whereas uptake of Mn and Cd was more dependent on soil acidity (Brown et al.,
1995a).
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III. PHYTOREMEDIATING PLANTS
Why do plants take up toxic metals?
To grow and complete the life cycle, plants must acquire not only macronutrients (N, P,
K, S, Ca, and Mg), but also essential micronutrients such as Fe, Zn, Mn, Ni, Cu, and Mo. Plants
have evolved highly specific mechanisms to take up, translocate, and store these nutrients. For
example, metal movement across biological membranes is mediated by proteins with transport
functions. In addition, sensitive mechanisms maintain intracellular concentration of metal ions
within the physiological range. In general, the uptake mechanism is selective, plants
preferentially acquiring some ions over others. Ion uptake selectivity depends upon the structure
and properties of membrane transporters. These characteristics allow transporters to recognize,
bind and mediate the trans-membrane transport of specific ions. For example, some transporters
mediate the transport of divalent cations, but do not recognize mono- or trivalent ions.
Many metals such as Zn, Mn, Ni and Cu are essential micronutrients. In common
nonaccumulator plants, accumulation of these micronutrients does not exceed their metabolic
needs (<10ppm). In contrast, metal hyperaccumulator plants can accumulate exceptionally high
amounts of metals (in the thousands of ppm). Since metal accumulation is ultimately an energy
consuming process, one would wonder what evolutionary advantage does metal
hyperaccumulation give to these species? P^ecent studies have srjown that metal accumulation in
the foliage may allow hyperaccumulator. species tc evade predatprs including caterpillarsrfungi
and bacteria (Boyd and Martens, 1994; Pollard and Baker, 1997).
Hyperaccumulator plants do not only accumulate high levels of essential micronutrients,
but can also absorb significant amounts of nonessential metals, such as Cd. The mechanism of
Cd accumulation has not been elucidated. It is possible that the uptake of this metal in roots is via
a system involved in the transport of another essential divalent micronutrient, possibly Zn2",
Cadmium is a chemical analogue of the latter, and plants may not be able to differentiate between
the two ions (Chaney et al, 1994).
What is a hyperaccumulator species?
Interest in phytoremediation has grown significantly following the identification of metal
hyperaccumulator plant species. Hyperaccumulators are conventionally defined as species
capable of accumulating metals at levels 100-fold greater than those typically measured in
common nonaccumulator plants. Thus, a hyperaccumulator will concentrate more than: 10 ppm
Hg; 100 ppm Cd; 1,000 ppm Co, Cr, Cu, and Pb; 10,000 ppm Ni and Zn. To date, approximately
400 plant species from at least 45 plant families have been reported to hyperaccumulate metals.
Most hyperaccumulators bioconcenrrate Ni, about 30 absorb either Co. Cu, and/or Zn, even
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fewer species accumulate Mn and Cd, and there are no known natural Pb-hyperaccumulators
(Reeves and Baker, 1999). Several hyperaccumulators and their bioaccumulation potential are
listed in Table 6.
Table 6. Several metal hyperaccuinulator species and their bioaccumulation potential
Plant species Metal Leaf content (ppm)
Thlaspi caerulescens
Ipomea alpina
Haumaniastram robertii
Astragalus racemosus
Sebertia aciiminata
Zn:Cd
Cu
Co
Se
Ni
39,600:1,800
12,300
10,200
14,900
25% bv wt dried sap
Reference
Reeves&Brooks(1983);Baker&Walker(1990)
Balcer&Walker(1990)
Brooks (1977)
Beathetal. (1937)
Jaffreetal.fi 9761
Possibly, the best-known metal hyperaccumulator is Thlaspi caerulescens (alpine
pennycress). While most plants show toxicity symptoms at Zn accumulation of about 100 ppm,
T. caerulescens was shown to accumulate up to 26,000 ppm without showing any injury (Brown
et al., 1995b). Possibly, hyperaccumulator plants may have a higher requirement for metals such
as Zn than non-accumulator'species (Hajar, 1997). In suppcn of this, many hyperaccumulators,
including T. caerulescens, have been shown co colonize metal-rich soils such as calamine soil
(soil enriched in Pb, Zn, and Cd). Because of this ability, considerable efforts have been directed
to identify hyperaccumulator plants endemic to metal rich soils (Baker and Proctor, 1990).
How do plants tolerate high metal concentration in soil?
Ecological studies have revealed the existence of specific plant communities, endemic
floras, which have adapted on soils contaminated with elevated levels of Zn Cu, and Ni.
Different ecbtypes of the same species may occur in areas uncontaminated by metals. To plants
endemic to metal-contaminated soils, metal tolerance is an indispensable property. In
comparison, in related populations inhabiting uncontaminated areas, a continuous gradation
between ecotypes with high and low tolerance usually occurs. Plants evolved several effective
mechanisms for tolerating high concentrations of metals in soil. In some species, tolerance is
achieved by preventing toxic metals uptake into root cells. These plants, coined excluders, have
little potential for metal extraction. Such an excluder is "Merlin," a commercial variety of red
fescue (Festuea rubrd). used to stabilize erosion-susceptible metal-contaminated soils. A second
group of plants, accumulators, does not prevent metals from entering the root. Accumulator
species have evolved specific mechanisms for detoxifying high metal levels accumulated in the
cells. These mechanisms allow bioaccumulation of extremely high concentration of metals. In
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addition, a third group of plants, termed indicators, shows poor, control over rnetal uptake and
transport processes. In these plants, the extent of metal accumulation reflects metal
concentration in the rhizospheric soil. Indicator species have been used for mine prospecting to
find new ore bodies (Raskin et al, 1994).
Mechanisms of metals uptake into roots and translocation to shoots
Because of their charge, metal ions cannot move freely across the cellular membranes,
which are lipophilic structures. Therefore, ion transport into cells must be mediated by membrane
proteins with transport functions, generically known as transporters. Transmembrane transporters
possess an extracellular binding domain to which the ions attach just before the transport, and a
transmembrane structure which connects extracelluar and intraeellular media. The binding
domain is receptive only to specific ions and is responsible for transporter specificity. The
transmembrane structure facilitates the transfer of bound ions from extracellular space through
the hydrophobic environment of the membrane into the cell. These transporters are characterized
by certain kinetic parameters, such as transport capacity (V^J and affinity for ion (KJ. Vmax
measures the maximum rate of ion transport across the cellular membranes. K,,, measures
transporter affinity for a specific ion and represents the ipr> concentration in the external solution
at which the transport rate equals Vraix/2. A low K^ value, hi^Jmhity, indicates that high levels
of ions are transported into the cells even at IqW^tenratjoicoRc'enfeation. By studying kinetic
parameters, K,,, and V^, plant biologists gkf ;^ighb to .specificityand^electivity of the
transport system. , '" r •"" " "^'"'"" "'"''
It is important to note that of the total amount of ions associated with the root, only a part
is absorbed into cells. A significant ion fraction is physically adsorbed at the extracellular
negatively charged sites (COO") of the root cell walls. The cell wall-bound fraction cannot be
translocated to the shoots and, therefore, cannot be removed by harvesting shoot biomass
(phytoextraction). Thus, it is possible that a plant exhibiting significant metal accumulation into
the root, to express a limited capacity for phytoex^r^^oii^Epr example, many plants accumulate
Pb in roots, but Pb translocation to shoot is very "low. In support of this, Blaylock and Huang
(1999) concluded that the limiting step for Pb phytoextraction is the long distance translocation
from roots to shoots.
Binding to the cell wall is not the only plant mechanism responsible for metal
immobilization into roots and subsequent inhibition of ion translocation to the shoot. Metals can
also be complexed and sequestered in cellular structures (e.g., vacuole) becoming unavailable for
translocation to the shoot (Lasat et al., 1998). In addition, some plants, coined excluders, possess
specialized mechanisms to restrict metal uptake into roots. However, the concept of metal
exclusion is not well understood (Peterson, 1983).
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Uptake of metals into root cells, the point of entry info living tissues, is a step of major
importance for the process of phytoextraction. However, for phytoextraction to occur metals
must also be transported from the root to the shoot. Movement of metal-containing sap from the
root to the shoot, termed translocation, is primarily controlled by two processes: root pressure
and leaf transpiration. Following translocation to leaves, metals can be reabsorbed from the sap
into leaf cells. A schematic representation of metal transport processes that take place in roots
and shoots is shown in Figure 1.
Figure 1. Metal uptake and accumulation in plants
1. A metal fraction is sorbed at root surface
2. Bioavailable metal moves across cellular membrane into root cells
3. A fraction of the metal absorbed into roots is immobilized in the vacuole
4. Intracellular mobile metal crosses cellular membranes into root vascular tissue
(xylem)
5. Metal is translocated from the root to aerial tissues (stems and leaves)
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Plant mechanisms for metal detoxification
Although micronutrients such as Zn, Mn, Ni and Cu are essential for plant growth and
development, high intracellular concentrations of these ions can be toxic. To deal with this
potential stress, common nonaccumulator plants have evolved several mechanisms to control the
homeostasis of intracellular ions. Such mechanisms include regulation of ion influx (stimulation
of transporter activity at low intracellular ion supply, and inhibition at high concentrations), and
extrusion of intracellular ions back into the external solution. Metal hyperaccumulator species,
capable of taking up metals in the thousands of ppm, possess additional detoxification
mechanisms. For example, research has shown that in T. goesingense, a Ni hyperacccumulator,
high tolerance was due to Ni complexation by histidine which rendered the metal inactive
(Kramer et al., 1997; Kramer et al., 1996). Sequestration in the vacuole has been suggested to be
responsible for Zn tolerance in the shoots of the Zn-hyperaecumulator T. caerulescens (Lasat et
al., 1996; Lasat et al., 1998). Several mechanisms have been proposed to account for Zn
inactivation in the vacuole including precipitation as Zn-phytate (Van Steveninck et al., 1990),
and binding to low molecular weight organic acids (Mathys, 1977; Tolra et al., 1996; Salt et al.,
1999). Complexation to low molecular weight organic compounds (<10 kD) was also shown to
play a role in tolerance to Ni (Lee et al., 1977). Cadmium, a potentially toxic metal; has been
shown to accumulate in plants, where it is detoxified by binding to phytochelatins. (Wagner 1984;
Steffens, 1990; Cobbett and Goldsbrough, 1999), a family of thi01(SH)-rich peptides
(Rauser,1990). Metallothioneins (MT), identified in numerous.animals and more recently, in
plants and bacteria (Kagi, 1991), are also compounds (proteins) with heavy metal-binding
properties (Tomsett et al., 1992).
Plant limitations
When the concept of phytoextraction was reintroduced (approximately two decades ago),
engineering calculations suggested that a successful plant-based decontamination of even
moderately contaminated soils would require crops able to concentrate metals hi excess of 1-2%.
Accumulation of such high levels of heavy metals is highly toxic and would certainly kill the
common nonaccumulator plant. However, in hyperaccumulator species, such concentrations are
attainable. Nevertheless, the extent of metal removal is ultimately limited by plant ability to
extract and tolerate only a finite amount of metals. On a dry weight basis, this threshold is around
3% for Zn and Ni, and considerably less for more toxic metals, such as Cd and Pb. The other
biological parameter which limits the potential for metal phytoextraction is biomass production.
With highly productive species, the potential for biomass production is about 100 tons fresh
weight/hectare. The values of these parameters limit the annual removal potential to a maximum
of 400 kg metal/ha/yr. It should be mentioned, however, that most metal hyperaccumulators are
14
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slow growing and produce little biomass. Thsse characteristics severely limit the use of
hyperaccumulator plants for environment cleanup.
Improving phytoremediating plants
It has been suggested that phytoremediation would rapidly become commercially
available if metal removal properties of hyperaccumulator plants, such as T. caerulescens, could
be transferred to high-biomass producing species, such as Indian mustard (Brassica juncea) or
maize (Zea mays) (Brown et al., 1995b). Biotechnology has already been successfully employed
to manipulate metal uptake and tolerance properties in several species. For example, ia tobacco
(Nicotiana tabacum) increased metal tolerance has been obtained by expressing the mammalian
metallothionem, metal binding proteins, genes (Lefebvre et al., 1987; Maiti et al., 1991).
Possibly, the most spectacular application of biotechnology for environmental restoration
has been the bioengineering of plants capable of volatilizing mercury from soil contaminated
with methyl-mercury. Methyl-mercury, a strong neurotoxic agents, is biosynthesized in Hg-
contaminated soils. To detoxify this toxin, transgenic plants (Arabidopsis and tobacco) were
engineered to express bacterial genes merB and merA. In these modified plants, merB catalyzes
the protonolysis of the carbon-mercury bond with the generation of Hg2", a less mobile mercury
species. Subsequently, MerA converts Hg(II) to Hg (0) a less toxic, volatile element which is
released into the atmosphere (Rugh et ai., 1996; Heaton et al., 1988). Although regulatory
concerns restrict tile us"& of plants modified with merA and merB, this research illustrates the
tremendous potential of biotechnology for environment restoration. In an effort to address-;
regulatory concerns related to phytovolatilization of mercury, Biziii et al. (1999) demonstrated
that plants engineered to express MerBpe (an organomercurial lyase under the control of a plant
promoter) may be used to degrade methyl-mercury and subsequently remove ionic mercury via
extraction. Despite recent advances in biotechnology, little is known about the genetics of metal
hyperaccumulation- in plants. Particularly, the heredity of relevant plant mechanisms, such as
metal transport and storage (Lasat et al., 2000) and metal tolerance (Ortiz et al., 1992; Ortiz et al.,
1995) must be better understood. Recently, Chaney et al. (1999) proposed the use of traditional
breeding approaches for improving metal hyperaccumulator species and possibly incorporating
significant traits such as metal tolerance and uptake characteristics into high-biomass-producing
plants. Partial success has been reported in the literature. For example, in an effort to correct for
small size of hyperaccumulator plants, Brewer et al. (1997) generated somatic hybrids between T.
caenilescens (a Zn hyperaccumulator) and Brassica napus (canola) followed by hybrid selection
for Zn tolerance. High biomass hybrids with superior Zn tolerance were recovered. These authors
have also advocated a coordinated effort to collect and preserve germplasm of accumulator
species.
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IV. PLANT-METAL INTERACTION IN THE RHIZOSPHERE
Metal bioavailability for uptake into roots
A major factor limiting metal uptake into roots is slow transport from soil particles to root
surfaces (Nye and Tinker, 1977; Barber, 1984). With the possible exception of volatile mercury,
for all other metals, this transport takes place in soil solution. In soil, metal solubility is restricted
due to adsorption to soil particles. Some of the soil binding sites are not particularly selective.
For example, they bind Cd as strong as Ca. Nonspecific binding occurs at clay cation exchange
sites and carboxylic groups associated with soil organic matter. Other sites are more selective and
bind Cd stronger than Ca. For example, most clay particles are covered with a thin layer of
hydrous Fe, Mn, and Al oxides. These selective sites maintain Cd activity in the soil solution at
low levels (Chaney, 1988). Lead, a major contaminant, is .notorious for the lack of soil mobility,
primarily due to metal precipitation as insoluble phosphates, carbonates and (hydr)oxides
(Blaylock and Huang, 1999). Thus, increasing metal solubility in the soil is an important
prerequisite to enhance the potential for Pb phytoextraction. This subject is detailed in the next
section.
Two mechanisms are responsible for metal transport from .the bulk soil to plant roots: 1)
convection or massjflow, and 2) diffusion (Corey et a!., 19&l^Barber. 1984). Due to..convection,
soluble metal ions move from soil solids to root surface. From the rhizosphere, water is absorbed
by roots to replace water transpired by leaves., Water uptake-from rhizosplaere creates a hydraulic
gradient directed from the bulk soil to the rooj surface. Some ions are absorbed by roots faster
than the rate of supply via mass flow. Thus; a depleted zone is created in soil immediately
adjacent to the root. This generates a concentration gradient directed from the bulk soil solution
and soil particles holding the adsorbed elements, to the solution in contact with the root surface.
This concentration gradient drives the diffusion of ions toward the depleted layer surrounding the
roots.
Plants have evolved specialized mechanisms to increase the concentration of metal ions
in soil solution. For example, at low ion supply, plants may alter the chemical environment of the
rhizosphere to stimulate the desorption of ions from soil solids into solution. Such a mechanism
is rhizosphere acidification due to FTextrusion from roots (Crowley et al., 1991). Protons
compete and replace metal ions from binding sites, stimulating their desorption from soil solids
into solution. In addition, some plants can regulate metal solubility in the rhizosphere by exuding
a variety of organic compounds, from roots. Root exudates complex metal ions keeping them in
solution available for uptake into roots (Romheld and Marschner, 1986).
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Effect of soil microorganisms on metal uptake
Root growth affects the properties of the rhizospheric soil and stimulates the growth of
the microbial consortium. To illustrate this, research has shown that the population of
microorganisms in the rhizosphere is several orders of magnitude greater than in the surrounding
soil (Anderson, 1997). In turn, rhizospheric microorganisms may interact symbiotically with
roots to enhance the potential for metal uptake. In addition, some microorganisms may excrete
organic compounds which increase bioavailability, and facilitate root absorption of essential
metals, such as Fe (Crowley et al, 1991) and Mn (Barber and Lee, 1974) as well as nonessential
metals, such as Cd (Salt et al., 1995). Soil microorganisms can also directly influence metal
solubility by altering their chemical properties. For example, a strain of Pseudomonas
maltophilia was shown to reduce the mobile and toxic Cr6" to nontoxic and immobile Cr3", and
also to minimize environmental mobility of other toxic ions such as Hg:~, Pb2~, and Cd2^ (Blake
et al., 1993; Park et al., 1999). In addition, it has been estimated that micrcbial reduction cf Hg2"
generates a significant fraction of global atmospheric Hg° emissions (Keating et al., 1977)
Effect of root exudates on metal uptake
Root exudates have an important role in the acquisition of several essential metals. For
example, some grass species can exude from roots a class of organic acids called siderophores
(mugineio &iid avenic acids), which were shown to significantly enhance the bioavailability of
soil-bound iron (Kanazav/a et al., 1995), and possibly zinc (Cakmak 1996a; 1996b). In addition,
root exudates have been shown to be involved in plant tolerance. In support of this, it has been
demonstrated that some plant species tolerate Al in the rbizosphere, by a mechanism involving
exudation of citric and malic acids (Pellet et al., 1995; Larsen et al., 1998). These organic acids
chelate rhizospheric Al3" which is highly phytotoxic to form a significantly less toxic complex.
V. OPTIMIZATION OF METAL PHYTOEXTRACTION WITH
AGRONOMIC PRACTICES
Plant selection
The selection of phytoremediating species is possibly the single most important factor
affecting the extent of metal removal. Although, the potential for metal extraction is of primary
importance, other criteria, such as ecosystem protection must be also considered when selecting
remediating plants. As a general rule, native species are preferred to exotic plants which can be
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invasive and endanger the harmony of the ecosystem. To avoid propagation of weedy species,
crops are in general preferred although some crops may be too palatable and pose a risk to
grazing animals.
The rate of metal removal depends upon the biomass harvested and metal concentration
in harvested biomass. Possibly, one of the most debated controversies in the field refers to the
choice of remediative species; metal hyperaccumulators vs. common nonaccumulator species.
Hyperaccumulator plants have the potential to bioconcentrate high metal levels. However, their
use may be limited by small size and slow growth. In common nonaccumulator species, low
potential for metal bioconcentration is often compensated by the production of significant
biomass (Ebbs et al., 1997). Chaney et al. (1999), analyzed the rate of Zn and Cd removal, and
reached the conclusion that non-accumulator crops will not remove enough meta! to support
phytoextraction. Furthermore, these authors argued that at many sites metal contamination is high
enough to cause toxicity to crop species and significant biomass reduction. In support of this,
several maize (one of the most productive crops) inbred lines have been identified which can
accumulate high levels of Cd (Hinesly et al., 1978). However, these lines were susceptible to Zn
toxicity and, therefore, could not be used to cleanup soils at the normal Zn:Cd ratio of 100:1
(Chaney et al., 1999). In addition, when appropriate disposal is an important regulatory concern,
the use of lower biomass producing hyperaccumulator species would be an advantage because
less contaminated biomass will have to be handled.
For Pb, a major soil contaminant, no hyperaccumulator species has been identified.
However, several species, such as hemp dogbane (Apocynum cannabinum),common ragweed
(Ambrosia artemisiifolia), nodding thistle (Carduus nutans), and Asiatic dayflower (Commelina
communis), were shown to have superior Pb accumulating properties (Berti and Cunningham,
1993). Practices have been developed to increase the potential of common nonaccumulator plants
for Pb phytoextraction. Particularly, the uptake-inducing properties of synthetic chelates open the
possibility of using high biomass producing crops for Pb phytoextraction. Under.chelate-induced
conditions, maize (Huang and Cunningham. 1.996) and Indian mustard (Blaylock et al., 1997)
have been successfully used to remove Pb from solution culture and contaminated soil,
respectively.
Physical characteristics of soil contamination are also important for the selection of
remediating plants. For example, for the remediation of surface-contaminated soils, shallow-
rooted species would be appropriate to use, whereas deep-rooted plants would be the choice for
more profound contamination.
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Soil fertilization, and conditioning
Phytoremediation is essentially an agronomic approach and its success depends ultimately
on agronomic practices applied at the site. The importance of employing effective agronomic
practices has been discussed by Chaney et al. (1999). These authors investigated the effect of soil
acidification on Zn and Cd phytoextraction and proposed the use of (NH4)2SO, as a soil additive
to provide nutrients (N and S) needed for high yield, and to acidify the soil for greater metal
bioavailability. It should be noted that there may be some negative side effects associated with
soil acidification. For example, due to increased solubility some toxic metals may leach into the
groundwater creating an additional environmental risk. Chaney et al. (1999) indicated that
following metal phytoextraction, soil can be limed to elevate the pH near a neutral value, so that
normal farm uses or ecosystem development could resume. However, premature liming may
increase soil capacity for metal binding and restrict the potential for phytbextiaction. A similar
effect can be expected following the addition of organic fertilizers. In addition, the raising of pH
may stimulate the formation of metal hydroxy ions, such as ZnOFT which is more strongly
sorbed to soil solids than the uncomplexed ions.
Phosphorus is a major nutrient, and plants respond favorably to the application of P
fertilizer by increasing biomass production. The addition of P fertilizer, however, can also inhibit
the uptake cf seme major metal contaminants, such as Pb, due to metal precipitation as
pyromorphite and chloropyromorphite (Chaney et al., 2000). This underlines the importance of
finding new approaches for P application. Such an alternative may be foliage application. This
method may lead :o improvement of plant P status-without inhibiting ?b mobility in soil.
Enhancing metal bioavailability with synthetic cheiators
For some toxic metals such as Pb, a major factor limiting the potential for phytoextraction
is limited solubility and bioavailability for uptake into roots. One way to induce Pb solubility is
to decrease soil pH (McEride, 1994). Following soil acidification, however, mobilized Pb can
leach rapidly below the root zone. In addition, soluble ionic lead has little propensity for uptake
into roots. The use of specific chemicals, synthetic chelates, has been shown to dramatically
stimulate the potential for Pb accumulation in plants. These compounds prevent Pb precipitation
and keep the metal as soluble chelate-Pb complexes available for uptake into roots and transport
within plant. For example, addition of EDTA (ethylene-diamine-tetraacetic acid), at a rate of 10
mmol/kg soil, stimulated Pb accumulation in shoots of maize up to 1.6 % (Blaylock et al., 1997).
In a subsequent study, Indian mustard exposed to Pb and EDTA in hydroponic solution was able
to accumulate more than l%Pb in dry shoots (Vassil et al., 1998). Another synthetic chelator,
HEDTA (hydroxyethyl- ethylenediamine-triacetic acid) applied at 2.0 g/kg soil contaminated
with 2,500 ppm Pb, increased Pb accumulation in shoots of Indian mustard from 40 ppm to
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10,600 ppm (Huang and Cunningham, 1996). Accumulation of elevated Pb levels is highly toxic
and can cause plant death. Because of the toxic effects, it is recommended that chelates be
applied only after a maximum amount of plant biomass was produced. Prompt harvesting (within
one week of treatment) is required to minimize the loss of Pb-laden shoots.
Blaylock et al. (1997), indicated that, in addition to Pb, chelate-assisted phytoextraction is
applicable to other metals. These authors indicated that application of EDTA also stimulated Cd,
Cu, Ni, and Zn phytoaccumulation. Chelate ability to facilitate phytoextraction was shown to be
directly related to its affinity for metals. For example, EGTA (ethylenebis (oxyethylenetrinitrilo)
tetraacetic acid) has a high affinity for Cd"", but does not bind Zn2". EDTA, HEDTA, and DTPA
(diethylene-triarnme-pentaacetic acid) are selective for Zn. In fact, zinc binding by DTPA is so
strong that plants cannot use Zn from this complex and potentially suffer from Zn deficiency.
Sowing
The extent of metal extraction depends on the amount of plant biomass produced. An
important factor that controls biomass production is plant density (number of plants/m2). Density
affects both yield/plant and yield/ha. In general, higher density tends to minimize yield per plant
and maximize yield per hectare. Density is also likely to affect the-pattem of plant growth and
development. For example, at higher stand density, plants will :ccmpete more strongly for light-.-
Thus, more resources (nutrients and energy) may be allocated for plant growth as opposed to
developmental processes (flowering). An extended growth period may be beneficial if plant
metal absorption and accumulation depend upon' growih processes-. Furthermolre, the distance
between plants is likely to affect the architecture of the-root system with possible further
implications on metal uptake. However, the effect of this interaction is unknown and awaits
investigation.
Crop rotation
Another agronomic principle, which has been neglected in phytoremediation research, is
crop rotation. Because of the proliferation of weeds, predators, and diseases, which can cause
significant yield reduction, crops, including those used for soil remediation, must be rotated. In
general, crops are rotated less frequently today than 30 years ago. From crop science, it can be
extrapolated that short-term (two to three years) monoculture (the use of the same species in
consecutive seasons), may be acceptable for metal phytoremediation. However, for longer-term
applications, as most metal phytoextraction projects are anticipated, it is unlikely that successful
metal cleanup can be achieved with only one remediative species used exclusively in
monoculture. Plant rotation is even more important when multiple crops per year are projected.
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Crop maintenance: pest control and irrigation
Weed control and irrigation are major crop maintenance practices. Weeds can be
controlled by mechanical or chemical methods. Herbicides can be applied before or after the
emergence of phytoremediating species. Application of pre-emergent herbicides ensures good
weed control, quick emergence, and establishment of selected plants. Post-emergent herbicides
control weeds that occur later in the growing season. Because metal uptake into roots depends
on the movement of soil solution from the bulk soil to root surface, maintaining an adequate soil
moisture is important. Depending on the local climate, irrigation may be required to achieve
adequate soil moisture. The volume of water delivered must be carefully considered. This
volume should compensate for losses due to evaporation and transpiration. Excessive water
delivery will not only inflate operational cost, but may also restrict root growth and depress metal
extraction rates. The method of irrigation must also be carefully considered. For example, when
delivered under low pressure directly to the soil, as dripping, losses due to evaporation are kept
to a minimum. In addition, this method will have little effect on air humidity. In contrast, water
delivered under pressure from a nozzle, will elevate air humidity and possibly inhibit leaf
transpiration. Since the movement of metal-containing sap from the root to the shoot depends on
transpiration, tran?port arid rate of me'ial accumulation in shoots may be affected. Furthermore,
when applied under pressure, water losses due to evaporation are significant and add to the
operational cost. ..,.
Handling 2;?d. disposal of contaminated waste
One concern associated with the application of phytotechnology is handling and disposal
of contaminated plant waste. The need to harvest contaminated biomass and possibly dispose of
it as hazardous waste subject to RCRA standards creates an added cost and represents a potential
drawback to the technology. One option is disposal of contaminated biomass to a regulated
landfill. To decrease handling, processing, and potential landfilling costs, waste volume can be
reduced by thermal, microbial, physical or chemical means. With some metals (Ni, Zn, and Cu)
the value of the reclaimed metal may provide an additional incentive for phytoextraction. Chancy
et al. (1999) proposed incineration of plant biomass to further concentrate the bio-ore. These
authors showed that the value of the metal recovered in the biomass was shown to offset the cost
of the technology. Furthermore, Watanabe (1997) showed that Zn and Cd recovered from a
typically contaminated site could have a resale value of S1,060/ha.
Cost and time projections
Cost analysis of metal phytoextraction is hampered by a lack of information. In support of
this, to date no metal-contaminated site has been completely remediated with plants. Therefore,
21
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available cost data are limited to short-term (two- to three-year-old) field studies. It is doubtful
that these results can be used to accurately estimate the cost of a full-scale project that can last as
long as 15 years. In addition, complexity argues against generic, and in favor of site-specific cost
analysis. Despite these limitations, several authors, have investigated the time-frame and cost of
metal phytoextraction. For example, Brown et al. (1995a) considered a soil contaminated with
400 mg kg"! Zn, and a desired cleanup level of 40 mg kg~!. These authors used T. caendescens in
their analysis and assumed a constant rate of uptake of 4,000 mg kg"1, and an annual yield of 10
t/ha. They estimated that it would take 18 growing seasons to remove excess Zn from the soil. In
a subsequent study, the cost of remediating a metal-contaminated soil by conventional
engineering techniques was estimated between S50 and S500 per ton (Cunningham and Ow,
1996). Thus, the price tag of remediating an acre of soil (3-foot-deep contamination), weighing
some 4,500 tons, would be in excess of S 250,000. These authors estimated that growing a crop
on an acre of land can be accomplished at cost ranging from two to four orders of magnitude less
than current cost for soil excavation and burial. Salt et al. (1995) estimated that using
phytoextraction to cleanup one acre of soil to a depth of 50 cm will cost 560,000-100,000
compared to at least 5400,000 for soil excavation and storage alone.
Research needs
There is a need to optimize the agronomic practices to maximize the cleanup potentialof
remediative plants. Since in many instances metal absorption in roots is limited by low solubility
in soil solution, it is important to further investigate, the use of chemical amendments to induce
metal bioavailability. Significant results have been obtained in this area. However, there is a need
to find cheaper, environmentally benign chemical compounds with metal chelating properties.
Research is also needed to identify phytoremediating species capable of being rotated to sustain
the rate of metal extraction. More information is also needed to optimize the time of harvest. .
Plants should be harvested when the rate of metal accumulation in plants declines. This will
minimize the duration of each growth cycle and allow more crops to be harvested in a growing
season. The current status of agronomic practices as they apply to metal phytoextraction and
further research needs are assessed in Table 7 .
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Table 7. Assessment of the current status of agronomic practices as they apply to metal phytoextraction and further
research needs.
Agronomic issue
Readiness1
Purpose
Current status
Research needs
Soil
mobili/ation
To control crop pests and condition
surface soil for seed germination.
Achieved via rototilling or plowing.
Information available on crop species
can be applied to remediating plants.
Research needed to determine whether
plowing may displace soil
contaminants to root inaccessible
depths.
Fertili/ation/
Conditioning
Fertilizers arc used to improve soil
nutrient supply and nutrient availability
for uptake into roots.
Conditioners are used to improve soil
aeration and water holding capacity.
Soil ferlili/ation has been researched
extensively and a wealth of
information is available in the
literature. However, with the exception
ofPb/P interaction little is known
about the effect of fertili/.ers/
conditioners on the mobility of toxic
metals and their bioavailability in soil.
The effect of ferlili/ers/conditioners
on the chemistry of soil metals and
plants' ability to absorb metals must
be belter understood. Of particular
concern is the application of synthetic
dictates (themselves potentially toxic).
Research should clarify the fate of
these compounds in soil and
grotmdwater, as well as the effect of
chclalc application on metal mobility
and uptake into plants.
Crop rotation
To control crop pests such as weeds,
insects and diseases, and to manage
nutrient soil supply.
Today crops are rotated less
frequently than 30 years ago. For
conventional crops, economics favor
the repeated use (rotation) of o;ie or
two profitable species. However, in
phytoremediation, where two or more
crops per year are expected, species
rotation may prove a valuable crop
maiiagcmcnt tool. Very little is known
about the rotation of phytorcmedialing
plants.
Research must identify metal
accumulator species that can be
rotated within a specific application.
23
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Sowing
The proper amount of seed iinisl he
incorporated in soil to obtain a plant
stand of desired density.
A wealth of information exists on
sowing of crop species. This
knowledge can be applied to related
pliyloremedinting species. l;or
example, similar techniques would be
employed to sow canola and Indian
mustard.
It is not clear how plant density will
affect the rate of metal phytocxtraction
and potential for biomass production.
These effects should be investigated.
Irrigation
To compensate for water loss due to
evaporation from soil and plant
transpiration.
A great deal of information exists on
the irrigation of crop species. This
information can be directly applied lo
plants grown for environmental
remediation.
The effect of various irrigation
methods on root growth and
expansion, and melal pliytoexlraclioii
rale must be better understood.
Weed control
To suppress the growth of weeds to the
point that its effect on the rate of metal
extraction is minimal.
Mechanical and chemical methods are
(lie most common approaches.
Biological control (the use of natural
enemies to reduce weed population) is
emerging as an alternative.
RI'Ms must identify the appropriate
weed control strategy (e.g., herbicides
rotation) to prevent '.he buildup of
weed populations and inaximi/e (he
rate of melal pliyloexlraclion.
Harvesting-
Remove plant biomass loaded with metal
contaminants.
In general, techniques and equipment
are readily available.
Research must be conducted to
determine when to harvest. It is
important lo harvest when the rale of
melal extraction starts declining. This
would allow more crops to be obtained
in a season and increase the amount of
metal removed. Appropriate liming
would also maximize the amount of
biomass removed at harvest.
" Readiness for field deployment: I) very little research is needed.jn this area; 2) practice is ready for field deployment although research may improve
its effectiveness; 3) significant research is needed to efficiently apply the practice; 4) practice has not been investigated for metal extraction.
24
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