Bioconcentration of heavy metals by plants

Ilya Raskin, PBA Nanda Kumar, Slavik Dushenkov and David E Salt
Rutgers University, N e w Jersey, USA

Certain plants can concentrate essential and non-essential heavy metals in

their roots and shoots to levels far exceeding those present in the soil.
Metal-accumulating plant species are invariably restricted to metalliferous
soils found in different regions around the world. The mechanisms of metal
accumulation, which involve extracellular and intracellular metal chelation,
precipitation, compartmentalization and translocation in the vascular system,
are poorly understood. Interest in these mechanisms has led to the
development of phytoremediation--a new technology to use plants to clean
up soil and water contaminated with heavy metals.

Current Opinion in 8iotechnology 1994, 5:285-290

Introduction
The notion that the elemental composition of plants is
very different from that of the soil in which they grow
is taken for granted. Most of these differences can be
attributed to a plant's ability to fix carbon from the air
and to absorb essential macronutrients and micronutri-
ents from the soil. In many ways, living plants can be
compared to solar driven pumps that can extract and
concentrate certain elements from their environment.
The root morphology of terrestrial plants is a good ex-
ample of h o w plants are able to produce a large surface
area to volume ratio in order to maximize this accumu-
lation. The total length of roots (including root hairs)
of a single pot-grown rye plant has been estimated to
be about 387 miles [1], and can be even larger for field-
grown plants,
The list of elements essential for plant nutrition is still
a subject of some controversy (an essential element is
usually defined as one without which plants cannot
complete their lifecycle). It is certainly true, however,
that plants are not only built of molecules and ions
that have functional or structural roles in their devel-
opment. Therefore, the elemental composition of any
field-grown plant exceeds that thought to be essential
for its survival. This is particularly true for many heavy
metals, which can be readily detected in field-grown
plants.
The term heavy metal is arbitrary and imprecise. For
reasons of simplicity, we will broadly define it here as
meaning any element that has metallic properties (duc-
tility, conductivity, density, stability as cations, ligand
specificity, etc.) and an atomic number >20. A more
biologically relevant but complex classification of met-
als based o n ligand-forming properties was proposed
by Nieboer and Richardson [2]. Heavy metals that are
considered essential for at least some forms of life in-
clude V, Cr, Mn, Fe, Co, Ni, Cu, Zn, and Mo. Heavy
metals required by plants include Mn, Fe, Cu, Zn, Mo,
and, possibly, Ni. The phytotoxicity of such relatively
c o m m o n heavy metals as Cd, Cu, Hg, and Ni is sub-
stantially greater than that of Pb and Zn. Hexavalent
Cr is much more toxic to plants than trivalent Cr.
Heavy metals are present in soils as natural com-
ponents or as a result of human activity. Metal-rich
mine tailings, metal smelting, electroplating, gas ex-
hausts, energy and fuel production, downwash from
p o w e r lines, intensive agriculture, and sludge dumping
are the most important human activities that contam-
inate soils and aqueous streams with large quantities
of toxic metals [3]. It should be noted, however, that
in some areas, natural background concentrations of
heavy metals in soils and waters exceed those that are
considered safe by the regulatory agencies [4°]. Natu-
ral mineral deposits containing particularly large quan-
tities of heavy metals are present in many regions of
the globe. These areas often support very characteris-
tic plant assemblages and species that thrive in these
metal-enriched environments. Examples of such dis-
tinct plant communities include serpentine (i.e. grow-
ing on Ni, Cr, Mn, Mg and Co rich soils), seleniferous
(i.e. growing on Se rich soil), uraniferous (i.e. grow-
ing on U rich soils), calamine (i.e. growing on Zn and
Cd rich soils) and Cr/Co floras. As a result of their
association with specific ore deposits, many metallo-
phyte plants are used as so-called indicator species
in prospecting for mineral deposits [5,6]. For exam-
ple, the copper flower (Haumaniastrum katangense)
is a classical indicator of Cr and Co ores in central
Africa [7"].
In this review, we summarize current knowledge con-
cerning metal accumulation in plants and the potential
commercial application of this p h e n o m e n o n in biore-
mediation.

© Current Biology Ltd ISSN 0958-1669 285

286 Environmentalbiotechnology
Plant responses to heavy metals
Plants employ three basic strategies for growing on
metalliferous soils [8]. Metal excluders effectively pre-
vent metal from entering their aerial parts over a broad
range of metal concentrations in the soil; however, they
can still contain large amounts of metal in their roots.
Metal non-excluders actively accunmlate metals in their
above-ground tissues and can be roughly divided into
two groups: indicators and hyperaccumulators. Metal
levels in the tissues of indicator species generally re-
flect metal levels in the soil. Hyperaccumulators can
concentrate metals in their above-ground tissues to
levels far exceeding those present in the soil or in the
non-accumulating species growing nearby. One defini-
tion proposes that a plant containing more than 0.1% of
Ni, Co, Cu, Cr and Pb or 1% of Zn in its leaves on a dry
weight basis is called a hyperaccumulator, irrespective
of the metal concentration in the soil [8,9].
Almost all metal-hyperaccumulating species known to-
day were discovered on metalliferous soils, either nat-
ural or man-made, often growing together with metal
excluders. Actually, almost all metal-hyperaccumulat-
ing plants are endemic to these soils, suggesting that
hyperaccumulation is an important ecophysiological
adaptation to heavy-metal stress and one of the man-
ifestations of heavy-metal resistance [9]. The majority
of hyperaccumulating species discovered so far are
restricted to a few specific geographical locations. Ni
hyperaccumulators are found in New Caledonia [9],
the Philippines [10], Brazil [11] and Cuba (AJM Baker,
personal communication), Ni and Zn accumulators are
found in southern and central Europe and Asia Minor
[5,6,9], and Cr and Co accumulators in Central Africa
[5,6,9]. We still k n o w very little about the biological
and evolutionary significance of metal hyperaccumu-
lation. Several hypotheses have been put forward, in-
cluding the following: tolerance or disposal of metal
from plants, drought resistance, inadvertent uptake,
and defense against herbivores or pathogens. Of these
hypotheses, present evidence favours most strongly
the function of hyperaccumulated metals as a defense
mechanism against herbivores [12,13].
A fair amount of phenomenological data on hyperac-
cumulating plants is available from extensive field col-
lections and herbarium studies [5,9]. The list of hyper-
accumulating plants is expanding rapidly. Ni hyperac-
cumulators are most numerous (numbering at least 277
taxa belonging to 36 different families) followed by Co,
Cu a n d . Z n hyperaccumulators (AJM Baker, personal
communications). Hyperaccumulators of Mn, Pb, Cd,
Cr and Se have also been described. So far, the largest
numbers of hyperaccumulating species in the temper-
ate zone belong to the Brassicaceae (nmstard family)
[9], but in the tropics, the Euphorbiaceae (spurge fam-
ily) is the best represented. A New Caledonian tree,
Sebertia acuminata, which has more than 11% of Ni
in its latex (on a dry weight basis) [13], is one of the
most striking examples of metal hyperaccumulation. A
large number of other Ni hyperaccumulators (contain-
ing Ni at a level over 1% of total dry weigh0 belong to
the genera Alyssum and Thlaspi. Many Thlaspi species
also accumulate Pb (up to 1% dry weight) and Zn (up
to 3% dry weigh0 [9]. Armeria maritima is another
prominent European Pb hyperaccumulator (over 1%
Pb in dried tissue) [9,13]. The African species Aeol-
lanthus biformifolius and Haumaniastrum katangense
may often contain more than 0.1% Cu or Co in dried
leaf tissue [7"]. Astragalus species growing on selenifer-
ous soils may accumulate close to 1% Se in dry matter
[61.
The specificity of metal uptake by hyperaccunmlating
plants is poorly understood and requires much more
research. Even so, the available field data suggest
that all hyperaccumulators may be divided into three
groups on the basis of their tendency to accumu-
late different metals: Cu/Co accunmlators, Zn/Cd/Pb
accumulators and Ni accunmlators (AJM Baker, per-
sonal comnmnications). The biochemical and molec-
ular bases of such group selectivity is unknown.
So far, the search for metal-accunmlating plants has
b e e n carried out by a few people in selected parts of
the globe. It is likely that many more as yet uniden-
tified metal-accumulating plants growing o n natural
and man-made metalliferous soils remain to be dis-
covered by plant scientists. It is vital that rare and en-
demic metal-accumulating plant species are identified
and preserved before they become extinct. To date,
few controlled laboratory experiments that quantify,
compare, and explain the n~etal-accumulating ability
of various plant species have been carried out. The
information is most needed in four areas: first, the
metal-accumulating ability of various species as a func-
tion of soil metal concentrations, physical and chemi-
cal soil properties, physiological state of the plant, et
cetera; second, the specificity of metal uptake, trans-
port and accumulation; third, the physiological, bio-
chemical and molecular mechanisms of accumulation
and hyperaccumulation; and fourth, the biological and
evolutionary significance of metal accumulation.
Mechanisms of metal accumulation
A large proportion of metals in soils are bound to
organic '(humus) soil constituents, inorganic (clay)
soil constituents, or alternatively, are present as in-
soluble precipitates. For plants to accumulate these
'soil-bound' metals, they must first mobilize them into
the soil solution. This mobilization of soil-bound metal
can b e achieved in a number of different ways. First,
metal-chelating molecules (phytosiderophores) can be
secreted into the rhizosphere to chelate and solubi-
lize soil-bound metal. For example, mugineic acid,
avenic acid and nicotianamine are phytosiderophores
that occur in graminaceous species [14"']. These phy-
tosiderophores are released in response to Fe and Zn
deficiency and can mobilize Cu, Zn and Mn from soil
[15]. Metal-chelating proteins, perhaps related to met-
allothioneins [16] or y-glutamylcysteinyl-isopeptides (3'
EC-isopepticles) [17], may also function as siderophores

in plants. Second, roots can reduce soil-bound metal
ions by specific plasma membrane bound metal re-
ductases. Pea plants deficient in Fe or Cu have an
increased ability to reduce Fe(III) and Cu(II) that is
coupled with an increased uptake of Cu, Mn, Fe and
Mg [18]. Third, plant roots can solubilize heavy metals
by acidifying their soil environment with protons ex-
truded from the roots. A lower pH solubilizes metal
precipitates and releases soil-bound metal ions into
the soil. A similar mechanism has been observed for
Fe mobilization in some Fe-deficient dicotyledonous
plants [19]. It should be noted here that all three of
the above processes might also b e performed by myc-
orrhizal fungi or root-colonizing bacteria. Therefore, it
is difficult to assess the individual contribution of root
cells and rhizosphere microorganisms in metal mobi-
lization by plants.
Solubilized metal ions may enter the root either via
the extracellular (apoplastic) or via intracellular (sym-
plastic) pathways. Apoplastic transport is limited by
the high cation exchange capacity of cell walls, un-
less the metal ion is transported as a non-cationic
metal chelate. Symplastic transport requires that metal
ions move across the plasma membrane, which usu-
ally has a large negative resting potential of ap-
proximately 170mV (negative inside the membrane).
This membrane potential provides a strong electro-
chemical gradient for the inward movement of metal
ions. Most metal ions enter plant cells by an energy-
dependent saturable process via specific or generic
metal ion carriers or channels [20]. Non-essential heavy
metals may effectively compete for the same trans-
membrane carriers used by essential heavy metals. This
relative lack of selectivity in transmembrane ion trans-
port may partially explain w h y non-essential heavy
metals can enter cells, even against a concentration
gradient. For example, kinetic data demonstrate that
essential Cu 2~ and Zn 2+ and non-essential Ni 2+ and
Cd 2+ compete for the same transmembrane carrier
[20]. Metal--chelate complexes may also be transported
across the plasma membrane via specialized carriers, as
is the case for Fe-phytosiderophore transport in gram-
inaceous species [19].
Once metal ions have entered the root, they can ei-
ther be stored or exported to the shoot. The vacuole
plays an important role in metal ion storage [21]. Metal
ions can be actively transported across the tonoplast
as free ions or as metal-chelate complexes. For exam-
ple, Cd is actively transported across the tonoplast of
oat roots as either a free ion via a Cd/H + antiport [22° ]
or as a Cd-('~C)3G/Cd-(TEC)2G (one-letter amino acid
code) metal-peptide complex energized by the direct
hydrolysis of ATP (DE Salt, WE Rauser, unpublished
data). Inside the vacuole, metal ions are chelated ei-
ther by organic acids, such as citric or malic acid, or
by enzymatically synthesized (yEC)-isopeptides, com-
monly called phytochelatins. The metal-binding role
of the proteins encoded by metallothionein-like genes
recently identified in plants remains unknown [23].
Under certain conditions, metal ions may form insol-
Bioconcentration of heavy metals by plants Raskin et al. 287
uble precipitates, for example Zn-phytate [24] and CdS
[25]. Compartmentalization, chelation and precipitation
of metal ions probably comprise the major resistance
mechanisms that protect plant cells from the damaging
effect of metals.
In hyperaccunmlating species, heavy metals accumu-
late both in the shoot and the root. In these plants,
metal transport to the shoot probably takes place in the
xylem. Metals may, however, redistribute in the shoot
via the p h l o e m [26]. Xylem cell walls have a high cation
exchange capacity that would be expected to retard
severely the movement of metal cations. Therefore,
metal-chelate complexes, such as Cd-citrate, should
facilitate metal movement in the transpiration stream
[27]. Analysis of the xylem sap of certain hyperaccu-
mulators has demonstrated the involvement of organic
acids in metal transport [9]. Recent work also sug-
gests that (TEC)-isopeptides may be involved in metal
binding in xylem sap [28]. Nicotianamine, involved in
phloem transport of Fe, can also bind Zn, Co, Ni and
Cu, and, therefore, may serve as a general heavy metal
transporter in the phloem [26]. In addition, metals may
be transported in the phloem chelated to either organic
acids, (yEC)-isopeptides or metallothioneins. Clearly,
more research is needed to understand the biologi-
cal mechanisms involved in heavy metal movement,
accumulation, and detoxification in plants. Hyperaccu-
mulating plants provide an excellent and almost com-
pletely unresearched model system for such studies.
Uses for metal-accumulating plants
For a long time, the ability of plants to accumulate met-
als was considered a detrimental trait. Being at the bot-
tom of many natural food chains, metal-accumulating
plants are directly or indirectly responsible for a large
proportion of the dietary uptake of toxic heavy met-
als by humans and other animals [29]. Although some
heavy metals are required for life, their excessive ac-
cumulation in living organisms is always toxic. The
danger of heavy metals is aggravated by their almost in-
definite persistence in the environment. The acute and
chronic toxic effects of consuming metal-contaminated
plants in wild animals, cattle and humans are very well
documented and lie outside the scope of this review.
Recently, several attempts have been made to produce
plants that can trap heavy metals in their roots, thus
inhibiting their translocation to the harvestable above-
ground parts. For example, transgenic tobacco plants
expressing the heavy metal chelating protein mouse
metallothionein I contained -24% lower and 5% higher
Cd levels in shoots and roots, respectively, than control
plants [30°]. In spite of the obvious benefits for human
health, currently, there are only a few ongoing efforts
to select or genetically engineer metal-excluding crop
plants or forage grasses. The need for such crops is
particularly great in the large areas of Eastern Europe
that were contaminated with radioactive fallout from
the Chernobyl nuclear reactor.
288 Environmentalbiotechnology
Only recently has the value of naetal-accumulating
plants for environmental remediation been fully real-
ized [13,31,32°',33]. Despite the ever-growing number
of toxic metal contaminated sites, the extremely costly
process of removal and burial, or simply isolation of
the contaminated sites, are still the most commonly
used methods for dealing with heavy metal pollution.
Water treatment facilities also do a relatively poor job
of removing toxic metals from residential and indus-
trial effluents, contributing to the overall problem [34].
The use of metal-accumulating plants for removal of
metals from contaminated soils and waters has a num-
ber of advantages: the lower costs, generation of a
recyclable metal-rich plant residue, applicability to a
range of toxic metals and radionuclides, minimal en-
vironmental disturbance, elimination of secondary air
or water-borne wastes, and public acceptance.
The application of plants in environmental cleanup is
an emerging technology that has been called phytore-
mediation (I Raskin, Grant Proposal #R81869, 1991).
Three subsets of this technology are being devel-
o p e d in our laboratory. First, phytoextraction, in which
metal-accumulating plants are used to transport and
concentrate metals from the soil into the harvestable
parts of roots and above-ground shoots. Second, rhi-
zofiltration, which uses plant roots to absorb, concen-
trate and precipitate toxic metals from polluted efflu-
ents. Finally, phytostabilization, which involves the use
of plants to eliminate the bioavailability of toxic metals
in soils. Since phytostabilization does not necessarily
involve bioaccumulation, w e do not discuss this con-
cept in this review.
In the phytoextraction process, several sequential
crops of laboratory-improved hyperaccumulating pl-
ants may be used to reduce soil concentrations of
heavy metals to environmentally acceptable levels.
Preliminary trials with Ni and Zn hyperaccumulator
plants from the Brassicaceae family were successful
in partially removing heavy metals from soils con-
taminated by long-terna application of heavy metal
containing sludges [32°°]. Dried, ashed or composted
plant residues, highly enriched in heavy metals may
be isolated as hazardous waste or recycled as bio-metal
ore. Although the most heavily contaminated soils do
not support plant growth, sites with light to moderate
toxic metal contamination might be suitable for grow-
ing hyperaccumulating plants for toxic metal cleanup.
Plants that accumulate toxic metals can be grown and
harvested economically, leaving the soil or water with
a greatly reduced level of toxic metal contamination.
Recently, there has been a growing interest in the use
of metal-accumulating roots and rhizomes of aquatic or
semiaquatic vascular plants for the removal of heavy
metals from contaminated aqueous streams. For exam-
ple, water hyacinth (Eicbbornia crassipes) [35], penny-
wort (Hydrocotyle umbellata) [36], duckweed (Lemna
minor) [37,38] and water velvet (Azolla pinnata) [39],
take up Pb, Cu, Cd, Fe, and Hg from contaminated
solutions. In a related development, cell suspension
cultures of Datura innoxia were found to remove
a wide variety of metal ions from solutions [39,40].
Most of the removed metals were tightly chelated by
unidentified components of cell walls in a process that
did not require metabolic activity. The observation that
hydroponically grown roots of terrestrial plants are ex-
tremely effective in removing Pb, Cr, Zn, Cd, Cu, and
Ni from water has laid the foundation for the develop-
ment of rhizofiltration in our laboratory. For example,
1.1 g dry weight of either sunflower (Helianthus an-
nuus) or Indian mustard (Brassica juncea) roots, im-
mersed in 400 ml of water containing 300 gtg ml-1 of Pb,
brought the Pb concentration to below 1 l.tg m1-1 in 8
hours (S Dushenkov, I Raskin, unpublished data). Dis-
appearance of Pb from the solution was accompanied
by a dramatic concentration of Pb in the root tissue,
over 10% on a dry weight basis. Initial analysis in-
dicates that, at least in some instances, rhizofiltration
may provide an attractive alternative to current meth-
ods of chemical and microbial precipitation of heavy
metals.
Conclusions
The bioconcentration of heavy metals by plants is a fas-
cinating area of research that should be able to provide
answers to some of the fundamental questions of plant
biochemistry, nutrition, and stress physiology. Study-
ing heavy metal-plant interactions should improve our
understanding of the mechanisms of ion uptake, accu-
mulation and resistance. The unique 'metal accumula-
tion' and 'metal resistance' genes of metal-accumulat-
ing plants may directly benefit world agriculture and
the environment. Phytoremediation, although still in
its infancy, may one day b e c o m e an established en-
vironmental cleanup technology. Further development
of phytoremediation requires an integrated multidisci-
plinary research effort that combines plant biology, soil
chemistry, soil microbiology, as well as agricultural and
environmental engineering. As a major renewable re-
source exploited by man, plants already give us food,
energy, construction materials, natural fibers, and var-
ious chemicals. The use of plants in environmental
cleanup may guarantee a greener and cleaner planet
for all of us.
Acknowledgements
The authors thank the US Department of EnvironmentalProtection,
New Jersey Commission for Science and Technology,New Jersey
Agricultural Experimental Station (NJAES) and PhytoTech Inc for
supporting the research related to the topic of this review. We are
also grateful to Dr AJM Baker for helpful discussions.

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I Raskin, PBAN Kumar, S Dushenkov and DE Salt, Center for Agricul-
tural Molecular Biology, Environmental and Natural Sciences Build-
ing, Rutgers, The State University of New Jersey, Cook College, PO
Box 231, New Brunswick, New Jersey 08903-0231, USA.