Phytoremediation: Principles and perspectives

Abstract

Human evolution has led to immense scientific and technological progress. Global development, however, raises new challenges, especially in the field of environmental protection and conservation. Technological ingenuity has enhanced the potential for improving industrial development and rapid progress is being made not only in the field of electronics but also in biological, medical and pharmaceutical applications. In recent decades, increasingly precise knowledge of basic biological functions has brought about biotechnological advances. The possibility to produce transgenic organisms has opened up new fields of experimentation and perspectives for scientific and technological development which go beyond the limits of natural evolution.

Full text

Human evolution has led to immense scientific and techno-
logical progress. Global development, however, raises new
challenges, especially in the field of environmental protec-
tion and conservation. Technological ingenuity has en-
hanced the potential for improving industrial development
and rapid progress is being made not only in the field of
electronics but also in biological, medical and pharmaceu-
tical applications. In recent decades, increasingly precise
knowledge of basic biological functions has brought about
biotechnological advances. The possibility to produce
transgenic organisms has opened up new fields of experi-
mentation and perspectives for scientific and technological
development which go beyond the limits of natural evolu-
tion.
In summary, the beginning of the XXI century is witness to
an irreversible dimension of power, with global concern, in
all fields: politics, economics, social and cultural affairs,
science and technology. Technological potential and deve-
lopment, however, have not always had beneficial effects
[15]. Social and cultural progress fall far behind technologi-
cal evolution and the over-exploitation of natural resources
with short-term, fast profit-oriented management systems
has severely damaged the environment.
Acute water and soil pollution are evident consequences
that call for rapid and efficient solutions. However, in addi-
tion, diffuse contamination of large expanses of land [56] is
an ever-growing problem that requires sustainable correc-
tion measures.
Remediation of contamination
The cleanup of soils contaminated by hazardous chemical
substances is a cost-intensive, technically complex proce-
dure. Conventional methods of in situ or ex situ remediation
are based on a number of techniques such as [122]:
• Leaching of pollutant by flushing with water or a
chelate. The leachate is recovered and treated on– or
off-site
• Solidification/stabilization by either physical inclusion
or chemical interactions between the stabilizing agent
and the pollutant.
• Vitrification using thermal energy for soil fusion, allow-
ing physical or chemical stabilization
• Electrokinetical treatment: ionic species of the pollutant
migrate to electrodes inserted into the soil.
• Chemical oxidation or reduction of the pollutant to at-
Abstract
Acute and diffuse contamination of soil and water by heavy
metals and metalloids cause wide, environmental and social
concern. Among the techniques used to cleanup affected
sites, phytoremediation has recently emerged as a new,
cost-effective, environment-friendly alternative. After a short
introduction to the types of plant-based cleanup techniques,
this review focuses on metal hyperaccumulator plants and
their potential use in phytoextraction technology.
Resum
Hi ha una preocupació social i científica creixent per la con-
taminació ambiental, aguda i difusa, dels sòls i de l’aigua
per metalls pesants. Entre la diversitat de tècniques disponi-
bles per a la neteja dels llocs afectats, la fitoremediació ha
emergit recentment com una nova alternativa, efectiva de
costos i sostenible ambientalment. Després d’una breu in-
troducció als diferents tipus de tècniques basades en les
plantes, aquesta revisió se centra principalment en les plan-
tes hiperacumuladores de metalls i considera el seu poten-
cial per a les tecnologies de fitoextracció.
CONTRIBUTIONS to SCIENCE, 2(3): 333-344 (2003)
Institut d’Estudis Catalans, Barcelona
Phytoremediation: principles and perspectives
Joan Barceló* and Charlotte Poschenrieder
Laboratori de Fisiologia Vegetal, Facultat de Ciències, Universitat Autònoma de Barcelona
Keywords: Contamination, heavy metal,
hyperaccumulator plant, phytoremediation.
* Author for correspondence: J. Barceló, Laboratori de Fisiologia
Vegetal, Facultat de Ciències, Universitat Autònoma de Barcelona.
08193 Bellaterra, Catalonia (Spain). Tel. 34 935811267. Fax: 34
935812003. Email: juan.barcelo@uab.es

334 Joan Barceló and Charlotte Poschenrieder
tain chemical species with lower toxicity that are more
stable and less mobile.
• Excavation and off-site treatment or storage at a more
appropriate site (“dig and dump”)
In most cases, these techniques are expensive and tech-
nically limited to relatively small areas. These technical diffi-
culties, together with improved knowledge of the mecha-
nisms of uptake, transport, tolerance and exclusion of heavy
metals and other potentially hazardous, contaminants in mi-
croorganisms and plants, have recently promoted the devel-
opment of a new technology, named bioremediation. Biore-
mediation is based on the potential of living organisms,
mainly microorganisms and plants, to detoxify the environ-
ment [2]. The capacity of plants to clean the environment
has been known since the XVIII century when experiments
by Joseph Priestley, Antoine Lavoissier, Karl Scheele and
Jan Ingenhousz demonstrated that, in light, plants purify the
atmosphere. The importance of green zones for the mainte-
nance of air quality is universally accepted, albeit not always
respected.
The use of plants for purifying contaminated soils and wa-
ter has been developed much more recently. In the 1970s,
reclamation initiatives of mining sites developed technolo-
gies for covering soil with vegetation for stabilization purpos-
es and reduction of visual impact [142]. It was not until the
1990s that the concept of phytoremediation emerged as a
new technology that uses plants for cleaning or decreasing
the toxicity of soils and surface and waste waters contami-
nated by metals, organic xenobiotics, explosives or radionu-
clides [4, 34, 36, 40, 47, 80, 89, 98, 115, 121]. In this com-
prehensive review, we mainly refer to phytoremediation of
soils contaminated by heavy metals.
Phytoremediation of soils contaminated by heavy
metals
Plants show several response patterns to the presence of
potentially toxic concentrations of heavy metal ions. Most
are sensitive even to very low concentrations, others have
developed resistance and a reduced number behave as hy-
peraccumulators of toxic metals [5, 16, 21, 27, 120, 123, 128].
This particular capacity to accumulate and tolerate large
metal concentrations has opened up the possibility to use
phytoextraction for remediation of polluted soils and waters
[58, 131].
Plants with metal resistance mechanisms based on exclu-
sion can be efficient for phytostabilization technologies. Hy-
peraccumulator plants, in contrast, may become useful for
extracting toxic elements from the soil and thus decontami-
nate and restore fertility in polluted areas. In 1885, the Ger-
man botanist A. Baumann had already found that the leaves
of certain plant species grown on soils with high Zn levels
concentrated high amounts of this metal. However, it was
not until the end of the last century that metal hyperaccumu-
lation was studied in detail. In recent years, improved knowl-
edge of the mechanisms of uptake, transport and tolerance
of high metal concentrations in these plants [3, 62, 74, 75,
81, 106, 107, 127, 135, 136] has opened up new avenues for
remediation by phytoextraction.
In practice, to be operative, phytoextraction requires the
fulfillment of several basic conditions. An ideal plant species
for remediation purposes should grow easily on soils conta-
minated by metals, have high soil-to-shoot transfer factors,
tolerate high shoot metal concentrations, and produce high
biomass quickly [1,12, 13, 17, 23, 24, 55, 59, 60,111].
Unfortunately, most metal hyperaccumulator plants grow
quite slowly and have a low biomass, while plants that pro-
duce a high biomass quickly are usually sensitive to high
metal concentrations. The energy costs of metal tolerance
mechanisms are responsible for this phenomenon (trade-off
hypothesis). There are, however, exceptions (e.g. the Ni hy-
peraccumulator Berkheya coddii) that indicate that the ca-
pacity to accumulate and tolerate high metal concentrations
in shoots and to produce high amounts of dry matter are not
always mutually exclusive [117]. The cost of Cu tolerance in
the non-hyperaccumulator Mimulus guttatus is very small
and no consistent effects on growth or competitiveness are
observed [64]. These data indicate that there is no intrinsic
reason why metal-tolerant plants produced for phytoremedi-
ation should be competitively inferior or slow-growing [90].
As this is a crucial point for developing efficient plants for
metal extraction from polluted soils, the mechanisms of met-
al tolerance in hyperaccumulators are addressed later in
more detail.
Phytoremediation of soils contaminated by heavy metals
can be achieved by a number of techniques [122]:
1. Phytoextraction: This technique reduces soil metal
concentrations by cultivating plants with a high capac-
ity for metal accumulation in shoots. Plants used for
Table 1. Some examples of metal hyperaccumulators. Detailed in-
formation can be found in refs. [2, 8, 29, 46]
Species Shoot metal Reference
concentration
µg g–1
Arabidopsis halleri 13,600 Zn Ernst, 1968[44]
(Cardaminopsis halleri)
Thlaspi caerulescens 10,300 Zn Ernst, 1982[46]
Thlaspi caerulescens 12,000 Cd Mádico et al., 1992[91]
Thlaspi rotundifolium 8,200 Pb Reeves & Brooks, 1983 [116]
Minuartia verna 11,000 Pb Ernst 1974[1974]
Thlaspi goesingense 12,000 Ni Reeves & Brooks, 1983[116]
Alyssum bertholonii 13,400 Ni Brooks & Radford, 1978[31]
Alyssum pintodasilvae 9,000 Ni Brooks & Radford, 1978[31]
Berkheya codii 11,600 Ni Brooks, 1998[27]
Psychotria douarrei 47,500 Ni Baker et al., 1985[6]
Miconia lutescens 6,800 Al Bech et al., 1997[24]
Melastoma
malabathricum 10,000 Al Watanabe et al., 1998[7]

Phytoremediation: principles and perspectives 335
this purpose should ideally combine high metal accu-
mulation in shoots and high biomass production. Many
hyperaccumulator species fulfill the first (see Table 1
for examples), but not the second condition.
Therefore, species that accumulate lower metal con-
centrations but are high biomass producers may also
be useful (for examples see Table 2). When plants are
harvested, the contaminants are removed from the
soil. Recovery of high-price metals from the harvested
plant material may be cost effective (e.g. phytomining
[30] of Ni, Tl or Au.). If not, the dry matter can be
burnt and the ash disposed of under controlled con-
ditions.
2. Rhizofiltration: This technique is used for cleaning con-
taminated surface waters or wastewaters by adsorp-
tion or precipitation of metals onto roots or absorption
by roots or other submerged organs of metal-tolerant
aquatic plants. For this purpose, plants must not only
be metal-resistant but also have a high adsorption sur-
face and must tolerate hypoxia [42, 65]. Some exam-
ples are listed in Table 2.
3. Phytostabilization: Plants are used for immobilizing
contaminant metals in soils or sediments by root up-
take, adsorption onto roots or precipitation in the rhi-
zosphere. By decreasing metal mobility, these
processes prevent leaching and groundwater pollu-
tion. Bioavailability is reduced and fewer metals enter
the trophic web.
4. Phytodegradation: Elimination of organic pollutants by
decomposition through plant enzymes or products.
5. Rhizodegradation: Decomposition of organic pollu-
tants by means of rhizosphere microorganisms [138].
6. Phytovolatilization: Organic pollutants absorbed by
plants are released into the atmosphere by transpira-
tion, either in their original form or after metabolic mod-
ification. In addition, certain metals can be absorbed
and volatilized by certain organisms. Several species
of the genus Astragalus accumulate and volatilize Se.
Uptake and evaporation of Hg is achieved by some
bacteria. The bacterial genes responsible have al-
ready been transferred to Nicotiana or Brassica
species, and these transgenic plants may become
useful in cleaning Hg-contaminated soils [13, 101].
7. Hydraulic control: This technique uses plants that ab-
sorb large amounts of water and thus prevent the
spread of contaminated wastewater into adjacent un-
contaminated areas. Phreatophytes can be used for
cleaning saturated soils and contaminated aquifers
[113]
8. Phytorestauration: Revegetation of barren areas by
fast-growing resistant species that efficiently cover the
soil, thus preventing the migration of contaminated soil
particles and soil erosion by wind and surface water
run-off. This technique reduces the spread of contami-
nants and also visual impact. However, previous soil
conditioning is required (e.g. liming or berengerite-
amendments) to enable plants to colonize the polluted
substrate [102, 133, 134,]
In recent years, the scientific and social interest in phy-
toremediation techniques has increased substantially for
several reasons: extensive soil contamination, advanced
scientific knowledge of the mechanisms and functions of liv-
ing organisms and ecosystems, the pressure of public opin-
ion, and political and economical concerns. Twenty years
ago, studies on this subject were scarce, while today many
scientists, especially in the USA and Europe are involved in
basic and applied research projects aimed to make phytore-
mediation a commercially viable technique. Given the inher-
ent limitations of biological systems and the diversity of
problems present at polluted sites, it is unrealistic to con-
ceive phytoremediation as an instant, high-profit, universal
solution for contaminated soil. However, site-specific adap-
tation of general strategies developed in basic scientific re-
search programs can provide sustainable, environment-
friendly solutions for the cleanup of contaminated soils and
sediments. The challenge of contamination cleanup and the
crucial contribution of research in this field can be put into
perspective by considering some statistic and economic
data. In 1998, the European Environmental Agency estimat-
Species Extracted contaminant/substrate References
Salix Heavy metals/soil, water Greger and Landberg, 1999 [60]
Populus Ni/ soil, water, groundwater Punshon and Adriano, 2003[112]
Brassica napus, B. juncea, B. nigra Radionuclides, heavy metals, Se/soil Brown, 1996[32], Bañuelos et al.,1997[12]
Cannabis sativa Radionuclides, Cd/soil Ostwald 2000[105]
Helianthus Pb,Cd /soil EPA, 2000[130] Elkatib et al., 2001[43]
Typha sp. Mn, Cu, Se/mine wastewater Horne, 2000 [65]
Brassica juncea Se/saline drainage effluent Bañuelos et al.1997[14]
Phragmites australis Heavy metals/mine tailings-wetland Massacci et al., 2001[95]
Glyceria fluitans Heavy metals/mine tailings-wetland MacCabe and Otte, 2000[97]
Lemna minor Heavy metals/water Zayed et al., 1998[143]
Table 2. Some examples of high biomass-producing species with potential use in phytoextraction or rhizofiltration

336 Joan Barceló and Charlotte Poschenrieder
ed a total of 1,400,000 contaminated sites in Western Eu-
rope. A comparison of the economic costs between conven-
tional, physical-chemical, decontamination procedures and
new, plant-based, phytoremediation technology clearly fa-
vors the latter. According to several authors [39, 100], con-
ventional procedures raise the average cost per contaminat-
ed hectare of soil from 0.27 to 1.6 million $, while
phytoremediation costs from about 10 to 1000 times less.
From the period 1998-2000 to 2005, the market for phytore-
mediation in the USA is estimated to increase from $16-29
million to $214-370 million The time factor is by far the most
critical point in plant-based cleanup techniques. However,
the long persistence of heavy metal contamination in soils
(residence times of thousands of years) makes even long-
term cleaning strategies attractive.
The complex scenario of acute or diffuse soil contamina-
tion by heavy metals therefore deserves attention by the sci-
entific, economic, social, and politic authorities in order to
provide the means to study successful mechanisms for soil
remediation. Only with a sustainable focus of this kind will
harmony between nature and human evolution be restored.
Metal hyperaccumulator plants
At least 400 species distributed in 45 botanical families are
considered metal hyperaccumulators [27]. By definition, hy-
peraccumulators are herbaceous or woody plants that accu-
mulate and tolerate without visible symptoms a hundred
times or greater metal concentrations in shoots than those
usually found in non-accumulators. Baker and Brooks estab-
lished 0.1% as the minimum threshold tissue concentrations
for plants considered Co, Cu, Cr, Pb or Ni hyperaccumula-
tors, while for Zn or Mn the threshold is 1% [5, 7].
Hyperaccumulators are metallophytes and belong to the
natural vegetation of metal-enriched soils [48, 49, 108].
These species have evolved internal mechanisms that allow
them to take up and tolerate large metal concentrations that
would be extremely toxic to other organisms [37, 79]. These
plants are perfectly adapted to the particular environmental
conditions of their habitat and high metal accumulation may
contribute to their defense against herbivores and fungal in-
fections [26, 94, 127]. However, usually, the metabolic and
energetic costs of their adaptation mechanisms do not allow
them to compete efficiently on uncontaminated soil with non-
metallophytes.
Metal hyperaccumulation has evolved in plants all over
the world and important sites for collecting germplasm are,
among others, New Caledonia, Australia, Central and South
Europe, the Mediterranean Area, South-East Asia, Cuba,
Dominican Republic, California, Zimbabwe, Transvaal in
South Africa, Goiàs in Brazil, Hokkaido in Japan, and New-
foundland in Canada [5, 45].
Several hypotheses have been proposed to explain the
mechanisms of metal hyperaccumulation and the evolution-
ary advantage of this strategy.
1. Complex formation and compartmentation: Hyperac-
cumulators synthesize chelators that detoxify metal
ions by complex formation. The soluble, less-toxic,
organic-metal complex is transported to cell compart-
ments with low metabolic activity (cell wall, vacuole)
where it is stored in the form of a stable organic or in-
organic compound. [18, 19, 20, 54, 62, 129, 135,
136]
2. Deposition hypothesis: Hyperaccumulators separate
metals from the root, accumulating them in plant parts
that are abscised (old leaves), leached by rain (epider-
mis, hairs) or burnt.
3. Inadverted uptake: Hyperaccumulation of the metal is
thought to be the by-product of an adaptation mecha-
nism to other adverse soil characteristics (e.g. Ni hy-
peraccumulation in serpentinophytes)
4. Hyperaccumulation as a defense mechanism against
abiotic or biotic stress conditions. Metal effectiveness
against certain pathogenic fungi and bacteria and on
leaf-consuming herbivores has been reported [25].
Phloem parasites [53], however, are unaffected, prob-
ably because of low phloem mobility of the metals.
High metal concentrations in leaves can act as feeding
deterrents or, after ingestion, may reduce the repro-
duction rate of herbivores or poison them. Trade-off of
organic defenses by metal hyperaccumulation may
also confer advantage [127]. It has been suggested
that high leaf metal concentrations may be used in os-
motic adjustment under drought stress [8, 109]. Exten-
sive studies in Alyssum murale (Ni hyperaccumulator)
and Thlaspi caerulescens (Zn hyperaccumulator)
have not confirmed this hypothesis [141].
At present, the adaptive advantages of the hyperaccumu-
lation strategy are unclear. The strategy probably involves
complex interactions of diverse factors and mechanisms
that cannot be ascertained by a single, reductionistic inter-
pretation. From both the scientific and practical viewpoint,
however, the way in which plants achieve metal hyperaccu-
mulation is of much greater interest than the raison d’être of
the mechanism. Only by studying the basic mechanisms of
hyperaccumulation will phytoextraction technologies be
successfully developed.
Mechanisms of metal tolerance and
hyperaccumulation in plants
Metal hyperaccumulators are highly specialized models of
plant mineral nutrition. Seventeen elements are considered
essential for all higher plants (C, H, O, N, S, P, K, Ca, Mg, Fe,
Mn, Cu, Zn, B, Mo, Cl, and Ni). Macronutrients are those
necessary in high concentrations (mM level) while micronu-
trients are required only in µM tissue concentrations. Hyper-
accumulators concentrate, in a specific way, certain trace
metals or metalloids that may be essential (Cu, Mn, Zn, or Ni)
or not (e.g. Cd, Pb, Hg, Se, Al, As) at amounts that would be

Phytoremediation: principles and perspectives 337
extremely toxic to other plants [3, 5, 27, 62, 67, 93, 110,128].
Some examples are given in Table 1.
Most metallophytes that can colonize metal-polluted soils
base their metal resistance on efficient exclusion of metal
ions from root tip meristems and shoots [16, 18, 20, 50, 54,
82]. In contrast, hyperaccumulators, preferentially accumu-
late the metal in shoots and, in the case of hyperaccumula-
tion of essential trace elements, this capacity is frequently
accompanied by plant requirement for unusually high sub-
strate availability of the metal in order to avoid deficiency
[128, 129]. This may be the result of a constitutively active
mechanism that inactivates incoming metal ions in hyperac-
cumulators. Rapid complexation and compartmentation not
only detoxify metal ions but also make them less available
for essential metabolic processes. To understand the physi-
ological, biochemical and molecular mechanisms that un-
derlie metal hyperaccumulation in plants, it is necessary to
consider metal uptake, transport and metabolic processes
involved in the entire organism, from the rhizosphere to the
leaf cell compartment.
Rhizosphere interactions
The availability of metals in the soil around roots is strongly
affected by root exudates and root depositions (mucilage
and border cells) but also by microbial activities such as
siderophore release and redox reactions [41, 70, 84, 93].
The mutual influence of plants and soil microorganisms and
the selective force of soil metal concentrations on microbe
populations make research on this topic very difficult. Given
the possible environmental problems associated with metal
mobilization by synthetic chelators [22, 61, 85, 110, 118],
enhancement of metal availability in the rhizosphere by hy-
peraccumulators would be a useful mechanism for improv-
ing phytoextraction technologies. At present, however, data
on the role of root exudates from hyperaccumulators in met-
al mobilization are contradictory. While in acidic and cal-
careous soils some authors found that Thlaspi caerulescens
tends to decrease rhizosphere pH [63, 71], others observed
a decrease [99]. However, pH changes do not appear to be
a relevant mechanism for metal mobilization by this kind of
plant. Release of histidine and citrate into the rhizosphere
may play a crucial role in the reduction of Ni uptake and tox-
icity in the Ni sensitive Thlaspi arvense, while in the Ni hyper-
accumulator Thlaspi goesingense no Ni-enhanced release
of these chelators into the rhizosphere was found [119]. The
involvement of bacteria in Zn mobilization in the rhizosphere
of Thlaspi caerulescens has been reported [140]; bacterial
production of siderophores may be responsible for en-
hanced bioavailability [83].
Metal uptake and root-to-shoot transport
Metal resistance in species with exclusion strategy is fre-
quently based on reduced metal uptake into roots, preferen-
tial storage of metals in root vacuoles and restricted translo-
cation into shoots. Hyperaccumulators, in contrast, take up
more metals, store a lower proportion of them in root vac-
uoles, and export higher amounts to shoots (Figure 1).
Comparative studies on Zn uptake and transport in the
non-hyperaccumulator Thlaspi arvense and the Zn– hyper-
accumulator Thlaspi caerulescens indicate that these differ-
ences are caused by altered tonoplast Zn transport in roots
and stimulated Zn uptake into leaves [77, 78]. Stimulation of
ZNT1 expression, a genes that encodes a Zn transporter
that belongs to the ZIP family of plant micronutrient trans-
porters, was higher in Thlaspi caerulescens than in Thlaspi
arvense. Other transporter systems of special interest in hy-
peraccumulators are the cation diffusion facilitators (CDF
type), the ABC transporters for phytochelatins and the metal
chaperones [38, 81, 103, 104, 106, 125, 132]. Detailed stud-
ies on the characterization and the differential distribution of
these transporter systems in hyperaccumulators and non-
hyperaccumulators will clarify the basic genetic and molec-
ular mechanisms responsible for metal hyperaccumulation.
The results from this research may lead to the development
of specific strategies to produce efficient plants for metal ex-
Zn
Zn
Epidermis/cortex Xylem
parenchyma
XYLEM
To Shoot
Endodermis with
Casparian band
Thlaspi arvense
vacuole
Zn
Zn
Epidermis/cortex Xylem
parenchyma
XYLEM
To Shoot
Endodermis with
Casparian band
Thlaspi
caerulescens
vacuole
Figure 1. Differences in Zn transport in roots of the Zn hyperaccu-
mulator Thlaspi caerulescens and the non-accumulator Thlaspi ar-
vense. Thin arrows, low transport; thick arrows, high transport.
(modified after Lasat and Kochian, 2000 [76].)

338 Joan Barceló and Charlotte Poschenrieder
traction technologies by molecular engineering and conven-
tional breeding [10].
The pathways of radial root transport of metals and their
entrance into the vascular cylinder are a further issue of de-
bate in hyperaccumulation research. It has been proposed
that apoplastic transport of Zn to the xylem is required for
sustaining rapid Zn transport to the leaves [106], while facto-
rial experiments with Zn and Cd [86, 92] support the hypoth-
esis of a common, but Cd-preferent, symplastic transport
system for Zn and Cd to the xylem [52].
Formation of less toxic metal complexes is essential in
metal hyperaccumulation in plants. The toxicity of metal
cations is mainly due to their tendency to form organic com-
plexes with distinct ligands, which interfere with membrane
functions, enzyme reactions, electron transport etc. Uptake
and root-to-shoot transport of high metal concentrations is
only possible when these toxic interactions are avoided by
the synthesis of strong chelators that efficiently bind the met-
als in a non-toxic form, thereby allowing flux to and through
the xylem up to the leaves. Organic acids, aminoacids, phy-
tochelatins have been implied in metal detoxification The Zn
and Cd hyperaccumulator Thlaspi caerulescens contains
constitutively high organic acid levels [129]. Many Al hyper-
accumulators contain high concentrations of either or both
organic acids and flavonoid-type phenolics, which form
strong complexes with the metal [18, 19]. High concentra-
tions of organic acid anions in leaf tissues are a crucial,
widely distributed, mechanism that allows plants to maintain
cation/anion homeostasis under conditions of excess ion
stress. The ability to accumulate high organic acid levels in
tissues may be considered a prerequisite, but is not suffi-
cient, for metal tolerance [18, 129].
The sulfhydryl-rich phytochelatins (PC) have high affinity
for binding Cd, Hg, Cu or even As. Arabidopsis cad1 mu-
tants, deficient in PC synthase, are very sensitive to Cd [66].
Nonetheless, treatment of Cd-exposed plants with BSO, an
inhibitor of PC synthesis, increased Cd sensitivity only in
plants that lacked Cd hypertolerance. These results indicate
that Cd hypertolerance is not based on PC-mediated se-
questration. In contrast, PC-based sequestration may be es-
sential in both constitutive As tolerance and As hypertoler-
ance [124].
A metal can be bound by a number of ligands within dis-
tinct plant organs and compartments. In this regard, in 1975,
Mathys [96] and Ernst [51] proposed the malate shuttle hy-
pothesis. According to this hypothesis, in Zn-resistant plants
excess Zn is bound to malate in the cytoplasm and, after
transport to the vacuole, a ligand exchange occurs. Zn
forms more stable complexes with citrate, oxalate or other
ligands, while malate returns to the cytoplasm. Recent re-
search shows that ligand exchange can also play a critical
role in hyperaccumulators. Studies with the Al hyperaccu-
mulator Fagopyrum esculentum revealed the importance of
ligand exchange (oxalate-citrate-oxalate) during Al trans-
port from the rhizosphere through roots to the leaves [88].
Furthermore, in the Ni hyperaccumulator Thlaspi goesin-
gense, Ni is bound by several ligands. Cytoplastic Ni seems
to be detoxified by binding to histidine, while vacuolar stor-
age of Ni is probably in the form of citrate [74].
Data indicate that metal hyperaccumulation is not based
on the capacity of a plant to produce a high concentration of
a particular chelator, but that it involves the coordinated ac-
tion of a number of ligands and differential distribution of di-
verse metal transport systems. Current research is begin-
ning to identify the components that interact in metal
hyperaccumulation. However, the regulatory processes re-
sponsible for this particularly altered metal homeostasis in
hyperaccumulators remains to be elucidated. Progress in
this field of basic research is essential to identify or produce
effective plants for phytoextraction technologies.
Conventional breeding and genetic engineering
for efficient phytoextraction
As stated at the beginning of this review, the development of
commercial phytoextraction technologies requires plants
that produce high biomass and that accumulate high metal
concentrations in organs that can be easily harvested, i.e. in
shoots. There are two main approaches to this problem:
(1) Domestication and breeding of improved hyperaccu-
mulator species [33] and
(2) Application of genetic engineering to develop fast-
growing high biomass plants with improved metal uptake,
translocation and tolerance [69, 73]. Some examples are
shown in Table 2.
The first approach, mainly developed in the USA by the
group headed by Chaney, involves several crucial steps: se-
lection of hyperaccumulator plants that are likely to be do-
mesticated, collection of seeds from wild plants and bioas-
say of their phytoextraction utility, breeding for improved
cultivars and development of adequate soil and crop man-
agement practices [33]. The usefulness of this system has
been shown for Co and Ni and has obtained a utility patent
[35]. However, the authors recognize that in situations where
available hyperaccumulator species are too small to afford
economic cleanup procedures, biotechnology may be re-
quired to combine hyperaccumulation and high biomass
production.
The V Framework Research Program of the European
Community includes two projects [68] on the production of
genetically modified plants for phytoremediation, PHYTAC
and METALLOPHYTES. The former (http://www.uku.fi/~ater-
vaha/PHYTAChome.htm) aims to transfer genes from the hy-
peraccumulator Thlaspi caerulescens to the high biomass–
producing Brassica or tobacco. While the METALLO-
PHYTES project is devoted to engineering Festuca for im-
proved metal tolerance and or accumulation
(http://biobase.dk/~palmgren/metallophytes.html).
To date, the most successful approach has been the
transformation of plants using modified bacterial merA gene
(mer A9) for detoxifying Hg (II). The merA gene codifies for a
mercuric ion reductase that removes Hg from stable thiol
salts by reducing it to volatile metallic Hg. When grown in a 5

Phytoremediation: principles and perspectives 339
µM Hg(II) solution, transformed Arabidopsis plants express-
ing the merA9 gene volatilized 10 ng of metallic Hg per
minute and mg plant tissue. Hg reductase has also been
successfully transferred to Brassica, tobacco and yellow
poplar trees [101].
There are, however, ecological, social, and legal objec-
tions to the practical application of genetically modified or-
ganisms in the field. The potential of transgenic plants to effi-
ciently cleanup contaminated sites may help to change
adverse public opinion. Nonetheless, future research should
address not only the “know-how” of producing efficient
plants for phytoremediation and their integration into sus-
tainable cropping and management systems, but should
also clarify the potential impact of transgenic plants on the
target habitat and the fate of the introduced genes in the sur-
rounding environment.
Concluding remarks
Phytoremediation is a new, attractive technique that has
emerged over recent years. This technique offers excellent
perspectives for the development of plants with the potential
for cleaning metal-contaminated soils, at least under certain,
favorable conditions and for using adequate crop manage-
ment systems. Advances in molecular biology and genetic
engineering of plants have been indispensable for this
progress. However, this spectacular development would not
have been possible without the invaluable contribution of a
small group of researchers. More than thirty years ago they
showed extraordinary scientific insight by recognizing the
enormous potential of plants that can colonize metal-conta-
minated soils and they dedicated many years of conscien-
tious research to the geobotany and ecophysiology of metal-
lophytes. Exploratory studies of this kind are still necessary
and should be supported in order to preserve the immense
natural genetic resources of metallipherous habitats and to
increase our basic knowledge about the natural adaptation
mechanisms of hyperaccumulators.
In this regard, we dedicate this review to Prof. W.H.O
Ernst, an eminent pioneer in research into metallophytes.
Acknowledgements
Part of the authors work cited in this paper was supported by
the Generalitat de Catalunya (Autonomous Government of
Catalonia) (2001 SGR-00200), the European Union (ICA4-
CT-2000-30017), and the Spanish Government (DGICYT,
BFI2001-2475-CO2-01).
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344 Joan Barceló and Charlotte Poschenrieder
About the authors
Joan Barceló was born in Palma de
Mallorca in 1938. He graduated in
Pharmacy from the University of
Barcelona in 1964 and obtained Ph.D.
in 1970 with his work on the effect of
UV radiation on plants. In 1973 he be-
came an associate lecturer at the Fac-
ulty of Pharmacy of the same university
and in 1976 he was appointed Full Pro-
fessor of Plant Physiology at the Com-
plutense University of Madrid. From
1980 to 1982 he held the post of Pro-
fessor of Plant Physiology at the Uni-
versity of the Balearic Islands, where
he was also the Dean of the Science
Faculty. Since 1982 Joan Barceló has
been a Professor and Head of the
Plant Physiology Laboratory at the Sci-
ence Faculty of the Autonomous Uni-
versity of Barcelona. He is full member
of the Royal Academy of Pharmacy of
Catalonia.
Charlotte Poschenrieder was born in
1954 in Munich (Germany). She ob-
tained her degree in Pharmacy and her
Ph.D. from the Complutense University
of Madrid in 1978 and 1980, respec-
tively. From 1980 to 1982 she held the
post of assistant lecturer at the Science
Faculty of the University of the Balearic
Islands. Since 1982, she has been
teaching and carrying out research in
the Plant Physiology Laboratory at the
Autonomous University of Barcelona;
at present she holds the post of
tenured lecturer.
The authors research mainly focus-
es on stress physiology in plants, and
is reflected in more than 250 publica-
tions, many in peer-reviewed interna-
tional journals. The team headed by
Joan Barceló and Charlotte Poschen-
rieder is involved in several national
and international, research projects.
The mechanisms of heavy metal stress
and tolerance and the adaptation of
maize to acid soil conditions in the
tropics are the main subjects of their
current research.
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