remediation of heavy metal contaminated soils ... replacement. because biological ... soil washing...

Critical Reviews in Environmental Science and Technology, 39:622–654, 2009Copyright © Taylor & Francis Group, LLCISSN: 1064-3389 print / 1547-6537 onlineDOI: 10.1080/10643380701798272

Remediation of Heavy Metal ContaminatedSoils: Phytoremediation as a Potentially

Promising Clean-Up Technology

ANA P. G. C. MARQUES, ANTONIO O. S. S. RANGEL,and PAULA M. L. CASTRO

Escola Superior de Biotecnologia, Universidade Catolica Portuguesa, Rua Dr. AntonioBernardino de Almeida, 4200-072 Porto, Portugal

Increased soil pollution with heavy metals due to various humanand natural activities has led to a growing need to address en-vironmental contamination. Some remediation technologies havebeen developed to treat contaminated soil, but a biology-based tech-nology, phytoremediation, is emerging. Phytoremediation includesphytovolatilization, phytostabilization, and phytoextraction usinghyperaccumulator species or a chelate-enhancement strategy. Toenhance phytoremediation as a viable strategy, microbiota fromthe rhizosphere can play an important role, but the use of geneticengineering can also increase the success of the technique. Here wereview the key information on phytoremediation, addressing bothpotential and limitations, resulting from the research establishedon this topic.

KEY WORDS: phytoremediation, heavy metals, rhizospherecommunity, genetic engineering

INTRODUCTION

Because it is at the interface between the atmosphere and the earth’s crust,as well as being the substrate for natural and agricultural ecosystems, thesoil is open to inputs of heavy metals from many sources (Alloway, 1990).Due to their immutable nature, metals are a group of pollutants of muchconcern. The danger of toxic metals is aggravated by their almost indefinite

Address correspondence to Paula M. L. Castro, Escola Superior de Biotecnologia, Univer-sidade Catolica Portuguesa, Rua Dr. Antonio Bernardino de Almeida, 4200-072 Porto, Portugal;Tel.: + 351 22 558 00 59; Fax: + 351 22 509 03 51; E-mail: [email protected]

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Remediation of Heavy Metal Contaminated Soils 623

persistence in the environment (Garbisu & Alkorta, 2001). Heavy metalscannot be destroyed but can only be transformed from one oxidation stageor organic complex to another.

Pollution of the biosphere with toxic metals has accelerated dramaticallysince the beginning of the industrial revolution (Nriagu, 1979). The primarysources of this pollution are the burning of fossil fuels, mining and smeltingof metalliferous ores, metallurgical industries, municipal wastes, fertilizers,pesticides, and sewage (Alloway, 1990). In addition to sites contaminated byhuman activity, natural mineral deposits containing particularly large quanti-ties of heavy metals are present in many regions of the globe (Memon et al.,2000).

In response to a growing need to address environmental contamination,many remediation technologies have been developed to treat contaminatedsoil (Riser-Roberts, 1998), mainly mechanically or physio-chemically basedremediation methods. The most commonly used techniques are listed inTable 1. However, these technologies are usually expensive and soil disturb-ing, sometimes rendering the land useless as a medium for further activitiessuch as plant growth. Consequently, a biologly-based emerging technologyis gaining the attention of both soil remediation scientists and the generalpublic—phytoremediation. Phytoremediation makes use of the naturally oc-curring processes by which plants and their microbial rhizosphere organismssequester, degrade or immobilize pollutants for cleaning not only soils butalso water matrices contaminated with heavy metals or organic pollutants(Pilon-Smits, 2005).

PHYTOREMEDIATION OF HEAVY METAL CONTAMINATED SOILS

The basic idea that plants can be used for environmental remediation isvery old and cannot be traced to any particular source (Raskin et al., 1997).Nevertheless, an interdisciplinary research approach combined with a seriesof fascinating scientific discoveries have allowed the development of thisidea into an emerging technology, phytoremediation, which uses plants andtheir associated rhizospheric microorganisms to remove, degrade, or immo-bilize various contaminants from polluted soils, but also from sediments,groundwater, or surface water. Early research indicates that phytoremedi-ation is a promising clean-up solution for a wide variety of contaminatedsites, although it has its restrictions. Many of the limitations and advantagesof phytoremediation are a direct result of the biological aspect of this typeof treatment system (Singh et al., 2003). Plant-based remediation technolo-gies can function with minimal maintenance after its establishment, as thecosts of growing a crop are minimal compared to those of soil removaland replacement. Because biological processes are ultimately solar-driven,phytoremediation is on average ten-fold cheaper than engineering-based


624 A. P. G. C. Marques et al.

TABLE 1. Technologies for remediation of heavy metal contaminated soil (Hamby, 1996;Khan et al., 2004; Mulligan et al., 2001; Ottosen and Jensen, 2005)

Soil washing The process separates coarse soil (sand and gravel) from finesoil (silt and clay), where contaminants tend to bind andsorb. This soil fraction must be further treated with othertechnologies.

Soil vapor extraction Involves the installation of wells in the area of contamination.Vacuum is applied through the wells to evaporate thevolatile constituents of the contaminated mass, which aresubsequently withdrawn through an extraction well.Afterward, the extracted vapors are adequately treated.

Soil flushing “Floods” contaminated soils with a solution that moves thecontaminant to an area where they can be removed. Soilflushing is accomplished by passing an extraction fluidthrough soils using an injection or infiltration process.Recovered fluids with the absorbed contaminants mayneed further treatment.

Solidification Encapsulates the waste materials in a monolithic solid of highstructural integrity.

Stabilization/immobilization Reduces the risk posed by a waste by converting thecontaminant into a less soluble, immobile, and toxic form.

Vitrification Uses a powerful source of energy to “melt” soil at extremelyhigh temperatures (1600–2000oC), immobilizing mostinorganics into a chemically inert, stable glass product anddestroying organic pollutants by pyrolysis.

Electrokinetics Removes contaminants from soil by application of an electricfield.

Thermal desorption Contaminated soil is excavated, screened, and heated totemperatures such that the boiling point of thecontaminants is reached, and they are released from thesoil. The vaporized contaminants are often collected andtreated by other means.

Encapsulation Physical isolation and containment of the contaminatedmaterial. The impacted soils are isolated by lowpermeability caps or walls to limit the infiltration ofprecipitation.

remediation methods, such as soil excavation, soil washing or burning, orpump-and-treat systems (Glass, 1999). The fact that phytoremediation is car-ried out in situ contributes to its cost-effectiveness and may reduce exposureof the polluted substrate to humans, wildlife, and the environment (Pilon-Smits, 2005). However, it is not always the best solution to a contaminationproblem. The use of phytoremediation is limited by the climatic and geo-logical conditions of the site to be cleaned, such as temperature, altitude,soil type, and the accessibility for agricultural equipment (Schmoger et al.,2000). On one hand, phytoremediation is far less disruptive to the environ-ment, but, on the other, other problems may arise (e.g., contaminants canbe accumulated in wood that can be used as fuel; the pollutants collectedin leaves can be released again into the environment during litter fall; seeSchmoger et al., 2000). One way to summarize many of the limitations of



phytoremediation is that contaminants must be available to a plant and itsroot systems (i.e., the plants that mediate the clean-up have to be where thepollutant is and have to be able to act on it; see Pilon-Smits, 2005).

Plants have a range of potential mechanisms at the cellular level thatmight be involved in the detoxification and tolerance to heavy metal stress.These all appear to be involved primarily in avoiding the build-up of toxicconcentrations at sensitive sites within the cell, thus preventing the dam-aging effects (Hall, 2002). When metals accumulate in tissues they oftencause toxicity, both directly by damaging cell structure and indirectly viareplacement of other essential nutrients (Taiz & Zeiger, 2002). The strategiesfor avoiding heavy metal build-up are diverse (Hall, 2002; Marschner, 1995;Mejare & Bulow, 2001). One way of avoiding metal accumulation can bethe restriction of its movement to roots with the help of mycorrhizal fungi.As an example, Huang et al. (2002) reported an exclusion strategy of Znin arbuscular mycorrhizal Zea mays. Reduction of the influx across plasmamembrane as well as binding to cell wall and root exudates can also bepossible avoidance strategies, as shown by Marques et al. (2007b) for reten-tion of Zn in the cell walls of Solanum nigrum. Other mechanisms used byplants to avoid metal build-up can be the stimulation of the efflux of met-als into the apoplast. As an example, Benaroya et al. (2004) demonstratedthat this stimulation occurred, and that the apoplastic accumulation of Pbwas very significant in Azolla filiculoides-, or the chelation in cytosol byvarious ligands. Ligands such as phytochelatins and metalotheins promotethe detoxification abilities of metals in the plant, as shown for the engi-neered Nicotiana tabacum (Mejare & Bulow, 2001). A possible avoidancestrategy is that transport and accumulation of metals in the vacuole—the Nihyperaccumulator Thlaspi goesingense—enhances its Ni tolerance by trans-porting and compartmentalizing most of the intracellular leaf Ni into thevacuole (Kramer et al., 2000) in order to restrict metal accumulation in ar-eas of the cell where the occurrence of metals will be damaging to cellfunctions.

Various phytoremediation strategies are possible for the remediation ofheavy metal contaminated soils (Salt et al., 1998). Different phytotechnolo-gies make use of different plant properties (Pilon-Smits, 2005). The maintreatment streamlines are described in Figure 1 and can be regarded as oneof the following:

� phytovolatilization: contaminants taken up by the roots pass through theplants to the leaves and are volatized through stomata, where gas ex-change occurs (Vroblesky et al., 1999);

� phytostabilization: plants are used to reduce the mobility and bioavail-ability of environmental pollutants (Vangronsveld et al., 1995); or

� phytoextraction: plant roots take up contaminants and store them in stemsand leaves (harvestable regions) (Kumar et al., 1995).



FIGURE 1. Types of soil phytoremediation (adapted from Sing et al., 2002; Suresh andRavishankar, 2004).

Phytovolatilization of Heavy Metals

The chemical conversion of toxic elements into less toxic and volatile com-pounds is a possible strategy for detoxification of metal ion contaminants,resulting in the removal of specific harmful volatile elements (e.g., Hg andSe) from soil and plant foliage to the atmosphere (Raskin et al., 1997).

For example, the volatilization of Se involves the assimilation of inor-ganic Se into the organic selenoaminoacids selenocysteine and selenome-thionine. The latter can be biomethylated to form dimethylselenide, whichis volatile and can be lost to the atmosphere (Terry et al., 2000). Bras-sica juncea was identified as a valuable plant for removing Se from soils(Banuelos & Meek, 1990; Banuelos et al., 1993) via Se volatilization. Mer-cury in its elemental form is also easily volatilized, as it is liquid at roomtemperature. However, because of its high reactivity, Hg in the environmentexists mainly as a divalent cation Hg2+; bacteria can catalyze the reductionof the mercuric ion to elemental Hg and enhance the volatilization abilitiesof associated plants (Fox & Walsh, 1982). The volatilization of As has alsobeen demonstrated for microorganisms but, as for Hg, this does not appearto be a significant process in plants (Rugh et al., 1996).

The practicality of using plants able to volatilize metals for environmen-tal remediation seems questionable, however: if a toxic volatile compoundis emitted by plants during phytoremediation, the fate of the gas in the



atmosphere should be determined as part of risk assessment (Pilon-Smits,2005). Such a study was done for volatile Se and Hg, and it was reported thatthe pollutants were dispersed and diluted to such an extent that volatilizationdid not pose a threat (Lin et al., 2000; Meagher et al., 2000). Therefore, thework developed in this area points to a new environmental use of plants.

Phytoextraction of Heavy Metals

The term “phytoextraction” mainly concerns the removal of heavy metalsfrom soil by means of plant uptake. This technology is based on the capacityof the roots of plants to absorb, translocate, and concentrate toxic metals fromsoil to the aboveground harvestable plant tissues. The concentration processresults in a reduction of the contaminated mass and also in the transfer of themetal from an aluminosilicate-based matrix (soil) to a carbon-based matrix(plants). The carbon in the plant material can be oxidized to carbon dioxide,further decreasing (and concentrating) the mass of material to be treated,disposed, or recycled (Blaylock & Huang, 2000).

Metals can exist in the soil as discrete particles or can be associatedwith different soil components, including free metal ions and soluble metalcompounds in the soil solution, exchangeable ions sorbed onto inorganicsolid phase surfaces, nonexchangeable ions and precipitated or insoluble in-organic metal compounds (carbonates, phosphates, etc.), metals complexedby soluble or insoluble organic material, and metals bound in silicate miner-als. Contamination events are usually indicated by discrete particles or highconcentrations found in the first four components; the fifth component isindicative of background or indigenous soil metal concentrations (Ramoset al., 1994). The metals considered available for plant uptake are those thatexist as soluble components in the soil solution or are easily desorbed orsolubilized by root exudates or other components of the soil solution, theseportions representing often only a small part of the total metal content ofthe soil (Blaylock & Huang, 2000). A major factor driving up the availabilityof metallic ions, solubility (Petrangeli et al., 2001), depends on various soilphysicochemical factors, such as the degree of complexation with solubleligands, the type and density of the charge on soil colloids, the reactive sur-face area (Norwell, 1984), and also the soil pH (Harter, 1983). Soil colloidalparticles provide large interface and specific surface areas, which play animportant role in regulating the concentrations of many trace elements andheavy metals in natural soils. In the soil, metal availability to plant roots de-creases as the soil pH increases, as shown by Wang et al. (2005) for Thaspicaerulescens growing in a Cd- and Zn-contaminated soil. Additionally, thepresence in the soil of particles with a high specific surface area may alsoreduce the soluble concentration of specific metals in the contaminated soil.However, this seems to be metal-specific—McBride and Martinez (2000) have



reported that the solubility of Mo, As, Cd, Pb, and Cu was decreased by theaddition of an amendment consisting of hydroxides with high reactive sur-face area, whereas the solubility of Zn and Ni remained unchanged. Thesephysiochemical factors are dependent upon soil properties, including metalconcentration and form, particle size distribution, quantity and reactivity ofhydrous oxides, mineralogy, and degree of aeration and microbial activity(Magnuson et al., 2001). It is thus clear that the soil factors influencing theconcentration, form, and plant availability of metals are highly complex.The supply of ions from the soil is controlled by the kinetics of solubiliza-tion of ions absorbed to its solid phase (Chaney et al., 1997). The limitedbioavailability of various metallic ions, due to their low solubility in waterand strong binding to soil particles, restricts their uptake/accumulation byplants. The plant itself can enhance metal bioavailability. For example, plantscan extrude H+ via ATPases, which replace cations at soil cation exchangecapacity (CEC) sites, making metal cations more bioavailable (Taiz & Zeiger,2002).

Plant species vary significantly in the ability of accumulating metalsfrom contaminated soils, as a balance between the uptake of essential metalions to maintain growth and development and the ability to protect sen-sitive cellular activity and structures from excessive levels of essential andnon-essential metals is required (Garbisu & Alkorta, 2001). Generally, metalsenter the plants primarily via absorption of the available metal ions fromthe soil solution into the root symplasm, driven by the electrical chemi-cal potential gradient across the plasma membrane of root cells (Blaylock& Huang, 2000). Once inside the plant, most metals are too insoluble tomove freely in the vascular system, so they usually form phosphate, sul-phate, or carbonate precipitates. These precipitates are then immobilized inthe apoplastic (extracellular)—cellular walls and intercellular spaces—andsymplastic (intracellular) compartments, such as vacuoles. Unless the metalion is transported as a non-cationic metal chelate, apoplastic transport is fur-ther limited by the high CEC of cell walls (Raskin et al., 1997). Some metalsmay be transported to the shoots by the transpiration stream complexed toorganic acids, mainly citrate (Senden et al., 1992). Taking into account thefeatures of the uptake and translocation mechanisms cited above, the idealplant to be used in phytoextraction should have the following characteristics:

� be tolerant to high levels of the metal;� have a profuse root system;� have a rapid growth rate;� have the potential to produce a high biomass in the field; and� accumulate high levels of the metal in the harvestable parts, as generally

the harvestable portion of most plants is limited to the aboveground parts(although the roots of some crops may also be harvestable).



However, the capacity of plants to concentrate metals in their harvestabletissues may be considered by some authors as a detrimental trait, as someplants can be directly (via plant consumption) or indirectly (via animal con-sumption and further contamination of the food chain) responsible for aportion of the dietary intake of toxic metals by humans, which can havedamaging effects on human health (Brown et al., 1995b; Ow, 1996). Twodifferent approaches have been generally proposed for the phytoextrationof heavy metals, based on the different characteristics required for a plantto be useful for this application: the use of natural hyperaccumulator plantswith exceptional metal-accumulating capacities, and the utilization of highbiomass plants with a chemically (chelate) enhanced method of phytoex-traction (Salt et al., 1998).

HYPERACCUMULATION OF HEAVY METALS

Some naturally occurring plants, termed metal hyperaccumulator plants, canaccumulate in their harvestable tissues abnormally high levels of some met-als. According to Reeves and Baker (2000), the term hyperaccumulator,describing a plant with a highly abnormal level of metal accumulation, ap-pears to have been first applied by Jaffre et al. (1976), who reported high Niconcentrations in the New Caledonian plant Sebertia acuminate. The spe-cific use of the term to denote a defined concentration (>1000 mg Ni/kg)was introduced by Brooks et al. (1977) in discussing Ni concentrations inspecies of Homalium and Hybanthus from various parts of the world—hyperaccumulation was used to describe accumulation of Ni to >1000 mgkg−1 in dry leaf tissue, because it was a level 100 to 1000 times higher thanthat normally found in plants growing on soils non-contaminated with Ni,and 10 to 100 times higher than that found for most other plants growingon Ni-rich soils. An attempt to give greater precision to the definition ofhyperaccumulation was made by Reeves (1992) for Ni: “A hyperaccumulatorof Ni is a plant in which a Ni concentration of at least 1000 mg kg −1 hasbeen recorded in the dry matter of any aboveground tissue in at least onespecimen growing in its natural habitat.”

The definition of hyperaccumulation has extended to elements otherthan Ni. Brooks et al. (1980), Malaise et al. (1978), and Reeves and Brooks(1983) applied the 1000 mg kg−1 criterion for, respectively, Co, Cu, and Pbaccumulation. For Zn, normally present at higher and more widely rang-ing concentrations, a 10,000 mg kg−1 threshold was suggested by Bakerand Brooks (1989). The present definition of an hyperaccumulator is moreextensive and should meet the following requirements:

� the concentration of the metal in the shoot must be higher than: 1.0% forZn and Mn, 0.1% for Al, As, Se, Ni, Co, Cr, Cu, and Pb, and 0.01% for Cd(Baker and Brooks, 1989);



� the shoot to root concentrations ratio must be invariably higher than 1(McGrath & Zhao, 2003), indicating an efficient ability to transport metalsfrom roots to shoots and, most likely, the existence of tolerance mecha-nisms to cope with high concentrations of metals; and

� the shoot to soil concentration ratio must be higher than 1, indicatinghigher metal concentrations in the plant than in the soil, which emphasizesthe degree of plant metal uptake (McGrath & Zhao, 2003).

Despite these requirements, hypertolerance seems to be the key propertythat makes hyperaccumulation possible. The apparent tolerance of plantsto increasing levels of toxic elements can result from the exclusion of toxicelements or their metabolic tolerance to specific elements. The major mech-anism in tolerant plant species appears to be compartmentalization of metalions (i.e., sequestration in the vacuolar compartment or cell walls), whichexcludes them from cellular sites where processes such as cell division andrespiration occur, thus providing an effective protective mechanism (Chaneyet al., 1997). This is consistent in reports of histochemical localization of met-als in several plants, namely A. maritime ssp. halleri (Heumann, 2002), Silenevulgaris (Harmens et al., 1993), Avicenia marina (MacFarlane & Burchett,2002), and Solanum nigrum (Marques et al., 2007b). There are studies in-dicating that hypertolerance in known hyperaccumulators, such as the Zn-hyperaccumulator Thlaspi caerulescens, is due to an alteration of these mech-anisms (Vazquez et al., 1994).

The majority of hyperaccumulating species discovered so far are re-stricted to tropical areas (Baker & Brooks, 1989; Baker et al., 1993; Brookset al., 1993; Ma et al., 2001). More than 430 taxa to date were reportedto hyperaccumulate heavy metals, ranging from annual herbs to perennialshrubs and trees (Whiting et al., 2002), and some species, such as Sedumalfredii, show the capacity of accumulating two or more elements (He et al.,2002; Yang et al., 2002, 2004). For example, one of the best known Zn hy-peraccumulators is Thlaspi caerulescens. While most plants exhibit toxicitysymptoms at Zn concentrations of about 100 mg kg−1, T. caerulescens wasshown to accumulate up to 26,000 mg kg−1 without showing any damageto the plant (Brown et al., 1995b). In addition, this species extracted up to22% of soil exchangeable Cd from a contaminated site (Gerard et al., 2000).Unfortunately, T. caerulescens can be described as a low biomass plant, asit typically produces 2–5 t ha−1 of shoot dry matter (McGrath et al., 2002).Indeed, hyperaccumulator species tend to grow slowly and to have lowbiomass yields (Chen et al., 2004; Raskin et al., 1997); the annual yields inbiomass of hyperaccumulators are generally one to two orders of magnitudelower than those of robust crop plants (Ow, 1996). However, some authors(Chaney et al., 1997; McGrath & Zhao, 2003) keep defending that naturalmetal hyperaccumulator phenotype appears to be much more important



TABLE 2. Abnormal metal accumulation levels registered for the abovegroundsections of some plant species growing in contaminated soils

Plant species MetalMaximum concentrationin the plant (mg kg−1)

Psycotria vanhermanni Ni 35720Psycotria glomerata Ni 10250Psycotria osseana Ni 12780Garcinia bakeriana Ni 7440Streptanthus polygaloydes Ni 14800Thlaspi tatrense Zn 20100Cardaminopsis halleri Zn 13620Dichapetalum gelonioides Zn 30000Viola calaminaria Zn 10000Minuarti vernia Pb 20000Armeria maritime Pb 1600Agrostis tenuis Pb 13490Alyxia rubricalis Mn 14000Maytenus bureaviana Mn 33750Lecythis ollaria Se 18200Astragalus racemosus Se 14920Aeollanthus subacaulis Cu 13700Haumaniastrum robertii Co 10232

Adapted from Reeves and Baker (2000).

than high biomass yield when using plants to treat metal contaminated soilsvia phytoextraction. Nevertheless, Long et al. (2002) have recently reporteda large biomass plant species, Sedum alfreddi, growing in some ancientPb-Zn mine areas in Eastern China, which can also hyperaccumulate Zn.Examples of other plants accumulating metals and metalloids are diverse.Ma et al. (2001) reported the first As hyperaccumulator in terrestrial plants,the brake fern Pteris vittata, which can produce a relatively large biomassin favourable climates; it can accumulate up to 22,000 mg As kg−1 in thefrond (dry weight), although phytotoxicity occurs when shoot arsenic levelis higher than ca. 10,000 mg kg−1 (Wang et al., 2002). Hyperaccumulationof Mg has also been reported, namely in Vaccinium myrtillus (Denayer-DeSmet, 1966). Other examples are listed in Table 2.

CHELATE-ASSISTED PHYTOEXTRACTION OF HEAVY METALS

Chelate-enhanced phytoextraction is based on the fact that the applicationof metal-chelating agents to a contaminated soil may enhance metal accu-mulation by plants (Garbisu & Alkorta, 2001). In the majority of cases, metaluptake into roots occurs from the aqueous phase (Lasat, 2002). In soil, somemetals occur primarily as soluble or exchangeable, readily available form.Nevertheless, other metals occur as insoluble precipitates that are largely un-available for plant uptake (Pitchel et al., 1999). Binding and immobilizationwithin the soil matrix can significantly restrict the potential for soil phytoex-traction (Lasat, 2002). In general, for any given heavy metal, only a fraction



is bioavailable, and thus, potentially, it is only this fraction that can be takenup by the plants. More of the metal could be converted to the bioavailablefraction as it is gradually removed by the plant, but the extent to which thishappens and the kinetics of such processes are soil-specific (Khan et al.,2000). The addition of chelating agents and the consequent formation ofmetal-chelate complexes prevents precipitation and sorption of the metals inthe soil, thereby maintaining their availability for plant uptake. The additionof chelates to the soil can also bring metals into solution through desorp-tion of sorbed species and dissolution of precipitated compounds (Norwell,1984). Additionally, the application of certain chelates to the soil increasesthe translocation of heavy metals into the shoots (Blaylok et al., 1997). Luoet al. (2004) reported that the application of chelating agents increased theroot-to-shoot ratios of the metals Cu, Pb, Zn, and Cd in Zea mays and Phase-olus vulgaris.

Many studies concerning chelate-assisted phytoextraction havebeen reported, with the use of chelating agents such as CDTA(trans-1,2-diaminocyclohexane-N,N,N’,N’-tetraacetic acid), HEIDA [N-(2-hydroxyethyl)iminodiacetic acid] (Chiu et al., 2006), HEDTA (N-hydroxyethylenediaminetriacetic acid) DTPA (diethylenetriaminepentaaceticacid) (Chiu et al., 2006; Huang et al., 1997), EGTA [ethyleneglycol-bis(β-aminoethyl ether),N,N,N′, N-tetraacetic acid], or EDDHA (ethylenediamine-di-o-hydroxyphenylacetic acid) (Huang et al., 1997). Other components—namely, the malic (Chiu et al., 2006; Wu et al., 2004), citric (Chiu et al.,2006; Quartacci et al., 2006; Wu et al., 2004), and nitriloacetic acids(Chiu et al., 2006; Quartacci et al., 2006)—have been proposed as use-ful for enhanced-phytoextraction. Nevertheless, the majority of the re-ports indicate EDTA (ethylenediaminetetraacetic acid) and/or EDDS (SS-ethylenediaminedissucinic acid) as the main chelates applied in these studies,being those that more successfully improve heavy metal uptake by plants(Chen et al., 2004; Grcman et al., 2003; Huang et al., 1997; Lai & Chen,2004, 2005; Luo et al. 2005, 2006; Marques et al, 2007c; Wu et al., 2004). Asan example, Marques et al. (2007c) reported that the addition of EDTA tocontaminated soils promoted an increase in the concentration of Zn accu-mulated by Solanum nigrum of up to 231% in the leaves, 93% in the stems,and 81% in the roots, while EDDS application enhanced the accumulationin leaves, stems, and roots up to 140, 124, and 104%, respectively, with theplants accumulating up to 8267 mg Zn kg−1 in the stems.

Despite the possible usefulness of this technology, some concerns havebeen expressed regarding the potential inherent risk of leaching of metals togroundwater. The addition of chelates to a metal-contaminated matrix canincrease the levels of the water extractable metals. For example, the appli-cation of EDTA to a metal-contaminated soil has been reported to increasesignificantly the concentrations of Cd, Zn, and Pb in the soil solution (Lai &Chen, 2004, 2005). High concentrations of heavy metals in soil solution could



pose an environmental risk in the form of groundwater contamination (Lombiet al., 2001). A similar trend is also reported for EDDS. However, EDDS has areduced risk of metal leaching in the application to metal contaminated soils(Grcman et al., 2003; Luo et al., 2006; Marques et al., 2007c). Marques et al.(2007c) reported that the Zn concentration in water-extracts of soils collectedat the time of plant harvest were significantly increased by the addition ofEDTA or EDDS, by up to 4.0- and 3.1-fold, respectively. EDDS thus seemsa safer option when the application of chelate-assisted phytoextraction isconsidered. Additionally, synthetic chelating agents at high concentrationscan also be toxic to plants. Chlorosis, necrosis, and impairment of plantgrowth have been reported for plants growing in soils amended with EDTA(Chen et al., 2004; Luo et al. 2005, 2006; Wu et al., 2004), EDDS (Luo et al.,2005, 2006), NTA (Kulli et al., 1999; Quartacci et al., 2006), and citric acid(Quartacci et al., 2006).

Moreover, the presence of these chelates can reduce the occurrenceand number of microorganisms in the rhizosphere (Marques et al., 2007c).Chelates, especially EDTA (Grcman et al., 2003), can greatly reduce thenumber of microbivorous nematodes (Romkens et al., 2002) and increasethe stress index of microbial populations. Marques et al. (2007c) reportedthat the addition of EDDS, and especially of EDTA, to Zn-contaminated soilspromoted a decrease in the root colonization of Solanum nigrum by AMF.Grcman et al. (2003) showed that EDTA and EDDS addition to a metal-contaminated soil induced fungi stress. Chen et al. (2004) indicated lowerAMF colonization of Zea mays when grown in EDTA-treated soil. Sudovaet al. (2007) also reported a reduced AMF colonization of Nicotiana tabacumwhen growing in EDDS-treated soil.

The ability of plants to accumulate metals in the harvestable tissuesis not the only factor influencing or determining the ability of phytoex-traction, either by using hyperaccumulators or by adding chelating agents,to effectively remediate a metal-contaminated site. Other important factorssuch as the adequate selection of a site conductive to phytoextraction, aswell as metal solubility and availability for uptake, should be taken into ac-count (Blaylock & Huang, 2000). Soil clean-up criteria are also important inconsidering phytoextraction as a remedial option. The regulatory goals andtimeline must be indicative that phytoremediation is an applicable solution.Therefore, treatability studies should be conducted to evaluate a particularsite, especially the suitability of the soil for a phytoextraction treatment, withthe evaluation of metal solubility as an essential step in the study (Blaylock& Huang, 2000).

Phytostabilization of Heavy Metals

Some soils are so heavily contaminated that the use of plants for removingmetals would not be an adequate approach and would take an unrealistic



amount of time. Nevertheless, without some remediation effort, these con-taminated areas remain barren, and the contaminated soil remains exposedto human and animal contact and to erosion that may carry contaminantsoff site. An alternative means of decreasing the environmental risk posedby these metal-contaminated soils may be the use of plants to stabilize thesurface, thus reducing erosion and leaching to the soil deeper layers. Thisoption is called phytostabilization, and considers the use of metal-tolerantplant species to immobilize heavy metals belowground, decreasing metalmobility and reducing the likelihood of metals entering into the food chain(Wong, 2003). Phytostabilization is hence used where phytoextraction is notpossible or desirable (McGrath & Zhao, 2003). Additionally, it can also beapplied at sites where regulatory or technical constraints delay the selectionof the most appropriate techniques for site recovery as a provisional strategyto reduce environmental risk, by protecting barren contaminated areas fromcontinuous erosion or leaching (Berti & Cunningham, 2000). This techniquecan indeed be adapted to a variety of sites and situations, with differentconditions (e.g., soil pH, salinity, soil texture, metal levels, and contaminanttypes) through the careful selection not only of the appropriate plant speciesbut also of the applied amendments (Berti & Cunningham, 2000). There arethus two major components in the phytostabilization process: the plant itselfand the amendments added to the system.

Plants play an important role in phytostabilization, not only by pro-tecting the soil surface from human contact and rain impact with a densecanopy, but also by physically stabilizing the soil with dense root systems toprevent erosion. Plant roots also help to minimize water percolation throughthe soil, further reducing contaminant leaching (Berti & Cunningham, 2000).In addition, plant roots can also provide surfaces for sorption or precipitationof metal contaminants (Laperche et al., 1997). Consequently, the selection ofthe adequate plant species for phytostabilization should take into consider-ation the following:

� plants should be tolerant to the soil conditions;� plants must grow quickly to set up a ground cover;� plants should have dense rooting systems;� plants must be easy to establish and to maintain under field conditions;

and� plants must have a relatively long life or be able to self propagate (Berti

& Cunningham, 2000).

As phytostabilization is similar to establishing a meadow, soil amendmentssimilar to those used in agriculture can also be applied and assume a roleof great importance by helping to inactivate metal contaminants, prevent-ing plant uptake, and reducing biological activity. Ideally, soil amendments



should be easy to handle and to apply, safe to workers handling the amend-ment, non-toxic to the plants, easy to produce, and inexpensive—soil amend-ments that have little to no economic value, such as oyster shells for pHcorrection or manure for organic matter supplementation (Marques et al.,2007a), are preferred to more expensive materials (Berti & Cunningham,2000). Attention should also be given to the capacity of the amendmentsto reduce the leaching of metals, as this could be an important advantagein an in situ stabilization process and play an important role in ground-water protection and reduction of metal dispersion (Ruttens et al., 2006a).Marques et al. (2007a) have shown that the sole application of organic mat-ter amendments, such as manure or compost, to metal contaminated soil ledto a significant reduction in the amount of Zn leached through the soil; incombination with plants, the reduction in metal percolation ascended up to80%.

A range of organic and inorganic compounds (Adriano et al., 2004),such as lime, phosphate, and other low economical value organic mate-rials like biosolids, litter, compost, and manure, can be used. Liming hasbeen considered as an important management tool in reducing the toxi-city of metals in soils (Gray et al., 2006; Madejon et al., 2006). There isconclusive evidence for the mitigative value of both water-soluble (e.g.,diammonium phosphate) and water-insoluble (e.g., apatite) phosphate toimmobilize some metals in soils, thereby reducing their bioavailability forplant uptake (Brown et al., 1995a). Phosphate enhances the immobilizationof metals in soils through various processes, including direct metal adsorp-tion, phosphate anion-induced metal adsorption, and precipitation of metalswith solution phosphate as metal phosphates (Adriano et al., 2004). In fact,Bolan et al. (2003) reported that the sole application of lime or phosphate iseffective in reducing Cd in contaminated soils. The use of organic amend-ments, such as manure (Chiu et al., 2006; Clemente et al., 2006; Marqueset al., 2007a; Walker et al., 2004; Ye et al., 1999), compost (Cao & Ma,2004; Clemente et al., 2006; Marques et al., 2007a), and other bio-wastes(Karaca, 2004; Madejon et al., 2006) is a standing practice used for restora-tion of contaminated sites (Sopper, 1993). As examples, Walker et al. (2004)reported lower Zn tissue concentration in Chenopodium album L. plantswhen grown in compost or manure amended soils; Marques et al. (2007a)showed that the addition of manure or compost to the soils induced re-ductions in the Zn accumulation of Solanum nigrum of up to 80 and 40%while enhancing plant biomass yields; Ye et al. (1999) observed that Tri-folium repens tended to accumulate less Pb in the shoots when manure wasadded to the growing matrix. In fact, organic matter amendments are amongthe most promising additives, especially due to their low commercial costand consequent added value of their application for soil remediation pur-poses. Their application provides organic matter to improve soil physicalproperties, water infiltration, and water-holding capacity. They also contain



essential nutrients for plant growth. Immobilization of metals by such amend-ments is achieved through adsorption, complexation, and redox reactions(Adriano et al., 2004)—organic matter makes strong complexes with heavymetals (Krogstad, 1983). The addition of organic amendments has often beenshown to increase the CEC of soils (Marques et al., 2007a), increasing cationadsorption caused by the dissociation of H+ from the functional groups in or-ganic matter (Zhu et al., 1991). The presence of phosphates, Al compounds,and other inorganic minerals in some organic amendments is also believedto be responsible for the retention of metals (Adriano et al., 2004). Addi-tionally, amendment with organic matter and its resulting degradation maychange the soil pH and thereby indirectly affect the bioavailability of metals(Karaca, 2004), as it is well known that metal solubility is greatly determinedby the pH (Yoo & James, 2002). The research in soil amelioration usingmetal immobilizing amendments is now also focusing on the application ofother type of compounds, such as cyclonic ashes (Ruttens et al., 2006b),calcium carbonate (Lee et al., 2004), zeolites (Chlopecka & Adriano, 1996),steel shots (Ruttens et al., 2006b), beringite (Mench et al., 1994), red mud(also known as bauxite residue; Gray et al., 2006), or leonardite (Madejon etal., 2006), with positive effects on the reduction of soluble concentrations ofheavy metals in soils.

TOOLS TO IMPROVE THE POTENTIAL OF PHYTOREMEDIATIONOF HEAVY METALS

The Role of the Microbial Community of the Rhizospherein the Phytoremediation of Heavy Metals

Metal uptake by plants can be influenced by soil microorganisms that asso-ciate with the plant roots to form the rhizosphere community (Shilev et al.,2001). It is well known that mycorrhizal fungi are a major component ofthe rhizosphere and form mutualistic associations with most plant species(Azcon-Aguillar & Barea, 1992). In all, 90–95% of all land plants form sometype of mycorrhizal associations so that the symbiotic association, the my-corrhiza, seems to be the chief organ of nutrient uptake in the majority ofplants (Bago et al., 2000; Entry et al., 2002). Of the existent mycorrhizalassociations—ectomycorrhizas, arbuscular mycorrhizas, ericaceous mycor-rhizas, and orchid mycorrhizas (Entry et al., 2000)—the arbuscular mycor-rhizas (AM) associations between arbuscular mycorrhizal fungi (AMF) andthe roots of terrestrial plant species are by far the most widespread (Smith &Read, 1997).

Arbuscular mycorrhizal fungi can benefit plants in numerous ways. Ben-efits to plants include improved nutrition (Clark & Zeto, 2000), through exten-sive extraradical hyphal networks, which explore the soil, absorb nutrients,



and translocate them to the roots (Giovannetti et al., 2002), and root systemmodifications, generally resulting in a more extensive length and increasedbranching and therefore in a more efficient nutrient absorption (Berta et al.,2002). The increase in the uptake of inorganic P (Harrison & van Buuren,1995; Smith & Read, 1997), K, Ca, S, Cu, Zn, Mg, Co, Ni, and N (Koide, 1991;Marschner & Dell, 1994; Smith & Read, 1997) are well-documented nutritionalbeneficial effects of AMF. In addition, the arbuscular mycorrhizal symbioticstatus changes the chemical composition of root exudates (Laheurte et al.,1990) and influences soil pH (Li et al., 1991), thus quantitatively affectingthe microbial populations in the rhizosphere (Azcon-Aguilar & Barea, 1992;Barea, 1997), protecting against soil-borne plant pathogens (Azcon-Aguilar& Barea, 1996; De la Pena et al., 2006), and improving soil structure (Rillig& Mummey, 2006). Other benefits include protection against insect herbi-vores (Gange & Brown, 2002), hormone regulation (Ludwig-Muller, 2000),and drought tolerance (Auge, 2001; Ruiz-Lozano et al., 2001).

Arbuscular mycorrhizal fungi have also been shown to enhance planttolerance to biotic and abiotic stresses, including the presence of high levelsof heavy metals (Leyval et al., 2002): as they are a direct link between soiland roots, they can be very important for heavy metal availability and toxicityto plants (Leyval et al., 1997). It has been reported that mycorrhizal fungican impact plant uptake or translocation of soil metals (Khan et al., 2000).When the host is exposed to metal stress, the role of AMF in the plant stressresponse is variable. Some studies indicate reduced metal concentrationsin plants due to mycorrhizal colonization (Heggo et al., 1990; Jentschkeet al., 1998). Huang et al. (2002) reported an exclusion strategy, showinglower Zn accumulation by AMF colonized Zea mays. However, other reportsindicate enhanced metal uptake and accumulation in plants due to AMFcolonization (Ahonen-Jonnarth & Finlay, 2001; Jamal et al., 2002; Joner &Leyval, 2001; Marques et al., 2006, 2007b). Citterio et al. (2005) have shownan enhanced growth and metal root to stem translocation on Cannabis sativaplants inoculated with the AMF Glomus mosseae, while Chen et al. (2005)observed that a mixed AMF inoculum enhanced Pb uptake and growthof Kummerowia striata, Ixeris denticulate, and Echinochloa crusgalli varmitis-, even resulting in metal levels toxic to plants (Weissenhorn & Leyval,1995). Other reports indicate that both effects can occur or even show noeffects exerted by AMF on the contaminant uptake and accumulation inthe host plants (Joner et al., 2004). The bulk of evidence seems thus tosuggest a species-specific effect of AM associations on plant metal uptakeand accumulation. As examples, Marques et al. (2007c) have shown that theinoculation with the AMF G. intraradices or G. claroideum protected the hostplant Solanum nigrum of excessive Zn, which was translated in a decreasein metal accumulation in AMF inoculated plants, whereas at lower Zn levelsin the growing matrix, there was an increase in the metal accumulation. Diazet al. (1996) have also reported similar tendencies for the uptake of Zn and



Pb by Lygeum spartum and Anthylis cytisoides inoculated with G. mosseae insoils with different levels of theses metals: at low doses, mycorrhizal plantshad equal or higher Zn or Pb concentrations than non-inoculated controls;at higher doses, however, metal concentrations in the plants inoculated withG. mosseae were lower than those found in the corresponding controls.

Plant growth-promoting bacteria (PGPR) communities naturally existingin the rhizosphere can also be an important tool in the decontaminationof metal-contaminated soils through plant use. PGPR can be divided intotwo groups according to their relationship with the plants: symbiotic bac-teria and free-living rhizobacteria (Khan, 2005). The enhancement of cropplant growth using PGPR is documented (Reed & Glick, 2004); more re-cently, these organisms have been used to reduce plant stress associatedwith phytoremediation strategies for metal contaminated soils (Reed & Glick,2005). The PGPR are able to enhance plant growth through various mech-anisms, such as reduction of ethylene production (thus allowing plants todevelop longer roots and better establish during early stages of growth;see Glick et al., 1998), nitrogen fixation, specific enzymatic activity (Khan,2005), supply of bioavailable phosphorous and other trace elements forplant uptake, and production of phytohormones such as auxins, cytokinins,and gibberelins (Glick et al., 1995). These microorganisms can also produceantibiotic and other pathogen-depressing substances such as siderophoresand chelating agents that protect plants from diseases (Kamnev & Lelie,2000) and can also increase plant tolerance to flooding (Grichko & Glick,2001), salt stress (Mayak et al., 2004a), and water deprivation (Mayak et al.,2004b).

Plant growth promoting bacteria have also shown to reduce plant stressat metal exposure. Brassica napus has shown reduced accumulation of Cuwhen inoculated with Pseudomonas putida UW4 (Reed & Glick, 2005; Reedet al., 2005) and lower Ni toxicity when in the presence of the bacteriaKluyvera ascorbata SUD165 (Burd et al., 1998). Nevertheless, other studiesindicate a PGPR-driven increase in the availability of heavy metals in soil,thus enhancing metal accumulation by plants, as reported for Zn accumu-lation by Thlaspi caerulescens (Whiting et al., 2001) and uptake of Ni byAlyssum murale and Thlaspi goesingense (Abou-Shanab et al., 2003; Idriset al., 2004) and of Se by Brassica juncea (De Souza et al., 1999). Althoughthe employment of PGPR is potentially important in phytoremediation ex-periments, research in this area is not as extensive as for the AMF use, andfurther investigation is needed to better understand the prospects of PGPRapplication in such strategies.

Different microorganisms may play assorted roles in plant growth and/ormetal tolerance via different mechanisms, so it can be beneficial for the de-sign of a phytoremediation plan to select appropriate multifunctional micro-bial combinations, which may include AMF and PGPR. Further examples ofthe contribution of selected microorganisms in phytoremediation—namely,bacteria, fungi, and a combination of the two—are described in Table 3.


TA

BLE

3.

Exa

mple

sofphyt

ore

med

iatio

nex

per

imen

tsusi

ng

bac

teria,

AM

F,an

dth

eco

nju

gatio

nofboth

mic

roorg

anis

ms

Pla

nts

Mic

roorg

anis

ms

Hea

vym

etal

sEffec

tsRef

eren

ce

Bra

ssic

aju

nce

aA

zoto

bact

erch

rooc

ocu

mH

KN

-5B

aci

llus

meg

ate

riu

mH

KP-1

Ba

cillu

sm

uci

lagi

nos

us

HK

K-1

Pb,Zn

-St

imula

tion

ofpla

ntgr

ow

th-

Pro

tect

ion

ofpla

ntfr

om

met

alto

xici

ty

Wu

etal

.,20

06

Pse

ud

omon

as

sp.PsA

4B

aci

llus

sp.B

a32

Cr

Raj

kum

anet

al.,

2006

Va

riov

ora

xpa

rad

oxu

sR

odoc

occu

ssp

.Fl

avo

bact

eriu

msp

.

Cd

-St

imula

tion

ofro

otel

onga

tion

Bel

imov

etal

.,20

05

Ba

cillu

ssu

btil

isSJ

-101

Ni

-St

imula

tion

ofpla

ntgr

ow

th-

Incr

ease

ofN

iac

cum

ula

tion

by

pla

nts

Zai

diet

al.,

2006

Bra

ssic

an

apu

sP

seu

dom

ona

spu

tid

aU

W4

Ni

-St

imula

tion

ofsh

ootbio

mas

sfo

rmat

ion

-In

crea

seofto

lera

nce

toN

i

Farw

ellet

al.,

2007

Ba

cillu

ssp

.RJ1

6Cd

-St

imula

tion

ofgr

ow

th-

Incr

ease

ofm

etal

accu

mula

tion

insh

oottis

sues

Shen

gan

dX

ia,20

06

Lyco

pers

icon

escu

len

tum

Met

ylob

act

eriu

mor

yza

lCB

MB

20B

urk

ohd

eria

sp.O

BM

B40

Ni,

Cd

-St

imula

tion

ofpla

ntgr

ow

th-

Dec

reas

eofm

etal

accu

mula

tion

insh

ootan

dro

ottis

sues

Mad

hai

yan

etal

.,20

07

Tri

foli

um

pra

ten

seG

lom

us

mos

sea

eZn

-D

ecre

ase

ofm

etal

accu

mula

tion

insh

ootan

dro

ottis

sues

Biet

al.,

2003

Chen

etal

.,20

03H

elia

nth

us

an

nu

us

Glo

mu

sin

tra

rad

ices

Cr

-In

crea

seofm

etal

accu

mula

tion

inro

ots

Dav

ies

etal

.,20

01

Zea

ma

ysG

lom

us

ma

nih

otis

Cu

-In

crea

seofm

etal

accu

mula

tion

inro

otan

dsh

oottis

sues

Liao

etal

.,20

03

Glo

mu

sin

tra

rad

ices

PH

5Pb

-In

crea

seofm

etal

accu

mula

tion

inro

otan

dsh

oottis

sues

Mal

cova

etal

.,20

03

Tri

foli

um

repe

ns

Glo

mu

sm

osse

ae

Bre

viba

cillu

ssp

.(B

-I)

Zn

-St

imula

tion

ofpla

ntgr

ow

th-

Dec

reas

eofm

etal

accu

mula

tion

Viv

aset

al.,

2006

639



Genetic Engineering of Plants for the Phytoremediationof Heavy Metals

Phytoremediation by natural plant species can be limited in several ways.These limitations could be overcome by using conventional plant breed-ing practices; however, conventional approaches can take decades. Geneticengineering, on the other hand, has the potential to produce plant popula-tions with superior traits for phytoremediation in a relatively short time andeven transfer genes form organism that can not be crossed by conventionalbreeding methods (Berken et al., 2002).

Many genes are involved in metal uptake, translocation, and seques-tration, and the transfer of any of these genes into candidate plants is apossible strategy for genetic engineering of plants for improved phytoreme-diation traits (Eapen & D’Souza, 2005). In genetic engineering of plants, aforeign piece of DNA is stably inserted into the genome of a cell, whichis regenerated into a mature transgenic plant; the piece of DNA can comefrom any organism, from bacteria to mammals, or other plants. When thetransformed plant is propagated, the foreign gene is inherited by its offspring(Pilon-Smits & Pilon, 2002).

The ideal plant species to engineer for phytoremediation purposes isone that has high biomass production, is sufficiently hardy and competitivein the climate where it is to be used, has a good phytoremediation capacityto start with (Pilon-Smits & Pilon, 2002), has the ability to accumulate metalspreferably in the aboveground parts, has a widespread, highly branched rootsystem, is easy to harvest, and is amicable for genetic transformation (Eapen& D’Souza, 2005). Preferably, crop plants should not be used.

Classic genetic studies have shown that only a few genes (up to three)are responsible for metal tolerance (Macnair et al., 2000). According toEapen and D’Souza (2005), the possible areas of genetic manipulation asfollows:

� metallotioneins: the transfer of human metallotionein gene in tobaccoresulted in plants with enhanced Cd tolerance (Misra & Gedamu, 1989),and pea metallotionein gene transfer to Arabidopsis thaliana resulted inincreased Cu accumulation (Evans et al., 1992);

� phytochelatins: transgenic Brassica juncea overexpressing different en-zymes involved in phytochelatin synthesis were shown to extract moreCd, Cr, Cu, Pb, and Zn than wild plants (Zhu et al., 1999a, 1999b);

� organic acids: the overexpression of citrate synthase has shown to pro-mote enhanced Al tolerance;

� phytosiderophores: the overexpression of nicotianamine aminotransferase(NAAT) in rice resulted in the overproduction of the iron-chelator deoxy-mugineic acid, a phytosiderophore, and consequently promoted a moreefficient growth in iron-deficient soils (Takahashi et al., 2001);



� ferritin: the overexpression of the iron-binding protein ferritin has shownto increase up to 1.3-fold higher the iron level in tobacco leaves (Gotoet al., 1998);

� metal transporters: transfer of Zn transporter-ZAT gene from Thlaspigoesingense to Arabidopsis thaliana resulted in two-fold higher Zn ac-cumulation in its roots (Van der Zaal et al., 1999);

� alteration of metabolic pathways: transfer of Escherichia coli ars C andγ -ECS genes to Arabidopsis plants resulted in individuals that could trans-port oxyanion arsenate to aboveground tissues, reduce to arsenite, andsequester it to thiol peptide complexes (Dhankher et al., 2002);

� alteration of oxidative stress mechanisms: overexpression of glutathione-S-transferase and peroxidase in Arabidopsis plants resulted in enhancedAl tolerance (Ezaki et al., 2000); and

� alteration in biomass: increasing phytohormones synthesis can increasebiomass of transgenic plants, as reported by Eriksson et al. (2000) for treeswith genetically induced increase in giberellin biosynthesis that presentedenhanced growth and biomass production.

Although no practical applications of transgenic plants are reported and thetheoretical risk of escape of the genes from the transgenic plants has beencalculated as negligible (Meagher et al., 2000), risk assessment of any useof these transgenic species should be carefully undertaken before any fieldtesting or further application is to be planned (Wolfenbarger & Phifer, 2000).Some of the possible risks involved are biological transformation of metalsinto forms that are more bioavailable, enhanced exposure of wildlife andhumans to metals, uncontrolled spread of transgenic plants due to higherfitness or general weedy nature, and/or uncontrolled spread of the transgenicplants by interbreeding with populations of wild relatives (Pilon-Smits &Pilon, 2002). These risks have to be assessed and weighed not only againstthe benefits of the technique, but also against the risks of doing nothingor using other methods. If the adequate prevention measures are taken,these new developments in plant genetic engineering may lead to fruitfulapplications in environmental cleanup.

CONCLUSIONS

Phytoremediation is emerging as a bio-based and low-cost alternative in thecleanup of heavy metal-contaminated soils. The application of a vegetationcover can limit the local effects and the spreading of the contamination,or even remove via phytoextraction or phytovolatilization the metals fromthe polluted soil. The future of this technique is still mainly in the researchphase, and the optimization and greater understanding of the process bywhich plants absorb, translocate, and metabolize heavy metals needs to be



addressed. There is still much fundamental and applied and field researchneeded. The potential role of both free living and symbiotic soil microbesin the rhizosphere of plants growing in metal-contaminated soils in enhanc-ing the phytoremediation process can be an important tool to support thetechnology. The outcome of undergoing genetic engineering investigationconcerning plants applicable in phytoremediation may also lead to a betterunderstanding of metal metabolism in plants, which can result in importantcontributions for the implementation of phytoremediation as a feasible soilremediation technology. A multidisciplinary research effort that integratesthe work of plant biologist, soil chemists, microbiologists, geneticists, andenvironmental engineers thus seems essential for the success of phytoreme-diation as a soil cleanup technology.

ACKNOWLEDGMENTS

This work was supported by Fundacao para a Ciencia e a Tecnologia andFundo Social Europeu (III Quadro Comunitario de apoio), a research grantof Ana Marques (SFRH/BPD/34585/2007), and funded by the Project MI-COMETA - POCI/AMB/60131/2004, financed by Medida V.4-Accao V.4.1 ofPrograma Operacional Ciencia e Inovacao 2010 (Fundacao para a Ciencia eTecnologia).

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remediation of heavy metal contaminated soils ... replacement. because biological ... soil washing...

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