土壤重金属的生物修复技术进展

Review Recent findings on the phytoremediation of soils contaminated with environmentally toxic heavy metals and metalloids such as zinc, cadmium, lead, and arsenic I. Alkorta1, J. Herna´ndez-Allica2, J.M. Becerril3, I. Amezaga3, I. Albizu2 & C. Garbisu2,* 1Unidad de Biofı´sica, Centro Mixto UPV/EHU, Apdo. 644, E-48080 Bilbao, Spain; 2NEIKER, Basque Institute of Agricultural Research and Development, Berreaga 1, E-48160 Derio, Spain; 3Department of Plant Biology and Ecology, University of the Basque Country, Apdo. 644, E-48080 Bilbao, Spain (*author for correspondence: phone: +34-94-403-43-00; fax: +34-94-403-43-10; e-mail: cgarbisu@neiker.net) Key words: metalloids, metals, phytochelatins, phytoextraction, phytoremediation, transgenic plants Abstract Due to their immutable nature, metals are a group of pollutants of much concern. As a result of human activities such as mining and smelting of metalliferous ores, electroplating, gas exhaust, energy and fuel production, fertilizer and pesticide application, etc., metal pollution has become one of the most serious environmental problems today. Phytoremediation, an emerging cost-effective, non-intrusive, and aesthet- ically pleasing technology, that uses the remarkable ability of plants to concentrate elements and com- pounds from the environment and to metabolize various molecules in their tissues, appears very promising for the removal of pollutants from the environment. Within this field of phytoremediation, the utilization of plants to transport and concentrate metals from the soil into the harvestable parts of roots and above- ground shoots, i.e., phytoextraction, may be, at present, approaching commercialization. Improvement of the capacity of plants to tolerate and accumulate metals by genetic engineering should open up new possibilities for phytoremediation. The lack of understanding pertaining to metal uptake and translocation mechanisms, enhancement amendments, and external effects of phytoremediation is hindering its full scale application. Due to its great potential as a viable alternative to traditional contaminated land remediation methods, phytoremediation is currently an exciting area of active research. 1. Environmental metal pollution and phytoremediation Soil pollution has recently been attracting consid- erable public attention since the magnitude of the problem in our soils calls for immediate action (Garbisu & Alkorta 2003). As a result of human activities such as mining and smelting of metallif- erous, electroplating, gas exhaust, energy and fuel production, fertilizer and pesticide application, etc., metal pollution has become one of the most serious environmental problems today. Due to their immutable nature, metals are a group of pollutants of much concern. In fact, although several metals are essential for biological systems and must be present within a certain concentration range (Garbisu & Alkorta 2003), at high concen- trations, metals can act in a deleterious manner by blocking essential functional groups, displacing other metal ions, or modifying the active confor- mation of biological molecules (Collins & Stotzky 1989). Metal toxicity for living organisms involves oxidative and/or genotoxic mechanisms (Briat & Lebrun 1999). Based on their chemical and physical proper- ties, three different molecular mechanisms of heavy metal toxicity can be distinguished: (i) pro- duction of reactive species by autooxidation and Fenton reaction (Fe, Cu), (ii) blocking of essential functional groups in biomolecules (Cd, Hg), and (iii) displacement of essential metal ions from biomolecules (Schutzendubel & Polle 2002). Reviews in Environmental Science and Bio/Technology 3: 71–90, 2004. � 2004 Kluwer Academic Publishers. Printed in the Netherlands. 71 Metal-contaminated soils are notoriously hard to remediate. Current technologies resort to soil excavation and either landfilling or soil washing followed by physical or chemical separation of the contaminants. Although highly variable and dependent on the contaminants of concern, soil properties, site conditions, and so on, the usually enormous costs associated with the removal of metals from soils by means of traditional physi- cochemical methods explain why most companies tend to ignore the problem. Due to the fact that very often large areas are affected by heavy metal contamination, a removal is certainly difficult. Therefore, some methods are developed to keep the metals in the soil but reduce the risks related to this presence (e.g., by decreasing bioavailability by in situ immobilisation processes) (Diels et al. 2002). One way to facilitate such immobilisation is by altering the physicochemical properties of the metal-soil complex by introducing a multipurpose anion, such as phosphate, that enhances metal adsorption via anion-induced negative charge and metal precipitation (Bolan et al. 2003a). Heavy metals cannot be destroyed biologically (no ‘‘degradation’’, change in the nuclear struc- ture of the element, occurs) but are only trans- formed from one oxidation state or organic complex to another (Garbisu & Alkorta 2001). Although microorganisms that use metals as ter- minal electron acceptors or reduce them as part of a detoxification mechanism can be used for metal remediation (Garbisu & Alkorta 1997), when considering the remediation of metal-pol- luted soil, metal-accumulating plants offer numerous advantages over microbial processes since plants can actually extract metals from the polluted soils, theoretically rendering them clean (metal-free) (Garbisu & Alkorta 2001; Garbisu et al. 2002). Phytoremediation, the use of plants to extract, sequester, and/or detoxify pollutants, has been reported to be an effective, non-intrusive, inex- pensive, aesthetically pleasing, socially accepted technology to remediate polluted soils (Alkorta & Garbisu 2001; Weber et al. 2001; Garbisu et al. 2002). Phytoremediation is widely viewed as the ecologically responsible alternative to the envi- ronmentally destructive physical remediation methods currently practiced (Meagher 2000). The US phytoremediation market is expected to ex- pand more than ten-fold between 1998 and 2005, to over $214 million (Evans 2002). In the last few years, some excellent reviews have been published focusing on different aspects of phytoremediation (Salt et al. 1995a, 1998; Chaney et al. 1997; Raskin et al. 1997; Chaudhry et al. 1998; Wenzel et al. 1999; Meagher 2000; Navari- Izzo & Quartacci 2001; Lasat 2002; McGrath et al. 2002;McGrath&Zhao 2003;McIntyre 2003; Singh et al. 2003). In any case, and in contrast to its many positive aspects, phytoremediation does have cer- tain disadvantages and limitations (Table 1). Table 1. Advantages and limitations of the phytoremediation technology Advantages Limitations Applicable to a wide variety of inorganic and organic contaminants. Limited by depth (roots) and solubility and availability of the contaminant. Reduces the amount of waste going to landfills. Although faster than natural attenuation, it requires long time periods (several years). Does not require expensive equipment or highly specialized personnel. Restricted to sites with low contaminant concentration. It can be applied in situ. Reduces soil disturbance and the spread of contaminants. Plant biomass from phytoextraction requires proper disposal as hazardous waste. Early estimates of the costs indicate that phytoremediation is cheaper than conventional remediation methods. Climate and season dependent. It can also lose its effectiveness when damage occurs to the vegetation from disease or pests. Easy to implement and maintain. Plants are a cheap and renewable resource, easily available. Introduction of inappropriate or invasive plant species should be avoided (non-native species may affect biodiversity). Environmentally friendly, aesthetically pleasing, socially accepted, low-tech alternative. Contaminants may be transferred to another medium, the environment, and/or the food chain. Less noisy than other remediation methods. Actually, trees may reduce noise from industrial activities. Amendments and cultivation practices may have negative consequences on contaminant mobility. 72 Within the field of phytoremediation, different categories have been defined such as, among others, phytoextraction, phytofiltration (rhizofiltration, blastofiltration), phytostabilization, phytovolatil- ization, phytodegradation (phytotransformation), plant-assisted bioremediation (plant-assisted deg- radation, plant-aided in situ biodegradation, phyt- ostimulation, enhanced rhizosphere degradation, rhizodegradation), etc. (Table 2). Plants for phytoextraction, i.e., metal removal from soil, should have the following characteris- tics: (i) tolerant to high levels of the metal, (ii) accumulate reasonably high levels of the metal, (iii) rapid growth rate, (iv) produce reasonably high biomass in the field, and (v) profuse root system (Garbisu et al. 2002). The idea of using plants to remediate metal polluted soils came from the discovery of ‘‘hyper- accumulators’’ (Table 3), defined as plants, often endemic to naturally mineralized soils, that accu- mulate high concentrations of metals in their fo- liage (Baker & Brooks 1989; Raskin et al. 1997; Brooks 1998). In fact, plants growing on metal- liferous soils can be grouped into three categories according to Baker (1981): (i) excluders, where metal concentrations in the shoot are maintained, up to a critical value, at a low level across a wide range of soil concentration; (ii) accumulators, where metals are concentrated in above-ground plant parts from low to high soil concentrations; and (iii) indicators, where internal concentration reflects external levels (McGrath et al. 2002). The criterion for defining Ni hyperaccumulation is 1000 lg Ni g)1 on a dry leaf basis (Brooks et al. 1977), whereas for Zn and Mn the threshold is 10,000 lg g)1 and for Cd 100 lg Cd g)1. Finally, the criterion for Co, Cu, Pb and Se hyperaccu- mulation is also 1,000 lg g)1 in shoot dry matter (Brooks 1998; Baker et al. 2000; McGrath et al. 2002). In general terms, metal concentrations in hyperaccumulators are about 100–1000-fold high- er than those found in normal plants growing on soils with background metal concentrations, and about 10–100-fold higher than most other plants growing on metal-comtaminated soils (McGrath et al. 2002). Hyperaccumulators are also charac- terized by a shoot-to-root metal concentration ratio of >1 (i.e., hyperaccumulator plants show a highly efficient transport of metals from roots to shoots), whereas non-hyperaccumulators usually have higher metal concentrations in roots than in shoots (Baker 1981; Gabbrielli et al. 1990; Homer Table 2. Categories of phytoremediation Term Definition Phytoextraction The use of plants to remove pollutants (mostly, metals) from soils. Phytofiltration The use of plants roots (rhizofiltration) or seedlings (blastofiltration) to absorb or adsorb pollutants (mostly, metals) from water. Phytostabilization The use of plants to reduce the bioavailability of pollutants in the environment. Phytovolatilization The use of plants to volatilize pollutants. Phytodegradation The use of plants to degrade organic pollutants Phytotransformation Phytostimulation The use of plant roots in conjunction with their rhizospheric microorganisms to remediate soils contaminated with organics.Enhanced rhizosphere degradation Rhizodegradation Plant-assisted bioremediation Plant-asssisted degradation Plant-aided in situ biodegradation Table 3. Examples of hyperaccumulators Metal Species Zinc (Zn) T. caerulescens Cadmium (Cd) T. caerulescens Nickel (Ni) Berkheya coddii Selenium (Se) Astragalus racemosa Thallium (Tl) Iberis intermedia Copper (Cu) Ipomoea alpina Cobalt (Co) Haumaniastrum robertii Arsenic (As) P. vittata 73 et al. 1991; Baker et al. 1994a,b; Brown et al. 1995; Kra¨mer et al. 1996; Shen et al. 1997; Zhao et al. 2000; McGrath et al. 2002). Hyperaccumulation of heavy metal ions is in- deed a striking phenomenon exhibited by <0.2% of angiosperms (Baker & Whiting 2002). Most recently, a plethora of papers are being published in an attempt to dissect the mechanisms of metal uptake, transport and accumulation, both at the physiological and molecular level (Baker & Whiting 2002). The ‘‘model’’ hyperaccumulator Thlaspi caerulescens has been much screened in the search for new and more extreme ecotypes (McGrath et al. 2001; Lombi et al. 2002). Our understanding of the internal processes that confer the hyperaccumulation phenotype is advancing in leaps and bounds, and the mechanisms of trans- port, tolerance and sequestration in some species, at least in the genera Thlaspi and Alyssum, are partially elucidated (Lasat 2002). Trees have also been considered for phyto- remediation of heavy metal-contaminated land, with willow and poplar being promising candi- dates, among others, in this respect (Pulford & Watson 2003). According to some authors, trees potentially are the lowest-cost plant type to use for phytoremediation (Stomp et al. 1994). Many trees can grow on land of marginal quality, have mas- sive root systems, and their above-ground biomass can be harvested with subsequent resprouting without disturbance of the site (Stomp et al. 1994). Following the harvest of metal-enriched plants, the weight and volume of the contaminated material can be further reduced by ashing or composting (Garbisu & Alkorta 2001; Garbisu et al. 2002). Metal-enriched plants can be disposed of as hazardous material or, if economically fea- sible, used for metal recovery (Salt et al. 1998). Recently, some studies have reported on the utili- zation of pyrolysis to separate heavy metals from hyperaccumulators (Koppolu & Clements 2003). Although plants acquire essential minerals such as Fe, Cu, Ni, Zn and Se from the soil, for reasons that are not yet clear, they also have the ability to acquire and detoxify non-essential elements such as As, Cd, Cr and Pb (Salt et al. 2002). Certain themes in the physiology and biochemistry of trace element accumulation by plants appear common (Salt et al. 2002). Most phytoremediation studies have consid- ered metal extraction efficiency in relation to metal concentration of bulk soil samples or metal con- centration of the soil solution, but little is known about the effect of various metal-bearing solids on metal extraction by hyperaccumulators. In fact, it has been shown that it is essential to consider the nature of the metal-bearing solids to better predict the efficiency of plant extraction (Dahmani-Muller et al. 2001). Besides, it is also important to con- sider that metal bioavailability changes between the bulk soil and the rhizosphere, the latter being a microbiosphere which has quite different chemical, physical and biological properties from bulk soils (Wang et al. 2002b). In this respect, recently, it has been reported that root growth is a more sensitive endpoint of metal availability than chlorophyll assays (Morgan et al. 2002). In order to improve phytoremediation of heavy metal polluted sites, the speciation and bioavailability of the metals in the soil, the role of plant-associated soil microor- ganisms and fungi in phytoremediation, and that of plants have to be elucidated (Kamnev & van der Lelie 2000). Phytoremediation has been used in mined soil restoration, since these soils are sources of air and water pollution, by means of phytostabilization and phytoextraction techniques to stabilize toxic mine spoils and remove toxic metals from the spoils, respectively (Wong 2003). Some higher plant species have developed heavy metal tolerance strategies which enable them to survive and reproduce in highly-metal contam- inated soils. Dahmani-Muller et al. (2000) inves- tigated metal uptake and accumulation strategies of two absolute metallophyte species and one pseudometallophyte. In the former two species, real hyperaccumulation in the leaves as well as metal immobilisation in roots and/or a detoxifi- cation mechanism by leaf fall were found as possible strategies to deal with the high metal concentrations. By contrast, the strategy of the pseudometallophyte, i.e., Agrostis tenuis, pre- sented a significant metal immobilisation by the roots. Most plants have mycorrhizal fungi associated with them, providing their hosts with an increased capacity to absorb water and nutrients from the soil. The formation and function of mycorrhizal relationships are affected by anthropogenic stressors including metals (Entry et al. 2002). Arbuscular mycorrhizal fungi are of interest for their reported roles in alleviation of diverse 74 soil-associated plant stressors, including those in- duced by metals, so it has been claimed that the evaluation of the efficacy of plant-mycorrhizal associations to remediate metal-polluted soils de- serves increased attention (Entry et al. 2002). In addition, phytoextraction practices, e.g., the choice of plant species and soil amendments, may have a great influence on the quantity and species composition of glomalean propagules as well as on arbuscular mycorrhizal fungi functioning during long-term metal-remediation treatments (Paw- lowska et al. 2000). A unique testing system, the target-neighbour method, has been described to allow evaluation of how planting density influences metal uptake, so that the information needed to manipulate plant density for optimization of metal removal could be obtained (Shann 1995). Finally, most recently, phytoremediation has been combined with electrokinetic remediation, applying a constant voltage of 30 V across the soil, concluding that the combination of both tech- niques represents a very promising approach to the decontamination of metal polluted soils (O0Connor et al. 2003). 2. New findings on the phytoextraction of some of the most relevant environmentally toxic heavy metals (zinc, cadmium, lead) and metalloids (arsenic) This section is divided into four different sub- headings. The first three correspond to three of the most environmentally relevant heavy metals, i.e., Zn, Cd, and Pb. The fourth sub-heading deals with As, a well-known toxic metalloid. It is important to emphasize here that very often information regarding one metal appears under a different, apparently wrong, sub-heading. Since many of the reviewed publications deal with more than one metal at the same time, it has been preferred to present them as part of the same research, despite the fact that section structure could not be main- tained as desired. 2.1. Zinc Zinc and Cd are ubiquitous pollutants that tend to occur together at many contaminated sites. While Zn is often phytotoxic, Cd rarely inhibits plant growth. In T. caerulescens, an integrated molecular and physiological investigation of the fundamental mechanisms of heavy metal accumulation was conducted (Pence et al. 2000). A metal transporter cDNA, znt1 (expressed at very high levels in roots and shoots of this plant), was cloned from T. caerulescens through functional complementa- tion in yeast and was shown to mediate high- affinity Zn uptake as well as low affinity Cd uptake. Alteration in the regulation of znt1 gene expression by plant Zn status results in the over- expression of this transporter and in increased Zn influx in roots, even when intracellular Zn levels are high. Thus, specific alterations in Zn-respon- sive elements (e.g., transcriptional activators) possibly play an important role in Zn hyperaccu- mulation in T. caerulescens (Pence et al. 2000). In this respect, Lasat et al. (1998) found that the en- hanced root-to-shoot Zn transport in T. caerules- cens was, at least partly, achieved through an altered Zn compartmentation in the root sym- plasm, which reduces Zn sequestration in root vacuoles. A further step at elucidating the mech- anisms underlying Zn hyperaccumulation was gi- ven thanks to the cloning of metal transporter genes