PHYTOREMEDIATION AND PHYTOTECHNOLOGIES: A REVIEW FOR THE PRESENT AND THE FUTURE

PHYTOREMEDIATION AND

PHYTOTECHNOLOGIES: A REVIEW FOR THE

PRESENT AND THE FUTURE

Nelson Marmiroli, Marta Marmiroli and Elena Maestri University of Parma, Department of Environmental Sciences, Parco Area delle Scienze 11/A,

43100 Parma, Italy

Abstract: Research on phytoremediation and phytotechnologies is proceeding at a different pace in the EU than in the USA. In fact although EU researchers have closed the gap on basics and fundamentals of phytoremediation with overseas researchers, a large gap of application exists in the extent of cases to which phytotechnologies are applied. The purpose of this paper is to review weaknesses and strengths of European contribution in the field. Research in the field of phytoremediation can be classified into two main groups: understanding of basic mechanisms and knowledge implementation for applications. Many groups are actively working to elucidate the basic genetic, molecular and cellular mechanisms underlying accumulation, transport and tolerance of plants to heavy metals. In comparison, interest towards organic pollutants is less widespread. Excellence in European research concerns the basic enzymology of phytodegradation of PAHs, TNT, dyes, herbicides, studied by means of in vitro cultures or in whole plants. As far as applications are concerned, the favorite phytotechnology in Europe is definitely the constructed wetland, often planted with Phragmites australis, which has been used for treatment of mine wastes, explosives, agricultural and municipal wastewaters. Other favored phytotechnologies are phytostabilization and phytoextraction. Phytotechnologies are particularly useful for preservation of agricultural soil. Cutting downstream the flow of pollutants into the food chain is a strategy also for conserving food safety and human health. Since research on phytotechnologies involves hundreds of scientists and dozens of research institutions and private companies; communication and networking are very important. Training and formation represent also an important aspect in developing a greater comprehension and acceptance of phytoremediation.

Key words: phytoremediation; Europe; research activities; new trends; networking

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1. INTRODUCTION

Phytoremediation, defined as the use of vegetation for in situ treatment of contaminated soils, sediments and water, is an environmental biotechnology that has attracted recently the interest of scientists, public opinion, regulators, and public administration. Several extensive reviews and books have been written on this topic. As a book, a very extensive treaty is “Phytoremediation: Transformation and Control of Contaminants” edited in 2003 by McCutcheon and Schnoor, and as a review the most recent is by Elizabeth Pilon-Smits (2004). The purpose of this paper will be to review the most recent advances in this field, as they have been gleaned by meetings of the EC COST Action 859 “Phytotechnologies to promote sustainable land use management and improve food safety”, which took place from late 2004 through spring 2005 in Europe. In these meetings, the richness and diversity of research in European phytoremediation has emerged, a field in which US research has been one step ahead.

1.1 Phytotechnologies and Their Application

The traditional term of phytoremediation has been recently supplanted by the term “phytotechnologies”, used to indicate all applications in which plants are used to manage and control pollutants, even without removing or destroying it (ITRC, 2001).

Phytotechnologies are based upon the basic physiological mechanisms taking place in higher plants and associated microorganisms, such as transpiration, photosynthesis, metabolism, and mineral nutrition. Plants dig their roots in soils, sediments and water, and roots can take up organic compounds and inorganic substances; roots can stabilize and bind substances on their external surfaces, and when they interact with microorganisms in the rhizosphere. Uptaken substances may be transported, stored, converted, and accumulated in the different cells and tissues of the plant. Finally, aerial parts of the plant may exchange gases with the atmosphere allowing uptake or release of molecules. A series of six phytotechnologies have been identified (ITRC, 2001) which may address different contaminants in different substrates, and which rely on one or more of the plant properties as in the exemplification listed above: 1. phytotransformation, ideal for organic contaminants in all substrates 2. rhizosphere bioremediation, applied to organic contaminants in soil 3. phytostabilisation, for organic and inorganic contaminants in soil 4. phytoextraction, useful for inorganic contaminants in all substrates 5. phytovolatilisation, which concerns volatile substances 6. evapotranspiration, to control hydraulic flow in the contaminated

environment

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From knowledge of the basic phytoremediation, applications (phyto-technologies) have been designed which consider the appropriate combination of plant choice and technology to address a specific contaminated site. This list has been taken from the document prepared by ITRC in 2001, even if different authors may identify a different set of methodologies: 1. vegetative covers for control of infiltration 2. vegetative covers for soil remediation 3. hydraulic barriers 4. tree stands for remediation of soil and groundwater 5. treatment wetlands 6. riparian buffers 7. hydroponic systems

These approaches differ in purposes and goals, which can be remediation, decontamination, control of water movement and leaching of the contaminant, containment, stabilization. Once the goal is identified, the technique is applied by choosing the appropriate plants and the relevant phytotechnologies. Examples and case studies describing these applications may be found in the specialised literature in books and in journals.

1.2 Phytotechnologies in Europe

Use of phytotechnologies in Europe is limited, as compared with USA and Canada. In those countries, private companies have been formed with the purpose to apply plant resources to control pollution (van der Lelie et al., 2001). European normative and public opinion are still precautionary in the use of phytoremediation. The following constraints have been evidenced:

due to limited knowledge and poor dissemination, there are doubts in the public opinion and limited acceptance, the current regulations, which do not clearly consider phytoremediation within the current applicable technologies unfavorable competition with standard clean-up methods, which can provide a long-standing record of success lack of sufficient investments, for encouraging the private initiative proprietary rights, which may hinder application of approaches already proved as successful in USA One of the main constraints, however, rests on the lack of fundamental

and applied research. In fact, research on the basic mechanisms of phytotechnologies and case studies are still needed in order to use these technologies in a coherent way. Moreover, in the last Framework Programme of the European Commission there was no topic really suitable for presentation of projects on phytotechnologies, and most of the European

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scientists have to rely on small budgets obtained from National or Regional sources. Nevertheless, the EC has recently funded projects on this topic, as listed in the CORDIS web site (www.cordis.lu), which have avoided a complete blockage of research in phytoremediation. Another tool which was instrumental to maintain interest in phytoremediation and phytotechnologies has been the COST Action 837 “Plant biotechnology for the removal of organic pollutants and toxic metals from wastewaters and contaminated sites” and the new one, 859, previously mentioned.

A survey, carried out in April 2005, enlisted all scientists involved in phytotechnology research in Europe which could be found in publications indexed by ISI (Current Contents) and/or those who participated to the COST meetings (see COST Actions www sites, lbewww.epfl.ch/COST837/ and www.gre.ac.uk/cost859/). The countries involved were 29, including Israel and Turkey which participate to European projects. About 350 research groups were involved, of which 60% were in Universities, 30% in research institutes and 10% in private companies. Indeed this distribution of efforts may be biased by industrial research which, for competition problems, is limited in dissemination through journals.

From papers published in 2001-2004, the distribution of topics for research is the same in Europe as in the USA, with most of the efforts concentrated on understanding the basic principles of phytotechnologies. A new trend is in the field of management and sustainability of enabling technologies. At this purpose different papers concern risk assessment and utilization of Decision Support Systems.

Here follows a short review of recent results obtained by European scientists, focused on the two main topics of principles of phytotechnologies and implementation and use of phytotechnologies.

2. BASIC PRINCIPLES OF

PHYTOTECHNOLOGIES

2.1 Basic Principles – Heavy Metals and Inorganic

Contaminants

2.1.1 Uptake and Transport

Interaction among plants and metals starts in the root environment. All phytotechnologies can be applied only if the contaminant is in contact with roots, and most of them rely on contaminant uptake by roots. This is the

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reason why plasma membrane transporters are a subject of research for phytotechnology implementation. Heavy metals uptake involves the same kind of transporters which otherwise provide macro- and micronutrients entrance. Recently, Perfus-Barboech et al. (2002) have demonstrated the involvement of Ca channels in Cd uptake in Arabidopsis thaliana. The possibility of Cd mimicking Ca in plant cells can also justify its toxicity with perturbation of metabolism and homeostasis of this vital element.

Studies performed with plant cell protoplasts have tried to ascertain if differences in transport in sink tissues could explain the different behavior of hyperaccumulator plants (Cosio et al., 2004). The results obtained with A.

halleri and Thlaspi caerulescens show that plasma membranes of leaf cells do not account for differences in transport. Therefore, it has been hypothesized that other mechanisms may be active to direct the metals to their subcellular compartments, where they are stored: vacuoles and lignocellulosic material such as cell wall may be among these.

Studies of metal transport, and especially in the case of radionuclides, can benefit from autoradiographic techniques, as shown by Soudek et al. (2004) with Cs. Imaging techniques allowed comparison between different species for uptake efficiency, but they also revealed potential sink tissues, providing useful information for implementation of phytoextraction.

2.1.2 Accumulation and Sequestration

In recent years, several authors have attempted to describe the differences between hyperaccumulator taxa and non-accumulator congeners by exploiting analytical techniques which give information on speciation and localisation of metals in plant tissues. To understand the molecular bases of the hyperaccumulation capacity and to define the storage strategies will be instrumental in developing and implementing phytoextraction. Analytical techniques based on X-ray emission (scanning electron microscopy and microanalysis) have been used to show Ni accumulation in the leaf trichomes of Alyssum bertolonii, in contrast with the non accumulator Alyssum montanum which stores Ni in the roots (Marmiroli et al., 2004). Arabidopsis halleri, a Zn and Cd hyperaccumulator, has been studied by Sarret et al. (2002). Zn is mainly sequestered in vacuoles of leaf trichomes and mesophyll cells. The authors determined through EXAFS that the two main forms of Zn in the plant roots were malate and phytate (or possibly phosphate), whereas in trichomes it is coordinated by C atoms, presumably belonging to organic acids. Careful computations however suggest that Zn in trichomes, even if highly concentrated, cannot constitute the major sink. Another interesting result concerns Zn binding in the non accumulator Arabidopsis lyrata, in which phosphate species were predominantly

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involved. With a similar approach based on EXAFS it has been possible to show that Pb can be accumulated in roots of walnut trees by coordination with C atoms of cellulose and lignin (Marmiroli et al., 2005).

2.1.3

To unravel the molecular and biochemical mechanisms of hyperaccumulation, the search for genes and proteins is being carried on with genomics and proteomics approaches. Genomic efforts have been promoted by the EC in research projects studying the properties of the hyperaccumulators, and results are showing how different genes are induced by metals in these plants and their congeners (van de Mortel et al., 2004). Comparative genomics and proteomics also contribute knowledge on orthologous genes, novel sequences, and molecular markers (Tuomainen et al., 2004).

Bernard et al. (2004) have shown how ectopic expression of Thlaspi

genes in yeast led to the isolation of a new gene function involved in Cd transport and probably also hyperaccumulation, a P-type ATPase.

Genetic mapping is the method of choice in the case of quantitative traits, and some authors are building maps of Quantitative trait Loci (QTLs) for hyperaccumulation and tolerance in model plants and in hyperaccumulators. Due to phylogenetic relationships with known hyperaccumulators in the family Brassicaceae, Arabidopsis thaliana is the best model plant available, due to the complete knowledge of the genomic sequence and to the genetic knowledge. The group led by Martin Broadley has recently mapped QTL involved in Cs accumulation of A. thaliana (Payne et al., 2004). Several accessions were analysed for Cs accumulation, leading to the description of a 2-fold variation in Cs concentration. Crosses among contrasting phenotypes and analysis in segregating progenies led to mapping of putative QTLs on several chromosomes; the existence of two QTLs on chromosomes I and V was confirmed from the analysis of segregating populations from independent crosses. Mapping of candidate genes in these regions will lead to new hypotheses about the structure and function of these QTLs.

2.1.4 Genetic Bases of Tolerance

Classical genetic studies have been exploited in order to address the issue of genetic bases of tolerance and/or accumulation in addition to genomic and proteomic approaches towards gene identification. For this purpose, model plants of choice have traditionally been Arabidopsis halleri and Thlaspi

caerulescens, two hyperaccumulators that can be crossed with non-

Identification of Genes and Proteins Involved In Tolerance

and Accumulation

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hyperaccumulator ecotypes or congeners for studies of traits segregation. Genecological observations have demonstrated that tolerance to one heavy metal is a trait which is independent from accumulation of the same metal, and also that tolerance is controlled by few major genes. Interspecific crosses between A. halleri and A. lyrata ssp. petraea have contributed information about tolerance and hyperaccumulation to Cd (Bert et al., 2003): tolerance and hyperaccumulation segregate as independent characters, whereas Cd tolerance co-segregates with Zn tolerance. Moreover Cd and Zn hyperaccumulation seem to be co-regulated or controlled by the same genes.

The same approach has been pursued in Thlaspi with ecotypes differing in accumulation capacity (Zha et al., 2004). Segregation results suggest that 2 genes at least are responsible for Zn accumulation, whereas for Cd accumulation more than one gene could be involved. Correlation between accumulation of Zn, Cd and Mn is consistent with a multiple transporter with simultaneous specificity for the three metals. Also in Thlaspi, Cd tolerance and accumulation segregate as independent characters.

2.1.5 New Contaminants

Phytotechnologies have traditionally been applied to heavy metals, nutrients and radionuclides. New contaminants of interest include arsenic: only recently have hyperaccumulators for this element been described: the fern Pteris vittata (Zhao et al., 2003) and other species of the same genus (Zhao et al., 2002). Phytochelatins play a role in As tolerance, as reviewed in Zhao et al. (2003), but in Pteris As is mainly in an inorganic form as arsenite in the vacuole of leaf cells. The authors suggest that phytochelatins may have a role in binding the small As quantities found in the cytoplasm.

Mercury is also attracting new interest, and there are indications that plants may be able to volatilize it as metallic mercury (Ernst et al., 2005).

2.2 Basic Mechanisms – Organic Contaminants

2.2.1 Mechanisms of Genetic Controls – Candidate Genes

As in the case of inorganic contaminants, also for organic contaminants researches are focusing on gene identification with a genomic approach. Recent advances concern specific candidate genes, coding for enzymes which are known to be involved in the metabolism of xenobiotics. The best known example is glutathione transferase (GST), a multigenic family recently reviewed by Frova (2003) and studied by the same author in rice (Soranzo et al., 2004). Plant genomes may contain between 25 and 60 GST

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genes. Five different classes are recognised in plant taxa, with two of them plant specific, Phi and Tau. Their main interest for phytotechnologies is the formation of glutathione-xenobiotic conjugates, in phase II of the contaminant metabolism. Genomic approaches have led to the isolation and identification of GST genes in important species, such as Arabidopsis, rice, maize, soybean. Rice contains 61 genes, 55% of which seem to be expressed based on EST libraries. The Tau class is the most expressed. However, many GST enzymes are inducible and expressed only in the presence of specific factors. As far as genome structure is concerned, 30 GST genes cluster on chromosome 10 of rice; this feature is in common with other plant species. Evolutionary studies suggest that the plant-specific families Phi and Tau evolved in response to stress challenges by toxic compounds. Co-evolution of GST and other enzymes involved in xenobiotic metabolism, cytochromes P450 and ABC transporters, will be of great interest to understand the biochemical and physiological resources that plants can play to deal with contaminants.

2.2.2 Analysis and Identification of Enzymes and Proteins

For identification of new enzymes involved in xenobiotic metabolism proteomic approaches are also pursued. A recent example is the isolation of a new glucosyltransferase from Arabidopsis thaliana, responsible for the detoxification of 3,4-dichloroaniline (Loutre et al., 2003). After purification from in vitro cell cultures, the enzyme was characterised with MALDI-TOF MS and cloning of the gene was possible based on sequence information. Inducibility of the enzyme by herbicide safeners may contribute to elucidate the interactions among xenobiotics and plant metabolism.

2.2.3 Transgenic Approaches

Metabolic modification and degradation of a xenobiotic molecule may depend on a single enzyme. A transgenic approach for modifying or improving this enzyme with benefit for the relevant phytotechnology, is therefore conceivable. Examples reported in literature concern engineering herbicide tolerance, since these compounds are completely assimilable to environmental xenobiotics. For instance, Diderjean et al. (2002) reported of a successful transgenic approach with a gene for cytochrome P450, involved in Phase I of the metabolism. The gene chosen is inducible by chemical stress (metals and drugs) in Jerusalem artichoke, and it conferred resistance to phenylurea upon transfer in the sensitive species tobacco and Arabidopsis. This gene may further be considered a useful tool for phytotransformation application in case of contamination by herbicides in soils and water.

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The following example is not concerned strictly phytoremediation, but rather phytomonitoring of organic compounds with a transgenic approach. The Danish company Aresa Biotechnology has developed a GM plant of Arabidopsis thaliana which can detect nitrogen dioxide emitted by explosives and signal this contact by changing to red color (Anonymous, 2003). The proposed application would be that of growing plants in areas affected by anti-personnel mines in order to contribute to decontamination of the site.

2.2.4

Cell cultures are utilized for bioassessment studies before practical application in constructed wetlands, especially in the case of complex contaminants, for which knowledge of the degradative patterns are not accurate or existing at all. Species differ in their uptake and metabolic capacities, and it has been shown that Carex (sedge) has negligible uptake of TNT, different from Juncus, Phragmites or Typha (Gerth and Hartmut, 2004).

3. IMPLEMENTATION OF KNOWLEDGE FOR

APPLICATION

3.1.1 Constructed Wetlands

Application of constructed wetlands for treatment of contaminated waters is receiving growing interest in Europe. Several examples have been described in different meetings. EC has supported the implementation of a 10000 m2 wetland in Portugal, to treat industrial effluents containing aniline, nitrobenzene and sulfanilic acid (Ramos et al., 2004). Other examples, which cannot be cited here for space limitations, can be found in the abstracts books of COST (www.gre.ac.uk/cost859/).

3.1.2 Short Rotation Coppice Forestry

Short rotation coppice is a plantation of trees, poplars or willows, which are kept for less than 15 years and produce plant biomass for several purposes in the paper and pulp industry. In particular, coppicing consists in cutting the trunk at the base at intervals of 2-3 years, and new shoots emerge from the stump. This type of forestry also represents a source of renewable energy, constituting at the same time a sink for atmospheric carbon. Utilizing

In Vitro Studies for Implementation

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plants which can take up heavy metals, consume CO2 and produce biomass, combines forestry with phytotechnologies. Several authors are therefore studying metal uptake in willow and poplar, in order to assess the biodiversity existing among cultivars, clones and accessions. As a recent example, poplar clones were analyzed for uptake of several metals, showing how Cd, Zn, Al were taken up with high efficiency (Laureysens et al., 2004).

3.1.3 Interactions with Microorganisms

It is well known that plant-microorganisms interactions play important roles in phytoremediation. In recent years discovery of the role of endophytic bacteria in phytoremediation has led to several interesting considerations. Engineering endophytic bacteria of the species Burkholderia

cepacia with plasmid pTOM increased degradation of toluene in yellow lupine plants (Barac et al., 2004), at the same time lowering toxicity to the plant.

3.1.4 Atmosphere Contaminants

Phytotechnologies have traditionally been limited to contaminants accessible through plant roots, either in soil and sediments or in water. However, contaminants may enter the plant also from the atmosphere, and a new application of the phytotechnologies may be the removal of pollutants from the troposphere (Morikawa et al., 2003). Nitrogen dioxide is a pollutant which may be taken up through stomata and incorporated into organic compounds. There is extensive variability among plant taxa in this regard, and a survey of about 300 species showed that the most efficient plant is Eucalyptus viminalis, 657 times more efficient than Tillandsia, the less efficient taxon. These plants could be used to assembly “green walls”, covering the vertical surfaces of building where plants are able to assimilate NO2 in great quantities. Recently the authors have described a positive effect of NO2 on plant biomass growth, defining it as a “plant vitalisation signal” (Morikawa et al., 2005), but this still waits for a confirmation.

4. NEW TRENDS

4.1 Natural Remediation

A new trend in use of phytotechnologies is favour encountered by the so-called “Assisted Natural Remediation”, clearly a non conventional

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application (Adriano et al., 2004). In Assisted Natural Remediation, amendments are added to the soil in order to accelerate natural processes of remediation. In the case of metals, amendments contribute to immobilization with complexation, adsorption, precipitation and chemical reactions: the main purpose is to lower the bioavailability of the metal, and not its total concentration. The literature reviewed by Adriano et al. (2004) includes successful examples to which Assisted Natural Remediation has been applied.

4.2 Biofortification

Studies on the interaction between plant tissues, heavy metals and/or trace elements have led to the concept of biofortification, in which plants enriched in micronutrient content are seen as an aid against malnutrition. Differently from phytoaccumulation of metals, which is considered as a risk for the food chain, biofortification of crops with specific elements may become advantageous (Welch and Graham, 2004). Fortified crops are suitable for growth on micronutrient-poor soil because their bioconcentration capacity will lead to higher content of micronutrients in edible tissues. Knowledge of mechanisms controlling metal accumulation is a prerequisite for elucidating the biochemical basis of these phenomena. Information is also requested for those antinutrients that decrease element availability: examples include phytic acid, fibres, and polyphenols.

4.3 Glucosinolates and Biofumigation

Biofumigation is a recent application of the properties of plant chemicals. In particular, several Brassicaceae are exploited in the fight towards pests and pathogens in agriculture due to the production of specific secondary metabolites called glucosinolates (Mithen, 2001). These sulfur-containing compounds have an anticarcinogenic activity in man, they contribute to the characteristic flavor of cruciferous plants, and their degradation products can deter herbivores and inhibit microorganisms. From this derives the use of Brassicaceae as “green manure” to be added to the soil during preparation, in order to decrease the load due to pathogens and pests. This is a sustainable substitute to the use of chemical fumigants. An interesting feature still to be explored is the possible connection between production of specific glucosinolates in the plant and the presence of heavy metals in the environment which may act as inducers or repressors. Since glucosinolates contain sulfur, like metallothioneins and phytochelatins, they could have an impact in the sulfur metabolism of these heavy metal sequestering peptides.

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This in turn may determine an interlock during the process of pest resistance and heavy metal resistance.

5. CONCLUSIONS

Research on phytoremediation and phytotechnologies is thriving in Europe even despite lack of funding. Few EC funded projects and different National or regional activities has prevented research in this field to collapse. Networking activities such as those promoted within COST are also of extreme importance for building interactions and collaboration among scientists within and outside Europe. Other initiatives targeted at dissemination, education and training, should be activated in order to increase the familiarity and confidence of the public opinion and of stakeholders in these new sustainable technologies.

ACKNOWLEDGEMENTS

The authors wish to thank Dr Andrea Pirondini, doctoral fellow in the Division of Genetics and Environmental Biotechnologies for the literature search. The project was carried out within the framework and activities of COST Action 859 and the NATO/Russia Council-CCMS project “development of a prototype system for sharing information related to acts of terrorism to the environment, agriculture and water systems”.

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