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Biotechnology Advances 18 (2000) 23–34

Research review paper

Exploitation of plants for the removal of organics in

environmental remediation

T. Macek

a,

_{*, M. Macková}

b

_{, J. Ká}

b

a_{Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,}

Flemingovo n. 2, 166 10 Prague, Czech Republic

b_{Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology,}

ICT Prague, Technicka 3, 166 28 Prague, Czech Republic

Abstract

This review concentrates on the description of various phytoremediation technologies, paying spe-cial attention to removal of organics and the application of in vitro systems for basic research in the role of plants for the remediation of contaminated sites or flows, and in the improvement of their ef-fectiveness. Various aspects of xenobiotic metabolism in plant cells, the role of enzymes involved, and the cooperation with rhizospheric microorganisms accelerating remediation of organics are shown. Application of this approach as well as the possibility of introduction of foreign genes into plant ge-nome that can enhance the rate of the bioremediation are discussed. © 2000 Elsevier Science Inc. All rights reserved.

Keywords: Phytoremediation; Xenobiotics; Polychlorinated biphenyls; Plant cells

1. Introduction

Phytoremediation is defined, according to Cunningham and Berti [1] and Cunningham et al. [2], as the use of green plants to remove, contain, or render harmless environmental con-taminants. It is important to note that this includes the use of vegetation for in situ treatment of water, sediments, soils, and air. In this process specially selected or engineered plants can be used for extraction of toxic metals from soil or water, including removal of radioactive el-ements, removal of toxic organic compounds, and, if possible, their mineralization.

sˇ

* Corresponding author. Fax: 1420-2-24310090.

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24 T. Macek et al. / Biotechnology Advances 18 (2000) 23–34

Cost comparisons of phytoremediation to other remediation technologies have recently been made. The consensus cost of phytoremediation has been estimated at $25–$100 per ton of soil treatment and $0.60–$6.00 per 1000 gallons for treatment of aqueous waste streams. In both cases, the remediation of organic contaminants can be expected to fall at the lower end of these ranges and remediation of heavy metals to fall at the higher end. In each ap-proach discussed, the expenses of phytoremediation represent less than half of the price needed for any other effective treatment. According to 1997 U.S. EPA estimates, the cost of using phytoremediation in the form of an alternative cover (vegetative cap) ranges from $10000 to $30000 per acre, which is thought to be two- to five-fold less expensive than tradi-tional capping [3].

2. Advantages of phytoremediation

Phytoremediation has made tremendous gains in market acceptance in recent years. In ad-dition to its favorable economics, according to various authors [1,4–6] the main advantages of phytoremediation in comparison with classical remediation methods can be summarized as follows:

It is far less disruptive to the environment. There is no need for disposal sites.

It has a high probability of public acceptance. It avoids excavation and heavy traffic.

It has potential versatility to treat a diverse range of hazardous materials.

Considering these factors and the much lower cost expected for phytoremediation, it ap-pears that it may be used in much larger scale clean-up operations than is possible by other methods. The process is relatively inexpensive, because it uses the same equipment and sup-plies that are generally used in agriculture.

3. Disadvantages of phytoremediation

Like other methods of environmental remediation, phytoremediation has its disadvan-tages. The use of phytoremediation is limited by the climatic and geological conditions of the site to be cleaned, temperature, altitude, soil type, and accessibility by agricultural equip-ment. Other disadvantages vary with the application and type of contamination for which the method will be used. As discussed by others [1,4–6], the main concerns come from the fol-lowing problems:

Formation of vegetation may be limited by extremes of environmental toxicity.

Contaminants collected in leaves can be released again to the environment during litter fall. Contaminants can be accumulated in fuel woods.

The solubility of some contaminants may be increased, resulting in greater environmental damage and/or pollutant migration.

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T. Macek et al. / Biotechnology Advances 18 (2000) 23–34 25

4. Uptake and biotransformation of pollutants in plants

Phytoremediation is currently divided into following areas as summarized by Salt et al. [7].

• Phytoextraction: the use of pollutant-accumulating plants to remove metals or organics from soil by concentrating them in the harvestable parts.

• Phytodegradation: the use of plants and associated microorganisms to degrade organic pollutants.

• Rhizofiltration: the use of plant roots to absorb and adsorb pollutants, mainly metals, from water and aqueous waste streams.

• Phytostabilization: the use of plants to reduce the bioavailability of pollutants in the en-vironment.

• Phytovolatilization: the use of plants to volatilize pollutants. • The use of plants to remove pollutants from air.

Various mechanisms are involved in each of the processes listed above. Plants remediate organic compounds by direct uptake of contaminants, as summarized by Schnoor et al. [5], followed by subsequent transformation, transport, and their accumulation in a nonphytotoxic form (which does not necessarily mean nontoxic for humans). In addition, plants support bioremediation by release of exudates and enzymes that stimulate both microbial and bio-chemical activity in the surrounding soil and mineralization in the rhizosphere. The use of plants as a final water treatment step and for the disposal of sludge resulting from waste wa-ter treatment is centuries old [8]. These processes either ‘decontaminate’ the soil, or ‘stabi-lize’ the pollutant within it (i.e. preventing its migration to a site of actual danger to human health). Specifically, two subsets of phytoremediation are nearing commercialization. First is phytoextraction, in which high biomass metal-accumulating plants and appropriate soil amendments are used to transport and concentrate metals from the soil into the harvestable part of roots and aboveground shoots, which are harvested with conventional agricultural methods [2,7]. The other is rhizofiltration, in which plant roots grown in water absorb, con-centrate, and precipitate toxic metals and organics from polluted effluents [2,7].

5. Role of rhizospheric microbial communities

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wastes from soil or water through their roots. Although it is not discussed in this review, plants have a great capacity to bind organic compounds from the air [7]. Classes of organic compounds that are more rapidly degraded in the rhizosphere than in bulk soil include poly-cyclic aromatic hydrocarbons [12,13], total petroleum hydrocarbons [14], chlorinated pesti-cides (PCP, 2,4-D), other chlorinated compounds like polychlorinated biphenyls (PCBs) [15,16], TCE [17], explosives [2,4,6,-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), dinitrotoluene (DNT)], organophosphate insecticides (diazinon, parathion), and surfactants (detergents). Burken and Schnoor [18] discussed beneficial impacts of root exudates and biomass on mineralization of atrazine by poplar trees.

Betts [19] reported a field trial in which a substantial decrease in total petroleum hydrocar-bon (TPH) content using bermuda grass, rye grass, white clover, and tall fescue was ob-served in plots at a large fuel facility in the United States. Recently, it was shown that some compounds present in root exudates can serve not only as nutrients for microorganisms [11,20], but they can also induce the bacterial degradation of PCBs [21–23]. These com-pounds (phenolics, flavonoids, terpenes) served as growth substrates and they stimulated PCB degradation in the same manner as biphenyl.

6. Metabolism

Vegetative caps at landfill sites and other contaminated areas are used as alternative tech-nologies to help contain toxic chemicals and reduce their movement to other sites. The dif-ferent mechanisms mentioned earlier can be involved. Organic compounds can be translo-cated to other plant tissues [7] and subsequently volatilized, they may undergo partial or complete degradation, or they may be transformed to less toxic, especially less phytotoxic, compounds and bound in plant tissues. In general, most organics appear to undergo some de-gree of transformation in plant cells before being sequestered in vacuoles or bound to insolu-ble cellular structures such as lignin. Metabolism of herbicides and pesticides was exten-sively studied many years ago [24–27]. During recent years metabolism of nonagricultural xenobiotics such as trichloroethylene (TCE), TNT, glyceroltrinitrate (GTN), polyaromatic hydrocarbons (PAHs), PCBs [12–14,16,17] and other chlorinated compounds has been stud-ied [7,28]. It was shown that most of these compounds are metabolized but only a few chem-icals appear to be fully mineralized. Some plant metabolites of pollutants may be more toxic than the original compounds, making plants less attractive compared with bacteria, which to-tally degrade organic pollutants.

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conjugated, have been identified, together with some dechlorinated products. Wilken et al. [35] studied the metabolism of 10 different congeners of PCBs in 12 cell cultures of different plant species (one culture of each species). The authors observed that metabolism of defined PCBs congeners was dependent on plant species, detecting various monohydroxylated and dihydroxylated compounds after acid hydrolysis of polar metabolites.

Macková et al. [16,36,37] studied the ability of cultures of various species cultivated in vitro to degrade Delor 103 (a mixture of PCBs commercially produced in the former Czech-oslovakia until the mid-1980s) [38,39]. The PCB mixture contained about 59 congeners with differing degrees of chlorination, with an average number of three chlorines per biphenyl molecule. The analytical procedure was optimized by Burkhard et al. [40]. About 40 axenic cell cultures of different plant species were screened for the ability to transform PCBs. When the PCB degradative ability in relation to the origin and morphology of the cultures was eval-uated, the results showed a great variability in the capability to convert PCBs within different cultures of the same plant species [41,42]. The best results were obtained with Solanum

ni-grum (black nightshade) hairy root clone SNC-9O. Such cultures, obtained after

transforma-tion of plant cells by Agrobacterium rhizogenes, proved to be a very useful tool in basic re-search for phytoremediation purposes [37,43,44]. Metabolism of individual congeners of all three monochlorobiphenyls were studied and various monohydroxylated and dihydroxylated chlorobiphenyls were detected. 4-Hydroxy derivatives were identified as the major products in all three cases of monochlorobiphenyls transformation [16].

7. Enzymes involved

In his review Cole [45] showed that oxygenation is a common process in pesticide and herbicide metabolism and it is an important initial mode of attack when organisms encounter what are often highly lipophylic compounds. This step serves to increase water solubility and provides an opportunity for conjugation via glycosidic bond formation. In several instances mixed function oxidases have been implicated, similar to those in mammals and insects [45]. The xenobiotics can be oxidized by cytochrome P450 and peroxidases may also be important [46,47]. Many early examples of plant metabolic sequences of transformation, conjugation, and compartmentation (three phases of xenobiotic transformation in plants) reactions have been summarized and compared with animal metabolite patterns [48]. Recent studies have shown that plants appear to contain sets of specific metabolic isoenzymes and the corre-sponding genes. Similarities between animal and plant metabolic pathways exist; however, plant metabolism may often be more complex and an important difference from animal me-tabolism appears to exist especially in the formation of bound residues (phase III). Enzymes involved in xenobiotic metabolism were reviewed by Sandermann [48,49]. He showed that cytochrome P450, peroxygenases, and peroxidases are involved in plant oxidations of xeno-biotics. Other enzyme classes like gluthathione S-transferases, carboxylesterases, O-gluco-syltransferases O-malonyltransferases, N-glucoO-gluco-syltransferases, and N-malonyltransferases are associated with xenobiotic metabolism in plant cells, transport of intermediates, and compartmentation processes.

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metabolism were studied. The effect of PCBs on the changes of the level of peroxidase activ-ity and the pattern of peroxidase isoenzymes was also followed [36,50,51]. Lee and Fletcher [52] suggested that cytochrome P450, rather than peroxidases, is involved in the PCB degra-dation pathway. Nevertheless, we found a significant positive correlation between peroxidase content and PCB disappearance during incubation of cultures in the presence of PCB [51].

8. Plant cells cultivated in vitro as a tool for phytoremediation experiments

Most experiments used to establish phytoremediation techniques were done with normal soil-grown or hydroponically grown plants. Recently, as more and more effort is directed to-ward research to understand and improve the performance of plants in remediation technolo-gies, the number of results obtained with the help of in vitro plant cell and tissue cultures is rapidly increasing. The concept is not new—in vitro cultivated plant cells have been used in studies of herbicide resistance and metabolism for many years. Other organic xenobiotics have also been studied, as in the case of pentachlorophenol, which was shown to be metabo-lized by wheat and soybean cell suspension cultures yielding glucosides and nonextractable residues as described by Langebartels and Harms [28]. The ability of plant cells to metabo-lize PCBs was demonstrated 10 years ago by Groeger and Fletcher [53]. Transformation of TNT by a hairy root culture of Catharanthus roseus was investigated by Hughes et al. [54]. They did not detect mineralization of TNT; instead, products such as 4-amino-2,6-dinitrotol-uene and 2-amino-4,6-dinitrotol4-amino-2,6-dinitrotol-uene were identified.

Following transformation of 3,4-dichloraniline by leaves and suspension cultures of soy-bean, it was observed that both systems almost completely metabolize this compound during 48 h. Mostly N-glucosyl and N-malonyl conjugates were analyzed in leaves. These conju-gates were bound to cell wall structures. Cells of suspension cultures produced soluble N-malonylconjugates, which were excreted into the medium. It was proven that axenic cell cultures are able to metabolize certain compounds by common metabolic pathways. Plant cell tissue culture can be a useful system with some advantages in comparison with intact plants [55]. These advantages include: (1) the material can be grown under standard labora-tory conditions, (2) the growth is independent of the weather or climate, and (3) in vitro cul-tures often grow more rapidly. The exploitation of transformed hairy root culcul-tures is espe-cially rewarding, as discussed by Macková et al. [15,37], Hughes et al. [54], Macek et al. [43,56], and Betts [20]. Plant roots transformed by Agrobacterium rhizogenes exhibit all fea-tures of normal plant roots and grow rapidly under defined aseptic conditions in vitro [57], thus allowing the distinction to be made between the plant metabolism itself and the effect of the complex interaction between plants and microbial communities in the rhizosphere [58]. Metabolism of many other organic compounds has been addressed with the help of plant cell and tissue cultures, because in addition to the above advantages this model facilitates obtain-ing results with much lower analytical expenses.

9. Practical approaches

Phytoremediation has already been successfully implemented. Trees of the Salicaceae

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erance and fast growth. Schnoor et al. [5] used the hybrid poplar Imperial Carolina for in-creasing soil suction and dein-creasing downward migration of pollutants. This system was used to control agricultural runoff along a small creek in Iowa, and as final cap on a landfill in Oregon. The University of Washington has developed multiple hybrid poplar clones, some with growth rates of 10–15 feet per year. These trees were found to have remarkable ability to take up and degrade certain halogenated organic solvents. In laboratory conditions the poplar tissues were used to examine the metabolism of trichloroethylene. Phytotransforma-tion of perchlorate using parrot-feather (Myriophyllum aquaticum) was described by Susarla et al. [59]. This plant has already been successful in the remediation of soils contaminated with TNT as well as other contaminants (e.g. TCE, PCP). There are numerous Department of Defence sites across the USA with explosives contaminated groundwater. The U.S. Army Environmental Centre is developing technologies to effectively clean up groundwater con-taminated with residues of explosives like TNT, RDX, octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine (HMX), and DNT. Current groundwater cleanup technologies, such as granular activated carbon and advanced oxidation, have proven to be labor-intensive and costly. One potential treatment alternative is phytoremediation using constructed wetlands [60]. The U.S. EPA National Exposure Research Laboratory identified, in bench scale testing, a plant nitroreductase, which in cooperation with other plant enzymes is able to degrade TNT, RDX, and HMX. An artificial wetland used to demonstrate the feasibility of using selected plants to clean up explosive-contaminated groundwater was constructed at the Milan Army Ammu-nition Plant, and experiments were begun in 1995. A pilot-scale plant lagoon system was constructed and operated by the Georgia Institute of Technology. Results indicated that the TNT removal in plant cells matched laboratory batch study predictions. Removal percent-ages relative to TNT loading for all species ranged from 85.4 to 99.7%.

In addition, the U.S. Air Force is trying to clean TCE from groundwater using poplar trees and the U.S. Army is endeavoring to clean 2,4,6-trinitrotoluene and RDX from contaminated wetlands, using a variety of plants. Pradhan et al. [12] used phytoremediation as a primary remediation technology and as a final polishing step for treatment of soil contaminated with PAHs. Three plant species, alfalfa (Medicago sativa), switch grass (Panicum virgatum), and little bluestem grass (Schizachyrium scoparium) were successfully used. A 57% reduction in total PAH concentration was observed after 6 months of treatment. Phytoremediation was also tested in pot experiments in soil contaminated with petroleum hydrocarbons during the Gulf War. Three domestic plants, broad beans (Vicia faba), alfalfa (Medicago sativa), and ryegrass (Lolium perenne) were tested. This study also described the rhizospheric effects of the leguminous plants alfalfa and broad bean, and demonstrated high degradation rates due to plant-microbe interactions. In this study the selection of plants was completely random and further studies are needed to optimize the technology and to identify key parameters. The determination of the most efficient plant species to degrade a particular compound is the most important step in this technology [14].

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Force Base) eastern cottonwood trees were planted to clean up trichloroethylene from a shal-low, thin aerobic aquifer. The organism was chosen, because “they [eastern cottonwoods] are indigenous to the site, they are members of the same genus as poplar trees and grow at similarly rapid rates”! The trees were planted to intercept and reverse the flow gradient and to determine if cottonwoods can metabolize TCE and its daughter compounds under field conditions [62]. The system decreased the TCE content; however, the reasons leading to the choice of eastern cottonwood seems to be quite unsatisfactory and lacking in support of re-search results, including the risk of survival problems for plants after long-term exposure to the contaminants. The above approach cannot generally be considered as optimal. This is of-ten the problem of agencies funding remediation experiments, where time pressure leads sometimes to support of projects lacking the time-consuming attempts to explain experimen-tally molecular mechanisms involved in the desired activity.

Many of these problems can be addressed by the selection of the proper type of plants. Phytoremediation has a number of inherent technical limitations. The contaminant must be within, or drawn toward, the root zones of plants (usually the top 3–6 feet of soil). This im-plies water, depth, nutrient, atmospheric, and physical and chemical limitations. In addition, the site must be large enough to make farming techniques appropriate. It must not present an eminent danger to human health or further environmental harm [8]. There may also be a con-siderable delay in the time needed for obtaining satisfactory cleanup results between phy-toremediation and ‘dig and dump’ techniques. Although plants have upward of 100 million miles of roots per acre, their root system may not extend deeply enough to eliminate all of the contamination [61].

10. Future prospects

Only the future can tell whether phytoremediation will become a widely accepted technol-ogy. A growing knowledge of the factors important to phytoremediation can provide a basis for genetic modification of plants for improved performance. Breeders have been modifying agronomically important plant traits for years. However, in these instances yield and aesthet-ics were usually the criteria for selection of a plant trait. As Cunningham et al. [2] state, ‘phytoremediation requires a new paradigm in which plants are valued based on what they adsorb, sequester, destroy and tolerate.’ All of these traits can be specifically targeted by tra-ditional breeding as well as molecular biology.

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bacteria to degrade anthropogenic toxins [21]. Plant-fungal interactions would also seem ripe for exploitation in this area, particularly mycorrhizal associations [65]. Alternatively, trans-genic plants can be transformed to harbor microbial genes for biodegradation. This is already a routine practice in the engineering of many herbicide-resistant plants and their field testing, product development, and registration are well advanced. The concept could be extended to ad-dress additional xenobiotics. Biodegradative microbial strains are notoriously unreliable in their ability to compete with native microorganisms when released into the natural environ-ment. In addition, such release faces general public opposition. In contrast to herbicide resis-tance, the degradation of xenobiotics is rarely performed by one single enzyme. For the degra-dation of organic xenobiotics, the concerted action of many enzymes is needed, and introduction of all of the necessary genes into a plant is a formidable task.

11. Conclusions

Phytoremediation is fast becoming recognized as a cost-effective method for remediating sites contaminated with toxic metals, radionuclides, and hazardous organics at a fraction of the cost of conventional technology. Hybrid technologies such as phyto-vapor extraction are being actively investigated for technical and economic feasibility. The use of plant roots as ‘biocurtains’ or ‘bio-filters’ for the passive remediation of shallow groundwater is also an active area of research [8]. The establishment of vegetation on a site also reduces soil erosion by wind and water, which helps to prevent the spread of contaminants and reduces exposure of humans and animals.

These hybrid technologies will undoubtedly provide both research opportunities and po-tential field applications in the short term. In the longer term, use of such hybrid technologies will probably have significantly greater costs than is likely achievable by phytoremediation utilizing advanced plant selection and genetic engineering techniques.

There have been about two dozen field tests of phytoremediation to date, with many new ones appearing. Yet in many ways this technology is still in its infancy. Obtaining credible, sci-entifically valid cost estimates of phytoremediation is a critical element in the acceptance of phytoremediation in the market and should be a major goal of the demonstration projects now underway [3]. A comforting thought for plant biologists is that much of the research effort will center on a deeper understanding of basic plant processes [8]. New innovative methods are needed to solve some of our worst pollution problems. The concept of manipulating plant genes that regulate toxic metal uptake is cutting-edge research. The likelihood of public accep-tance and the fact that permits for field testing of genetically engineered plants now vastly out-strip permits for genetically altered microbes represent good reasons for moderate optimism. And finally, many accepted agricultural techniques for cultivating, harvesting, and processing plants have now been adapted for phytoremediation. The application of phytoremediation is being driven by its technical and economic advantages over conventional approaches.

Acknowledgments

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