Acta Biotechnol. 23 (2003) 2 – 3, 281 – 288
Short Review Biotechnological Approaches for Enhancing Phytoremediation of Heavy Metals and Metalloids TERRY*, N., SAMBUKUMAR, S. V., LEDUC, D. L.
Department of Plant and Microbial Biology 111 Koshland Hall University of California Berkeley, CA 94720-3102 USA
*
Corresponding author Phone: +1 510 642 3510 Fax: +1 510 642 3510 E-mail:
[email protected]
Summary Phytoremediation, the use of plants and their associated microbes to accumulate, detoxify and/or stabilise contaminants, is an environment-friendly and sustainable means of remediating contaminated soil and water. Phytoremediation has been an important aspect of constructed wetlands, which have been used successfully to detoxify large volumes of wastewater with dilute concentrations of contaminants. The usefulness of phytoremediation appears to extend to a wide variety of contaminants, and a recent study demonstrated its possible application for selenocyanate. Genetic engineering approaches are currently being used to optimise the metabolic and physiological processes that enable plants to phytoremediate sites contaminated with heavy metals and metalloids. Over-expressing enzymes catalysing rate-limiting steps in the sulphate assimilation and phytochelatin synthesis pathways has been shown to confer increased tolerance to and the ability to accumulate higher concentrations of selenium and cadmium, respectively. Recent research in our laboratory is aimed at determining the regulatory genes involved in heavy metal accumulation and detoxification in hyperaccumulating plants that could then be transferred to fast-growing, high biomass plant species for phytoremediation. Microbes isolated from highly contaminated environments represent another potentially huge reservoir of new genes and unique metabolic capabilities that could be transferred to plants to enhance their phytoremediation potential.
Introduction
Contamination of soil and water with heavy metals and metalloids such as cadmium (Cd), lead (Pb), mercury (Hg), arsenic (As) and selenium (Se) is an increasing environmental problem worldwide. Due to the acute toxicity of these contaminants, there is an urgent need to develop low-cost, effective and sustainable methods to remove them © WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, 2003 0138-4988/03/02-3-07-0281 $ 17.50+.50/0
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from the environment or to convert them to less toxic forms. Phytoremediation is just such a technology [1]. Plants naturally take up heavy metals and metalloids from soil and water but normally suffer toxicity effects at concentrations lower than would be required for efficient phytoremediation. A small number of plant species, referred to as “hyperaccumulators”, possess a unique ability to accumulate metals to extremely high concentrations without suffering any toxic effects. Although the potential of hyperaccumulators for use in phytoremediation is limited by their slow growth rate and low biomass [2], they serve as an important resource for research into the unique biochemical and genetic mechanisms responsible for trace element hyperaccumulation and detoxification. Additionally, hyperaccumulators can provide a source of genes for enhancing phytoremediation. Transforming fast-growing plant species, such as Brassica juncea (Indian mustard), with hyperaccumulator genes, particularly those involved in detoxification, could combine the unique capabilities of a hyperaccumulator with those of high biomass plants to enhance phytoremediation. Furthermore, a better understanding of the role of rhizospheric microbes associated with hyperaccumulating plants may eventually be used to develop other means of enhancing phytoremediation potential. Constructed Wetlands and Biological Volatilisation
Constructed wetlands have been used to detoxify municipal wastewater and many other types of polluted waters in the U.S. and Europe [3]. They comprise a complex ecosystem of plants, microbes and sediment that together act as a biogeochemical filter, efficiently removing dilute contaminants from very large volumes of water, as is the case with Se-contaminated agricultural and industrial wastewater containing high concentrations of selenate (SeO4–2) and selenite (SeO3−2), respectively [4]. Although constructed wetlands offer a less expensive alternative to other water treatment methods, much remains to be understood to make it a highly efficient and reproducible method for the remediation of Se. A major target for optimisation is to increase Se volatilisation by plants and microbes. Because of the chemical similarity of sulphur (S) and Se, plants and microbes are able to take up inorganic and organic forms of Se and metabolise them to volatile forms via the S assimilation pathway. Biological volatilisation has the advantage of removing Se from a contaminated site in relatively non-toxic forms, such as dimethylselenide (DMSe), which is 500–700 times less toxic than SeO4–2 or SeO3−2 [5−7]. Thus, biological volatilisation of Se is important because it can lead to its safe removal from the food chain. Although the volatilised Se may eventually be redeposited in other areas, this is not a problem in California where much of the state is deficient in Se with respect to the nutrition of animals, which require Se in low concentrations [8]. Uptake and Detoxification of SeCN−
Phytoremediation has a great potential for the cleanup of a wide variety of contaminants, including highly toxic ones such as selenocyanate, SeCN−. This was demonstrated for two species that have been identified as excellent candidates for phytoremediation, i.e., Brassica juncea (Indian mustard) and Chara canescens (muskgrass). Indian mustard is useful for phytoremediation of contaminated upland soils because it
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extracts and accumulates relatively high concentrations of contaminants, particularly Se; it grows quickly, produces a high biomass and is salt tolerant [9]. Studies have shown that Indian mustard can remove 50% of the total Se present in the soil during a single growth cycle (50−55 days) [10, 11]. Indian mustard is also an efficient producer of volatile Se. Muskgrass is an excellent species for phytoremediation because it produces a large biomass under wetland conditions [12] and can accumulate metals to high concentrations [13]. Research in our laboratory has shown that Indian mustard and muskgrass are suitable for the phytoremediation of SeCN− in upland and wetland conditions, respectively [14]. Growth of Indian mustard treated with 200 µM SeCN− was not significantly different from growth on 200 µM SeO4–2. When compared with 200 µM SeO3−2, Indian mustard treated with 200 µM SeCN− had significantly higher fresh weights and no significant difference in root lengths. This suggests that Indian mustard would be able to tolerate SeCN− concentrations present in the actual contaminated soils [14]. The study also showed that muskgrass could be used in constructed wetlands treating SeCN−-contaminated wastewater, although it was less efficient in extracting Se over a short period of time [14]. X-ray absorption spectroscopy revealed that the Se accumulated by Indian mustard plants supplied with SeCN− was mainly in the form of organic Se with no Se remaining in the form of SeCN− [14]. Although the Indian mustard plants accumulated large amounts of organic Se in their tissues, they did not produce large amounts of volatile Se [14]. This indicates that one or more biochemical steps in the conversion of Se to DMSe could be rate limiting, for example, the methylation of selenomethionine (SeMet) by methionine methyltransferase. Although the actual mechanisms for the uptake and degradation of SeCN− are unknown, we proposed one possible model based on knowledge of the metabolism of the S analogue, thiocyanate (SCN−) [14]. Plants naturally detoxify SCN− by converting it to dimethylsulphide and, by analogy with sulphur, it is possible that plants are also able to convert SeCN− to DMSe. The first step of this pathway is the uptake of SeCN−, which is most likely taken up actively by the plant (as are SeO4–2 and SeMet) [15, 16]. SeCN− may then be degraded to selenide, Se−2, and OCN− by an as yet undefined enzyme similar to that suggested for bacteria [17]. OCN− may then be converted to ammonia and CO2 by cyanase. Alternatively, Se−2 could be generated via the pathway of thiocyanate degradation, in which thiocyanate hydrolase mediates the conversion of thiocyanate to ammonia and carbonyl sulphide [18−20]. The carbonyl sulphide is then degraded to CO2 by an unknown enzyme. In the third proposed step, Se− 2 enters the pathway described for the assimilation of inorganic Se to DMSe [7].
Genetic Modification of Plants to Enhance Phytoremediation
Recent research in our laboratory has shown that genetic modification of plants can provide a powerful method of improving the capacity of plants for the remediation of various contaminants such as Se and Cd [21−23]. Identifying candidate genes for transfer and/or over-expression is a key step and a useful approach is to over-express enzymes catalysing rate-limiting steps. For example, ATP sulphurylase, which facili-
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tates the reduction of sulphate (SeO4–2) to sulphite (SeO3−2) in the S assimilation pathway, is rate limiting with respect to the production of reduced, organic S compounds. The over-expression of Arabidopsis ATP sulphurylase (APS) in Indian mustard resulted in an increased reduction of (supplied) SeO4–2 to organic Se forms in the APS transgenics, while wild type plants accumulated mainly SeO4–2 [21]. This in turn led to increased uptake and reduction of SeO4–2, as well as to an increased tolerance to selenate, in the APS transgenics compared to wild type [21]. The reduction of SeO4–2 is carried out almost exclusively in the shoots [21], as is the case with SeO4–2 reduction [24]. The APS plants also contained higher levels of glutathione (GSH), indicating that ATP sulphurylase is also rate limiting for GSH synthesis. GSH plays an important role in plant resistance to oxidative stress and is the precursor of phytochelatins (PCs), which bind, detoxify and sequester metal ions to the vacuole [25, 26]. As such, it is likely that the APS plants could also have enhanced tolerance to other toxic metals [21]. The approach of over-expressing genes that catalyse rate-limiting steps can also be used for the phytoremediation of heavy metals. GSH (γ-Glu-Cys-Gly) plays an essential role in heavy metal detoxification by plants. GSH is the direct precursor of PCs, which are metal-binding peptides involved in heavy metal tolerance and sequestration [27]. Additionally, GSH is a major component of the active oxygen scavenging system of the cell [28] and can protect the plant cell from Cd-induced oxidative stress [29, 30]. It is also possible that GSH detoxifies Cd by directly forming a GSH-Cd complex such as that reported for yeast [31]. The role of GSH and PCs in heavy metal tolerance is illustrated by the Cd-hypersensitivity of Arabidopsis mutants defective in GSH and PC biosynthesis [32]. γ-glutamylcysteine synthetase (γ-ECS) catalyses the first step in the ATP-dependent synthesis of GSH. This is considered to be the rate-limiting step in the biosynthesis of GSH since the activity of this enzyme is subject to feedback regulation by GSH and is dependent upon the availability of cysteine [27]. ZHU et al. [33] studied the effect of over-expression of E. coli γ -ECS, targeted to the chloroplasts of Indian mustard. The transgenic plants had 2- to 3-fold higher levels of γ-EC as well as GSH and PC when subjected to Cd [33]. Their increased Cd tolerance was almost certainly due to their higher production of PCs or GSH. In addition to conferring tolerance to Cd, over-expression of γ-ECS led to an increase in total shoot S suggesting an added advantage of enhanced S assimilation [33]. Similar results were also obtained in the case of poplar plants over-expressing γ-ECS [34, 35]. Over-expression of glutathione synthetase in Indian mustard also led to enhanced levels of GSH and PC2 in the presence of heavy metals [22]. In another study, the over-expression of glutathione reductase (GR) in the plastids of Indian mustard led to enhanced Cd tolerance at the chloroplast level; compared to wild type plants, the transgenic plants had higher levels of chlorophyll fluorescence and exhibited no signs of chlorosis [23]. However, the tolerance to Cd at the whole plant level was not increased in comparison to the wild type plants [23]. Differences in Cd tolerance and accumulation were attributed to increased root GSH levels, which could have reduced membrane damage in the transgenic plants and resulted in more efficient Cd exclusion by the root membranes [23]. The plants over-expressing GR in the cytosol were, however, not tolerant to Cd [23] suggesting that the intracellular location of the detoxifying enzyme could be an important factor in devising strategies to improve phytoremediation.
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Another approach using genetic engineering to enhance phytoremediation potential is to transform fast-growing host plants with unique genes from natural hyperaccumulators. One such gene encodes the enzyme selenocysteine methyltransferase (SMT), which has been cloned from the Se-hyperaccumulator, Astragalus bisulcatus [36]. SMT converts the amino acid, selenocysteine, to the non-protein amino acid, methylselenocysteine (MetSeCys). By doing so, it diverts the flow of Se from the Se-amino acids that may otherwise be incorporated into protein, leading to alterations in enzyme structure and function and possible toxicity. Additionally, SeCys may also cause oxidative damage. Transgenic plants over-expressing SMT show enhanced tolerance to Se, particularly selenite, and produced 3- to 7-fold more biomass than the wild type plants (T ARUN et al., unpublished). These transgenic plants, which accumulated Se mainly as MetSeCys, exhibited increased rates of Se volatilisation rates when treated with different forms of selenocompounds (TARUN et al., unpublished).
Use of Microorganisms in the Remediation of Toxic Metals
The diversity and adaptability of microorganisms allows them to thrive in harsh, toxic environments that prevent the growth of higher plants. For example, solar evaporation ponds, which are used to collect Se-contaminated agricultural drainage water, have extremely high concentrations of salt, Se and other toxic trace elements. The specific composition of the microbial communities present in these ponds may themselves be useful for the bioremediation of Se since bacteria are able to produce volatile Se [37]. Additionally, they may serve as reservoirs of unique genes involved in tolerance and volatilisation of Se. Identification of the genes involved in these processes could pave the way for generating highly efficient plants by transferring these genes to the plants [38]. We have characterised the total bacterial diversity in an aerobic hypersaline Secontaminated pond using 16S rDNA analysis; the results show that a previously unaffiliated group of uncultured bacteria belonging to the order Cytophagales was dominant [38], followed by a group of culturable γ-protobacteria closely related to Halomonas sp. These results, taken together with earlier studies [39, 40], suggest that bacteria can exist and dominate in some extreme environments that were previously assumed to contain mainly Archaea. It is possible that the predominant group of bacteria (Cytophagales) provides a source of novel genes involved in Se volatilisation. Unfortunately, these bacteria could not be cultured in the laboratory. However, it might be possible to clone the genes involved without culturing the bacteria using recently devised methods. Interestingly, the bacteria were able to volatilise Se in the presence of sulphate in the pond water, indicating that they may have transporters with a high specificity for SeO4–2 or that are highly expressed even in the presence of high concentrations of SeO4–2 [38]. Conclusions and Future Prospects
Recent research has shown that phytoremediation can be an effective method for removing and detoxifying heavy metals and metalloids such as Cd, Se, and As from
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contaminated soil and water. Transgenic plants over-expressing genes catalysing ratelimiting steps in the S assimilation pathway and in PC biosynthesis were able to tolerate and accumulate higher levels of Se or Cd than wild type plants. The identification of unique genes from natural Se hyperaccumulators and their subsequent transfer to fastgrowing species is another promising approach as demonstrated by our recent success with SMT transgenic plants. Further increases in the efficiency of phytoremediation may be achieved by overcoming multiple rate-limiting steps in the pathway or by using modified enzymes that are resistant to feedback inhibition and repression. Another area of potential research is to elucidate the molecular mechanisms by which bacteria have adapted to heavily contaminated environments and then transfer the genes responsible for such tolerance to plants.
Received 9 December 2002 Received in revised form 23 June 2003 Accepted 1 July 2003
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