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Biotechnological potential of aquatic plantmicrobe interactions

L Stout1 and K Nusslein2
The rhizosphere in terrestrial systems is the region of soil surrounding plant roots where there is increased microbial activity; in aquatic plants, this denition may be less clear because of diffusion of nutrients in water, but there is still a zone of inuence by plant roots in this environment [1]. Within that zone chemical conditions differ from those of the surrounding environment as a consequence of a range of processes that were induced either directly by the activity of plant roots or by the activity of rhizosphere microora. Recently, there are a number of new studies related to rhizospheres of aquatic plants and specically their increased potential for remediation of contaminants, especially remediation of metals through aquatic plantmicrobial interaction.
Addresses 1 Department of Biology, Southern Connecticut State University, New Haven, CT 06515, United States 2 Department of Microbiology, University of Massachusetts, Amherst, MA 01003-9298, United States Corresponding authors: Stout, L (stoutl1@southernct.edu) and Nusslein, K (nusslein@microbio.umass.edu)

zoltration and are summarized in several reviews [4,5,6]. Recent studies have applied some of these technologies to phytoremediation of heavy metals in aquatic or wetland systems. For example, Murakami et al. [7] used rice cultivars that accumulated high levels of cadmium to remove metal from contaminated paddy elds. In these conditions where rice was well-adapted to growth, one rice cultivar accumulated 10 times as much Cd as Thlaspi caerulescens, a terrestrial plant well known for its ability to accumulate heavy metals. Rhizoltration, the use of plants to remove heavy metals from aqueous environments, has been extensively tested as a way to remove contaminants from solutions, and can include aquatic or terrestrial plants in hydroponic systems. Recently, sunower and bean plants were tested for their abilities to remove U from contaminated groundwater. In laboratory batch experiments bean plants removed more than 70% and sunower removed more than 80% of U but when sunower was tested in a continuous rhizoltration system, U removal was greater than 99% [8]. Aquatic and wetland plants including the water hyacinth Eichhorinia crassipes [9,10], the invasive reed Phragmites australis [11], the duckweeds Spirodela polyrrhiza [12], Lemna minor [13,14], and Lemna gibba [15,16], the aquatic fern Azolla pinnata [17], and yellow velvetleaf Limnocharis ava [18] have recently been studied for their abilities to remove metals from aquatic systems and show promising results. For instance, A. pinnata was found to remove as much as 94% of Hg from a solution [17], while Eichhornia crassipes was found to accumulate Cr in its shoots at 223 times the concentration in the water [9], and removed 84% of Cr from water and 95% of Zn from water [10]. While metals negatively affected growth of Lemna gibba, the plants were able to remove 90% of Cd from solution after six to eight days [16].

Current Opinion in Biotechnology 2010, 21:339345 This review comes from a themed issue on Environmental biotechnology Edited by Sharon Borglin and John van der Meer

0958-1669/$ see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2010.04.004

Application, advantages, and limitations of plant-based remediation

Phytoremediation, the use of plants and their associated microbial communities to remove or inactivate pollutants from the environment, includes any of several technologies for detoxifying the environment with genetically modied or wild-type plants [2]. Phytoremediation of aquatic environments may be used as an alternative or in addition to conventional remediation methods including ion exchange resins and electrodialysis, chemical precipitation, sedimentation, microltration, and reverse osmosis [3]. Biological remediation techniques offer effective alternative treatments that are often less costly and are considered more environmentally friendly and publicly acceptable than conventional technologies. Various phytoremediation technologies include phytoextraction, phytovolatilization, phytostabilization, and rhiwww.sciencedirect.com

Metal tolerance and resistance: the bacterial side

Bacteria have demonstrated elevated tolerance to metals using many diverse mechanisms. They may maintain metal homeostasis, keeping concentrations of essential metals such as Zn from reaching toxic levels within cells [19] or they may contain resistance systems, active mechanisms for removing or sequestering metals [20]. For heavy metals including Cd, Zn, Ni, Cr, Co, and Cu, there are several types of resistance systems, including efux pumps to remove metals from the cell, and sequestration mechanisms to bind metal inside the cell. The two known types of efux systems are ATPases, which pump out metals using ATP to drive the reaction, and proton
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antiports, which use the proton gradient to pump metals across the cell membrane [21]. Another mechanism of bacterial metal resistance, best known in cyanobacteria, is sequestration by metallothioneins. Metallothioneins bind metals to sulfhydryl groups of cysteine residues [22,23]. Pages et al. recently described cadmium tolerance in Stenotrophomonas maltophilia, and found not only a Cd efux pump but also accumulation of CdS particles [24]. Some bacteria expressing metal resistance produce more extracellular polymeric substances (EPS), which bind the metal, perhaps making the microenvironment around the plant less toxic. Production of EPS has been shown to increase with increased metal resistance [25]. In different strains of Rhizobium leguminosarum, Cd tolerant strains showed increased levels of glutathione, indicating that this tripeptide allows the bacterium to deal with heavy metals, rather than an efux system [26]. The biomarker glutathione is an important antioxidant that may protect against metal toxicity associated with oxidative stress. Another mechanism of dealing with toxic metals may involve polyphosphates, long chains of orthophosphates, which may sequester metals [27]. Perrin et al. recently reported that Ni exposure promoted biolm formation in Escherichia coli cultures, which may serve as a protective tolerance mechanism. Ni appeared to be involved in adherence by inducing transcription of genes encoding curli, the adhesive structures necessary for biolm formation [28]. The protective quality of EPS or these other mechanisms provided by root-associated bacteria suggests that enriching for certain bacteria may replace the technique of amending plant root zones with synthetic cross-linked polyacrylates and hydrogels to protect roots from heavy metal toxicity [29].

late extremely high concentrations of metals are termed hyperaccumulators. There have been more than 400 plant species identied as such [5], including crop species [32], and the number of hyperaccumulators among aquatic and wetland plants is rising [33]. Hyperaccumulators may be dened based on bioconcentration factor (BCF), or the ability to accumulate metals in plant tissues. For instance, the ability to accumulate greater than 1000 times the concentration of Cd (based on concentration of metal in dry weight of plant) than that in the surrounding medium would be considered hyperaccumulation [34]. One of the most studied hyperaccumulators is the terrestrial plant T. caerulescens, which is a Cd/Zn hyperaccumulator [35]. Several aquatic plants have been found to have similar abilities, including Salvinia minima [36], Potamogeton natans [37], Ceratophyllum demersum [38], and S. polyrrhiza [39]. Metal hyperaccumulation is an adaptive process between microbes exposed to heavy metals and plants, requiring continuous interactions among the co-occurring organisms. A recent proteomics study by Farinati et al. [40] indicated that the presence of a rhizosphere microbial population, adapted to heavy-metal-polluted sites, greatly enhanced the accumulation of metals in shoots of the hyperaccumulator Arabidopsis halleri. Aquatic environments include not only macrophytes, but also algae that may interact with microbes to remove contaminants from the environment. Algae can be produced in articial systems and used to remove contaminants. Loutseti et al. used a dried mixture of microalgae and bacteria to remove Cu and Cd from wastewater [41]. Munoz et al. successfully examined the combination of the bacterium Ralstonia basilensis and the microalga Chlorella sorokiniana on adsorption of Cd, Cu, Ni, and Zn [42]. Moreover, mycorrhizal fungi associated with plants can enhance uptake of metals when essential metal concentrations are low and, vice versa, when metal quantities are too high mycorrhizae can be effective in alleviating metal toxicity decreasing plant uptake [43].

Metal tolerance and resistance: the plant side

In plants, metal accumulation in the cells may be regulated by glutathionephytochelatin-mediated resistance. In this system, glutathione, the same cysteinecontaining tripeptide described above in Rhizobium sp., which also has several functions in plant cells including dealing with toxic oxygen species and amino acid transport, is used to synthesize phytochelatins, which chelate heavy metals by formation of a thiolate. Thiolates can be transported to vacuoles for heavy metal storage [30]. Phytochelatins are activated by heavy metals and scavenge heavy metals in plant cells [31]. For a review of plant metal tolerance mechanisms, see [6]. While some plants deal with moderate levels of toxic metals by chelation, other plants have the ability to accumulate extremely high levels of heavy metals and sequester them in their tissues. Plants that can accumuCurrent Opinion in Biotechnology 2010, 21:339345

Mechanisms for how aquatic plantmicrobe interactions affect phytoremediation processes

While many plants and bacteria have their own mechanisms for dealing with heavy metal contaminants, the interaction of plants and microorganisms may increase or decrease heavy metal accumulation in plants, depending on the nature of the plantmicrobe interaction. Because phytoremediation is a relatively new technology, understanding mechanisms of plantmicrobe interactions in removing contaminants from the environment is still not well characterized. There have, however, been
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 Biotech potential of plantmicrobe interaction Stout and Nusslein 341

Figure 1

Mechanisms of aquatic plantmicrobe associations that support metal phytoremediation.

several ideas about the nature of plantmicrobe interactions in metal accumulation (Figure 1). In their paper describing bacterial enhancement of Se and Hg uptake by wetland plants, De Souza et al. proposed several possible mechanisms, including bacterial stimulation of plant metal uptake compounds such as siderophores; bacterial root growth promotion increasing the root surface area; bacterial transformation of elements into more soluble forms; or bacterial stimulation of plant transporters that may transport essential elements as well as heavy metals (in the case of selenate, the sulfate transporter) [44]. Van der Lelie related the basis of this plantmicrobe interaction to bacterial metal resistance, since the bioavailability of metals could be altered by bacterial expression of resistance systems [25].
Plant growth promotion

presence of heavy metals [45,46]. Bacterial production of siderophores may protect plants from heavy metal toxicity, increasing plant growth by providing the plants with sufcient iron and allowing them to overcome the toxic effects of heavy metals. Another plant growth promoting compound that has been studied in relation to heavy metals is 1-aminocyclopropane-1-carboxylic acid deaminase (ACC deaminase). ACC is an intermediate of ethylene produced by plants under stress, and bacteria that produce ACC deaminase can lower the levels of ethylene in plants, promoting plant growth [47]. Belimov et al. found that bacteria containing ACC deaminase improve plant growth in metal-polluted conditions [48]. Bacteria as well as plants can produce the auxin indole-3acetic acid (IAA). Rajkumar and Freitas [49] suggested that IAA indirectly promotes metal accumulation in plants by increasing plant biomass. Grandlic et al. found that 76% of plant growth promoting isolates from plants grown in mine tailings were able to produce IAA when supplemented with tryptophan, and these bacteria could promote growth of plants growing in mine tailings for phytostabilization [50].
Current Opinion in Biotechnology 2010, 21:339345

One idea is that bacteria promote plant growth, thus increasing surface area of the plant and allowing more metal uptake. Certain compounds produced by bacteria have been shown to promote plant growth, including siderophores. Siderophores, Fe-chelating compounds, have been shown to promote plant growth even in the
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Bacterial metal resistance/chelation/EPS

Metal resistance has been described as a necessity for plant-associated bacteria in contaminated environments [51]. Van der Lelie related plant metal uptake to bacterial metal resistance, since the bioavailability of metals could be altered by expression of bacterial metal resistance systems [25]. Faisal and Hasnain inoculated sunower plants with Cr resistant bacteria, and found that plant growth in Cr was improved by inoculation, although inoculated plants accumulated less Cr than uninoculated plants [52]. Kunito et al. examined rhizosphere bacteria from Phragmites grown in Cu. They found EPS production was greater for rhizosphere bacteria compared to nonrhizosphere bacteria. Because of Cu binding to bacterial EPS, rhizosphere soil may become less toxic to bacteria and also to plants [53].
Alteration of rhizosphere pH

Another possibility could be the lowering of pH in the rhizosphere by bacteria, which would make metals more soluble. The rhizosphere pH could be lowered by processes listed above, or other mechanisms, such as bacterial metal resistance systems. Bravin et al. pointed out that rhizosphere pH can also be raised by biological activity; in extremely low pH environments, roots alkalized the rhizosphere, making Cu less bioavailable [54]. Abou-Shanab et al. hypothesized that rhizosphere bacteria of Alyssum murale lowered rhizosphere pH, solubilizing metal for hyperaccumulation of Ni by the plant [55].
Phytoprotection

ant to and accumulated more Cd [56]. Che et al. engineered transgenic cottonwood trees (Populus deltoides) to express bacterial merB genes, and found that plants were more resistant to organic Hg compounds than wild-type plants [57]. Hussein et al. engineered transgenic tobacco plants through the chloroplast genome with both merA and merB genes, and saw fewer toxic effects of Hg and more Hg accumulation than in wild-type plants [58]. Plantmicrobe symbioses also have been exploited in transgenics. Wu et al. manufactured a synthetic phytochelatin analog that was expressed in Pseudomonas putida to increase Cd binding, and this engineered bacterium was then added to sunower roots to increase Cd accumulation and lessen Cd toxicity in plants [59]. While few studies so far have focused on engineering aquatic plants for decreased metal toxicity and increased metal removal, a recent study by Moontongchoon et al. focused on water spinach (Ipomoea aquatica). Expression of genes from sulfate assimilation pathways in these transgenes provided elevated Cd tolerance, and could be useful for remediation of metals in high sulfate environments [60].

Future directions
In the coming years, projects involving aquatic plant microbe interactions for the removal of heavy metal contaminants may become increasingly viable options, especially in shallow wetland and estuary environments. Use of constructed wetlands for ltration and remediation of water is currently a popular method, and understanding the nature of plantmicrobe interactions may improve this process. Developing new methods to support microbial activity to either enhance (for phytoextraction) or reduce (for phytostabilization) the bioavailability of metal contaminants in the rhizosphere could signicantly improve the efciency of these remediation techniques. Plants that hyperaccumulate metals have tremendous potential for application in remediation of metals in the environment. Signicant progress in phytoremediation has been made with metals and radionuclides. This process involves raising plants hydroponically and transplanting them into metal-polluted waters where they absorb and concentrate metals in their roots and shoots, and can be harvested for disposal. Root exudates lead to selective recruitment and accumulation of a diverse range of bacterial species associated with plants. Besides heavy metals, organic contaminants can also be removed by aquatic plants and their associated microbial communities. Unlike metals, which cannot be degraded and tend to be accumulated by plants, there is evidence that many organic contaminants are degraded in the rhizosphere by plant-associated bacteria and are transformed there rather than inside the plant. For instance, the duckweed S. polyrrhiza and its associated bacteria may degrade the aromatic compounds phenol, aniline, and 2,4dichlorophenol [61], and the reed Phragmites australis and
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As illustrated above, two possibilities may exist for plant microbe interactions in relation to metals. Bacterial mechanisms could lead to increased accumulation of metals in plants, or bacteria may keep metals from being accumulated by plants at high concentrations that are toxic to the plant. This may be the case for chelation or EPS production by bacteria [52]. Salt suggested that rhizobacteria promote precipitation of Cd at the root surface, causing plants to take up less metal [51]. While the studies of plantmicrobe interactions in the rhizosphere have been carried out in mainly terrestrial systems (although some wetland plants have been used), these principles could also apply to aquatic systems, where bacteria are still closely associated with plant roots.

Transgenic plants
The biotechnological potential of plantmicrobe relationships for use in phytoremediation has included research on transgenic plants. Much of this research has focused on terrestrial plants, and many engineered plant systems for phytoremediation have been used to degrade organic contaminants. However, several recent studies have focused on transgenic plants and metals. Parkash Dhankher et al. found that tobacco plants expressing the bacterial arsenate reductase gene, arsC, were more tolerCurrent Opinion in Biotechnology 2010, 21:339345

 Biotech potential of plantmicrobe interaction Stout and Nusslein 343

its associated rhizosphere bacteria might degrade bisphenol [62]. The marine eelgrass Zostera marina and its associated bacteria may degrade PAHs and PCBs [63]. Applications have recently been extended to commercial phytomining, the recovery of precious metals such as gold, silver, platinum, and palladium in mining [64]. The attractiveness of phytomining should increase if combined with other technologies such as biofuel production. Results reported in the literature indicate that plantmicrobial associations can signicantly increase metal uptake and accumulation. Many possibilities exist for the large-scale application of bacterialplant systems for removal of metals from aquatic environments. Large, shallow areas with low levels of contamination are ideal sites for these techniques, and use of transgenic plants and microbes may allow plants not only to survive, but also to accumulate metals in areas with higher levels of contaminants.

Such knowledge would increase the number of potentially widespread applications and their impact such as the treatment of heavy metals from industrial efuents in natural and constructed wetlands, or a wastewater metal stripping phase using rhizoltration. A directed functional analysis should investigate plant microbe interactions at full biological hierarchy, starting with the genomic, transcriptomic, and proteomic analysis of plant-associated bacteria [40] and their extracellular enzyme activities, all the way to biochemical processes and cycling that are active within the bacterially inuenced rhizosphere. With this understanding the plant microbe system could be implemented at eld-scale, using naturally adapted indigenous microbes that have been cultured and enriched in the laboratory. Such a multidisciplinary and integrated approach may gain aquatic metal phytoremediation the commercial signicance in environmental biotechnology it deserves.

References and recommended reading Conclusions

Aquatic phytoremediation of metals, the use of plants to extract, contain and immobilize, or remove hazardous substances from aqueous environments is a very promising area, and several highly efcient examples have shown the applicability of this process to clean industrial waste streams, to concentrate heavy metals, and to preserve drinking water and aquatic biodiversity. Limitations to this technology do exist and must also be considered. The application of rhizoltration is limited by metal availability, concentration, and phytotoxicity. Environmental factors like light, salinity, temperature, pH, and presence of multiple heavy metals may affect metal uptake. Further limitations of phytoremediation technology are seasonal growth of aquatic plants and contaminated biomass disposal issues. Phytoremediation focused on dissolved metals can be based on the application of both dead or live plant material or on the cultivation of aquatic plants. Not only aquatic macrophytes, but also algae and fungi, represent a cost-effective and eco-friendly technology for environmental cleanup, a green solution often preferred in political decision-making. Rhizosphere microbes can reduce metal toxicity and enhance plant tolerance to dissolved metals, and can therefore be applied to supply increased phytoprotection from harmful effects of the metals on plants. In a direct extension of this idea the bacterial genes coding for metal resistance can be transplanted into the plant genome to confer elevated metal tolerance to plants. Further research in aquatic phytoremediation is needed to advance understanding of microbeplant interactions.
www.sciencedirect.com Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1. Christensen PB, Revsbech NP, Sand-Jensen K: Microsensor analysis of oxygen in the rhizosphere of the aquatic macrophyte Littorella uniora (L.) Ascherson. Plant Physiol 1994, 105:847-852.

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Kraemer U: Phytoremediation: novel approaches to cleaning up polluted soils. Curr Opin Biotechnol 2005, 16:1-9. Interesting recent review discussing phytoextraction and transgenic plants, and modern genetic approaches to phytoremediation. Rai PK: Heavy metal phytoremediation from aquatic ecosystems with special reference to aquatic macrophytes. Crit Rev Env Sci Technol 2009, 39:697-753. Detailed review of phytoremediation focusing on heavy metals and aquatic plants. Highlights advantages of phytoremediation for use in aquatic systems over conventional technologies and several hypotheses as to why plants hyperaccumulate metals. 4. Flathman PE, Lanza GR: Phytoremediation: current views on an emerging green technology. J Soil Contam 1998, 7:415-432.

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Prasad MNV, Freitas HMD: Metal hyperaccumulation in plants biodiversity prospecting for phytoremediation technology. Electron J Biotechnol 2003, 6:285-321. Review of phytoremediation technologies that includes list of known bacteria, algae, lichens, fungi, and plants that show resistance to metals and have potential applications for metal removal from the environment.

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Jabeen R, Ahmad A, Iqbal M: Phytoremediation of heavy metals: physiological and molecular mechanisms. Bot Rev 2009, 75:339-364. Detailed review on phytoremediation and mechanisms used by plants to accumulate and tolerate heavy metals including bioavailability, translocation, exclusion, compartmentalization, plant metallothioneins, and phytochelatins, and discussion of how to improve phytoremediation through transgenic plants and addition of chelators. Murakami M, Nakagawa F, Ae N, Ito M, Arao T: Phytoextraction by rice capable of accumulating Cd at high levels: reduction of Cd content of rice grain. Environ Sci Technol 2009, 43:5878-5883. This paper shows that rice plants, adapted to aquatic/wetland environments, outperform the terrestrial hyperaccumulator Thlaspi caerulescens, which is not well-adapted to this environment, in metal accumulation. Current Opinion in Biotechnology 2010, 21:339345

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Lee M, Yang M: Rhizoltration using sunower (Helianthus annuus L.) and bean (Phaseolus vulgaris L. var. vulgaris) to remediate uranium contaminated groundwater. J Hazard Mater 2010, 173:589-596. Agunbiade FO, Olu-Owolabi BI, Adebowale KO: Phytoremediation potential of Eichornia crassipes in metal-contaminated coastal water. Bioresour Technol 2009, 100:4521-4526.

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10. Mishra VK, Tripathi BD: Accumulation of chromium and zinc  from aqueous solutions using water hyacinth (Eichhornia crassipes). J Hazard Mater 2009, 164:1059-1063. This article shows that water hyacinth, Eichhornia crassipes, is very effective at removing chromium and zinc from aquatic systems. 11. Ghassemzadeh F, Yousefzadeh H, Arbab-Zavar MH: Removing  arsenic and antimony by Phragmites australis: rhizoltration technology. J Appl Sci 2008, 8:1668-1675. This paper shows the potential for arsenic and antimony removal by the aquatic invasive reed Phragmites australis, suggesting that these nuisance plants may have phytoremediation potential. 12. Rahman MA, Hasegawa H, Ueda K, Maki T, Okumura C, Rahman MM: Arsenic accumulation in duckweed (Spirodela polyrhiza L.): a good option for phytoremediation. Chemosphere 2007, 69:493-499. 13. Hou W, Chen X, Song G, Wang Q, Chi Chang C: Effects of copper  and cadmium on heavy metal polluted waterbody restoration by duckweed (Lemna minor). Plant Physiol Biochem 2007, 45:62-69. Points out that duckweed (Lemna minor) would be a suitable candidate for phytoremediation but also studies mechanisms of toxic effects of cadmium and copper on the plants. 14. Uysal Y, Taner F: Effect of pH, temperature, and lead concentration on the bioremoval of lead from water using Lemna minor. Int J Phytoremed 2009, 11:591-608. 15. Khellaf N, Zerdaoui M: Phytoaccumulation of zinc by the aquatic plant, Lemna gibba L.. Bioresour Technol 2009, 100:6137-6140. 16. Megateli S, Semsari S, Couderchet M: Toxicity and removal of heavy metals (cadmium, copper, and zinc) by Lemna gibba. Ecotoxicol Environ Saf 2009, 72:1774-1780. 17. Rai PK, Tripathi BD: Comparative assessment of Azolla pinnata  and Vallisneria spiralis in Hg removal from G.B. Pant Sagar of Singrauli industrial region, India. Environ Monit Assess 2009, 148:75-84. This work compares the mercury removal abilities of two aquatic plants; Azolla, a free-oating fern, and Vallisneria, a submerged macrophyte. While both were able to accumulate mercury, Azolla was able to remove more than Vallisneria. 18. Abhilash PC, Pandey VC, Srivastava P, Rakesh PS, Chandran S, Singh N, Thomas AP: Phytoltration of cadmium from water by Limnocharis ava (L.) Buchenau grown in free-oating culture system. J Hazard Mater 2009, 170:791-797. 19. Coombs JM, Barkay T: New ndings on evolution of metal homeostasis genes: evidence from comparative genome analysis of bacteria and archaea. Appl Environ Microbiol 2005, 71:7083-7091. 20. Gadd GM: Metals and microorganisms: a problem of denition. FEMS Microbiol Lett 1992, 79:197-203. 21. Nies DH: Efux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol Rev 2003, 781:1-27. 22. Huckle JW, Morby AP, Turner JS, Robinson NJ: Isolation of a prokaryotic metallothionein locus and analysis of transcriptional control by trace metal ions. Mol Microbiol 1993, 7:177-187. 23. Ybarra GR, Webb R: Effects of divalent metal cations and resistance mechanisms of the cyanobacterium Synechococcus sp. strain PCC 7942. J Hazard Subst Res 1999, 2:1-9. 24. Pages D, Rose J, Conrod S, Cuine S, Carrier P, Heulin T, Achouak W: Heavy metal tolerance in Stenotrophomonas maltophilia. PLoS One 2008, 3:e1539. Current Opinion in Biotechnology 2010, 21:339345

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hyperaccumulation is an adaptive process requiring continuous interactions among co-occurring organisms. 41. Loutseti S, Danielidis DB, Economou-Amilli A, Katsaros C,  Santas R, Santas P: The application of a micro-algal/bacterial biolter for the detoxication of copper and cadmium metal wastes. Bioresour Technol 2009, 100:2099-2105. This study is a good example how microbial biomass of dried microalgae and bacteria can be applied to abiotically detoxify aqueous metal wastewaters by sorption. Subsequent acidic desorption reached 100% efciency. 42. Munoz R, Alvarez MT, Munoz A, Terrazas E, Guieysse B, Mattiasson B: Sequential removal of heavy metals ions and organic pollutants using an algalbacterial consortium. Chemosphere 2006, 63:903-911. 43. Frey B, Zierold K, Brunner I: Extracellular complexation of Cd in the Hartig net and cytosolic Zn sequestration in the fungal mantle of Picea abiesHebeloma crustuliniforme ectomycorrhizas. Plant Cell Environ 2000, 23:1257-1265. 44. De Souza MP, Huang CPA, Chee N, Terry N: Rhizosphere  bacteria enhance the accumulation of selenium and mercury in wetland plants. Planta 1999, 209:259-263. One of the rst studies that demonstrated the role of rhizosphere bacteria in facilitating increased heavy metal accumulation in wetland plants. 45. Tripathi M, Munot HP, Shouche Y, Meyer JM, Goel R: Isolation and functional characterization of siderophore-producing lead- and cadmium-resistant Pseudomonas putida KNP9. Curr Microbiol 2005, 50:233-237. 46. Burd GI, Dixon DG, Glick BR: Plant growth-promoting bacteria that decrease heavy metal toxicity in plants. Can J Microbiol 2000, 46:237-245. 47. DellAmico E, Cavalca L, Andreoni V: Analysis of rhizobacterial communities in perennial Graminaceae from polluted water meadow soil, and screening of metal-resistant, potentially plant growth-promoting bacteria. FEMS Microbiol Ecol 2005, 52:153-162. 48. Belimov AA, Safronova VI, Sergeyeva TA, Egorova TN, Matveyeva VA, Tsyganov VE, Borisov AY, Tikhonovich IA, Kluge C, Preisfeld A et al.: Characterization of plant growth promoting rhizobacteria isolated from polluted soils and containing 1aminocyclopropane-1-carboxylate deaminase. Can J Microbiol 2001, 47:642-652. 49. Rajkumar M, Freitas H: Effects of inoculation of plant-growth promoting bacteria on Ni uptake by Indian mustard. Bioresour Technol 2008, 99:3491-3498. 50. Grandlic CJ, Mendez MO, Chorover J, Machado B, Maier RM: Plant growth-promoting bacteria for phytostabilization of mine tailings. Environ Sci Technol 2008, 42:2079-2084. 51. Salt DE, Benhamou N, Leszczyniecka M, Raskin I: A possible role for rhizobacteria in water treatment by plant roots. Int J Phytoremed 1999, 1:67-79. 52. Faisal M, Hasnain S: Chromate resistant Bacillus cereus augments sunower growth by reducing toxicity of Cr(VI). J Plant Biol 2005, 48:187-194. 53. Kunito T, Saeki K, Nagaoka K, Oyaizu H, Matsumoto S: Characterization of copper-resistant bacterial community in rhizosphere of highly copper-contaminated soil. Eur J Soil Biol 2001, 37:95-102.

54. Bravin MN, Tentscher P, Rose J, Hinsinger P: Rhizosphere pH gradient controls copper availability in a strongly acidic soil. Environ Sci Technol 2009, 43:5686-5691. 55. Abou-Shanab RA, Delorme TA, Angle JS, Chaney RL, Ghanem K, Moawad H, Ghozlan HA: Phenotypic characterization of microbes in the rhizosphere of Alyssum murale. Int J Phytoremed 2003, 5:367-379. 56. Dhankher OP, Shasti NA, Rosen BP, Fuhrmann M, Meagher RB: Increased cadmium tolerance and accumulation by plants expressing bacterial arsenate reductase. New Phytol 2003, 159:431-441. 57. Che D, Meagher RB, Heaton ACP, Lima A, Rugh CL, Merkle SA: Expression of mercuric ion reductase in Eastern cottonwood (Populus deltoides) confers mercuric ion resistance. Plant Biotechnol J 2003, 1:311-319. 58. Hussein HS, Ruiz ON, Terry N, Daniell H: Phytoremediation of mercury and organomercurials in chloroplast transgenic plants: enhanced root uptake, translocation to shoots, and volatilization. Environ Sci Technol 2007, 41:8439-8446. 59. Wu CH, Wood TK, Mulchandani A, Chen W: Engineering plant  microbe symbiosis for rhizoremediation of heavy metals. Appl Environ Microbiol 2006, 72:1129-1134. The idea for an effective cleanup technology is demonstrated by combining expression of a metal-binding peptide in a rhizobacterium with sunower roots. Both organisms had signicantly improved growth characteristics and cadmium tolerance, and the plant had 40% higher metal uptake. 60. Moontongchoon P, Chadchawan S, Leepipatpiboon N, Akaracharanya A, Shinmyo A, Sano H: Cadmium-tolerance of transgenic Ipomoea aquatica expressing serine acetyltransferase and cysteine synthase. Plant Biotechnol 2008, 25:201-203. 61. Toyama T, Yu N, Kumada H, Sei K, Ike M, Fujita M: Accelerated aromatic compounds degradation in aquatic environment by use of interaction between Spirodela polyrrhiza and bacteria in its rhizosphere. J Biosci Bioeng 2006, 101:346-353. 62. Toyama T, Sato Y, Inoue D, Sei K, Chang YC, Kikuchi S, Ike M:  Biodegradation of bisphenol A and bisphenol F in the rhizosphere sediment of Phragmites australis. J Biosci Bioeng 2009, 108:147-150. This recent study of plantmicrobe interactions in phytoremediation could successfully show that interactions between an aquatic macrophyte (P. australis) and specic root-associated bacteria accelerate the removal of bisphenols from sediment. Here the rhizosphere bacteria involved have been studied. This nding goes beyond previous studies by this group where mere rhizosphere oxidation, the ability of plants to release oxygen into the rhizosphere, was thought to be the driver for the biodegradation of organic compounds. 63. Huesemann MH, Hausmann TS, Fortman TJ, Thom RM, Cullinan V: In situ phytoremediation of PAH- and PCBcontaminated marine sediments with eelgrass (Zostera marina). Ecol Eng 2009, 35:1395-1404. 64. Sheoran V, Sheoran AS, Poonia P: Phytomining: a review. Miner  Eng 2009, 22:1007-1019. An excellent recent review of various aspects of phytomining along with the advantages, limitations, and future feasibility to employ plants (macrophytes and microcosms) to bioharvest desired metals from terrestrial environments, particularly those associated with sub-economic potential. Examples discussed in detail are Ni, Co, Ti, Au, and Ag.

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Current Opinion in Biotechnology 2010, 21:339345