15 The Role of Bacteria in the Phytoremediation of Heavy Metals D. van der Lelie, P. Corbisier, L. Diels, A. Gilis, C. Lodewyckx, M. Mergeay, S. Taghavi, N. Spelmans, and J. Vangronsveld CONTENTS Introduction Effects of Siderophore Production by Rhizosphere Bacteria on Plant Growth Interactions of Siderophores with Heavy Metals Metal-Resistant Soil Bacteria Bacterial Heavy Metal Resistance: The czc (Cadmium, Zinc, Cobalt) Operon Possible Effects of Heavy Metal-Resistant Soil Bacteria on Heavy Metal Speciation Applications of A. eutrophus CH34 Heavy Metal Resistances for the Removal of Heavy Metals Heavy Metal Biosensors Based on Bacterial Heavy Metal Resistance Genes Use of Biomet Sensors to Predict Heavy Metal Phytotoxicity Acknowledgment References INTRODUCTION The metabolic capacity of plant-associated bacteria may be used to develop new phytoremediation strategies. In the rhizosphere, many pesticides as well as trichloro- ethylene, polycyclic aromatic compounds, and petroleum hydrocarbons are degraded at accelerated rates (Hsu and Bartha, 1979; Nichols et al., 1997). Scientists are using plants for phytoextraction and phytoimmobilization of heavy metals from polluted soils and wastewater. Although plant-associated bacteria have dynamic and varied metabolic capacities, current strategies do not capitalize on the physiology of these microbes in phytoremediation processes. Plants stimulate the growth of microorgan- isms due to secretion of organic molecules by their roots. This results in higher population densities of bacteria in the rhizosphere (Anderson and Coats, 1995; Rovira et al., 1979). In addition, endophytic bacteria colonize the interior of root and stem tissues. In this chapter, the potential impact of soil bacteria on heavy metal Copyright © 2000 by Taylor & Francis extraction by plants is discussed. We have given special attention to heterologous expression of heavy metal resistance genes by plant associated baceria. In addition, the development of reporter gene systems to determine the level of available heavy metals is explored, this in relation to phytotoxicity. EFFECTS OF SIDEROPHORE PRODUCTION BY RHIZOSPHERE BACTERIA ON PLANT GROWTH Rhizosphere bacteria, including those which carry plasmid-borne resistances to heavy metals, may be expected to play an important role in the bioavailability of metals to the plant. They can produce metal-chelating agents, like siderophores, that possess a high affinity for Fe 3+ . In oxygenated environments such as the soil, iron is basically unavailable for uptake and limiting to microbial growth, even though it is the fourth most abundant element in the Earth’s crust (Lindsay, 1979). Therefore, aerobic and facultative anaerobic microorganisms have evolved various systems to overcome the low solubility of external iron (reviewed by Neilands, 1981; Crichton and Charloteaux-Wauters, 1987; Crosa, 1989; Guerinot, 1994). Siderophores produced by plant growth-promoting rhizobacteria (PGPR) and bacterial biological control agents are associated with improved plant growth, either through a direct effect on the plant, through control of noxious organisms in the soil, or via some other route (Kloepper et al., 1980). The PGPR include members of the genera Arthrobacter, Alcaligenes (Ralstonia), Serratia, Pseudomonas, Rhizo- bium, Agrobacterium, and Bacillus (O’Sullivan and O’Gara, 1992). The most prom- ising group for application in the biocontrol of plant diseases is that of the fluorescent pseudomonads, especially P. fluorescens and P. putida (O’Sullivan and O’Gara, 1992). Several mechanisms are known to account for the growth-promoting and disease-suppressing impact of fluorescent siderophores on the plant. One mechanism by which antagonists control disease is by competition for iron, mediated by fluo- rescent siderophores, which have an extremely high affinity for ferric iron. Produc- tion of siderophores by antagonists further limits the already limited supply of iron in the environment (Neilands, 1981; Neilands and Leong, 1986). Growth of patho- gens is therefore inhibited in the presence of siderophore-producing strains due to an inadequate supply of iron. Some plants utilize microbial ferric iron-siderophore complexes. Microbial hydroxamate type siderophores were used by plants, such as sunflower and sorghum, for the uptake of iron (Cline et al., 1982, 1983, 1984). Furthermore, siderophores from fluorescent pseudomonads have also been implicated in iron uptake by tomato (Duss et al., 1986), carnations, barley (Duijff et al., 1991), and in the reversion of lime-induced chlorosis by peanut (Jurkevitch et al., 1988). In contrast, the fluorescent siderophore from Pseudomonas sp. B10 inhibited iron uptake by peas and maize plants (Becker et al., 1985). Copyright © 2000 by Taylor & Francis INTERACTIONS OF SIDEROPHORES WITH HEAVY METALS Siderophore production can be stimulated by the presence of heavy metals. Since most siderophores also show a lower but significant affinity for bivalent heavy metal ions, they affect the bioavailability of the heavy metals. For instance, in Azotobacter vinelandii siderophore production is increased in the presence of zinc (Huyer and Page, 1988). Schizoken production in Bacillus megaterium is increased by exposure to copper, chromate, cadmium, zinc, and aluminium (Beyers et al., 1967; Hu and Boyer, 1996). Zinc, cadmium, nickel, and aluminium were found to increase sid- erophore production in P. aeruginosa strains (Gilis, 1993; Hassan, 1996). The same effect was found for zinc and aluminium in P. fluorescens ATCC17400 (Gilis, 1993). In the plant growth beneficial strain P. aeruginosa 7NSK2, zinc was found to stimulate pyoverdine production even in the presence of iron (Höfte et al., 1993). Cadmium was shown to have a similar effect on P. aeruginosa PAO and P. fluorescens strain 6.2 (Mergeay et al., 1978). Different explanations have been offered for the stimulating effect of metals other than iron on siderophore production. First, the metal may be directly involved in the siderophore biosynthesis pathway or its regulation. For example, in P. aeruginosa 7NSK2, a site-specific recombinase (sim- ilar to XerC of E. coli) encoded by a gene called sss (stress-induced siderophore synthesis) was found to be involved in the regulation of pyoverdine synthesis by zinc (Höfte et al., 1994). Alternatively, the free siderophore concentration in the medium may be reduced by complex formation with metal ions other than Fe(III). Because iron limitation stimulates siderophore production, more siderophores would be produced. Zinc- and cadmium-induced pyoverdine production in a family of heavy metal-resistant P. aeruginosa strains (Sss + phenotype; Hassan, 1996). These strains showed very high resistance levels for zinc and cadmium, with MIC values for these metals of 8.0 and 2.0 mM, respectively. Genes for resistance to zinc and cadmium are encoded by the czr operon and are induced by these metals. Thus, induction of siderophore biosynthesis genes may act in combination with metal resistance genes to overcome heavy metal toxicity. The siderophore alcaligin E produced by the soil bacterium Alcaligenes eutro- phus CH34 effects the bioavailability of cadmium (Gilis et al., 1996; 1997). The addition of exogenous alcaligin E overcomes the inhibition of growth of a sidero- phore minus mutant (Sid - ) by cadmium. The presence of 1mM cadmium was suffi- cient to inhibit growth of the Sid - strain AE1595, when no exogenous source of alcaligin E was added. In contrast, when 0.8 or 8 μm alcaligin E was added the bacteria grew at higher concentrations of cadmium, 4 or 8 mM, respectively (Figure 15.1). The same beneficial effect of alcaligin E is observed with cadmium under iron-limiting and -replete conditions, indicating that alcaligin E directly interacted with cadmium, thereby decreasing the availability of cadmium and consequently its toxic effects. Although cadmium-uptake studies showed that alcaligin E does not effect the cellular concentration of cadmium, other evidence suggests that alcaligin E alters the bioavailability of cadmium. A reporter gene system designated to determine the level of cadmium available biologically demonstrated that cadmium availability Copyright © 2000 by Taylor & Francis decreases in the presence of alcaligin E (Figure 15.2). Alcaligin E also shifted the toxicity of cadmium to higher concentrations, while altering the morphology of precipitated cadmium crystals observed by SEM. These data suggest that alcaligin E may provide protection against heavy metal toxicity as well as function as an iron transport vehicle for A. eutrophus. It is not clear what role siderophore production by rhizosphere bacteria have on plant growth in the presence of heavy metals. Siderophores might directly interact with the heavy metals, making them less bioavailable for the plants or may help plants to overcome metal-induced iron limitation. In addition, production of sidero- phores might protect plant roots from pathogens. Siderophore production might contribute to a competitive advantage resulting in dominance of pseudomonads in the rhizosphere. Importantly, the heavy metal resistance mechanisms of these bac- teria combined with post-efflux metal binding might decrease the bioavailable metal fraction and, consequently, the metal toxicity for the plants. METAL-RESISTANT SOIL BACTERIA In addition to other benefits that rhizosphere bacteria confer to their hosts, plant- associated bacteria may protect plants from heavy metal toxicity. A prerequisite in protecting plants from heavy metals is resistance of the bacteria to these metals. Metal-tolerant bacteria are isolated from many metal-rich biotopes, of either manmade or natural origins. The bacteria isolated from metal-rich biotopes of anthropogenic origin (mining and industrial sites) mainly belong to the genus Ral- stonia (Yabuuchi et al., 1995), of which the former species Burkholderia pickettii, FIGURE 15.1 Influence of alcaligin E on the growth of the alcaligin E-deficient strain AE1595 in the presence of cadmium in precipitating conditions, after 44 h of growth in Schatz lactate medium. Due to the high phosphate content in this medium, most cadmium will be present in the form of CdHPO 4 . (From Gilis, A., P. Corbisier, W. Baeyens, S. Taghavi, M. Mergeay, and D. van der Lelie, J. Ind. Microbiol. Biotechnol. 20, 61-68, 1998. With permission.) Copyright © 2000 by Taylor & Francis B. solanacearum (formerly Pseudomonas solanacearum), and A. eutrophus are members. Metal-tolerant A. eutrophus strains are a strongly related group that are well adapted to environments polluted by heavy metals and/or organic xenobiotics. Sim- ilar bacteria are isolated from desertified soils (Maatheide) and low-grade ore depos- its in Belgium and Zaïre (Diels and Mergeay, 1990). All these strains carry one or two megaplasmids which contain genes for multiple resistances to heavy metals. In addition, they exhibit similar resistance and substrate utilization patterns. The type- strain of this family is A. eutrophus strain CH34 (Mergeay et al., 1985). A similar strain, A. eutrophus strain KT02, was isolated from the wastewater treatment plant of Göttingen, and carries three plasmids (Schmidt et al., 1991). A. eutrophus strain 31A was isolated from the metal-working industry in Holzminden. It is an organ- otrophic bacterium highly resistant to nickel and carries two plasmids (Schmidt and Schlegel, 1994). A. denitrificans strain 4a-2 was isolated from the wastewater treat- ment plant of Dransfeld (Kaur et al., 1990). It is highly resistant to nickel and carries the heavy metal resistance determinants on the chromosome (Stoppel et al., 1995; Stoppel and Schlegel, 1995). The bacteria isolated from natural metal-rich biotopes belonged to various gen- era. Bacteria resistant to nickel were isolated from soils of two kinds of ecosystems, both naturally laden with nickel. Soils from both ecosystems developed from nick- eliferous rocks. One ecosystem is characterized by serpentine or ultramafic soils which originate from weathering of serpentine rocks frequently enriched with nickel, FIGURE 15.2 Bioluminescence profile of the ale-1595 (Sid - ) cadmium, zinc, and lead bio- sensor (AE2350) in the presence of increasing concentrations of cadmium in precipitating conditions, with and without addition of alcaligin E-containing supernatant. ( • ) without alcaligin E, ( Ⅺ) with approximately 1.6 μm alcaligin E-containing supernatant, and ( * ) with approximately 8 μm alcaligin E-containing supernatant. The light production, expressed in RLU (relative light units) was measured after 4 h and related to the OD 660 of the culture. (From Gilis, A., P. Corbisier, W. Baeyens, S. Taghavi, M. Mergeay, and D. van der Lelie, J. Ind. Microbiol. Biotechnol. 20, 61-68, 1998. With permission.) Copyright © 2000 by Taylor & Francis chromium, and cobalt. They are inhabited with endemic plant species (herbs) resis- tant to nickel. Nickel-resistant bacteria were isolated from such soils collected in Scotland and California. The other ecosystem in New Caledonia is also characterized by rocks rich in iron and manganese and enriched with nickel, chromium, and cobalt. The soils originating from such rocks are inhabited by many nickel-hyperaccumu- lation plants, among them many trees and shrubs. The most outstanding example is the tree Sebertia acuminata. It contains a blue-green milk with 25% nickel (weight/dry weight) and leaves with 1 to 2% nickel. The nickel is transiently stored in the vacuoles and released into the soil during decay of the leaves. The soil contains about 10 mg nickel per gram soil. The soil is healthy with respect to humus content and structure and contains nickel-resistant bacteria of all physiological groups belonging to the indigenous bacteria of a good humus soil. Most bacteria isolated from this soil are able to grow on nickel concentrations up to 20 mM (Stoppel and Schlegel, 1995). Members of the genus Burkholderia make up the majority of nickel- resistant strains collected. Hafnia alvei, Pseudomonas mendocina, Acinetobacter, Comamonas acidovorans, and Agrobacterium tumefaciens are other nickel-resistant strains collected. All the strains collected among New Caledonian soil samples are physiologically different from the European strains. Gram-positive bacteria were isolated from a soil sample (serpentine) in California. They were identified as Arthrobacter ramosus strain 60-6 and Arthrobacter aurescens strain 59-6 (Stoppel and Schlegel, 1995). BACTERIAL HEAVY METAL RESISTANCE: THE czc (CADMIUM, ZINC, COBALT) OPERON Among the heterotrophic bacteria, members of the β-Proteobacteria have the highest levels of resistance to heavy metals. A. eutrophus is a member of this group. The type strain A. eutrophus CH34 was originally isolated from a decantation tank of a zinc factory; the genetics, physiology, and biochemistry of this organism are the best studied. Strain CH34 harbors two endogenous megaplasmids encoding multiple heavy metal resistance genes. Plasmid pMOL28 is 180 kb and codes for resistance to cobalt, nickel, chromate, mercury, and thallium. Resistance genes are organized into operons with the chr and mer operons, coding for resistances to chromate and mercury, respectively. The mer operon coding for mercury resistance is derived from Tn4378. Resistance genes for both cobalt and nickel are present in the cnr operon (Mergeay et al., 1985; Taghavi et al., 1997). In addition to these operons, the tllA locus is involved in thallium resistance (Collard et al., 1994). The second plasmid from strain CH34, pMOL30, is 240 kb and is responsible for resistance to some of the same heavy metals for which plasmid pMOL28 has resistance genes. On this plasmid, resistance genes are also organized into operons. The mer (this time from Tn4380), cop, and pbr operons encode resistance to mercury, copper, and lead. The czc operon encodes for cadmium, zinc, and cobalt resistance. Again, a single locus, tllB, is involved in thallium resistance (Mergeay et al., 1985; Collard et al., 1994). Copyright © 2000 by Taylor & Francis These resistance genes have been used as probes to detect of a large number of related strains with resistance to heavy metals in mining areas or industrial sites in Congo and Belgium (Diels and Mergeay, 1990). The czc operon of A. eutrophus CH34 is the most completely studied heavy metal resistance operon of A. eutrophus. The CzcABC structural resistance proteins form an efflux pump that functions as a chemiosmotic cation/proton antiporter (Figure 15.3) (Nies, 1995; Nies and Silver, 1995). The proteins involved have become the prototype for a new family of three-component chemiosmotic exporters, includ- ing members that efflux toxic cations or organic compounds (Diels et al., 1995a; Dong and Mergeay, 1994). CzcA, the central component of the system, functions as an inner membrane transport protein. CzcA is a chemiosmotic cation/proton antiporter belonging to the RND (resistance/nodulation/division) family (Nies and Silver, 1995; Nies, 1995). Such exporters, which are not driven by ATP and are thus different from traffic FIGURE 15.3 Schematic presentation of the Czc efflux system and a working model for heavy metal uptake, processing by efflux, and post-efflux metal fixation on polysaccharides and proteins. (From Taghavi, S., M. Mergeay, D. Nies, and D. van der Lelie, Res. Microbiol. 148, 536-551, 1997. With permission.) Copyright © 2000 by Taylor & Francis ATPases, transport various metabolites, antibiotics, or drugs to the extracellular space (Saier et al., 1994). CzcC is thought to function as an outer membrane protein. The N-terminal part of CzcC is typical of a signal for peptide secretion (Diels et al., 1995a). Additionally, there are sequence homologies and topological similarities between CzcC and other accessory proteins of ABC exporter systems. High homology is seen with a family of outer membrane factors (OMF; Diels et al., 1995a; Dong and Mergeay, 1994). CzcC is required in the process to complete the efflux of cadmium to the extracellular medium. Transport data (Nies and Silver, 1989) suggest that the export of the very toxic cadmium to the extracellular medium requires the participation of CzcC, which is dispensable for the efflux of zinc. Since zinc is an essential metal, it is conceivable that a chromosomal OMF involved in the homeostasis of zinc can act to replace CzcC for the efflux of zinc. CzcB appears to function as a membrane fusion protein (MFP), that bridges the inner and outer cell membrane, probably facilitating the export of ions across both membranes without release in the periplasm (Diels et al., 1995a). It might also function in providing specificity for heavy metals (Nies et al., 1989). In addition, CzcB displays some homology with calphotin, a metal “mobilizing” protein that removes calcium cations from the cytoplasm (Ballinger et al., 1993). The transcriptional regulation of the czc operon is currently being studied. The czc operon is inducible by zinc, cadmium, and to a lesser extent by cobalt and is controlled by two regulatory loci that are located both upstream and downstream of czc (van der Lelie et al., 1997). The cnr and ncc operons are similar to czc. The cnr operon of the A. eutrophus CH34 plasmid pMOL28 ensures the inducible efflux of cobalt and nickel (Varma et al., 1990; Sensfuss and Schlegel, 1988). It is the most thoroughly studied nickel resistance determinant (Siddiqui et al., 1989; Liesegang et al., 1993; Collard et al., 1993). Sequencing analysis revealed that cnr consists of 6 ORFs, cnrYXHCBA (Liesegang et al., 1993; Stoppel and Schlegel, 1995). The cnrCBA structural genes are arranged in the same order and determine proteins of similar molecular weights as czcCBA (Liesegang et al., 1993). Although the structural cnr and czc genes are very similar, the regulation of both operons is completely different. Upstream of the cnrCBA genes, three loci, cnrY, cnrX, and cnrH, grouped in an operon-like structure, were found to be required for regulation of cnr (Liesegang et al., 1993). The cnrH gene product seems to be a member of a novel sigma factor group, belonging to the extracytoplasmic (ECF) subfamily of sigma 70 (Liesegang et al., 1993). Some regulators involved in siderophore biosynthesis are also belonging to this subfamily. The CnrY protein has been suggested to function as an (auto)repres- sor: inactivation of cnrY led to highly increased constitutive cobalt and nickel resistance and resulted in zinc resistance (ZinB phenotype; Collard et al., 1993; Liesegang et al., 1993). This observation led to the conclusion that the cnr system may also be involved in zinc efflux in the wildtype strain but at levels too low to confer a detectable resistance phenotype. This hypothesis was confirmed by zinc Copyright © 2000 by Taylor & Francis efflux studies after induction of the cnr operon by nickel. These data show that the stuctural genes of the cnr and czc systems are fundamentally the same. The role of the cnrX locus is not clear. However, recent data suggest that the CnrX protein is secreted into the periplasm where it might function as a metal sensor (C. Tibazarwa, personal communication). The ncc operon of A. eutrophus 31A is very similar to the cnr operon (Schmidt and Schlegel, 1994). In addition to the six genes, nccYX- HCBA, whose homologs were also present in the cnr operon, a seventh gene nccN was identified. The expression of nccN is required for full nickel resistance: when nccN is deleted, the nickel resistance drops from 30 to 5 mM, the same resistance level as found with the cnr operon. Downstream of ncc, a second nickel resistance determinant, nre, was identified. Similar three component efflux systems were also identified in E. coli where they encode silver resistance (sil operon; S. Silver) and copper resistance (cur operon; T. V. O’Halloran). Also, a three-component efflux system similar to czc that con- ferred resistance to Cd and Zn was identified and cloned form P. aeruginosa CMG103 (Hassan, 1996). The three structural genes, czrCBA, were very similar to czcCBA. However, czr regulation seems to be under control of a classical two- component regulatory system. Despite the similarity in structural genes, the heterologous expression patterns of ncc-nre, czr, and czc are quite different. This might be a consequence of the differences between the regulatory loci associated with the structural resistance genes. Many of these genes have been expressed in heterologous organisms, however. The ncc-nre determinant could be expressed in E. coli, Sphingobacterium heparium, Rhodobacter sphaeroides, Thiobacillus versutus (Q. Dong, personal communica- tion), and the plant associated bacteria Pseudomonas putida, Pseudomonas stutzeri, Burkholderia cepacia, and Herbaspirillum sp. (S. Taghavi and C. Lodewyckx, per- sonal communication). The ncc-nre determinant was also introduced on a broad host-range plasmid into an activated sludge system. Natural transfer of this plasmid into the activated sludge system resulted in a rapid adaptation of the system to treat nickel-containing wastewater (Q. Dong, personal communication). Analysis of the nickel-resistant strains that were isolated after gene transfer indicated that the ncc - nre determinant could be expressed in a range of Gram - and Gram + species. The czr operon of P. aeruginosa could be expressed in A. eutrophus (Hassan, 1996), Herbaspirillum sp., and in some P. fluorescens strains (S. Taghavi and C. Lodewyckx, personal communication). The czc operon has never been expressed outside A. eutrophus. The broad range of organisms which express the ncc-nre determinant opens the possibility to increase the nickel (and cadmium and cobalt) resistance of plant- associated bacteria, while the czr determinant could be used to increase resistance to cadmium and zinc of plant-associated Pseudomonas sp. The presence of heavy- metal-resistant, plant-associated bacteria might have beneficial effects on plant growth in the presence of heavy metals. The speciation of the heavy metals and, consequently, their bioavailability for plants might alter due to the activity of bacteria expressing heavy metal resistance. Copyright © 2000 by Taylor & Francis POSSIBLE EFFECTS OF HEAVY METAL-RESISTANT SOIL BACTERIA ON HEAVY METAL SPECIATION A. eutrophus effects the concentration of heavy metal ions present in culture and may alter the species present in the environment. The presence of A. eutrophus-like bacteria in soils contaminated with heavy metals might have long-term effects on the speciation of the heavy metals and consequently their bioavailability and toxicity for plants. In cultures of A. eutrophus CH34, grown in the presence of high concentrations of cadmium (2 mM) or zinc (5 mM), metal concentrations decreased drastically (up to 99%) in the late log phase. This effect was always accompanied by a progressive pH increase (up to 9), and precipitation and sequestration of metals (Diels, 1990). The alkalization is thought to be a consequence of the proton influx during the czc- mediated proton antiporter efflux of cations (Diels et al., 1995a). The mechanisms of the bioprecipitation and biological sequestration are still poorly understood. X- ray diffraction spectroscopy of the precipitated material showed that carbonates precipitate with cadmium ions to form Cd(HCO 3 ) 2 and CdCO 3 (Diels et al., 1995a). The precipitation of cadmium seems to occur at defined nucleation foci around the cell surface as shown by transmission electron spectroscopy (Diels et al., 1995a). It is thought that extracellular polysaccharides (which are able to bind high amounts of metals and whose concentration has been shown to increase as a consequence of cadmium resistance) and outer membrane proteins are important post-efflux func- tions, which avoid re-entry of metal ions in the cell (Diels et al., 1995a). APPLICATIONS OF A. eutrophus CH34 HEAVY METAL RESISTANCES FOR THE REMOVAL OF HEAVY METALS The observation of bioprecipitation as a physiological consequence of plasmid- mediated efflux of cations led to the possible application of metal sequestration/bio- precipitation as a tool to remove heavy metals from polluted effluents. A special reactor was developed involving immobilization of cells, a crystal collection system for collecting the heavy metal precipitates, and a nutrient system to keep bacteria alive. This resulted in the design of a novel reactor named BICMER (Bacteria Immobilized Composite MEmbrane Reactor; Diels et al., 1993a; Diels et al., 1995b). Once operational, this reactor provides a final residual concentration of zinc or cadmium (output) lower than 1 ppm. A simple modification of the growth medium allowed the efficient sequestration of cobalt, nickel, and copper ions, only after induction by cadmium or zinc (Diels et al., 1993a,b, 1995b). Treatment of soils contaminated by heavy metals is another potential application for metal-resistant A. eutrophus CH34 and related bacteria. In a BMSR (Bacteria Metal Sludge Reactor), polluted sandy soils and bacteria were mixed together, and the fate of the heavy metals was studied. The bacterial treatment modified the colloidal behavior of sandy soil suspensions in such a way that soil particles were sedimenting much faster than soils without bacterial treatment. This allowed the easy separation of the bacterial suspension from the soil particle fraction (Diels, 1997). In this way, 30 to 70% of the metals from these polluted soils could be Copyright © 2000 by Taylor & Francis [...]... Comparison of the abilities of hydroxamate, synthetic and other organic acids to chelate iron and other ions in nutrient solution Soil Sci Soc Am J 46, 115 8-1 164, 1982 Cline, G.R., P.E Powell, P.J Szaniszlo, and C.P.P Reid, Comparison of the abilities of hydroxamate and other organic acids to chelate iron and other ions in soil Soil Sci 136, 14 5-1 57, 1983 Cline, G.R., C.P.P Reid, P.E Powell, and P.J Szaniszlo,... 90, 153 6-1 540, 1993 Becker, J.O., R.W Hedges, and E Messens, Inhibitory effects of pseudobactin on the uptake of iron by higher plants Appl Environ Microbiol 54, 109 0-1 093, 1985 Beyers, B.R., M.V Powell, and C.E Lankfort, Iron-chelating hydroxamic acid (schizoken) active in initiation of cell division in Bacillus megaterium J Bacteriol 93, 28 6-2 94, 1967 Cline, G.R., P.E Powell, P.J Szaniszlo, and C.P.P... as mUnits/g dry-weight) and the fraction of zinc, as was determined using the Biomet Copyright © 2000 by Taylor & Francis ACKNOWLEDGMENT This work was supported by the European Commission as part of the Environment and Climate program projects ENV4-CT9 5-0 141, Biomet Sensors, and ENV4CT9 5-0 083, PHYTOREHAB Part of the work was carried out as collaboration between VITO and LUC in the frame of an EFRO/OVAM... assessment of solid wastes Environ Toxicol Water Qual 11, 17 1-1 77, 1996 Corbisier, P., E Thiry, A Masolijn, and L Diels, Construction and development of metal ion biosensors, in Bioluminescence and Chemoluminescence: Fundamentals and Applied Aspects, Campbell, A.K., Cricka, L.J., and Stanley, P.E., Eds., John Wiley & Sons, Chichester, 1994 Crichton, R.R and M Charloteaux-Wauters, Iron transport and storage... nutrition of plants, in Abstr 6th Int Fe Symp., 31, 1991 Duss, F., A Moazfar, J.J Oertli, and W Jaeggi, Effect of bacteria on the iron uptake by axenically-cultured roots of Fe-efficient and Fe-inefficient tomatoes (Lycopersicon esculentum Mill.) J Plant Nutr 9, 58 7-5 98, 1986 Gilis, A., Interactie tussen verschillende potentieel toxische metalen (Zn, Cd, Ni en Al) en siderofoor-afhankelijke ijzer-opname... Huyer, M and W Page, Zn 2+ increases siderophore production in Azotobacter vinelandii Appl Environ Microbiol 54, 262 5-2 631, 1988 Jurkevitch, E., Y Hadar, and Y Chen, Involvement of bacterial siderophores in the remedy of lime-induced chlorosis in peanut Soil Sci Soc Am J 52, 103 2-1 037, 1988 Kaur, P., K Ross, R.A Siddiqui, and H.G Schlegel, Nickel resistance of Alcaligenes denitrificans strain 4a-2 is... 48 5-5 06, 1987 Crosa, J.H., Genetics and molecular biology of siderophore-mediated iron-transport in bacteria Microbiol Rev 53, 51 7-5 30, 1989 Copyright © 2000 by Taylor & Francis Diels, L., Accumulation and precipitation of Cd and Zn ions by Alcaligenes eutrophus strains, in Biohydrometallurgy 89, Proc Int Symp Jackson Hole, Wyoming,1990, Salby, J., McCready, R.G.I., and Wichlacz, P.Z., Eds., 36 9-3 77,... 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Part of the work was carried out as collaboration between VITO and LUC