The worldwide occurrence of sites contaminated with toxic metals and the associated high costs of remediating them using chemical and mechanical methods have led to calls to develop inexpensive and sustainable approaches based on the use of plants that naturally accumulate large amounts of metals in their tissues. The ability of plants to remediate metals has been rigorously studied and some species have been identified as excellent phytoremediators. However, the growth of phytoremediators is often retarded under high soil metal concentrations, rendering them ineffective. Meanwhile, some plants do not have remediating abilities but are capable of growing in contaminated environments with little or no sign of stress. Despite the volume of research dedicated to the screening and evaluation of phytoremediators, major questions remain about why some plants survive but do not remediate while the growth of phytoremediators is mostly hindered. The growth and metal-remediating efficiency of plants exposed to toxic concentrations of metals can be enhanced by inoculating phytoremediating plants with certain bacteria but the mechanisms behind this process remain unclear. Furthermore, the use of leguminous plants to improve the growth of a target plant under a mixed planting system has long been recognised as an effective yield-enhancing cropping system. However, the possibility of a non-remediating but tolerant leguminous plant conferring metal tolerance to a phytoremediator has not been explored. This thesis reports results from repeated glasshouse and lab-based growth experiments on the phytoremediating plant Brassica juncea exposed to 400 – 600 mg Zn kg-1. The aim was to investigate the abilities of two plant growth promoting bacteria (PGPB) species Pseudomonas brassicacearum and Rhizobium leguminosarum, and a leguminous plant Vicia sativa to promote B. juncea growth and enhance remediation of Zn-contaminated soil. B. juncea plant roots were analysed using synchrotron based micro-focus X-ray Fluorescence (μXRF) imaging and X-ray Absorption Near Edge Structure (μXANES) analysis to probe Zn speciation. P. brassicacearum exhibited the poorest plant growth promoting ability, while R. leguminosarum alone and in combination with P. brassicacearum significantly enhanced B. juncea growth and Zn bioaccumulation. X-ray Absorption Spectroscopy (XAS) analysis showed that reduced plant growth was due to root accumulation of Zn as Zn sulphate, Zn oxalate and Zn polygalacturonic acids. The better growth and increased metal accumulation observed in plants inoculated with R. leguminosarum and its combination with P. brassicacearum was attributed to root storage of Zn in the chelated forms of Zn phytate and Zn cysteine. A subcellular analysis of plant root also showed that the PGPB enhanced tolerance to Zn contamination by enhancing epidermal Zn compartmentalisation depending on the nature of root colonization, and induced changes in Zn speciation to less toxic Zn species in the epidermis and endodermis of plant root. The thesis therefore identifies enhanced Zn compartmentalization at the root epidermis and bacterial mediated changes in Zn toxicity through changes in Zn speciation as key complimentary mechanisms of plant growth promotion and enhanced Zn accumulation in plants by PGPB. Further experiments investigating alternative phytoremediation strategies showed that the use of the leguminous plant V. sativa in a mixed planting system with B. juncea plants completely out performed the effects of bacteria in promoting the growth and remediation potential of B. juncea under Zn contamination. By combining PGPB with mixed planting, B. juncea recovered full growth while also achieving maximum phytoremediation efficiency. The novel legume assistedmicrobial phytoremediation method that is reported in this thesis is the first to demonstrate complete plant growth recovery in plants exposed to 400 – 450 mg kg-1 soil Zn contamination for 5 weeks. Survival of V. sativa was attributed to its root storage of Zn in the chelated forms of Zn histidine and cysteine whereas in the roots of stunted B. juncea plants the majority of Zn was present as Zn oxalate and toxic Zn sulphate. Although the use of natural and synthetic chelates has been reported to enhance phytoremediation, this thesis recommends a legume-assisted-microbialphytoremediation system as a more sustainable method for Zn bioremediation.

Excessive metal deposition in soil is of major concern to the environment due to the toxicity of metals to animals and plants. Since metals do not degrade, reducing risk of exposure relies in either removing the metals from soil, or changing their speciation which leads to changes in bioavailability, mobility and toxicity. Plants have been shown to provide a cheap alternative to chemical methods for both removing and changing metal speciation, particularly when augmented with plant growth promoting bacteria. The focus of this thesis was to investigate whether the form (speciation) in which a metal contaminant is introduced to soil affects both plant health and the efficiency of metal remediation by the plant, using the well-known hyperaccumulator Brassica juncea (L.) Czern and zinc (Zn) as the metal contaminant. This study also examined the role of plant growth promoting bacteria in changing metal speciation, impact on metal toxicity and phytoremediation efficiency. Brassica juncea was grown in pots containing soil spiked with equal amounts (600 mg Zn kg-1) of soluble Zn (ZnSO4) and nanoparticulate ZnS and ZnO. Plant height, number of leaves, root length, plant biomass and chlorophyll content of Brassica juncea were used to assess Zn toxicity. Zn localisation and speciation in soil and plant tissues was studied using transmission electron microscopy (TEM), synchrotron micro-X-ray fluorescence elemental mapping (μXRF) and synchrotron X-ray absorption spectroscopy (XAS). Growth parameters showed that ZnSO4 was the most toxic form of Zn whilst ZnS and ZnO effects were not statistically different. These differences were linked to differences in Zn content in root and shoot biomass, which was higher in ZnSO4 treatments. Inoculation with Rhizobium leguminosarum and Pseudomonas brassicacearum enhanced plant growth, Zn concentration in plant biomass and translocation of Zn in all Zn treatments. XAS analysis showed that Zn speciation was altered in roots of plants inoculated with bacteria, with Zn cysteine as the most dominant form of Zn in all inoculated Zn treatments, suggesting a role for cysteine in ameliorating Zn toxicity. By also assessing Zn speciation changes across the soilrhizosphere- plant interface, this study established that Rhizobium leguminosarum modified Zn speciation at the rhizosphere. Through this thesis work, metal speciation is a major factor in determining the efficiency of metal phytoremediation and plant tolerance. Hence, this research provides useful information on Zn speciation which will contribute to effective implementation of Zn phytoremediation.

HudBay Minerals (formerly the Hudson Bay Mining and Smelting Co., Limited) has operated a Zn and Cu processing facility in Flin Flon, MB since the 1930’s. Located in the Boreal Shield, the area surrounding the mine complex has been severely impacted by both natural (forest fires) and the anthropogenic disturbance, which has adversely affected recovery of the local forest ecosystem. Zinc is one of the most prevalent smelter-derived metals in the soils and has been identified as a key factor limiting natural revegetation of the landscape. Because metal toxicity is related more to speciation than to total concentration, Zn speciation in soils from the impacted landscape was characterized using X-ray absorption fine structure, X-ray fluorescence mapping and µ-X-ray absorption near edge structure. Beginning with speciation at a micro-scale and transitioning to bulk speciation was able to determine Zn speciation and link it to two distinct landform characteristics: (1) soils stabilized by metal tolerant grass species—in which secondary adsorption species of Zn (i.e., sorbed to Mn and Si oxides, and as outer-sphere adsorbed Zn) were found to be more abundant; and (2) eroded, sparsely vegetated soils in mid to upper slope positions that were dominated almost entirely by smelter derived Zn minerals, specifically Franklinite (ZnFe2O4). The long-term effect of liming on pH and Zn speciation was examined using field sites limed by a community led organization over a ten year period. Upon liming to a pH of 4 to 4.5, the eroded, sparsely vegetated soils where found to form a Zn-Al-Hydroxy Interlayer Material (HIM) co-precipitate, reducing the phytotoxicity of both Zn and Al and allowed for boreal forest vegetation to recovery quickly in these areas. The grass stabilized soils experienced a steady pH increase, as compared to a sporadic pH increase in the heavily eroded soils, as the buffering capacity was overcome allowing for a transition between multiple adsorption species based upon the point of zero charge of reactive soil elements. Ultimately reaching a near neutral pH after ten years, this allowed for the formation of stable Zn-Al-layered double hydroxide (LDH) soil precipitates and significantly reduced concentrations of plant available Zn.

En Lorraine, l’Orne, un affluent de la Moselle, a été affecté par une activité minière et industrielle qui s’est intensifiée depuis le milieu du XIXe siècle et au cours du XXe siècle. Les barrages, créés pour les besoins en eau de l’industrie, ont favorisé l’accumulation de dépôts sédimentaires contaminés en métaux. En effaçant les barrages qui ont perdu leur fonctionnalité première, le cours d’eau devrait retrouver un fonctionnement hydrologique plus naturel, requis par la directive cadre européenne sur l’eau (DCE 2000/60/CE). Les travaux de recherche présentés ont mis en évidence les différents dépôts sédimentaires dans la partie d’aval de l’Orne. Des sédiments ont été prélevés en surface et carottés afin d’être précisément caractérisés d’un point de vue minéralogique et géochimique. Ces analyses ont permis de mettre en évidence le caractère fortement contaminé des dépôts sédimentaires en présents en amont des barrages. De plus, il a été possible, de distinguer les contributions industrielles et naturelles. Ces contributions industrielles mettant en évidence une forte contribution de boues sidérurgiques. L’étude de la minéralogie du fer et de la spéciation du zinc a mis en évidence des marqueurs minéralogiques qui devraient permettre de tracer les sédiments contaminés au sein de la colonne d’eau lors de leur remise en suspension. L’étude de la spéciation chimique du Zn a montré que cet élément était essentiellement stocké sous forme de sulfures. La prédominance de la taille nanométrique à sub-micrométrique de ces sulfures renforce leur probabilité de remobilisation lors d’opérations de réaménagement du cours d’eau ou lors d’évènements hydrologiques intenses (crues) / The Orne River is a tributary of the Moselle River, located northeastern France. During the last two centuries, the Orne watershed was highly industrialized. The introduction of wastes or by-products into the river is highly anticipated. Based on industrial needs, some small dams were built. However, the Directive 2000/60/EC of the European parliament strongly incite the removal of engineered structures (such as dams). This raises the question about the fate of contaminated sediment remobilization. The aims of this work are to identify the different sediment deposits along the Orne River. Surface sediments and sediment cores were collected along the Orne River. The sediments were then analyzed for water content, grain size distribution, pH, major and trace chemical composition using ICP-OES and ICP-MS, respectively, major crystalline minerals (XRD), micrometric (light microscope and SEM) and sub-micrometric (TEM) mineralogy, and Zn speciation at a molecular level (XANES). The chemical and mineralogical composition of the surface sediments revealed lithogenic as well as anthropogenic contributions. Interestingly, the sediment layers of the core collected upstream of the Beth dam showed fingerprints of the former steelmaking facilities. Those deposits were highly enriched in Fe, Zn and Pb, and were fingerprinted by crystalline iron minerals, and by newly formed Fe-aluminosilicates. TEM-EDXS and XANES at the Zn K-edge observations evidenced that Zn was mainly carried as sulfides, and to a lesser extent associated to Fe oxy-hydroxides and Fe-aluminosilicates. The remobilization of the contaminated sediments can then be traced by the unique mineralogical composition