Dissertations / Theses on the topic 'Nickel hyperaccumulator plants'

Soil contamination with heavy metal(loid)s is a major environmental problem that requires effective and affordable remediation technologies. The utilisation of plants to remediate heavy metal(loid)s contaminated soils has attracted considerable interest as a low cost green remediation technology. The process is referred to as phytoremediation, and this versatile technology utilises plants to phytostabilise and/or phytoextract heavy metal(loid)s from contaminated soils, thereby effectively minimising their threat to ecosystem, human and animal health. Plants that can accumulate exceptionally high concentrations of heavy metal(loid)s into above-ground biomass are referred to as hyperaccumulators, and may be exploited in phytoremediation, geobotanical prospecting and/or phytomining of low-grade ore bodies. Despite the apparent tangible benefits of utilising phytoremediation techniques, a greater understanding is required to comprehend the ecophysiological aspects of species suitable for phytoremediation purposes. A screening study was instigated to assess phytoremediation potential of several fern species for soils contaminated with cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni), lead (Pb) and zinc (Zn). Hyperaccumulation was not observed in any of the studied species, and in general, species excluded heavy metal uptake by restricting their translocation into aboveground biomass. Nephrolepis cordifolia and Hypolepis muelleri were identified as possible candidates in phytostabilisation of Cu-, Pb-, Ni- or Zn-contaminated soils and Dennstaedtia davallioides appeared favourable for use in phytostabilisation of Cu- and Zn-contaminated soils. Conversely, Blechnum nudum, B. cartilagineum, Doodia aspera and Calochlaena dubia were least tolerant to most heavy metals and were classified as being least suitable for phytoremediation purposes Ensuing studies addressed the physiology of arsenic (As) hyperaccumulation in a lesser known hyperaccumulator, Pityrogramma calomelanos var. austroamericana. The phytoremediation potential of this species was compared with that of the well known As hyperaccumulator Pteris vittata. Arsenic concentration of 3,008 mg kg–1 dry weight (DW) occurred in P. calomelanos var. austroamericana fronds when exposed to 50 mg kg–1 As without visual symptoms of phytotoxicities. Conversely, P. vittata was able to hyperaccumulate 10,753 mg As kg–1 DW when exposed to 100 mg kg–1 As without the onset of phytotoxicities. In P. calomelanos var. austroamericana, As was readily translocated to fronds with concentrations 75 times greater in fronds than in roots. This species has the potential for use in phytoremediation of soils with As levels up to 50 mg kg–1. Localisation and spatial distribution of As in P. calomelanos var. austroamericana pinnule and stipe tissues was investigated using micro-proton induced X-ray emission spectrometry (µ-PIXE). Freeze-drying and freeze-substitution protocols (using tetrahydrofuran [THF] as a freeze-substitution medium) were compared to ascertain their usefulness in tissue preservation. Micro-PIXE results indicated that pinnule sections prepared by freeze-drying adequately preserved the spatial elemental distribution and tissue structure of pinnule samples. In pinnules, µ-PIXE results indicated higher As concentration than in stipe tissues, with concentrations of 3,700 and 1,600 mg As kg–1 DW, respectively. In pinnules, a clear pattern of cellular localisation was not resolved whereas vascular bundles in stipe tissues contained the highest As concentration (2,000 mg As kg–1 DW). Building on these µ-PIXE results, the chemical speciation of As in P. calomelanos var. austroamericana was determined using micro-focused X-ray fluorescence (µ-XRF) spectroscopy in conjunction with micro-focused X-ray absorption near edge structure (µ-XANES) spectroscopy. The results suggested that arsenate (AsV) absorbed by roots was reduced to arsenite (AsIII) in roots prior to transport through vascular tissues as AsV and AsIII. In pinnules, AsIII was the predominant species, presumably as aqueous-oxygen coordinated compounds. Linear least-squares combination fits of µ-XANES spectra showed AsIII as the predominant component in all tissues sampled. The results also revealed that sulphur containing thiolates may, in part sequester accumulated As. The final aspect of this thesis examined several ecophysiological strategies of Ni hyperaccumulation in Hybanthus floribundus subsp. floribundus, a native Australian perennial shrub species and promising candidate in phytoremediation of Ni-contaminated soils. Micro-PIXE analysis revealed that cellular structure in leaf tissues prepared by freeze-drying was adequately preserved as compared to THF freeze-substituted tissues. Elemental distribution maps of leaves showed that Ni was preferentially localised in the adaxial epidermal tissues and leaf margin, with concentration of 10,000 kg–1 DW in both regions. Nickel concentrations in stem tissues obtained by µ-PIXE analysis were lower than in the leaf tissues (1,800 mg kg–1 vs. 7,800 mg kg–1 DW, respectively), and there was no clear pattern of compartmentalisation across different anatomical regions. It is possible that storage of accumulated Ni in epidermal tissues may provide Ni tolerance to this species, and may further act as a deterrent against herbivory and pathogenic attack. In H. floribundus subsp. floribundus seeds, µ-PIXE analysis did not resolve a clear pattern of Ni compartmentalisation and suggests that Ni was able to move apoplastically within the seed tissues. The role of organic acids and free amino acids (low molecular weight ligands [LMW]) in Ni detoxification in H. floribundus subsp. floribundus were quantified using high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC). Nickel accumulation stimulated a significant increase in citric acid concentration in leaf extracts, and based on the molar ratios of Ni to citric acid (1.3:1–1.7:1), citric acid was sufficient to account for approximately 50% of the accumulated Ni. Glutamine, alanine and aspartic acid concentrations were also stimulated in response to Ni hyperaccumulation and accounted for up to 75% of the total free amino acid concentration in leaf extracts. Together, these LMW ligands may complex with accumulated Ni and contribute to its detoxification and storage in this hyperaccumulator species. Lastly, the hypothesis that hyperaccumulation of Ni in certain plants may act as an osmoticum under water stress (drought) was tested in context of H. floribundus subsp. floribundus. A 38% decline in water potential and a 68% decline in osmotic potential occurred between water stressed and unstressed plants, however, this was not matched by an increase in accumulated Ni. The results suggested that Ni was unlikely to play a role in osmotic adjustment in this species. Drought stressed plants exhibited a low water use efficiency which might be a conservative ecophysiological strategy enabling survival of this species in competitive water-limited environments.

Doctor of Philosophy(PhD)
Soil contamination with heavy metal(loid)s is a major environmental problem that requires effective and affordable remediation technologies. The utilisation of plants to remediate heavy metal(loid)s contaminated soils has attracted considerable interest as a low cost green remediation technology. The process is referred to as phytoremediation, and this versatile technology utilises plants to phytostabilise and/or phytoextract heavy metal(loid)s from contaminated soils, thereby effectively minimising their threat to ecosystem, human and animal health. Plants that can accumulate exceptionally high concentrations of heavy metal(loid)s into above-ground biomass are referred to as hyperaccumulators, and may be exploited in phytoremediation, geobotanical prospecting and/or phytomining of low-grade ore bodies. Despite the apparent tangible benefits of utilising phytoremediation techniques, a greater understanding is required to comprehend the ecophysiological aspects of species suitable for phytoremediation purposes. A screening study was instigated to assess phytoremediation potential of several fern species for soils contaminated with cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni), lead (Pb) and zinc (Zn). Hyperaccumulation was not observed in any of the studied species, and in general, species excluded heavy metal uptake by restricting their translocation into aboveground biomass. Nephrolepis cordifolia and Hypolepis muelleri were identified as possible candidates in phytostabilisation of Cu-, Pb-, Ni- or Zn-contaminated soils and Dennstaedtia davallioides appeared favourable for use in phytostabilisation of Cu- and Zn-contaminated soils. Conversely, Blechnum nudum, B. cartilagineum, Doodia aspera and Calochlaena dubia were least tolerant to most heavy metals and were classified as being least suitable for phytoremediation purposes Ensuing studies addressed the physiology of arsenic (As) hyperaccumulation in a lesser known hyperaccumulator, Pityrogramma calomelanos var. austroamericana. The phytoremediation potential of this species was compared with that of the well known As hyperaccumulator Pteris vittata. Arsenic concentration of 3,008 mg kg–1 dry weight (DW) occurred in P. calomelanos var. austroamericana fronds when exposed to 50 mg kg–1 As without visual symptoms of phytotoxicities. Conversely, P. vittata was able to hyperaccumulate 10,753 mg As kg–1 DW when exposed to 100 mg kg–1 As without the onset of phytotoxicities. In P. calomelanos var. austroamericana, As was readily translocated to fronds with concentrations 75 times greater in fronds than in roots. This species has the potential for use in phytoremediation of soils with As levels up to 50 mg kg–1. Localisation and spatial distribution of As in P. calomelanos var. austroamericana pinnule and stipe tissues was investigated using micro-proton induced X-ray emission spectrometry (µ-PIXE). Freeze-drying and freeze-substitution protocols (using tetrahydrofuran [THF] as a freeze-substitution medium) were compared to ascertain their usefulness in tissue preservation. Micro-PIXE results indicated that pinnule sections prepared by freeze-drying adequately preserved the spatial elemental distribution and tissue structure of pinnule samples. In pinnules, µ-PIXE results indicated higher As concentration than in stipe tissues, with concentrations of 3,700 and 1,600 mg As kg–1 DW, respectively. In pinnules, a clear pattern of cellular localisation was not resolved whereas vascular bundles in stipe tissues contained the highest As concentration (2,000 mg As kg–1 DW). Building on these µ-PIXE results, the chemical speciation of As in P. calomelanos var. austroamericana was determined using micro-focused X-ray fluorescence (µ-XRF) spectroscopy in conjunction with micro-focused X-ray absorption near edge structure (µ-XANES) spectroscopy. The results suggested that arsenate (AsV) absorbed by roots was reduced to arsenite (AsIII) in roots prior to transport through vascular tissues as AsV and AsIII. In pinnules, AsIII was the predominant species, presumably as aqueous-oxygen coordinated compounds. Linear least-squares combination fits of µ-XANES spectra showed AsIII as the predominant component in all tissues sampled. The results also revealed that sulphur containing thiolates may, in part sequester accumulated As. The final aspect of this thesis examined several ecophysiological strategies of Ni hyperaccumulation in Hybanthus floribundus subsp. floribundus, a native Australian perennial shrub species and promising candidate in phytoremediation of Ni-contaminated soils. Micro-PIXE analysis revealed that cellular structure in leaf tissues prepared by freeze-drying was adequately preserved as compared to THF freeze-substituted tissues. Elemental distribution maps of leaves showed that Ni was preferentially localised in the adaxial epidermal tissues and leaf margin, with concentration of 10,000 kg–1 DW in both regions. Nickel concentrations in stem tissues obtained by µ-PIXE analysis were lower than in the leaf tissues (1,800 mg kg–1 vs. 7,800 mg kg–1 DW, respectively), and there was no clear pattern of compartmentalisation across different anatomical regions. It is possible that storage of accumulated Ni in epidermal tissues may provide Ni tolerance to this species, and may further act as a deterrent against herbivory and pathogenic attack. In H. floribundus subsp. floribundus seeds, µ-PIXE analysis did not resolve a clear pattern of Ni compartmentalisation and suggests that Ni was able to move apoplastically within the seed tissues. The role of organic acids and free amino acids (low molecular weight ligands [LMW]) in Ni detoxification in H. floribundus subsp. floribundus were quantified using high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC). Nickel accumulation stimulated a significant increase in citric acid concentration in leaf extracts, and based on the molar ratios of Ni to citric acid (1.3:1–1.7:1), citric acid was sufficient to account for approximately 50% of the accumulated Ni. Glutamine, alanine and aspartic acid concentrations were also stimulated in response to Ni hyperaccumulation and accounted for up to 75% of the total free amino acid concentration in leaf extracts. Together, these LMW ligands may complex with accumulated Ni and contribute to its detoxification and storage in this hyperaccumulator species. Lastly, the hypothesis that hyperaccumulation of Ni in certain plants may act as an osmoticum under water stress (drought) was tested in context of H. floribundus subsp. floribundus. A 38% decline in water potential and a 68% decline in osmotic potential occurred between water stressed and unstressed plants, however, this was not matched by an increase in accumulated Ni. The results suggested that Ni was unlikely to play a role in osmotic adjustment in this species. Drought stressed plants exhibited a low water use efficiency which might be a conservative ecophysiological strategy enabling survival of this species in competitive water-limited environments.

L’agromine est une filière destinée à valoriser des métaux dispersés dans des sols ou autres matrices, à l’aide de plantes hyperaccumulatrices (HA). La première étape consiste à cultiver ces plantes pour obtenir des rendements élevés en métaux et la seconde, à produire des composés métalliques d’intérêt à partir de la biomasse. L’agromine a surtout été développée pour valoriser le nickel (Ni). Jusqu’à présent, la biomasse était brûlée pour concentrer le métal et éliminer les matières organiques. L’enjeu de cette recherche est de concevoir des procédés de récupération du Ni par extraction directe depuis la biomasse, sans brûler la plante. Il s’agit de comprendre les processus impliqués lors de l’extraction du Ni de la biomasse sèche à l’aide d’un solvant et déterminer les formes chimiques des espèces en solution. A partir de là seront mises en œuvre des opérations de séparation adaptées, pour isoler le Ni sous une forme intéressante pour des applications ultérieures. Les expériences de lixiviation à l’eau à 20 °C, menées avec deux HA contrastées, ont démontré qu’il était possible de transférer en solution jusqu’à 80% du Ni présent dans les tissus des plantes. Celui-ci est accompagné des ions majeurs et de composés organiques. L’analyse des composés et la modélisation des équilibres chimiques en solution ont montré que le Ni était complexé à plus de 95% par des ligands organiques, acides carboxyliques, porteurs du Ni dans la plante, ainsi que des complexants plus forts. A partir de ces résultats, des procédés de séparation ont été sélectionnés : la précipitation sélective et l’adsorption sur résine complexante. Ils ont permis de récupérer respectivement 75 et plus de 95% du nickel sous forme sulfure ou composé carboxylique. En revanche, la purification à l’aide de décanoate n’a pas permis d’isoler le Ni. Ainsi, ce travail a permis de mieux comprendre l’extraction du Ni directement à partir de plantes, la spéciation du Ni en solution multiconstituant en présence de ligands organiques, et de valoriser le nickel par des voies jusqu’alors inexplorées avec ce type de matière première
Agromining is a chain allowing the recovery of metals dispersed in soils or other matrices, using hyperaccumulator plants (HA). The first step is to grow these plants to achieve high yields of metals and the second to produce metal compounds of interest from the plant biomass. Agromining has mainly been developed to value nickel (Ni). Until now, biomass was burnt to concentrate the metal and remove organic matter. The challenge of this research is to design processes for Ni recovery by direct extraction from biomass, without burning the plant. It will allow a better understanding of the processes involved in the extraction of Ni from dry biomass using a solvent and the determination of the the speciation in the solution. Then, appropriate separation operations will be implemented to isolate the Ni in an interesting form for subsequent applications.Water leaching experiments, run at 20 ° C with two contrasted HAs, demonstrated that up to 80% of Ni could be transferred from the plant tissues to the solution. Ni is accompanied by major ions and organic compounds. The analysis of these compounds and the modeling of the chemical equilibria in solution showed that more than 95% of Ni was complexed by organic ligands, carboxylic acids (Ni carriers in the plant) as well as stronger complexing agents. From these results, separation processes were selected: selective precipitation and adsorption on complexing resin. They made it possible to recover respectively 75 and more than 95% of the nickel in sulphide or carboxylic compound forms. In contrast, purification with decanoate did not isolate the Ni.Thus, this work has made it possible to better understand the extraction of Ni directly from plants, the speciation of Ni in a multicomponent solution in the presence of organic ligands, and to valorize nickel by ways previously unexplored with this type of material

L’agromine concerne la récupération de métaux stratégiques dans les sols métallifères par la culture de plantes hyperaccumulatrices de métaux (et metalloïdes). Le moteur de cette recherche était d'évaluer le potentiel des ressources végétales mexicaines pour le développement de l'agromine. Les principaux objectifs étaient d'identifier et d'étudier quelques espèces de plantes hyperaccumulatrices de métaux au Mexique, et d'évaluer l'agronomie d'une de ces espèces avec des caractéristiques prometteuses pour l’agromine. Nous avons d'abord effectué des explorations dans trois régions ultramafiques riches en nickel (Ni) du centre et du sud du Mexique. Malgré la disponibilité du nickel dans le sol et les conditions climatiques, aucune hyperaccumulation de Ni n'a été trouvée dans ces régions. Une deuxième stratégie basée sur la phylogénie végétale comme outil de prédiction de l'hyperaccumulation des métaux a été suivie. Au total, dix espèces hyperaccumulatrices de métaux ont été identifiées au cours de cette recherche (Rubiaceae et Violaceae) dans des sols riches en Ni influencés par l'activité volcanique, dans le sud-est du Mexique ; la majorité d’entre elles n’était pas identifiée comme hyperaccumulatrices. Nos études ont révélé deux des hypernickelophores les plus puissants détectés jusqu'à présent (>4% wt Ni) et deux nouveaux genres hyperaccumulateurs de nickel (Orthion et Mayanaea). Une attention particulière a été accordée à l'hypernickelophore Blepharidium guatemalense. Le phloème des feuilles, des racines, des tiges et des pétioles de cette plante est très riche en Ni, ce qui suggère un mécanisme de redistribution via le phloème. Différentes pratiques agronomiques ont été testées pour cette plante. La fertilisation inorganique a fortement augmenté l'absorption du Ni sans modifier la croissance ou la biomasse de la plante, tandis que la fertilisation organique a augmenté la biomasse de la plante avec un effet négligeable sur les concentrations de Ni dans les parties aériennes. Une parcelle avec une culture de 5 ans, qui a ensuite été récolté deux fois par an, produit le rendement maximal en Ni de 142 kg ha⁻¹ an⁻¹. Blepharidium guatemalense est un candidat de choix pour l'agromine du Ni en raison de ses caractéristiques appréciables : absorption extrêmement efficace du Ni, production élevée de biomasse, taux de croissance rapide, et facilité de reproduction
Agromining technology involves the recovery of strategic metals from metalliferous soils through the cultivation of metal(loid) hyperaccumulator plants. The impetus of this research was to evaluate the potential of Mexican plant resources for the future development of agromining. The main objectives were then to identify and to study some metal hyperaccumulator plant species in Mexico, and to assess the agronomy of one promising “metal crop” for agromining. We first undertook field explorations in three nickel-rich ultramafic regions of central and southern Mexico. Despite the availability of soil and climatic conditions, no nickel (Ni) hyperaccumulation was found in any of these regions. A second strategy based on plant phylogeny as a prediction tool for metal hyperaccumulation was followed. In total, ten plant metal hyperaccumulator species were identified during this research (Rubiaceae and Violaceae) in Ni-enriched soils influenced by volcanic activity in Southeastern Mexico; most of them were priorly unknown. Our studies revealed two of the strongest hypernickelophores reported so far (>4%wt Ni) and two new Ni hyperaccumulator genera (Orthion and Mayanaea). Special focus was given to the hypernickelophore tree Blepharidium guatemalense. The phloem on leaves, roots, stems and petioles of this plant are the richest in Ni suggesting an unusual re-distribution mechanism via the phloem. Different agronomic practices were tested for this plant. Synthetic fertilization strongly increased nickel uptake without any change in plant growth or biomass, whereas organic fertilization enhanced plant shoot biomass with a negligible effect on foliar Ni concentrations. A 5-year-old stand which was subsequently harvested twice per year produced the maximum Ni yield tree⁻¹ yr⁻¹, with an estimated total nickel yield of 142 kg ha⁻¹ yr⁻¹. Blepharidium guatemalense is a prime candidate for Ni agromining on the account of its valuable traits: extremely efficient Ni uptake, high biomass production, fast growth rate, and easy to reproduce

Les plantes hyperaccumulatrices peuvent accumuler des concentrations extraordinaires de métaux dans leurs parties aériennes (e.g. Ni, Zn et Cd). Cette thèse a été entreprise afin d’élucider : 1) comment le Ni est absorbé par les racines des hyperaccumulateurs, et 2) comment il circule dans les différents organes via le xylème et le phloème. Les travaux ont utilisé des cultures hydroponiques avec l’hyperaccumulateur de Ni et Zn Noccaea caerulescens en présence de concentrations faibles et élevées de Ni et Zn et en interaction avec Fe et Co ; des analyses isotopiques et d’expression de gènes ont été conduites. Les résultats ont montré que l’hyperaccumulateur N. caerulescens possède un système de transport du Ni à faible affinité et à haute efficacité. L’absorption du Ni semble impliquer principalement les transporteurs de Zn et Fe. Le transport par le xylème est la principale voie d'accumulation du Ni dans les jeunes feuilles et les feuilles âgées. Mais la translocation par le phloème est aussi une source importante de Ni pour les jeunes feuilles. Dans le phloème, le Ni est principalement chélaté par des acides organiques, de type malate. La thèse ouvre des perspectives pour l’optimisation des procédés de phytoextraction et d’agromine des sols contaminés
Hyperaccumulating plants are capable of accumulating extraordinary concentrations of heavy metals, e.g. Ni, Zn and Cd, in their shoots. This thesis was conducted to assess: 1) how roots of hyperaccumulators absorb Ni, and 2) how Ni circulates in different organs via xylem and phloem. Methods used were hydroponic cultures with the Ni/Zn hyperaccumulator Noccaea caerulescens in the presence of low and high Ni and Zn solutions, and in competition with Fe, Co, and Rb and Sr. Isotope fractionation in the plant, and gene expression of the Zn transporter ZIP10 and the Fe transporter IRT1 were studied. Results showed that the hyperaccumulator N. caerulescens takes up Ni mainly via low-affinity transport system, which seemed to be Zn and Fe transporters. Xylem transport is the main source for Ni accumulation in both young and old leaves, while phloem translocation also acts as an important source for young leaves. Ni is enriched in phloem sap and mainly chelated by organic acids especially malate during phloem translocation

Phylogenetic studies are providing powerful new insights into the evolution of complex traits. Metal hyperaccumulation is an unusual and complex physiological trait found in about 500 plant species and is associated with an exceptionally high degree of tolerance of metalliferous soils. Alyssum L. (Brassicaceae) is the largest known hyperaccumulator genus, comprising approximately 188 species distributed throughout the Mediterranean region and south-west Asia. Approximately one-quarter of these are largely restricted to areas of serpentine soils and have the ability to accumulate nickel to high concentrations in shoot tissue. This genus provides a good example in which to study the origins of a complex physiological trait, but its phylogeny is currently poorly understood. To produce a well-resolved phylogenetic tree to investigate the number and timing of origins of nickel hyperaccumulation within Alyssum, DNA sequences were generated for four chloroplast regions (matK, rps16–trnK, trnD–T and trnL–F) from 170 of 255 species in the tribe Alysseae. Additional sequencing was carried out for the chloroplast genes ndhF and rbcL and the nuclear gene PHYA. A Bayesian analysis employing a relaxed uncorrelated lognormal molecular clock and multiple fossil-age calibration points was carried out to reconstruct a time-calibrated phylogeny of this tribe using appropriate outgroups. Optimization of the nickel hyperaccumulation trait onto the resulting phylogenetic tree suggests that nickel hyperaccumulation arose twice in the Alysseae in the late Miocene/early Pliocene: 3.3–8.3 Mya in Alyssum and 6.3–8.8 Mya in Bornmuellera. The single origin in Alyssum is strongly associated with a significant acceleration in net species diversification rate, suggesting the ability to hyperaccumulate nickel could have provided a key evolutionary innovation facilitating rapid range expansion and subsequent species diversification. The scattered distribution of nickel hyperaccumulators across small island-like patches of serpentine soil suggests that allopatric speciation may have driven rapid diversification in this clade.

Certaines plantes, dites hyperaccumulatrices, ont la capacité de se développer sur des sols riches en métaux et d’accumuler ces métaux à des concentrations élevées. L’incinération de la biomasse produit des cendres qui contiennent de 10 à 25% en masse de Ni. Ce travail s’inscrit dans la continuité d’une recherche menée par l’équipe depuis plusieurs années, qui a donné lieu notamment à un brevet sur la production du sel double sulfate de nickel et d’ammonium hexahydraté (ANSH) à partir de la biomasse d’Alyssum murale. Le manuscrit comprend d’abord une synthèse bibliographique sur la phytomine, allant des hyperaccumulateurs aux procédés de valorisation, essentiellement centrée sur le nickel. Ensuite, ont été comparées quinze plantes hyperaccumulatrices (des genres Alyssum, Leptoplax et Bornmuellera) provenant d’Albanie ou de Grèce, en vue de leur application pour la phytomine. Les teneurs en nickel ont été mesurées dans les différents organes des plantes et dans les cendres obtenues par combustion. Les trois genres ont de l’intérêt pour l’application, les plantes contiennent 1 à 3% en masse de nickel et les cendres 15 à 20 %. Le procédé hydrométallurgique de production d’ANSH a été étudié étape par étape en vue d’optimiser chaque étape pour produire un sel très pur tout en économisant matière et énergie et minimisant la production d’effluents et de déchets. Ce travail a conduit à l’amélioration du procédé de départ. Enfin, de nouvelles pistes ont été proposées pour conduire à de nouveaux procédés et produits du nickel. Les résultats obtenus et la dynamique actuelle autour de la phytomine montrent l’intérêt de cette approche et annoncent son développement imminent
Some plants, known as hyperaccumulators, are able to develop on metal containing soils and to accumulate these metals at high concentrations in shoots. Biomass incineration leads to ash containing 10 to 25 wt % nickels, greater than in some mineral ores. This work follows a research that has been carried out by the team for several years, which has resulted in a patent on the hydrometallurgical production of the double salt ammonium and nickel hexahydrate (ANSH) from the biomass of Alyssum murale. It aims at improving the synthesis method of this salt in order to upscale it at the pilot scale and explore new methods leading to new products. The manuscript begins with a bibliographic review on phytomining from hyperaccumulators to metal recycling processes, essentially focused on nickel. Then ca 15 hyperaccumulator plants (genus Alyssum, Leptoplax and Bornmuellera) collected in Greece or Albania have been compared, in the objective of phytomining. Nickel concentrations were measured in the plant organs and in the ashes after combustion. The three types of plants are of great interest for the technology, they contain 1 to 3 wt % of nickel and the ashes 15 to 20%. The hydrometallurgical process of ANSH production was investigated step by step to optimize each step to produce a salt of high purity, to decrease materials and energy consumption and to minimize effluent and waste production. The process was thus improved. Eventually, new ideas have been tested for new processes and nickel products. The obtained results and the current dynamics prove the interest of phytomining and announce its imminent development

Phytoextraction du nickel dans les sols ultramafiques d’Albanie La phytoextraction minière est un procédé de récupération des métaux des sols minéralisés naturels ou pollués à l’aide de plantes hyperaccumulatrices. Elle est une alternative à l’agriculture vivrière des zones ultramafiques. L’objectif de la thèse est le développement d’une technologie de phytoextraction extensive du Ni avec Alyssum murale sur les Vertisols ultramafiques. Pour cela, il s’agissait : i) d’identifier les plantes hyperaccumulatrices les plus efficaces dans le prélèvement du Ni et comprendre les relations entre le prélèvement du métal et sa biodisponibilité, ii) de déterminer les types de sols adaptés à la phytoextraction du Ni et iii) de définir et optimiser un itinéraire agronomique adapté pour l’espèce retenue et pour les conditions édaphiques. Dans ce but, des prospections géobotaniques ont été conduites en Albanie et en Grèce. Puis une étude in situ des facteurs qui influencent la biodisponibilité du Ni et le comportement des plantes sur une toposéquence ultramafique a été mise en place. Enfin un essai agronomique de quatre années sur un site ultramafique d’Albanie (Pojske) a permis de tester la fertilisation, le contrôle des adventices par herbicide et la date de récolte pour optimiser le rendement d’extraction du Ni. Les résultats montrent que parmi l’ensemble des espèces présentes naturellement sur les serpentines des Balkans, A. markgrafii et A. murale ont le plus fort taux d’accumulation du Ni. Les Vertisols ultramafiques présentent une disponibilité élevée du Ni favorable à la phytoextraction minière. La biomasse d’A murale est augmentée de 0,2 t ha-1 à 6,0 t ha-1 à partir des traitements agronomiques et le rendement de phytoextraction de Ni par A. murale est de 23 à 69 kg ha-1. Alyssum murale peut être envisagée comme une culture pérenne et la fertilisation permet d’augmenter la compétitivité de la plante sans affecter les concentrations de Ni dans les parties récoltées
Phytomining is a process for recovering metals with hyperaccumulating plants from natural or polluted soils. It is an alternative to conventional farming in ultramafic areas. The aim of the thesis is the development of an extensive phytoextraction technology with Alyssum murale on ultramafic Vertisols. Therefore, work was conducted to i) identify the most effective Ni hyperaccumulators, and understand the relationship between metal uptake and bioavailability, ii) identify soil types suitable for phytoextraction, and iii) define and optimize agronomic practices adapted to the plant species and the edaphic conditions. Hence, geobotanical surveys were conducted in Albania and Greece. Then an in situ study was run on an ultramafic toposequence to assess the factors that influence Ni bioavailability and behavior of plants. Finally a four-year field trial was carried out on an ultramafic site in Albania (Pojske) where fertilization, weed control by herbicide, and harvest date were tested to optimize the efficiency of Ni extraction. The results showed that A. markgrafii and A. murale exhibit the highest rate of Ni accumulation among all species of Balkan serpentines. The ultramafic Vertisols have a high Ni availability phytoextraction and are favourable for phytomining. A. murale biomass increased from 0.2 t ha-1 to 6.0 t ha-1 due to optimization of agronomic treatments, and performance of phytoextraction from 23 to 69 kg ha-1. Alyssum murale can be seen as a perennial crop, and fertilization increases the competitiveness of the plant without affecting the Ni concentrations in the harvested parts

Due to the high nickel concentrations, serpentine soils provide a very restrictive and selective environment for plant life. Some plants, termed “Ni-hyperaccumulators”, are adapted to live on these heavy-metal-enriched soils without toxicity symptoms. Ni-hyperaccumulators are increasingly important for research on metal tolerance, homeostasis and biotechnological applications. This project aims to investigate nickel accumulation in taxa and populations of Odontarrhena, a genus of tribe Alysseae (Brassicaceae) that includes over 85 species many of which are Ni-hyperaccumulators. Based on a previous systematic study conducted on poorly-known populations of Odontarrhena native to Albania we performed a molecular study to characterize taxa and populations of this genus. To this purpose we used DNA sequencing and the AFLP-fingerprint technique to reconstruct the species phylogenetic relationships and the population differentiation patterns in relation to their distribution, ploidy level, intensity of anthropic site disturbance, altitude, soil type and metal concentration population (Ni, Cr, Co, Ca, Mg). We found significant population differentiation, dominance of within-population variation, no isolation by geographic distance and existence of six genetic groups variously represented across the six taxa possibly due to hybridization especially in disturbed sites. Next, we compared metal concentrations in native Odontarrhena populations from Albania in relation to their soil of origin. We determined the concentration of the most important trace metals (Ni, Co, Cr, Mg, Ca, K, Fe and Mn) in soil, plant roots and shoots of five taxa from 20 different outcrops. We found large differences in mineral element concentrations in soils and also between the plants; shoot Ni concentrations in Albanian Odontarrhena taxa depend on soil Ni concentrations but not on species identity. For O. chalcidica, the most widely distributed species, this “environmental fingerprint” was found not only for Ni, but also for Ca and Mg. After these investigations on native populations from the natural environment, we designed an experimental study in controlled conditions. Plant seedlings of seven taxa and 11 populations of Odontarrhena from serpentine and non-serpentine sites of the Balkan peninsula and Italy were cultivated in hydroponics with increasing NiSO4 concentrations to determine plant growth and Ni accumulation. These plantlets were analyzed to test inter- and intra-specific differences in nickel tolerance and accumulation, in relation to Ni levels in the soils and in wild plants. We found a metal stimulatory effect on growth that was present in the low-dose zone and significantly fitted the Brain-Cousens hormetic model. Taxa showed broad variation in tolerance, with the most tolerant plants requiring the highest Ni concentration for optimal growth. Our data suggested that tolerance is associated with hyperaccumulation ability. Among the obligate and facultative serpentinophytic species of Odontarrhena that have been investigated we found a notable exception, O. sibirica, a facultative serpentinophyte in which accumulation ability was enigmatic from previous studies. We addressed this issue using observational and experimental methods as in our previous researches. We found that Ni-concentrations in the native populations sampled on serpentine soils in Greece were always much lower than the hyperaccumulation threshold. When cultivated together with other Ni-accumulating Odontarrhena species on the same natural ultramafic soil, O. sibirica was the only one unable to accumulate the metal. When grown in hydroponics at different NiSO4 levels Ni-accumulation occurred only at higher concentrations which, however, had a toxic effect. This peculiar combination of Ni-response traits could be the result of a partial evolutionary loss of ability with respect to all other Ni-accumulating congeneric species. For its unique characteristics, O. sibirica could therefore represent a unique model system for further studies on the evolutionary dynamics, physiological mechanisms and genetic control of metal accumulation and homeostasis. In a parallel study, we investigated photosynthesis responses of the same plants using an experimental approach. In non-hyperaccumulator plants, toxicity symptoms to above 10 μg g-1 DW nickel concentrations in soils can include inhibition of photosynthesis, impaired nitrogen assimilation and disturbed enzyme activity. However, there is a complete lack of information about how Ni-hyperaccumulators reconcile that extraordinary amount of metal in their shoots with an efficient photosynthetic activity, or at least on which photosynthetic parameters the excess of Ni impacts less in these plants. We measured Ni effects on growth, root and shoot metal accumulation and several photosynthetic parameters, such as gas exchange, chlorophyll fluorescence analyses and pigments content in three Odontarrhena taxa (two hyperaccumulators, one not) grown in hydroponics and exposed to three NiSO4 treatments. We found that Ni-hyperaccumulators species are photosynthetically more efficient under Ni excess in respect to the non-accumulating species. In fact, Ni treatment in O. chalcidica increased not only the photochemical efficiency of PSII and the CO2 assimilation rate, but also the stomatal conductance. Finally, this project focused on the determination of the activity of the enzyme urease, the only Nimetalloenzyme known so far in plants, in selected Odontarrhena taxa. The hypothesis to test was whether the high basal requirement for this micronutrient in these plants could be linked to a depletion of the Ni cytosolic pool at low external metal concentration, due to hyperaccumulation mechanism and impairing urease activity. To this purpose, enzyme activity and Ni shoot concentration were determined in plants of accumulating and non-accumulating taxa of Odontarrhena cultivated on Ni-rich serpentine soil and on garden soil, as well as in samples of O. bertolonii cultivated in hydroponics at increasing Ni concentrations. Odontarrhena hyperaccumulators showed similar urease activity when grown on both kinds of soils, with no relation between the enzyme activity and the leaf Ni accumulation. Contrarily, clear indications came from the experiment in controlled conditions, where the presence of Ni determined a progressive stimulation, in respect to control samples, of the activity of the enzyme, associated with an increase in shoot metal concentration. A significant relationship was found between the levels of urease activity and the amount of Ni accumulated in the leaves. Therefore, the already known Ni-stimulated growth of O. bertolonii at increasing metal concentrations in the low-dose zone could be explained by a Ni-induced activity of urease, associable to an enhanced nitrogen metabolism, unless other still unknown physiological functions of Ni in hyperaccumulating plants.

Reliable, high-throughput and low-cost next-generation sequencing technologies have invigorated genetic research into non-model organisms over the last decade. In this work, RNA-Seq was employed to obtain the first-ever transcriptomes of two groups of closely related plant taxa possessing distinctive complex physiological traits, namely metal hyperaccumulation and C4 photosynthesis. Metal hyperaccumulator plants possess an extraordinary ability to take up trace elements from the soil and accumulate them to high concentrations in their shoots, probably to serve as a type of elemental defence against natural enemies. Taxonomically, the most common form of metal hyperaccumulation, nickel hyperaccumulation, is encountered on nickel-rich ultramafic (serpentine) soils, and is found with the highest frequency (ca. 51 species) in the genus Alyssum (family Brassicaceae). Here, the genetic basis and evolutionary history of nickel tolerance and hyperaccumulation was investigated in the Alyssum serpyllifolium Desf. species complex, which contains both serpentine and non-serpentine populations of unresolved phylogenetic relationships on the Iberian Peninsula. Genome scans for outlier loci and differential expression analyses identified a number of candidate hyperaccumulator genes common to two serpentine populations found in Portugal and Spain, but the majority of adaptive variation was of local origin. Phylogenetic and population-genetic inferences based on neutral and putatively adaptive loci suggested that the key genes for the nickel hyperaccumulation trait evolved once and spread across serpentine populations early in the history of this species, with no genetic isolation but continued recent gene flow between serpentine and non-serpentine populations. To test the power of next-generation sequencing for analysing the genetic basis of a separate complex trait, a cross-species comparison was performed using RNA-Seq of two congeneric tropical species, the C4 plant Alternanthera pungens Kunth and the C3 plant Alternanthera philoxeroides (Mart.) Griseb. f. angustifolia Suess. (family Amaranthaceae). These species were cultivated at two different temperatures and showed significant differences in levels of overall gene expression plasticity and isoform switching in certain photosynthesis genes, which it is proposed may explain the observed difference in the ability of these two species to acclimate to low and high growth temperatures.

Selective accumulation of certain metals (elements) to exceptionally high concentrations in plants is intriguing. Approximately 425 species of so-called metal hyperaccumulators are currently known, of which about 75% hyperaccumulate nickel. Stackhousia tryonii Bailey (Stackhousiaceae) - a rare, herbaceous, serpentine-endelnic species - is one of the three nickel hyperaccumulators reported from Australia. This thesis reports research aimed at two broad aspects: propagation and ecophysiology of Ni hyperaccumulation in S. tryonii. Protocols were developed for seed germination, vegetative propagation and micropropagation and with the view to producing sufficient plants for use in the current study. Four-year-old S. tryonii seeds had poor germination (< 25%). However, this species was relatively easy to propagate via stem cuttings and micropropagation methods, as it possessed very high regenerative capacity (one explant produced up to 18 shoots within 4 weeks). Micropropagated shoots also responded well to ex vitro rooting, and were successfully hardened under controlled conditions. These propagation protocols could be useful to underpin conservation programs and mine site revegetation. The examination of natural populations of S. tryonii for arbuscular mycorrhizal colonisation suggested that S. tryonii is a favourable host. A moderately high colonisation (29-39%) of roots by arbuscular mycorrhizal fungi suggested a possible role of these fungi in improved nutrition of S. tryonii in typically nutrient-poor serpentine soils. A positive relationship between root colonisation and leaf Ni concentration suggested that mycorrhizal fungi might be involved in increased influx of Ni into the roots, which is readily transported and localised in the tissues. Spore density was very low (3-4 spores 100 g-¹dry soil, for two depths) in the associated serpentine soils and the dominant mycorrhizal species were: Glomus albidum, aggregatum, G. intraradices and G. tenebrosum. Based on five key soil characteristics (viz. pH, Ca, Mg, Ni and P), the study sites were segregated into four groups using hierarchical cluster analysis. Considerable variation existed in tissue Ni (and other elements) concentrations, both within and between populations and followed the order: leaf> root> stem. Localisation and spatial distribution of nickel, within both vegetative (leaf and stem) and reproductive (fruit) tissues were investigated using two microanalytical techniques [viz., micro-proton-induced x-ray emission spectrometry (micro-PIXE; nuclear microprobe) and scanning electron microscope with energy-dispersive x-ray spectroscopy (SEM-EDXS)]. In leaf and stem tissues, Ni was localised within epidermal and sub-epidermal tissues, palisade/mesophyll tissues, vascular bundles and/or pith. In contrast, in fruits, this metal was partitioned to the fruit wall (pericarp), while endospermic and cotyledonary tissues contained very little Ni. Accumulation of higher levels of Ni within the pericarp does not appear to inhibit seed germination in S. tryonii. To elucidate physiological mechanisms o fNi detoxification in S. tryonii, organic acids (leaf tissue) and free amino acids (xylem sap) were quantified using HPLC. Nickel concentration in the leaf tissues increased from 3695 g g-¹to 13,717 g g-¹with soil nickel supplementation, of which > 60% was extracted with dilute acid (0.025 M HCI). Oxalic, citric and malic acids were detected and quantified in the leaf tissue. Malic acid was the dominant organic acid, and based on a Ni to malic acid ratio (between 0.2:1 and 1:1), malic acid appears to play a major role in detoxification/transport and storage of Ni in S. tryonii. The total amino acid concentrations in the xylem sap decreased with nickel treatment. Glutamine was the major amino acid in both the low- and high- nickel treated plants. A role of amino acids in nickel complexation and transport in S. tryonii could not be established. The possibility of hyperaccumulated Ni acting as an osmoticum under waterstress (drought) in serpentine soils was also investigated. Drought severely affected the growth and overall biomass of the plants. However, survival of plants at the lowest levels of soil moisture (i. e. 20% of field capacity) suggested that it possesses an efficient water regulation mechanism. The results indicated possible involvement of Ni in osmotic adjustment under drought stress.