Les hydrocarbures pétroliers sont expédiés et utilisés dans pratiquement toutes les parties du monde et sont devenus l’un des contaminants parmi les plus communs et les plus répandus dans les sols. Les éléments traces sont parfois associés aux hydrocarbures, ce qui peut augmenter la toxicité d’un site, compliquer les voies possibles de remédiation, et augmenter les coûts de traitements traditionnels. Dans les régions nordiques, où des déversements sont recensés dans de nombreux sites, le temps de nettoyage des contaminants et les coûts des méthodes d’assainissement traditionnelles peuvent s’avérer considérablement plus élevés que dans les régions tempérées. Des recherches alternatives visant une remédiation plus rapide et moins coûteuse, adaptée à des climats subarctiques, sont nécessaires et l’utilisation de plantes et champignons indigènes à l’écosystème local sont des approches prometteuses. En parallèle, les gouvernements de multiples pays visent à réduire les émissions de gaz à effet de serre, notamment par la réduction des déchets putrescibles voués à l’enfouissement. En utilisant ces matières résiduelles fertilisantes obtenues localement dans le processus de décontamination, une opportunité se présente pour valoriser ces matières tout en décontaminant des sites. Cette recherche doctorale vise à développer des méthodes de bioremédiation adaptées aux conditions locales, avec des intrants indigènes aux régions d’étude, pour des sols contaminés aux HCP au sein de trois projets de recherche à grande échelle sur le terrain, dont un également contaminé aux éléments traces. Deux projets ont été effectués dans un climat subarctique au 60° parallèle (Whitehorse, Yukon), en utilisant une approche novatrice de phytoremédiation assistée de champignons et compost municipal; nous qualifions cette technique intégrée d’approche par « microsystème écologique ». Comme le volume de sol affecte beaucoup l’efficacité des méthodes de bioremédiation, des volumes considérables de 0.15 m3 et 1 m3 ont été utilisés (au premier et deuxième site, respectivement), pour maximiser la pertinence des résultats lors de transfert d’échelles futures. Au premier site, l’efficacité des différentes composantes du microsystème a été évaluée en bacs dans quatre différentes combinaisons et comparée au traitement de base habituel (fertilisant) dans un sol contaminé par un déversement accidentel de diésel. La plante choisie était le saule Salix planifolia et le champignon Pleurotus ostreatus. Les résultats indiquent qu’après une saison de traitement, le microsystème était le traitement avec le taux d’élimination du diésel le plus rapide. Après trois saisons, les traitements contenant un ou plusieurs éléments du microsystème avaient des taux de contamination sous les normes pour des sols agricoles et étaient plus efficaces que le traitement au fertilisant ou que l’atténuation naturelle. Le deuxième site était une fosse à huiles usées située sur une pile de stériles miniers au cœur d’un ancien dépotoir. Le microsystème a été implanté dans des cellules au sol avec une doublure à l’épreuve du lessivât de contaminants. Les saules Salix alaxensis et Salix planifolia furent utilisés en combinaison avec le champignon Trametes versicolor. Cette approche, dans un sol hautement contaminé, a réussi à diminuer de façon considérable les hydrocarbures pétroliers (plus de 65 à 75 %). Le potentiel d’accumulation des métaux dans les tissus aériens des plantes a également été mesuré. Les deux espèces de saules ont démontré des taux d’accumulation d’éléments traces distincts et des stratégies racinaires différentes. Un champignon de la famille des Psathyrellaceae fut observé pour la première fois sur un site si hautement contaminé, puis est apparu de façon récurrente sur les cellules du microsystème pendant quatre ans. Les deux premiers sites répondent à un besoin de développer des méthodes de bioremédiation efficaces, passives et applicables en climat subarctique. Le troisième site de recherche porte sur l’utilisation de deux matières résiduelles fertilisantes (bois raméal fragmenté et drêche) et de fumier pour la dégradation de l’huile à moteur dans un climat continental humide à une latitude moyenne (Neuville, Québec). Des mésocosmes de 0.76 m3 avec aération contrôlée furent utilisés. L’utilisation de bois raméal fragmenté et de drêche de brassage à cette échelle, de même que l’acquisition des matières résiduelles dans un rayon très rapproché du centre de traitement afin d’explorer une approche d’économie circulaire dans un tel contexte, constituent les volets novateurs de cette étude. Les résultats démontrent que le fumier est plus efficace que le traitement habituel au fertilisant. La drêche de brassage et le bois raméal fragmenté sont utiles pour conserver l’humidité dans les sols (un paramètre de bioremédiation important). Cette étude, menée en collaboration avec un partenaire industriel, s’intègre dans la politique canadienne et québécoise de réductions des gaz à effets de serre et la philosophie de l’économie circulaire en valorisant des matières organiques résiduelles locales. Cette thèse a permis de démontrer qu’une combinaison de stratégies de bioremédiation avec des composantes locales est une méthode efficace dans un climat subarctique. Deux nouvelles espèces de saules (Salix planifolia et Salix alaxensis) ont été utilisées avec succès pour la bioremédiation d’hydrocarbures pétroliers et pour l’accumulation de certains éléments traces. Lors d’une collaboration avec un partenaire industriel, il a été possible de démontrer l’applicabilité de certains concepts d’économie circulaire et d’approche écosystémique en bioremédiation. / Petroleum hydrocarbons are shipped and used in virtually all parts of the world and have become one of the most common and widespread contaminants in soils. Trace elements are sometimes associated with them, and they can increase the toxicity of a site, complicate remediation, and increase the costs of traditional treatments. In northern areas where spills occur at multiple sites, the clean-up time and cost of traditional remediation methods can be significantly higher than in temperate regions. Alternative research aimed at faster and cheaper remediation adapted to subarctic climates is needed and the use of native plants and fungi integrated into the local ecosystem are promising approaches. Concurrently, governments in multiple countries aim to reduce greenhouse gas emissions, namely through the reduction of putrescible waste destined for landfills. By using locally-sourced residual fertilizing materials for decontamination processes, an opportunity arises to valorize these materials while restoring soils. This doctoral research aims to develop locally-adapted bioremediation methods, with indigenous plant and fungal inputs, to treat petroleum hydrocarbon contaminated soils in three large-scale field research projects, including one also contaminated with trace elements. Two projects were carried out in a subarctic climate at the 60 ° parallel (Whitehorse, Yukon), using an innovative approach of phytoremediation assisted by mushrooms and municipal compost; we call this integrated technique the "ecological microsystem" approach. Because soil volume has significant impacts on the efficiency of bioremediation methods, considerable volumes of 0.15 m3 and 1 m3 were used (at the first and second sites, respectively) to maximize the relevance of results in the event of a scale-up operation. At the first site, the effectiveness of the various components of the microsystem were evaluated in four different combinations, as well as compared to the usual basic treatment (fertilizer) in soil contaminated by an accidental diesel spill. The plant species chosen was Salix planifolia and Pleurotus ostreatus was selected for the fungus. Results indicate that after one treatment season, the microsystem was the treatment with the fastest diesel removal rates. After three seasons, treatments containing one or more elements of the microsystem were below standards for agricultural soils and were more effective than fertilizer treatment or natural attenuation. The second northern site was a waste oil pit located on top of a mine waste rock pile, at the heart of an old landfill. The microsystem was implanted into ground-level cells with a contaminant-proof leachate liner. The plant species Salix alaxensis and Salix planifolia were used in combination with the fungus Trametes versicolor. This approach in a highly contaminated soil was able to significantly reduce petroleum hydrocarbons (65 to 75%). The potential for metal accumulation in aerial plant tissues was also measured. Both willow species demonstrated distinct trace element accumulation patterns and different rooting strategies. A fungus of the Psathyrellaceae family was observed for the first time at a site so highly contaminated and was recurrent on the cells of the microsystem for 4 years. The first two sites contribute to the development of efficient and passive bioremediation methods applicable in subarctic climates. The third research site focused on the use of two residual fertilizing materials (fragmented rameal wood and brewer’s spent grain) and manure for the degradation of motor oil in a humid continental climate at a medium latitude (Neuville, Quebec). Mesocosms of 0.76 m3 with controlled aeration were used. Innovative aspects of this project include the use of rameal wood and spent grain at this scale, as well as the acquisition of residual materials in a very close radius of the treatment center to explore how a circular economy approach could apply in such a context. Results indicate that the addition of manure is more effective than the usual fertilizer treatment alone. Brewer’s spent grain and fragmented rameal wood were useful for maintaining soil moisture (an important bioremediation parameter) but did not increase degradation. This study, conducted in close collaboration with an industrial partner, falls under the objectives set by Canadian and Quebec policy on greenhouse gas reductions and circular economy approaches by working on the valorization of local residual organic matter. This thesis has demonstrated that combining strategies with local biological components is an effective bioremediation method in a subarctic climate. Two new willow species (Salix planifolia and Salix alaxensis) have been successfully used for the bioremediation of petroleum hydrocarbons and for the accumulation of certain trace elements. In collaboration with an industrial partner, it has been possible to demonstrate the applicability of certain circular economy concepts and the ecosystemic approach to bioremediation.

L'intensification des activités industrielles et les besoins en énergie font des hydrocarbures pétroliers (HP) un enjeu majeur mondial mais augmentent aussi considérablement les risques environnementaux dans divers écosystèmes. La phytoremédiation est une phytotechnologie qui a fait ses preuves en tant que solution verte pour faire face aux contaminations des sols par des HP. La phytoremédiation des sols contaminés par les HP repose principalement sur l’activité des communautés microbiennes associées aux racines des plantes au niveau de la rhizosphère, qui peuvent non seulement favoriser la croissance des plantes hôtes mais aussi augmenter leur tolérance à divers stress biotiques et abiotiques. Parmi les défis majeurs de la phytoremédiation des sols contaminés par les HP, on compte la forte toxicité de certains composés des HP qui entravent la croissance des plantes et par conséquent l’efficacité de la phytoremédiation. Cependant, la croissance des plantes peut être positivement stimulée par la présence de rhizobactéries favorisant leur croissance (PGPR) qui sont capables d'atténuer le stress des plantes par divers mécanismes. Dans cette thèse, un total de 438 bactéries PGPR dégradant les hydrocarbures pétroliers, ont été isolées de la rhizosphère et du sol de deux espèces de plantes, Salix purpurea et Eleocharis obusta, dans un site d'une ancienne raffinerie pétrochimique à Varennes, QC, Canada. Les isolats bactériens ont été classés en 62 genres, appartenant aux phylums Actinobacteria, Bacteroidetes, Firmicutes et aux sous-groupes Alpha-, Beta- et Gamma-Proteobacteria. De plus, cette collection de cultures contient 438 isolats bactériens avec de multiples caractéristiques de dégradation et de stimulation de croissance (PGPR), représentant une diversité fonctionnelle de dégradation des HP et de caractéristiques PGPR qui pourraient être utilisées dans la phytoremédiation assistée par les bactéries, des sols contaminés par les HP. Parmi ces 438 isolats bactériens, 50 isolats représentant une large diversité taxonomique, ont été sélectionnées pour une caractérisation approfondie supplémentaire concernant leur capacité à favoriser la croissance des plantes en présence de différentes concentrations de n-hexadécane (0%, 1%, 2%, 3%) dans des conditions contrôlées. Les résultats ont indiqué que les isolats bactériens Nocardia sp. (WB46), Pseudomonas plecoglossicida (ET27), Stenotrophomonas pavanii (EB31), Bacillus megaterium (WT10) et Gordonia amicalis (WT12) ont significativement augmenté la croissance des plantes cultivées dans 3% de n-hexadécane par rapport au traitement témoin. De plus, ces isolats possèdent plusieurs traits favorisant la croissance des plantes (PGPR) tels que l'activité 1-aminocyclopropane-1-carboxylate (ACC) désaminase (ACCD), la production d'acide indole-3-acétique (IAA) et la fixation de l'azote. De plus, ces isolats étaient capables d'utiliser le n-hexadécane comme seule source de carbone et possédaient des gènes cataboliques liés à la dégradation des hydrocarbures tels que le gène de l'alcane monooxygénase (alkB), le cytochrome P450 hydroxylase (CYP153) et le gène de la naphtalène dioxygénase (nah1). Nocardia sp. isolate WB46, a été sélectionné pour le séquençage de son génome afin de déterminer sa diversité génétique et fonctionnelle relatives à la dégradation des HP et les potentiels PGPR. Les résultats ont indiqué que, sur la base des analyses du gène de l'ARNr 16S, l'hybridation ADN-ADN in silico (DDH) et l'identité moyenne des nucléotides (ANI), Nocardia sp. isolate WB46 représente une nouvelle espèce bactérienne. De plus, l'annotation fonctionnelle de son génome révèle que celui-ci contient de nombreux gènes responsables de la dégradation des hydrocarbures pétroliers tels que l'alcane 1-monooxygénase (alkB) et la naphtalène dioxygénase (ndo) ainsi que d'autres gènes liés à ses potentiels PGPR. En conclusion, la rhizosphère des espèces S. purpurea et E. obusta poussant dans un site fortement pollué par les HP représente un biotope diversifié et comprenant des bactéries PGPR avec de multiples potentiels de dégradation des HP. De plus, plusieurs isolats bactériens tels que Nocardia sp. (WB46), Pseudomonas plecoglossicida (ET27) et Stenotrophomonas pavanii (EB31) démontrent un potentiel d'utilisation comme bioinoculants pour de futures études de phytoremédiation à grande échelle. / Petroleum hydrocarbons (PHCs), as a result of intensification of industrial activities, are a global environmental issue especially in soil environments. Phytoremediation represents an ideal solution to tackle this global crisis. Phytoremediation of PHC-contaminated soils proceeds mainly through the activities of microbial communities that colonize the plant rhizosphere which might promote host plants growth and increase its tolerance to various biotic and abiotic stresses. A main challenge in phytoremediation of PHC-contaminated soils is the high toxicity of PHCs which hinder plant growth and reduce the efficiency of phytoremediation. However, plant growth may be positively stimulated by the presence of plant growth-promoting rhizobacteria (PGPR) that are able to alleviate stresses in plants through various mechanisms. In this thesis, a total of 438 petroleum hydrocarbons degrading-PGPR bacterial isolates were recovered from the rhizosphere and the surrounding bulk soil of Salix purpurea and Eleocharis obusta plants from the site of a former petrochemical plant in Varennes, QC, Canada. Bacterial isolates were classified into 62 genera, belonging to the phyla Actinobacteria, Bacteroidetes, Firmicutes and the Alpha, Beta and Gamma-subgroups of Proteobacteria. Additionally, this culture collection holds 438 bacterial isolates with multiple degradative and PGP features, representing a rich reservoir of metabolically versatile PGPR-PHC degraders that could be used in holistic, bacterial-aided phytomanagement of PHC-contaminated soils. Among the above 438 bacterial isolates, 50 bacterial strains representing a wide phylogenetic range were selected for an additional in-depth characterization regarding their ability to promote plant growth under the presence of different concentrations of n-hexadecane (0%, 1%, 2%, 3%) under gnotobiotic conditions. Results indicated that bacterial isolates Nocardia sp. (WB46), Pseudomonas plecoglossicida (ET27), Stenotrophomonas pavanii (EB31), Bacillus megaterium (WT10) and Gordonia amicalis (WT12) significantly increased the growth of plants grown in 3% n-hexadecane compared with the control treatment. Additionally, these isolates possess several plant-growth-promoting (PGP) traits such as 1-aminocyclopropane-1-carboxylate (ACC) deaminase (ACCD) activity, indole-3-acetic acid (IAA) production and nitrogen fixation. Also, these isolates were able to use n-hexadecane as sole source of carbon and have catabolic genes related to hydrocarbon degradation such alkane monooxygenase (alkB) gene, the cytochrome P450 hydroxylase (CYP153) and the naphthalene dioxygenase (nah1) gene. The isolate that showed the highest growth stimulation of plants grown in 3% n-hexadecane under gnotobiotic conditions, Nocardia sp. isolate WB46, was selected for de novo genome sequencing to unveil its genetic versatility and the mechanisms of PHCs biodegradation and PGP potentials. Results indicated that based on the 16S rRNA gene analyses, in silico DNA-DNA hybridization (DDH) and average nucleotide identity (ANI) Nocardia sp. isolate WB46 is a new species. Additionally, the functional annotation of the genome of Nocardia sp. isolate WB46 reveals that its genome contains many genes responsible for petroleum hydrocarbon degradation such as alkane 1-monooxygenase (alkB) and naphthalene dioxygenase (ndo) as well as other genes related to its PGP potentials. In conclusion, S. purpurea and E. obusta growing in a site highly polluted with PHCs are rich reservoir of diverse PGPR with multiple PHC-degradation and PGP potentials. In addition, several bacterial isolates such as Nocardia sp. (WB46), Pseudomonas plecoglossicida (ET27) and Stenotrophomonas pavanii (EB31) demonstrate potential for use as bioinoculants in future large-scale phytoremediation studies.

Submitted by Biblioteca de Pós-Graduação em Geoquímica BGQ (bgq@ndc.uff.br) on 2016-05-02T17:58:54Z No. of bitstreams: 1 GALLOTTA_30_06_14.pdf: 62695484 bytes, checksum: 33335ee3affc2bcffb11fc10caf9ae3f (MD5) / Made available in DSpace on 2016-05-02T17:58:54Z (GMT). No. of bitstreams: 1 GALLOTTA_30_06_14.pdf: 62695484 bytes, checksum: 33335ee3affc2bcffb11fc10caf9ae3f (MD5) / Universidade Federal Fluminense. Instituto de Química. Programa de Pós-Graduação em Geociências-Geoquímica. Niterói, RJ / Os hidrocarbonetos presentes no ambiente consistem em misturas complexas de compostos derivados de múltiplas fontes. Os combustíveis fósseis representam a principal contribuição, devido à taxa e escala espacial, em que o petróleo tem sido usado como fonte de energia e matéria-prima para a indústria química. O objetivo deste estudo foi avaliar os níveis de concentração e identificar fontes de hidrocarbonetos na Bacia do Alto Iguaçu e, em especial, na área de influência da Refinaria Presidente Getúlio Vargas (REPAR). Além dos fatores de poluição crônica, a área de estudo foi alvo de um derrame acidental de petróleo em julho de 2000. Diversos indicadores em diferentes compartimentos ambientais (água superficial, sedimento, solo e água subterrânea) foram avaliados na fase pós-derrame e no monitoramento ambiental ao longo de várias campanhas por mais de uma década. Os esforços de avaliação foram concentrados nos seguintes indicadores: os nalcanos, os alcanos isoprenoides, os hidrocarbonetos policíclicos aromáticos (HPA), os biomarcadores de petróleo e ainda o total de hidrocarbonetos de petróleo (THP). Os resultados identificaram como principal aporte de matéria orgânica para as águas superficiais dos rios Barigüi e Iguaçu, os fluxos materiais originados em região a montante do acidente, refletindo a contribuição antropogênica crônica da cidade de Curitiba. Em 2007 e 2008, as concentrações de THP e HPA nas águas superficiais e sedimentos dos rios Barigüi e Iguaçu refletem uma expressiva melhoria nas condições desses rios em relação a 2000. Na área interna da refinaria (Ponto Zero), observou-se uma nítida diminuição (atenuação) natural das concentrações de THP no solo em todas as profundidades dos perfis amostrados ao longo do tempo nos Banhados 1 e 4 e, em particular, uma diminuição importante das concentrações entre 2004 e 2007. Para a identificação das fontes de hidrocarbonetos foram utilizadas razões diagnósticas e quimiometria. As razões diagnósticas calculadas a partir de concentrações de HPA sugeriram que, na maioria dos sedimentos dos Rios Barigüi e Iguaçu coletados nas campanhas de 2000 e 2001, a fonte petrogênica é a principal. Somente na estação a montante do acidente no Rio Barigüi, a fonte pirolítica predominou nestas duas campanhas. As razões diagnósticas que apresentaram maior eficiência na identificação de fontes de hidrocarbonetos nos sedimentos dos rios Barigüi e Iguaçu foram: ΣC1-Fenantrenos/Fenantreno; e (ΣHPA parentais de 3-6 anéis)/(Σ5 séries de HPA alquilados). A identificação de fontes através de razões diagnósticas calculadas a partir de áreas e alturas de picos cromatográficos demonstrou sua aplicabilidade verificando a relação entre os compostos encontrados em amostras de solo da área interna da refinaria com a amostra de petróleo derramado no acidente, após quase uma década da ocorrência do vazamento. A identificação de fontes através método de quimiométrico baseado na análise de componentes principais (ACP) de seções pré-processadas e combinadas dos Cromatogramas de Íons Selecionados (CIS) mostrou que as amostras mais contaminadas estão na área interna da refinaria. Essas amostras apresentam um padrão de distribuição petrogênica e diferentes graus de intemperismo. As amostras da área externa à refinaria (Guajuvira, General Lúcio e Balsa Nova) são menos ou não contaminadas e/ou contém uma mistura de contribuições diagenéticas, pirolíticas e petrogênicas onde predominam diferentes proporções. Os locais mais distantes da atividade industrial (Balsa Nova) contem, como esperado, os níveis mais baixos de contaminação por HPA. Os resultados de biomarcadores demonstraram que não há evidências para concluir que as amostras da área externa à refinaria e o óleo Cusiana vazado tenham a mesma origem. Os resultados ao longo dos rios Barigüi e Iguaçu e do Ponto Zero demonstraram que as ações de emergência para a contenção do óleo foram adequadas para os rios, e que a contaminação decorrente do derrame ficou predominantemente contida no Ponto Zero e diminuiu significativamente após uma década. / Hydrocarbons present in the environment consist of complex mixtures of compounds derived from multiple sources. The main contribution lies on fossil fuel inputs due to the rate and spatial scale by which petroleum has been used as an energy source and chemical feedstock. The aim of this study was to assess the concentration levels and identify sources of hydrocarbons in the Upper Iguaçu Watershed and, in particular, in the area of influence of the President Getulio Vargas Refinery (REPAR). In addition to the factors of chronic pollution, the study area was the scenario of an acute accidental oil spill in July 2000. Numerous indicators in different environmental compartments (surface water, sediment, soil and groundwater) were assessed in the post spill phase and during the environmental monitoring programs over the course of several campaigns for more than a decade. Assessment efforts were concentrated on the following indicators: n-alkanes, alkanes isoprenoids, polycyclic aromatic hydrocarbons (PAH), petroleum biomarkers and total petroleum hydrocarbons (TPH). The results identified as the main contribution of organic matter to surface waters of the Barigüi and Iguaçu Rivers the materials flows originated in the region upstream of the accident, reflecting chronic anthropogenic contribution of the city of Curitiba. In 2007 and 2008, the TPH and PAH concentrations in surface waters and sediments of the Barigüi and Iguaçu Rivers revealed a significant improvement in the conditions of these rivers when compared with 2000. Inside the refinery area (Point Zero), it was observed a clear natural decrease (attenuation) of the concentrations of TPH in the soil at all depths sampled over time in Marshes 1 and 4 and, in particular, an important decrease of concentrations between 2004 and 2007. Diagnostic ratios and chemometrics were used to identity hydrocarbon sources. The diagnostic ratios calculated from the concentrations of PAH suggested that, in the majority of sediments from the Barigüi and Iguaçu Rivers collected in 2000 and 2001 campaigns, the main source is petrogenic. Only in the station upstream the accident in the Barigüi River, the pyrolytic source predominated in these two campaigns. The diagnostic ratios that presented higher efficiency in identifying sources of hydrocarbons in sediments of the Barigüi and Iguaçu Rivers were: ΣC1- Phenantrenes/Phenanthrene; and (Σ3-6 rings parental PAH)/(Σ5 alkylated PAH series). The source identification through diagnostic ratios calculated from heights and areas of chromatographic peaks demonstrated its applicability establishing a relationship between the compounds found in soil samples of the internal area of the refinery and the sample of the oil spilled in the accident, after nearly a decade of occurrence of the spillage. The source identification through chemometric method based on principal components analysis (PCA) of preprocessed and combined sections of Selected Ion Chromatograms (SIC) showed that the most contaminated samples are inside the refinery area. These samples present a petrogenic pattern and different weathering degrees. Samples from outside the refinery area (Guajuvira, General Lúcio e Balsa Nova) are either less or not contaminated, and/or contain mixtures of diagenetic, pyrogenic and petrogenic inputs where different proportions predominate. The locations farthest away from industrial activity (Balsa Nova) contain, as expected, the lowest levels of PAH contamination. The biomarkers results do not show any evidences to conclude positive matches between the samples from outside the refinery area and the spilled Cusiana oil. The results along Barigüi and Iguaçu rivers and Point Zero demonstrated that emergency actions to contain the oil were appropriate for the rivers, and that the contamination resulting from the spill was mostly contained in the Point Zero and decreased significantly after one decade