The aqueous photochemistry of 4-hydroxy-3,5-dimethoxybenzoic acid (syringic acid) has been studied as a model humic substance in order to better understand the reactions that compounds of this type undergo in the natural environment. Syringic acid was chosen since it has been identified as a component of humic substances in the environment and bears many of chemical moieties found in structures of this type. In addition, there has been speculation that humic substances are responsible for some of the production of halomethanes that are released into the environment. Photolysis of these compounds in marine and estuarine waters may be responsible for the release of halomethanes which are known stratospheric ozone depleters. Photochemical product studies of syringic acid and related compounds along with UV-Vis spectrometry, laser flash photolysis and membrane introduction mass spectrometry were carried out in aqueous solutions to study its photochemical transformations. Syringic acid was found to form methanol at a 0.01 quantum yield upon its photolysis in basic solution. Other major photoproducts included 3-methoxygallic acid and 3,5-dimethoxybenzoic acid. Chloromethane was identified as a minor photoproduct in chloride enriched solution by following its production via membrane introduction mass spectrometry. The proposed mechanism for the formation of these photoproducts involves an initial photoprotonation of the benzene ring, resulting in a carbocation that can facilitate the nucleophilic attack by water or chloride, to produce methanol or chloromethane, respectively. The formation of 3,5-dimethoxybenzoic acid is via a novel pathway that involves the loss of the hydroxy group from the aromatic ring after the photoprotonation.

The aqueous photochemistry of 4-hydroxy-3,5-dimethoxybenzoic acid (syringic acid) has been studied as a model humic substance in order to better understand the reactions that compounds of this type undergo in the natural environment. Syringic acid was chosen since it has been identified as a component of humic substances in the environment and bears many of chemical moieties found in structures of this type. In addition, there has been speculation that humic substances are responsible for some of the production of halomethanes that are released into the environment. Photolysis of these compounds in marine and estuarine waters may be responsible for the release of halomethanes which are known stratospheric ozone depleters. Photochemical product studies of syringic acid and related compounds along with UV-Vis spectrometry, laser flash photolysis and membrane introduction mass spectrometry were carried out in aqueous solutions to study its photochemical transformations. Syringic acid was found to form methanol at a 0.01 quantum yield upon its photolysis in basic solution. Other major photoproducts included 3-methoxygallic acid and 3,5-dimethoxybenzoic acid. Chloromethane was identified as a minor photoproduct in chloride enriched solution by following its production via membrane introduction mass spectrometry. The proposed mechanism for the formation of these photoproducts involves an initial photoprotonation of the benzene ring, resulting in a carbocation that can facilitate the nucleophilic attack by water or chloride, to produce methanol or chloromethane, respectively. The formation of 3,5-dimethoxybenzoic acid is via a novel pathway that involves the loss of the hydroxy group from the aromatic ring after the photoprotonation.

Chloromethane is the most abundant halocarbon in the environment, and responsible for substantial ozone destruction in the stratosphere. Sources and sinks of chloromethane are still poorly constrained. Although synthesized and used industrially, chloromethane is mainly produced naturally, with major emissions from vegetation and especially the phyllosphere, i.e. the aerial parts of plants. Some phyllosphere epiphytes are methylotrophic bacteria which can use single carbon compounds such as methanol and chloromethane as the sole source of carbon and energy for growth. Most chloromethane-degrading strains isolated so far utilize the cmu pathway for growth with chloromethane which was characterized by the team. The main objective of this work was to investigate whether epiphytes may act as filters for plant emissions of chloromethane, by using a laboratory bipartite system consisting of the model plant Arabidopsis thaliana, known to produce chloromethane mainly by way of the HOL1 gene, and the reference chloromethane-degrading bacterial strain Methylobacterium extorquens CM4, possessing the cmu pathway and of known genome sequence. Three A. thaliana Col-0 variants with different levels of expression of HOL1, i.e. the wild-type strain, its homozygous HOL1 knockout mutant hol1 and an HOL1-OX HOL1 overexpressor, were selected using PCR and qRT-PCR. Chloromethane-degrading strains were isolated from the A. thaliana phyllosphere, and shown to contain the cmu pathway. A plasmid-based bacterial bioreporter for chloromethane was constructed which exploits the promoter region of the conserved chloromethane dehalogenase gene cmuA of strain CM4. It yields rapid, highly sensitive, specific and methyl halide concentration-dependent fluorescence. Application of the bioreporter to the three A. thaliana variants differing in expression of HOL1 investigated in this work suggested that they indeed synthesize different levels of chloromethane. Analysis by qPCR and qRT-PCR of metagenomic DNA from the leaf surface of these variants showed that the relative proportion and expression of cmuA in this environment paralleled HOL1 gene expression. Taken together, the results obtained indicate that even minor amounts of chloromethane produced by A. thaliana in the face of large emissions of methanol may provide a selective advantage for chloromethane-degrading methylotrophic bacteria in the phyllosphere environment. This suggests that chloromethane-degrading epiphytes may indeed act as filters for emissions of chloromethane from plants. Further experiments are envisaged to further assess the adaptation mechanisms of chloromethane-degrading bacteria in the phyllosphere, building upon the comparative genomic analysis of chloromethane-degrading strains which was also performed in this work, and on the preliminary investigations using high-throughput sequencing that were initiated.

Made available in DSpace on 2014-12-17T15:01:54Z (GMT). No. of bitstreams: 1 LeopoldoOAR_TESE.pdf: 2477409 bytes, checksum: 8a0937fc273e62c20239012357028d11 (MD5) Previous issue date: 2012-03-02 / Amorphous silica-alumina and modified by incipient impregnation of iron, nickel, zinc and chromium were synthetized in oxide and metal state and evaluated as catalysts for the chloromethane conversion reaction. With known techniques their textural properties were determined and dynamics techniques in programmed temperature were used to find the acid properties of the materials. A thermodynamic model was used to determine the adsorption and desorption capacity of chloromethane. Two types of reactions were studied. Firstly the chloromethane was catalytically converted to hydrocarbons (T = 300 450 oC e m = 300 mg) in a fixed bed reactor with controlled pressure and flow. Secondly the deactivation of the unmodified support was studied (at 300 ?C and m=250 g) in a micro-adsorver provided of gravimetric monitoring. The metal content (2,5%) and the chloromethane percent of the reagent mixture (10% chloromethane in nitrogen) were fixed for all the tests. From the results the chloromethane conversion and selectivity of the gaseous products (H2, CH4, C3 and C4) were determined as well as the energy of desorption (75,2 KJ/mol for Ni/Al2O3-SiO2 to 684 KJ/mol for the Zn/Al2O3-SiO2 catalyst) considering the desorption rate as a temperature function. The presence of a metal on the support showed to have an important significance in the chloromethane condensation. The oxide class catalyst presented a better performance toward the production of hydrocarbons. Especial mention to the ZnO/Al2O3-SiO2 that, in a gas phase basis, produced C3 83 % max. and C4 63% max., respectively, in the temperature of 450 oC and 20 hours on stream. Hydrogen was produced exclusively in the FeO/Al2O3-SiO2 catalysts (15 % max., T = 550 oC and 5,6 h on stream) and Ni/SiO2-Al2O3 (75 % max., T = 400 oC and 21,6 h on stream). All the catalysts produced methane (10 ? 92 %), except for Ni/Al2O3-SiO2 and CrO/Al2O3-SiO2. In the deactivation study two models were proposed: The parallel model, where the product production competes with coke formation; and the sequential model, where the coke formation competes with the product desorption dessorption step. With the mass balance equations and the mechanism proposed six parameters were determined. Two kinetic parameters: the hydrocarbon formation constant, 8,46 10-4 min-1, the coke formation, 1,46 10-1 min-1; three thermodynamic constants (the global, 0,003, the chloromethane adsorption 0,417 bar-1, the hydrocarbon adsorption 2,266 bar-1), and the activity exponent of the coke formation (1,516). The model was reasonable well fitted and presented a satisfactory behavior in relation with the proposed mechanism / S?lica alumina amorfa e modificada por impregna??o incipiente de precursores de ferro, n?quel, zinco e cromo foram sintetizados na forma de ?xido e reduzidos, tendo sido posteriormente avaliados como catalisadores na convers?o de clorometano em hidrocarbonetos. Propriedades texturais e t?cnicas din?micas em temperatura programada foram usadas para a determina??o das propriedades ?cidas dos materiais. Um modelo termodin?mico para representar a capacidade de adsor??o e dessor??o de clorometano foi avaliado. Dois tipos de rea??es foram abordados. No primeiro, o clorometano foi convertido cataliticamente a hidrocarbonetos (T = 300 450 oC e m = 300 mg) em um reator de leito fixo catal?tico com controle de fluxo, press?o e vaz?o m?ssica e, no segundo, com o suporte puro (T = 300 oC e m = 250 mg) foi estudado a rea??o de desativa??o do catalisador pelo coque em um micro adsorvedor com monitoramento gravim?trico. O teor de metal no suporte (2,5 %) e o percentual de clorometano na mistura (10 %) foram mantidos constantes durante os experimentos. Com os resultados obtidos foram avaliadas a convers?o e a seletividade dos produtos gasosos, ou seja, H2, CH4, C3 e C4 bem como a energia da etapa de dessor??o (75,2 KJ/mol para o Ni/Al2O3-SiO2 e 684 KJ/mol para o catalisador Zn/Al2O3-SiO2), considerando a taxa de adsor??o do g?s em fun??o da temperatura. A presen?a do metal no suporte mostrou uma import?ncia significativa em termos de atividade na rea??o de condensa??o do clorometano. Os catalisadores na forma de ?xido apresentaram melhor desempenho para obten??o de hidrocarbonetos. ? poss?vel destacar o ZnO/Al2O3-SiO2 que , em termos de constituinte gasoso, produziu apenas C3 (M?x. de 83 %) e C4 (M?x. de 63%), respectivamente, na temperatura de 450 oC e 20 horas de rea??o,. O g?s hidrog?nio foi formado exclusivamente com os catalisadores FeO/Al2O3-SiO2 (M?x. de 15 %, T = 550 oC e tempo de rea??o de 5,6 h) e Ni/SiO2-Al2O3 (M?x. 75 %, T = 400 oC e tempo de rea??o de 21,6 h). Todos os catalisadores produziram o g?s metano (10 ? 92 %), salvo os do tipo Ni/Al2O3- SiO2 e CrO/Al2O3-SiO2. No estudo com o suporte puro foram propostos dois modelos de iii desativa??o, do tipo paralelo, onde a forma??o do produto compete com a forma??o de coque e o sequencial, onde a etapa de dessor??o ? a competidora. Com as equa??es do balan?o de massa baseadas no mecanismo proposto foram determinados duas constantes cin?ticas (a primeira de valor 8,01?10-4 min-1, relacionada ? etapa da forma??o de hidrocarbonetos e a segunda, 1,46?10-1 min-1, ao coque depositado no s?tio do material), tr?s constantes de equil?brio (a global 0,003, relativa ao clorometano 0,417 bar-1, e a de forma??o de hidrocarbonetos 2,266 bar-1) e o do perfil do fator de atividade (1,516). Em termos de ajustes, o modelo representou um comportamento satisfat?rio em rela??o ao mecanismo proposto

Le chlorométhane est le plus abondant des composés organo-halogénés dans l’atmosphère et il est impliqué dans la destruction de l’ozone dans la stratosphère. Les sources et les puits de chlorométhane restent mal évalués. Bien que synthétisé et utilisé de manière industrielle, il est principalement produit naturellement, avec comme sources majeures les émissions provenant des végétaux et plus particulièrement de la phyllosphère, qui correspond aux parties aériennes des plantes. Certaines bactéries épiphytes de la phyllosphère sont des méthylotrophes capables d’utiliser des composés organiques sans liaison carbone-carbone comme le méthanol et le chlorométhane comme unique source de carbone et d’énergie pour leur croissance. La plupart des bactéries chlorométhane-dégradantes isolées jusqu’à présent utilisent une voie métabolique pour leur croissance sur chlorométhane appelée voie cmu (pour chloromethane utilisation), caractérisée par l’équipe. L’objectif principal de cette thèse a été de déterminer si des bactéries de la phyllosphère peuvent jouer le rôle de filtre pour l’émission de chlorométhane par les plantes. Dans ce but, un modèle de laboratoire a été mis en place, constitué de la plante Arabidopsis thaliana connue pour produire du chlorométhane par une réaction impliquant le gène HOL1, et la bactérie Methylobacterium extorquens CM4, souche de référence pour l’étude du métabolisme de dégradation du chlorométhane, qui possède la voie cmu et dont le génome complet a été séquencé et analysé. Des variants d’A. thaliana avec différents niveaux d’expression du gène HOL1 (le type sauvage, le mutant homozygote « knock-out » hol1 et un variant HOL1-OX avec surexpression) ont été sélectionnés par PCR et qPCR. Des souches bactériennes chlorométhane-dégradantes ont été isolées à partir de la phyllosphère d’A. thaliana, dont il a été montré qu’elles possèdent la voie cmu. Un bio-rapporteur bactérien pour le chlorométhane a été construit à l’aide d’un plasmide exploitant la région promotrice du gène conservé de la déshalogénase (cmuA) de la souche M. extorquens CM4. Il présente une réponse fluorescente rapide, sensible, et spécifique aux méthyl-halogénés de manière concentration-dépendante. L’application du bio-rapporteur aux trois variants d’A. thaliana étudiés suggère des niveaux d’émissions de chlorométhane différents. L’analyse, par qPCR et qRT-PCR, de l’ADN métagénomique extrait de la surface des feuilles a montré une corrélation entre la proportion relative de bactéries portant le gène cmuA et l’exprimant dans cet environnement, et l’expression du gène HOL1. Ces résultats indiquent qu’une production de chlorométhane, même très modeste par rapport aux fortes émissions de méthanol par A. thaliana, confère un avantage sélectif pour les bactéries épiphytes chlorométhane-dégradantes. Ces dernières pourraient ainsi bien jouer un rôle de filtre pour les émissions de chlorométhane de la phyllosphère vers l’atmosphère. En perspective, de nouvelles expériences complémentaires, basées sur l’analyse par génomique comparative des souches chlorométhane-dégradantes également effectuée dans le cadre du projet et sur une analyse par séquençage à haut-débit initiée dans ce travail, sont proposées pour améliorer la compréhension des mécanismes d’adaptation des bactéries chlorométhane-dégradantes dans la phyllosphère. / Chloromethane is the most abundant halocarbon in the environment, and responsible for substantial ozone destruction in the stratosphere. Sources and sinks of chloromethane are still poorly constrained. Although synthesized and used industrially, chloromethane is mainly produced naturally, with major emissions from vegetation and especially the phyllosphere, i.e. the aerial parts of plants. Some phyllosphere epiphytes are methylotrophic bacteria which can use single carbon compounds such as methanol and chloromethane as the sole source of carbon and energy for growth. Most chloromethane-degrading strains isolated so far utilize the cmu pathway for growth with chloromethane which was characterized by the team. The main objective of this work was to investigate whether epiphytes may act as filters for plant emissions of chloromethane, by using a laboratory bipartite system consisting of the model plant Arabidopsis thaliana, known to produce chloromethane mainly by way of the HOL1 gene, and the reference chloromethane-degrading bacterial strain Methylobacterium extorquens CM4, possessing the cmu pathway and of known genome sequence. Three A. thaliana Col-0 variants with different levels of expression of HOL1, i.e. the wild-type strain, its homozygous HOL1 knockout mutant hol1 and an HOL1-OX HOL1 overexpressor, were selected using PCR and qRT-PCR. Chloromethane-degrading strains were isolated from the A. thaliana phyllosphere, and shown to contain the cmu pathway. A plasmid-based bacterial bioreporter for chloromethane was constructed which exploits the promoter region of the conserved chloromethane dehalogenase gene cmuA of strain CM4. It yields rapid, highly sensitive, specific and methyl halide concentration-dependent fluorescence. Application of the bioreporter to the three A. thaliana variants differing in expression of HOL1 investigated in this work suggested that they indeed synthesize different levels of chloromethane. Analysis by qPCR and qRT-PCR of metagenomic DNA from the leaf surface of these variants showed that the relative proportion and expression of cmuA in this environment paralleled HOL1 gene expression. Taken together, the results obtained indicate that even minor amounts of chloromethane produced by A. thaliana in the face of large emissions of methanol may provide a selective advantage for chloromethane-degrading methylotrophic bacteria in the phyllosphere environment. This suggests that chloromethane-degrading epiphytes may indeed act as filters for emissions of chloromethane from plants. Further experiments are envisaged to further assess the adaptation mechanisms of chloromethane-degrading bacteria in the phyllosphere, building upon the comparative genomic analysis of chloromethane-degrading strains which was also performed in this work, and on the preliminary investigations using high-throughput sequencing that were initiated.

The main purpose of this Thesis is the experimental characterization of the electronic and geometric structures of objects called clusters. A cluster consists of a finite group of bound atoms or molecules. Due to its finite size, it may present completely different properties than those of the isolated atom and the bulk. The clusters studied in this work are constituted by rare-gas atoms, organic molecules, and metal atoms. Intense cluster beams were created using either an adiabatic expansion source or a gas-aggregation source, and investigated by means of synchrotron radiation based photoelectron spectroscopy. The reports presented in this Thesis may be divided into three parts. The first one deals with results concerning homogeneous molecular clusters (benzene- and methyl-related clusters) highlighting how molecular properties, such as dipole moment and polarizability, influence the cluster structure. The second part focuses on studies of solvation processes in clusters. In particular, the adsorption of polar molecules on rare-gas clusters is studied. It is shown that the doping method, i.e. the technique used to expose clusters to molecules, and the fraction of polar molecules are important factors in determining the location of the molecules in the clusters. Finally, a summary of investigations performed on metal clusters is presented. The applicability of solid state models to analyse the cluster spectra is considered, and the differences between the atomic, cluster and solid electronic structures are discussed.

Le chlorométhane (CH3Cl) est un composé organique volatile responsable de plus de 15 % de la dégradation de l’ozone stratosphérique due aux composés chlorés. Il est produit majoritairement par les plantes vivantes ou en décomposition. Les bactéries capables d’utiliser le CH3Cl comme source de carbone pour leur croissance peuvent jouer un rôle de filtre dans les émissions de CH3Cl vers l'atmosphère. Ce processus biologique reste à quantifier dans l'environnement, notamment pour les sols forestiers considérés comme un puits majeur de ce composé.Dans les études environnementales, le gène cmu A est utilisé comme biomarqueur de la dégradation bactérienne du CH3Cl. Il code une chlorométhane méthyltransférase essentielle à la croissance bactérienne avec le CH3Cl parla voie cmu (pour chloromethane utilisation), la seule caractérisée à ce jour. Mon projet de thèse avait un double objectif : i) l’approfondissement des connaissances de l’adaptation au CH3Cl chez une bactérie méthylotrophe modèle, Methylobacterium extorquens CM4; ii) l’exploration de la diversité des bactéries CH3Cl-dégradantes de sols forestiers. L’étude RNAseq chez la souche CM4 a montré que la croissance avec le CH3Cl s'accompagne de différences dans la transcription de 137 gènes de son génome (6.2 Mb) par rapport à sa croissance sur le méthanol (CH3OH). Les gènes de la voie cmu, ainsi que d’autres gènes impliqués dans le métabolisme de cofacteurs essentiels à l’utilisation du CH3Cl par cette voie et eux aussi portés par le plasmide pCMU01 de la souche, en font partie. Les paralogues de ces gènes localisés sur le chromosome ne sont quant à eux pas différentiellement exprimés. En revanche, d’autres gènes du chromosome, potentiellement impliqués dans l’excrétion de protons produits lors de la déshalogénation (hppA), la régénération du NADP+ (pnt), ou le métabolisme du cofacteur tétrahydrofolate(gènes gcvPHT), le sont. L’étude de la diversité des bactéries CH3Cl-dégradantes de sol forestier de la réserve naturelle de Steigerwald (Allemagne) a été réalisée sur des microcosmes par une approche de « Stable Isotope Probing ». Les microorganismes capables d’assimiler le CH3Cl marqué au [13C] incorporent cet isotope lourd du carbone dans leur ADN. L'analyse des séquences amplifiées par PCR des gènes codant l’ARN 16S des fractions d'ADN enrichies en [13C] a permis de mettre en évidence de nouveaux phylotypes, du genre Methylovirgula et de l’ordre des Actinomycetales, distincts de ceux auxquelles les souches dégradant le CH3Cl isolées jusqu'ici sont affiliées. En revanche, les séquences du gène cmuA et d’autres gènes du métabolisme méthylotrophe obtenues par PCR à partir de l'ADN enrichi en [13C] sont très proches de celles des souches CH3Cl-dégradantes connues. Les résultats obtenus suggèrent ainsi que des bactéries ayant acquis par transfert horizontal les gènes de dégradation de la voie cmu ou ne possédant pas de gène cmuA contribuent au filtrage biologique du CH3Cl des sols forestiers. A l'avenir, le couplage de différentes méthodes moléculaires et des approches culturales visera à découvrir de nouvelles voies microbiennes de l’utilisation du CH3Cl, et à caractériser l’abondance et la diversité des métabolismes impliqués dans la dégradation du CH3Cl dans les sols et d'autres compartiments environnementaux. / Chloromethane (CH3Cl) is a volatile organic compound responsible for over 15% of stratospheric ozone degradation due to chlorinated compounds. It is mainly produced by living and decaying plants. Bacteria utilizing CH3Cl as sole carbon and energy source for growth were shown to be involved in the filtering of CH3Cl emissions to the atmosphere. This biological process remains to be quantified in the environment, especially for forest soil, a major CH3Cl sink. The cmuA gene is used as a biomarker of bacterial CH3Cl degradation in environmental studies. It encodes a CH3Cl methyltransferase essential for bacterial growth by the cmu (chloromethane utilization) pathway for growth with CH3Cl and the only one characterized so far. My thesis project had a double aim: i) In depth studies of CH3Cl adaptation of a model methylotrophic bacterium, Methylobacterium extorquens strain CM4; ii) Exploration of bacterial CH3Cl-utilizers in forest. An RNAseq study of strain CM4 has shown that growth with CH3Cl leads to a difference of transcription of 137 genes in its 6.2 Mb genome compared to growth with methanol (CH3OH). Among those, genes of the cmu pathway and other genes involved in the metabolism of essential cofactors for CH3Cl utilization by this pathway, are all plasmid pCMU01-encoded. Paralogous genes located on the chromosome were not differentially expressed. On the other hand, other chromosomal genes potentially involved in extruding protons generated during CH3Cl deshalogenation (hppA), NADP+ regeneration (pnt), or in the cofactor tetrahydrofolate metabolism (gcvPHT) were differentially expressed. The diversity of CH3Cl-degrading bacteria in forest soil of the German natural park of Steigerwald was studied in microcosms using stable isotope probing. Microorganisms able to assimilate labeled [13C]- CH3Cl incorporate this heavy carbon isotope in their DNA. Sequence analysis of the PCR-amplified 16S RNA encoding gene from [13C]-DNA fractions uncovered phylotypes of the genus Methylovirgula and of the order of the Actinomycetales, which were not associated with bacterial CH3Cl degradation so far. In contrast, PCR-amplified sequences of cmuA and other genes of methylotrophic metabolism were closely related to known CH3Cl-degrading isolates. These results suggest that bacteria containing genes of the cmu pathway acquired by horizontal gene transfer as well as bacteria lacking the cmu pathway contribute to biological filtering of CH3Cl in forest soil. Future experiments coupling molecular and culture methods will aim to discover new CH3Cl-degrading pathways and to characterize the abundance and diversity of CH3Cl-degradation metabolism in soil and other environmental compartments.