Fruits and flowers house microbial communities that are unique from the rest of the plant. While a great deal is known about a handful of symbiotic microbes associated with roots and leaves, the microbial communities of fruits and flowers have received considerably less attention. Fruits are reproductive tissues that house, protect, and facilitate the dispersal of seeds, and thus they are directly tied to plant reproductive success. Fruit and flower microbial communities may, therefore, also impact plant fitness. This dissertation examines how fruit bacterial communities, as determined using the 16S rRNA gene marker, are shaped across spatial and environmental gradients and the role of pollinators in shaping floral bacterial communities among natural populations of Solanum carolinense. There have been limited studies on how spatial distance influences bacterial communities found in and on fruit tissue and the role of pollinators in shaping floral bacterial communities. The first study addresses how bacterial communities in fruit change across similar environmental conditions at fine spatial scales (2 to 450 m). Overall, no differences were found in observed richness or bacterial community composition. Next, the role that generalist pollinators might play in shaping these communities was tested using pollinator exclusion cages. Here we found that generalist pollinators do not play a large role in shaping floral bacterial communities in Solanum carolinense. Comparing bacterial community diversity between caged and uncaged flowers, via PCoA we found no significant clustering of samples. In contrast, significant clustering was detected between flowers and bee pollen baskets. Together these results suggested that environmental factors may be more important in shaping floral bacterial communities. To test this, we sampled 9 populations along a 337 km latitudinal transect and again used the 16S rRNA gene to characterize bacterial communities. We did not identify a significant correlation between distance and bacterial community composition in either the total nor endophytic community in the fruit. Results from these studies suggest that while there is some evidence for environmental effects shaping fruit and flower communities, other factors such as host selection (e.g., secondary compounds in fruit) also likely play an important role in shaping bacterial communities. / Doctor of Philosophy / Fruit and flowers are directly tied to plant reproduction, yet little is known about the bacterial communities associated with these important organs, especially compared to other plant tissues. This dissertation aims to address gaps in our knowledge regarding how spatial distance and pollinators influence fruit and floral bacterial communities. Specifically, how does bacterial community composition (what bacteria taxa are present or not and how abundant the bacterial taxa are) change based on spatial distance. Studies were conducted at both fine (under 0.5 km) and broad (337 km) scales to determine if a pattern was observed between increasing distance and how distinct the bacterial community composition is. There was no significant correlation between spatial distance and bacterial community composition at fine spatial distances, however there were high levels of dissimilarity in the bacterial communities sampled across fine spatial scales. This led to the investigation of pollinators, which directly interact with flowers and may act as a source of bacterial community transmission in the flower and fruit bacterial communities as they move around the landscape. To address the role that pollinators play in shaping bacterial communities in the fruits an exclusion cage study was used to prevent pollinators from interacting with a subset of flowers. Pollen baskets (pollen that was collected by the pollinator and is found on the legs of pollinator) were also collected from pollinators to determine what bacteria may be present on the pollinator. Pollen baskets may also represent what bacteria may have been picked up by the pollinator at the site. It was observed that caged and uncaged samples were similar to one another, which suggest that pollinators were not adding new bacteria nor changing the bacterial composition in the flowers. Yet, the pollen baskets (from the pollinator) were unique from the caged and uncaged floral samples. To further investigate what factors may be at play in shaping the fruit bacterial community the transect was expanded to 337 km to determine if there was an observable relationship between spatial distance and bacterial community composition. The relationship between spatial distance and bacterial community composition was not significant in either the endophytic (internal bacterial community) or the total fruit bacterial community (which represented external and internal bacterial communities). Similar to the fine spatial scale study, there were high levels of dissimilarity in the bacterial community that was observed across sites. Results from these studies may suggest that there are other factors that play a larger role in shaping bacterial communities in fruit and flower tissue. These could include the host plant and the production of secondary compounds, which in some cases can act as antimicrobial compounds, and enhance or inhibit the growth of specific bacterial taxa. Environmental factors such as wind and rainfall may have influenced the bacterial community composition. It is likely that environmental factors play a role in shaping floral and fruit bacterial communities. However, it is still unclear what factors shape fruit and floral bacterial communities are. This study provides the foundation for future studies to address additional factors that shape fruit and flower bacterial communities.

<p>Treatment wetlands play an important role in reducing nitrogen content in wastewater and agricultural run-off water. The main removal process is denitrification and the removal efficiency depends on the hydrological and microbiological features of the wetland, especially in terms of water residence times and denitrification rates. The aim of this thesis was to increase the understanding of the coupled hydrological and microbiological processes regulating the denitrification capacity. This was done by applying a broad spectrum of analyses methods, including tracer experiment, water flow modeling, denitrification rate measurements, and analyses of the microbial community structures. The tracer experiment and flow modeling revealed that the wetland design, especially the vegetation, largely can affect the water residence time distributions in wetlands. In the investigated wetland, vegetation dominated the water flow, explaining 60-80% of the variance in water residence times, whereas basin shape only explained about 10% of the variance, but also mixing phenomena significantly affected the residence times and could considerably delay solutes. Measured potential denitrification rates in the wetland exhibited significant spatial variations, and the variations were best described by concentration of nitrogen in sediments and water residence time. Analyses of the denitrifying bacteria populations indicated that a few key populations dominated and that the community diversity increased with decreasing nutrient levels and increasing water residence times. Moreover, it was found that denitrification rates in terms of Menten and first order kinetics can be evaluated by fitting a mathematical expression, considering denitrification and other nitrogen transforming processes to measured product formation in nitrate limited experiments.</p>

Treatment wetlands play an important role in reducing nitrogen content in wastewater and agricultural run-off water. The main removal process is denitrification and the removal efficiency depends on the hydrological and microbiological features of the wetland, especially in terms of water residence times and denitrification rates. The aim of this thesis was to increase the understanding of the coupled hydrological and microbiological processes regulating the denitrification capacity. This was done by applying a broad spectrum of analyses methods, including tracer experiment, water flow modeling, denitrification rate measurements, and analyses of the microbial community structures. The tracer experiment and flow modeling revealed that the wetland design, especially the vegetation, largely can affect the water residence time distributions in wetlands. In the investigated wetland, vegetation dominated the water flow, explaining 60-80% of the variance in water residence times, whereas basin shape only explained about 10% of the variance, but also mixing phenomena significantly affected the residence times and could considerably delay solutes. Measured potential denitrification rates in the wetland exhibited significant spatial variations, and the variations were best described by concentration of nitrogen in sediments and water residence time. Analyses of the denitrifying bacteria populations indicated that a few key populations dominated and that the community diversity increased with decreasing nutrient levels and increasing water residence times. Moreover, it was found that denitrification rates in terms of Menten and first order kinetics can be evaluated by fitting a mathematical expression, considering denitrification and other nitrogen transforming processes to measured product formation in nitrate limited experiments. / QC 20101110

Ecosystems currently face widespread biodiversity losses and other environmental disturbances, such as climate warming, related to increased anthropogenic activities. Within this context, scientists consider the effects of such changes on the biodiversity, and hence on the activity, of soil microorganisms. Indeed, soil microorganisms mediate a wide range of soil processes. Currently, knowledge on soil microbial diversity is still limited, partially due to technical limitations. The advent of molecular-based analyses now allows studying the soil microbial diversity. These advances in the study of soil microbial communities have lead to a growing evidence of the critical role played by the microbial community in ecosystem functioning. This relationship is supposed to be relevant for narrow processes, regulated by a restricted group of microorganisms, such as the nitrification process. This PhD thesis aimed at studying ammonia oxidizing bacteria (AOB) community structure and richness as an integrated part of soil functioning. This research aimed at investigating the effect of aboveground plant diversity on ammonia oxidizing bacteria diversity and function in forest and grassland soils with focus on the influence of (a) functional group identity of grassland plants (legumes, grasses, forbs), (b) grassland plant species richness and (c) tree species, on AOB diversity and function. Another objective of this research was to study the effect of a 3°C increase in air temperature on AOB diversity and function. The link between AOB diversity and function (potential nitrification) is also investigated. For grassland ecosystems, a microcosm experiment was realized. An experimental platform containing 288 assembled grassland communities was established in Wilrijk (Belgium). Grassland species were grown in 12 sunlit, climate controlled chambers. Each chamber contained 24 communities of variable species richness (S) (9 S=1, 9 S=3 and 6 S=9).The grassland species belonged to three functional groups: three species of each grasses (Dactylis glomerata L., Festuca arundinacea SCHREB., Lolium perenne L.), forbs (non-N-fixing dicots; Bellis perennis L., Rumex acetosa L., Plantagolanceolata L.), and legumes (N-fixing dicots; Trifolium repens L., Medicago sativa L., Lotus corniculatus L.). Half of these chambers were exposed to ambient temperature and the other half were exposed to (ambient +3°C) temperature. One ambient and one (ambient+3°C) chambers were destructively harvested 4, 16 and 28 months after the start of the experiment. The influence of plant functional group identity on the nitrification process and on AOB community structure and richness (AOB diversity) was assessed in soils collected from the first two destructive amplings (chapter 2). The effect of plant species richness on AOB diversity and function was considered for soils sampled after 16 and 28 months (chapter 3). AOB function was determined by potential nitrification. AOB community structure and richness were assessed by polymerase chain reaction followed by denaturing gradient gel electrophoresis (DGGE) and sequencing of excised DGGE bands. I found that functional group identity can affect AOB community structure. In particular, the presence of legumes, both in monoculture or in mixture with forbs and grasses, lead to AOB community composition changes towards AOB clusters tolerating higher ammonium concentrations. This change in AOB community structure was only linked to increased potential nitrification under monocultures of legumes, when ammonium was supposed to be not limiting. This study revealed that physiological attributes of AOB and resource availability may be important factors in controlling the nitrification process. This research showed that the impact of plant species richness on the nitrification process could be mediated by the interactions between plants and AOB, through competition for substrate. A 3°C increase in air temperature did not affect AOB community structure, richness or function. In forest ecosystems, we studied the effect of tree species in forest sites located in Belgian and in the Grand-Duchy of Luxembourg covered each by several deciduous or coniferous tree species (Fagus sylvatica L., Quercus petraea (Mattuschka) Lieblein, Picea abies (L.) Karst, Pseudotsuga menziesii (Mirbel) Franco). We investigated the influence of these tree species on microbial processes (chapter 5) related to C and N cycling, particularly with emphasize on the nitrification process and on the diversity of AOB (chapter 6). The results showed that the effect of tree species on net N mineralization was likely to be mediated through their effect on soil microbial biomass, reflecting their influence on organic matter content and carbon availability. Influence of tree species on nitrification (potential and relative) might be related to the presence of ground vegetation through its influence on soil ammonium and labile C availability. AOB community structure was more site-specific than tree specific. However, within sites, AOB community structure under broadleaved trees differed from the one under coniferous trees. The effect on tree species on AOB was likely to be driven by the influence of tree species on net N mineralization, which regulates the substrate availability for AOB. The results also demonstrated that the relationship between AOB diversity and function might be related both to AOB abundance and AOB community structure and richness. This thesis showed no clear relationship between AOB community structure or richness and AOB function. However, we revealed that aboveground grassland plant richness, grassland plant functional groups and tree species influence AOB community structure and richness. Actuellement, les écosystèmes sont soumis à dimportantes pressions anthropiques et environnementales, pouvant aboutir à des pertes massives de biodiversité. Les scientifiques sinterrogent sur limpact de ces perturbations sur la diversité et, par conséquent, sur lactivité des microorganismes du sol. En effet, ceux-ci régulent de nombreux processus du sol. Actuellement, de nombreuses lacunes subsistent dans la connaissance de la diversité microbienne du sol. Celles-ci peuvent être partiellement attribuées aux difficultés méthodologiques associées à l'étude des micro-organismes du sol. Lavènement des techniques moléculaires nous permet de combler ces lacunes. Les avancées réalisées dans l'étude des communautés microbiennes du sol ont mis en évidence le rôle crucial joué par les communautés microbiennes dans le fonctionnement des écosystèmes. De plus, il semblerait que les processus régulés par un groupe restreint dorganismes, tel le processus de nitrification, soient plus sensibles à toute altération de la communauté. Lobjectif de cette thèse était détudier la structure de la communauté ainsi que la richesse (nombre de bandes DGGE) des bactéries oxydant lammoniac (AOB) comme une partie intégrante du fonctionnement des sols. Notre étude se focalisait sur linfluence de (a) différents groupes fonctionnels de plantes (graminées, légumineuses, dicotylédones), (b) communautés de plantes présentant une richesse spécifique croissante et (c) différentes essences forestières, sur la diversité (structure de la communauté et richesse des AOB) et la fonction des AOB. Cette recherche étudiait également limpact dune augmentation de température de 3°C sur ces paramètres. Létablissement dun lien éventuel entre la diversité et la fonction (nitrification potentielle) des AOB a aussi été envisagé. Concernant les écosystèmes prairiaux, nous avons réalisé une étude en microcosmes. Une plateforme expérimentale comprenant 288 communautés artificielles de plantes a été établie à Wilrijk (Belgique). Cette plateforme consistait en 12 chambres, dont une moitié était à température ambiante et la seconde était à température ambiante augmentée de 3°C. Chaque chambre contient 24 communautés de plantes de richesse spécifique variable (9 S=1, 9 S=3 et 6 S=9). Les communautés de plantes sont créées avec 9 espèces de plantes appartenant à trois groupes fonctionnels : 3 espèces de graminées (Dactylis glomerata L., Festuca arundinacea SCHREB., Lolium perenne L.), de légumineuses (dicotylédones fixatrices dazote ;Trifolim repens L., Medicago sativa L., Lotus corniculatus L.), et de dicotylédones non fixatrices dazote (Bellis perennis L., Rumex acetosa L., Plantago lanceolata L.). Les sols issus dune chambre à température ambiante et dune chambre à température ambiante augmentée de 3°C ont été échantillonnés, respectivement, 4, 16 et 28 mois après le début de lexpérimentation. Linfluence des groupes fonctionnels de plantes sur le processus de nitrification ainsi que sur la structure de la communauté et la richesse des AOB a été mesuré sur les sols issus des deux premiers échantillonnages (chapitre 2). Nous avons mesuré leffet de la richesse croissante en plantes sur la diversité et lactivité des AOB sur les sols échantillonnés après 16 et 28 mois dexpérimentation (chapitre 3). La structure de la communauté ainsi que la richesse des AOB ont été évaluées à laide dune amplification spécifique par réaction de polymérisation en chaîne (PCR) de lADN génomique extrait du sol suivie par une séparation par électrophorèse sur gel dacrylamide en présence dun gradient dénaturant (DGGE). Nous avons identifié les différentes AOB présentes par séquençage des bandes DGGE excisées. Nos résultats ont montré que les différents groupes fonctionnels peuvent affecter la structure de la communauté des AOB. En particulier, la présence de légumineuses, aussi bien en monoculture quen mélange avec des graminées ou des dicotylédones non fixatrices dazote, provoque des changements au sein de la structure de la communauté des AOB, favorisant la présence de clusters tolérants des concentrations en ammonium plus élevées. Ces changements de la structure de la communauté des AOB sont liés à des augmentations de la production potentielle de nitrates (nitrification potentielle) quand lammonium est supposé être non limitant. Cette étude révèle que la physiologie des AOB ainsi que la disponibilité en substrat peuvent être des facteurs majeurs intervenant dans le contrôle du processus de nitrification. Cette recherche montre que linfluence de la richesse spécifique des plantes sur la nitrification pourrait dépendre des interactions entre les plantes et les AOB via la compétition pour le substrat. Une augmentation de la température de lair de 3°C na pas influencé les richesse, structure de la communauté ou les fonctions des AOB. Pour les écosystèmes forestiers, nous aborderons leffet de différentes essences forestières (Picea abies (L.) KARST, Fagus sylvatica L., Quercus petraea LIEBLEIN ; Pseudotsuga menziezii (MIRB.) FRANCO) dans différents peuplements au Grand Duché du Luxembourg et en Belgique. Nous avons étudié l'influence de ces essences forestières sur les processus microbiens (chapitre 5) liés aux cycles du carbone et de lazote, en particulier leur effet sur le processus de nitrification et la diversité des AOB (chapitre 6). Notre étude révèle que linfluence des essences forestières sur la minéralisation nette de lazote pourrait être attribuable à leur effet sur la biomasse microbienne, reflétant ainsi leur effet sur la teneur en matière organique et la disponibilité en carbone. Limpact des essences forestières sur la nitrification (à la fois sur la nitrification relative et sur la nitrification potentielle) serait conditionné par la présence de végétation au sol, en raison de linfluence de celle-ci sur la disponibilité en ammonium et en carbone labile. Nous avons observé que la structure de la communauté des AOB était plus spécifique aux sites quaux essences forestières. Cependant, au sein dun site, elle différait sous feuillus et sous conifères. Les essences forestières influenceraient la structure de la communauté des AOB au travers de limpact quelles ont sur la minéralisation nette de lazote qui régule, quant à elle, la disponibilité en ammonium. Cette recherche démontre que le lien observé entre la diversité et la fonction dépendrait la fois de labondance, de la structure de la communauté et de la richesse des AOB. Cette thèse na révélé aucune relation claire entre la structure de la communauté ou la richesse des AOB et leur fonction. Par contre, nous avons observé que la richesse spécifique et les groupes fonctionnels de plantes prairiales et les essences forestières affectent la structure de la communauté et la richesse des AOB.

Grâce au développement des technologies de métagénomique au cours des dix dernières années, il a été constaté que les micro-organismes représentent la plus grande ressource de diversité métabolique et génétique sur Terre. En effet, un gramme de sol contient 109 cellules bactériennes et 103-104 différentes espèces bactériennes. Certaines sont en mesure de réaliser des réactions enzymatiques conduisant à la dégradation complète de certains polluants toxiques pour l’environnement comme les composés organiques tels que la quinoléine. Cependant, l'immense réservoir de molécules et enzymes microbiennes n'a pas encore été exploité, car plus de 99% d'entre elles ne sont, pour l’instant, pas cultivables in vitro. Mon travail s’inscrit dans le cadre d’une collaboration entre l’Université SJTU (Shanghai Jiao Tong Université en Chine) et le groupe de G. M.E (Génomique Microbienne Environmentale) du laboratoire Ampère à l’Ecole Centrale de Lyon. Nos partenaires à l’Université SJTU ont construit un réacteur de dénitrification à l'échelle du laboratoire capable de dégrader la quinoléine en retirant la demande chimique en oxygène. Un nouvel outil appelé "Genefish" a été developpé dans notre laboratoire comme une méthode alternative de la métagénomique pour aider à la découverte de nouveaux gènes d’intérêt industriel ou environnemental. A la suite des premiers travaux réalisés dans notre laboratoire, ma thèse présentée ici comporte deux parties.Dans la première partie de ce travail, nous avons étudié le potentiel de dégradation de la quinoléine présente dans les bactéries d’un sol de référence largement étudié au laboratoire. Pour cela nous avons mis en place des expériences de microcosme qui visent à révéler la diversité potentielle des bactéries responsables de la dégradation de la quinoléine. Des analyses comparatives des profils RISA (Ribosomal Intergenic Spacer analysis) nous ont permis de mettre en évidence des changements dans la structure de la communauté des bactéries du sol incubé en conditions aérobie et anaérobie en présence de quinoléine. La dégradation de la quinoléine a été confirmée par technique de GC/MS (Gas Chromatography-Mass Spectrometry). Les travaux futurs seront de vérifier la communauté de bactéries responsables de la dégradation de quinoléine en utilisant la technique de NGS (Next Generation Sequencing).Le deuxième objectif de ma thèse a été d'utiliser Genefish dont la finalité est de capturer des gènes ciblés (le gène bcr qui serait responsable de la degradation de quinoléine dans le réacteur de nos partenaires) dans l'ADN métagénomique extrait du sol. Genefish consiste à élaborer une souche d’E.coli incluant un plasmide de capture permettant de pêcher les gènes recherchés dans un échantillon d’ADN metagénomique par recombinaison homologue. Le plasmide de capture comprend une cassette de deux gènes toxiques pour la souche qui activés par induction chimique vont permettre la sélection positive directe des clones recombinants, et deux sites multiples de clonage dans lesquels sont insérées les zones de recombinaison qui vont jouer le rôle d’hameçons. Nous avons testé la capacité de Genefish à capturer des produits PCR du gène bcr, l'efficacité de recombinaison reste faible à cause de la persistance de plusieurs copies du plasmide suicide dans la cellule après l’ évenement de recombinaison. Par conséquent, trois stratégies ont été essayées pour améliorer l’efficacité: la co-électroporation, la ségrégation de plasmide et la construction de plasmide suicide en mono-copie. Finalement, la stratégie de la ségrégation plasmidique fonctionne mais l'efficacité de recombinaison est encore trop faible peut-être due à l’incertitude des modèles de recombinaison homologue. Les travaux futurs se concentreront sur l'amélioration des fréquences de recombinaison par transfert de fragments du plasmide de capture dans le chromosome de la souche Genefish. / As the development of metagenomic technologies in the past ten years, it is unquestionned that microorganisms encompass the largest resource of metabolic and genetic diversity in the world. Actually, one gramme of soil contains more than 109 bacteria and 103-104 species. Some of their members are able to carry out enzymatic reactions leading to the complete degradation of pollutants (such as quinoline). So, the biodegradation of some highly toxic or organic compounds by microorganisms will be a general trend for pollutant treatment. However, the huge reservoir of molecules and enzymes from microorganisms still need to be explored because more than 99% of microorganisms cannot be cultivated in vitro.My work was based on collaboration between the University SJTU and Ecole Centrale de Lyon. Our partners at the University SJTU have built a laboratory scale denitrification reactor which was capable of degrading quinoline by removing the chemical oxygen demand. A new tool called "Genefish" has been developed in our laboratory as an alternative method for metagenomics which aims to discover novel industrial or environmental genes of interest. Following the early work in our laboratory, my thesis is presented here in two parts.In the first part, we set up a quinoline microcosm experiment both under aerobic and anaerobic condition using reference soil extensively studied in the laboratory at Ecole Centrale de LYON. This work aimed to reveal the potential bacterial diversity and even genes responsible for quinoline degradation. We used RISA(Ribosomal intergenic Spacer analysis) to analyze the bacteria community structure changes and GC/MS (Gas Chromatography-Mass Spectrometry) was also used to detect the quinoline degradation and reveal potential quinoline metabolic pathways under aerobic and anaerobic condition. Results showed great bacteria community structure changes and high quinoline degradation activity after the quinoline addition under aerobic condition. The future work is to investigate the bacteria community which may be responsible for quinoline degradation using the technique of NGS (Next Generation Sequencing).The second object of my thesis was to use the Genefish tool to capture targeted genes (the bcr gene responsible for the quinoline degradation in the wastewater treatment bioreactor) from the soil metagenome. The aim was to construct an E.coli strain containing a capture plasmid and Red system for capturing targeted genes from metagenomic DNA by homologous recombination. The capture plasmid includes a toxic cassette consisting of two suicide genes which can be activated by chemical induction, finally support the positive recombinants selection. It also contains two multiply cloning sites in which highly conserved sequences were inserted and works as the bait during recombination. We have tested the capacity of Genefish to capture the PCR products of bcr gene; the efficiency was low because of the persistence of several copies of the capture plasmid into the Genefish strain after recombination events. So, three strategies were tried to improve the recombination efficiency: co-electroporation, plasmid segregation and mono-copy capture plasmid construction. Finally, the strategy of plasmid segregation works but the recombination efficiency was still low maybe caused by the uncertain model of homologous recombination. The further research will focus on the transfer of the toxic cassette and homologous arms into the host strain chromosome, this new strategy will exclude the bad effect of low copy number capture plasmid, uncertain model of λ Red induced homologous recombination and the homologous arms site in the capture plasmid which are the most important factors influencing the homologous recombination efficiency in Genefish.