Deep Hypersaline Anoxic Basin (DHAB) are extreme hypersaline environments located on the seafloor which, despite conditions that are hardly favourable to life, can support thriving ecosystems and demonstrate unique microbial communities. Information on the diversity and functioning of microbial communities living in these harsh environments remains limited, partly because of access to samples and the difficulty of maintaining the sampled microorganisms once back in the laboratory. The brine seawater interface (BSI) is the most active part of DHAB ecosystems and exhibits different successive microbial communities. This study focuses on the Afifi DHAB, a relatively shallow site described in 2020 and located near the eastern coast of the Red Sea, and uses the DNA-Stable Isotope Probing (DNA-SIP) method to link microorganisms’ identity and their associated functions. Seawater layers of different salinities (50 to 250 PSU) from the Afifi BSI were sampled and incubated under anaerobic conditions with different carbon sources: glucose, pyruvate and acetate. Through this approach, the study successfully identified carbon consumers in different layers of the Afifi DHAB and constructed a putative carbon flow among the microorganisms present. Specifically, Vibrionaceae and Halanaerobiaceae are the predominant glucose utilizers, while Thalassospiraceae, Desulfovibrionaceae and Desulfobacteraceae are the main pyruvate and acetate utilizers. Several sulfate-reducing bacteria groups are also identified as potential secondary glucose and pyruvate utilizers, i.e., they utilize the metabolites produced by glucose and pyruvate first consumers. Layers specificities are also revealed, e.g., glucose is consumed by Deferribacteraceae, Rhodobacteraceae and Pseudoalteromonadaceae in the upper, less salty layer, and by Acetothermiia, Melioribacteraceae and Spirochaetacea in the more saline underlying layer. This study provides an initial functional characterization of the microbial community within the Afifi DHAB, highlighting insights into the ecological dynamics and metabolic activities of microorganisms in this unique ecosystem.

Cellulose is the most abundant organic compound on the planet, and is found in nearly every ecosystem. Cellulose is also the most abundant waste product produced by human activity. These enormous stores of natural cellulose and cellulose-containing wastes are a potential renewable energy source. The hydrolysis of cellulose is referred to as cellulolysis and is carried out by cellulase enzymes, which are members of certain glycoside hydrolase families. For most of its history, the microbiology of organisms like those that hydrolyze cellulose was based solely on the testing of physiological and biochemical behaviour of isolated organisms in pure cultures. Despite having gained an important foundation of knowledge in the characterization of microorganisms, cultivation-based techniques introduce major bias into understanding the role that specific microorganisms play because the majority of microorganisms are not readily cultured. Two of the most powerful culture-independent approaches for accessing microbial communities are DNA stable-isotope probing (DNA-SIP) and metagenomics. Though each methodology has been used on its own, it is a combination of these two approaches that has enormous potential to generate results for industrial applications and to help characterize biogeochemical cycling. This thesis presents the first research combining DNA-SIP and metagenomics using cellulose, and the first to target glycoside hydrolase genes from Arctic tundra. For this research, two-month DNA-SIP incubations were carried out with 200 mg of 13C-labelled cellulose as a substrate. Denaturing gradient gel electrophoresis (DGGE) provided evidence indicating the successful enrichment of microorganisms able to metabolize cellulose. Multiple displacement amplification (MDA) was applied to both the bulk-soil samples and DNA-SIP samples. Following MDA, all DNA samples were subjected to Illumina sequencing, including DNA from a cellulose-degrading enrichment. Functional annotation for each Illumina library was done using the SwissProt database within MG-RAST. The DNA-SIP enrichment resulted in a ~3 fold increase in the relative abundance of glycoside hydrolases and cellulase enzymes in relation to bulk soil samples. A cellulose degrading enrichment contained the highest relative abundance of glycoside hydrolases and cellulase enzymes, with a five fold increase relative to the DNA-SIP enrichment. The enrichment culture had a much lower relative diversity, which was measured using the Shannon Index. An unrooted neighbor-joining tree constructed using Bray-Curtis similarity coefficients for each sample demonstrated that as a result of a considerably higher proportion of cellulase gene sequences and a lower diversity the enrichment culture was the most distinct library, with the DNA-SIP library most closely related to it. DGGE provided initial evidence that MDA introduced bias into the amplification of DNA from the DNA-SIP sample. This was confirmed following sequencing and annotation as the proportion of glycoside hydrolase enzymes sequences decreased 67% following MDA of DNA-SIP enriched DNA and the mean G+ C content of libraries decreased. This research provides evidence indicating that DNA-SIP enrichment prior to the construction of metagenomic libraries increases the abundance of targeted gene sequences, which should enable greater access to functional genes of active microorganisms for potential industrial applications.

Cellulose is the most abundant organic compound on the planet, and is found in nearly every ecosystem. Cellulose is also the most abundant waste product produced by human activity. These enormous stores of natural cellulose and cellulose-containing wastes are a potential renewable energy source. The hydrolysis of cellulose is referred to as cellulolysis and is carried out by cellulase enzymes, which are members of certain glycoside hydrolase families. For most of its history, the microbiology of organisms like those that hydrolyze cellulose was based solely on the testing of physiological and biochemical behaviour of isolated organisms in pure cultures. Despite having gained an important foundation of knowledge in the characterization of microorganisms, cultivation-based techniques introduce major bias into understanding the role that specific microorganisms play because the majority of microorganisms are not readily cultured. Two of the most powerful culture-independent approaches for accessing microbial communities are DNA stable-isotope probing (DNA-SIP) and metagenomics. Though each methodology has been used on its own, it is a combination of these two approaches that has enormous potential to generate results for industrial applications and to help characterize biogeochemical cycling. This thesis presents the first research combining DNA-SIP and metagenomics using cellulose, and the first to target glycoside hydrolase genes from Arctic tundra. For this research, two-month DNA-SIP incubations were carried out with 200 mg of 13C-labelled cellulose as a substrate. Denaturing gradient gel electrophoresis (DGGE) provided evidence indicating the successful enrichment of microorganisms able to metabolize cellulose. Multiple displacement amplification (MDA) was applied to both the bulk-soil samples and DNA-SIP samples. Following MDA, all DNA samples were subjected to Illumina sequencing, including DNA from a cellulose-degrading enrichment. Functional annotation for each Illumina library was done using the SwissProt database within MG-RAST. The DNA-SIP enrichment resulted in a ~3 fold increase in the relative abundance of glycoside hydrolases and cellulase enzymes in relation to bulk soil samples. A cellulose degrading enrichment contained the highest relative abundance of glycoside hydrolases and cellulase enzymes, with a five fold increase relative to the DNA-SIP enrichment. The enrichment culture had a much lower relative diversity, which was measured using the Shannon Index. An unrooted neighbor-joining tree constructed using Bray-Curtis similarity coefficients for each sample demonstrated that as a result of a considerably higher proportion of cellulase gene sequences and a lower diversity the enrichment culture was the most distinct library, with the DNA-SIP library most closely related to it. DGGE provided initial evidence that MDA introduced bias into the amplification of DNA from the DNA-SIP sample. This was confirmed following sequencing and annotation as the proportion of glycoside hydrolase enzymes sequences decreased 67% following MDA of DNA-SIP enriched DNA and the mean G+ C content of libraries decreased. This research provides evidence indicating that DNA-SIP enrichment prior to the construction of metagenomic libraries increases the abundance of targeted gene sequences, which should enable greater access to functional genes of active microorganisms for potential industrial applications.

La France est dépendante en gaz naturel dont elle importe 98% de sa consommation. Comme pour plusieurs autres pays (Etats-Unis, Canada, Grande Bretagne, Autriche, Allemagne, etc.), le stockage de gaz est principalement réalisé afin de pallier aux variations saisonnières de consommation. Grâce aux spécificités géologiques de notre territoire, ce stockage se fait essentiellement aux niveaux d’aquifères très profonds (-500 à 1000 mètres). Le gaz naturel contient en majorité du méthane mais également des traces d’autres composés tels que les BTEX (Benzène, Toluène, Ethylbenzène et les trois isomères du Xylène) qui sont connus pour leur toxicité. Ces hydrocarbures monoaromatiques peuvent se solubiliser dans l’eau de formation aux niveaux des interfaces eau/gaz. Leur biodégradation est bien moins rapide en anaérobiose qu’en aérobiose mais un potentiel d’atténuation naturelle des BTEX par les communautés microbiennes indigènes a déjà pu être démontré lors de travaux antérieurs. Cependant, bien que de nombreuses études aient été réalisées sur le sujet, les voies de dégradation anaérobie ne sont que partiellement connues et les connaissances concernant les microorganismes impliqués sont réduites, voire inexistantes. Le développement de biomarqueurs moléculaires in situ doit permettre d’évaluer rapidement le potentiel de dégradation des microorganismes d’un aquifère. Pour atteindre cet objectif, il est indispensable d’acquérir une meilleure compréhension des mécanismes de dégradation et donc, d’isoler les microorganismes impliqués dans la dégradation de ces composés. Au cours de cette étude, des communautés microbiennes provenant d’échantillons d’eau de formation issue de trois aquifères distincts (nommés dans ce travail A, C et D) ont été étudiées à l’aide de trois approches différentes de microbiologie environnementale. L’ensemble de ces résultats ainsi que ceux de la littérature suggèrent l’ubiquité des bactéries sulfato-réductrices à Gram positif tels que les Desulfotomaculum dans les environnements profonds. Les résultats obtenus lors de ce travail de doctorat suggèrent le rôle prépondérant de microorganismes affiliés au genre Desulfotomaculum dans la dégradation des BTEX en aquifères très profonds et représentent une avancée dans la compréhension des phénomènes d’atténuation naturelle des BTEX dans ce type d’environnement. / France is dependent on natural gas and imports 98% of its consumption. Like in many other countries (The United States of America, Canada, Great Britain, Austria, Germany, etc.), gas storage is primarily performed to compensate for seasonal variations in consumption. Geological characteristics of our territory allow to store essentially natural gas into deep aquifers (-500 to 1000 meters). Natural gas contains mostly methane, but also traces of other compounds such as BTEX (Benzene, Toluene, Ethylbenzene and the three isomers of Xylene) which are known to be toxic. These mono-aromatic hydrocarbons are soluble in water. Anaerobic biodegradation is much slower than aerobic processes however potential for anaerobic BTEX natural attenuation by indigenous microbial communities has already been shown previously. Although many studies have been done on the topic, the anaerobic degradation pathways are only partially known and the knowledge of microorganisms involved is low or nonexistent. The development of in situ molecular biomarkers will allow rapid evaluation of the potential degradation of aquifer microorganisms. To achieve this goal, a better understanding of the mechanisms of degradation is crucial and requires isolation of microorganisms involved in the degradation of these compounds. In this study, microbial communities sampled from formation waters of three distinct aquifers (named in this work A, C and D) were studied using three different environmental microbiology approaches. All these results and those from the literature suggest the ubiquity of sulfate-reducing bacteria such as Gram positive Desulfotomaculum in deep environments. The results obtained during this PhD suggest the importance of microorganisms related to the genus Desulfotomaculum in BTEX degradation in deep aquifers. This work represents a step forward in understanding the phenomenon of natural attenuation of BTEX in this kind of environment.

La compréhension des interactions qui associent les plantes et les microorganismes du sol est une étape incontournable pour une gestion durable de nos écosystèmes notamment en agriculture. Parmi les services écosystémiques résultant de leurs interactions, on peut citer la productivité végétale répondant, en partie, aux besoins alimentaires de la population mondiale et la régulation des cycles biogéochimiques. Les services écosystémiques, qui émergent de telles interactions, reposent sur des liens trophiques pouvant être représentés par un compromis entre coûts et bénéfices pour les différents partenaires de l’interaction. Les plantes, organismes autotrophes ou producteurs primaires, sont des organismes clefs qui font entrer le carbone dans l’écosystème, via la photosynthèse. Une partie de ce carbone est libérée sous forme de molécules plus ou moins complexes, au niveau de leurs racines, par le processus de rhizodéposition. Ces composés servent de molécules signal et de nutriments pour les microorganismes du sol, essentiellement hétérotrophes, c’est l’effet rhizosphère. Ce processus est donc coûteux pour la plante mais bénéfique aux microorganismes. Les microorganismes contribuent, en retour, à la nutrition et la santé des plantes ce qui est coûteux mais leur assure une source bénéfique de nutriments. Ces échanges trophiques reposent néanmoins sur un équilibre dépendant des conditions biotiques et abiotiques qui affectent chaque partenaire. La biodiversité microbienne, de par la multitude d’interactions au sein des communautés microbiennes, est un facteur biotique important. Parmi les facteurs abiotiques, le contexte environnemental actuel, soumis aux changements globaux, est propice à une déstabilisation de ces interactions. L’objectif de ce travail est donc de comprendre comment vont varier les coûts et bénéfices, pour chaque partenaire, suite à des modifications de l’environnement affectant l’un ou l’autre. L’intérêt étant de savoir si les bénéfices pour les plantes et les microorganismes, qui permettent les services écosystémiques, seront affectés. Pour répondre à cet objectif, un cadre d’interaction plantes-microbes simplifié a été choisi et une déstabilisation, au niveau de la plante, a été effectuée au moyen d’une augmentation en CO2 atmosphérique. L’interaction entre Medicago truncatula et Pseudomonas fluorescens a ainsi été étudiée. Les interactions ont ensuite été complexifiées en utilisant une communauté microbienne dans son ensemble et, cette fois, la modification a été appliquée au compartiment microbien soumis à une dilution de sa diversité. L’effet du gradient de diversité microbienne obtenu a été mesuré sur la croissance et la reproduction de trois espèces végétales modèles (Medicago truncatula, Brachypodium distachyon et Arabidopsis thaliana). Enfin, l’analyse s’est focalisée sur la communauté microbienne en identifiant la part active, c'est-à-dire les microorganismes qui utilisent les composés libérés par la plante. Ces microorganismes, qui interagissent réellement avec la plante, ont été détectés grâce à une analyse ADN SIP utilisant l’isotope 13C. Les principaux résultats observés, que la modification affecte l’un ou l’autre des partenaires, sont une déstabilisation des coûts et bénéfices. La première étude montre une variation temporaire des interactions en faveur de la plante en condition de CO2 augmenté. Dans le cas d’une dilution de la diversité microbienne, les coûts pour la plante sont conditionnés par la dépendance naturelle des plantes vis-à-vis des microorganismes symbiotiques qui interagissent avec le reste de la communauté. Cela est confirmé par la dernière expérimentation qui met en évidence les interactions microbes-microbes qui conditionnent la structure de la communauté microbienne interagissant avec la plante. [...] / Understanding the interactions that bind plants and soil microorganisms is an essential step for the sustainable management of ecosystems, especially in agriculture. The ecosystem services resulting from such interactions include plant productivity which responds, in part, to the food requirements of the world's population and the regulation of biogeochemical cycles. These ecosystem services depend on trophic links between the two partners in the interaction and can be represented by a tradeoff between the costs and benefits for each partner. Plants, being autotrophic organisms or primary producers, are key organisms which introduce carbon into the ecosystem, through photosynthesis. Part of this carbon is released as more or less complex molecules at the roots level, thanks to the rhizodeposition process. These compounds act as signal molecules and nutrients for soil microorganisms, which are mainly heterotrophic, in the so-called rhizosphere effect. This process is costly for the plant but beneficial to the microorganisms. In return, microorganisms contribute to plant nutrition and health, which is costly but provides them with a beneficial source of nutrients. These trophic exchanges, however, are based on a balance which depends on the biotic and abiotic conditions that affect each partner. Microbial biodiversity, through the multitude of interactions occurring within microbial communities, is a significant biotic factor. Among the abiotic factors, the current environmental context, subject to global change, is tending to destabilize these interactions. The objective of this work was to understand how environmental changes affect the costs and benefits for each partner by applying changes to one or the other, the aim being to determine whether these changes would affect the benefits for plants and microorganisms that provide ecosystem services. To achieve this objective, a simplified framework for plants-microbes interaction was first chosen. Destabilization at the plant level was carried out by increasing the atmospheric CO2 and studying the interaction between Medicago truncatula and Pseudomonas fluorescens. The interactions were then made more complex by using a whole microbial community but this time the change was applied to the microbial compartment by subjecting it to diversity dilution. The effect of the resulting microbial diversity gradient was measured on the growth and reproduction of three model plant species (Medicago truncatula, Brachypodium distachyon and Arabidopsis thaliana). Finally, the microbial community was subjected to a DNA SIP analysis, with the isotope 13C, to identify the active portion, i.e., those microorganisms which really interacted with the plant and used compounds released by it. The main result, when the change affected one or other partner, was a destabilization of the costs and benefits. The first study showed a transient variation in the interactions in favour of the plant under increased CO2 conditions. In the case of a dilution of microbial diversity, the costs for the plant are conditioned by the natural dependency of plants on symbiotic microorganisms that interact with the rest of the community. This was confirmed by the last experiment that highlighted the between-microbes interactions which determined the composition of the microbial community that interacted with the plant. This work has helped to clarify the functioning of relationships between plants and soil microbes and the factors that contribute to their maintenance which is essential to the functioning of ecosystems. These studies also provide ways for predicting the impacts of global change on ecosystems. The conservation or restoration of ecosystem services is essential for human well-being

Agricultural management is essential for achieving optimum crop production and maintaining soil quality. Soil microorganisms are responsible for nutrient cycling and are an important consideration for effective soil management. The overall goal of the present research was to better understand microbial communities in agricultural soils as they relate to soil management practices. For this, we evaluated the differential impact of two contrasting drainage practices on microbial community composition and characterized active denitrifiers from selected agricultural sites. Field drainage is important for crop growth in arable soils. Controlled and uncontrolled tile drainage practices maintain water in the field or fully drain it, respectively. Because soil water content influences nutrient concentration, moisture, and oxygen availability, the effects of these two disparate practices on microbial community composition was compared in paired fields that had diverse land management histories. Libraries of the 16S rRNA gene were generated from DNA from 168 soil samples collected from eight fields during the 2012 growing season. Paired-end sequencing using next-generation sequencing was followed by read assembly and multivariate statistical analyses. Results showed that drainage practice exerted no measureable effect on the bacterial communities. However, bacterial communities were impacted by plant cultivar and applied fertilizer, in addition to sampled soil depth. Indicator species were only recovered for depth; plant cultivar or applied fertilizer type had no strong and specific indicator species. Among indicator species for soil depth (30-90 cm) were Chloroflexi (Anaerolineae), Betaproteobacteria (Janthinobacterium, Herminiimonas, Rhodoferax, Polaromonas), Deltaproteobacteria (Anaeromyxobacter, Geobacter), Alphaproteobacteria (Novosphingobium, Rhodobacter), and Actinobacteria (Promicromonospora). Denitrification in agricultural fields transforms nitrogen applied as fertilizer, reduces crop production, and emits N2O, which is a potent greenhouse gas. Agriculture is the highest anthropogenic source of N2O, which underlines the importance of understanding the microbiology of denitrification for reducing greenhouse gas emissions by altered management practices. Existing denitrifier probes and primers are biased due to their development based mostly on sequence information from cultured denitrifiers. To circumvent this limitation, this study investigated active and uncultivated denitrifiers from two agricultural sites in Ottawa, Ontario. Using DNA stable-isotope probing, we enriched nucleic acids from active soil denitrifiers by exposing intact replicate soil cores to NO3- and 13C6-glucose under anoxic conditions using flow-through reactors, with parallel native substrate controls. Spectrophotometric chemistry assays and gas chromatography confirmed active NO3- depletion and N2O production, respectively. Duplicate flow-through reactors were sacrificed after one and four week incubation periods to assess temporal changes due to food web dynamics. Soil DNA was extracted and processed by density gradient ultracentrifugation, followed by fractionation to separate DNA contributed by active denitrifiers (i.e., “heavy” DNA) from that of the background community (i.e., “light” DNA). Light and heavy DNA samples were analyzed by paired-end sequencing of 16S rRNA genes using next-generation sequencing. Multivariate statistics of assembled 16S rRNA genes confirmed unique taxonomic representation in heavy fractions from flow-through reactors fed 13C6-glucose, which exceeded any site-specific or temporal shifts in putative denitrifiers. Based on high relative abundance in heavy DNA, labelled taxa affiliated with the Betaproteobacteria (71%; Janthinobacterium, Acidovorax, Azoarcus, Dechloromonas), Alphaproteobacteria (8%; Rhizobium), Gammaproteobacteria (4%; Pseudomonas), and Actinobacteria (4%; Streptomycetaceae). Metagenomic DNA from the original soil and recovered heavy fractions were subjected to next-generation sequencing and the results demonstrated enrichment of denitrification genes with taxonomic affiliations to Brucella, Ralstonia, and Chromobacterium in heavy fractions of flow-through reactors fed 13C6-glucose. The vast majority of heavy-DNA-associated nitrite-reductase reads annotated to the copper-containing form (nirK), rather than the heme-containing enzyme (nirS). Analysis of recovered nirK genes demonstrated low sequence identity across common primer-binding sites used for the detection and quantification of soil denitrifiers, indicating that these active denitrifiers would not have been detected in molecular surveys of these same soils.