Environmental gradients exist at global and local scales and the variable conditions they encompass allow the coexistence of different microbial assemblages. Studying gradients and the selection forces they enclose can reveal the spatial succession and interactions of microorganisms and, therefore, how they are assembled in functionally stable communities. By combining high-throughput sequencing technology and laboratory experimental approaches, I investigated the factors that influence the microbial community assemblages in two types of environmental gradients in the Red Sea. I have studied the communities in the chemoclines occurring at the transition zones along the interfaces between seawater and the Deep Hypersaline Anoxic Brines (DHABs) at the bottom of the Red Sea. Across these chemoclines salinity increases of 5-10 times respect to the overlying seawater. I compared the microbial community diversity and metabolisms in the chemoclines of five different DHABs, finding different microbial community compositions due to the different DHABs characteristics, but the same succession of metabolisms along the five interfaces. From the Suakin Deep brine, I assembled the genome of a novel bacterial phylum and revealed the metabolic features that allow this organism to cope with the challenging variable conditions along the chemocline. In an alternative environmental system, I studied the effect of different thermal regimes on the microbiome of coastal sediment exposed to different yearly ranges of temperature variation. Sediment bacterial communities living under larger temperature variations are more flexible and can grow under a larger range of thermal conditions than communities experiencing narrower temperature ranges. My results highlight the large metabolic flexibility of microorganisms and their capacity to efficiently self-organize in complex functional assemblages under extreme ranges of environmental conditions.

Thesis (Ph.D.)--Joint Program in Oceanography (Massachusetts Institute of Technology, Dept. of Civil and Environmental Engineering; and the Woods Hole Oceanographic Institution), 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Extensive genetic diversity has been discovered in the microbial world, yet mechanisms that shape and maintain this diversity remain poorly understood. This thesis investigates to what extent populations of the gamma-proteobacterial family, Vibrionaceae, are ecologically specialized by investigating the distribution across a wide range of environmental categories, such as marine invertebrates or particles in the water column. Additionally, it seeks to determine whether in situ population distributions directly result from a competitive advantage over other Vibrio populations. This was investigated by in vitro competition assays on mixtures of native, sterilized particles. Generalist populations were found to dominate the associations with marine invertebrates, consistent with a model of high migration dominated population assembly. A majority of populations occurred broadly within and among the different types of invertebrates sampled, with one population being a near perfect generalist with regard to seasons, host taxa and body regions. High variability across host individuals, consistent with a scenario of stochastic clonal expansion, was especially pronounced in crab and zooplankton samples. Specialization, demonstrated by specific and reproducible association with different particle types in the water column, is more common than specialization within invertebrate hosts. / (cont.) Co-existing Vibrio species show strong preferences for different types of particulate matter in the water column suggesting that competition for limited resources influences their evolution. While populations show different growth profiles on particle derived substrates, relative growth advantages of specialist populations in competition with other Vibrio populations on native particles may not be sufficient to explain observed environmental distributions. Instead, populations may gain an advantage on these particles by colonizing the living plant or zooplankton prior to death and degradation into particulate matter. In summary, although vibrios are known commensals of marine invertebrates, evidence suggests that population structure within animals is fairly weak compared to suspended particles in the water column. This highlights the importance of comparing multiple environmental categories and migration among them to investigate population structure and adaptation. / by Sarah Pacocha Preheim. / Ph.D.

Understanding the response of soil microbial communities to various environmental stresses is of current interest, because of their pivotal role in nutrient cycling, soil organic matter mineralization and influence on plant growth. Determining the affect of several biotic and abiotic factors on soil microbial communities is the overall objective of the study. The specific goals are to determine 1) the response of microbial communities to water deficit in soil and 2) how the presence of a rich biotic community determines the direction of microbial community development in cultures. Both goals are novel and unique contributions to understanding microbial ecology in soil. Dynamics in water potentials due to drying and rewetting of soil impose significant physiological challenges to soil microorganisms. To cope with these fluctuations, many microorganisms alter the chemistry and concentration of their cytoplasmic contents. The aim of this research is to understand how the microbial biomass and their cytoplasm change in response to water potential deficits under in situ soil conditions. To address this objective we characterized intracellular and extracellular metabolites in moist, dry and salt stressed soils. Our results provided the first direct evidence that microbial communities in soil in situ utilize sugars and sugar alcohols to cope with low water potential. While the cultivation and isolation of microorganisms is essential to completely explore their physiology and ecology, 99% of soil microbes resist growing in cultures. Presence of very unnatural conditions in the culture plates was considered as main reason for low cultivability. Thus, a culture-based study was conducted whereby microorganisms were grown in association with their native habitat with an objective of mimicking native conditions to promote the growth of previously uncultivated microorganisms. Moreover, the importance of biotic communities (microbe-microbe) and abiotic soil effects were assessed on bacterial growth. Our results strongly indicate that the presence of living microbial community in the vicinity of the target culture resulted in the cultivation of novel members of rare bacterial taxa from phyla Verrucomicrobia, Bacteroidetes, Proteobacteria, and Planctomycetes. These results emphasize the need to develop new culturing methods to tap the hidden microbial potential for emerging anthropogenic needs.

The nature and extent of microbial biodiversity remain controversial with persistent debates over patterns of distributions (i.e. cosmopolitanism vs. endemism) and the processes that structure these patterns (neutrality vs. selection). We used culture-independent approaches to address these issues focusing on two groups of ciliates, the Oligotrichia (Spirotrichea) and Choreotrichea (Spirotrichea). To assess the diversity of these ciliates, we designed primers specific to SSU rDNA of ciliates within these clades, and investigated (1) geographic and temporal distributions along three coastal sites in the Northwest Atlantic; (2) the relationship between ciliate communities in the benthos and the plankton along the New England coast; and (3) diversity in ciliate communities across an environmental gradient at six stations in Long Island Sound spanning the frontal region that separates the fresher Connecticut River outflow plume from the open Sound. Each collection had its own distinct assemblage of rare and abundant ciliate haplotypes, and genealogical analyses of our samples combined with published sequences from identified morphospecies reveal that haplotype diversity at these sites is greatest within the genus Strombidium, in the Oligotrichia. Clustering of phylogenetic types indicates that benthic assemblages of oligotrichs and choreotrichs appear to be more like those from spatially distinct benthic communities than the ciliate communities sampled in the water above them. Neither ciliate diversity nor species composition showed any clear relationship to measured environmental parameters (temperature, salinity, accessory pigment composition, and chorophyll), although we observed that diversity decreased moving from nearshore to offshore. We find no strong fit of our communities to log series, geometric, or log normal distributions, though one of the 3 clusters is most consistent with a log series distribution. These analyses suggest that Oligotrich and Choreotrich communities in coastal environments may be distributed in a neutral manner. We investigated the effectiveness of molecular approaches in characterizing ciliate diversity in our samples. Estimates of diversity based on molecular markers are similar to estimates from morphological observations for Choreotrich ciliates, but much greater for Oligotrich ciliates. Sediment and plankton subsamples differed in their robustness to repeated subsampling. Sediment gave variable estimates of diversity while plankton subsamples produced consistent results.

No description available.

Continental shelves located along eastern boundary currents occupy relatively small volumes of the world’s oceans, yet are responsible for a large proportion of global primary production. The Oregon coast is among these ecosystems. Recent analyses of dissolved oxygen at shallow depths in the water column has suggested increasing episodes of hypoxia and anoxia, events that are detrimental to larger macro-faunal species. Microbial communities, however, are metabolically diverse, capable of utilizing alternative electron donors and acceptors, and can withstand transient periods of low dissolved oxygen. Understanding the phylogenetic and metabolic diversity of microorganisms in these environments is important for assessing the impact hypoxic events have on local and global biogeochemistry. Several molecular ecology tools were used to answer questions about the distribution patterns and activities of microorganisms residing along the coast of Oregon in this dissertation. Ribosomal rRNA fingerprinting and sequence analyses of samples collected during 2007-2008 suggested that bacterial community structure was not substantially influenced by changes in dissolved oxygen. However, substantial depth dependent changes were observed, with samples collected in the bottom boundary layer (BBL) displaying significant differences from those collected in the surface layer. Phylogenetic analyses of bacterial rRNA genes revealed novel phylotypes associated with this area of the water column, including groups with close evolutionary relationships to putative or characterized sulfur oxidizing bacteria (SOB). Analysis of metagenomes and metatranscriptomes collected during 2009 suggested increasing abundances of chemolithoautrophic organisms and their activities in the BBL. Thaumarchaea displayed significant depth dependent increases during the summer, and were detected at maximal frequencies during periods of hypoxia, suggesting that nitrification maybe influenced by local changes in dissolved oxygen. Metagenomic analysis of samples collected from 2010 revealed substantial variability in the metabolic potential of the microbial communities from different water masses. Samples collected during the spring, prior to upwelling clustered independently of those collected during the summer, during a period of upwelling, and did not display any clear stratification. Samples collected during the summer did cluster based on depth, consistent with previous observations, and increases in the relative abundances of chemolithotrophic gene suites were observed in the BBL during stratified conditions, suggesting that the metabolic potential for these processes is a repeatable feature along the Oregon coast. Overall, these observations suggest that depth impacts microbial community diversity, metabolic potential, and transcriptional activity in shallow areas of the Northeast Pacific Ocean. The increase in lithotrophic genes and transcripts in the BBL suggests that this microbial community includes many organisms that are able to use inorganic electron donors for respiration. We speculate that the dissolved organic material in the BBL is semi-labile and not available for immediate oxidation, favoring the growth for microorganisms that are able to use alternative electron donors. / Graduation date: 2012

With rising concentrations of CO₂ in the Earth's atmosphere causing concern about climate change, many solutions are being presented to decrease emissions. One of the proposed solutions is to sequester excess CO₂ in geological formations such as basalt. The deep subsurface is known to harbor much of the microbial biomass on earth and questions abound as to how this deep life is going to respond to the injection of CO₂. Many studies have used model microorganisms to demonstrate the ability of microbes to aid in the safe, permanent sequestration of CO₂ in the subsurface. The objective of this research is to characterize the microbial community present in the basalts at the Wallula pilot carbon sequestration well prior to the injection of CO₂ and then perform laboratory studies to determine how the native microbial community will respond to carbon sequestration conditions. Six samples were collected from the Wallula pilot well prior to the injection of CO₂ into the system. The microorganisms in these samples were characterized by pyrosequencing of 16S rRNA genes, revealing a community dominated by the Proteobacteria, Firmicutes, and Actinobacteria. The organisms detected were related to microbes known to metabolize hydrogen, sulfur, and single carbon compounds. These microorganisms may be stimulated in formations located at the fringe of the pool of injected CO₂. Laboratory studies revealed that the native microbial community suffered a two order of magnitude loss of population upon exposure to CO₂ under carbon sequestration conditions. The community also shifted from being dominated by Proteobacteria prior to CO₂ exposure to being dominated by Firmicutes after exposure. Specifically, the genus Alkaliphilus, which was previously undetected, appeared after CO₂ exposure and became dominant. The dominance of Alkaliphilus, along with other rare organisms which did not compose a majority of the population prior to the introduction of CO₂ to the system, indicates that members of the rare biosphere may be better adapted to changing environmental conditions specific to CO₂ sequestration than other indigenous cells. Thus, the rare biosphere should be examined closely as part of any environmental study, as these minority microorganisms may be the first indication of perturbation or impact. / Graduation date: 2013

La fertilisation phosphatée est très répandue dans les pratiques agricoles Nord-Américaines. Bien que généralement très efficace pour augmenter la production végétale, son utilisation peut engendrer certaines contaminations environnementales. Afin de diminuer ce problème, plusieurs pratiques de gestion sont envisagées. Parmi celles-ci, on retrouve l’intéressante possibilité de manipuler la flore microbienne car cette dernière est reconnue pour son implication dans bons nombres de processus fondamentaux liés à la fertilité du sol. Cette étude a démontré que lors d’essais en champs, la forme de fertilisant ajouté au sol ainsi que la dose de phosphore (P) appliquée avaient un impact sur la distribution des microorganismes dans les différentes parcelles. Une première expérience menée sur une culture de luzerne en prairie semi-aride a montré que les échantillons provenant de parcelles ayant reçu différentes doses de P présentaient des différences significatives dans leurs communautés bactériennes et fongiques. La communauté de CMA est restée similaire entre les différents traitements. Une deuxième expérience fut menée pendant trois saisons consécutives afin de déterminer l’effet de différentes formes de fertilisation organiques et minérale ajustées selon une dose unique de P sur les populations bactériennes et fongiques d’une culture intensive de maïs en rotation avec du soja. Les résultats des analyses ont montrés que les populations varient selon le type de fertilisation reçu et que les changements sont indépendants du type de végétaux cultivé. Par contre, les populations microbiennes subissent une variation plus marquée au cours de la saison de culture. La technique de DGGE a permis d’observer les changements frappant la diversité microbienne du sol mais n’a permis d’identifier qu’une faible proportion des organismes en cause. Parallèlement à cette deuxième étude, une seconde expérience au même site fut menée sur la communauté de champignons mycorhiziens à arbuscules (CMA) puisqu’il s’agit d’organismes vivant en symbiose mutualiste avec la majorité des plantes et favorisant la nutrition de même que l’augmentation de la résistance aux stress de l’hôte. Ceci permit d’identifier et de comparer les différents CMA présents dans des échantillons de sol et de racines de maïs et soja. Contrairement aux bactéries et aux champignons en général, les CMA présentaient une diversité très stable lors des différents traitements. Par contre, au cours des trois années expérimentales, il a été noté que certains ribotypes étaient significativement plus liés au sol ou aux racines. Finalement, l’ensemble de l’étude a démontré que la fertilisation phosphatée affecte la structure des communautés microbiennes du sol dans les systèmes évalués. Cependant, lors de chaque expérience, la date d’échantillonnage jouait également un rôle prépondérant sur la distribution des organismes. Plusieurs paramètres du sol furent aussi mesurés et ils présentaient aussi une variation au cours de la saison. L’ensemble des interactions possibles entre ces différents paramètres qui, dans certains cas, variaient selon le traitement appliqué, aurait alors probablement plus d’impact sur la biodiversité microbienne que la seule fertilisation. / Phosphorus fertilization is a widespread practice in North American agriculture. Although it is generally efficient to increase yields, its use can also induce some environmental contaminations. Several management practices are considered in order to decrease this problem. Among these possibilities there is the challenging one of manipulating microbial flora, which is well known for its implication in many processes related to soil fertility. We have demonstrated in field trials that both the form of fertilizer added to soil and the applied P amounts impact microbial distribution in plots. A first experiment performed on alfalfa monocultures in semi-arid prairie conditions demonstrated that samples coming from plots that had received different doses of P fertilizer presented significant differences on their bacterial and fungal communities. AMF population remained stable between treatments. A second experiment was conducted over three growing season of an intensive maize/soybean rotation cropping system. It aimed to determine the effect of different organic and mineral fertilizers containing equal P amount on bacterial and fungal populations. It was demonstrated that these communities varied according to the fertilizer type applied. Changes are independent from the grown crop. However, microbial populations have undergone greater variation within each growing season. DGGE approach allowed to observe changes occurring in soil microbial diversity but have only permit to identify a small proportion of organisms. A second experiment in the latter study was performed on the same site and focused on arbuscular mycorrhizal fungi (AMF) as they are organisms living in a mutualistic symbiosis with most land plants and increasing host nutrition and resistance to stresses. It led to the identification and comparison of the different AMF found in maize and soybean soil and root samples. In opposition to what was observed with bacteria and fungi previously, AMF presented a very stable diversity between the different treatments. However, some ribotypes were significantly more present in soil or roots during each growing season. Finally, our whole project demonstrated that P fertilization affected microbial community structure on studied sites. Nevertheless, in each experiment, sampling time also played a substantial role in the organism distribution. Many soil parameters were also monitored and presented a seasonal variation. The sum of possible interactions between these parameters, which in some cases varied according to treatment, would thus have more impact on microbial diversity that the sole fertilization.

L'ancienne mine de plomb et de zinc de Carnoulès (Gard, France) a généré 1.5 Mt de déchets d'où émerge un drainage de mine aux eaux acides alimentant un ruisseau, le Reigous. Ce site fournit un exceptionnel exemple d'adaptation à un environnement extrême en raison des eaux acides (pH~3) et de très fortes concentrations en métaux et métalloïdes, particulièrement en As. Dans les 30 premiers mètres du ruisseau, l'activité bactérienne conduit à un phénomène de remédiation naturelle avec la co-précipitation de 20 à 60% de l'As dissous avec du fer. Les bactéries présentes dans les sédiments du ruisseau ont dans un premier temps été décrites par clonage/séquençage du gène de l'ARNr 16S, puis les membres actifs des communautés bactériennes ont été révélés par une approche de métaprotéomique. L'étude des Archaea au sein des sédiments a révélé la présence de groupes impliqués dans la méthanogénèse ou dans l'oxydation de l'ammoniac qui pourraient participer au cycle du carbone ou de l'azote. Les eucaryotes ont été caractérisés pour la première fois sur ce site par pyroséquençage, mettant en évidence une forte proportion de champignons (60%). Enfin, l'étude des variations spatiotemporelles des populations bactériennes dans les eaux a conduit à l'identification de 6801 OTUs dont des phyla encore jamais identifiés sur ce site. La concentration en arsenic, la température et le potentiel redox semblent jouer un rôle dans la structuration de ces communautés. Ce travail de thèse a ainsi contribué à une meilleure connaissance des microorganismes présents (Bactéries, Archaea, Eucaryotes) et de leurs dynamiques spatiotemporelles en relation avec les paramètres physicochimiques du milieu. / Acidic mining drainage generated at Carnoulès, a former Pb-Zn mine (Gard, France) coincides with the spring of the Reigous Creek. This site provides an exceptional example of adaptation to extreme environments due to its acidic water (pH~3) and very high concentrations of metals and metalloids, particularly arsenic. During the first 30 m of downflow in Reigous Creek, natural remediation occurred, with co-precipitation of 20 to 60% of the dissolved arsenic with iron, mediated by bacteria. Bacterial communities inhabiting the creek sediments were first described by cloning/sequencing of the 16S rRNA genes and the active members were identified by a metaproteomic approach. A survey of the archaeal community in the sediment highlighted the presence of sequences phylogenetically related to methanogenic Archaea and to ammonia oxidizers, which could be involved in carbon and nitrogen biochemical cycling. The Eukaryotic communities were studied for the first time at this site by pyrosequencing, revealing that around 60% of the sequences belonged to Fungi. Finally, the study of the spatiotemporal variations of the water bacterial communities allowed the identification of 6801 OTUs including sequences of taxa never detected before. The environmental variables significantly correlated with bacterial community dynamics appear to be arsenic concentration, temperature and Eh. This PhD work has contributed to a better understanding of the spatiotemporal dynamics of microorganisms (Bacteria, Archaea, Eukaryotes) in relation with the physicochemical parameters of their environment.

Sedimentos de mar profundo são ambientes estáveis e homogêneos, apesar disso, eles apresentam uma grande variedade de habitats disponíveis, possibilitando uma alta diversidade microbiana. A distribuição espacial dos micro-organismos é influenciada por fatores locais e regionais. Os fatores locais são associados à estrutura do ambiente e os fatores regionais, a limitação na dispersão dos micro-organismos que compõem as comunidades e eventos históricos que eventualmente podem modificar o ambiente. Eventos como a liberação de hidrocarbonetos das camadas mais profundas do sedimento para superfície podem alterar os padrões de distribuição espacial das comunidades microbianas, devido o aumento na disponibilidade de carbono e consequentemente selecionando as espécies capazes de degradá-los. Esses eventos são denominados de exsudações de asfalto e foram encontradas na região de estudo. Considerando a falta de conhecimento e a importância dos micro-organismos em sedimentos de mar profundo do oceano Atlântico Sul, o objetivo desse estudo foi compreender os padrões de diversidade microbiana nessas regiões e também investigar como o óleo proveniente da exsudação de asfalto influência as comunidades de micro-organismos no seu entorno. Esse estudo foi realizado na região do Platô de São Paulo que foi dividido em duas regiões, norte e sul. A diversidade microbiana foi estudada em 14 amostras de sedimento de mar profundo, nove amostras na região norte e cinco na sul. A exsudação de asfalto foi encontrada na região norte, influenciando diretamente três das nove amostras. As comunidades foram estudadas através dos métodos de eletroforese em gel de gradiente desnaturante (DGGE), PCR quantitativa (qPCR) e sequenciamento de última geração (Ion Torrent). A distribuição espacial das comunidades foi analisada em diferentes escalas espaciais: verticalmente, variando com a profundidade do sedimento (≤ 4 cm), localmente, em cada uma das regiões amostradas (1-34 Km) e regionalmente, comparando as regiões norte e sul (> 250 Km). O perfil da comunidade obtido com a técnica de DGGE mostrou que as comunidades microbianas foram menos similares entre as regiões e apresentaram relação com a distância geográfica para arqueia e bactéria. Os valores de similaridade foi maior localmente do que regionalmente. A similaridade obtida nas camadas de profundidade analisadas foi alta e não houve relação com a distância geográfica. O número de células entre as camadas de profundidades foi diferente, com tendência de diminuição com o aumento da profundidade. As classes bacterianas mais abundantes foram Alphaproteobacteria (30%), Acidimicrobiia (18%), Gammaproteobacteria (16%), Deltaproteobacteria e Gemmatimonadetes (3%). A composição das comunidades influenciadas pela exsudação de asfalto não teve relação com a presença do óleo ou com as camadas de profundidade. A distância geográfica e a exsudação de asfalto foram importantes fatores para determinação da distribuição geográfica das comunidades microbianas em sedimento marinhos profundos do Platô de São Paulo. Apesar da ausência de relação entre o óleo proveniente da exsudação de asfalto e a composição das comunidades, a alta abundância de Alphaproteobacteria e a importância da distância dentro da região norte são indicativos do aumento da heterogeneidade causado pela exsudação de asfalto. Mais estudos procurando compreender a composição geoquímica dos sedimentos e do óleo são necessários para explicar como esses fatores influenciam a estruturação das comunidades microbianas estudadas. / Deep-sea sediments are stable and homogeneous environments, however, they have a high variety of available habitats, allowing a high microbial diversity to occur. Microbial spatial distribution is determined by local and regional factors. Local factors are associated to environment structure and regional factors, to microbial dispersal limitation and historical events that may cause environmental changes. Historical events such as hydrocarbon emanation from sub-seafloor to seafloor may change the patterns of microbial spatial distribution, due to an increase of carbon, thus, selecting species capable to degrade them. These events are denominated as asphalt seep and they were found on the studied region. Considering the lack of knowledge and the importance of microorganisms on deep-sea sediments from South Atlantic ocean, this study aims to understand the patterns of microbial spatial distribution and how the oil from asphalt seep influence the microbial communities. This study was realized in São Paulo Plateau region. The plateau was divided in two regions, north and south. Microbial diversity was studied from 14 deep-sea sediment samples, nine samples from north region and five from south region. The asphalt seep was found in north region from São Paulo Plateau, directly affecting three of the nine samples. The communities were studied through denaturing gradient gel electrophoresis (DGGE), quantitative PCR (qPCR) and next generation sequencing (Ion Torrent). The spatial distribution of the communities was analyzed at different spatial scales: sediment depth (≤ 4 cm), local (1-34 Km) and regional (> 250 Km). Microbial communities were less similar between regions and showing relation with geographic distance to achaea and bacteria. Similarity values within regions were higher then between them, but the geographic distance was also important to both domains, despite samples being closer. Similarity values between sediment depths were high and have no relation with geographic distance. The cell number between sediment depths was different, with tendency to decrease with depth increase. The most abundant classes were Alphaproteobacteria (30%), Acidimicrobiia (18%), Gammaproteobacteria (16%), Deltaproteobacteria e Gemmatimonadetes (3%). The communities composition influenced by asphalt seep have no relation with oil presence and sediment depths. Geographic distance and asphalt seep were important factors to determine the spatial distribution of microbial communities in deep-sea sediments from São Paulo Plateau. Despite the absence of relation between oil from asphalt seep and communities composition, the high abundance of Alphaproteobacteria and the importance of distance within north region are indicative of heterogeneity increase caused by asphalt seep. More studies aiming to understand the geochemical composition from sediments and oil are necessary to explain how these factors influence the communities structure.