From acid seeps and deep-sea thermal vents to glacial ice and hypersaline lakes, extreme environments contain relatively simplified communities consisting of extremophiles that have evolved to survive and thrive under adverse abiotic conditions. In more neutral environments, microorganisms use dormancy as a common life history strategy to weather temporal fluctuations of resources or stresses until more 'optimal' conditions are present. It is unclear if dormancy is an essential survival mechanism for microorganisms in extreme environments; however, recent studies suggest that extreme environments may create stable conditions for extremophiles to the extent that dormancy is of less ecological importance. Using lake salinity levels as measurements of "extreme," we evaluated the dormancy of bacterial and archaeal phyla and lake chemistry in five hypersaline and five freshwater lakes across the western United States. Dormancy was calculated using targeted metagenomics to analyze 16S rDNA and rRNA tag sequences. It was hypothesized that bacteria and archaea in hypersaline lake communities would exhibit lower levels dormancy than bacterial and archaeal communities in geologically similar freshwater lake controls. It was also hypothesized that microbial dormancy would decrease as the dominant extreme environmental variable increased in the lakes. As hypothesized, overall dormancy decreased at least 2-fold in hypersaline compared to freshwater lakes for both bacteria and archaea. Of the predominant phyla and subclasses, Firmicutes, Bacteroidetes, and Gammaproteobacteria each demonstrated at least a seven-fold decrease in dormancy in hypersaline lakes compared to freshwater lakes. Specifically, species within the genus Clostridium were responsible for 85% of the dormancy observed in the phylum Firmicutes. Also as hypothesized, microbial dormancy decreased as salinity increased in the lakes. Lower dormancy in hypersaline lakes correlated with increasing salinity while lower dormancy in freshwater lakes correlated with increasing total phosphorus levels. These results suggest that dormancy is a less common life history strategy for microorganisms in extreme environments; it is proposed that this is due to the relatively stable environment in hypersaline lakes and the reduced number of available microbial niches. These results also suggest that the dominant extreme stress (i.e., salinity) may override other driving factors in an environment to ultimately determine microbial community composition, diversity and richness.

Les aminopeptidases représentent un groupe d’enzymes qui possèdent une fonction cellulaire clef dans les mécanismes physiologiques et pathologiques. Elles interviennent dans la cascade enzymatique après l’action des endoprotéases, dans l’homéostasie au travers le renouvellement du pool d’acides aminés, dans le métabolisme énergétique, la régulation de l’activité des peptides bioactifs, la présentation antigénique ainsi dans une diversité de mécanismes pathologiques tels que les maladies neurologiques et les infections virales et parasitaires. Les aminopeptidases TETs sont capables de former des macro-assemblages tétraédriques comprenant douze sous-unités. En vue de mieux comprendre leur fonction biologique et leur mode d'action, nous avons étudié les propriétés fonctionnelles et structurales de deux nouveaux complexes TETs issus d'archées hyperthermophiles. L'archée hyperthermophile Methanocaldococcus jannaschii ne possède qu'une version de TET (MjTET) qui a été produite dans Escherichia coli et purifiée sous forme de dodécamère. La recherche de son activité enzymatique et de ses substrats peptidiques par des tests chromogéniques et fluorogéniques, ainsi que des études par HPLC en phase inverse, montre que cette enzyme est une leucine aminopeptidase activée par le cobalt se distinguant des autres aminopeptidases M42 par son très large spectre d'action qui s'étend aux résidus aromatiques. Une structure complète de cette aminopeptidase a été résolue en combinant la cristallographie (2.4 Å) et la cryo-EM (4,1 Å). L'analyse de la poche de spécificité de MjTET permet de mieux comprendre les bases structurales de la discrimination de substrat chez les TETs. De plus, l'analyse de la structure interne de la particule permet de proposer un nouveau mécanisme de navigation des peptides à l’intérieur des particules tétraédriques de la famille TET.L'archée hyperthermophile Pyrococcus horikoshii comporte trois types de complexes TETs. L'étude d'une protéine présentant ~20 % d'identité avec ces systèmes, nous a permis d'identifier une quatrième version du système TET dans cet organisme : PhTET4. La protéine recombinante a été purifiée. Elle forme un complexe dodécamérique tétraédrique. Les études biochimiques révèlent que l'enzyme possède une spécificité très étroite dirigée exclusivement vers l'hydrolyse des résidus glycines de l'extrémité N-terminale des peptides. De plus, elle estactivée par le nickel. Ces caractéristiques permettent de proposer que, chez les archées, la multiplication et la spécialisation des enzymes TETs seraient associées au caractère hétérotrophes alors que le système des archées autotrophes se réduirait à une TET unique apte à assurer une fonction de « ménage ». / Aminopeptidases represent a group of enzymes displaying key cellular function inphysiological and pathological mechanisms. They are involved in the enzymatic cascade beyond the action of endoproteases, in homeostasis through the renewal of the amino acid pool, in the energy metabolism, in the regulation of bioactive peptide activities, in the antigen presentation and in a diversity of pathological mechanisms such as neurological diseases as well as viral and parasitic infections. Aminopeptidases TET are able of forming tetrahedral macro-assemblies built by twelve subunits. In order to better understand their biological function and their mode of action, we studied the functional and structural properties of two novel TET complexes derived from hyperthermophilic archaea. The hyperthermophilic archaeon Methanocaldococcus jannaschii has only one version of TET (MjTET) that was produced in Escherichia coli and purified as dodecameric macromolecule. The search for its enzymatic activity and peptide substrates by using chromogenic/fluorogenic assays and reverse phase HPLC studies, demonstrated that this enzyme is a cobalt-activated leucine aminopeptidase, discriminated from other M42 aminopeptidases by its very broad activity spectrum, that extends to aromatic residues. Complete structure of this aminopeptidase was determined by combining X-ray crystallography (2.4 Å) and cryo-electron microscopy (4.1 Å). Analysis of MjTET specificity pocket indicated possible molecular bases for substrate discrimination in TET peptidases. In depth investigation of the particle internal structure allowed to propose a novel peptide trafficking mechanism for the TET family tetrahedral particles. Three types of TET complexes are present in the hyperthermophilic archaea, Pyrococcus horikoshii. The study of an unassigned protein displaying ~20% identity with the PhTETs systems allowed us to identify a fourth version of TET complex in this organism: PhTET4. The recombinant protein was purified. It formed tetrahedral dodecameric complex. Biochemical studies indicated that the enzyme has a very narrow hydrolytic specificity directed exclusively toward the peptide N-terminal glycine residues. In addition, this enzyme is activated by nickel ions. These features allowed proposing that, in archaea, the multiplicity of specialized TET systems could be associated with heterotrophy while unique TET system displaying “housekeeping” function is present in autotrophic organisms.

É notório o fato de que o interesse pela detecção de vida fora da Terra tenha aumentado no mundo científico. A existência de possíveis traçadores biológicos na atmosfera terrestre, a presença de material vivo ou de material orgânico decomposto em superfícies e no mar aumentam as possibilidades de sucesso das pesquisas. A vida na Terra é encontrada em todas as partes, e o planeta está saturado de suas manifestações. O objetivo da presente tese é o desenvolvimento de uma metodologia que ofereça ideias para a detecção de bioassinaturas fora da Terra, particularmente em exoplanetas. Em termos de biologia molecular, a principal assinatura de vida é o DNA (ácido desoxirribonucleico), que organiza e sedia o código genético de todos os seres vivos. Outras moléculas que denunciam a presença de vida são a clorofila, carotenoides, fragmentos de DNA, moléculas orgânicas, etc. Na tentativa de se encontrar bioassinaturas na atmosfera de exoplanetas, um requisito deve ser atendido: a identificação de bandas características que permitam a detecção de um sinal que esteja associado a uma biomolécula complexa, preferencialmente na região do infravermelho, vista em meio aos picos de gases atmosféricos. Sendo assim, uma série de análises espectrais foram realizadas para amostras de DNA/células do micro-organismo extremófilo Halobacterium salinarum, com a finalidade de serem comparadas ao espectro infravermelho, obtido de forma direta da atmosfera terrestre. A pesquisa por marcadores específicos foi realizada a fim de determinar os picos que permitam a detecção desses componentes singulares quando suspensos em gases atmosféricos. Os resultados da pesquisa mostram que a atmosfera terrestre está contaminada com moléculas complexas. Existem 37 absorções em comum quando se compara o espectro da atmosfera terrestre com os espectros de células ou DNA (por exemplo 966, 936, 924, 886 e 866 cm-1). Entre elas, os picos centrados em aproximadamente 1018, 996, 900 e 840 cm-1, denunciam a presença de estruturas biogênicas ligadas à presença de ácidos nucleicos (riboses e grupos fosfatos). Investigou-se, também, a possibilidade de certas assinaturas biológicas serem mascaradas pela presença de gases quando observadas remotamente. A conclusão é que as bandas de gases como o SO2 (1136 cm-1), O3 (1042 e 1124 cm-1) e C2H6 (826 cm-1), podem tornar a detecções de algumas bioassinaturas uma tarefa árdua e até mesmo impossível. Outras perguntas de pesquisa ligadas à determinação da quantidade mínima de material biológico capaz de oferecer um sinal que possa ser identificado e atribuído a um material biológico específico, assim como a possibilidade de determinação de uma banda de absorção apta a servir como fator de calibração foram abordadas. Conclui-se que há possibilidades de se detectar alguma característica biológica mesmo após diluições cobrindo 5 ordens de magnitude (variação de 760 a 0.076 ng/µl). A banda centrada em 893 cm-1 pode ser utilizada para futuras calibrações, pois responde linearmente com a variação da quantidade de material biológico. O resultado da pesquisa mostrou que existem bandas moleculares em comum entre a atmosfera e o material biológico, sendo atribuídas a potenciais marcadores moleculares que, possivelmente, poderão ser detectados de forma remota em futuras missões espaciais. / It is notorious that interest in detecting life beyond Earth has increased in the scientific world. The existence of possible biological markers the terrestrial atmosphere, the presence of living material or organic material decomposed on surfaces and at the sea increase the chances of success of the research. Life on Earth is everywhere and the planet is saturated with its manifestations. The objective of this thesis is the development of a methodology that offers ideas for the detection of bioassinatures outside the Earth, particularly in exoplanets. In terms of molecular biology, the main signature of life is DNA (desoxyribonucleic acid), which organizes and hosts the genetic code all of the living things. Other molecules that denounce the presence of life are chlorophyll, carotenoids, DNA fragments, organic molecules, etc. In an attempt to find bioassinatures in the atmosphere of exoplanets, a requirement must be met: the identification of characteristic bands that allow the detection of a signal associated with a complex biomolecule, preferably in the infrared region, seen in the middle of the peaks of gases atmospheric conditions. Thus, a series of spectral analyzes were performed for DNA samples/cells of the extremophilic microorganism Halobacterium salinarum, to be compared to the infrared spectrum obtained directly from the Earths atmosphere. The search for specific markers was performed in order to determine the peaks that allow the detection of these singular components when suspended in atmospheric gases. The results of the research show that the Earths atmosphere is contaminated with complex molecules. There is a total of 37 common absorptions found in the spectrum of the Earths atmosphere and in Cells or DNA spectra (for example 966, 936, 924, 886 and 866 cm-1). Among them, the peaks centered at approximately 1018, 996, 900 and 840 cm-1, denote the presence of biogenic structures linked to the presence of nucleic acids (riboses and phosphate groups). It was also investigated the possibility of certain biological signatures being masked by the presence of gases when observed remotely. The conclusion is that gas bands such as SO2 (1136 cm-1), O3 (1042 and 1124 cm-1) and C2H6 (826 cm-1) can make the detection of some bioassinatures a difficult task. Other research questions related to the determination of the minimum quantity of biological material can providing a signal capable of being identified and assigned to a specific biological material, as well as the possibility of determining an absorption that could a serving as a calibration factor were addressed . It is concluded that it is possible to detect a same biological characteristic after dilutions covering 5 orders of magnitude (ranging from 760 to 0.076 ng/µl). The band centered at 893 cm-1 can be used for future calibrations because it responds linearly with the variation of the amount of biological material. The research results showed that there are molecular bands in common between the atmosphere and biological material and are attributed to potential molecular markers that may possibly be detected remotely in future space missions.

É notório o fato de que o interesse pela detecção de vida fora da Terra tenha aumentado no mundo científico. A existência de possíveis traçadores biológicos na atmosfera terrestre, a presença de material vivo ou de material orgânico decomposto em superfícies e no mar aumentam as possibilidades de sucesso das pesquisas. A vida na Terra é encontrada em todas as partes, e o planeta está saturado de suas manifestações. O objetivo da presente tese é o desenvolvimento de uma metodologia que ofereça ideias para a detecção de bioassinaturas fora da Terra, particularmente em exoplanetas. Em termos de biologia molecular, a principal assinatura de vida é o DNA (ácido desoxirribonucleico), que organiza e sedia o código genético de todos os seres vivos. Outras moléculas que denunciam a presença de vida são a clorofila, carotenoides, fragmentos de DNA, moléculas orgânicas, etc. Na tentativa de se encontrar bioassinaturas na atmosfera de exoplanetas, um requisito deve ser atendido: a identificação de bandas características que permitam a detecção de um sinal que esteja associado a uma biomolécula complexa, preferencialmente na região do infravermelho, vista em meio aos picos de gases atmosféricos. Sendo assim, uma série de análises espectrais foram realizadas para amostras de DNA/células do micro-organismo extremófilo Halobacterium salinarum, com a finalidade de serem comparadas ao espectro infravermelho, obtido de forma direta da atmosfera terrestre. A pesquisa por marcadores específicos foi realizada a fim de determinar os picos que permitam a detecção desses componentes singulares quando suspensos em gases atmosféricos. Os resultados da pesquisa mostram que a atmosfera terrestre está contaminada com moléculas complexas. Existem 37 absorções em comum quando se compara o espectro da atmosfera terrestre com os espectros de células ou DNA (por exemplo 966, 936, 924, 886 e 866 cm-1). Entre elas, os picos centrados em aproximadamente 1018, 996, 900 e 840 cm-1, denunciam a presença de estruturas biogênicas ligadas à presença de ácidos nucleicos (riboses e grupos fosfatos). Investigou-se, também, a possibilidade de certas assinaturas biológicas serem mascaradas pela presença de gases quando observadas remotamente. A conclusão é que as bandas de gases como o SO2 (1136 cm-1), O3 (1042 e 1124 cm-1) e C2H6 (826 cm-1), podem tornar a detecções de algumas bioassinaturas uma tarefa árdua e até mesmo impossível. Outras perguntas de pesquisa ligadas à determinação da quantidade mínima de material biológico capaz de oferecer um sinal que possa ser identificado e atribuído a um material biológico específico, assim como a possibilidade de determinação de uma banda de absorção apta a servir como fator de calibração foram abordadas. Conclui-se que há possibilidades de se detectar alguma característica biológica mesmo após diluições cobrindo 5 ordens de magnitude (variação de 760 a 0.076 ng/µl). A banda centrada em 893 cm-1 pode ser utilizada para futuras calibrações, pois responde linearmente com a variação da quantidade de material biológico. O resultado da pesquisa mostrou que existem bandas moleculares em comum entre a atmosfera e o material biológico, sendo atribuídas a potenciais marcadores moleculares que, possivelmente, poderão ser detectados de forma remota em futuras missões espaciais. / It is notorious that interest in detecting life beyond Earth has increased in the scientific world. The existence of possible biological markers the terrestrial atmosphere, the presence of living material or organic material decomposed on surfaces and at the sea increase the chances of success of the research. Life on Earth is everywhere and the planet is saturated with its manifestations. The objective of this thesis is the development of a methodology that offers ideas for the detection of bioassinatures outside the Earth, particularly in exoplanets. In terms of molecular biology, the main signature of life is DNA (desoxyribonucleic acid), which organizes and hosts the genetic code all of the living things. Other molecules that denounce the presence of life are chlorophyll, carotenoids, DNA fragments, organic molecules, etc. In an attempt to find bioassinatures in the atmosphere of exoplanets, a requirement must be met: the identification of characteristic bands that allow the detection of a signal associated with a complex biomolecule, preferably in the infrared region, seen in the middle of the peaks of gases atmospheric conditions. Thus, a series of spectral analyzes were performed for DNA samples/cells of the extremophilic microorganism Halobacterium salinarum, to be compared to the infrared spectrum obtained directly from the Earths atmosphere. The search for specific markers was performed in order to determine the peaks that allow the detection of these singular components when suspended in atmospheric gases. The results of the research show that the Earths atmosphere is contaminated with complex molecules. There is a total of 37 common absorptions found in the spectrum of the Earths atmosphere and in Cells or DNA spectra (for example 966, 936, 924, 886 and 866 cm-1). Among them, the peaks centered at approximately 1018, 996, 900 and 840 cm-1, denote the presence of biogenic structures linked to the presence of nucleic acids (riboses and phosphate groups). It was also investigated the possibility of certain biological signatures being masked by the presence of gases when observed remotely. The conclusion is that gas bands such as SO2 (1136 cm-1), O3 (1042 and 1124 cm-1) and C2H6 (826 cm-1) can make the detection of some bioassinatures a difficult task. Other research questions related to the determination of the minimum quantity of biological material can providing a signal capable of being identified and assigned to a specific biological material, as well as the possibility of determining an absorption that could a serving as a calibration factor were addressed . It is concluded that it is possible to detect a same biological characteristic after dilutions covering 5 orders of magnitude (ranging from 760 to 0.076 ng/µl). The band centered at 893 cm-1 can be used for future calibrations because it responds linearly with the variation of the amount of biological material. The research results showed that there are molecular bands in common between the atmosphere and biological material and are attributed to potential molecular markers that may possibly be detected remotely in future space missions.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-19T15:54:36Z No. of bitstreams: 2 Tese - Emmanuel Bezerra D’Alessandro - 2017.pdf: 5468492 bytes, checksum: 142fd12ec077386e23484e261e25c7ea (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-19T15:55:07Z (GMT) No. of bitstreams: 2 Tese - Emmanuel Bezerra D’Alessandro - 2017.pdf: 5468492 bytes, checksum: 142fd12ec077386e23484e261e25c7ea (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-04-19T15:55:07Z (GMT). No. of bitstreams: 2 Tese - Emmanuel Bezerra D’Alessandro - 2017.pdf: 5468492 bytes, checksum: 142fd12ec077386e23484e261e25c7ea (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-03-31 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Global energy needs are steadily increasing, driven by the rise in both industrialization and population. The major sources of energy are non-renewable and highly polluting. Therefore, the demand for renewable fuels is increasing. One source of renewable fuels is biomass, such as that of microalgae. Microalgae biomasses are a source of lipids and antioxidant pigments and have great potential for biodiesel production and lutein, which have high commercial value. Therefore, in this study I tested the feasibility of producing biodiesel and lutein from microalgae of extremophile environmental of Central Brazil. The freshwater microalgae T. minimum, P.morum, D. communis, N. digitus, E. planctonicus and A. obliquus were collected, isolated, and cultivated in the laboratory, comparing their lipid productivity with that of soybean and lutein productivity with that of Marigold, which is the main raw material for this pigment . The percentage of Fatty Acid Methyl Esters (FAME) of microalgae varied from 9.6 to 21.3%, being A. obliquus the species with the highest content of fatty acids, E. planctonicus with the highest content of lutein and D. communis the one with the highest productivity of biomass, biodiesel and lutein. The D. communis fatty acid profile was also suitable for biodiesel production, since it produced up to 21 times more biodiesel than soybean, but consuming up to 4.8% of the area and 42 times less water. Microalgae cultivation for biodiesel production can be done with less complex and low-cost culture media, including waste water from biodiesel plants. Thus, although the BBM medium had higher biodiesel productivity, the less nutritive media (DAF + OGR and DAF) also yielded adequate biodiesel amounts, being 72% cheaper than BBM. Thus, the biodiesel yield of A. obliquus grown in DAF + OGR was 46 times higher than that of soybeans, using 7.2% of water and 2.2% of the area. These results demonstrate the potential of microalgae in the biodiesel production chain / A demanda por energia aumenta continuamente em nível mundial, impulsionadas tanto pelo aumento da industrialização como também pelo aumento populacional, sendo grande parte dessas necessidades ainda supridas fontes não renovável e altamente poluente e, portanto, limitados às reservas existentes. Assim, a procura por combustíveis derivados de fontes renováveis vêm aumentando a cada dia, sendo intensa a busca por biomassa tais como as microalgas, as quais, por serem fontes de lipídios e pigmentos antioxidantes, vêm sendo estudadas com respeito a seu potencial para produção de biodiesel e luteína, que possui alto valor comercial. Dessa forma, este estudo buscou avaliar a viabilidade da utilização de microalgas de ambientes extremófilos do Brasil Central na produção de biodiesel e luteína. As microalgas dulcícolas T. minimum, P.morum, D. communis, N. digitus, E. planctonicus e A. obliquus foram coletadas em ambientes extremófilos, isoladas e cultivadas em laboratório, tendo sua produtividade lipídica comparada com a da semente de soja e a produtividade de luteína comparada com as flores de Marigold, que é a principal matéria-prima para tal pigmento. As microalgas extremófilas apresentaram potencial para biodiesel, gerando porcentagem de ésteres metílicos de ácidos graxos variando de 9,6 a 21,3%, sendo A. obliquus com maior teor de ácidos graxos, E. planctonicus com maior teor de luteína e D. communis com maior destaque na produtividade de biomassa, biodiesel e luteína. D. communis também apresentou perfil de ácidos graxos adequados para biodiesel, além de produzir até 21 vezes mais biodiesel que a semente de soja. Através das estimativas comparativas concluiu-se que D. communis consumiria até 4,8% da área e 42 vezes menos água do que a usada pelo cultivo de soja para produzir a mesma quantidade de biodiesel. Verificou-se que o cultivo de microalgas para a produção de biodiesel pode ser feito com o uso de meios de menor complexidade e custo, usando inclusive águas residuais que podem ser encontradas em usinas de biodiesel. Assim, apesar do meio de cultivo convencional BBM apresentar maior produtividade em biodiesel, os meios com menos nutrientes (DAF+OGR e DAF), também proporcionaram adequados rendimentos em biodiesel, sendo 72% mais econômicos que o meio BBM. Assim, verificou-se que para A. obliquus cultivada no meio de menor custo, DAF+OGR, a produtividade em biodiesel foi 46 vezes superior ao da soja, com uso de 7,2% de água e 2,2% da área usada no cultivo de soja para produção de mesma quantidade de biodiesel, mostrando o potencial das microalgas para a cadeia de produção e uso de biodiesel.

Les systèmes hydrothermaux associés à la serpentinisation sont anoxiques et riches en $H_2$, $CH_4$ et molécules organiques. Ces composants alimentent des micro-organismes qui colonisent les systèmes serpentinisés, et ce en dépit d’un pH élevé et de faibles concentrations en accepteurs d'électrons et en carbone dissous. Dans ce travail, les communautés microbiennes ont été étudiées en se focalisant sur Prony, un écosystème serpentinisé côtier de Nouvelle-Calédonie, puis, en comparant différents écosystèmes serpentinisés, pour faire émerger des similarités taxonomiques et fonctionnelles. À Prony, nos analyses de métabarcoding ont mis en évidence l'importance d’une biosphère rare. L'analyse de métagénomes a permis de reconstruire 82 génomes procaryotes. Un de ces génomes est phylogénétiquement proche des espèces du genre Serpentinomonas, bactéries chimiolithotrophes isolées du site serpentinisé The Cedars, qui détiennent le record d’alcalophilie. Ces espèces et d'autres phylotypes, tels que les taxons affiliés aux Lost City Methanosarcinales, ont été trouvés dans plusieurs sites serpentinisés et pourraient contribuer à la définition d'une signature biologique des phénomènes de serpentinisation. En ciblant spécifiquement les métabolismes enrichis dans les milieux serpentinisés, nous avons pu mettre en évidence l'importance du métabolisme de l'hydrogène, des mécanismes cellulaires de réponse aux stress et d’une voie de dégradation des phosphonates, reposant sur l’activité d'une C-P lyase. Cette voie métabolique, qui a un rôle clé dans l'assimilation du phosphore et la libération de molécules organiques, vient enrichir les modèles écologiques des systèmes serpentinisés. / Serpentinizing hydrothermal systems are anoxic and enriched in $H_2$, $CH_4$ and organic molecules. These compounds support microbes that colonize serpentinizing systems, despite high pH and low concentrations of electron acceptors and dissolved inorganic carbon. In this work, two axes were explored to study the microbial communities. On the one hand, we focused on Prony, a coastal serpentinizing site in New Caledonia, and on the other hand we compared different serpentinizing systems to reveal taxonomic and functional similarities. At Prony, our metabarcoding analyses highlighted the importance of the rare biosphere. Moreover, 82 prokaryotic genomes were successfully reconstructed using five metagenomes from Prony. One of these genomes was phylogenetically close to the species of the genus Serpentinomonas, chemolithotrophic bacteria isolated at the serpentinizing site The Cedars that are capable of growth up to pH 12.5. These species, and other phylotypes, such as taxa affiliated with Lost City Methanosarcinales were identified in several serpentinizing sites and could contribute to the definition of a biological signature associated with serpentinization. By specifically targeting enriched metabolisms in serpentinizing environments, we highlighted key functions associated with hydrogen metabolism and environmental stress response mechanisms. The comparison of serpentinizing metagenomes revealed the importance of a phosphonate degradative pathway, based on the activity of a C-P lyase. This metabolic pathway, which plays a key role in the uptake of phosphorus and the release of organic molecules, was integrated into the ecological models of serpentinizing systems.

Vulcões ativos na Antártica contrastam com a paisagem predominantemente gelada do continente. Eles fornecem condições únicas capazes de selecionar uma grande variedade de adaptações microbianas. A Ilha Deception localiza-se na região da Península Antártica e difere de outros vulcões antárticos especialmente pela influência marinha e temperaturas mais elevadas. Foram coletadas amostras de sedimentos associados a fumarolas e geleiras em dois sítios geotermais de Deception, com temperaturas variando entre 0°C a 98°C. Diferentes técnicas independentes de cultivo foram empregadas com o intuito de entender como as comunidades microbianas respondem as variações ambientais extremas produzidas pela atividade vulcânica. Os resultados indicaram que a co-ocorrência de arqueias hipertermófilas e suas adaptações com micro-organismos metabolicamente diversos adaptados a regiões geladas representa uma estrutura de comunidades única para ecossistemas antárticos. Este trabalho forneceu dados ineditos sobre questoes centrais de diversidade e adaptacao microbiana a ambientes geotermais polares. / Active volcanoes in Antarctica contrast with the predominately icy landscape. They harbor unique conditions capable to select an extreme range of microbial adaptations. Deception Island is located in the Antarctic Peninsula region and differs from other Antarctic volcanoes specially by its higher temperatures and marine influence. We collected sediment samples associated to active fumaroles and glaciers on two geothermal sites of Deception Island, with temperatures ranging from 0°C to 98°C. Different cultivation-indepedent techniques were used to understand how microbial communities respond to extreme environmental variations produced by volcanic activity. The results indicate that co-occurrence of hyperthermophiles and their specific adaptations with metabolically diverse cold-adapted micro-organisms represents a unique community structure for antarctic ecosystems. This study provided primordial data on central questions about microbial diversity and adaptation to polar geothermal environments.

Polyhydroxyalkanoates (PHA) are a family of biodegradable polyesters naturally synthesised by some bacteria and archaea. PHA have high industrial value as bioplastics for packaging and biomedical applications. However, their broader use is hindered by high production costs and uncontrolled variation of polymer properties. The extreme halophile Haloferax mediterranei shows bioprocess advantages that can be exploited for the low cost production of the PHA copolymer Poly(3-hydroxbutyrate-co-3-hydroxyvalterate) (PHBV). The focus of this thesis is to identify process variables responsible for the uncontrolled variation of PHA properties in order to progress towards the robust production of PHBV using H. mediterranei. The outcome of the investigation is a novel cultivation strategy for the reliable synthesis of PHBV copolymers with controlled composition and microstructure showing minor differences in material characteristics. Initially, growth kinetics and PHBV synthesis were characterised under nitrogen-excess and nitrogen-limiting conditions in ammonium and for the first time, nitrate. The nitrogen source and concentration influenced PHBV accumulation and variations in polymer composition were observed with ammonium, highlighting the importance of the control of cultivation conditions. Volatile fatty acids (VFA) were found to be a more direct approach to determine PHBV composition and for the first time were used as substrates in H. mediterranei cultures. When the cells were grown in C4:0/C5:0 mixtures, the 3HV fraction in the PHBV was proportional to the percentage of C5:0 in the feed mixture, allowing the synthesis of copolymers with a predefined composition ranging from pure PHB to pure PHV. The cultivation strategy proved effective for the synthesis of HV rich PHBV, which is not easily obtained due to the 3HV precursor toxicity. The polymer microstructure was controlled using different feeding strategies: co-feeding generated random copolymers, while sequential feeding created block and blend copolymers. The synthesis of block copolymers is of interest because the materials show enhanced yield strength and mechanical strength, making such materials more suitable for commodity uses. Bespoke random, block, and blend copolymers with 0−100 mol% 3HV were synthesized and their thermal and mechanical properties studied. Lastly, high temperature cultivation and several surfactants were tested to enhance the production of bespoke PHBV from VFA. PHBV productivity and accumulation was greatly improved in a fed-batch bioreactor fermentation at 37°C with Tween-80 and the maximum PHBV content 58.9% was obtained. The polymers from shake-flasks and from bioreactors showed minor variations in their material properties, demonstrating the scalability and the robustness of the process developed. Further understanding of the different process variables affecting polymer synthesis and composition was gained in this thesis. It is now possible to produce PHBV with controllable composition, microstructure and minor differences in material characteristics. The novel and robust production strategy developed address the bioprocess challenge of minimising the uncontrolled variation of polymer characteristics that is currently hindering the wider use of PHA hence allowing the production of high quality polymers for commodity goods, packaging and biomedical applications.

Vulcões ativos na Antártica contrastam com a paisagem predominantemente gelada do continente. Eles fornecem condições únicas capazes de selecionar uma grande variedade de adaptações microbianas. A Ilha Deception localiza-se na região da Península Antártica e difere de outros vulcões antárticos especialmente pela influência marinha e temperaturas mais elevadas. Foram coletadas amostras de sedimentos associados a fumarolas e geleiras em dois sítios geotermais de Deception, com temperaturas variando entre 0°C a 98°C. Diferentes técnicas independentes de cultivo foram empregadas com o intuito de entender como as comunidades microbianas respondem as variações ambientais extremas produzidas pela atividade vulcânica. Os resultados indicaram que a co-ocorrência de arqueias hipertermófilas e suas adaptações com micro-organismos metabolicamente diversos adaptados a regiões geladas representa uma estrutura de comunidades única para ecossistemas antárticos. Este trabalho forneceu dados ineditos sobre questoes centrais de diversidade e adaptacao microbiana a ambientes geotermais polares. / Active volcanoes in Antarctica contrast with the predominately icy landscape. They harbor unique conditions capable to select an extreme range of microbial adaptations. Deception Island is located in the Antarctic Peninsula region and differs from other Antarctic volcanoes specially by its higher temperatures and marine influence. We collected sediment samples associated to active fumaroles and glaciers on two geothermal sites of Deception Island, with temperatures ranging from 0°C to 98°C. Different cultivation-indepedent techniques were used to understand how microbial communities respond to extreme environmental variations produced by volcanic activity. The results indicate that co-occurrence of hyperthermophiles and their specific adaptations with metabolically diverse cold-adapted micro-organisms represents a unique community structure for antarctic ecosystems. This study provided primordial data on central questions about microbial diversity and adaptation to polar geothermal environments.

The objective of this study is to assess the uncultured bacterial diversity in the snowpack of the Mont Blanc (MtBl) glacier containing Saharan dust deposited during four dust events during the period 2006 – 2009 by means of molecular phylogenetics. The final goal is to discover the bacteria that could be involved in the establishment of snow microbiota. Bacterial diversity was evaluated using rybotyping and subsequent sequencing of partial (V3-V5) and full-length 16S rRNA genes. For comparison purpose we also studied following samples: “clean” MtBl snow containing no Saharan dust; Saharan sand collected in Tunisia; Saharan dust collected in Grenoble (200 m a.s.l.) and recovered later on MtBl (4250 m a.s.l.). In order to verify possible microbial activity in situ, both rDNA and rRNA approaches were implemented for the “clean” snow sample. To evaluate the survival/colonization abilities of bacterial phylotypes recovered in snow samples with Saharan dust, we analyzed their closest strain physiology as well as sources of environmental clones using a threshold of ≥98% sequence similarity. For the result interpretation, we also used data on dust elemental composition and dust particles size distribution. As a result 8 clone libraries (including rRNA-based one) were constructed using V3-V5 16S rRNA gene sequences for 5 snow samples (4 with Saharan dust and one “clean”), sample of Saharan dust collected in Grenoble and Saharan sand sample. Furthermore, 4 clone libraries were generated using full-length 16S rRNA gene amplicons obtained from 4 of the above snow samples (three with Saharan dust and one ‘clean'). Species content and dominant phylotypes and their assigning to major divisions varied significantly in alpine snow on a Mont Blanc glacier associated with four depositions of Saharan dust over a 3-year. Dominant phylotypes revealed are belonged to Actinobacteria, Proteobactreia, Firmicutes, Deinococcus-Thermus, Bacteroidetes and Cyanobacteria. Such variability was detected by both partial and full-length 16S rRNA gene sequencing and seems to be caused more by conditions of dust transport than bacterial load from the original dust source. Also the preservation period of dust in snowpack could affect the species composition. Thirteen icy phylotypes as candidates into snow microbiota establishing were recognized in snow containing Saharan dust and only two in “clean” snow sample. Of them, both dominant and minor phylotypes of Cyanobacteria, Proteobacteria, Actinobacteria и Firmicutes were revealed. Data on the closest strain physiology of recognized icy phylotypes suggests that representatives of genera Massilia (Betaproteobacteria), Tumebacillus (Firmicutes), Phormidium and Stigonema (both Cyanobacteria) are most relevant findings in terms of propagation in snow. By analyzing 16S rRNA from the “clean” snow containing no Saharan dust and comparing the data with those obtained for 16S rDNA library, it has been shown that Stigonema-like cyanobacterium identified could be propagating in snow at subzero temperature. Among all identified phylotypes, 10% were categorized as HA-phylotypes based on their con-specificity (≥98% similarity) with normal (non-pathogenic) human microbiome representatives. Furthermore, 11% out of all phylotypes showed less than 90% similarity with known taxa, thus, presenting novel taxa. Sequencing of both partial (V3-V5) and full-length 16S rRNA genes permitted to describe microbial diversity more fully and get more detailed picture. / The objective of this study is to assess the uncultured bacterial diversity in the snowpack of the Mont Blanc (MtBl) glacier containing Saharan dust deposited during four dust events during the period 2006 – 2009 by means of molecular phylogenetics. The final goal is to discover the bacteria that could be involved in the establishment of snow microbiota. Bacterial diversity was evaluated using rybotyping and subsequent sequencing of partial (V3-V5) and full-length 16S rRNA genes. For comparison purpose we also studied following samples: “clean” MtBl snow containing no Saharan dust; Saharan sand collected in Tunisia; Saharan dust collected in Grenoble (200 m a.s.l.) and recovered later on MtBl (4250 m a.s.l.). In order to verify possible microbial activity in situ, both rDNA and rRNA approaches were implemented for the “clean” snow sample. To evaluate the survival/colonization abilities of bacterial phylotypes recovered in snow samples with Saharan dust, we analyzed their closest strain physiology as well as sources of environmental clones using a threshold of ≥98% sequence similarity. For the result interpretation, we also used data on dust elemental composition and dust particles size distribution. As a result 8 clone libraries (including rRNA-based one) were constructed using V3-V5 16S rRNA gene sequences for 5 snow samples (4 with Saharan dust and one “clean”), sample of Saharan dust collected in Grenoble and Saharan sand sample. Furthermore, 4 clone libraries were generated using full-length 16S rRNA gene amplicons obtained from 4 of the above snow samples (three with Saharan dust and one ‘clean'). Species content and dominant phylotypes and their assigning to major divisions varied significantly in alpine snow on a Mont Blanc glacier associated with four depositions of Saharan dust over a 3-year. Dominant phylotypes revealed are belonged to Actinobacteria, Proteobactreia, Firmicutes, Deinococcus-Thermus, Bacteroidetes and Cyanobacteria. Such variability was detected by both partial and full-length 16S rRNA gene sequencing and seems to be caused more by conditions of dust transport than bacterial load from the original dust source. Also the preservation period of dust in snowpack could affect the species composition. Thirteen icy phylotypes as candidates into snow microbiota establishing were recognized in snow containing Saharan dust and only two in “clean” snow sample. Of them, both dominant and minor phylotypes of Cyanobacteria, Proteobacteria, Actinobacteria и Firmicutes were revealed. Data on the closest strain physiology of recognized icy phylotypes suggests that representatives of genera Massilia (Betaproteobacteria), Tumebacillus (Firmicutes), Phormidium and Stigonema (both Cyanobacteria) are most relevant findings in terms of propagation in snow. By analyzing 16S rRNA from the “clean” snow containing no Saharan dust and comparing the data with those obtained for 16S rDNA library, it has been shown that Stigonema-like cyanobacterium identified could be propagating in snow at subzero temperature. Among all identified phylotypes, 10% were categorized as HA-phylotypes based on their con-specificity (≥98% similarity) with normal (non-pathogenic) human microbiome representatives. Furthermore, 11% out of all phylotypes showed less than 90% similarity with known taxa, thus, presenting novel taxa. Sequencing of both partial (V3-V5) and full-length 16S rRNA genes permitted to describe