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It has been already noticed, by comparing several studies, that the possibilities to identify microbial activity in deep subsurface environments is not always in agreement with the known limitations for life concerning pressure, salinity and temperature. For instance, it is very unlikely to find living microorganisms when temperature and salinity are higher than 70°C and 120 g/L respectively, which demonstrates a combined negative effect of salinity and temperature. In order to understand the different stress effects and the specificity of these parameters, eight selected microbial strains (bacteria and archaea), representative of the subsurface metabolism (methanogenesis, sulfate reduction, thiosulfate reduction, fermentation), have been tested on different temperatures (40, 55 and 70°C), pressures (1, 90 and 180 bar) and salinities (13, 50, 110, 180 and 260 g/L). Experiments were realized accordingly to a set of full factorial plans, and, for each test, the microbial activities were estimated by dosing the main expected metabolic products (CH4, H2S and acetic acid), the pH and the optical density. Results show variable response depending of the strains. Curiously, some species that were not supposed to be piezophile (i.e. surface species) have demonstrated a positive reactivity when the pressure was increased. On the other hand, fermenters and thiosulfate reducers were proven to be the most tolerant metabolisms to the overall range of exposition compared to other strains. Nevertheless, general trends have been also identified: the combined increase of pressure and temperature as well as the combined increase of pressure and salinity did globally provide synergetic effect on the strains activities. This leads to ask whether it is possible that the symmetrical behavior of these stresses is due to the sensitivity of one common structural (membranes) or metabolic target (compatible solutes) that is implicated in the microbial mechanisms of resistance and/or adaptation.

La prospection des environnements de subsurface permet un accès à la compréhension des cycles biogéochimiques se déroulant in-situ. Malgré les récentes avancées dans le domaine de la géomicrobiologie, la composition, la distribution et l'organisation microbienne des écosystèmes terrestres profonds restent encore mal connues. L'écologie moléculaire, bien qu'étant un outil puissant, se heurte à la faible biomasse contenue dans les roches profondes, rendant complexe l'accès à l'ADN. Dans cette étude, différentes méthodes d'extraction d'ADN ont été entreprises afin d'optimiser et d'adapter cette étape à des environnements extrêmes. La qualité des ADN extraits a été validée par l'amplification du gène codant pour l'ARNr 16S et la réalisation d'empreintes moléculaires (DGGE). Quatre échantillons de roches issues du Trias ont été collectés entre 1725 et 2000 mètres de profondeur à l'occasion d'un forage réalisé par l'ANDRA (programme TAPSS-FORPRO II). Le cœur de chaque carotte a été pulvérisé en conditions anoxiques et stériles. Trois protocoles d'extraction directe d'ADN génomique ont été comparés à partir des quatre matrices environnementales. Malgré la faible quantité d'ADN extrait, les amplifications du gène codant pour l'ARNr 16S bactérien se sont révélées positives montrant la bonne qualité des ADN extraits pour la suite des travaux de microbiologie moléculaire. De plus, une purification supplémentaire des ADN extraits par dialyse a permis l'augmentation significative des rendements d'amplification et l'acquisition d'empreintes moléculaires de qualité. Les profils de diversité obtenus à partir des différents extraits d'ADN seront présentés et discutés. Cette étude méthodologique est la première réalisée sur des environnements terrestres profonds. Les résultats démontrent la nécessité de tester et d'optimiser les procédures d'extraction d'ADN afin d'améliorer l'expertise des systèmes profonds pauvres en biomasse lors de futurs programmes de valorisations du sous-sol.

The retina and other tissues need iron to survive. However, the normal iron metabolism in rodent retinas had not been characterized. This study was intended to investigate iron and iron homeostasis protein (ferritin, transferrin [Tf] and transferrin receptor [Tf-R]) distribution in 20- to 55-day-old rat retinas.Iron was revealed on retinal sections directly by proton-induced x-ray emission (PIXE) and indirectly by electron microscopy (EM). Ferritin, Tf, and Tf-R proteins were localized by immunohistochemistry. Transferrin expression was localized by in situ hybridization (ISH). Transferrin and ferritin proteins and mRNA were analyzed by Western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR), respectively.Iron is widely and unevenly distributed throughout the adult rat retina. The highest concentration was observed by PIXE in the choroid and the retinal pigmented epithelial cell (RPE) layer, and in inner segments of photoreceptors (IS). Outer segments of photoreceptors (OS) also contain iron. EM studies suggested the presence of iron inclusions inside the photoreceptor discs. Choroid, RPE, and IS showed a strong immunoreactivity for ferritin. Transferrin accumulated mainly in the IS and OS areas and in RPE cells but can also be detected slightly in retinal capillaries. Western blot analysis for Tf and ferritin confirmed their presence in the adult neural retina. By RT-PCR, H- and L-chains of ferritin and Tf mRNAs were expressed in neural retina, but the main sites of Tf synthesis observed by ISH were the RPE and choroid cell layers. Tf-R immunoreactivity was detected in the ganglion cell layer, inner nuclear layer, outer plexiform layer, IS, RPE, and choroid. These results were similar for all stages studied.For the first time, the present study characterized both iron and iron homeostasis proteins in rodent retinas. In the outer retina, iron and ferritin shared the same distribution patterns. In contrast, Tf, mainly synthesized by RPE cells and detected in OS and IS areas, probably helps to transport iron to photoreceptors through their Tf-R. This is a likely pathway for filling iron needs in the outer retina.