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  • Publication
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    Genome Sequence of Kosakonia radicincitans Strain YD4, a Plant Growth-Promoting Rhizobacterium Isolated from Yerba Mate (Ilex paraguariensis St. Hill.)
    (2015)
    Bergottini, Veronica M.
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    Junier, Thomas
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    Johnson, Shannon
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    Chain, Patrick S.
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    Otegui, Monica B.
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    Zapata, Pedro D.
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    Kosakonia radicincitans strain YD4 is a rhizospheric isolate from yerba mate (Ilex paraguariensis St. Hill.) with plant growth-promoting effects on this crop. Genes involved in different plant growth-promoting activities are present in this genome, suggesting its potential as a bioinoculant for yerba mate.
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    MLgsc: A Maximum-Likelihood General Sequence Classifier
    We present software package for classifying protein or nucleotide sequences to user-specified sets of reference sequences. The software trains a model using a multiple sequence alignment and a phylogenetic tree, both supplied by the user. The latter is used to guide model construction and as a decision tree to speed up the classification process. The software was evaluated on all the 16S rRNA gene sequences of the reference dataset found in the GreenGenes database. On this dataset, the software was shown to achieve an error rate of around 1% at genus level. Examples of applications based on the nitrogenase subunit NifH gene and a protein-coding gene found in endospore-forming Firmicutes is also presented. The programs in the package have a simple, straightforward command-line interface for the Unix shell, and are free and open-source. The package has minimal dependencies and thus can be easily integrated in command-line based classification pipelines.
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    Genetic diversity of Rhizobium present in nodules of Phaseolus vulgaris L. cultivated in two soils of the central region in Chile
    (2014-4-1)
    Although Phaseolus vulgaris L. is native from the Americas and is currently cultured in diverse areas, very little is know about the diversity of symbiotic nitrogen fixing Rhizobium (mycrosymbiont) in many of those cultures. Therefore, the aim of this study was to assess the genetic diversity of Rhizobium present in nodules of P. vulgaris in the central region of Chile. A method to extract DNA from surface-sterilized nodules was applied to two populations of the same seed variety grown in different fields. The 16S rRNA and nifH genes were amplified directly from the DNA extracted. DGGE analysis and clone libraries showed a restricted genetic diversity of the microsymbiotic populations that nodulate P. vulgaris. Both molecular markers revealed the presence of a microsymbiont closely related to Rhizobium etli in all the plants from the soils studied, indicating that the populations of Rhizobium spp. nodulating P. vulgaris in the central region of Chile displayed an extremely low genetic diversity. The level of genetic diversity in microsymbiont populations in plants grown in soils with different origin suggested that other factors rather than the indigenous soil rhizobial populations play a major role in the selection of the symbiotic partner in P. vulgaris.
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    Detection of active oxalate-carbonate pathway ecosystems in the Amazon Basin: global implications of a natural potential C sink
    (2013-12-24)
    The oxalate-carbonate pathway (OCP) is a biogeochemical process, which has been described in Milicia excelsa tree ecosystems of Africa. This pathway involves biological and geological parameters at different scales: oxalate, as a by-product of photosynthesis, is oxidized by oxalotrophic bacteria leading to a local pH increase, and eventually to carbonate accumulation through time in previously acidic and carbonate-free tropical soils. Former studies have shown that this pedogenic process can potentially lead to the formation of an atmospheric carbon sink. Considering that 80 % of plant species are known to produce oxalate, it is reasonable to assume that Milicia excelsa is not the only tree that can support OCP ecosystems. The search for similar conditions on another continent led us to South America, in an Amazon forest ecosystem (Alto Beni, Bolivia). This area was chosen because of the absence of local inherited carbonate in the bedrock, as well as its expected acidic soil conditions. Eleven tree species and associated soils were tested positive for the presence of carbonate with a more alkaline soil pH close to the tree than at distance from it. A detailed study of Pentaplaris davidsmithii and Ceiba speciosa trees showed that oxalotrophy impacted soil pH in a similar way to at African sites (at least with 1 pH unit increasing). African and South American sites display similar characteristics regarding the mineralogical assemblage associated with the OCP, except for the absence of weddellite. The amount of carbonate accumulated is 3 to 4 times lower than the values measured in African sites related to Milicia excelsa ecosystems. Still, these secondary carbonates remain critical for the continental carbon cycle, as they are unexpected in the acidic context of Amazonian soils. Therefore, the present study demonstrates the existence of an active OCP in South America. The three critical components of an operating OCP are the presence of : i) local alkalinisation, ii) carbonate accumulations, and iii) oxalotrophic bacteria, which were identified associated to the oxalogenic tree Ceiba speciosa. If the question of a potential carbon sink related to oxalotrophic-oxalogenic ecosystems in the Amazon Basin is still pending, this study highlights the implication of OCP ecosystems on carbon and calcium biogeochemical coupled cycles. As previously mentioned for Milicia excelsa tree ecosystems in Africa, carbonate accumulations observed in the Bolivian tropical forest could be extrapolated to part or the whole Amazon Basin and might constitute an important reservoir that must be taken into account in the global carbon balance of the Tropics.
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    Assessment of a biological approach for the protection of copper alloys artefacts
    (Edinburgh: Historic Scotland and International Council of Museums, 2013) ;
    Letardi, Paola
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    ; ; ; ;
    Wörle, Marie
    We propose an innovative treatment by which the protection of copper-based artefacts can be provided by naturally occurring microorganisms. The properties of some fungi were exploited for the transformation of existing corrosion patinas into copper oxalates. The latter are known to be insoluble and chemically stable. Within the earlier EU-ARTECH and BAHAMAS projects, very promising results were obtained with an almost 100% conversion from copper hydroxysulfates and hydroxychlorides into copper oxalates. A fungal strain was used which had been isolated from vineyard soil heavily contaminated with copper. Further scientific investigations were carried out to determine the parameters of the process and allowing the formation of a reproducible and homogeneous patina of copper oxalates, called biopatina to highlight its biological origin. Particular attention was paid to the efficacy, durability and impact on colour of the newly developed treatment. Different copper and bronze coupons with either urban or marine patinas were prepared. Several analytical techniques were used for the characterisation of the patinas: Fourier Transform InfraRed microspectroscopy (µFTIR), colourimetry and Electrochemical Impedance Spectroscopy (EIS). The coupons were treated with either the biological treatment or reference materials (wax: Cosmoloid H80; silane: Dynasylan® F8263) and exposed to atmospheric corrosion (ISMAR-SMS Genoa Harbour, corrosivity class 5) in December 2011. The long-term behaviour and performance of the treatments under study was monitored and compared over a one year period using the same complement of analytical techniques used for the characterisation of the original patinas. These first measurements suggested a different weathering behaviour of the biopatina. In fact, in comparison to the silane and wax treatments the biopatina showed a lower colour variation, and a corrosion stabilisation process seemed to be in progress. A deeper analysis of colour and corrosion rate variation from different application methods was also achieved. The complete assessment of the different treatments will be finished this year.
  • Publication
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    Diversity of endospore-forming bacteria in sediment as a proxy for environmental lake history
    (2013)
    Wunderlin, Tina
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    Les lacs une grande importance écologique et économique, mais ils sont aussi très vulnérables aux pressions anthropogéniques (pollution; surpêche), aux changements climatiques et à l'eutrophisation (hautes concentrations de nutriments). Pour une gestion environnementale et la mise en place de mesures de conservation, les dynamiques des écosystèmes lacustres doivent être connues et les conditions biologiques de référence doivent être établies afin de mesurer dans le futur la santé écologique des lacs. Les conditions biologiques de références, comme les connaissances de la biodiversité et les réponses des écosystèmes aux perturbations environnementales sont fournies par les sédiments, qui sont des archives idéales des conditions du passé.
    Cette thèse présente la recherche sur la détection et la diversité des bactéries sporulantes et leur usage comme indicateurs des conditions écologiques du lac Léman (France-Suisse) au cours des 100 dernières années. Les endospores sont des structures résistantes qui sont produites par un groupe de bactéries dans des conditions de stress. Les endospores dormantes sont déposées avec le sédiment et sont des capsules biologiques qui reflètent les conditions environnementales au moment de leur sédimentation.
    Les approches métagénomiques sont des études de séquençage du métagénome entier qui se trouve dans un échantillon environnemental. Une approche ciblée est réalisée uniquement sur une partie de la communauté (fraction ciblée). Ces approches métagénomiques ciblées augmentent la couverture de séquençage et également la résolution de détection de taxons. Elles résolvent donc les problèmes connus des approches métagénomiques globales. Deux méthodes de métagénomique ciblée ont été développées au cours de cette thèse pour étudier la diversité des bactéries sporulantes dans les sédiments.
    La première méthode est basée sur des amorces moléculaires dessinée pour amplifier un fragment du gène spo0A, gène spécifique aux bactéries sporulantes codant pour le facteur de transcription de la sporulation. De plus, une méthode optimisée d'extraction d'ADN pour les bactéries sporulantes a été développée. En appliquant ces méthodes, la diversité des cellules végétatives des bactéries sporulantes ainsi que les endospores dans le sédiment peut être étudiée.
    La deuxième méthode de métagénomique ciblée est une méthode de traitement avec chaleur et agents chimiques pour détruire les cellules végétatives, qui sont fragiles, comparé aux endospores qui résistent au traitement. Avec cette méthode, la diversité seule des endospores peut être évaluée. Le traitement pour détruire les cellules végétatives est efficace, 90% des séquences détectées sont classifiées comme bactéries sporulantes. Avec une approche globale, seulement 10% des séquences détectées sont classifiées comme bactéries sporulantes. De plus, la résolution a été augmentée, en détectant jusqu'à 10 fois plus de taxon. La meilleure résolution permet de détecter 34 genres de bactéries sporulantes non révélés avec l'approche globale, dont certain genres qui ont été définis comme étant asporogénique, comme Ethanoligenens et Trichococcus. D'autres séquences ont pu être classifiées au niveau de l'espèce comme Bacillus longiquaesitum ou Clostridium bowmanii.
    Les méthodes métagénomiques ciblées ont été appliquées à une carotte de sédiment du Lac Léman, qui couvre la période de 1921 à 2010. Une diversité extraordinaire de bactéries sporulantes a été observée dans ces sédiments. Il y a des fluctuations importantes dans la diversité au cours des 100 dernières années. Prioritairement les changements de diversité peuvent être liée à l'eutrophisation du lac de 1960 à 1990, mais aussi au métabolisme du soufre, la charge de matière organique d'origine terrestre ou à des évènements climatiques. La communauté bactérienne dans la période eutrophe a changé vers une dominance de bactéries anaérobies comme les Clostridia, qui reflètent les conditions anoxiques de la surface du sédiment pendant cette période.
    Les avantages du traitement pour séparer les endospores des cellules végétatives sont que ces deux fractions peuvent être analysées individuellement. Avec ces différentiations, des genres actifs dans le sédiment comme Clostridium et Heliobacterium peuvent être relevés. Par contre, il y a toute une fraction diversifiée d'endospores dormantes qui est présente en faible nombre, jusqu'à ce que les conditions environnementales changent en leur faveur ou elles deviennent dominantes. Des exemples de ce phénomène ont été observés pour les genres Desulfotomaculum, Sporomusa et Brevibacillus.
    Les nouvelles méthodes de métagénomique ciblée qui ont été développées dans le cadre de cette thèse sont des améliorations importantes pour étudier la diversité des bactéries sporulantes à haute résolution. Les données acquises pendant cette recherche amènent de nouvelles informations sur le rôle des bactéries sporulantes dans les sédiments et en général sur les bactéries des sédiments lacustres. Cette étude est aussi la première étude sur la diversité des bactéries sporulantes par des méthodes de séquençage et la première étude d'utilisation des endospores comme indicateur paléolimnologique., Freshwater systems are of high ecological and economical importance but at the same time subjected to anthropogenic pressure such as input of pollutants, overfishing, changes in climate regime, and eutrophication (high nutrient input). For environmental management and the implementation of conservation measures, the dynamics of freshwater ecosystems need to be known and biological reference conditions have to be established against which future changes can be measured. Baseline knowledge about biodiversity and ecosystem responses to environmental perturbations in lakes can be obtained from the sediments that provide an ideal environmental archive of past conditions.
    This thesis presents research on the diversity detection of bacterial endospores, and its use as proxy to reconstruct the environmental history of the last 100 years of Lake Geneva at the border of France and Switzerland.
    Endospores are resistant structures formed when bacteria are under stress. Once these endospores are deposited in the sediment they remain dormant and serve as natural biological time capsules, archiving the conditions at the time of sedimentation. To infer the diversity of endospores, two specific methods for targeted metagenomics were developed and validated in sediments. Metagenomics is a sequencing approach of the entire genetic pool directly retrieved from an environmental sample. Similarly, targeted metagenomics is based on a targeted genetic pool, for example a sub-community of the sample. Targeted metagenomics increases the sequencing coverage and resolution of detection, circumventing common problems of traditional metagenomics studies. For this work the endospore-forming bacterial community was targeted.
    The first targeting method was based on a molecular marker for endospore-forming bacteria, the global transcription regulator of sporulation (spo0A). After an optimized DNA extraction method for endospores in sediment, where biomass was separated from the sediment particles (indirect DNA extraction), and the spo0A gene fragment was amplified and sequenced. With this method, the diversity of the endospore-forming bacteria (vegetative cells and endospores) in sediments was determined.
    The second targeting method consisted of a treatment to separate the endospores from vegetative cells, prior to DNA extraction and sequencing. The goal of the treatment was to destroy vegetative cells that are generally more fragile, while leaving the more resistant endospores intact. With this method, the diversity of only the endospores in sediment was detected. The treatment to separate endospores was successful, as shown by an enrichment of endospore-forming bacteria from 10% abundance in the global approach to over 90% abundance in the targeted metagenome. Also the resolution was improved to up to 10-fold increase in detected endospore-forming taxa. The better resolution led to the detection of 34 genera unique to the targeted metagenome, including some supposedly asporogenic groups like Ethanoligenens and Trichococcus and high numbers of sequences that could be classified to a species level such as Bacillus longiquaesitum or Clostridium bowmanii.
    The application of targeted metagenomics to a sediment core retrieved from Lake Geneva spanning a time period from 1921 to 2010, revealed substantial diversity of endospore-forming bacteria in sediments. The diversity fluctuated significantly in the last 100 years, reflecting the eutrophication period from 1960 to 1990 as well as sulphate metabolism, input of terrestrial organic matter, and specific climate events. The shift in the community composition during eutrophication was linked to a dominance of anaerobic Clostridia-like members that reflect anoxic sediment conditions during this time.
    The advantage of the treatment is that the communities in vegetative cell state can be differentiated from the communities present as dormant endospores. Using this differentiation we report activity of selected endospore-forming bacteria in sediment, for example members from genus Clostridium and Heliobacterium at the sediment surface.
    In contrast, a small fraction of dormant endospores present at high diversity represent the microbial seed bank. This group of bacteria is inactive for long periods of time but selected members can propagate and become dominant if the environmental conditions change to their favour, as was observed in this study for Desulfotomaculum, Sporomusa or Brevibacillus.
    The novel targeted metagenomics approaches developed here provide a significant experimental improvement to explore the diversity of endospore-forming bacteria at high resolution. The data provides knowledge on the role of endospore-forming bacteria in freshwater sediment and on freshwater sediment bacteria in general. It is also the first report of metagenomics to reveal the diversity of endospores in sediment and the use of endospores as paleolimnological proxies.
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    Uranium speciation and stability after reductive immobilization in aquifer sediments
    (2011)
    Sharp, Jonathan O
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    Lezama-Pacheco, Juan S
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    Schofield, Eleanor J
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    Ulrich, Kai-Uwe
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    Chinni, Satya
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    Veeramani, Harish
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    Margot-Roquier, Camille
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    Webb, Samuel M
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    Tebo, Bradley M
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