La produzione di biogas è un tematica di forte impatto globale per due ragioni principali: il prossimo esaurimento dei combustibili fossili e l’inquinamento ambientale dovuto allo smaltimento di scarti organici. La Digestione Anaerobica (DA) è un processo biologico che permette la risoluzione di entrambi i problemi, producendo energia (in forma di biogas) e convertendo gli scarti organici in metano e anidride carbonica. Tale processo è basato su una complessa catena sintrofica tra consorzi microbici che produco il substrato per la fase finale di metanogenesi. Il siero di latte è uno scarto altamente inquinante derivante dal processo di lavorazione del formaggio e per questo è stato ampiamente investigato come substrato per la DA. Tuttavia esiste uno scarto meno noto prodotto dalle fasi di porzionatura e grattugia del formaggio a lunga stagionatura. Il presente studio analizza il microbioma di digestori anaerobici processanti scarti dell’industria lattiero-casearia, quali letame bovino, siero di latte e scarto del formaggio a pasta dura. In particolare, viene analizzato l’effetto dei parametri di processo, delle diverse configurazioni dei reattori e del tipo di scarto, su tale microbioma. L’obiettivo è raggiunto tramite tecniche biomolecolari che permettono di quantificare e identificare le principali specie presenti nei reattori, insieme alla differente espressione genica in seguito all’iniezione di idrogeno a scopo di upgrading del biogas. / Biogas production is a hot topic, which has globally gained interest from many researchers over the past years. This fact is mainly due to the depletion of fossil fuels and environmental concerns regarding wastes disposal. Anaerobic Digestion (AD) represents a biological way to obtain both energy (in form of biogas) and waste discard, by converting the polluting organic matter. The overall process relies on a syntrophic chain where different microbial consortia produce the feed necessary for the final methanogenic step. Cheese whey has been largely investigated for AD treatment, since is a high polluting waste derived from the cheese-making process. However, there is a less-known waste originating from the portioning and shaving phases of long-ripened hard-cheese. This study aimed to investigate the microbiome of anaerobic digesters processing dairy industry wastes, such as cattle manure, cheese whey and hard-cheese powder wastes. In particular, the effects of process parameters, reactor configurations and type of dairy wastes, on the microbial populations, have been analyzed. The goal was achieved by means of culture-independent methods and high throughput sequencing, which allowed quantifying and identifying the main species present, as well as their differential gene expression in relation to hydrogen injection for biogas upgrading purposes.

With its unique consortium of microorganisms from all domains of life, termite gut is considered one of the most efficient lignocellulose degrading systems in nature. Recently, host diet and taxonomy as well as gut microenvironmental conditions have emerged as main factors shaping microbial communities in termite guts. The aim of this thesis was to investigate this highly efficient lignocellulolytic system at holobiont level, with a particular focus on gut microbiome function and composition in relation to the host diet. As a starting point, we optimised a complete framework for an accurate termite gut prokaryote-oriented metatranscriptomics, which was at the basis of all subsequent sequencing assay designs and analyses performed in the course of the work. Afterwards, we characterised the compositions and functions of biomass-degrading bacterial communities in guts of plant fibre- and soil-feeding higher termites, proving the existence of functional equivalence across microbial populations from different termite hosts. We also showed that each termite is a reservoir of unique microorganisms and their accompanying genes. We further extended above approach to metagenomics and bacterial genomes reconstruction and we applied it to explore the process of biomass digestion in the different sections of the highly compartmented gut of soil feeding Labiotermes labralis. We showed that primarily cellulolytic activity of the termite host was restricted to foregut and midgut, while bacterial contribution was most pronounced in P1 and P3 hindgut compartments and included activities targeting broad range of lignocellulose components. Finally, we investigated the adaptation of a laboratory-maintained grass-feeding higher termite colony of Cortaritermes spp. to Miscanthus diet at host and symbiont levels. A natural system of a termite gut was shown to progressively change in composition to yield a consortium of microbes specialised in degradation of a specific biomass. Overall, the integrative omics approach proposed here provide a framework for a better understanding of a complex lignocellulose degradation by a higher termite gut system and pave a road towards its future bioprospecting. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Les populations naturelles sont confrontées à des changements environnementaux. Pour y faire face, différentes réponses ont été sélectionnées au cours de l'évolution. Parmi elles se trouvent la plasticité phénotypique et l'adaptation génétique. Etudier les liens existants entre elles est une manière de comprendre les dynamiques des populations et de prévoir leurs réponses à un environnement changeant. Dans la présente étude, je me suis attaché à étudier ces liens à plusieurs échelles (intra- et interspécifique), chez le complexe d'espèces cryptiques de la micro-algue Alexandrium minutum, et ce à la fois in vitro et in situ. En ce qui concerne la plasticité phénotypique, ces deux espèces proches montrent de profondes différences, soulignant les liens entre divergence génétique et écologique. Au niveau intraspécifique, il apparaît que face à des variations de facteurs abiotiques, les populations ajustent les niveaux d'expression de certains gènes (notamment impliqués dans des fonctions de motilité et d'interactions intercellulaires dans des environnements froids à faible salinité). D'autre part, les populations montrent de la différentiation génétique à la fois à faible échelle spatiale, au cours du temps, et lorsque la communauté change. Pour conclure, il existe une interaction directe entre divergence génétique et changements d'expression de gènes. En plus de poser de nombreuses questions quant aux capacités de réponse des populations, ces résultats soulignent comment plasticité phénotypique et changements génétique sont liés et interagissent. Ils offrent une perspective nouvelle sur les mécanismes qui sous-tendent les réponses des populations à leur environnement. / Natural populations face environmental changes. In this context, different responses were evolutionnary selected. Among them are phenotypic plasticity and genetic adaptation. Studying the links between these two types of response is a way to understand population dynamics and to predict how they may respond to a changing environment. In the present Ph.D thesis, I focused on studying these links at several scales (intra- and interspecific), in the cryptic species complex of the microalga Alexandrium minutum, both in vitro and in situ. With respect to phenotypic plasticity, these two closely related species show profound differences, highlighting the links between genetic and ecological divergence. At the intraspecific level, it appears that, when facing abiotic factors variations, populations adjust the expression levels of certain genes (notably involved in motility related functions and intercellular interactions under low-salinity and cold environments). On the other hand, populations show genetic differentiation at both small spatial scale, over time, and when the community changes. To conclude, there is a direct interaction between genetic divergence and changes in gene expression. In addition to asking many questions about the response capabilities of populations, these results highlight how phenotypic plasticity and genetic changes are linked and interact. They offer new perspectives on the mechanisms underlying population responses to their environment.

Deadwood is a hotspot of microbial diversity and its decomposition contributes to carbon and nitrogen cycling in temperate forests. The historically recognized importance of fungi in the decomposition of deadwood has recently been complemented by the description of bacterial functions thanks to the rapid progress of culture-independent methods based on the analysis of nucleic acids. To study different aspects of deadwood decomposition, a temperate mixed forest in Zofinsky prales National Nature Reserve was selected as a site with rich historical forestry data where deadwood decomposition represents an important process in wood turnover. The aim of this thesis is to describe role of bacteria in deadwood decomposition at fine scale resolution with respect to community composition, enzyme transcription, and metabolic potential of dominant species. Effects of deadwood age together with pH and water content on the bacterial community composition proved to be more important than tree species identity. Bacteria showed distinct composition between early and late community in decomposing deadwood. The bacterial community was also under a significant influence of fungal community composition. Despite being in a close contact, bacterial and fungal communities differed significantly between deadwood and the...

Pelagic aggregates function as biological carbon pumps for transporting fixed organic carbon to sediments. In iron-rich (ferruginous) lakes, photoferrotrophic and chemolithoautotrophic bacteria contribute to CO2 fixation by oxidizing reduced iron, leading to the formation of iron-rich pelagic aggregates (iron snow). The significance of iron oxidizers in carbon fixation, their general role in iron snow functioning and the flow of carbon within iron snow is still unclear. Here, we combined a two-year metatranscriptome analysis of iron snow collected from an acidic lake with protein-based stable isotope probing to determine general metabolic activities and to trace 13CO2 incorporation in iron snow over time under oxic and anoxic conditions. mRNA-derived metatranscriptome of iron snow identified four key players (Leptospirillum, Ferrovum, Acidithrix, Acidiphilium) with relative abundances (59.6–85.7%) encoding ecologically relevant pathways, including carbon fixation and polysaccharide biosynthesis. No transcriptional activity for carbon fixation from archaea or eukaryotes was detected. 13CO2 incorporation studies identified active chemolithoautotroph Ferrovum under both conditions. Only 1.0–5.3% relative 13C abundances were found in heterotrophic Acidiphilium and Acidocella under oxic conditions. These data show that iron oxidizers play an important role in CO2 fixation, but the majority of fixed C will be directly transported to the sediment without feeding heterotrophs in the water column in acidic ferruginous lakes.

La dégradation des fibres alimentaires est une fonction essentielle des écosystèmes digestifs microbiens. Chez le ruminant, elle est assurée par des bactéries, champignons et protozoaires capables de produire de nombreuses enzymes nécessaires à l’hydrolyse des polysaccharides de paroi végétale. Parmi les facteurs susceptibles d’influencer l’efficacité de dégradation des fibres, qui est une composante importante de la productivité et de la santé animales, des additifs tels que des levures probiotiques apparaissent comme un levier intéressant. Afin d’approfondir les connaissances sur les facteurs de modulation de l’activité fibrolytique, une biopuce ADN fonctionnelle, outil moléculaire haut-débit, ciblant les gènes codant les enzymes clés de la dégradation de la cellulose et des xylanes dans les écosystèmes digestifs a été développée. Aussi, une méthode efficace dédiée à des échantillons ruminaux pour la soustraction des ARNr à partir des ARN totaux a été mise au point afin d’accroitre la sensibilité de l’outil. La biopuce fonctionnelle a été validée sur échantillons de complexité croissante et démontre d’excellents caractères de spécificité et de sensibilité tout en étant exploratoire et quantitative. Des régulations différentielles de l’arsenal des gènes de la fibrolyse de la bactérie du rumen Fibrobacter succinogenes ont pu être montrées. De même, les résultats sur échantillons de rumen suggèrent un rôle des microorganismes eucaryotes dans la fibrolyse pouvant être plus important qu’initialement envisagé. Cette approche métatranscriptomique dirigée pourra in fine continuer d’être appliquée dans l’étude de l’impact de facteurs biotiques et abiotiques sur la fonction fibrolytique microbienne chez les animaux d’élevage. / Dietary fibre degradation is an essential function of microbial digestive ecosystems. In ruminants, this function is ensured by bacteria, fungi and protozoa, producing a large array of enzymes able to degrade plant cell wall polysaccharides. Among factors likely to influence the efficiency of fibre degradation, which is an important component in animal productivity and health, dietary additives such as probiotic yeasts appear as an interesting tool. To provide more insight on factors modulating fibrolytic activity, we designed a functional DNA microarray targeting genes coding for key enzymes involved in cellulose and xylan degradation by digestive microbiota. Also, an efficient method dedicated to rumen samples for removing microorganisms’ rRNA from total RNA samples was developed to increase the sensitivity of the tool. The DNA microarray was validated using targets of increasing complexity and demonstrated sensitivity and specificity as well as explorative and quantitative potential. Differential expression of genes involved in fibrolysis was evidenced in the rumen bacterium Fibrobacter succinogenes. Moreover, results on rumen samples suggest a more important role of eucaryotes in fibre degradation than previously thought. This targeted metatranscriptomic approach will be further applied to the study of the impact of biotic and abiotic factors on the microbial mechanisms of fibre degradation in livestock.

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