With consideration to the crude glycerol surplus driven by the growth in biodiesel manufacturing, alternative uses for biodiesel derived glycerol have become increasingly essential. Anaerobic digestion of the glycerol reduces the chemical oxygen demand of the glycerol waste product, while capturing biogas as an energy source that can be used on site to reduce heating cost at a biodiesel facility. In this thesis, kinetic parameters are extracted from batch experimental data and applied to steady state equations. A flow sheet for the application of anaerobic digestion to biodiesel derived glycerol is developed and explored. The economic analysis of the scenario tracks the capital cost, operating cost, and savings associated with implementing the system.

Les Archaea hyperthermophiles sont des micro-organismes particulièrement adaptés à la vie à très haute température. Les températures élevées sont responsables de la création de diverses lésions à l’ADN. Pourtant, le taux de mutations spontanées chez l’Archaea Sulfolobus solfataricus est proche de celui mesuré chez des organismes mésophiles. Ceci laisse suggérer que les Archaea doivent être équipées de mécanismes spécialisés efficaces dans la reconnaissance et réparation des dommages à l’ADN. La réplication de l’ADN est un mécanisme fonctionnellement conservé dans les trois domaines du Vivant assuré par des enzymes clefs, les ADN polymérases. A ce jour, chez Pyrococcus abyssi (Pab), Euryarchaea hyperthermophile, deux familles de pol ont été identifiées, une pol réplicative de la famille B, présente dans les trois règnes du Vivant et une pol réplicative la famille D, spécifique de l’embranchement des Euryarchaea. Tous les membres de la famille B des pols archéennes possèdent une signature structurale unique impliquée dans la reconnaissance spécifique des bases endommagées (uracile et hypoxanthine). Ce doctorat a permis de démontrer que cette poche pouvait également accommoder les bases canoniques (A, T, C, G) contribuant ainsi à la haute fidélité de ces enzymes. Des structures cristallographiques à très haute résolution de l’ADN polymérase B (PabPolB) ont permis d’identifier la présence d’un ion métallique divalent situé à proximité de cette poche de reconnaissance. Il a été suggéré que cet ion modulerait la reconnaissance des bases désaminées et le glissement de l’ADN polymérase au cours de la synthèse d’ADN. De plus, nous avons montré que les ions catalytiques divalents ont un rôle central dans la modulation des propriétés intrinsèques de PabPolB. L’ion calcium est utilisé par PabPolB pour mener à bien la synthèse d’ADN mais engendre des caractéristiques cinétiques différentes de celles conférées par l’ion magnésium universel. D’autre part, nous avons déterminé pour la première fois chez un organisme archéen la concentration intracellulaire de dNTPs et NTPs. Leurs concentrations sont relativement plus élevées que celles mesurées par exemple chez l’eucaryote Saccharomyces cerevisiae, suggérant un mécanisme constitutif de réaction de protection à des agressions de l’ADN. L’ensemble de ces résultats participe à la compréhension des mécanismes moléculaires qui régissent la maintenance de l’intégrité du génome et, donc, de la vie des organismes hyperthermophiles. / Hyperthermophilic Archaea that thrive under harsh environments (elevated temperature, pH shifts, andionizing radiations) are supposed to be exposed to massive DNA damages. However, the mutations frequencies in hyperthermophilic Archaea are comparable with those of other microorganisms (1) indicating they are equipped with unique and efficient molecular mechanisms to ensure their genome integrity. DNA replication is an essential and conserved process among the three domains of life. DNA polymerases are central enzymes involved in the joining of deoxyribonucleoside 5′-triphosphates (dNTPs) to form the growing DNA chain. In Pyrococcus abyssi (Pab), two familiesDNA polymerases have been described as replicases, one family B (PabPol B) with structural diversity and common mechanisms among all organisms in the three domains of life, and one family D found exclusively in Euryarchaea. All members of archaeal family B DNA polymerases possess a unique structural feature involved in the recognition of deaminated bases (uracil and hypoxanthine) called uracil-pocket. During my PhD, it has been shown that not only deaminated but also canonical bases (A, T, C, G) could enter this pocket in PabPolB. We therefore renamed it as the base-checking pocket and proposed a role in the high fidelity of PabPolB. High-resolution crystal structures of PabPolBhig hlighted the presence of divalent metal ion to the proximity of the base-checking pocket. It has been suggested that thismetal ion may modulate the recognition of deaminated bases and the translocation of PabPolB on DNA during DNAsynthesis. Moreover, the crucial role of metal ion on DNA synthesis and exonuclease activity by PabPolB has beenevaluated. DNA polymerisation in the presence of calcium was as effective as the universal magnesium ion while showing different time-course kinetics. For the first time, intracellular concentrations of nucleotides (dNTPs and NTPs) have been determined in an archaeal microorganism. dNTPs and NTPs concentrations seem rather elevated for Pab than in the eukaryotic Saccharomyces cerevisiae cells, suggesting a constitutive mechanism for protecting the genome from DNAdamage. Overall, these results contributed to unravel specific molecular mechanisms involved in the maintenance of the genome integrity with a particular emphasis on molecules (DNA polymerases, dNTPs and NTPs) crucial for cellular life.

Les organismes vivants doivent reproduire et transmettre ne variatur l’information contenue dans les chromosomes. Ainsi, la conservation de l’intégrité du génome est un processus biologique fondamental. La maintenance génomique constitue l’ensemble des processus biologiques impliqués dans la conservation, la duplication et la transmission de l’information génétique contenue dans les chromosomes. La machinerie réplicative des Archaea est décrite comme une version simplifiés de celle connue chez les Eucaryotes faisant des Archaea un excellent modèle d’étude de la réplication. Contrairement à la réplication, les processus Archaea de réparation de l’ADN sont encore mystérieux. En effet, plusieurs protéines essentielles de la réparation semblent absentes des génomes Archaea et ce même chez les espèces Hyperthermophiles (HA). Avec une température optimale de croissance proche de 100°C, ces Archaea doivent posséder des capacités considérables de réparation des dommages de l’ADN, catalysés à haute température. Ainsi les Archaea hyperthermophiles sont probablement dotées d’un système de réparation alternatif extrêmement efficace. Ce système et sa coordination avec la réplication sont inconnus. Un protocole de purification d’affinité couplée à la spectrométrie de masse des protéines a permis d’identifier les complexes protéiques impliqués dans la maintenance génomique de l’Archaea hyperthermophile P. abyssi. Les complexes identifiés sont compilées dans un réseau d’interaction. Soumis à une étude topologique le réseau révèle notamment de nouvelles interactions entre des protéines essentielles de la maintenance génomique, conservées avec les Eucaryotes. Plusieurs interactions sont vérifiées indépendamment où caractérisées fonctionnellement in vitro ou in vivo. Ces travaux mettent en lumière l’étroite collaboration entre la réplication et la recombinaison de l’ADN et révèlent de nouveaux aspects de la machinerie de transcription. / DNA replication, recombination and repair are central and essential mechanisms in all cells. Highly efficienthigh-fidelity chromosome replication is vital for maintaining the integrity of the genetic information and for theavoidance of genetic disease. Archaeal replisome is described as simplified version of the eukaryotic system.However, DNA repair is still enigmatic, as many essential repair proteins have not been identified in Archaealgenomes. The question of DNA repair is even more puzzling while many Archaea lives under extremetemperature that promotes DNA instability and catalyses nucleobase damages. Thus, HyperthermophilicArchaea (HA) must have solved a molecular problem (spontaneous loss of native DNA structure) at amagnitude that mesophilic organisms do not face. A highly adapted DNA maintenance system must operate inorder to maintain DNA integrity. Those mechanisms and their possible coordination with DNA replication arestill unknown. Here, I report the first protein-protein interaction network of genomic maintenance in HA. Using AP-MSapproach we identified new protein complexes potentially implicated in DNA replication, recombination andrepair of HA P. abyssi. Topological analysis of the network highlighted both known and unknown partners ofessential and conserved protein of genomic maintenance. From the network emerges multifunctional clustersintegrating both replication and recombination proteins and revealing new aspects of the transcriptionmachinery. I also provide experimental confirmation of some of the interactions we detected.I propose that the interactions we observe reflects the interplay between recombination and replicationmachineries that likely interfaces with regulatory elements involved in the control of the DNA damageresponse, as shown by the identification of a new factors, presumably involved in the coupling of DNArecombination and DNA synthesis at the replication fork.

Les archées sont principalement connues pour leur capacité à croître et survivre dans des conditions extrêmes de température, pression, pH, etc. qui sont hostiles à l’homme. Néanmoins, il est désormais clair que les archées sont aussi présentes de manière ubiquitaire dans divers environnements. L’étude détaillée des différents aspects de la biologie de ces microorganismes a amené à des découvertes pour le moins inattendues comme celle de la virosphère associée aux archées qui est unique. En effet, plusieurs virus infectant les archées ont été isolés et présentent une incroyable diversité tant au niveau morphologique que génomique et ne ressemblent aucunement aux virus connus de bactéries ou d’eucaryotes. Récemment, l’analyse en détails du cycle viral a mis à jour de nouveaux mécanismes d’interactions avec la cellule hôte. Au cours de mes travaux de thèse, nous nous sommes intéressés aux systèmes virus-hôtes présents dans les milieux hyperthermiques et acidophiles en sélectionnant les virus fusiforme et filamenteux SSV1 et SIRV2 en tant que modèles d’étude. Tout d’abord, nous avons défini une nouvelle classification des virus fusiformes en basée sur l’analyse comparative des protéines structurales et des génomes viraux. L’ensemble des virus considérés forme un réseau global malgré le fait qu’ils ont été isolés dans des environnements distincts ; qu’ils infectent des hôtes qui sont distant phylogénétiquement parlant et que certains de leurs virions présentent une certaine pléomorphicité. Ensuite, la caractérisation en détails de l’architecture des virions fusiformes de SSV1 a révélé qu’ils étaient enveloppés, composés de protéines de capside glycosylées et contenaient le complexe nucléoprotéique. Finalement, nous nous sommes concentrés sur la manière dont les virus d’archées interagissent avec la cellule hôte. Alors que les virions de SIRV2 semblent utiliser une stratégie pour l’entrée qui est similaire aux bactériophages dits flagellotrophiques ; on observe que les virions de SSV1 emploient un mécanisme de sortie qui rappelle le bourgeonnement des virus eucaryotes enveloppés. L’ensemble de ces recherches participent à une meilleure compréhension de la biologie des archées ainsi que de leurs virus et permettent de définir des cibles intéressantes pour de futures études. / Although, archaea were initially regarded as exotic microorganisms capable of growing in conditions which are hostile to humans, it became clear that they are ubiquitous and abundant in various environments. Detailed studies focusing on different aspects of archaeal biology have led to many unexpected discoveries, including the unique virosphere associated with archaea. Indeed, highly diverse viruses characterized by uncommon virion shapes and mysterious genomic contents have been isolated that typically do not resemble viruses of either bacteria or eukaryotes. Recent analysis of the sequential events of the viral cycle resulted in major breakthroughs in the field. In the framework of my PhD studies, I have focused on two model hyperthermo-acidophilic virus-host systems, the spindle-shaped SSV1 and rod-shaped SIRV2, both infecting organisms of the genus Sulfolobus. Initially, we defined structure-based lineages for all known spindle-shaped viruses isolated from highly divergent hosts and residing in very different environments. Then, we provided insights into the architecture of spindle-shaped viruses by showing that SSV1 virions are composed of glycosylated structural proteins and contain a lipid envelope. Finally, we focused on virus-host interplay. Whereas SIRV2 virions appear to use a similar entry strategy as flagellotrophic bacteriophages, SSV1 virions employ an exit mechanism reminiscent of the budding of eukaryotic enveloped viruses. Collectively, these studies shed light on the biology of archaeal viruses and help to define interesting targets that should be the focus of intensive research in the next future.

Orientador: Lucia Regina Durrant / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-18T08:43:54Z (GMT). No. of bitstreams: 1 Piubeli_FrancineAmaral_D.pdf: 6673876 bytes, checksum: 678469d11c710488c1e19eda0ee7d179 (MD5) Previous issue date: 2011 / Resumo: A água residuária da extração de petróleo é altamente salina e contém uma mistura complexa de hidrocarbonetos, muitos dos quais são altamente tóxicos. Com o aumento da preocupação em preservar o meio ambiente, as tecnologias voltadas para a recuperação e despoluição de áreas degradadas tem ganhado a atenção. A biorremediação tem sido utilizada como método de biodegradação natural através da otimização de processos biológicos por ser econômica, versátil e a que mais se aproxima de uma despoluição ecologicamente aceitável. No entanto, a biorremediaçao depende de alguns fatores tais como: pH, temperatura, salinidade e pressão. Condições extremas desses fatores podem matar ou inibir espécies que não estejam adaptadas a ambientes extremos, por isso o desenvolvimento e otimização de processos de biorremediação de ambientes extremos contaminados por é de grande relevância. Desta forma, este trabalho teve como objetivos o isolamento e caracterização de bactérias halofílicas presentes na água residuária da extração de petróleo, além do estudo molecular dos genes envolvidos na degradação de compostos aromáticos para que posteriormente utilizássemos essas linhagens na biorremediação desse efluente, outra vertente desse trabalho foi o estudo da diversidade microbiana presente nesta água para auxiliar o direcionamento de esforços na minimização dos impactos causados com a exploração de petróleo. Foram isoladas sete linhagens de bactérias halofílicas da água de produção de petróleo, sendo todas pertencentes a família Halomoneaceae e com potencial para degradação de compostos aromáticos. A linhagem que apresentou maior taxa de crescimento nos compostos testados (ácido benzóico, fenol, ácido p-hidroxibénzoico) foi a denominada df2. Com relação à taxa de degradação dos compostos aromáticos testados, a dp3 degradou cerca 90% do fenol disponível, seguida pela df2 que degradou 80% desse mesmo composto. Quando a fonte de carbono foi o ácido benzóico, a df1 e DP3 degradaram 99% do composto e 70% do ácido p-hidroxibenzóico foi degradado em 12 dias de experimento pela linhagem df2. Para a degradação desses compostos a linhagem Halomonas organivorans seguiu a via de degradação do catecol na rota do ß- cetoadipato compreendendo a codificaçao de diferentes enzimas por catRBCA. A comparação das seqüências da biblioteca de bactérias com as sequências do gene RNA ribossomal 16S presente nos bancos de dados GenBank e RDP revelou a presença de micro-organismos pertencentes aos gêneros Marinobacter e Halomonas e na comparação da técnica clássica de isolamento com a técnica independente de cultivo (biblioteca do gene RNA ribossomal 16S), foi encontrada uma maior diversidade na última. Finalmente de todos os testes realizados na tentativa de biorremediar a água de produção de petróleo, a bioestimulação foi a mais efetiva, sendo conseguida uma diminuição de 77% da carga orgânica com a adição de solução de fosfato e alanina. Estes resultados demonstraram o grande potencial destas linhagens para a degradação de compostos aromáticos, bem como para a biorremediação da água de produção de petróleo, além de descrever a via metabólica utilizada por membros da família Halomoneaceas na degradação de compostos aromáticos, auxiliando assim nos esforços para biorremediação de ambientes salinos contaminados / Abstract: The wastewater from oil extraction (produced water) is highly saline and contains a complex mixture of hydrocarbons, many of which are highly toxic. With increasing concern about preserving the environment, the technologies for recovery and remediation of degraded areas has been gaining attention. Bioremediation has been used as a method of natural biodegradation by optimizing biological processes because it is economical, versatile and it is the closest to a ecologically acceptable decontamination. However, bioremediation depends on factors such as pH, temperature, salinity and pressure. Extremes of these factors can inhibit or kill species that are adapted to extreme environments, so the development and optimization of bioremediation processes for contaminated extreme environments is of great importance. Thus, this work aimed at the isolation and characterization of halophilic bacteria present in the wastewater from oil extraction, and molecular studies of the genes involved with the degradation of aromatic compounds so that later we could use these strains in the bioremediation of this wastewater. Another aspect of this work was to study the microbial diversity present in this wastewater to assist in directing the efforts to minimize the impact of oil drilling. Seven strains of halophilic bacteria were isolated from of the produced water of oil production, all belonging to the family Halomoneaceae having potential for degradation of aromatic compounds. The strain that showed the highest growth rate in the tested compounds (benzoic acid, phenol, p-hydroxybenzoic acid) was strain DF2. Regarding the rate of degradation of the aromatic compounds tested, dp3 degraded about 90% of available phenol, followed by DF2 that degraded 80% of this compound. When the carbon source was benzoic acid, DP3 and df1 and degraded 99% and strain DF2 degraded 70% when the compound was p-hydroxybenzoic acid. For the degradation of these compounds the strain Halomonas organivorans followed the catechol degradation pathway ending in the formation of ß-Ketoadipate and was shown to produce various enzyme activities encoded by catRBCA. Comparison of the 16S rRNA gene sequences of the isolates with the sequences present in the GenBank and RDP databases revealed the isolates belonged to the genera Marinobacter and Halomonas. Comparing these results with those obtained by classical taxonomic techniques demonstrated that the 16S ribosomal RNA gene analysis (culture independent method), found greater diversity. Finally, of all tests performed to bioremediate the produced water, biostimulation by addition of nutrients was the most effective, and achieved a reduction of 77% of the organic load with the addition of phosphate combined with alanine. These results demonstrate the great potential of these strains for the degradation of aromatic compounds, as well as for the bioremediation of produced water from crude oil production, and describe the metabolic pathway used by members of the family Halomoneaceas for the degradation of aromatic compounds, thus assisting in efforts to develop methods for the bioremediation of contaminated saline environments / Doutorado / Ciência de Alimentos / Doutor em Ciência de Alimentos

Les archées halophiles sont des procaryotes vivant dans des conditions d’extrême salinité. Ces micro-organismes colonisent les environnements hypersalins et synthétisent des peptides antimicrobiens appelés halocines, qui pourraient leur conférer un avantage sélectif sur leurs compétiteurs. Parmi les peptides antimicrobiens d’archées halophiles décrits, on distingue l’halocine C8, initialement purifiée à partir de la souche halophile Natrinema sp. AS7092. Ce travail de thèse a permis de montrer que la production d’halocine C8 était conservée chez plusieurs archées halophiles appartenant aux genres : Natrinema, Haloterrigena, Haloferax et Halobacterium. Une activité antimicrobienne associée des particules supérieures à 100 kDa non infectieuses suggère que l’halocine C8 pourrait être localisée dans des vésicules membranaires. Ce travail présente des résultats qui déboucheront sur une meilleure compréhension des compétitions microbiennes dans les environnements hypersalins et du rôle écologique des halocines. / Halophilic archaea are prokaryotes living in extremely high salinity conditions. Those microorganisms thrive in hypersaline environments and produce antimicrobial peptides named halocins, which may confer them a selective advantage over competitors. Among the known antimicrobial peptides produced by halophilic archaea, halocin C8 had been initially purified from the halophilic strain Natrinema sp. AS7092. This work demonstrates that halocin C8 production is conserved among several halophilic archaea belonging to genera Natrinema, Haloterrigena, Haloferax and Halobacterium. An antimicrobial activity has been associated with non-infectious particles larger than 100 kDa, suggesting that halocin C8 could be localized in membrane vesicles. Results obtained from this work will lead to a better understanding of microbial competitions arising in hypersaline environments and the ecological role of halocins

Reconnu par leur effet toxique, inhibiteur et corrosif, les sulfures (S2-, SH-, SH2) sont un sous-produit indésirable de la digestion anaérobie des boues de station de traitement des eaux domestiques de la ville de Marrakech, Maroc (STEP). Ils proviennent essentiellement de la réduction "dissimilatrice" des composés soufrés (SO4 2-, SO3 2-, S2O4 2- ..) contenus dans ces boues. Ce processus est réalisé par un groupe bactérien anaérobie appelé bactéries sulfatoréductrices (BSR). Une fois dans le biogaz, les sulfures sous forme gazeuse réduisent en plus la durée de vie des installations et des équipements de la STEP. Elle est ainsi dotée d’installations biologiques et physico-chimiques lui permettant d’éliminer ces sulfures avant la transformation du biogaz en énergie électrique. Cependant, ces procédés sont onéreuses et grandes consommatrices d’énergies. D’où l’idée de minimiser la production des sulfures au sein même des digesteurs anaérobies. Pour cela, il était nécessaire d’abord de connaître les microorganismes à l'origine de la production des sulfures (BSR), ceux potentiellement impliqués dans leur élimination (bactéries sulfo-oxydantes), et d’un groupe de microorganismes fermentaires (Synergistetes) intervenant dans le bon fonctionnement de la digestion anaérobie. Ces études ont été menées à la fois sur des d'approches moléculaires et culturales. Les résultats obtenus, ont permis de comprendre comment ces groupes bactériens, d’intérêts écologique et économique importants, interviennent dans la digestion anaérobie des boues de la STEP permettant à la fois d’accélérer les processus d’oxydation de la matière organique combinée à la réduction des composés soufrés et de minimiser la concentration des en sulfure dans le biogaz. / Recognized by their toxic, inhibitory and corrosive effect, sulfides (S2-, SH-, H2S) are an undesirable by-product of the anaerobic digestion of from domestic wastewater treatment plants sludge in the city of Marrakech, Morocco (WWTP). They produced mainly by the dissimilatory reduction of sulfur compounds (SO42-, SO32-, S2O42-) contained in these sludges. This process is performed by an anaerobic bacterial group called sulfate reducing bacteria (SRB). Once in the biogas, the sulfides in gaseous form shorten in addition the lifetime of the installations and equipments of the WWTP. It is thus equipped with biological and physicochemical installations allowing it to eliminate these sulfides before the transformation of biogas into electrical energy. However, these processes are expensive and consume large amounts of energy. Hence the idea of minimizing the production of sulfides within anaerobic digesters. For this, it was first necessary to know the microorganisms originating of the production of sulfides (SRB), those potentially involved in their elimination (sulfur oxidizing bacteria), and a group of fermentative microorganisms (Synergistetes) involved in the good functioning of the anaerobic digestion. These studies were conducted on both molecular and cultural approaches. The results obtained allowed to understand how these bacterial groups, of great ecological and economic interest, are involved in the anaerobic digestion of sludge from the WWTP, which both accelerates the oxidation processes of the organic matter combined with the reduction of sulfur compounds and to minimize the concentration of sulfide in biogas.