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Dans l’environnement marin, les surfaces artificielles sont rapidement colonisées par des bactéries qui s’organisent en communautés appelées biofilms, s’entourant d’une matrice de substances polymériques extracellulaires (EPS). La formation d’un biofilm est une étape critique du processus nommé biofouling, c’est-à-dire l’accumulation de micro- et de macro-organismes sur une surface immergée, pouvant conduire à des conséquences néfastes dans le secteur marin. Dans cette étude, il s’agit d’identifier des souches bactériennes isolées de supports immergés en Mer Méditerranée et de les caractériser phénotypiquement par diverses approches. Leur capacité à former un biofilm in vitro a été évaluée dans différentes conditions avec une attention particulière portée sur leurs capacités à produire une matrice polymérique abondante riche en polysaccharides; l’objectif étant d’isoler des exopolysaccharides originaux à activité antifouling. Treize souches ont ainsi fait l’objet d’analyses phylogénétiques et d’une caractérisation phénotypique. Sept genres et douze espèces différentes ont été identifiés au sein desquelles deux isolats peuvent être affiliés à une nouvelle espèce, nommée Persicivirga mediterranea. Ce genre n’a jamais été décrit en Mer Méditerranée jusqu’à présent. L’extraction des EPS de chaque souche cultivée en biofilm a permis de déterminer leur composition générale en glucides, protéines, acides nucléiques et lipides. Une souche, Pseudoalteromonas ulvae TC14, se distingue par sa capacité à produire des exopolysaccharides en quantité importante. Il s’agit essentiellement de polymères du glucose dont les analyses chromatographiques et spectroscopiques ont révélé la diversité de taille (Mw ~ 1–4000 kDa), de charge (neutre ou anionique) et de fonction associée (lactate ou acétate). Les fractions d’EPS enrichies en polysaccharides inhibent la formation de biofilm par d’autres souches marines. Ces derniers sont également synthétisés par les bactéries en culture planctonique mais en proportions très différentes. / In marine environment, artificial surfaces are promptly colonized by biofilms, which are communities of bacteria surrounded by matrix of extracellular polymeric substances (EPS). Formation of biofilm is a critical step of biofouling development, which corresponds to the accumulation of micro and macro-organisms on immersed surfaces and which can have important negative ramifications in particular in the marine sector. In this study, bacteria isolated from the Mediterranean Sea have been identified and characterized using different phenotypical tools. Their capacity to form a biofilm in vitro has been studied in different conditions, with a particular focus on their ability to produce abundant carbohydrate-rich EPS, the overall objective of the study being the isolation of original antifouling-active exopolysaccharides. Thirteen strains have been phylogenetically and phenotypically characterized. Seven genera and twelve species were identified among which two isolates were affiliated to a new species, named Persicivirga mediterranea. This genus has never been described in the Mediterranean Sea. Extraction of EPS of each strain, grown in biofilm conditions, allowed the determination of their general composition in carbohydrates, proteins, nucleic acids and lipids. One strain, Pseudoalteromonas ulvae TC14, was able to produce large quantities of exopolysaccharides, comprising in majority polymers of glucose whose chromatographic and spectroscopic analyzes revealed a diversity in size (Mw ~ 1-4000 kDa), charge (neutral or anionic) and associated function (acetate or lactate). These polysaccharides inhibited biofilm formed by other marine strains isolated from the Mediterranean Sea. They can also be synthesized by planktonic TC14, but in very different proportions.

Orientadores: Anita Jocelyne Marsaioli, Valeria Maia de Oliveira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-08T05:19:46Z (GMT). No. of bitstreams: 1 Vasconcellos_SuzanPantarotode_D.pdf: 2236898 bytes, checksum: 015039f9f79ab3bebdcffa9003d44d0f (MD5) Previous issue date: 2006 / Resumo: Amostras de águas de formação de petróleo e óleos foram coletadas na Formação Pampo, situada na Bacia de Campos, através das quais, aplicando-se diferentes condições de cultivo, foram recuperadas 92 linhagens de bactérias aeróbias. Dentre os isolados, obtidos, 29 foram identificados por técnicas moleculares baseadas em DNAr 16S, observando-se a predominância de bactérias do gênero Bacillus.. De forma a constatar sobre a produção microbiana de metabólitos secundários com atividade inibitória ao desenvolvimento de outras espécies no reservatório de petróleo investigado, realizou-se análises de antibiogramas e Concentrações Inibitórias Mínimas (MIC), constatando Bacillus possuíam tal atividade. Sob condições anaeróbias foram obtidos consórcios microbianos, os quais foram caracterizados por técnicas independentes-de-cultivo, identificando-se bactérias do gênero Petrotoga como constituintes majoritários das comunidades presentes nos poços de petróleo amostrados. Duas espécies distintas de arquéias metanogênicas, Methanohalophilus sp. e Methanomethylovorans sp., também foram identificadas. Foram realizadas análises para a detecção da atividade biodegradadora da microbiota anaeróbia sobre biomarcadores do petróleo, onde foi observada forte tendência da comunidade microbiana investigada à biotransformação de compostos oxigenados (ácido octadecanóico; a e b-amirina). A biodegradação de biomarcadores do petróleo sob condições aeróbias revelou que a maioria das 29 bactérias avaliadas demonstrou tendência à biotransformação de ácidos carboxílicos de cadeia longa (ácido nonadecanóico). A linhagem de Micrococcus sp., entretanto, foi exceção a esta tendência, biotransformando preferencialmente hidrocarbonetos (fitano ediidrofenantreno), mesmo em presença de compostos oxigenados. A existência de interações simbióticas entre as comunidades microbianas aeróbias e anaeróbias nos processos de biodegradação de biomarcadores do petróleo, foi avaliada simulando atuação de ambas as microbiotas sobre uma mistura de hidrocarbonetos e compostos oxigenados. Os resultados obtidos sugeriram que a microbiota anaeróbia poderia sobreviver às custas dos metabólitos produzidos pela microbiota aeróbia durante a biodegradação do petróleo. A produção de exopolímeros microbianos com atividade bioemulsificante de hidrocarbonetos também foi determinada, verificando-se que microrganismos tidos com excelentes produtores de EPS não corresponderam às linhagens que apresentaram atividade biodegradadora de hidrocarbonetos do petróleo. Análises de triagens enzimáticas de alto desempenho (HTS) foram realizadas, e Dietzia sp. sobressaiu-se quanto às excelentes atividades de lipases, esterases e monooxigenases, enquanto Halomonas sp. apresentou o melhor desempenho quanto à atividade de epóxido hidrolase / Abstract: Samples of petroleum formation water and oils were collected from Pampo Formation (Campos Basin). Applying different conditions of microbial cultivation it was recuperated 92 bacterial strains from among 29 were identified by molecular techniques based in rDNA 16S. The predominance of Bacillus genus was observed in this recuperated bacterial community. The production of microbial secondary metabolites with antibiotic activity was investigated through analysis of antibiograms and Microbial Inibitory Concentrations (MIC) assays. Strains of Bacillus produced metabolites with microbial growth inhibition that can actuate in the petroleum reservoir in the selection of microbial species. Under anaerobic conditions microbial consortia were characterized by independent of cultivation techniques, identifying bacteria of Petrotoga genus as main constituent from the sampled reservoir. Two distinct species of methanogenic archaea, Methanohalophilus sp and Methanomethylovorans sp, were identified too. Analysis for the detection of the biodegrader activities of anaerobic microbiota under petroleum biomarkers were realized. Strong tendency of this microbial community to oxygenated compounds biotransformation was observed. The biodegradation of petroleum biomarkers under aerobic conditions was investigated as a form to determinate the microbial preferences for the petroleum compounds, in mixture or isolate, evaluating the formed products. The results presented the preference for the biotransformation of long chain carboxylic acids before the metabolism of hydrocarbons. Although, the strain of Micrococcus sp. preferred the biotransformation of hydrocarbons (phytane and dihydrophenanthrene), not biodegrading the fatty acid in strong levels. To investigate about the existence of symbiotic interactions between aerobic and anaerobic microbial communities in petroleum biodegradation processes, the actuation of the both microbiota under a mixture of hydrocarbons and oxygenated compounds was simulated. The obtained results suggest that the anaerobic microbiota can survive from the aerobic metabolites produced by the aerobic community. The production of microbial exopolymers with bioemulsifier activity of hydrocarbons was determined too. It was verified that strains presented as excellent producers of bioemulsifiers are not efficient petroleum biodegraders. Analysis of High Throughput Screening (HTS) were realized and Dietzia sp. presented strong activities of lipases, esterases and monooxygenases, while Halomonas sp. was the best result how the epoxide hydrolase activity / Doutorado / Quimica Organica / Doutor em Ciências

Après déshydration, les boues biologiques présentent en moyenne une siccité de 30%, ce qui pose d'importants problèmes environnementaux. Afin de comprendre les mécanismes physico-chimiques de rétention d'eau dans ces boues, nous les avons modélisées par une souche bactérienne pure présentant la particularité de produire ou non des surstructures, suivant sa température de culture. Les suspensions bactériennes ainsi obtenues ont été floculées par du polyéthylèneimine de différentes masses moléculaires. Des mesures de densité optique, mobilité électrophorétique, conductivité, pH, absorbance, granulométrie, ainsi que des observations en MET et AFM ont été effectuées. Cela nous a permis de caractériser la structure des agrégats ainsi obtenus et d'évaluer les influences relatives de la masse moléculaire du polymère ainsi que de sa concentration et de la présence/absence de surstructures sur l'agrégation de cellules bactériennes. Des mesures d'élasticité, de constante de raideur et de pression de turgescence ont été effectuées par spectroscopie de force, ce qui nous a permis de caractériser les propriétés nanomécaniques des agrégats. Dans un second temps, les suspensions floculées ont été déshydratées par centrifugation, procédé mis en oeuvre dans certaines station d'épuration. Des mesures d'élasticité et de viscosité (gradient de cisaillement infini) ont été effectuées par rhéologie. La corrélation de celles-ci avec des mesures de siccité nous a permis de conclure à un impact important des surstructures, de la masse moléculaire et de la concentration en polymère sur la déshydratation des boues biologiques / After dewatering, a biological sludge still contains an average water content of 70%, thus causing huge environmental problems. To achieve a better understanding of the mechanisms underlying physicochemical interactions inducing water retention in the sludge, the bacterial sludge was modeled by a pure strain producing or not surface appendages, depending on the growth temperature. These bacterial suspensions were flocculated by polyethyleneimine of various molecular weights. Measurements of optical density, electrophoretic mobility, conductivity, pH, absorbance, particle size, as well as AFM and TEM observations were performed. This allowed to characterize aggregates structures and to estimate the influence of molecular weight and concentration of polymer and presence/absence of surface appendages. Measurements of elasticity, spring constant and turgor pressure were carried out by force spectroscopy allowing to characterize nanomechanical properties of aggregates. In a second step, a dewatering of these flocculated suspensions was performed by centrifugation, process used in some wastewater treatment plants. Measurements of elasticity and viscosity were carried out by rheology. Correlations with dryness measurements allowed to conclude that the presence/absence of surface appendages, and also the molecular weight and concentration of the polymer have a significant impact on biological sludge dewatering

Le principal objectif de cette thèse était de mieux comprendre l’importance des Substances Polymériques Extracellulaires (SPE) dans la structuration et la formation des biofilms benthiques ; tout en s’inscrivant dans une étude plus globale des mécanismes écologiques impliqués dans le fonctionnement des vasières intertidales. La mise au point des dosages biochimiques a été effectuée sur le mucilage de l’algue Chaetomorpha aerea et a permis en parallèle de purifier un polysaccharide sulfaté riche en galactose, présentant une activité bactéricide sélective contre la souche Staphylococcus aureus (ATCC 25923). Les études biochimiques et écologiques menées sur les SPE extraits de la vasière charentaise ont ensuite permis de quantifier leur dynamique de production et leur composition, en fonction des conditions environnementales. La présence de désoxy-sucres et d’acides uroniques au sein des SPE capsulaires a laissé supposer que ces fractions jouaient un rôle important dans la formation et le devenir du biofilm microphytobenthique. La dernière partie des travaux a permis de caractériser les propriétés acide/base de Lewis et hydrophile/hydrophobe de la surface de la micro-algue Navicula jeffreyi, impliquée dans la formation de biofilms benthiques, par des méthodes classiques d’analyse. L’utilisation d’une nouvelle méthode, la Chromatographie Gazeuse Inverse (CGI), a permis d’obtenir des résultats intéressants et relativement similaires, confirmant le caractère prometteur de la CGI pour l’étude des propriétés de surface des micro-organismes. / The main goal of this thesis was to better understand the importance of Extracellular Polymeric Substances (EPS) in the structuring and formation of benthic biofilms; while considering a global conception of the ecological mechanisms involved in the functioning of intertidal mudflats. The development of the biochemical assays was done on the mucilage of the macroalgae Chaetomorpha aerea and allowed purifying a polysaccharide rich in galactose, showing a selective bactericidal activity against Staphylococcus aureus (ATCC 25923). Then, the biochemical and ecological studies concerning the EPS extracted from the local mudflat allowed studying their dynamic of production and composition in relation to environmental conditions. The presence of deoxy sugars and uronic acids in the bound EPS highlighted their important roles during the formation and the life of microphytobenthic biofilms. The last part of the work was used to characterize the acid/base of Lewis and hydrophilic/hydrophobic surface properties of the microalgae Navicula jeffreyi, involved in the formation of benthic biofilms, by using classical analysis methods. The use of a new method, named Inverse Gas Chromatography (IGC), allowed getting interesting and relatively similar results, confirming the potential of the method to study the surface properties of microorganisms.