A series of laboratory and field investigations were carried out to elucidate the importance of natural organic matter in aquatic systems, i.e., trace element scavenging (e.g., 234Th) by exopolymeric substances (EPS), formation of biofilms, as well as interactions with 129I. A method involving cross flow ultrafiltration, followed by a three-step cartridge soaking and stirred-cell diafiltration, was developed for isolating EPS from phytoplankton cultures, especially in seawater media. EPS isolated from a marine diatom, Amphora sp. was then subjected to semi-quantitative (e.g., carbohydrate, proteins) and quantitative analysis (e.g., neutral sugars, acidic sugars, sulfate). It appeared that Th (IV) binding by EPS was dominated by the acidic polysaccharides of fraction. For EPS of biofilms collected from polluted streams, hydrophobic proteins were the most abundant components in EPS, followed by more hydrophilic carbohydrates. However, chemical composition of carbohydrates or proteins, i.e., monosaccharides and amino acids, respectively, varied with environmental conditions and substrata applied, which suggests that the formation of biofilms on different substrates is regulated by specific properties of microorganisms, environmental conditions and nature of substratum. No correlation between relative hydrophobicity of substratum and development of biofilm was found in this study. A sensitive and rapid GC-MS method was developed to enable the determination of isotopic ratios (129I/127I) of speciated iodine in natural waters. At the F-area of the Savannah River Site (SRS), iodine species in the groundwater consisted of 48.8 percent iodide, 27.3 percent iodate and 23.9 percent organo-iodine. Each of these iodine species exhibited vastly different transport behavior in the column experiments using surface soil from the SRS. Results demonstrated that mobility of iodine species depended greatly on the iodine concentration, mostly due to the limited sorptive capacity for anions of the soil. EPS, especially enzymes (e.g., haloperoxidases) could facilitate the incorporation of iodide to natural organic carbon. At high input concentrations of iodate (78.7 μM), iodate was found to be completely reduced and subsequently followed the transport behavior of iodide. The marked reduction of iodate was probably associated with natural organic carbon and facilitated by bacteria, besides inorganic reductants (e.g., Fe2 ) in sediments and pore water.

Orientadores: Valéria Maia de Oliveira, Suzan Pantaroto de Vasconcellos / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T10:57:30Z (GMT). No. of bitstreams: 1 Dellagnezze_BrunaMartins_M.pdf: 2605800 bytes, checksum: 9f658e8eb7ad92524b8042afa7233e71 (MD5) Previous issue date: 2010 / Resumo: Bioemulsificantes ou biossurfactantes são compostos sintetizados por micro-organismos que aumentam a biodisponibilidade de hidrocarbonetos, promovendo a estabilização de substâncias com diferentes polaridades, como por exemplo, emulsões água-óleo existentes nos reservatórios de petróleo. As amostras utilizadas neste trabalho foram coletadas na Bacia Potiguar (RN), e empregadas como inóculo para a realização de enriquecimentos anaeróbios e aeróbios da microbiota do petróleo. A amostra de óleo selecionada (GMR 75) foi obtida a partir de um poço com nível de biodegradação moderado. Após o período de incubação dos enriquecimentos, foi realizada a extração de DNA dos mesmos e a construção das bibliotecas de fosmídios, utilizando-se kit de clonagem comercial. A avaliação da produção de emulsificantes pelos clones fosmidias provenientes das bibliotecas metagenômicas foi realizada através de dois tipos de ensaios de triagem, um teste baseado em atividade biológica e outro em similaridade de sequência. O teste funcional, denominado "colapso da gota", apresenta caráter qualitativo e foi utilizado para a triagem preliminar de 2.880 clones, gerando 2.104 resultados positivos. Para o teste baseado em seqüência, os clones que se mostraram positivos no teste funcional foram avaliados através de reações de PCR utilizando primers homólogos a regiões conservadas de genes para a síntese de biossurfactantes. Entretanto, nenhum sinal positivo de amplificação foi observado com os primers utilizados. Ensaios de tensiometria, utilizando meio mineral e hexadecano como única fonte de carbono e energia, foram realizados para a confirmação da produção dos emulsificantes pelos clones. Nove clones capazes de reduzir a tensão interfacial de 45 mN\m para 30 mN/m foram selecionados e submetidos aos ensaios de emulsificação utilizando petróleo, óleo diesel e hexadecano. Os resultados demonstraram a formação de emulsão, principalmente em óleo diesel, sugerindo poder emulsificante sobre este composto. As análises colorimétricas dos biossurfactantes produzidos pelos nove clones revelaram proporções similares de carboidratos, proteínas e ácidos urônicos, não permitindo resultados conclusivos sobre a composição destes compostos. / Abstract: Bioemulsifiers or biosurfactants are compounds produced by microorganisms which enhance the bioavailability of hydrophobic substances promoting the stabilization of substances with different polarities, like oil-in-water emulsion occurring in petroleum reservoirs. The samples used in this work were collected in the Potiguar Basin (RN, Brazil), and employed as inoculum for aerobic and anaerobic enrichments of the petroleum microbiota. The selected oil sample (GMR 75) was obtained from a reservoir presenting a moderate biodegradation level. After the incubation period of the microbial enrichments, total DNA was extracted and used for the assembly of the metagenomic libraries by using a comercial kit. The evaluation of the potential for emulsifier production by the fosmid clones derived from the metagenomic libraries was performed by means of two screening assays, one based on biological activity and another based on sequence similarity. The functional assay, named "drop collapse", has a qualitative character and was used for the preliminary screening of 2,880 clones, yielding 2,104 positive results. For the sequence-based assay, clones shown to be positive in the functional assay were evaluated through PCR reactions using primers homologous to conserved regions of genes for biosurfactant synthesis. However, no positive amplification signal was observed with the primers used. Tensitometric assays, using mineral medium and hexadecane as sole carbon and energy source, were performed to confirm the production of the biosurfactants by the clones. Nine clones able to reduce the interfacial tension from 45 mN\m to 30 mN/m were selected and subsequently submitted to emulsification assays using petroleum, diesel oil and hexadecane. Results demonstrated the formation of an emulsion, mainly in diesel oil, suggesting the emulsifier power of the biosurfactants over this substance. Colorimetric analyses of the biosurfactants produced by the clones revealed similar proportion of carbohydrates, proteins and uronic acids, not allowing conclusive characterization of their composition. / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

Les estuaires, de par leur position stratégique à l'interface entre les eaux continentales et les eaux marines jouent un rôle écologique de première importance et sont le siège de nombreuses activités humaines. L’estuaire de Seine est caractéristique des grands estuaires anthropisés. Le management à long terme de ces écosystèmes soumis à des pressions croissantes réside notamment en une meilleure connaissance de la dynamique spatiale et temporelle des réseaux trophiques estuariens. Le phytoplancton et le microphytobenthos sont les principaux contributeurs de la production primaire (PP) dans ces écosystèmes et sont à la base des réseaux trophiques. Ces compartiments sont souvent réduits à la teneur en chlorophylle du milieu et leur productivité n’a jamais été mesurée à l’échelle de l’estuaire de Seine. L’objectif de ces travaux a été d’estimer la PP de ces compartiments le long du gradient halin. Afin d’accéder à des mesures à une haute résolution spatiale, des mesures de fluorescence modulée (PAM) à haute fréquence ont été couplées à des mesures d’incorporation de carbone (13C) à basse fréquence. Les mesures de production primaire ont été mises en perspective avec la dynamique des paramètres physico-chimiques du milieu et la structure communautés phytoplanctoniques déterminés par différentes techniques (microscopie, cytométrie, biologie moléculaire). La dynamique du carbone excrété sous forme de TEP (Transparent Exopolymeric Particules) et EPS (Exopolymeric substances) a également été étudiée pour chacun des compartiments. Au-delà des méthodologies innovantes mises en place qui montre l’intérêt des mesures à haute fréquence dans ces écosystèmes très dynamiques, ce travail apporte une nouvelle vision de la dynamique du phytoplancton et de la richesse spécifique du microbiome estuarien et apporte une estimation fiable de la production primaire. / The estuaries play an important ecological role and are the site of many human activities because of their strategic position at the interface between continental and marine waters. Seine estuary is characteristic of large anthropized estuaries. Long-term management requires better knowledge of the spatial and temporal dynamics of the estuarine food webs. Phytoplankton and microphytobenthos are the main contributor of primary production (PP) in these ecosystems and are at the basis of trophic food webs. These compartments are often reduced to the chlorophyll concentration and their productivity has never been measured along the Seine estuary. The objective of this study was to estimate the PP of these compartments along the salinity gradient. In order to access to measurements at high spatial resolution, high-frequency measurements of modulated fluorescence (PAM) were coupled to low-frequency carbon (13C) incorporation measurements. The primary production measurements have been put into perspective with the dynamics of the physical and chemical parameters and the structure of phytoplankton communities determined by different techniques (microscopy, cytometry, molecular biology). The dynamics of carbon excreted as TEP (Transparent Exopolymeric Particles) and EPS (Exopolymeric substances) were also studied for each compartment. Beyond the innovative methodologies which demonstrate the interest of high-frequency measurements in these highly dynamic ecosystems, this work provides a new insight into the phytoplankton dynamics and the specific richness of the estuarine microbiome and provides a reliable estimate of primary production.

Although wastewater treatment plants are a line of defense in minimizing indiscriminate output of microconstituents to natural waters, we do not possess a fundamental understanding of the mechanisms involved in microconstituent removal during wastewater treatment. With this in mind, experiments were designed to investigate the factors that can influence the fate of four microconstituents, carbamazepine (CBZ), 17alpha-ethinylestradiol (EE2), iopromide (IOP), and trimethoprim (TMP), during biological suspended culture treatment. Specifically, the role that various ecological members of biological treatment systems play in biotransforming these compounds was evaluated. Sorption assays were performed with inactivated biomass samples (ammonia oxidizing bacteria (AOB), laboratory enriched heterotrophic cultures free of active nitrifiers with low (Ox⁻) or high (Ox⁺) oxygenase activity, and a nitrifying activated sludge (NAS) from a full-scale wastewater treatment plant) to determine whether partitioning dictates removal of individual microconstituents. No microconstituents sorbed to the AOB culture. Neither CBZ nor IOP sorbed to Ox⁻, Ox⁺ and NAS cultures; however, EE2 and TMP sorbed to the Ox⁻, Ox⁺ and NAS biomass. Sorption was positively influenced by the presence of exopolymeric substances (EPS) associated with the cultures. The protein content of EPS affected EE2 and TMP sorption more appreciably than the polysaccharide content of EPS. Further experiments were performed to investigate microconstituent biodegradation by AOBs, Ox⁻ and Ox⁺ cultures. The influence of growth state and oxygenase activity on biotransformation by each culture was also evaluated. Results indicate that EE2 was the only microconstituent that was amenable to biotransformation by batch cultured AOB and heterotrophic cultures. EE2 was biotransformed but not mineralized by AOB chemostat and batch cultures. TMP was not transformed by AOB batch or chemostat cultures; however both EE2 and TMP were transformed by Ox⁻ and Ox⁺ chemostat cultures. Radiolabeled studies showed that EE2 was mineralized by this culture. Kinetically, AOBs dominated EE2 transformation to monohydroxylated metabolites; however, both Ox⁻ and Ox⁺ cultures further degraded and mineralized EE2 and metabolites generated by AOBs. These results indicate that biotransformation of EE2 by NAS may be limited by heterotrophic activity whereas TMP fate may be a function of heterotrophic activity only. Oxygenase activity did not limit EE2 or TMP biotransformation in chemostat cultures. Subsequent experiments that were performed to identify the factors that influence heterotrophic degradation of EE2 and TMP indicated that the presence of readily biodegradable substrates slows EE2 and TMP biotransformation. The impact of slowly biodegradable substrates like EPS on EE2 and TMP degradation was unclear. These results suggest that EE2 and TMP are most amenable to biodegradation in bioreactors where endogenous conditions dominate. / Ph. D.

Les biofilms périphytiques, ou périphyton, sont des assemblages de microorganismes colonisant les surfaces d’une variété de substrats de la zone photique des habitats aquatiques. Retrouvés à la base des réseaux trophiques, ils présentent une grande importance écologique. Les microorganismes du périphyton sont maintenus les uns aux autres par une matrice extracellulaire composée de substances exopolymériques endogènes. Cette matrice permet la formation de microenvironnements aux propriétés physicochimiques distinctes du milieu extrapériphytique. En raison de la diversité microbienne qui les compose, de leur capacité à moduler l’environnement immédiat et leur propension à séquestrer puis à transformer les métaux, les biofilms périphytiques représentent des modèles de recherche des plus pertinents d’un point de vue biogéochimique. L’objectif général de la présente thèse est d’étudier le rôle des biofilms périphytiques, de leur matrice extracellulaire et de la matière organique qu’elle contient sur le cycle des métaux, plus particulièrement le mercure. Dans un premier temps, nous avons mené une étude sur la composition organique de la matrice extracellulaire du périphyton de lacs d’environnements non contaminés et sur la mobilité des métaux dans les différentes fractions de cette matrice. Nos résultats ont démontré que la matrice extracellulaire du périphyton était riche en matière organique fluorescente et nous y avons identifié deux composantes majeures d’origine périphytique. Nous avons observé une cooccurrence de certains métaux avec ces composantes dans les fractions mobiles et attachées de la matrice extracellulaire du périphyton. Nous avons constaté que l’une des composantes périphytiques entraînait une diminution des concentrations de métaux dans les cellules du périphyton, alors que l’autre était associée à des concentrations plus élevées de métaux essentiels. Notre étude a montré que la matière organique de la matrice extracellulaire joue un double rôle sur la mobilité des métaux et apporte un regard nouveau sur les mécanismes naturels de gestion des métaux du périphyton. Dans un deuxième temps, nous avons réalisé une étude de terrain dans une rivière altérée par des centrales hydroélectriques au fil de l’eau et par la création de milieux humides artificiels. Cette étude avait pour objectif de déterminer si le périphyton de ces habitats modifiés pouvait produire du méthylmercure. En combinant des mesures des taux de transformation in situ à des approches génomiques ciblant les gènes hgcAB, responsables de la méthylation du mercure, nous avons démontré que le périphyton des milieux artificiels avait la capacité de produire du méthylmercure. Nous avons mesuré des taux de méthylation positifs et nous avons détecté la présence du gène hgcA dans les communautés périphytiques des milieux humides. Nous avons majoritairement associé le gène hgcA aux bactéries réductrices de fer de la famille des Geobacteraceae. Notre travail a apporté de nouvelles informations sur la méthylation du mercure au sein du périphyton colonisant des rivières ayant subi des transformations d’habitats. Nos résultats ont également permis d'associer cette méthylation aux bactéries réductrices de fer, jusqu’alors rarement considérées comme potentielles méthylatrices dans le périphyton. Finalement, nous avons étudié les impacts de la mise en eau d’une rivière par des centrales hydroélectriques au fil de l’eau sur les dynamiques du mercure en nous attardant au périphyton et aux premiers niveaux des réseaux trophiques. La création de nouveaux habitats aquatiques favorise l’accumulation et la transformation du mercure en plus de fournir des conditions propices à l’établissement et à la croissance du périphyton. Nos résultats ont démontré que le périphyton de ces habitats pouvait accumuler d’importantes concentrations de méthylmercure et qu’il était une voie d’entrée efficace de ce contaminant pour les macroinvertébrés benthiques. Cette étude souligne les rôles clés que joue le périphyton dans le cycle du mercure des écosystèmes aquatiques. Les résultats de la thèse appuient la pertinence de considérer le périphyton comme un compartiment biologique de haute incidence sur les cycles biogéochimiques des métaux lors de l’étude ou de la gestion des écosystèmes aquatiques. / Periphytic biofilms, or periphyton, are collections of microorganisms that colonize the surfaces of a variety of substrates in the photic zone of aquatic habitats. Found at the base of trophic webs, they are of major ecological importance. Periphytic microorganisms are linked to each other by an extracellular matrix composed of endogenous exopolymeric substances. This matrix allows the formation of microenvironments with physicochemical properties distinct from the extraperiphytic environment. Because of their microbial diversity, their ability to modulate the immediate environment and their propensity to sequester and then transform metals, periphytic biofilms represent relevant research models from a biogeochemical perspective. The general objective of this thesis is to study the role of periphytic biofilms, their extracellular matrix and the organic matter they contain on the cycling of metals, more specifically mercury. First, we conducted a study on the organic composition of the periphyton extracellular matrix of lakes from uncontaminated environments and on the mobility of metals in the different fractions of this matrix. Our results showed that extracellular matrix of periphyton was rich in fluorescent dissolved organic matter and we identified two major organic components of periphyton origin. We observed co-occurrence of some metals with these components in the loosely- and tightly-bound fractions of the extracellular matrix. We found that one of the periphytic components resulted in decreased metal concentrations in periphyton cells, whereas the other was associated with higher concentrations of essential metals. Our study showed that the dissolved organic matter of the extracellular matrix plays a dual role on metal mobility and provides new insight into the natural mechanisms of metal management of periphyton in its immediate environment. Second, we conducted a field study in a river impacted by run-of-river hydroelectric power plants and the creation of artificial wetlands. The objective of this study was to determine if the periphyton of these modified habitats could produce methylmercury. By combining measurements of in situ transformation rates with genomic approaches targeting the hgcAB genes, associated with microbial mercury methylation, we demonstrated that the periphyton of the artificial wetlands had the capacity to produce methylmercury. We measured positive vi methylation rates and detected the presence of the hgcA gene in the periphytic communities of the wetland site. We mostly associated the hgcA gene with iron-reducing bacteria of the Geobacteraceae family. Our work provided new information on mercury methylation within periphyton colonizing rivers with altered habitats. Our results also allowed us to associate the methylation of mercury with iron-reducing bacteria, which are rarely considered as potential methylators in periphyton. Finally, we studied the impacts of river impoundment by run-of-river hydroelectric power plants on mercury dynamics by focusing on periphyton and lowertrophic web layers. The creation of new aquatic habitats promotes the accumulation and transformation of mercury and provides conditions for periphyton growth. Our results showed that periphyton in these habitats can accumulate important concentrations of methylmercury and is an efficient gateway to this contaminant for benthic macroinvertebrates. This study highlights the key roles that periphyton plays in the cycling of mercury in aquatic ecosystems. The results of this thesis support the relevance of considering periphyton as a biological compartment of high impact on biogeochemical metal cycles when studying or managing aquatic ecosystems.