Mikrobielle Prozesse spielen eine wichtige Rolle im Sediment von Talsperren und Seen. Demgegenüber stehen nur unzureichende Erkenntnisse über die Zusammensetzung mikrobieller Biozönosen in Sedimenten sowie deren Aktivität zur Verfügung. Das Ziel dieser Studie war die Untersuchung und der Vergleich der Zusammensetzung und der Struktur mikrobieller Gemeinschaften in Sedimenten um eine Abschätzung der mikrobiellen Diversität in Talsperrensedimenten unterschiedlicher Trophie zu erreichen. Durch die Kombination der in dieser Arbeit verwendeten Methoden (Vergleichende 16S rDNA Analyse, Fingerprinttechniken, klassische Methoden) konnte eine Charakterisierung der mikrobiellen Zusammensetzung der obersten 5 cm von den Talsperrensedimenten Neunzehnhain, Muldenberg, Quitzdorf und Saidenbach erzielt werden. Die vergleichende 16S rDNA Analyse offenbarte in 2541 analysierten rekombinanten Klonen 528 verschiedene Sequenztypen, welche zu 293 OTUs zusammengefaßt werden konnten. Obwohl die Gemeinschaften der verschiedenen Talsperren nur schwach auf der Ebene der phylogenetischen Gruppen differierten, konnte durch die Verwendung von Ähnlichkeitsindices gezeigt werden, dass jede Talsperre eine spezifische mikrobielle Sedimentgemeinschaft aufweist. Über 60% aller Klone zeigten Ähnlichkeiten von mehr als 97% zu 16S rDNA-Sequenzen kultivierter Organismen oder phylogenetisch eingeordneten Sequenzen (14 bekannte phylogenetische Gruppen). Alle anderen Klone zeigten hohe Sequenzhomologien zu unidentifizierten, phylogenetisch bisher nicht eingeordneten Bakterien. Diese Bakterien waren mit Anteilen zwischen 19,8% (Muldenberg) und 54,6% (Saidenbach) in den 16S rDNA Bibliotheken repräsentiert. Mittels Fingerprinttechniken (DGGE, T-RFLP, ARISA) konnten komplexe Muster der mikrobiellen Diversität erzeugt werden. Dabei konnten die Ergebnisse der 16S rDNA Analyse bestätigt werden. Durch die verwendeten Methoden konnte eine komplexe mikrobielle Diversität in den Sedimenten aufgedeckt werden und die Ergebnisse weisen darauf hin, dass die mikrobielle Diversität in Sedimenten wesentlich höher ist als bisher angenommen.

Tidal freshwater wetlands (TFW) are at significant risk of loss or alteration due to global climate change, and saltwater intrusion from sea level rise is of particular concern for these habitats due to their proximity to coastal areas. A space-for-time model was used to investigate the effects of saltwater intrusion on soil methanogen communities along naturally occurring salinity gradients on the Waccamaw, James, and Hudson Rivers. Amplification of the methyl coenzyme-M reductase (mcrA) functional gene was used in qPCR, reverse transcription qPCR, and T-RFLP to measure the abundance, activity, and community composition of soil methanogens. Both the abundance and activity of methanogens decreased with increasing salinity, and the both total and active methanogen community composition shifted in response to changes in salinity. This research demonstrates that saltwater intrusion will alter carbon cycling in TFWs, potentially altering their ability to sequester carbon and keep pace with rising sea level.

O uso de culturas GM (geneticamente modificadas) tem sido questionado quanto ao destino dos produtos derivados da transgenia no ambiente. Com a liberação de exsudatos das raízes das plantas e a decomposição dos resíduos culturais, aumenta-se a quantidade de DNA transgênico no ambiente, que pode ser adsorvido à superfície ativa das partículas do solo e/ou degradado pela ação de enzimas microbianas. A comunidade microbiana do solo pode entrar em contato direto com estes produtos, aumentando a probabilidade de transferência horizontal de fragmentos de DNA transgênico para os microrganismos. Também, alterações na composição dos exsudatos das plantas GM e mudanças em função das práticas de manejo, podem resultar em alterações na composição funcional e estrutural da comunidade microbiana. Assim, faz-se necessário avaliar a persistência dos produtos derivados da transgenia no solo e seus possíveis efeitos sobre a comunidade microbiana. Os objetivos deste estudo foram: avaliar a persistência dos fragmentos 35S-hsp70, hsp70-cry1Ab e cry1Ab-planta da construção gênica do milho Bt (evento MON810) em diferentes tipos de solo e temperaturas, em condições de microcosmo e de campo; e determinar a abundância do número de cópias dos gene 16S rRNA de Bacteria, Firmicutes, Verrucomicrobria e Archaea, e 18S rRNA de Fungo nas mesmas condições, e avaliar a estrutura da comunidade bacteriana em áreas agrícolas de cultivo de milho Bt. No primeiro estudo, o DNA do milho Bt MON810 foi adicionado em solos arenoso e argiloso. Como controle negativo, apenas água estéril foi misturada ao solo. Amostras de solo foram incubadas a 15 e 25ºC. Em campo, os solos foram amostrados em três áreas agrícolas em Fátima do Sul, MS, em dois anos consecutivos. Após extração de DNA, os fragmentos foram quantificados por qPCR. No segundo estudo, foram determinadas a abundância dos genes 16S rRNA de Bacteria, Firmicutes, Verrucomicrobria e Archaea e 18S rRNA de Fungo e avaliada a estrutura da comunidade bacteriana por T-RLFP. Os resultados mostraram que em condições de microcosmo, os fragmentos hsp70-cry1Ab e cry1Ab-planta persistiram até 291 dias, e o fragmento 35S-hsp70 até 180 dias. A temperatura e o tipo de solo não afetaram a persistência dos fragmentos. Em campo, o número de cópias desses fragmentos foi maior na segunda coleta. No segundo estudo, o número de cópias do gene 16S rRNA de Bacteria aumentou com adição de DNA do milho Bt nos microcosmos, e uma redução do número de cópias foi verificada para Archaea, Verrucomicrobia e Fungo. Para Firmicutes, os resultados não foram consistentes. As temperaturas não resultaram em efeito na abundância dos genes, enquanto o tipo de solo teve efeito apenas para Archaea e Verrucomicrobia. Áreas agrícolas com cinco anos de cultivo de milho Bt apresentaram variações na estrutura da comunidade bacteriana em nível de filo, e maior abundância de Fungos no segundo ano de amostragem, enquanto em área com um ano de cultivo, observouse uma redução da população de Firmicutes e Verrucomicrobia. Os maiores efeitos na comunidade microbiana foram verificados entre os anos de amostragem / The use of GM (genetically modified) crops has been questioned about the fate of transgenes is derived products on the environment. With the release of exudates from roots of GM plants and the decomposition of its residues, the amount of transgenic DNA in the environment increases, which can be adsorbed to the active surface of soil particles and/or be degraded by the action of microbial enzymes. Soil microbial communities can come into direct contact with these products, raising the probability of horizontal transfer of transgenic DNA fragments to soil microorganisms. Moreover, changes in exudates composition of GM plants and changes depending on the management practices may result in structural and functional alterations in the microbial community. Thus, it is necessary to evaluate the persistence of transgenes is derivatives in the soil and effects on microbial community. The objectives of this study were to assess the persistence of fragments 35S-hsp70, hsp70-cry1Ab and cry1Abplant from the genetic construct of Bt corn (event MON810) in different soil types and temperatures, in microcosm and field conditions; and to determine the abundance of 16S rRNA copy number of Bacteria, Firmicutes, Verrucomicrobria and Archaea and 18S rRNA of Fungi under the same conditions, and to evaluate the structure of bacterial communities in agricultural areas of Bt corn cultivation. In the first study, DNA from Bt corn MON810 was added to sandy and clay soils. As negative control, only sterile water was mixed with soil. Soil samples were incubated at 15 and 25°C. At the field, soils were sampled in three agricultural areas in Fátima do Sul, MS, in two consecutive years. After DNA extraction, fragments were quantified by qPCR. In the second study, the abundance of 16S rRNA of Bacteria, Firmicutes, Verrucomicrobria and Archaea and 18S rRNA of Fungi were determined and the structure of bacterial communities was evaluated by T-RFLP. The results showed that in microcosm conditions, hsp70-cry1Ab and cry1Ab-plants fragments persisted until 291 days and the 35S-hsp70 up to 180 days. The temperature and the type of soil did not affect the persistence of fragments. In field, the copy number of these fragments was greater in the second sampling. In the second study, the copy number of 16S rRNA of Bacteria increased with the addition of DNA from Bt corn in microcosm, and a reduction in copy number was observed for Archaea, Verrucomicrobia and Fungi. The results were not consistent for Firmicutes. Temperatures resulted in no effect in gene abundance, while the soil was effective only for Archaea and Verrucomicrobia. Agricultural areas with five years of Bt corn cultivation showed variations in bacterial community structure at the phylum level, and greater abundance of fungi in the second year of sampling, while in the area with a year of cultivation, a reduction in population of Firmicutes and Verrucomicrobia was observed. The largest effects on the microbial community were observed between the sampled years

As technology and engineering allow mankind to survey nature at finer scales, the importance of bacteria has been elucidated in their metabolic diversity, ability to transfer genetic information, involvement in biogeochemical cycling, and sheer abundance. With an individual domain of life unto themselves, this diverse group of microorganisms plays an integral role in facilitating life on land and in the oceans, and is second only to viruses in abundance on Earth. They carve niches in a wide range of environments, including those inhospitable to other life forms, and reside in concert or to the detriment of other microbes and/or hosts they inhabit. Solely culturing microorganisms has proven to severely underestimate microbial numbers, capturing less than 1% of marine microbes. However, the advent of molecular methods has revealed the ubiquity, abundance, and diversity of bacteria. Higher organisms have evolved varying degrees of ecological relationships with bacteria, ranging from mutualism to parasitism. As the microbial players are elucidated, determining the specificity and functional roles of these bacteria is a critical and exciting scientific question. The microbiome of corals is an interesting model of complexity, with the animal host striving to maintain a delicate symbiosis, and using its microbiota to assist in nutrient cycling and protection. A contrasting example to the well-studied cnidarians are ctenophores, gelatinous organisms that are globally distributed in the world's oceans, yet the literature contains few studies on microbiota associated with this unique group of animals. Since ctenophores are one of the earliest diverging, extant multicellular animals, these unique organisms could prove to be a better model system than cnidarians. The first project in this dissertation examined spatial structure of bacteria across wild, healthy colonies of the coral Montastraea annularis. Microscale heterogeneity of the bacterial community was observed in coral mucus samples collected tens of centimeters apart on the same coral colony, which has implications for sampling strategies in microbiological studies, and impacts the application of the bacterial community as a proxy for determining coral condition in coral restoration projects. The second project looked at the linkages between coral bacterial community composition and zooxanthellae clade in Pocillopora damicornis, and results suggested that clade is not a major factor in influencing coral bacterial community composition. Sample location was also considered in the P. damicornis bacterial surveys and determined to be driving community structure. The third project is the first study to describe bacteria associated with ctenophores (Mnemiopsis leidyi and Beroe ovata). The ctenophores contained bacterial communities that were distinct from the surrounding water column, and temporal variability was exhibited by bacteria associated with the ctenophores. Exploring microbial landscapes in cnidarians and ctenophores to understand microbial roles in health and disease is the uniting theme of the three separate projects that will be discussed in this dissertation.

Terminal restriction fragment length polymorphism (T-RFLP) analysis and pyrosequencing were used to characterize the microbiota of the tonsil of the soft palate of 126 unfit and 18 healthy pigs. The T-RFLP analysis method was first optimized for the study of the pig tonsil microbiota and the data compared with culture-based identification of common pig pathogens. Putative identifications of the members of the microbiota revealed that the phyla Firmicutes, Proteobacteria and Bacteroidetes were the most prevalent. A comparison of the T-RFLP analysis results grouped into clusters to clinical conditions revealed paleness, abscess, PRRS virus, and Mycoplasma hyopneumoniae to be significantly associated with cluster membership. T-RFLP analysis was also used to select representative tonsil samples for pyrosequencing. These studies confirmed Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Proteobacteria to be the core phyla of the microbiota of the tonsil of the soft palate of pigs. / OMAFRA Animal Health Strategic Investment

O uso de culturas GM (geneticamente modificadas) tem sido questionado quanto ao destino dos produtos derivados da transgenia no ambiente. Com a liberação de exsudatos das raízes das plantas e a decomposição dos resíduos culturais, aumenta-se a quantidade de DNA transgênico no ambiente, que pode ser adsorvido à superfície ativa das partículas do solo e/ou degradado pela ação de enzimas microbianas. A comunidade microbiana do solo pode entrar em contato direto com estes produtos, aumentando a probabilidade de transferência horizontal de fragmentos de DNA transgênico para os microrganismos. Também, alterações na composição dos exsudatos das plantas GM e mudanças em função das práticas de manejo, podem resultar em alterações na composição funcional e estrutural da comunidade microbiana. Assim, faz-se necessário avaliar a persistência dos produtos derivados da transgenia no solo e seus possíveis efeitos sobre a comunidade microbiana. Os objetivos deste estudo foram: avaliar a persistência dos fragmentos 35S-hsp70, hsp70-cry1Ab e cry1Ab-planta da construção gênica do milho Bt (evento MON810) em diferentes tipos de solo e temperaturas, em condições de microcosmo e de campo; e determinar a abundância do número de cópias dos gene 16S rRNA de Bacteria, Firmicutes, Verrucomicrobria e Archaea, e 18S rRNA de Fungo nas mesmas condições, e avaliar a estrutura da comunidade bacteriana em áreas agrícolas de cultivo de milho Bt. No primeiro estudo, o DNA do milho Bt MON810 foi adicionado em solos arenoso e argiloso. Como controle negativo, apenas água estéril foi misturada ao solo. Amostras de solo foram incubadas a 15 e 25ºC. Em campo, os solos foram amostrados em três áreas agrícolas em Fátima do Sul, MS, em dois anos consecutivos. Após extração de DNA, os fragmentos foram quantificados por qPCR. No segundo estudo, foram determinadas a abundância dos genes 16S rRNA de Bacteria, Firmicutes, Verrucomicrobria e Archaea e 18S rRNA de Fungo e avaliada a estrutura da comunidade bacteriana por T-RLFP. Os resultados mostraram que em condições de microcosmo, os fragmentos hsp70-cry1Ab e cry1Ab-planta persistiram até 291 dias, e o fragmento 35S-hsp70 até 180 dias. A temperatura e o tipo de solo não afetaram a persistência dos fragmentos. Em campo, o número de cópias desses fragmentos foi maior na segunda coleta. No segundo estudo, o número de cópias do gene 16S rRNA de Bacteria aumentou com adição de DNA do milho Bt nos microcosmos, e uma redução do número de cópias foi verificada para Archaea, Verrucomicrobia e Fungo. Para Firmicutes, os resultados não foram consistentes. As temperaturas não resultaram em efeito na abundância dos genes, enquanto o tipo de solo teve efeito apenas para Archaea e Verrucomicrobia. Áreas agrícolas com cinco anos de cultivo de milho Bt apresentaram variações na estrutura da comunidade bacteriana em nível de filo, e maior abundância de Fungos no segundo ano de amostragem, enquanto em área com um ano de cultivo, observouse uma redução da população de Firmicutes e Verrucomicrobia. Os maiores efeitos na comunidade microbiana foram verificados entre os anos de amostragem / The use of GM (genetically modified) crops has been questioned about the fate of transgenes is derived products on the environment. With the release of exudates from roots of GM plants and the decomposition of its residues, the amount of transgenic DNA in the environment increases, which can be adsorbed to the active surface of soil particles and/or be degraded by the action of microbial enzymes. Soil microbial communities can come into direct contact with these products, raising the probability of horizontal transfer of transgenic DNA fragments to soil microorganisms. Moreover, changes in exudates composition of GM plants and changes depending on the management practices may result in structural and functional alterations in the microbial community. Thus, it is necessary to evaluate the persistence of transgenes is derivatives in the soil and effects on microbial community. The objectives of this study were to assess the persistence of fragments 35S-hsp70, hsp70-cry1Ab and cry1Abplant from the genetic construct of Bt corn (event MON810) in different soil types and temperatures, in microcosm and field conditions; and to determine the abundance of 16S rRNA copy number of Bacteria, Firmicutes, Verrucomicrobria and Archaea and 18S rRNA of Fungi under the same conditions, and to evaluate the structure of bacterial communities in agricultural areas of Bt corn cultivation. In the first study, DNA from Bt corn MON810 was added to sandy and clay soils. As negative control, only sterile water was mixed with soil. Soil samples were incubated at 15 and 25°C. At the field, soils were sampled in three agricultural areas in Fátima do Sul, MS, in two consecutive years. After DNA extraction, fragments were quantified by qPCR. In the second study, the abundance of 16S rRNA of Bacteria, Firmicutes, Verrucomicrobria and Archaea and 18S rRNA of Fungi were determined and the structure of bacterial communities was evaluated by T-RFLP. The results showed that in microcosm conditions, hsp70-cry1Ab and cry1Ab-plants fragments persisted until 291 days and the 35S-hsp70 up to 180 days. The temperature and the type of soil did not affect the persistence of fragments. In field, the copy number of these fragments was greater in the second sampling. In the second study, the copy number of 16S rRNA of Bacteria increased with the addition of DNA from Bt corn in microcosm, and a reduction in copy number was observed for Archaea, Verrucomicrobia and Fungi. The results were not consistent for Firmicutes. Temperatures resulted in no effect in gene abundance, while the soil was effective only for Archaea and Verrucomicrobia. Agricultural areas with five years of Bt corn cultivation showed variations in bacterial community structure at the phylum level, and greater abundance of fungi in the second year of sampling, while in the area with a year of cultivation, a reduction in population of Firmicutes and Verrucomicrobia was observed. The largest effects on the microbial community were observed between the sampled years

A cana-de-açúcar é atualmente a cultura de maior importância agrícola do Estado de São Paulo, a partir da qual são gerados açúcar e etanol, além de vários outros subprodutos. No entanto, com a expansão das fronteiras agrícolas e alterações nas práticas de manejo, ocorre atualmente um momento de adequação de tal cultivo, que visa uma maior produtividade e sustentabilidade de produção. Para isto, dentre outros fatores, o papel da comunidade microbiana presente nos solos pode ter fundamental importância, auxiliando no melhor desenvolvimento da planta. No entanto, pouco se sabe sobre a comunidade microbiana existente nos solos cultivados com cana-de-açúcar. Dessa forma, este trabalho teve como objetivo avaliar a diversidade e a abundância de fungos em solos de cultivo de cana-deaçúcar no estado de São Paulo, em áreas sob diferentes atributos químicos, físicos e de manejo. Objetivou-se também, verificar a ocorrência de padrões biogeográficos na estruturação de tais comunidades. Para isso, foi realizada a análise da estrutura das comunidades fúngicas por polimorfismo de comprimento de fragmentos de restrição terminal (T-RFLP), juntamente com a quantificação destas comunidades por meio da PCR em tempo real (qPCR) em 476 amostras de solo, obtidas de 11 áreas de cultivo (usinas). Dentro deste conjunto de dados, temos que os atributos químicos, físicos e manejo explicam maiores valores de variância dentro de cada área amostra, mas pouco explicam da variância geral dos dados, sugerindo a ocorrência de padrões biogeográficos das comunidades de fungos neste ambiente. Tal ocorrência foi confirmada pela significância estatística da correlação entre distância e dissimilaridade das comunidades de fungos, dando suporte a geração dos primeiros mapas biogeográficos de fungos em tais solos. Adicionalmente, a abundância de fungos mostrou-se relacionada com a produtividade da cultura, indicando este ser um dos fatores que modulam a produtividade de cana-de-açúcar nas áreas avaliadas. / The sugarcane is nowadays, the most important crop in the State of São Paulo, serving as the raw material for the production of sugar and ethanol, besides many others by-products. Considering the expansion of agricultural barriers, and shifts in fields management, such cultivation is under a re-arrangement process, aiming to a higher productivity and sustainability. In order to achieve that, among other factors, the role of microbial communities present in soils can be essential to support plant development. However, a few is known about the microbial community under sugarcane crop production soils. Hence, this work intended to evaluate the fungi diversity and abundance in soils cultivated with sugarcane in the State of São Paulo, exploring areas under distinct chemical and physical attributes and also distinct management practices. It was also aimed to determine the occurrence of biogeographically patterns in the structure of such communities. Indeed, it was made the analysis of the fungal community structure by terminal restriction fragment length polymorphism (T-RFLP), together with the quantification of these communities by real time PCR (qPCR) in 476 soils samples, collected in 11 areas cultivated with sugarcane (mills). Within this dataset, it was found that chemical, physical and management attributes explain higher values of variance within each sampled area, but explain little about the total variance of data, suggesting the occurrence of biogeographically patterns in fungal communities in this environment. It was confirmed by the statistical significance of the correlation between distance and dissimilarity of fungal communities, supporting the generation of very first biogeographically maps in such soils. Additionally, the abundance of fungi revealed to be related with sugarcane productivity, indicating this issue as one of the factors modulating the sugarcane productivity in the evaluated areas.

A cana-de-açúcar é atualmente a cultura de maior importância agrícola do Estado de São Paulo, a partir da qual são gerados açúcar e etanol, além de vários outros subprodutos. No entanto, com a expansão das fronteiras agrícolas e alterações nas práticas de manejo, ocorre atualmente um momento de adequação de tal cultivo, que visa uma maior produtividade e sustentabilidade de produção. Para isto, dentre outros fatores, o papel da comunidade microbiana presente nos solos pode ter fundamental importância, auxiliando no melhor desenvolvimento da planta. No entanto, pouco se sabe sobre a comunidade microbiana existente nos solos cultivados com cana-de-açúcar. Dessa forma, este trabalho teve como objetivo avaliar a diversidade e a abundância de fungos em solos de cultivo de cana-deaçúcar no estado de São Paulo, em áreas sob diferentes atributos químicos, físicos e de manejo. Objetivou-se também, verificar a ocorrência de padrões biogeográficos na estruturação de tais comunidades. Para isso, foi realizada a análise da estrutura das comunidades fúngicas por polimorfismo de comprimento de fragmentos de restrição terminal (T-RFLP), juntamente com a quantificação destas comunidades por meio da PCR em tempo real (qPCR) em 476 amostras de solo, obtidas de 11 áreas de cultivo (usinas). Dentro deste conjunto de dados, temos que os atributos químicos, físicos e manejo explicam maiores valores de variância dentro de cada área amostra, mas pouco explicam da variância geral dos dados, sugerindo a ocorrência de padrões biogeográficos das comunidades de fungos neste ambiente. Tal ocorrência foi confirmada pela significância estatística da correlação entre distância e dissimilaridade das comunidades de fungos, dando suporte a geração dos primeiros mapas biogeográficos de fungos em tais solos. Adicionalmente, a abundância de fungos mostrou-se relacionada com a produtividade da cultura, indicando este ser um dos fatores que modulam a produtividade de cana-de-açúcar nas áreas avaliadas. / The sugarcane is nowadays, the most important crop in the State of São Paulo, serving as the raw material for the production of sugar and ethanol, besides many others by-products. Considering the expansion of agricultural barriers, and shifts in fields management, such cultivation is under a re-arrangement process, aiming to a higher productivity and sustainability. In order to achieve that, among other factors, the role of microbial communities present in soils can be essential to support plant development. However, a few is known about the microbial community under sugarcane crop production soils. Hence, this work intended to evaluate the fungi diversity and abundance in soils cultivated with sugarcane in the State of São Paulo, exploring areas under distinct chemical and physical attributes and also distinct management practices. It was also aimed to determine the occurrence of biogeographically patterns in the structure of such communities. Indeed, it was made the analysis of the fungal community structure by terminal restriction fragment length polymorphism (T-RFLP), together with the quantification of these communities by real time PCR (qPCR) in 476 soils samples, collected in 11 areas cultivated with sugarcane (mills). Within this dataset, it was found that chemical, physical and management attributes explain higher values of variance within each sampled area, but explain little about the total variance of data, suggesting the occurrence of biogeographically patterns in fungal communities in this environment. It was confirmed by the statistical significance of the correlation between distance and dissimilarity of fungal communities, supporting the generation of very first biogeographically maps in such soils. Additionally, the abundance of fungi revealed to be related with sugarcane productivity, indicating this issue as one of the factors modulating the sugarcane productivity in the evaluated areas.

Fluorotelomer alcohols (FTOHs, F(CF2)nCH2CH2OH) are emerging contaminants in the environment. Biodegradation of 6:2 and 8:2 FTOHs has been intensively studied using soils and activated sludge. However, little is known about the bacteria responsible for biotransformation of FTOHs. This study deciphered factors affecting biodefluorination of FTOHs and their metabolites, and developed three effective FTOH-degrading consortia. Two alkane-degrading Pseudomonas strains (P. oleovorans and P. butanovora) can defluorinate 4:2, 6:2 and 8:2 FTOHs, with a higher degree of defluorination for 4:2 FTOH. According to the identified metabolites, P. oleovorans transformed FTOHs via two pathways I and II. Pathway I led to formation of x:2 ketone (x = n-1), x:2 sFTOH and perfluorinated carboxylic acids (PFCAs). Pathway II resulted in the formation of x:3 polyfluorinated acid and relatively minor shorter-chain PFCAs. Conversely, P. butanovora transformed FTOHs by pathway I only. Mycobacterium vaccae JOB5 (a C1-C22alkane-degrading bacterium) and P. fluorescens DSM 8341 (a fluoroacetate-degrading bacterium) can transform 6:2 FTOH via both pathways I and II with the formation of odd-numbered short-chain PFCAs. In the presence of dicyclopropylketone or formate, P. oleovorans transformed 6:2 FTOH six times faster and produced odd-numbered PFCAs. P. butanovora, utilized both pathways I and II in the presence of lactate, and it also produced odd-numbered PFCAs. Unlike P. oleovorans, P. fluorescens DSM 8341 could slightly convert 5:3 polyfluorinated acid (a key metabolite during 6:2 FTOH degradation, [F(CF2)5CH2CH2COOH]) to 4:3 acid and PFPeA via one-carbon removal pathways. Three FTOH-degrading consortia transformed FTOHs, with enhanced removal of FTOHs in the presence of n-octane. A higher copy number of alkB gene was found to correspond to better removal of FTOHs, suggesting that alkane-degrading bacteria might be the key degraders in the enrichments. The three enrichment cultures showed a similar microbial community structure. This is the first study reporting that pure strains of alkane- and fluoroacetate-degrading bacteria can bio-transform FTOHs via different or preferred transformation pathways to remove multiple –CF2– groups from FTOHs to form shorter-chain PFCAs, and to other perfluorinated acids. The results of this study also suggest that enhanced FTOH biodegradation is possible through co-substrate addition and/or using enrichment cultures.

Inflammatory bowel disease (IBD), comprises Crohn’s disease (CD) and ulcerative colitis (UC), and is a chronic relapsing inflammation of gastrointestinal tract without any known cause or cure. Currently, it is accepted that IBD is a result of a dysfunctional immune response to commensal bacteria in a genetically susceptible host, and that environmental factors can trigger the onset or reactivation of the disease. This thesis considers the possibility of a specific pathogenic agent as well as an imbalance in the composition of the normal microflora in the pathogenesis of IBD. Gut biopsy tissues were taken from a population-based case-control tissue bank held at the University of Manitoba. Automated ribosomal intergenic spacer analysis (ARISA) and terminal restriction fragment length polymorphisms (T-RFLP) were employed to assess the diversity of gut microbiota. The phylogenetic, virulence and biochemical characteristics of Escherichia coli isolated from IBD biopsies were examined using multi-locus sequence typing (MLST), DNA microarray technology and API 20E system. Utilizing ARISA and T-RFLP, a remarkable increase in the order of unclassified Clostridia was detected in inflamed tissues, particularly in CD patients (P < 0.05). Moreover, species richness and diversity were the highest in non-inflamed IBD biopsies. Culture-based quantification detected a significantly higher number of E. coli in IBD tissues (P < 0.05). Phylogenetic analysis revealed the tendency of E. coli isolated from IBD patients to be grouped into separate clonal clusters based on their allelic profiles (P = 0.02). A link was detected between uropathogenic E. coli (UPEC) CFT073 and strains isolated from IBD, with regards to gene distribution and virulence, using microarray technology. Amino acid substitutions N91S and S99N in FimH, the adhesive subunit of E. coli type I fimbria, were significantly associated to IBD (P < 0.05). This study demonstrated an increase in the microbial diversity of non-inflamed IBD tissues and suggested a recruitment phase of bacterial adherence and colonization, before the inflammation sets in. Furthermore, E. coli isolated from IBD tissues were distinct from commensal strains in both clonal and virulence characteristics and shared remarkable traits with extraintestinal pathogenic E. coli. Features involved in bacterial adhesion to epithelial cells may hold the key to E. coli pathogenesis in IBD.