Submitted by MILENA TAVARES LIMA null (milena.tavares@hotmail.com) on 2018-02-22T20:53:15Z No. of bitstreams: 1 tese.docx: 10715432 bytes, checksum: 43d05c474d46b9a07105fe43fdc08920 (MD5) / Rejected by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br), reason: Solicitamos que realize correções na submissão seguindo as orientações abaixo: O arquivo para ser submetido no repositório deve ser em formato pdf como descrito no item 13 do tutorial do repositório: 13. Para enviar o arquivo contendo sua dissertação ou tese é necessário que:  o arquivo esteja no formato Portable Document Format (PDF);  o arquivo não esteja protegido;  o trabalho (dissertação ou tese) esteja reunido em um único arquivo, inclusive os apêndices e anexos. Agradecemos a compreensão on 2018-02-23T13:51:33Z (GMT) / Submitted by MILENA TAVARES LIMA null (milena.tavares@hotmail.com) on 2018-02-23T13:57:19Z No. of bitstreams: 1 Tese_Milena_Tavares_Lima_Constancio.pdf: 2839426 bytes, checksum: 0aaadc8cdfe45a78b2777dd26109dea2 (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-02-23T17:32:08Z (GMT) No. of bitstreams: 1 constancio_mtl_dr_jabo.pdf: 2839426 bytes, checksum: 0aaadc8cdfe45a78b2777dd26109dea2 (MD5) / Made available in DSpace on 2018-02-23T17:32:08Z (GMT). No. of bitstreams: 1 constancio_mtl_dr_jabo.pdf: 2839426 bytes, checksum: 0aaadc8cdfe45a78b2777dd26109dea2 (MD5) Previous issue date: 2018-01-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O interesse no estudo de comunidades microbianas de ambientes pouco avaliados, impactados e/ou extremos tem aumentado significativamente. Dentre os ambientes estudados, encontram-se as águas residuais de áreas de mineração. Com o grande número de cavas e lagoas de rejeitos gerados através da atividade mineradora e o pouco estudo deste tema no Brasil, foi desenvolvido este trabalho, onde avaliamos a taxonomia e capacidade funcional da comunidade microbiana. As amostras de água foram coletadas de uma cava de mineração e uma lagoa de rejeitos de uma região anteriormente utilizada para extração de minério de ferro no Centro de Biodiversidade (CeBio) da empresa VALE/SA, em Sabará/MG. Análises do perfil de atividade metabólica indicaram um excelente desempenho quanto à degradação de diferentes compostos. Além disso, o isolamento com métodos tradicionais de cultivo mostrou que algumas bactérias apresentam capacidade em produzir diferentes enzimas de interesse biotecnológico. O DNA foi extraído e sequenciado através de técnicas metagenômicas, o sequenciamento do gene de 16S rRNA foi realizado na plataforma Ion PGM™. Esta análise, além de revelar a taxonomia da comunidade presente na água da cava e lagoa, identificou o perfil funcional indicando enriquecimento de subsistemas importantes para ambas as amostras. Adicionalmente para a água da cava de mineração, foi realizado o sequenciamento do DNA total na plataforma Ion Proton™, que através de análises com o banco de dados SEED revelou genes associados à manutenção celular e ao ciclo do nitrogênio, sugerindo que a comunidade microbiana está bem adaptada ao processo de recuperação da área. Além disso, as ORfs preditas do metagenoma foram utilizadas para uma análise no banco de dados BacMet, que revelou uma abundância de genes relacionados a resistência a metais. Assim, nossos dados expandem os conhecimentos sobre a estrutura e capacidade funcional das comunidades microbianas de ambientes aquáticos impactados, fornecendo uma melhor compreensão do papel dos micro-organismos em processos de remediação natural. / The interest in the study of microbial communities of low evaluated, impacted or extreme environments has increased significantly. Among the studied environments, there is the wastewater from mining areas. Due to the large number of reject pits and lagoons generated by the mining activity and the lack of study of this theme in Brazil, this study was developed to evaluate the taxonomy and functional capacity of the microbial community. The water samples were collected from a mining pit and a reject lagoon from a region formerly used for iron extraction at the Biodiversity Center (CeBio) from VALE/SA, in Sabará/MG. Analyzes of the metabolic activity profile indicated an excellent performance regarding the degradation of different compounds. Moreover, the isolation with traditional methods of culture showed that some bacteria have the capacity to produce different enzymes of biotechnological interest. The DNA was extracted and sequenced using metagenomic techniques, the 16S rRNA gene sequencing was performed on the Ion PGM™ platform. This analysis, in addition to revealing the taxonomy of the community present in the water of the pit and the lagoon, identified the functional profile indicating enrichment of important subsystems for the studied samples. In addition to the water from the mining pit, the total DNA sequencing was carried out on the ION Proton™ platform, which, through analysis with the SEED database, revealed genes associated with cell maintenance and the nitrogen cycle, suggesting that the microbial community is well adapted to the recovery process of the area. Also, the predicted ORfs from metagenomic were used for an analysis in the BacMet database that revealed an abundance of genes related to resistance to metals. Thus, our data expand the knowledge about the structure and functional capacity of microbial communities in impacted aquatic environments, providing a better understanding of the role of microorganisms in natural remediation processes. / FAPESP: 2014/07592-3

The need for new antibiotics has been highlighted recently with the increasing pace of emergence of drug resistant pathogens (MRSA, XDR-TB, etc.). Modification of existing antibiotics with the additions of side chains or other chemical groups and genomics based drug targeting have been the preferred method of drug development at the corporate level in recent years. These approaches have yielded few viable antibiotics and natural products are once again becoming an area of interest for drug discovery. We examined the antimicrobial "Red Soils" of the Hashemite Kingdom of Jordan that have historically been used to prevent infection and cure rashes by the native peoples. Antimicrobial producing bacteria were present in these soils and found to be the reason for their antibiotic activity. After isolation, these bacteria were found to excrete their antimicrobials into the liquid culture media which we could then attempt to isolate for further study. Adsorbent resins were employed to capture the antimicrobial compounds and then elute them in a more concentrated solution. As part of a drug discovery program, we sought a way to quickly characterize other soils for potential antibiotic producing bacteria. The community level physiologic profile was examined to determine if this approach would allow for a rapid categorizing of soils based on their probability of containing antimicrobial producing microorganisms. This method proved to have a high level of variability that could not be overcome even after mixing using a commercial blender. The role of these antimicrobial producing bacteria within their natural microbial community has largely been confined to microbe-plant interactions. The role of antimicrobial-producing microorganisms in driving the diversity of their community has not been a focus of considerable study. The potential of an antimicrobial-producing bacterium to act as a driver of diversity was examined using an artificial microbial community based in a sand microcosm. The changes in the microbial assemblage indicate that antimicrobial-producing bacteria may act in an allelopathic manner rather than in a predatory role. / Ph. D.

The objective of this work was to understand microbial population density and diversity, both spatially and temporally, in wetland mesocosms to gain a better fundamental understanding for use in the optimization and design of constructed wetlands (CWs). A standardized community level physiological profiling (CLPP) data analysis protocol was adapted and utilized for CW mesocosms. A new one-dimensional metric was developed to track community divergence using BIOLOGTM ECO plate data. The method proved easy to use, did not require a background in multivariate statistics, and accurately described community divergence in mesocosm systems. To study mesocosm biofilm-bound bacterial communities an appropriate detachment protocol was required. Various shaking protocols were evaluated for their effectiveness in the detachment of bacteria from mesocosm pea gravel, with a focus on detachment of viable and representative bacterial communities. A protocol based on mechanical shaking with buffer and enzymes was identified as an optimal approach and used further in this study. The bacterial communities associated with the interstitial water, pea gravel media, and rhizospheric regions from both planted and unplanted CW mesocosms were profiled using the CLPP method and compared. Vertical community stratification was observed for all mesocosm systems. Rhizospheric communities were found to be significantly more active than their gravel-associated counterparts, suggesting that although rhizospheric bacteria were less abundant in the mesocosms they may play a more significant role in the removal and fate of water born contaminants. The start-up dynamics of CW mesocosms was investigated using the CLPP and standard CW characterization methods over an eight month period. All mesocosms showed a steep increase in interstitial community divergence until day 75-100, at which point a steady-state was reached. The interstitial communities were also characterized in terms of similarity based on experimental design treatments (planted/unplanted and origin of seeding inoculum). Four stages were identified during the start-up consisting of an initial stage where mesocosm communities were differentiated based on origin of the inoculum, a period where adjustments and shifts occurred in all mesocosm, a time where all mesocosm communities were quite similar, and a final state where community differentiations were made based plant presence in the mesocosms.

The objective of this work was to understand microbial population density and diversity, both spatially and temporally, in wetland mesocosms to gain a better fundamental understanding for use in the optimization and design of constructed wetlands (CWs). A standardized community level physiological profiling (CLPP) data analysis protocol was adapted and utilized for CW mesocosms. A new one-dimensional metric was developed to track community divergence using BIOLOGTM ECO plate data. The method proved easy to use, did not require a background in multivariate statistics, and accurately described community divergence in mesocosm systems. To study mesocosm biofilm-bound bacterial communities an appropriate detachment protocol was required. Various shaking protocols were evaluated for their effectiveness in the detachment of bacteria from mesocosm pea gravel, with a focus on detachment of viable and representative bacterial communities. A protocol based on mechanical shaking with buffer and enzymes was identified as an optimal approach and used further in this study. The bacterial communities associated with the interstitial water, pea gravel media, and rhizospheric regions from both planted and unplanted CW mesocosms were profiled using the CLPP method and compared. Vertical community stratification was observed for all mesocosm systems. Rhizospheric communities were found to be significantly more active than their gravel-associated counterparts, suggesting that although rhizospheric bacteria were less abundant in the mesocosms they may play a more significant role in the removal and fate of water born contaminants. The start-up dynamics of CW mesocosms was investigated using the CLPP and standard CW characterization methods over an eight month period. All mesocosms showed a steep increase in interstitial community divergence until day 75-100, at which point a steady-state was reached. The interstitial communities were also characterized in terms of similarity based on experimental design treatments (planted/unplanted and origin of seeding inoculum). Four stages were identified during the start-up consisting of an initial stage where mesocosm communities were differentiated based on origin of the inoculum, a period where adjustments and shifts occurred in all mesocosm, a time where all mesocosm communities were quite similar, and a final state where community differentiations were made based plant presence in the mesocosms.