Esta tese apresenta e discute os dados obtidos a partir de trabalho experimental projetado para avaliar comparativamente o desempenho de reatores desnitrificantes em batelada, tendo etanol, metanol e gás metano como doadores de elétrons. Os experimentos foram realizados em reatores em escala de bancada. Os ensaios com gás metano objetivaram verificar a efetividade deste sub-produto de reatores anaeróbios em substituir os doadores exógenos de elétrons comumente utilizados, tais como metanol e etanol. Para alcançar o objetivo principal deste trabalho, os parâmetros cinéticos de desnitrificação, para os doadores de elétrons ensaiados, foram determinados nas diferentes condições operacionais. Além disso, as alterações ocorridas na população microbiana, ao longo do período experimental, foram avaliadas em relação à diversidade microbiana, por meio de análises microscópicas (óptica, de fluorescência e eletrônica de varredura) e da técnica de Biologia Molecular de PCR/DGGE. A completa desnitrificação foi alcançada para todos os compostos testados, e o etanol foi o doador de elétrons mais eficiente para a desnitrificação. A melhor razão carbono-nitrogênio para a desnitrificação foi igual a 1,0. Contudo, este parâmetro foi encontrado ser inadequado para utilização no processo de desnitrificação, uma vez que não expressa a capacidade real do composto usado em doar elétrons. A desnitrificação com metano ocorreu tanto na presença como na ausência de oxigênio, embora a baixas velocidades quando comparado com os outros compostos. No entanto, a configuração do reator utilizado neste estudo não foi adequada para promover a efetiva dissolução do gás metano na fase líquida. Por essa razão, sugere-se o desenvolvimento de configurações de reatores apropriadas para minimizar as resistências à transferência de massa da fase gasosa para a líquida e também desta para a biomassa. / This thesis presents and discusses the data obtained from an experimental work designed to evaluate the comparative performance of denitrifying batch reactors utilizing ethanol, methanol and methane gas as electron donors. The experiments were carried out at using bench-scale reactors. The experiments using methane gas were meant to verify the effectiveness of such a by-product of anaerobic reactors instead of exogenous electron donors commonly used, such as methanol and ethanol. To achieve this main objective, the kinetic parameters of denitrification for the distinct electron donors assayed were determined in different operating conditions. Besides, the microbial population changes inside the reactors along the experimental time were evaluated in respect to the microbial diversity, by means of microscopy analysis (optical, fluorescent and electronic scanning) and the Molecular Biology technique, PCR/DGGE. Complete denitrification was achieved with all the compounds tested, and ethanol was the most effective electron donor for denitrification. The best carbon to nitrogen ratio for denitrification was 1.0. However, this parameter was found to be inadequate for using in denitrification process, since it does not express the real capacity of the compound used to donate electrons. Denitrification with methane occurred in the presence and also in the absence of oxygen, although at lower velocities compared to those with the other compounds. However, the reactor configuration utilized in this study was not adequate to promote the effective methane gas dissolution in the liquid phase. Therefore, it is suggested the development of appropriate reactor configurations to minimize mass transfer resistances from the gas to the liquid phase and also from that to the biomass.

O conhecimento das associações entre os microrganismos componentes da microbiota dos solos é de grande interesse no meio científico, principalmente relacionado aos microrganismos que se associam de forma benéfica com as plantas. Neste contexto, destaca-se a micorriza arbuscular, que é a associação entre os fungos micorrízicos arbusculares (FMAs) e uma amplitude de espécies vegetais. Além desta íntima interação entre estes organismos, torna-se necessário conhecer a importância dos demais componentes do sistema solo, como a microbiota formada por fungos e bactérias dos solos, para o estabelecimento desta interação. Os objetivos dos estudos componentes desta tese se concentraram na avaliação de FMAs (D. heterogama, R. clarus e Gi. rosea) inoculados em sistemas alterados quanto à composição das comunidades microbianas dos solos, promovidas pela metodologia de \'diluição para extinção\'. No primeiro estudo foram encontradas respostas diferenciais na capacidade de colonização micorrízica (%CM) de plantas de cana-de-açúcar pelos FMAs inoculados nos diferentes sistemas, além do efeito diferencial dos FMAs quanto às alterações nas comunidades de fungos e bactérias do solo. Num estudo mais detalhado, desenvolvido apenas com R. clarus em plantas de milho, foi verificado que a maiores diversidades microbianas do solo resultaram em maior colonização do hospedeiro, principalmente no período inicial de desenvolvimento das plantas. Neste experimento foi descrita a correlação direta da capacidade de colonização do FMA com a riqueza e a diversidade filogenética da microbiota dos solos. A descrição dos perfis metabólicos dos solos contendo diferentes comunidades microbianas revelou a capacidade diferencial destes solos em utilizar diferentes fontes de carbono, além de demonstrar um aumento no metabolismo (evidenciado pelo consumo total de fontes de carbono) devido à inoculação do FMA. Em conjunto, os dados obtidos nos dois estudos indicam que a micorrização das plantas depende da ação de outros microrganismos do sistema solo, que atuam como um terceiro fator nesta simbiose e que a microbiota do solo responde a inoculação de um organismo exógeno, primordialmente aumentando seu metabolismo. Deve-se, portanto, considerar que a degradação biológica dos solos, com perda de sua biodiversidade, pode ter papel determinante no funcionamento de interações específicas e benéficas às plantas. / Knowledge about the associations between microbial components of soil microbiota is of great interest in the scientific community, primarily related to microorganisms that are associated beneficially with plants. In this context, there is the arbuscular mycorrhiza, which is made of the association between mycorrhizal fungi (AMF) and a range of plant species. In this close interaction between these organisms, it is necessary to describe the importance of other components of the soil system, such as the microbiota formed by fungi and bacteria from the soil, to establish this interaction. The objectives of the studied that compose this thesis focused on the evaluation of AMF (D. heterogama, R. clarus and Gi. rosea) fitness, when inoculated in modified systems on the composition of the microbial communities in the soil, with alterations promoted by the \'dilution to extinction\' methodology. In the first study, differential responses were found in root colonization capacity (% CM) of sugarcane by AMF inoculated in different systems, and the differential effect of AMFs, changing the communities of fungi and bacteria of soil. In a more detailed study, designed only to R. clarus in corn plants, it was found that higher microbial diversity of soil resulted in higher colonization of the host, especially in the initial period of plant development. In this experiment it was described the direct correlation of AMF colonization capacity with richness and phylogenetic diversity of soil microbiota. The description of the metabolic profiles of soil containing various microbial communities revealed the differential ability of these soils utilize different carbon sources, in addition to demonstrating an increase in metabolism (as evidenced by the total consumption of carbon sources) due to inoculation of the AMF. Together, the data from the two studies indicate that colonization of plants by AMF depends on the action of other microorganisms soil system, which act as a third factor in this symbiosis, and that the soil microbes respond to inoculation of an exogenous organism, primarily increasing its metabolism. One should therefore consider that the biological degradation of the soil, with loss of biodiversity, may have crucial role in the functioning of specific and beneficial interactions to plants.

Esta tese apresenta e discute os dados obtidos a partir de trabalho experimental projetado para avaliar comparativamente o desempenho de reatores desnitrificantes em batelada, tendo etanol, metanol e gás metano como doadores de elétrons. Os experimentos foram realizados em reatores em escala de bancada. Os ensaios com gás metano objetivaram verificar a efetividade deste sub-produto de reatores anaeróbios em substituir os doadores exógenos de elétrons comumente utilizados, tais como metanol e etanol. Para alcançar o objetivo principal deste trabalho, os parâmetros cinéticos de desnitrificação, para os doadores de elétrons ensaiados, foram determinados nas diferentes condições operacionais. Além disso, as alterações ocorridas na população microbiana, ao longo do período experimental, foram avaliadas em relação à diversidade microbiana, por meio de análises microscópicas (óptica, de fluorescência e eletrônica de varredura) e da técnica de Biologia Molecular de PCR/DGGE. A completa desnitrificação foi alcançada para todos os compostos testados, e o etanol foi o doador de elétrons mais eficiente para a desnitrificação. A melhor razão carbono-nitrogênio para a desnitrificação foi igual a 1,0. Contudo, este parâmetro foi encontrado ser inadequado para utilização no processo de desnitrificação, uma vez que não expressa a capacidade real do composto usado em doar elétrons. A desnitrificação com metano ocorreu tanto na presença como na ausência de oxigênio, embora a baixas velocidades quando comparado com os outros compostos. No entanto, a configuração do reator utilizado neste estudo não foi adequada para promover a efetiva dissolução do gás metano na fase líquida. Por essa razão, sugere-se o desenvolvimento de configurações de reatores apropriadas para minimizar as resistências à transferência de massa da fase gasosa para a líquida e também desta para a biomassa. / This thesis presents and discusses the data obtained from an experimental work designed to evaluate the comparative performance of denitrifying batch reactors utilizing ethanol, methanol and methane gas as electron donors. The experiments were carried out at using bench-scale reactors. The experiments using methane gas were meant to verify the effectiveness of such a by-product of anaerobic reactors instead of exogenous electron donors commonly used, such as methanol and ethanol. To achieve this main objective, the kinetic parameters of denitrification for the distinct electron donors assayed were determined in different operating conditions. Besides, the microbial population changes inside the reactors along the experimental time were evaluated in respect to the microbial diversity, by means of microscopy analysis (optical, fluorescent and electronic scanning) and the Molecular Biology technique, PCR/DGGE. Complete denitrification was achieved with all the compounds tested, and ethanol was the most effective electron donor for denitrification. The best carbon to nitrogen ratio for denitrification was 1.0. However, this parameter was found to be inadequate for using in denitrification process, since it does not express the real capacity of the compound used to donate electrons. Denitrification with methane occurred in the presence and also in the absence of oxygen, although at lower velocities compared to those with the other compounds. However, the reactor configuration utilized in this study was not adequate to promote the effective methane gas dissolution in the liquid phase. Therefore, it is suggested the development of appropriate reactor configurations to minimize mass transfer resistances from the gas to the liquid phase and also from that to the biomass.

Graduation date: 2005

This study investigated the microbiota in ten different drinking water treatment pools, particles in the Biological Activated Carbon Filtration (BACF) bed, and two mimic columns in the Cheng-Ching Lake Water Treatment Plant. Assimilable organic carbon (AOC) is one of the main nutrition sources for microbes to survive in tap water. Over growing microbes not only decrease the water quality, but also contaminate the water treatment system and distribution system. In this study, we used two molecular biology techniques, the polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE), to analyze the dynamic microbial communities and biodiversities in the drinking water cleaning system and the micorbiota that exist in the BAC and anthracite filtration pellets. The bacterial 16S rDNA sequences resulted from PCR-DGGE were compared with the data in the Ribosomal Database Project Bank to construct a phylogenetic tree which allowed us to understand the microbial communities and biodiversities in the drinking water treatment pools and the filtration pellets. The total bacterial count and PCR-DGGE profiles showed that the drinking water quality had been improved during the treating processes and most of the microbes in raw water were removed. The scanning electron microscopy clearly indicated the biofilms were developed on the pellet surface. From the mimic column studies, the PCR-DGGE profiles suggested that various microbial communities were present on different depth of the columns samples. In comparing the 16S rDNA sequences with Gene Bank, many are new category bacteria were found and most of them are unculturable. Most of these microbes belong to the beta-proteobacterium. Although many bacteria were located on the surface of the filtration pellet, the BAC and anthracite could still absorb AOC efficiently to enhance the bacteria growth. The over growing bacteria might release out and contaminate the drinking water. Therefore, we suggest that it is important to backwash the filter bed frequently in order to diminish microbes of the filtration pellet and avoid re-contaminate the drinking water.

Understanding microbial evolution lies at the heart of microbiology and environmental sciences. Numerous studies have been dedicated to elucidating the underlying mechanisms that create microbial genetic diversity and adaptation. However, due to technical limitations such as the high level of uncultured cells in almost every natural habitat, most of current knowledge is primarily based on axenic cultures grown under laboratory conditions, which typically do not simulate well the natural environment. How well the knowledge from isolates translates to in-situ processes and natural microbial communities remains essentially speculative. The recent development of culture-independent genomic techniques (aka metagenomics) provides possibilities to bypass some of these limitations and provide new insights into microbial evolution in-situ. To date, most of metagenomic studies have been focused on a few reduced-diversity model communities, e.g., acid mine drainage. Highly complex communities such as those of soil and sediment habitats remain comparatively less understood. Furthermore, a great power of metagenomics, which has not been fully capitalized yet, is the ability to follow the evolution of natural microbial communities over time and environmental perturbations, i.e., times-series metagenomics. Although the recent developments in DNA sequencing technologies have enabled (inexpensive) time-series studies, the bioinformatics approaches to analyze the resulting data have clearly fallen behind. Taken together, to scale up metagenomics for complex community studies, three major challenges remain: 1) the difficulty to process and analyze massive short read sequencing data, often at the terabyte level; 2) the difficulty to effectively assemble genomes from complex metagenomes; and 3) the lack of methods for tracking genotypes and mutational events such as horizontal gene transfer (HGT) through time. Therefore, developing efficient bioinformatics approaches to address these challenges represents an important and timely issue. This thesis aimed to develop novel bioinformatics pipelines and algorithms for high performance computing, and, subsequently, apply these tools to natural microbial communities to generate quantitative insights into the relative importance of the molecular mechanisms creating or maintaining microbial diversity. The tools are not specific to a particular habitat or group of organisms and thus, can be broadly used to advance our understanding of microbial evolution in different settings. In particular, the comparative whole-genome analysis of 24 Escherichia isolates form various habitats, including human and non-human associated habitats such as freshwater ecosystems and beaches, showed that organisms with more similar ecologies tend to exchange more genes, which has important implications for the prokaryotic species concept. To more directly test these findings from isolates and quantify the patterns of genetic exchange among co-occurring populations, three years of time-series metagenomics data from planktonic samples from Lake Lanier (Atlanta, GA) were analyzed. For this, it was first important to develop bioinformatics algorithms to robustly assemble population genomes from complex community metagenomes, identify the phylogenetic affiliation of assembled genome and contig sequences, and detect horizontal gene transfer among these sequences. Using these novel algorithms, in situ bacterial lineage evolution was quantitatively assessed, especially with respect to whether or not ecologically distinct lineages evolve according to the recently proposed fragmented speciation model (Retchless and Lawrence, Science 2008). Evidence in support of this model was rarely observed. Instead, it appeared that rampant HGT disseminated ecologically important genes within the population, maintaining intra-population diversity. By expanding the previous approaches to include methods to assess differential gene abundance and selection pressure between samples, it was possible to quantify how soil microbial communities respond to a decade of warming by 2 0C, which simulated the predicted effects of climate change. It was found that the heated communities showed significant shifts in composition and predicted metabolism, reflecting the release of additional soil carbon compared to the unheated (control) communities, and these shifts were community-wide as opposed to being attributable to a few taxa. These findings indicated that the microbial communities of temperate grassland soils play important roles in mediating the feedback responses to climate change. Collectively, the findings presented here advance our understanding of the modes and tempo of microbial community adaptation to environmental perturbations and have important implications for better modeling the microbial diversity on the planet. The bioinformatics algorithms and approaches developed as part of this thesis are expected to facilitate future genomic and metagenomic studies across the fields of microbiology, ecology, evolution and engineering.

A molecular phylogenetic approach was used to characterize the composition of microbial communities from two gas hydrate sedimentary systems in the Gulf of Mexico. Nucleic acids were extracted from three distinct locales on surface breaching gas hydrate mounds, i.e., sediment overlaying gas hydrate, sediment/hydrate interface and sediment-free hydrate, and from three sediment depths, i.e., 0-2, 6-8 and 10-12 cm, in Beggiatoa sp. mat-associated sediments located several meters from exposed gas hydrate. Samples were collected from a research submersible (water depth 550-575 m) during two research cruises aboard the R/V Seward Johnson I and II funded by the NSF Life in Extreme Environments program. The 16S rRNA gene and 16S rRNA were amplified using PCR and reverse transcription-PCR, respectively, from DNA and RNA extracted from the total microbial community. The primers targeted microorganisms at the domain-specific, i.e., Bacteria and Archaea, and group-specific, i.e., sulfate-reducing bacteria (SRB) and putative anaerobic methane-oxidizing (ANME) archaea, level. Sequence analysis of the Bacteria clones revealed that the microbial communities were primarily dominated by Deltaproteobacteria. Other Proteobacteria classes, including Epsilon- and Gammaproteobacteria, represented a large fraction of the total microbial community isolated from the sediment overlying hydrate sample and the metabolically active fraction of the 0-2 cm sediment depth sampled from the Beggiatoa sp. mat-associated sediments. Sequence analysis indicated the majority of the archaeal clones were most closely related to Methanosarcinales, Methanomicrobiales and distinct lineages within the ANME groups. Several novel lineages were identified including a fourth ANME-2 clade, i.e., ANME-2D, and three clades with no closely related previously sequenced 16S rRNA gene clones or isolates, i.e., Unclassified Bacteria groups 1 and 2 and Unclassified Euryarchaeota. These studies represent the first 16S rRNA gene and 16S rRNA phylogenetic-based description of microbial communities extant in sediment-free gas hydrate and in methane-rich hydrate-associated and Beggiatoa sp.-associated sediments from a hydrocarbon seep region in the Gulf of Mexico.

Abstract Based on maintaining healthy soil for sustainable agriculture and enhancing banana disease resistance, Taiwan Banana Research Institute began to conduct organic cultivation on a trial basis in 1998. It had been proved that the morbidity of banana Fusarial wilt disease at organic cultivation plots was significantly lower than that of conventional farming. In order to study the differences of soil microbiota between the organic cultivation plots and the conventional farming areas, physical and chemical properties of the rhizosphere and non- rhizosphere soil samples were assayed during the period of Aug. 2010 to May 2011. The bacterial diversity was analyzed by molecular biology methods, including PCR-DGGE to separate the 16S rDNA V6 ~ V8 region of various bacteria and the recombinant DNA technology by using pGEM-T Easy Vector System to separate and sequence the DNA fragments. The results showed that organic plots was loam soil, but the conventional farming soil was sandy loam with higher sand content. The soil pH in 13 years organic area was mildly alkaline, but in conventional farming area was mildly acidic to slightly acidic. The content of various nutrients in organic 13-year area soil was not necessarily higher than the conventional farming area soil. The available nutrient contents in organic areas trend to be more stable than that in the conventional areas. Fertilization may affect the content of available nutrients in the soil. No bacterial DNA could be extracted from the organic fertilizer. The bacterial microbiota in soil was very stable, and was not related to the sampling seasons. The Banana strains had little effect on soil bacterial microbiota. There was no difference on the bacterial microbiota between the rhizosphere and non-rhizosphere soil samples. It is not sure whether there were any differences on the bacterial microbiota between the nearby soil of banana Fusarial wilt plants and the nearby soil of the healthy plants. By analyzing the DNA fragment clone library, 43 strains correspond to known category, of which 28 belonged to the Proteobacteria, and 34 were uncultured strains. The role of these microbial strains might involve in various element cycles, such as N cycles, C cycles, and S cycles (including some photosynthetic bacteria). The systematic cladogram showed that organic 13-year areas, organic 3-year areas and conventional farming areas represented three major categaries. The organic 13-year area and conventional area possessed the highest difference on the microbiota composition.

Constructed wetlands had been used for water treatment worldwide. The efficiency of wastewater treatment in a constructed wetland depends on its design, types of aquatic plants and microbial community present in this wetland. The goal of this study is to analyze the microbial populations in KaoPing River Rail Bridge constructed wetland which was designed to remove the polluted material from municipal sewage and industrial wastewater. Sediment and water samples were collected every 3 months from April, 2007 to April, 2008. The bacterial community diversities were analyzed by PCR-DGGE of the bacterial 16S rRNA gene. Results show approximately 60% BOD, 41% COD, 46% nitrate, 22% total nitrogen, and 97% coliforms were removed by this wetland system. DGGE profiles revealed the bacterial community diversities shifted progressively from the entry to the exit of both A and B systems in this wetland. The microbial populations in water, sediment, biofilms on plants, and soil were quite different from each others. The fecal indicator Escherichia coli was used as a marker to monitor the fecal contamination in all samples. From PCR-DGGE profiles, E. coli could be successfully removed by this wetland system. In conclusion, this constructed wetland is a very successful system for wastewater treatment and is able to remove most of the pollutants before they are discharged into KaoPing River. The results of this study provided useful suggestions for the government to assess the bacterial diversities and the efficiency of this wetland system, to protect people from hazardous risks, and to manage a constructed wetland in the future.

Water treatment facilities have been relying on indicator bacteria to assess the quality of water for decades. The purpose of this group of studies is to investigate the predictive capabilities of conventional and alternative indicators for pathogenic microorganisms in disinfection processes and treated wastewater effluents. In addition, the possibility that diversity of indicator bacteria, as well as overall bacterial diversity, correlate with fecal contamination in water bodies has been investigated. Indicator organisms (total coliforms, fecal coliforms, enterococci, C. perfringens, and coliphages) as well as pathogens (enteroviruses, Giardia, and Cryptosporidium) were enumerated from six wastewater treatment facilities at various stages of treatment. Statistical analyses were conducted to determine if the indicator organisms (individually or as a set) could predict the presence or absence of pathogens. Single indicator organism analysis failed to correlate with the occu rrence pathogens, thus monitoring a suite of indicator organisms may be a better measure to predict the presence of pathogens. The product of chlorine residual concentration and contact time (CT) was identified as a factor for determining the log10 reduction of enteric viruses in wastewater treatment facilities that used chloramines for disinfection.Samples were also collected from river waters and sediments in watersheds with different human population densities to identify the impact of anthropogenic activities on bacterial diversity. 16S rRNA restriction fragment length polymorphism (RFLP), ribotyping, and denaturing gradient gel electrophoresis (DGGE) were used to determine total coliform, Escherichia coli, and bacterial community population structures, respectively. The concentrations of indicator organisms were significantly different among the river sites in sediments, but not in water column. The population diversity measurements were not significantly different among the river sites; while the indicator population and bacterial community structures were dissimilar in water column vs. associated sediment samples. Accumulation curves demonstrated that greater than 20 isolates must be sampled at most of the sites to represent the dominant populations. A better understanding of the relationship between the indicator organisms and pathogens as well as knowledge of the ecology of indicator organisms in pristine and anthropogenically impacted waters may contribute to water quality restoration and public health protection.