Com as limitações para cultivar os micro-organismos, tornou-se necessário caracterizar molecularmente os recursos genéticos microbianos. Entretanto, como estes estudos são realizados através do DNA, podemos encontrar distintos resultados dependendo da metodologia de extração de DNA utilizada. Assim, essa pesquisa buscou aperfeiçoar uma técnica para extrair DNA de água do e para demonstrar a importância dos métodos de extração de DNA no estudo dos micro-organismos por métodos independentes de cultivo, foi caracterizada, através da técnica de DGGE, a diversidade de uma comunidade bacteriana marinha usando diferentes métodos de extração de DNA. O aperfeiçoamento da metodologia de extração de DNA de água do mar resultou em um protocolo com a qualidade do DNA similar à do protocolo original, com rendimento melhor e aproximadamente quatro vezes mais rápido. Em relação à caracterização, verificamos, pelo DGGE, distintos perfis da comunidade microbiana marinha, tanto em relação à presença e ausência das bandas de DNA, como em relação à sua intensidade. / With the limitations of microorganisms culture techniques, it has become necessary to use molecular techniques to characterize microbial genetic resources. However, these studies are done using extracted environmental DNA, therefore we can find different results depending on the methodology chosen for the DNA extraction. Thus, this study aimed to improve a technique for extracting DNA from seawater and to demonstrate the importance of the DNA extraction methods in the study of microorganisms by cultivation-independent methods, it was characterized, by DGGE technique, the diversity of a marine bacterial community using different methods of DNA extraction. The improved methodology of DNA extraction from seawater resulted in a protocol similar in DNA quality, with better yield and approximately four times faster than the protocol initially described. Regarding the characterization by different DNA extraction protocols, we found distinct marine microbial community profiles, in relation the absence and presence of DNA bands and their relative intensity.

O presente estudo visou investigar os efeitos da ressuspensão do sedimento e enriquecimento orgânico por diferentes microalgas na estrutura das comunidades microbianas do sedimento. Para tanto dois experimentos laboratoriais separados foram realizados (Nov-Dez/2009 e Abr-Maio/2011) com o intuito de simular as condições de ressuspensão e pulsos de produtividade primária observadas em campo e assim observar como a comunidade microbiana do sedimento é influenciada por esses eventos. Ambos os experimentos foram mantidos por um total de 30 dias após o tratamento, durante os quais amostras para análises sedimentares, densidade de procariotos e composição da comunidade bacteriana foram tomadas em seis períodos diferentes. A camada superficial do sedimento foi estudada mais detalhadamente e mostrou que tanto a chegada de material algal quanto a ressuspensão são responsáveis por mudanças significativas na densidade, metabolismo e composição da comunidade bacteriana do sedimento. Ainda, a chegada de diferentes tipos de algas ao sedimento (fitoflagelados e diatomáceas) levou a diferentes repostas tanto na densidade quanto na diversidade dos micro-organismos sedimentares. A estrutura vertical dos micro-organismos na coluna sedimentar também foi estudada. A chegada de material algal no sedimento não levou a grandes mudanças na estrutura da comunidade mais profundas da coluna sedimentar. A estabilidade criada pelo ambiente experimental parece ter levado a um aumento tanto da densidade quanto da diversidade microbiana na camada intermediária do sedimento, em ambos os tratamentos e no controle. Já a ressuspensão parece influenciar de forma mais efetiva a distribuição dos micro-organismos na coluna sedimentar, devido a mistura da coluna sedimentar e mudanças nas condições redox das camadas sedimentares. Concluindo, tanto a chegada de alimento no sedimento, como eventos de ressuspensão são responsáveis por mudanças significativas na comunidade microbiana dos sedimentos costeiros. / The present study aimed to investigate the effects of sediment resuspension and organic enrichment by different microalgae on the sedimentary microbial community structure. We run two separate laboratory experiments (Nov-Dec/2009 and Apr-May/2011) to simulate resuspension conditions and pulses of primary productivity observed in the field, and analyze how these events affect the microbial community. Both experiments were maintained for a total of 30 days following treatment when samples were taken for sedimentary analysis, prokaryotic density and bacterial community analyses at six different sampling times. The sediment surface layer was studied in more detail, and showed that both the input of algal material and resuspension are responsible for significant changes in density, metabolism and bacterial community composition. Also, the arrival of different types of algae to the sediment (phytoflagellates and diatoms) led to different responses in both density and diversity of sedimentary microorganisms. The vertical structure of microorganisms in the sediment column was also studied. The arrival of algal material in the sediment did not show important changes in community structure of the deeper sedimentary layers. The stability created by the experimental environment seems to increase both the density and diversity of microbes in the middle layer of sediment in both treatments and control. On the other hand resuspension seems to influence more effectively the distribution of microorganisms in the sedimentary column due to sediment mixing and changes in redox conditions of different layers. In conclusion, both the arrival of food on the sediment and resuspension events is responsible for important changes in the coastal sediment microbial communities.

O presente estudo visou investigar os efeitos da ressuspensão do sedimento e enriquecimento orgânico por diferentes microalgas na estrutura das comunidades microbianas do sedimento. Para tanto dois experimentos laboratoriais separados foram realizados (Nov-Dez/2009 e Abr-Maio/2011) com o intuito de simular as condições de ressuspensão e pulsos de produtividade primária observadas em campo e assim observar como a comunidade microbiana do sedimento é influenciada por esses eventos. Ambos os experimentos foram mantidos por um total de 30 dias após o tratamento, durante os quais amostras para análises sedimentares, densidade de procariotos e composição da comunidade bacteriana foram tomadas em seis períodos diferentes. A camada superficial do sedimento foi estudada mais detalhadamente e mostrou que tanto a chegada de material algal quanto a ressuspensão são responsáveis por mudanças significativas na densidade, metabolismo e composição da comunidade bacteriana do sedimento. Ainda, a chegada de diferentes tipos de algas ao sedimento (fitoflagelados e diatomáceas) levou a diferentes repostas tanto na densidade quanto na diversidade dos micro-organismos sedimentares. A estrutura vertical dos micro-organismos na coluna sedimentar também foi estudada. A chegada de material algal no sedimento não levou a grandes mudanças na estrutura da comunidade mais profundas da coluna sedimentar. A estabilidade criada pelo ambiente experimental parece ter levado a um aumento tanto da densidade quanto da diversidade microbiana na camada intermediária do sedimento, em ambos os tratamentos e no controle. Já a ressuspensão parece influenciar de forma mais efetiva a distribuição dos micro-organismos na coluna sedimentar, devido a mistura da coluna sedimentar e mudanças nas condições redox das camadas sedimentares. Concluindo, tanto a chegada de alimento no sedimento, como eventos de ressuspensão são responsáveis por mudanças significativas na comunidade microbiana dos sedimentos costeiros. / The present study aimed to investigate the effects of sediment resuspension and organic enrichment by different microalgae on the sedimentary microbial community structure. We run two separate laboratory experiments (Nov-Dec/2009 and Apr-May/2011) to simulate resuspension conditions and pulses of primary productivity observed in the field, and analyze how these events affect the microbial community. Both experiments were maintained for a total of 30 days following treatment when samples were taken for sedimentary analysis, prokaryotic density and bacterial community analyses at six different sampling times. The sediment surface layer was studied in more detail, and showed that both the input of algal material and resuspension are responsible for significant changes in density, metabolism and bacterial community composition. Also, the arrival of different types of algae to the sediment (phytoflagellates and diatoms) led to different responses in both density and diversity of sedimentary microorganisms. The vertical structure of microorganisms in the sediment column was also studied. The arrival of algal material in the sediment did not show important changes in community structure of the deeper sedimentary layers. The stability created by the experimental environment seems to increase both the density and diversity of microbes in the middle layer of sediment in both treatments and control. On the other hand resuspension seems to influence more effectively the distribution of microorganisms in the sedimentary column due to sediment mixing and changes in redox conditions of different layers. In conclusion, both the arrival of food on the sediment and resuspension events is responsible for important changes in the coastal sediment microbial communities.

No description available.

A mesocosm experiment was carried out in a Norwegian fjord near Bergen in May 2006, with the main objective being the study of the effects of increasing concentrations of atmospheric CO2 (and associated effects such as increased acidification) on blooms of natural marine coastal plankton. Three mesocosms were bubbled with CO2(g) to achieve a high (~700ppm) CO2 concentration (pH ~7.8) to simulate predicted future conditions as a result of rising atmospheric CO2 concentrations. Another three mesocosms were treated as controls and bubbled with ambient air to represent a near pre-industrial scenario (atmospheric CO2 concentration ~300ppm, surface seawater pH ~8.15). Blooms in the mesocosms were stimulated by the addition of nutrients at a near-Redfield ratio ([N:P] ≈ [16:1]), and scientific measurements and analyses were carried out over the course of the blooms for approximately one month. Of particular interest in this study were the autotrophic plankton. The diversity and activities of these microorganisms under the two treatments was therefore investigated. By designing and using new degenerate primers specifically targeting ‘Green-type’ (Form IA and IB), ‘Red-type’ (Form IC and ID) and Form II RuBisCO, analysis of primary producers was carried out using PCR and either gDNA or cDNA (mRNA) templates from key time points spanning the complete duration of the blooms throughout the mesocosm experiment. Over 1250 novel RuBisCO large subunit sequences have been fully annotated and deposited in the NCBI GenBank® database. These sequences revealed distinct changes in the diversity of primary producers both over the courses of the blooms and between treatments. Particularly striking was the effect of acidification on the community structure of the eukaryotic picoplankton, Prasinophytes. A clade of prasinophytes closely related to Micromonas pusilla showed a distinct preference for the high CO2 conditions; a laboratory-based experiment confirmed the high tolerance of Micromonas pusilla to lower pH. Conversely, a clade related to Bathycoccus prasinos was almost entirely excluded from the high CO2 treatments. Clades of form II RuBisCO-containing dinoflagellates were also abundant throughout the experiment in both treatments. The high similarity of some of these clades to the toxin-producing species Heterocapsa triquetra and Gonyaulax polyedra, and apparent high tolerance of some clades to high CO2 conditions, is perhaps cause for concern in a high CO2 world and demands further research. In parallel with the RubisCO work, new primers were designed that target the gene encoding the Fe protein of nitrogenase (NifH). 82 Bergen genomic nifH sequences have been annotated and submitted to GenBank®. These sequences include those from organisms related to Alpha, Beta, and Gammaproteobacteria, and Cluster II and Cluster III sequences that align most closely with anaerobic Bacteria, Gram positive, and/or sulphur-reducing Bacteria. The biggest surprise, however, was the apparent abundance and significance of a Rhodobacter sphaeroides-like microorganism throughout the duration of the experiment in both treatments. Whilst this clade was unsurprisingly absent in the RuBisCO cDNA libraries, all but two of 128 nifH cDNA clones analysed were identical to the gene from Rhodobacter sphaeroides. This shows that this clade was potentially fixing N2 throughout the entire experiment, even in the presence of combined N added to both sets of mesocosms at the start of the experiment. A group of Rhodobacter sphaeroides-like microorganisms present at Bergen may therefore have been an unexpected source of new N during the experiment and contributed to the maintenance of the mesocosm communities as nutrients became depleted. One organism dominated the autotrophic communities after the blooms in both treatments. Synechococcus spp. Form IA rbcL clones most closely related to the coastal strain Synechococcus sp. strain CC9902 were recovered throughout the experiment but were particularly numerous toward the end of the experiment and dominated the “Green-type” libraries at this time. Initially, rbcL clones from these cyanobacteria were mostly derived from the ambient CO2 mesocosms but were equally distributed between treatments by the end of the experiment. This suggests that cyanobacteria related to strain CC9902 may be less tolerant of elevated CO2 (which was greatest at the beginning rather than the end of the experiment). However, despite the mesocosms being Pi-limited at the end of the experiment, several Synechococcus species (including those related to strain CC9902 and another coastal strain, CC9311) thrived. Following on from this observation, Pi uptake and assimilation mechanisms in a Synechococcus species were investigated in the laboratory. This led to the sequencing and characterisation of a pstS gene from the marine cyanobacterium Synechococcus sp. WH 8103. Unlike conventional pstS, it was discovered that the pstS II gene in this organism is constitutively expressed and unresponsive to or only weakly regulated by Pi supply. The use of PstS/pstS as a marker for P-limitation in natural samples, therefore, should be interpreted with caution.