Phylogenetic and functional diversity of estuarine bacterioneuston

The water surface microlayer (SML) is characterized by the occurrence of high amounts of organic compounds, the accumulation of anthropogenic contaminants and is subjected to intensive solar radiation, extreme temperature changes and, in the case of estuaries, salinity fluctuations. These particular properties are probably modulating a bacterial community (bacterioneuston) with specific functional and phylogenetic properties. In this study, culture-dependent and culture-independent approaches were applied to evaluate the structure and dynamics of the bacterioneuston and bacterioplankton communities in three geographic locations along the Ria de Aveiro estuary. Additionally, we compared the phylogenetic diversity of specific groups (Aeromonas, Pseudomonas and Psychrobacter) present in bacterioneuston and bacterioplankton. Finally, both communities were compared in terms of prevalence and diversity of antibiotic resistant bacteria and resistance genes. Cultivable heterotrophic bacteria were enriched in the SML. Denaturing gradient gel electrophoresis (DGGE) allowed identifying SML-specific phylotypes. Also, cluster analysis of DGGE profiles from both UW and SML revealed a slight tendency for grouping according to sampled layer. Differences between both communities varied according to spatial and temporal factors. In terms of phylogenetic diversity of specific groups, consistent differences between SML and UW aeromonads communities were not identified. Regarding Pseudomonas, a cultivable operational taxonomic unit was consistently overrepresented in SML samples. Culture-dependent and cultureindependent methodologies revealed the presence of complex and very stable Psychrobacter populations in all sampling sites and dates, with significant differences between SML and UW Psychrobacter communities Strains representing putative new species of Psychrobacter were cultivated. In terms of antibiotic resistance, the prevalence of antibiotic-resistant bacteria was higher in the SML suggesting selection by SML conditions. It has to be emphasized that antibiotic resistance was uncommon among estuarine bacteria and the resistance mechanisms were predominantly intrinsic. Differences between bacterioneuston and bacterioplankton in Ria de Aveiro were detected at different levels: the structure of the total bacterial community, the diversity of specific groups, and in the ability to resist to antibiotics. By the innovative combination of culture-dependent and independent approaches we provide new and consistent information regarding the differentiation of both bacterial communities and some of the factors that contribute for their shaping. List of original publications This thesis includes results published in the articles listed below. Additionally, some unpublished results are presented. Azevedo JSN, Ramos I, Araújo S, Oliveira CS, Correia A, Henriques I (2012) Spatial and temporal analysis of estuarine bacterioneuston and bacterioplankton using culturedependent and culture-independent methodologies. Antonie van Leeuwenhoek, 101, 819 – 835. – Results presented in Chapter 3. Azevedo JSN, Correia A, Henriques I (2012) Molecular analysis of the diversity of genus Psychrobacter present within a temperate estuary. FEMS Microbiology Ecology (in revision). – Results presented in Chapter 4. Azevedo JSN, Araújo S, Oliveira CS, Correia A, Henriques I (2012) Analysis of antibiotic resistance in bacteria isolated from the surface microlayer and underlying water of an estuarine environment. Microbial Drug Resistance (accepted for publication). – Results presented in Chapter 5.