Spatial and temporal analysis of estuarine bacterioneuston and bacterioplankton using culture-dependent and culture-independent methodologies

Abstract

Bacterioneuston may play a key role in water-air exchange of gases and in processing organic matter and pollutants that accumulate at the sea-surface microlayer (SML). However, the phylogenetic diversity of bacterioneuston has been poorly characterized. We analyzed 24 samples each from the SML and underlying water (UW) at three sites in the Ria de Aveiro estuary, Portugal. Cultivation and culture-independent techniques were used to compare bacterioneuston and bacterioplankton. Culturable heterotrophic bacteria were enriched in the SML. The culturable community was dominated by Psychrobacter and Acinetobacter. The presence of high numbers of Psychrobacter was a notable result. Differences were confined to a few genera overrepresented in UW samples (Kocuria, Agrococcus and Vibrio). 16S rDNA DGGE profiles were highly stable in terms of number and position of bands between sampling sites and dates but cluster analysis revealed a slight tendency for grouping according to sampled layer. SML-specific DGGE bands affiliated with Actinobacteria, Cyanobacteria, Gammaproteobacteria and Bacteroidetes. Low similarity between nucleotide sequences of DGGE-bands and previously reported sequences suggest the occurrence of SML-specific populations. Enrichment of SML for Pseudomonas and Aeromonas was questioned and the diversity of both communities was analyzed. Consistent differences between SML and UW aeromonads communities were not identified. In terms of Pseudomonas, a culturable operational taxonomic unit was consistently overrepresented within SML samples. Taken together, our results indicate that the similarity between SML and UW communities depends on spatial and temporal factors.

Molecular analysis of the diversity of genusPsychrobacterpresent within a temperate estuary

“…Psychrobacter isolates represented almost 25% of a culture collection of heterotrophic bacteria previously obtained from Ria de Aveiro (Azevedo et al ., ). In the present study, these isolates ( n = 81) were categorized into nine OTUs based on an identity criteria of 99% of the 16S rRNA gene sequence (Table ).…”

“…Samples were collected at three sites: Cais do Chegado (CC), where the main contamination sources are industrial effluents; Costa Nova (CN), mainly impacted by urban effluents, aquacultures and run‐off from agriculture fields; and Cais do Sporting (CS), subjected to anthropogenic pressure mainly due to the presence of harbour facilities. Water samples were retrieved as previously described (Azevedo et al ., ). Briefly, samples from SML were collected by adherence to glass and acrylic plates, and UW samples were collected by submerging a sterilized brown glass bottle and opening it at a depth of c .…”

“…Isolates were obtained as described by Azevedo et al . (). In summary, isolates were retrieved from estuarine agar (EA; Weiner et al ., ) and glutamate starch phenol red agar (GSP; Pseudomonas / Aeromonas selective agar) plates.…”

“…In summary, isolates were retrieved from estuarine agar (EA; Weiner et al ., ) and glutamate starch phenol red agar (GSP; Pseudomonas / Aeromonas selective agar) plates. DNA was purified as previously described (Azevedo et al ., ). Eighty‐one isolates (42 from SML and 39 from UW samples) were affiliated to Psychrobacter by sequencing the 16S rRNA gene.…”

“…During a previous study based on culture‐dependent methods, the genus Psychrobacter was, unexpectedly, the most frequently retrieved genus from the sea‐surface microlayer (SML) and the underlying water (UW) of a temperate estuary (Ria de Aveiro, Portugal) during a warm season (May–July; Azevedo et al ., ). SML refers to the thin (1–1000 μm) film that forms between the hydrosphere and atmosphere (Azevedo et al ., ).…”