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Culturability and coexistence of bacterioplankton exhibiting different life strategies were investigated in the Baltic Sea and Skagerrak Sea. Bacterial numbers were estimated using a dilution-to-extinction culturing assay (DCA) and calculated as the most probable number, based on six different methods to detect bacterial growth in the DCA. Irrespective of the method used to detect growth, the fraction of multiplying cells never exceeded 10%, using the total count of 4′,6′-diamidino-2-phenylindole (DAPI)-stainable cells as a reference. Furthermore, the data also showed that non-colony-forming bacteria made up the majority of the viable cells, confirming molecular results showing dominance of non-colony-forming bacteria in clone libraries. The results obtained are in agreement with previous observations, indicating that bacterial assemblages in seawater are dominated by small, active subpopulations coexisting with a large group of inactive cells. The ratio of colony-forming to non-colony-forming bacteria was approximately 10 to 20 times higher in the brackish Baltic Sea than in the Skagerrak Sea. These two sea areas differ in (for example) their levels of bacterial production, dissolved organic carbon, and salinity. We suggest that the relative importance of colony-forming versus non-colony-forming bacterioplankton may be linked to environmental characteristics.

The coupling between the presence of predominant bacterial species and particular biogeochemical processes is of primary interest in current microbial ecology. Molecular methods such as microarrays and real-time polymerase chain reaction (PCR) may be used to estimate presence or expression of different genes (e.g., 16S rDNA and nifH) in environmental samples; however, to be quantitative, these methods require a reproducible and efficient DNA extraction protocol. Using picogreen DNA quantification, real-time PCR, and an internal DNA standard, we step-wise examined and optimized a protocol for DNA extraction from centrifuged or filtered seawater samples (vol. 2 to 300 mL). Sample volume had a pronounced effect on DNA extraction efficiency showing that comparison of samples with different volumes is problematic. The duration of enzyme treatment (lysozyme and proteinase K) significantly influenced the DNA extraction efficiency. Dissolved DNA contributed significantly to total DNA when extracted from small filtering volumes (

The Baltic Sea is one of the largest brackish environments on Earth. Despite extensive knowledge about food web interactions and pelagic ecosystem functioning, information about the bacterial community composition in the Baltic Sea is scarce. We hypothesized that due to the eutrophic low-salinity environment and the long water residence time (>5 years), the bacterioplankton community from the Baltic proper shows a native “brackish” composition influenced by both freshwater and marine phylotypes. The bacterial community composition in surface water (3-m depth) was examined at a single station throughout a full year. Denaturing gradient gel electrophoresis (DGGE) showed that the community composition changed over the year. Further, it indicated that at the four extensive samplings (16S rRNA gene clone libraries and bacterial isolates from low- and high-nutrient agar plates and seawater cultures), different bacterial assemblages associated with different environmental conditions were present. Overall, the sequencing of 26 DGGE bands, 160 clones, 209 plate isolates, and 9 dilution culture isolates showed that the bacterial assemblage in surface waters of the central Baltic Sea was dominated by Bacteroidetes but exhibited a pronounced influence of typical freshwater phylogenetic groups within Actinobacteria , Verrucomicrobia , and Betaproteobacteria and a lack of typical marine taxa. This first comprehensive analysis of bacterial community composition in the central Baltic Sea points to the existence of an autochthonous estuarine community uniquely adapted to the environmental conditions prevailing in this brackish environment.

Formation of a biofilm or colony by bacteria is a behavior that allows prompt utilization of the substrate. The combined exoenzymatic action of a growing colony provides benefits to the individual cells when there is a rich food supply. Alternatively, an oligotrophic bacterioplankton growing slowly in a nutrient-poor environment may have a life strategy in which dispersal is promoted to optimize cell access to substrates. In the latter scenario the formation of colonies is probably not adaptive. These two different life strategies may explain the thousand-fold difference between the total number of bacteria and the number of CFU found in typical seawater samples. Over the years the discrepancy in these numbers has puzzled many authors (13). Clearly, dead and dying bacterial cells cannot form colonies; thus, the effects of virus infection and nutrient starvation have been inferred to be reasons for low viable counts (14, 19, 24). Furthermore, different states of bacterial cell physiology, such as dormancy and the so-called viable but not culturable state, have been suggested to be explanations for the lack of growth on solid media (20, 26). However, the behaviors of bacteria with different life strategies have not been considered in relation to the formation of colonies. In this study we used the dilution-to-extinction technique (6) to isolate bacterial strains unable to grow and form colonies on conventional culture media. In an attempt to understand this inability to grow and form colonies, we explored the growth behavior of three marine bacterioplankton species by using a novel assay. Bacteria were grown on seawater agar slides containing a fluorescent stain, which allowed direct observation by epifluorescence microscopy of living cells.