Cultivation of bacterioplankton from an oligotrophic freshwater lake using a dillution-to-extinction approach

초록

Although the importance of bacterioplankton in freshwater ecosystem is well recognized, still the major groups of freshwater bacteria playing dominant roles in biogeochemical processes remain uncultivated; these freshwater bacterial clades include the LD12 of the Alphaproteobacteria, LD28 of the Betaproteobacteria, acI of the Actinobacteria, several uncultured bacterial clades such as the OPB35 clade of the Verrucomicrobia and subdivision 3 of the Acidobacteria. In an effort to cultivate the uncultured yet abundant bacterial populations present in freshwater system, we report the application of dilution-to-extinction culturing to freshwater bacterioplankton community in this study. A high throughput cultivation approach based on dilution-to-extinction culturing has shown to be a great potential to isolate uncultured oligotrophic marine bacteria. The water of Soyang Lake, an oligotrophic lake, was examined using dilution-to-extinction culturing in small volumes of very low nutrient-amended lake water as culture media. Of the 313 cultivated strains grown from 720 multi-wells, 73 formed colonies onto 1/3R2A and/or 1/10R2A agar plates. Most of the extinction cultures belonged to the Betaproteobacteria and Alphaproeteobacteria, while very small number of isolates affiliated with the Actinobacteria and Gammaproteobacteria were retrieved. All of the colony formers belonged to the Alphaproteobacteria and Betaproteobacteria, while isolates of the Actinobacteria, Bacteroidetes and Gammaproteobacteria did not form colonies. Interestingly, we could successfully isolate several major freshwater bacterial groups, particularly acI of the Actinobacteria; Limnohabitans and Polynucleobacter of the Betaproteobacteria. Although two strains belonging to the acI clade of the Actinobacteria were found to grow up to ~106 cells/ml in the initial extinction-culturing wells, their viability decreased continuously in the following successive culturing procedures, resulting in the failure of re