Isolation of Brackish Water Bacterioplankton from Saemangeum by High-Throughput-Culturing Method Based on Cell-sorter Inoculation

초록

Cultivation of aquatic bacteria is necessary to understand the physiology and ecological importance of diverse bacterial groups. High-Throughput- Culturing (HTC) based on dilution-to-extinction has been successfully applied to isolate representative bacterial groups. In this study, we applied HTC to a brackish water sample collected from the just inside of the Saemangeum reclamation wall. Inoculation of bacteria was performed using a FACS instrument. Bacterial cells were sorted into 9 fractions according to forward and side scatter, and were inoculated directly from the nozzle into low nutrient media, with a final cell density of 3 cells per well. After 7 weeks of incubation at 20°C, microbial growth was detected in approximately 10% of 864 inoculated wells. Phylogenetic analyses of 16S rRNA genes showed that FACS-HTC isolates were distributed in the diverse phyla/classes such as Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacteroidetes, Planctomycetes, Actinobacteria and Verrucomicrobia, and included major bacterial groups representing either marine or freshwater habitats. Particularly, an uncultured subgroup of marine SAR11 clade was isolated. These results showed successful cultivation of previously uncultured marine and freshwater bacterial groups from brackish environment by application of HTC adopting the FACS inoculation. [This work was supported by a grant from the Marine Biotechnology Program (PJT200620) funded by the MOF, Korea.