HIGH THROUGHPUT CULTIVATION OF MAJOR MARINE BACTERIAL LINEAGES FROM THE EAST SEA USING VARIOUS CULTURE CONDITIONS

초록

High-throughput cultivation based on dilution-to-extinction method has been successfully used to cultivate marine uncultured bacterial lineages from Oregon coast and the Sargasso Sea. In this study, we applied a modified version of high-throughput cultivation to cultivate major uncultured marine bacterial lineages in the East Sea for one year. One to 10 cells from sampled seawater were inoculated into each well of 48-well plates, which contained low-nutrient heterotrophic media prepared by adding diverse combinations of organic compounds to filtered seawater. After 4 to 8 weeks of incubation at 10-15°, microbial growth was screened by using epifluorescence microscope and flow cytometer. Positive growth was detected in 1446 wells among 7872 inoculated wells and 325 colony-forming isolates were obtained on agar plates by transferring extinction cultures. Based on phylogenetic analyses of 16S rRNA gene sequences from pure isolates, cultured strains were generally assigned to the SAR11 groups 1 and 3, Roseobacter clade and SAR92 group. The isolates belonging to the OM42, OM60, OM182, K189A clades and the phylum Lentisphaerea were also recovered. Interestingly many strains were affiliated to genera of Fucophilus, Coraliomargarita and Pelagicoccus in the phylum Verrucomicrobia. This study suggests that efficient cultivation of novel members of major marine bacterial groups could be facilitated by a long-term approach using various culture conditions mimicking natural environments.