Culturing the ‘Uncultured’ Bacteria by Dilution-to-Extinction Method in the East Sea and Study on the Diversity of Bacterial Community Using Next Generation Sequencing

초록

High-throughput cultivation (HTC) based on dilution-to-extinction has been successful in the cultivation of abundant bacterial groups in the ocean. However, previous studies have been limited in the number of samples and culture conditions. In this study, we performed HTC for a multitude of water samples collected from the East Sea using various culture conditions, resulting in the isolation of 1,457 pure cultures (700 colony-formers) from 17,280 inoculated wells. Phylogenetic analyses showed that cultured bacteria included major marine bacterial lineages, such as SAR11, Roseobacter clade, OM43, oligotrophic marine Gammaproteobacteria. A large number of verrucomicrobial strains of the class Opitutae was also isolated in late summer when a massive proliferation of Verrucomicrobia was found by pyrosequencing of seawater sample. In addition, pyrosequencing-based bacterial community analyses of seawater samples and HTC extinction cultures implicated that several abundant bacterial groups, including SAR86, OM1, and SAR406, might have grown in our culture conditions, even though they were not purely isolated. Diverse bacterial strains isolated in this study would be useful resources for future ‘omics’-based studies, enhancing the understanding about the roles of bacterioplankton in marine environments.