Dilution-to-Extinction Culturing under Anaerobic Conditions Yielded Diverse Previously- uncultured Bacterial Isolates from Ganghwa Tidal Flat

초록

Tidal flats are important ecosystems where a large amount of organic matter is cycled through various biogeochemical processes. Sulfate-reducing bacteria is one of the major microbial groups that contribute up to > 30% of microbial assemblages in anaerobic zone of tidal flats. In this study, high-throughput dilution-to-extinction culturing (HTC) was conducted twice using sediment samples collected from a tidal flat in Ganghwa Island. Sediment samples obtained at a depth of 15 cm were homogenized with sterilized seawater. Following the determination of cell counts by flow cytometry, cells were diluted with liquid media and dispensed into multi-well plates. Two kinds of liquid media (each based on natural or artificial seawater) were used for HTC. Sulfate (20 mM) was added to artificial seawater as an electron acceptor and organic acids (such as pyruvate, lactate, and acetate) were added to both media as electron donors. After a month of anaerobic incubation, microbial growth was detected in approximately 33.1% of 3,456 inoculated wells. Phylogenetic analyses of 16S rRNA genes showed that many cultured isolates were affiliated with several previously-uncultured marine anaerobic bacterial groups, such as Sva1033 clade of Desulfuromonadales, BD2-2 clade of Bacteroidales, IheB3-7 clade of Ignavibacteriales, and R76- B128 clade of Kiritimatiellae. This study suggests that more diverse anaerobic bacterial groups could be isolated by the application of dilution-to-extinction culturing.