Cultivation of Anaerobic Bacteria from Tidal Flat Sediment Using a Dilution-to-extinction Approach

초록

Tidal flats are important ecosystems where a large amount of organic matter is cycled through various biogeochemical processes. Microorganisms equipped with diverse metabolic potentials play a major role in shaping material cycles in tidal flats. Sulfate-reducing bacteria (SRB) is one of major tidal flat microbial groups that can contribute up to >30% of microbial assemblages in anaerobic zone of tidal flats. In this study, we performed high-throughput dilution-to-extinction culturing (HTC) for sediment samples collected from a tidal flat in Ganghwa Island, South Korea, to cultivate novel anaerobic microorganisms with a focus on SRB. 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 (25 cells/well). The two liquid media (each based on natural or artificial seawater) contained >20 mM of sulfate as electron acceptor and organic acids (such as pyruvate, lactate, and acetate) as electron donors. After a month of incubation under anaerobic condition, microbial growth was detected in approximately 51.1% of 1,152 inoculated wells. Phylogenetic analyses of 16S rRNA genes showed that a total of 271 putatively pure HTC isolates were distributed among diverse phylogenetic groups such as the Bacteroidetes, Deltaproteobacteria, and Chlorobi. Interestingly, a number of cultured isolates were affiliated with several previously-uncultured marine anaerobic bacterial groups, such as Sva1033 clade of Desulfuromonadales, BD2-2 clade of Bacteroidales, and IheB3-7 clade of Ignavibacteriales. These results suggests that more diverse anaerobic sediment bacterial groups could be successfully isolated by the application of dilution-to-extinction culturing.