Isolation of bacterial strains from water samples collected nearby the Saemangeum Seawall by high-throughput-culturing method based on cell-sorter inoculation

초록

High-Throughput-Culturing (HTC) based on dilution-to-extinction has been proven to be highly e퍼cient in isolating representative bacterial groups. In this study, HTC based on cell-sorter inoculation was applied to water samples collected inside and outside of the Saemangeum Seawall to isolate novel bacterial strains and to compare seawater and brackisb water bacterioplankton. 8rackish water samples inside of tbe seawall were collected in September 2015 and Apri12016, and a seawater sample was collected in April 2016 from the outside of the seawall. Cells in water samples were sorted into 3 or 9 fractions using a FACS instrument based on forward and side scatter, and were inoculated directly from the nozzle into low nutrient media aliquoted into multiwall plates, witb a final cell density of 1 or 3 cells per well. The number of inoculation wells were 864 per each sample. Culture plates were incubated at 18- 200C for 7 weeks, and microbial growth was measured using a flow cytometer Proportion of growth-positive (즈 105 cells/ml) wells ranged between 10-23% Pbylogenetic analyses of 16S rRNA genes amplified from growtb-positive wells showed tbat seawater isolates were distributed in tbe diverse phyla/classes such as Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Flavobacteriia and Verrucomicrobia. 8rackish water isolates were also affiliated with diverse phyla/classes sucb as Aψhaproteobacteria ， Betaproteobacteria, Gammaproteobacteria, Bacteroidetes, Planctomycetes, Actinobacteria and Verrucomicrobia, and included bacterial groups representing botb marine and freshwater babitats. Particularly, a previously uncultured subgroup of marine SARll c1ade was isolated. These results sbowed that diverse marine and freshwater bacteria can be isolated from aquatic ecosystem by using HTC based on cell-sorter inoculation.