Summary of Three Years of High Throughput Cultivation in the Pacific and Atlantic Ocean

초록

Here we report the comprehensive results of high throughput culturing (HTC) of marine prokaryotes from different seawaters. To cultivate novel uncultured marine microoorganisms, HTC methods based on dilution-to-extinction in oligotrophic seawater media were developed. One to five cells were inoculated to each well and incubated at 16? for three weeks. The microbial growth was checked by epifluorescence microscopy using a custom-made 48-well blotter. Approximately 4,000 individual wells had been examined during the course of three years. Genomic DNA was extracted from only 200 ? of cultures and residual cultures were stored in liquid nitrogen. The 16S rDNA sequences obtained were aligned in the ARB database and phylogenetic analyses were carried out. Up to 15% of cells from coastal and pelagic seawaters were cultured using this method. Among the cultured marine bacteria are many unique phylogenetic lineages that have been classified a new phylum, orders, families, and genera. Representatives belonging to cosmopolitan and previously uncultured bacterial lineages, including SAR11, OM60, OMG, RCA, OM43, and NAC, were successfully recovered from the study. Many of isolates cultured in this method however could not grow on standard marine agar. Eighteen genomes of novel isolates are now being fully sequenced by Craig Venter Institute funded by Moore Foundation.