Isolation of SAR11 marine bacteria from cryopreserved seawater

Abstract

In this study, we sought a means to increase current culture collections of SAR11 marine bacteria by testing the use of seawater cryopreserved with glycerol as an inoculum. In July 2017, raw seawater was collected outside of K[a]ne ohe Bay, Hawai i, in the tropical Pacific Ocean. A portion of this sample was diluted in seawater-based growth medium to create 576 x 2 mL dilution cultures containing 5 cells each and incubated for a high-throughput cultivation experiment, while another portion was cryopreserved in 10% glycerol. After ten months, a cryopreserved aliquot of seawater was thawed, diluted in seawater-based growth medium, and distributed to create a second high-throughput cultivation experiment of 480 x 2 mL dilution cultures containing 5 cells each and 94 cultures containing 105 cells each. The raw seawater cultivation experiment resulted in the successful isolation of 54 monocultures and 29 mixed-cultures, while cryopreserved seawater resulted in 59 monocultures and 29 mixed cultures. Combined, the cultures included 51 SAR11 isolates spanning 11 unique 16S rRNA gene amplicon sequence variants (ASVs) from raw seawater inoculum and 74 SAR11 isolates spanning 13 unique ASVs from cryopreserved seawater. A vast majority (115 of 125) of SAR11 isolates from the two HTC experiments were members of SAR11 subclade Ia, though isolates of subclades IIIa and Va were also recovered from cryopreserved seawater and subclade Ib was recovered from both. The four most abundant SAR11 subclade Ia ASVs found in the initial seawater sample used to create both culture experiments were isolated by both approaches. ImportanceHigh-throughput dilution culture has proved to be a successful approach to bring some difficult-to-isolate planktonic microorganisms into culture, including the highly abundant SAR11 lineage of marine bacteria. While the long-term preservation of bacterial isolates by freezing in the presence of cryoprotectants such as glycerol has been shown to be an effective method of storing viable cells over long time periods (i.e. years), to our knowledge it had not previously been tested for its efficacy in preserving raw seawater for later use as inoculum for high-throughput cultivation experiments. We found that SAR11 and other abundant marine bacteria could be isolated from seawater that was previously cryopreserved for nearly 10 months, at a rate of culturability similar to that of the same seawater used fresh, immediately after collection. Our findings expand the potential of high-throughput cultivation experiments to include opportunities where immediate isolation experiments are impractical, allow for targeted isolation experiments from specific samples based on analyses such as microbial community structure, and enable cultivation experiments across a wide range of other conditions that would benefit from having source inoculum available over extended periods of time.