Isolation of SAR11 marine bacteria from cryopreserved seawater

Abstract

While marine microorganisms are frequently studied in their natural environment, laboratory cultures of isolated strains are invaluable resources that can be used in controlled experiments to confirm and expand upon direct observations from natural systems. Here, 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 for a high-throughput dilution culture experiment. In July 2017, raw seawater was collected outside of Kāneʻohe Bay, Hawaiʻi, in the tropical Pacific Ocean. A portion of this sample was diluted in sterile seawater-based growth medium to create 576 × 2 mL dilution cultures containing 5 cells each and incubated for a high-throughput cultivation experiment, while another portion was cryopreserved in 10% glycerol and stored at -80 ºC. After ten months, a 1.5 mL cryopreserved aliquot of seawater was thawed, diluted in sterile seawater-based growth medium, and distributed to create a second high-throughput cultivation experiment of 480 × 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. This approach should allow for the isolation of marine bacteria, including SAR11, via high-throughput cultivation from wherever in the global ocean that seawater can be collected and cryopreserved.