Abstract
While marine microorganisms are frequently studied in their natural environment, isolated strains are invaluable resources that can be used in controlled experiments to expand upon direct observations from natural systems. Here, we sought a means to enhance culture collections of SAR11 marine bacteria by testing the use of seawater cryopreserved with glycerol as an inoculum. Using a raw seawater sample collected from the tropical Pacific Ocean, a subsample was diluted in seawater growth medium to create 576 2-ml dilution cultures containing 5 cells each and incubated for a high-throughput culturing (HTC) experiment, while another portion was cryopreserved in 10% glycerol. After 10 months, a cryopreserved aliquot was thawed and used to create a second cultivation experiment of 480 2-ml cultures containing 5 cells each and 470 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 the raw seawater inoculum and 74 SAR11 isolates spanning 13 unique ASVs from cryopreserved seawater. A vast majority (92%) of SAR11 isolates from the two HTC experiments were members of SAR11 subclade Ia, though 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 environmental sample were isolated by both approaches.IMPORTANCE High-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 them 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 an 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 (i) expand the potential of high-throughput cultivation experiments to include testing when immediate isolation experiments are impractical, (ii) allow for targeted isolation experiments from specific samples based on analyses such as microbial community structure, and (iii) enable cultivation experiments across a wide range of other conditions that would benefit from having source inocula available over extended periods of time.
Highlights
While marine microorganisms are frequently studied in their natural environment, isolated strains are invaluable resources that can be used in controlled experiments to expand upon direct observations from natural systems
Using seawater sampled outside KaneÔohe Bay on the island of OÔahu, HawaiÔi (Fig. 1), two high-throughput cultivation experiments were conducted: one that used fresh seawater as an inoculum, labeled HTC2017, and one that used a cryopreserved sample of the same seawater ;10 months later (HTC2018) (Fig. 2)
The primary goal of this study was to test the hypothesis that SAR11 marine bacteria can be isolated from cryogenically preserved seawater
Summary
While marine microorganisms are frequently studied in their natural environment, isolated strains are invaluable resources that can be used in controlled experiments to expand upon direct observations from natural systems. IMPORTANCE High-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. Several different isolation methods and strategies have been developed in order to coax recalcitrant environmental microorganisms into laboratory culture (see, e.g., references 12 to 15) Among these novel approaches is an isolation technique based on the dilution-to-extinction culturing methodology first developed by Button and colleagues [16]. For over a decade since its initial discovery in 1990, the marine planktonic bacterial lineage known as SAR11 served as a notorious example of an abundant and widespread microorganism in nature that was recalcitrant to cultivation as an isolated strain in a controlled laboratory setting [21, 22]. While five of these subclades contain cultivated representatives (Ia, Ib, IIa, IIIa, and Va), the vast majority of strains are concentrated within subclade Ia and leave much of the genetic diversity of SAR11 lacking cultivated models
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