Isolation of Microbes from Lake Vostok Accretion Ice

Bacteria from seven Lake Vostok accretion and two deep glacialVostok ice core sections were characterized. The cell concentrationswere low, but many of the cells were viable. From the hundredsof cultures, 18 unique bacterial rRNA gene phylotypes were determined.Lake Vostok may contain a complex microbial ecosystem.

INTRODUCTION

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INTRODUCTION

Subglacial Lake Vostok, the eighth largest lake on Earth (area= 14,000 km², volume = 5,600 km³) (9, 16), is covered by a 4-kilometer-thicklayer of glacial ice. As the glacier traverses the lake overa period of 18,000 years, ice freezes (or accretes) to the bottomsurface of the glacier, eventually forming a 200-m layer ofaccretion ice that has retained a linear and temporal recordof the contents of the upper surface of the lake. The glacierpasses over a shallow embayment, near an island (or peninsula),and then over part of the main lake basin. As the glacier passesthrough the embayment, initially it is grounded on the lakebed,and partly because of this, it collects mineral inclusions,making the ice silty (termed type I accretion ice) (13). Meltingand freezing in this area, as well as a possible influx of materialfrom a river system and/or from hydrothermal activity, may contributeto the characteristics of the type I ice (3, 14). The glacieris suspended over open water in portions of the embayment andover most of the main parts of the lake. The ice that formsover open water contains far fewer inclusions and lower concentrationsof ions, organic carbon, and biomass (6, 10, 14). This ice isvery clear ice and has been termed type II accretion ice (13).The top section (from 3,538 to 3,595 m), which accreted withinand near the embayment, primarily consists of type I ice, althoughthere are some regions of type II ice (2). Bacteria from thisice, including potentially psychrophilic and psychrotolerantspecies as well as the molecular signature of a thermophilicbacterium, have been reported (1, 3, 4, 5, 7, 10). Within thelake, temperatures average –2°C, pressures approach400 atmospheres, high oxygen levels exist, there are low nutrientlevels, and it is completely dark.

We isolated and characterized microbes from Lake Vostok typeI and II accretion ice from the embayment and the main basinas well as from glacial ice immediately above the accretionice layers. Sequence results from the rRNA small subunit genesand internal transcribed spacers indicate that at least 18 speciesare represented in the accretion ice. All are psychrotolerantin that they grew at 4°C, although optimal growth was oftenat higher temperatures.

Descriptions and cell concentrations.

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Descriptions and cell...

Nine Vostok 5G ice core sections were assayed. Five sectionsrepresented ice that accreted over the shallow embayment (depthsof 3,540, 3,563, 3,582, and 3,584 m, all type I ice, and 3,591m, type II ice) (2) approximately 3,800 to 5,100 years ago (2,6, 13, 14), two accreted 3,400 to 3,500 years ago over the mainlake basin (3,606 m, type I ice, and 3,610 m, type II ice),and two were glacial ice cores near the bottom of the glacier(3,501 and 3,520 m, approximately 1 to 2 million years old)(13, 14). The surfaces of all of the ice core sections weredecontaminated prior to melting, as described previously (12).Ice core meltwater initially was analyzed using a live/deadstain (BacLight viability kit; Molecular Probes, Eugene, OR)to count cells from 10 1-ml samples for each core section byusing fluorescence microscopy. The concentrations (means ±standard deviations) of viable and nonviable cells ranged from2.33 ± 0.29 to 12.33 ± 9.58 cells/ml (Fig. 1).Considering the partition coefficient (0.56) (6), for ice versuswater, the concentrations in the lake are approximately 1.78times higher than these values (4.15 to 21.95 cells/ml). Thenumber of viable cells in each ice core section varied fromnearly 0 to a mean of 6.56 cells/ml (Fig. 1). The mean concentrationsof nonviable cells were from 1.28 to 5.58 cells/ml. The concentrationsof viable cells in glacial ice primarily were lower, between1.00 and 2.00 cells/ml. This is expected, given the fact thatthe glacial ice that was examined was between 1 and 2 millionyears old, while the accretion ice was only 3,400 to 5,100 yearsold (6, 13, 14).

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FIG. 1. Cell concentrations (upper panel), colonies of viable cells, and numbers of unique sequences (lower panel) in the ice core sections. Cell concentrations were based on cell counts of fluorescently stained cells visualized via microscopy from 10 ml of meltwater from two glacial and seven accretion ice core sections. The graph also shows the region of the lake that is represented in each ice core section. The highest concentrations of cells, viable cells, cultures, and unique sequences are in the areas near the transitions between type I and type II accretion ice.

A 5-ml sample from each core section also was examined by scanningelectron microscopy (SEM) (Fig. 2). After filtration, the filterwas fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer(pH 7.2), rinsed, dehydrated with ethanol, dried (Samdri 780Acritical point dryer), sputter coated with gold-palladium (PolaronE500 SEM coating unit), and viewed using an SEM (Hitachi S-2700).Many of the cells in the glacial ice were distorted (Fig. 2),which is consistent with the low cell viabilities indicatedby the fluorescence microscopy assays (Fig. 2). Cells in theshallow embayment type I ice also exhibited damage. However,cells in type II ice of the embayment exhibited lower levelsof distorted cells, and the diversity of cell shapes was higher.The same pattern was observed for type I and type II ice fromthe main lake basin, but the numbers of cells and the levelof cell diversity were lower than in the ice from the embayment.

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FIG. 2. Fluorescence and scanning electron micrographs of cells found in the glacial (3,501 and 3,520 m) and accretion ice (3,540, 3,563, 3,582, 3,584, 3,591, 3,606, and 3,610 m) core sections. Cells in the fluorographs were stained with BacLight (Molecular Probes, Eugene, OR). Green fluorescence indicates possible viable cells, based on membrane integrity. Red fluorescence indicates possible dead cells based on intracellular staining. Bars represent 5 µm on fluorographs and 1 µm on electron micrographs. 3501, cells characteristic of eubacteria, possibly eukaryotes (in one case, middle right); 3520, probably bacteria (upper two cells; upper right potentially viable), germinating spore (lower left; bacterial or fungal), and unknown (lower right); 3540, all apparently coccoid bacteria; 3563, all apparently bacteria (all nonviable); 3582, filamentous bacteria or fungi (upper portion; some structures may be oogonia [or similar structures]), coccoid bacteria (middle), and a mix of Bacillus-type cells, a spiral-shaped cell, and three linear and angular cells (lower portion; the one on the lower right might be related to one type of green algae, and the two angular cells are similar to those reported to occur in core section 3593 [5]); 3584/5 (the cell on the far left in the fourth row may represent a small Caulobacter-like cell); 3591, all three apparently bacteria; 3606 and 3610, cells are similar to those in 3582 and 3584/5.

Cultures and sequence analyses.

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Cultures and sequence analyses.

Approximately 2,000 meltwater aliquots (200 µl each) werespread on agar plates containing 13 different media and wereincubated for weeks to months at four temperatures (4, 8, 15,and 22°C). Portions of the rRNA loci were amplified by PCR(as in reference 12, with primers described in reference 11)and sequenced, followed by BLAST searches, CLUSTAL alignment,and phylogenetic analysis (as in reference 8). A total of 665colonies resulted from the seven Vostok 5G accretion ice coresections, and an additional 22 were isolated from the glacialice immediately above the accretion ice. The bacteria isolatedrepresent a variety of taxa (Fig. 3), all of which are psychrotolerant(data not shown). All are related to taxa that are aquatic and/orlive in lake sediments, soils, or rocks (Fig. 3). In additionto the bacteria, a dozen unique fungi were isolated (data notshown).

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FIG. 3. Maximum-parsimony phylogram (determined using PAUP, version 4 [Sinaur Academic Publishers]) of portions of the small subunit genes from bacterial isolates in this study (GI, glacial isolate numbers) as well as comparable sequences from bacteria that were closest to these sequences in BLAST searches. Brief descriptors are provided for each of the sequences determined by BLAST searches to be the taxa closest to the sequences from this research. NCBI (GenBank) accession numbers are shown for each sequence. Bootstrap values are shown on branches with >50% support.

Conclusions about Lake Vostok.

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Conclusions about Lake Vostok.

The assembly of microbes that were found in the Lake Vostokaccretion ice samples indicates that the lake has a diversepopulation of microorganisms and potentially a complex ecosystem.Nonetheless, the concentrations of microbes in the subglaciallake are lower than those in most environments on Earth (7).Some have suggested that Lake Vostok is sterile, since partsof the lake may be extremely oligotrophic (3). However, allof our data indicate that the lake supports a diverse microbialassembly, as has been concluded elsewhere (6, 7, 10, 15). Thereappear to be distinct ecological zones, either spatially ortemporally, since different sets of microbes were isolated fromeach of the four zones (type I and II ice from the shallow embaymentand type I and type II ice from the main basin), representingdifferent ages of ice (Fig. 1). Our results indicate that thehighest concentrations of viable cells are located close tothe transition zones between type I and type II ice, which wouldcorrespond to the shoreline of the lake near the grounding lineof the glacier. While most research on the accretion ice hasfocused on bacteria, some fungi have been described (3), andfungi were isolated and photographed in this study (Fig. 2).Therefore, heterotrophs may be present in Lake Vostok. If so,the Lake Vostok ecosystem is more complex than previously thought.

ACKNOWLEDGMENTS

This work was supported by the National Science Foundation Officeof Polar Programs (ANT-0536870).

We are very appreciative of the help from the National Ice CoreLaboratory (Denver, CO), which provided the ice core sectionsused in this study. We thank Marilyn Cayer for vital help withthe fluorescence and scanning electron microscopy. We thankthe reviewers of this article, who contributed their time andeffort to greatly improve the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: 217 Life Sciences Building, Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403. Phone: (419) 372-2333. Fax: (419) 372-2024. E-mail: srogers{at}bgsu.edu

Published ahead of print on 13 June 2008.

REFERENCES

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