Life at the Wedge: the Activity and Diversity of ArcticIce Wedge Microbial Communities

Roland C. Wilhelm,1,* Kristin J. Radtke,2,* Nadia C.S. Mykytczuk,2 Charles W. Greer,3 and Lyle G. Whyte2

Abstract

The discovery of polygonal terrain on Mars underlain by ice heightens interest in the possibility that this water-bearing habitat may be, or may have been, a suitable habitat for extant life. The possibility is supported by therecurring detection of terrestrial microorganisms in subsurface ice environments, such as ice wedges foundbeneath tundra polygon features. A characterization of the microbial community of ice wedges from the highArctic was performed to determine whether this ice environment can sustain actively respiring microorganismsand to assess the ecology of this extreme niche. We found that ice wedge samples contained a relativelyabundant number of culturable cells compared to other ice habitats (*105 CFU$mL - 1). Respiration assays inwhich radio-labeled acetate and in situ measurement of CO2 flux were used suggested low levels of microbialactivity, though more sensitive techniques are required to confirm these findings. Based on 16S rRNA genepyrosequencing, bacterial and archaeal ice wedge communities appeared to reflect surrounding soil commu-nities. Two Pseudomonas sp. were the most abundant taxa in the ice wedge bacterial library (*50%), while taxarelated to ammonia-oxidizing Thaumarchaeota occupied 90% of the archaeal library. The tolerance of a varietyof isolates to salinity and temperature revealed characteristics of a psychrotolerant, halotolerant community. Ourfindings support the hypothesis that ice wedges are capable of sustaining a diverse, plausibly active microbialcommunity. As such, ice wedges, compared to other forms of less habitable ground ice, could serve as a reservoirfor life on permanently cold, water-scarce, ice-rich extraterrestrial bodies and are therefore of interest to astro-biologists and ecologists alike. Key Words: Ice wedge—Cryo-environments—Astrobiology—Microbial respira-tion—Biodiversity. Astrobiology 12, 347–360.

1. Introduction

Our expanding characterization of the past andpresent climate on Mars, despite its outward appearance

as an arid, cold, lifeless planet, is challenging preconceptionsand propelling investigations of its habitability and astro-biological relevance (Barfoot et al., 2010). In 2008, the Phoenixspacecraft landed on the northern pole of Mars and began itssearch for microbial habitat by analyzing soil from polygonalterrain, a hallmark feature for the presence of subsurface iceon Earth (Smith et al., 2009). As anticipated, Phoenix discov-ered a heterogeneous composition of icy soil just underlyingthe topsoil (mean depth of 4.6 cm) with similarities to what isfound in certain types of terrestrial wedges (Mellon et al.,2009; Smith et al., 2009). On Earth, microorganisms have beenisolated from similar ice wedges (Katayama et al., 2007), andcellular respiration is known to occur within the ice matrix of

subsurface soil wedges (Lacelle et al., 2011). The confirmationof subsurface ice on Mars and the presence of ice-bound life onEarth are compelling evidence and affirm the need for studyof the ecology and physiological capabilities of microbial lifein ice wedges and an assessment as to how such a niche mightsustain life on Earth or in analogous martian environments.

Polygon wedges occur beneath the troughs of tundra po-lygonal features visible at the soil surface (see Fig. 1) andcause dome-like protrusions in the polygon centers( Jorgenson et al., 2006). The V-shaped, subsurface wedgesform over thousands of years of repeated influx of meltwateror debris, or both, into millimeter-sized cracks created by thethermal contraction of the surrounding soil. Depending onmoisture levels and soil composition, wedges are composedof a variety of infill materials, with some wedges consistingpredominantly of water (ice wedges), small particulatematter such as sand (sand wedges), or a mixture of both,

1Microbiology and Immunology, University of British Columbia, Vancouver, Canada.2Natural Resource Sciences, McGill University, Montreal, Canada.3National Research Council of Canada, Biotechnology Research Institute, Montreal, Canada.*Co–first authors.

ASTROBIOLOGYVolume 12, Number 4, 2012ª Mary Ann Liebert, Inc.DOI: 10.1089/ast.2011.0730

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such as ice-cemented soil (sublimation wedges) (Levy et al.,2009). In wet environments, like our study site, ice wedgesform at the permafrost table (soil frozen for greater than twoconsecutive years) and are typically between 2 and 4 macross at the top, forming over thousands of years. An icewedge can be overlain by 5–15 cm of soil, referred to as the‘‘transient layer,’’ which typically undergoes annual thaw(active layer) but can remain frozen depending on inter-annual temperature variation (Shur et al., 2005).

Polygonal terrain is common on the martian surface and isbroadly distributed at latitudes > 30�. According to a recentsurvey based on satellite photography, the majority of mar-tian polygon morphotypes resemble either sand or subli-mation wedges, such as those found in the McMurdo DryValleys, which are consistent with the arid conditions onMars (Levy et al., 2009). These observations are supported bythe Phoenix lander’s discovery of ice-cemented rock at thepolygon site instead of larger ice deposits (Smith et al., 2009).However, satellite photographs also display a number ofstark, high-rimmed polygons that are characteristic of ter-restrial ice wedge polygons (Levy et al., 2009). This discov-ery, together with growing evidence for a more dynamichydrology on Mars (i.e., liquid brine on the martian surface),improves the probability of finding ice-rich subsurfacewedges and the ongoing hydrological processes involved intheir formation (McEwen et al., 2011). Martian ice wedges arehigh-priority targets in the search for life beyond Earth, andto expand our understanding of the habitability of such iceenvironments, we elected to study analog ice wedge envi-ronments from the Canadian high Arctic.

The microbial communities of a variety of terrestrial cryo-environments have been studied, such as those found inglacier ice (Skidmore et al., 2000; Miteva et al., 2004), sub-

glacial environments (Yde et al., 2010), cryoconite holes(Sawstrom et al., 2002), Arctic permafrost soil (Steven et al.,2009; Wilhelm et al., 2011), Antarctic Dry Valley soils andendoliths (Pointing et al., 2009), cryopegs (Gilichinsky, 2002),ice shelves (Bottos et al., 2008), high Arctic saline perennialspring environments (Perreault et al., 2007; Niederbergeret al., 2009, 2010), and various other ice environments (La-celle et al., 2011). These studies reveal complex cold-tolerantmicrobial communities, some of which exhibit active me-tabolism at in situ temperatures in ice and brine (- 5�C)(Niederberger et al., 2010; Bakermans and Skidmore, 2011)and permafrost (- 15�C) (Steven et al., 2008). Ground ice,such as ice wedges, is one of the most inhospitable of cryo-environments due to a lack of sunlight, scarcity of liquidwater, scarcity of energy sources, and subzero temperatures,and may in fact be a poor microbial habitat (Steven et al.,2008). However, ice vapor or ice brine may provide thenecessary solvent for life in habitats beyond Earth, with icecommonly found in environments within the Solar System(Prockter, 2005): on planetary bodies such as Europa, En-celadus, and Mars (Rathbun et al., 1998; Fairen et al., 2010),and on satellites such as Tempel 1 (Sunshine et al., 2006).

On Earth, microbial life has been isolated from ice envi-ronments, such as glacier ice, ground ice, and snow envi-ronments, many of which were thousands of years old (Table1). Ice contains a network of liquid water veins (brine veins)that can transport soluble and insoluble particles and sup-port life within the ice (Mader et al., 2006). Isotopic analysesof gas trapped in ancient ice wedges have demonstrated thatactive microbial respiration occurs within the ice structure(Lacelle et al., 2011). Two preliminary studies have examinedthe culturable microbial community and the total diversity ofice wedges: one study involved a 25,000-year-old Alaskan ice

FIG. 1. Comparison of the morphologicalfeatures of Arctic (A) and martian (B and C)polygons where trough formations concealunderlying ice wedges. Image (A) depictsthe Axel Heiberg field sampling site, image(B) a polygon field on the south pole ofMars (image PIA05021), and image (C) apolygon sampled by the Phoenix lander.Image (D) depicts the drilling of ice wedgesamples at the Axel Heiberg field site,and image (E) exhibits the vertical ice stri-ations indicative of ice wedge formation.Color images available online at www.liebertonline.com/ast

348 WILHELM ET AL.

wedge (Katayama et al., 2007) that contained a relativelyabundant population (105 to 106 CFU$g - 1 ice), and anotherinvolved a 5,000- to 6,000-year-old Yukon ice wedge (Lacelleet al., 2011) that yielded a relatively diminutive population(*103 CFU$mL - 1 melted ice). In comparison, a variety ofother ground ice types have not yielded any culturable cells(Table 1). Yet despite a relative abundance of culturable cells,the Alaskan ice wedge bacterial 16S rRNA clone library hadlow taxonomic diversity, which consisted almost entirely ofGammaproteobacteria (93%) and is, to date, the only as-sessment of ice wedge bacterial diversity. Two studies ofsimilar environments, a high Arctic massive ground ice(Steven et al., 2008) and a Yukon pingo (Lacelle et al., 2011),discovered a markedly different diversity of ice-inhabitingmicroorganisms, with taxa belonging predominantly to Fir-micutes (*60%). There appears to be distinct variability inthe microbiota of ice-rich environments, which suggests thatconditions within ice can significantly differ.

Detection of cellular respiration is an important line of ev-idence for whether cryo-environments, ice wedges in partic-ular, can support active life or whether they merely preserveit. Microbial respiration in ice wedges has already been in-ferred based on isotopic ratios of Ar-O2-CO2 (Lacelle et al.,2011), though these methods did not determine the timeframe

of the microbial activity, that is, whether respiration occurredduring the initial stages of ice crystallization or it occurredperiodically or steadily over time. The mineralization ofradioisotope-labeled substrates at in situ temperatures hasbeen used to detect microbial respiration with high sensitivityin a number of cryo-environment samples, including groundice (Steven et al., 2007, 2008), and were used in the presentstudy to detect activity in ice wedge samples.

From an astrobiological perspective, whether ice wedgessupport active microbial communities will be important inidentifying possible refugia beyond our hospitable planet.Based on two ice wedge cores obtained from high Arcticpolygon terrain, our objective was to determine the microbialcomposition along a depth gradient, using culture-dependentand culture-independent approaches, and determine whetherice wedge microbial communities are metabolically activein situ. The conditions within terrestrial ice wedges, whilesevere, are inviting to life when compared to the physicalconditions expected in martian wedge environments, wheremicroorganisms would have to manage water scarcity, drasticdaily temperature fluctuation, low pressure, and high levels ofradiation (Gomez et al., 2010). Therefore, the characterization ofin situ respiration and the microbial community of terrestrial icewedge habitats represents a best-case scenario in the search for

Table 1. Summary of Culturable and Total Microbial Enumerations and Dominant Community Members

by Phylum or Class, Where Available, for Various Snow, Ice, and Ground Ice Environments

Sample origin Age (years)

Dominantbacterialisolates

Dominantbacterial

taxa (16S rDNA)Viable

cell countsTotal cell

counts References

Arctic lake ice — — — 102 106 Junge et al., 2004

Lake Vostok accreted ice 110–240 K$ya — — 0* 102 Karl et al., 1999; Poglazovaet al., 2001

Antarctic snow — — D.T. — 103 Carpenter et al., 2000

Spitsbergen snow — a-Prot — 0** 104 Amato et al., 2007; Laroseet al., 2010

Yukon firnified snow bank 25 K$ya a-Prot/Act — 0 106 Lacelle et al., 2011;Radtke, 2010

Eureka massive ground ice 5–6 K$ya — Fir 0 104 Steven et al., 2008

Cryoconite ice — — — — 104 Sawstrom et al., 2002

Mt. Everest glacier 1.5–2 K$ya Fir c-Prot 0 103 Zhang et al., 2010

Greenland glacier (clear ice) 57–68 K$ya Act Fir/Act 103 105 Miteva et al., 2009

Greenland glacier (silty ice) 57–68 K$ya Act c-Prot — 107 Miteva et al., 2004, 2009

Yukon buried glacier 100 K$ya Act — 0** 104 Lacelle et al., 2011;Radtke, 2010

Yukon pingo 15 K$ya Act Fir 102 105 Lacelle et al., 2011;Radtke, 2010

Alaskan ice wedge 25 K$ya Act c-Prot 106 — Katayama et al., 2007

Yukon MC ice wedge 5–6 K$ya Act — 103 107 Lacelle et al., 2011;Radtke, 2010

Yukon OC ice wedge 6–6 K$ya Act — 103 106 Lacelle et al., 2011;Radtke, 2010

Axel Heiberg ice wedge 1 > 4 K$ya Act c-Prot 105 108 Current study

Axel Heiberg ice wedge 2 > 4 K$ya — — 104 108 Current study

Viable cell counts are measured in CFU$mL - 1 or CFU$gdw - 1 (colony-forming units per gram dry weight), while total cell counts are incells$mL - 1 or cells$gdw - 1. Act, Actinobacteria; Fir, Firmicutes; Prot, Proteobacteria; D.T., Deinococcus-Thermus.

*Mineralization of [1 - 14C] acetate was observed.**Obtained isolates by alternate culturing methods.

MICROBIAL ACTIVITY AND DIVERSITY OF ARCTIC ICE WEDGE CORES 349

life on Mars and will broadly improve our understanding ofthe ecology of ice-rich habitats.

2. Methods

2.1. Description of site and sampling methods

Sampling was conducted in July 2009 on polygonal terrainlocated near the McGill Arctic Research Station on AxelHeiberg Island, Nunavut, Canada. The mean annual airtemperature for the region, recorded at the closest weatherstation in Eureka, NU, was - 17.6�C in 2009. Ice wedgesamples were taken in a polygon field characterized by acidicsoil (pH 4.5–5) adjacent to an acidic lake (pH 3.7), with pHdriven by iron-sulfur chemistry (Buttle and Fraser, 1992). Twotroughs running alongside a soil polygon, a feature createdby the pressure exerted during ice wedge formation, werechosen for coring (AH1: 79�24.963¢N, 90�45.759¢W; AH2:79�24.965¢N, 90�45.802¢W). Both troughs were cracked downthe center, which indicates that the ice wedges beneath are stillactively forming. The active layer was 45 cm deep at the timeof sampling, while the transient layer was 10 cm thick over-lying the ice wedge. Cores were taken by using a combinationof a Hilti hammer drill fitted with a 3-inch corer and a Pioneerportable hydraulic drill (Multi-Power Products Ltd., Ke-lowna, BC, Canada). Each corer was sterilized with ethanolprior to extraction of each sample. The AH1 borehole reacheda depth of 345 cm, whereas the AH2 hole reached 155 cmdeep. Cores were transported in sterile Whirl-Pak bags (NascoLaboratory Sampling Products, Modesto, CA, USA) and keptfrozen at - 20�C until processing.

The interior of each core was subsampled to ensure thematerial used in our analyses was not subject to contami-nation. This was performed according to the process outlinedby Juck et al. (2005) with minor changes. Fluorescent micro-spheres were not used, but rather the exterior of each corewas painted with a liquid culture (*108 cells$mL - 1 in TSB)of a kanamycin-resistant, green fluorescent protein-labeled(gfp) strain of Pseudomonas, strain Cam1-gfp2. To verify thatthere had been no contamination, a portion of the subsamplewas melted and plated on LB agar + 50 lg/mL kanamycin toverify that no kanamycin-resistant colonies grew. Ad-ditionally, following each DNA extraction, a polymerasechain reaction (PCR) with primers specific to the gfp gene(gfpF, gfpR) was performed to verify the absence of gfp geneartifacts in the sample; PCR conditions and primers were aspreviously described. The percentage of solid material in theice wedge was calculated based on mass, where 15 g of icefrom AH1 at a depth of 245–260 cm was dried at 100�C forapproximately 24–32 h and weighed. Conductivity, salinity,total dissolved solids, and oxidation/reduction potentialwere also measured from this sample with the YSI 556 MultiProbe System (YSI Incorporated, Yellow Springs, OH, USA).

2.2. Community DNA extraction and 16S rRNAgene 454 pyrosequencing analysis

An ice wedge sample (AH1 2.2.5) from the deepest portionof recovered ice (325 cm) was chosen for comprehensivecommunity analysis by 16S rRNA gene pyrosequencing. Toconcentrate biomass prior to DNA extraction, 15 mL ofmelted ice was passed through a Sterivex GP 0.22 lm filter(Millipore, Billerica, MA, USA). The filter was then sectioned

into 10, and DNA was extracted by using the UltraClean SoilDNA Isolation Kit’s (Mo Bio, Carlsbad, CA, USA) alternativeprotocol for maximum yields. The 10 individual extractionswere pooled onto a single spin column prior to the finalelution step to maximize DNA yields. DNA samples weresent to Research and Testing Laboratories, LLC (Lubbock,TX, USA) for tag-encoded FLX-titanium amplicon pyro-sequencing (Roche 454). 16S rRNA gene taxonomic profilesfor Bacteria and Archaea were created with primers ‘‘Assayb.2: 28F-519R,’’ for Bacteria (28F: 5¢-GAG TTT GAT YMTGGC TC & 519R: 5¢-GWA TTA CCG CGG CKG CTG), and‘‘Assay a.1: 340F-806R,’’ for Archaea (5¢-CCC TAC GGGGCG CAG CAG & 5¢-GGA CTA CCA GGG TAT CTA AT).The Ribosomal Database Project (RDP) pyrosequencingpipeline was used to pre-process 16S rRNA sequences (en-suring only high-quality sequences were used in analysis)and to provide the diversity estimates and classification of alltaxa (at 97% sequence similarity) (Cole et al., 2009).

2.3. Total cell and culturable heterotrophic cell counts

Total cell counts were made on the transient layer (55–56 cm) and shallow ice wedge samples (125–135 cm) fromboth cores with a 4,6-diamidino-2-phenylindole (DAPI) cellstain and fluorescence microscopy. DAPI was added to0.5 mL replicate melted ice sample to a final concentration of100 ng/mL. The stained samples were then filtered onto whitepolycarbonate filters, washed with 2 mL of ddH2O to removebackground stain, and visualized (k = 461 nm) with a Nikon Y-FL epifluorescence microscope at 1000 · magnification. Cellcounts were averaged over 10 randomly chosen fields of view.

Culturable heterotrophic cell counts were performed on 14sections of the AH1 and AH2 ice cores by using a variety ofmedia and at two incubation temperatures, 25�C and 5�C.Samples were serially diluted in 0.1% sodium pyrophos-phate and plated in triplicate onto R2A (7.2 – 0.2), R2Asupplemented with 5% w/v NaCl, R2A (pH 5.5 – 0.1), andTSA (7.2 – 0.2). Incubation times varied and were left until nonew growth was observed, corresponding to 14–21 days forincubations at 25�C and 40–60 days for incubations at 5�C.Samples were plated on LB agar with 50 lg/mL kanamycinto test for contamination from the Pseudomonas Cam1-gfp2tracer strain; any sample with a positive contamination wasreprocessed. A one-way ANOVA was performed comparingcolony-forming units (CFU) per core (AH1, AH2) and tem-perature (5�C and 25�C) at each depth with ezANOVA(http://www.cabiatl.com/mricro/ezanova/index.html).

2.4. Selection, identification, and characterizationof bacterial isolates

Isolates were characterized from ice wedge core AH1 fromall media types and incubation conditions based on colonymorphology, color, growth media, incubation temperature,and time of appearance. Each colony was streaked for iso-lation three times to ensure purity. The classification ofbacterial isolates was made by PCR amplifying and se-quencing 16S rRNA genes according to Steven et al. (2008)with the following primers: 27F (5¢- AGA GTT TGA TCCTGG CTC AG-3¢) and 758R (5¢- CTA CCA GGG TAT CTAATC C-3¢). PCR product was sequenced by Sequencage La-val (University of Laval, Quebec City, QC, Canada) with an

350 WILHELM ET AL.

ABI Prism 3130XL genetic analyzer. Isolates were tested forgrowth on R2A at various temperatures (- 5�C, 5�C, 25�C,37�C) and salt amendments (1%, 2%, 3%, 4%, 5% w/v NaCl).

2.5. Detection of microbial activity at coldtemperatures

The microbial activity of ice wedge, active layer, andtransient layer communities was compared by using radio-respiration assays at low temperatures (- 10�C, - 5�C, and5�C) as described for high Arctic permafrost samples by Ste-ven et al. (2007, 2008). Due to a limited supply of ice sample,only one radio-labeled substrate was used, [1 - 14C]acetate,which has been used successfully in radio-respiration assaysof accretion ice over Lake Vostok in Antarctica, permafrostbacteria, and glacial microorganisms. Four samples wereanalyzed: active layer from above AH1 (depth of 30 cm);transient layer from above AH1 (55–56 cm); and two icewedge samples—shallow (AH2: 60–75 cm) and deep (AH1:245–260 cm). These were prepared in triplicate with sterilecontrols for each temperature. A total of 5 mL of sample wasadded to each microcosm along with radio-labeled acetate,which yielded an average of 57,285 disintegrations per minuteper microcosm. Non-radio-labeled acetate was included to afinal concentration of 15 mM to stimulate growth. Incubationswere sampled periodically over a period of 164 days. Thepercentage of radio-labeled acetate mineralized to 14CO2 wascompared between sample and controls by using an unpairedt test.

2.6. In situ measurements of CO2 flux from surfaceof field sites

Net CO2 flux was measured from the soil surface at thefield site with a LiCor LI 8100 Field Survey Unit (LI-CORBiosciences, Lincoln, NE, USA), according to manufacturer’sinstructions. Flux measurements recorded with the LiCorsystem accounted for variation in surface soil temperature(*5 cm deep) and atmospheric CO2 concentration at the timeof sampling. Measurements were performed during 2 daysin the summer (mid July, 2009) and late winter (late April,2010). Readings of net CO2 flux (3 · 3 min replicates) weremeasured at three different sites on the surface of polygontroughs, where ice wedge samples were extracted, and onthe surface of polygon centers, underlain by permafrost.Mean CO2 flux values were compared per sampling year/season by using a student t test for the polygon center versustrough surface soil.

2.7. Nucleotide sequence accession numbers

The accession numbers for the nucleotide sequences de-posited in the GenBank database are as follows: bacterialisolates ( JN221486–JN221547), bacterial 16S rRNA gene li-brary ( JN217243–JN219938), and archaeal 16S rRNA genelibrary ( JN219939–JN221485).

3. Results

3.1. Ice wedge characteristics and DNA extraction

The age of the ice wedges is estimated to be at least 4000years before present, based on the Holocene deposits inwhich they have formed (Aitken and Bell, 1998) and ice

wedge dating at regionally proximal sites (Fortier and Al-lard, 2004). Vertical cracking patterns within the ice corestructures were apparent and are consistent with the processof ice wedge formation (Fig. 1). Other ground ice structuresobserved included either horizontal layering with no stria-tions or horizontal layering with random striations (resultingfrom the crystallization of snow, i.e., firnification). The in situtemperature of the ice wedge was - 16�C during the summersampling period. Measurements of pH for all ice wedge andtransient layer sections ranged from 5.2 to 5.8. The salinity,conductivity, oxidation/reduction potential, and total dis-solved solids of the ice wedge sample were as follows:0.20 ppt, 373 lS$cm - 1, + 186.0 mV, and 0.274 g/L. On aver-age, the ice wedge contained extremely low percentages ofsolid mass (*0.2% w/v – 0.07). The PCR screen to detect theintentional contaminant strain, Pseudomonas Cam1-gfp2, wasnegative during all molecular analyses, which indicatessample purity.

3.2. Abundance of total and culturable cells

Total cell counts ranged across one order of magnitude,from 8.4 · 107 to 9.7 · 108 cells$mL - 1, while culturable cellcounts were highly variable, ranging from 70 CFU$mL - 1 to1.1 · 106 CFU$mL - 1 (Fig. 2). The greatest abundance of cul-turable aerobic heterotrophs were cultivated at 5�C from theAH1 transient layer soil. Culturable counts at 5�C were sig-nificantly higher ( p = < 0.1) than those at 25�C for nearly allprofile depths, which indicates that the heterotrophic icewedge community consists predominantly of cold-adaptedbacteria. Culturable cell counts along the core profile re-vealed an inverse relationship between the number of cul-turable heterotrophs and the sampling depth. Culturableversus microscope-based ‘‘total’’ cell counts differed, at mostby a factor of 104, which indicates the presence of a largelyuncultured population. No clear delineation was found be-tween the abundance of culturable and total cells betweenthe transient layer soil and ice wedges, since both enumer-ations were greater in the ice wedge for core AH2, while theopposite was true of AH1. The type of solid growth mediaused, that is, R2A, R2A + 5% NaCl, R2A + pH 5.5, and TSA,influenced the recovery of culturable cells. In all cases, thegreatest growth was observed on R2A plates, and these cellcounts form the basis for Fig. 2.

3.3. Isolate identification and characterization

A total of 25 transient layer and 37 ice wedge isolates wereselected from AH1 growth plates for 16S rRNA gene se-quencing and characterization. Arthrobacter sp., Paenibacillussp., Shigella sp., and Curtobacterium sp. were the mostabundant (each constituted *10% of total isolates) and mostcosmopolitan species isolated and occurred in both transientlayer and ice wedge samples (Table 2). A number of isolateswere also detected in the 16S rRNA gene bacterial library (inbold type in Table 2), including one Pseudomonas sp. (P. mi-gulae) that constituted *50% of all bacterial sequences. Allisolates were capable of growth at 25�C, and nearly all iso-lates could grow at 5�C. Three isolates were capable ofgrowth at 37�C, two of which (Pseudomonas sp. and Ar-throbacter sp.) exhibited the broadest temperature toleranceand grew at 5�C as well. Isolates incubated at - 5�C did notgrow after 2 month’s incubation. The majority of isolates

MICROBIAL ACTIVITY AND DIVERSITY OF ARCTIC ICE WEDGE CORES 351

FIG. 2. Heterotrophic plate countson R2A growth medium at 5�C and25�C along the depth profile of thetwo ice wedge cores (AH1 and AH2).Error bars represent standard erroracross triplicates. Note that the y axisis not to scale. Color images availableonline at www.liebertonline.com/ast

Table 2. Summary of Isolate Classifications and Growth Characteristics

Growth temperature

SourceIsolate

accession # Classification% similarity totop BlastN hit Min Max

MaximumNaCl Amendment

Ice wedge& transient layer

JN221490 Arthrobacter sp. 99% 5 37 3% to ‡ 5%JN221510 Bradyrhizobium sp.* 98% 5 25 1–2%JN221500 Curtobacterium sp. 97% 5 25 1–4%JN221488 Paenibacillus sp. 98% 5 25 0–3%JN221492 Paenibacillus sp. 98% 5 25 3–4%JN221499 Paenibacillus sp. 94% 5 25 1% to ‡ 5%JN221529 Shigella sp. 97% 25 25 4% to ‡ 5%JN221519 Sphingomonas sp. 98% 5 25 4% to ‡ 5%JN221513 Streptomyces sp. 100% 5 25 3–4%

Ice wedge JN221503 Arthrobacter sp. 99% 5 25 1%JN221543 Cohnella sp. 98% 5 25 N/AJN221535 Leifsonia sp. 99% 5 25 N/AJN221516 Leifsonia sp. 99% 5 25 ‡ 5%JN221534 Micrococcus sp. 99% 5 25 N/AJN221512 Micromonospora sp.* 99% 5 25 1%JN221544 Paenibacillus sp. 99% 5 25 N/AJN221502 Paenisporosarcina sp. 100% 5 25 ‡ 5%JN221540 Pseudomonas sp. 99% 5 25 N/AJN221506 Pseudomonas sp. 97% 5 37 ‡ 5%JN221497 Shigella sp. 99% 5 25 ‡ 5%JN221505 Staphylococcus sp. 99% 5 25 1%

Transient layer JN221493 Brachybacterium sp.* 99% 25 37 ‡ 5%JN221526 Cellulomonas 96% 5 25 2%JN221533 Methylobacterium sp. 98% 5 25 2%JN221486 Mycobacterium sp. 99% 5 25 2%JN221522 Streptomyces sp. 99% 5 25 4% to ‡ 5%

Classifications are based on the Ribosomal Database Project and top cultured BlastN hits, which corresponded for the majority of isolates.Isolates with greater than 95% sequence similarity were aggregated and, as a result, growth temperatures and salt tolerance values representthe broadest range observed for each aggregated genotype. Isolates in bold share 97% similarity to sequences found in the 16S rRNA genelibrary.

*Classification derived solely from top cultured BlastN hit.N/A, not available.

352 WILHELM ET AL.

(65%) demonstrated mild halotolerance (3–5% NaCl), while33% were capable of growth at 5% NaCl.

3.4. 16S rRNA gene community analysis

Archaeal and bacterial 16S rRNA gene pyrosequencingwas performed on core subsection 2.2.5, the deepest icewedge sample, to provide a description of the abundanceand community structure therein. The bacterial communitylibrary was composed of 2690 sequences and exhibited highdiversity based on the Chao (872) and Shannon indices(4.10). The bacterial library provided reasonable coverage ofthe diversity of organisms present in the ice wedge sample.A total of 591 operational taxonomic units (OTUs) werepresent at 97% sequence similarity with a Good’s percentcoverage suggesting an estimated sampling of 75.2% of thecommunity (Good, 1953). The community was dominated byGammaproteobacteria, classified as Pseudomonas, and com-prised *50% of the total of classifiable Bacteria. The re-mainder of the taxa were distributed across 10 other phyla,the most abundant of which were Actinobacteria (15.2%),Acidobacteria (8.3%), and Alphaproteobacteria (7.9%) (Fig.3). The majority of 16S rRNA sequences classified as Pseu-domonas belong to a single OTU, the closest relative of whichis Pseudomonas migulae (98% similarity), a pseudomonadisolated from a low-nutrient mineral spring in France (Ver-hille et al., 1999). This type strain is capable of denitrification,possesses a flagella, and grows at 4�C. In analyzing all OTUsoccupying greater than 0.5% of the total library, the follow-ing environments were represented: Antarctic and Arcticsoils, aquatic environments (lakes, springs, wastewater),permafrost, alpine soils, agricultural soil, and human skin. Asubstantial proportion of the bacterial 16S rRNA gene library(12.4%) consisted of unclassifiable sequences.

The archaeal pyrosequencing data contained only 21 ar-chaeal sequences and consequently exhibited very low di-versity, according to Chao (18) and Shannon indices (2.14).Out of the total 1547 ‘‘archaeal’’ sequences, 1526 sequenceswere classified as bacterial. According to the experiences ofthe pyrosequencing facility, poor archaeal primer specificityresults when the abundance of archaeal sequences is minor

relative to bacterial sequences. The 21 archaeal sequencesgrouped into 11 OTUs at 97% similarity, the majority ofwhich (9) were related to Crenarchaeota of the class Ther-moprotei (clustering to marine Group 1.3b). The closestcultured match (95%) was with a member of Thaumarch-aeota, Nitrososphaera viennensis, an ammonia-oxidizing soilarchaeon (Tourna et al., 2011). The remaining two OTUs wereunclassifiable beyond the level of Euryarchaeota, with topcultured relatives from the genera Methanococcus andMethanosarcina.

Similarities in community structure were apparent fol-lowing direct comparison of our data to 16S rRNA gene li-braries from another ice wedge (Katayama et al., 2007) andfrom the active layer and permafrost at the same Axel Hei-berg field site (Wilhelm et al., 2011). The major Pseudomonassp. phylotype (*50% of sequences, n = 2690) in the AxelHeiberg ice wedge was also present in the Alaskan ice wedgelibrary (97% similarity), though at a relatively lower abun-dance (6%, n = 79); this was the only overlap between the twoice wedge libraries. A low degree of overlap was also seenwith the libraries obtained from the same field site, where 18bacterial (3% overlap) and 3 archaeal (14% overlap) OTUswere common to both. Members of the bacterial familiesBradyrhizobiaceae, Chitinophagaceae, Acidobacteriaceae,Gemmatimonadaceae, and Acidothermaceae were shared;three OTUs of unclassified Bacteria were also shared. Allthree archaeal OTUs were classifiable to class Thermoproteiand constituted the major archaeal taxa found in bothlibraries.

3.5. Microbial activity of ice wedge samplesby radio-labeled acetate mineralization

The relative mineralization rates of trough and ice wedgesamples are summarized in Fig. 4. Both ice wedge samples,shallow and deep, did not show any significant respiration atany temperature. At 5�C, the transient layer demonstratedthe greatest total mineralization (17.3%) compared to theactive layer (10.6%). The active layer ( p = 0.01) and transientlayer ( p = 0.01) were significantly different than controls,while both ice wedge samples were not ( p > 0.05; p > 0.05). At

FIG. 3. Distribution of bacterial taxa, at adepth of 325 cm in the ice wedge, displayingthe predominance of Proteobacteria andrange of bacterial diversity (n = 2396). Thephylum Proteobacteria was comprised oftwo major classes: Gammaproteobacteria(83%) and Alphaproteobacteria (12%). Un-classifiable 16S rRNA gene sequences werenot included in the analysis (12% of all se-quences).

MICROBIAL ACTIVITY AND DIVERSITY OF ARCTIC ICE WEDGE CORES 353

subzero temperatures, - 5�C and - 10�C, no sample differedsignificantly from its control after 164 days of incubation.

3.6. Microbial activity by in situ net CO2 flux

Carbon dioxide flux was measured to estimate in situmicrobial activity (respiration) at different locations alongpolygon and trough surfaces in summer (2009) and latewinter (2010) (Fig. 5). Active flux of CO2 was observedduring both sampling periods at significantly higher levelsabove atmospheric values, successfully identifying theproduct of microbial respiration in situ. Variability in CO2

flux was greater in summer and showed significantly higher

flux between trough versus polygon active layer surfaces,averaging 2118 – 805 mg/m2/day and 853 – 548 mg/m2/day, respectively ( p = 0.01). In late winter, flux values for thepolygon (1637 – 579 mg/m2/day) were similar ( p > 0.05) tothe trough (1682 – 446 mg/m2/day). Throughout the mea-surement period, the maximal single flux value observedwas 4600 mg/m2/day at the trough surface. The greatestaverage flux value was measured at the surface of an icewedge positioned directly over a borehole with a depth of155 cm (2604 – 456 mg/m2/day) (Fig. 5) ( p < 0.01). Duringsummer flux analyses, the soil temperature profile rangedfrom 1�C at a depth of 45 cm to 14�C in the upper 5 cm ofthe soil. During late-winter flux analyses, the ground was

FIG. 4. Percentage of radiolabeledacetate mineralized to CO2 at 5�Cfor active layer soil (depth of 30 cm);transient layer soil (55–65 cm); andtwo ice wedges—shallow (AH2: 60–75 cm) and deep (AH1: 245–260 cm).Transient layer and active layersamples were significantly differentfrom their controls. The notation ( - )denotes sterile negative controls,and error bars represent standarderror across triplicates.

FIG. 5. Mean net CO2 flux from three site types during summer (2009) and late winter (2010): polygon center, polygontrough (ice wedge), and the ice wedge surface above a borehole. Error bars represent the standard deviation of the mean fromthe total data collected per site type.

354 WILHELM ET AL.

completely frozen, and soil temperatures were measuredat - 16�C at 40 cm deep and - 9�C in the upper 5 cm ofsoil.

4. Discussion

Pursuing evidence of extraterrestrial life in ice environ-ments on Mars, or elsewhere, is possible through character-izations of analogous environments on Earth (Fairen et al.,2010). Terrestrial analogue environments provide improvedlogistical foresights, reveal priority targets like hotbeds ofbiological activity, and are useful in broader criteria devel-opment for assessing habitable zones on planetary bodies. Bystudying the microbial community capable of surviving theconditions in ice wedge environments, where liquid water isat its limit of stability, we gain insight into (i) the physio-logical limits of cold-tolerant life, (ii) characterizing the visualfeatures of extremophilic microorganisms, and (iii) studyingthe biosignatures left by certain types of microbial activity.Community differences may also be used to infer geochem-ical and geological differences in ice environments, andprovide insight into more habitable versus less habitable iceconditions.

Our characterization of Axel Heiberg ice wedges indicatedthat an extant, diverse, and low-activity microbial commu-nity exists within the ice wedge habitat. Ice wedges yield thegreatest number of culturable heterotrophic cells comparedto other ice environments, including glaciers, pingos, lakeice, ground ice, and snow (Table 1). It is still unclear whetherthis difference in abundance is due to relatively more hos-pitable conditions in ice wedges or a reflection of the pop-ulation sizes in the source of water. It is a reasonableassumption that glacier and snow ice, composed of cellsderived from particles carried by wind, may have lower cellabundances compared to ice wedges, which result fromwater percolating through soil (Xiang et al., 2009). Theabundance of culturable heterotrophs in the Axel Heiberg icewedges is corroborated by findings from an Alaskan icewedge, which demonstrates that ice wedge populations re-main high in the more extreme conditions of the high Arctic.The suitability of the Axel Heiberg ice wedge habitat formicrobial life is apparent when contrasted to massive groundice, which is defined as horizontally extensive subsurface icegreater than 2 m thick (Steven et al., 2008). This proximal highArctic site yielded zero culturable cells despite significantparallels in culturing technique (Steven et al., 2008). Thehigher abundance of culturable cells in ice wedges may re-sult from the diffusion of annual influxes of meltwater fromthe center of the ice wedge through the network of ice veinsthat carry nutrients and oxygenated water (Mader et al.,2006). The uniformity of the ice wedge culturable cell counts,which vary only 10-fold along the 3 m gradient, may be in-dicative of the effect this diffusion has on supporting themicrobial community throughout the ice wedge. In com-parison, permafrost soils show differences of 100- to 1000-fold in abundances across a much shorter distance (*1 m)(Steven et al., 2006; Gilichinsky et al., 2008). However, it isimportant to note that the uniformity of ice wedge commu-nities may also reflect the fluctuation or constancy in thedeposition rate of annual influxes, an effect that has beencorrelated to microbial abundances in the formation of gla-cier ice (Xiang et al., 2009).

The pattern of microbial diversity observed in the AxelHeiberg ice wedge at a depth of 3 m was consistent withother ice environments for both culture-based and 16SrRNA gene-based assays, namely, relatively high bacterialand low archaeal diversity (Steven et al., 2009; Wilhelmet al., 2011). In nearly all studies of the culturable com-munity of ice environments, including the Axel Heibergice wedge, Actinobacteria have been the most commonlycultured phylum and Arthrobacter sp. the most commongenus. Arthrobacter sp. are known to be hardy, cosmo-politan organisms capable of surviving long periods ofhigh stress such as starvation, exposure to radiation,and temperature shifts, and are well adapted for living inthe extreme conditions of ice wedges and other cryo-environments (Nelson and Parkinson, 1978; Mongodinet al., 2006). Arthrobacter psychrolactophilus, a psychrotolerantorganism, demonstrated an impressive hardiness when ex-posed to Mars-like conditions; from a starting population of109 cells$g - 1, a population of 105 cells$g - 1 survived 30days of fluctuating diurnal temperature ( - 40�C to 24�C),low pressure (13.3 mbar), and high background UV ra-diation ( Johnson et al., 2011). Bacterial diversity estimatesbased on 16S rRNA gene libraries placed the Axel Heibergice wedge community (H’: 4.10) at a similar level ofdiversity as Axel Heiberg permafrost (H’: 4.00) (Wilhelmet al., 2011) and an Arctic ice shelf microbial mat (H’: 4.41)(Bottos et al., 2008). However, the marked abundance ofPseudomonas sp. in the Axel Heiberg library (50%) indi-cates an uneven distribution of taxa with a few speciesthat dominate the community. Similar findings were re-ported in the Alaskan ice wedge ( > 60% Pseudomonas spp.)(Katayama et al., 2007) and a glacier on Mt. Everest(Zhang et al., 2010). For cryo-environments that preserveextracellular DNA, it is important to gauge diversityestimates carefully, as the species richness can be skewedby singleton sequences that may represent remnants ofancient DNA, sequencing noise (Tedersoo et al., 2010), orperhaps low-abundance organisms of the rare biosphere(Huse et al., 2010).

The abundance of Pseudomonas sp. in all ice wedgesstudied to date is compelling evidence and affirms theneed for examination of their particular role in this niche.The two most predominant Pseudomonas spp. in our 16SrRNA gene library are both most closely related to Pseu-domonas miguale (98%). Previous characterizations of P.miguale do not suggest a ready explanation as to why itmight be so abundant in ice (Verhille et al., 1999); how-ever, it is a member of the P. fluorescens group (Anzaiet al., 2000), known for their capacity to induce ice nucle-ation (Obata et al., 1987). This phylogenetic evidence im-plies an adaptive advantage for ice-nucleating bacteria inice wedge habitats. The benefits conferred by ice nucle-ation would be two-fold: (i) heat is released during thecrystallization of water molecules, (ii) ice nucleation cancontrol the size and growth of ice crystals and therebyprotect the cell from physical damage (Zachariassen andKristiansen, 2000). Ice nucleation has been observed byintact cells at subzero temperatures as low as - 4�C(Yankofsky et al., 1981) and is not affected by low pH( ‡ 5.0) (Obata et al., 1987), such as that of the Axel Heibergice wedges. The predominance of pseudomonads closelyrelated to ice-nucleating varieties in both Axel Heiberg and

MICROBIAL ACTIVITY AND DIVERSITY OF ARCTIC ICE WEDGE CORES 355

Alaskan ice wedges (Katayama et al., 2007) suggests aplausible role for this activity.

The detection of Archaea, which are known for their ad-aptation to extreme temperature and salinity, was limited bythe minimal data recovered during pyrosequencing analyses.The inability to recover archaeal sequences reflects the rela-tively low abundance of Archaea compared to Bacteria.Based on the sequences recovered, the community wascomposed almost entirely of Crenarchaeota, which are phy-logenetically related to marine Group 1.3b (90%). The samehigh levels of Crenarchaeota were reported in a study of theneighboring Axel Heiberg wetland soil (Wilhelm et al., 2011)and have been found in a wide variety of cryo-environments,such as Ellesmere Island permafrost (Steven et al., 2008),Norwegian Arctic wetland soil (Hoj et al., 2005), Arctic andAntarctic oceans (Kalanetra et al., 2009), and soils from theRoss Sea region of Antarctica ( > 99% of clones) (Ayton et al.,2010). The top matching cultured representative to the Crenar-chaeota detected in the Axel Heiberg ice wedge is a soil ar-chaeon, Nitrososphaera viennensis (95%), which is capable ofautotrophic ammonia-oxidation (Tourna et al., 2011). Am-monia-oxidizing Archaea are thought to contribute signifi-cantly to nitrogen cycling on a global scale in both terrestrialand marine environments (Tourna et al., 2011). The recur-rence of ammonia-oxidizing Archaea in cryo-environmentsand in ice suggests their possible ecological importance,though their activity at subzero temperatures is unknown.

The constituency of an ice wedge community is expectedto include a significant proportion of immigration from thelocal environment, as soil particles and meltwater from thesurface and active layer percolate through the soil duringthermal cracking (Fortier and Allard, 2005). The Axel Hei-berg archaeal and bacterial libraries reflect this in the largeproportion of taxa that are not present in the Alaskan icewedge library (Katayama et al., 2007) but are abundant inlibraries made from active layer soil at the same study site(Wilhelm et al., 2011). Members of Acidobacteriaceae andGemmatimonadaceae, for example, were not detected in theice Alaskan wedge, yet they were prevalent in the AxelHeiberg ice wedge, 18% and 1.9%, respectively, and weredominant community members of soil from the same site(*50 m of site separation), 30% and 14%, respectively (Wil-helm et al., 2011). A number of sequences recovered from thesoil and ice wedge were common to both sites at the specieslevel, which demonstrates that the microbial community, inpart, originates from the surrounding environment.

The use of in situ CO2 flux and lab-based radio-labeledsubstrate experiments to test for active respiration yieldedtentative evidence for current-day microbial respiration inthe ice wedge environment. Ice wedge samples showedslightly higher levels of radio-respiration than controlsbut were not statistically significant. The levels of radio-respiration in our ice wedge samples were extremely lowand, taken alone, do not indicate active respiration over themeasurement period. However, together with the trends innet CO2 flux observed in situ, which will be discussed in thefollowing paragraph, the low levels of radio-respiration maybe demonstrative of activity, albeit inconclusive. The radio-respiration experiment likely requires longer incubationtimes, and it is also possible that our methods were not ad-equately adapted for ice, as they were originally designed forsediments and soils (Steven et al., 2007). The amendment of

radio-labeled acetate to melted ice prior to refreezing mayhave resulted in concentrations too dilute for readily de-tectable rates of respiration, or the acetate may have beenfrozen in the bulk ice and inaccessible to microorganismsthat inhabited microstructures within the ice. An additionalconfounding factor may have been the loss of microstruc-tures, or brine veins, known to be important for microbial life(Mader et al., 2006) during the brief thaw necessary to in-corporate the radio-labeled acetate.

The use of CO2 flux as an index for microbial respiration isa common environmental method (Singh et al., 2011) and hasrecently been used on Antarctic Dry Valley soil (Ball et al.,2009). Ball et al. (2009) found that the relatively scant popu-lations of plant and soil invertebrates in Antarctic soilssimplified the correlation between CO2 flux and microbialactivity, a result that is applicable to the high Arctic. Abovethe Axel Heiberg ice wedges, net production of CO2 abovecontrol levels occurred in late winter, which demonstratesthat ice wedge communities continue to respire duringsubzero soil temperatures (between - 9�C and - 16�C). Thedetection of significantly higher CO2 flux in polygon troughs,underlain by ice wedges, compared to polygon centers is astrong indication of the effects of ice wedge formation onphysical and microbiological parameters. The observedbuildup of CO2 in the ice wedge, evident in the high rate offlux from above the ice wedge borehole, affirms the previousfinding of biogenic CO2 gas released from ice wedges (La-celle et al., 2011). The CO2 flux rates obtained from the sur-face fit within the range of previously reported values fortundra in summer, 960–2520 mg/m2/day (Lee et al., 2010),and are closer to dry, summer tundra soil flux, 958–2660 mg/m2/day, than wet conditions, 3180–3220 mg/m2/day ( Joneset al., 1998). Previous studies of in situ CO2 flux from thesurface of polygon troughs and centers report a negativeflux: - 3800 and - 4300 mg/m2/day, respectively (Sommer-korn et al., 1999), highlighting the range of possible polygontypes, some of which have protruding, drier centers, whileothers may exhibit a wetter environment produced by ther-mokarst development (see Fig. 1). Our data suggest that thelow levels of respiration activity in ice wedges necessitatemore sensitive methods of detection; however, the goal ofusing laboratory and in situ techniques to validate results hasbeen illustrated here and provides evidence for the capacityof ice wedge habitats for supporting an active microbialcommunity.

For life to adapt and persist in ice, the ice environmentwould have to be relatively stable over evolutionary time-scales. On Earth, the Neoproterozoic Era (‘‘Snowball Earth’’)is believed to have played a significant role in the develop-ment of cold-adapted life (Vincent et al., 1999; Corsetti et al.,2006) over a period of *35 million years (Kirschvink et al.,2000). In the 700–900 million years following the formation ofplanet Mars, it is believed to have been a cold, wet planet.Remnants of ice-sculpted surface features indicate the pastpresence of large amounts of surface ice (Fairen et al., 2010).Under current atmospheric conditions on Mars, it is possiblethat remnants of ground ice could have persisted since thelast major ice age 400,000 years ago (Bryson et al., 2008) andprovided a plausible refugia for the development of ice-adapted life that may persist today.

Currently, there is no data on the physical conditions be-neath martian polygons or the diversity of martian wedge

356 WILHELM ET AL.

morphotypes, which limits any specific comparison of theirhabitability to terrestrial wedges. In general, it is certain thatany extant life in martian wedges would be subject to con-siderably harsher conditions than those on Earth, with morearid conditions, drastic daily temperature fluctuation, lowpressure, and high levels of radiation (Gomez et al., 2010).Temperatures near the equator (Opportunity landing site)oscillate between - 93�C and 17�C during the warmest timeof year (Ulrich et al., 2010), and mean surface temperaturesdecrease significantly from the equator ( - 43�C) to the poles( - 113�C) (Tillman et al., 1994). In comparison, the terrestrialice wedge environment is less dynamic, with temperaturefluctuations that occur on a seasonal basis and in situ tem-peratures that vary from - 15�C to 0�C (Christiansen, 2005).Martian soils are expected to mediate temperature fluctua-tions with some surface features and regions possessing ahigh volumetric heat capacity and low albedo (Mellon et al.,2000); however, specific information regarding polygonalfeatures and a temperature-depth profile is unavailable.

The Axel Heiberg ice wedges contained sizeable popula-tions of culturable cells with characteristics of a cold-adapted,halotolerant community. Isolates were not capable of signifi-cant growth at subzero temperatures and were not tested atthe levels of salinity (*23% NaCl w/v) necessary for liquidbrines to exist at martian temperatures (Ulrich et al., 2010).However, eight isolates did demonstrate halophilic charac-teristics and required 4% NaCl w/v or higher for growth.Modeling the surface temperatures and the expected salinityof common mineral ions on Mars, Ulrich et al. (2010) reportedthat the most extremophilic terrestrial microorganisms (met-abolically active at - 20�C) could be seasonally active inmartian climes. While our predominantly psychrotolerantisolates would not be active at such low temperatures, similarpsychrotolerant chemoorganoheterotrophs have demon-strated surprising hardiness under simulated martian condi-tions ( Johnson et al., 2011), including a member of genusArthrobacter that was abundant in our isolates. Furthermore,our selection of isolates does not likely represent the mostextremophilic, cold-adapted organisms present in the icewedge, since significant technical challenges exist for mim-icking in vitro ice conditions. Yet we were able to culture oneof the most abundant taxa, a Pseudomonas sp., which dem-onstrated high salt tolerance. Developing more advancedculturing methods for ice-based life should be a priority forastrobiologists, since an account of the true limits to terrestriallife is likely incomplete.

The major questions have yet to be answered regardingthe ecological function or limitations of microbial life in iceand ice wedge environments, though this research providesthe groundwork for what we might expect. The in situ re-spiratory activity of heterotrophic ice wedge communitieswill be very minimal due to both limited access to nutrientsand low diffusion of gases through ice. The characterizationof terrestrial ice wedges as habitable niches with a plausiblyactive microbial community has been strengthened by ourresults. This research also lays the groundwork for futurestudies at the field site on Axel Heiberg, which is considereda prime analogue for the climate during Mars’ second age,when the planet transitioned from a warmer, wetter planet toan increasingly drier and colder one (Pollard et al., 2009;Fairen et al., 2010). Ice wedges are an enticing habitat for thestudy of terrestrial extremophiles and are likely to contribute

insight for developing useful criteria for searching for life inice-rich environments.

Acknowledgments

Logistical and financial support was provided by theNatural Sciences and Engineering Research Council(NSERC) Discovery Grant, Canada Research Chair Program,Canadian Foundation for Innovation (CFI), the CanadianSpace Agency Canadian Analogue Research Network Pro-gram (CARN), the Department of Indian and NorthernAffairs–Northern Scientific Training Program (NSTP), PolarContinental Shelf Project (PCSP), and McGill University’sHigh Arctic Research Station. We would like to thankDr. Helen Vrionis for assistance with the radio-labeledmineralization assay.

Abbreviations

CFU, colony-forming units; DAPI, 4,6-diamidino-2-phe-nylindole; gfp, green fluorescent protein-labeled; OTUs, op-erational taxonomic units; PCR, polymerase chain reaction.

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Address correspondence to:Lyle G. Whyte

Department of Natural Resource SciencesMcGill University

21,111 LakeshoreSte. Anne de Bellevue, Quebec

H9X 3V9Canada

E-mail: lyle.whyte@mcgill.ca

Submitted 23 January 2012Accepted 27 February 2012

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