INTRODUCTION

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Microbial activity has been found in ice-sediment communities in the surface layers of perennial and permanent lake ice at a depth of 2 m (30, 31). The lake ice microbial consortia are dependent on photosynthesis to provide energy for growth. In subglacial environments bacterial populations have been found beneath alpine glaciers (35) and in subglacially accreted ice above Lake Vostok, Antarctica (20, 29). However, the biogeochemical function of these populations has not been determined, and it remains to be demonstrated that they are active at in situ subglacial temperatures and under in situ conditions. Clearly, active subglacial microbial populations would have to be chemotrophic (nonphotosynthetic). Demonstrating the viability and geochemical function of subglacial microbial populations has a number of important consequences.

First, the presence of viable chemotrophic microbial activity at low temperatures (0 to 4°C) has important implications for global carbon cycling calculations. Traditionally, it has been thought that continental glaciation results in cessation of geochemical processes beneath the ice (13), and the impact of subglacial microbially mediated weathering has received little consideration. However, during the last glacial maximum, ice sheets covered approximately 20% of the continental northern hemisphere (16, 23, 24), including the area that is currently covered by the boreal forest, the world's largest store of soil carbon, whose size is estimated to be 330 Pg (40). A similar distribution of vegetation has been proposed for the last interglacial period (Eemian), as recorded in glacially overridden soils and peat (10, 32) and paleosols in unglaciated terrain beyond the ice margin (26). These overridden soils and peat deposits provide a large source of organic carbon beneath the midlatitude ice sheets. A number of the current carbon cycle models assume that the carbon accumulated during the Eemian in areas covered by the ice sheets was returned to the atmosphere by the last glacial maximum (1, 12, 40), but there has been no explanation as to how this was achieved. Ice is an efficient erosive agent (2, 15), and, therefore, it is likely that some of the carbon may be moved by physical transport to the ice sheet margins, either in ice, in deforming sediments, or in meltwater. However, active biogeochemical oxidation or reduction of the remaining carbon beneath warm-based sectors of midlatitude ice sheets may have a significant impact on carbon budget calculations for continental regions during the glacial phase of a glacial-interglacial cycle. Moreover, low-temperature respiration and/or fermentation of organic carbon in the subsurface environments of periglacial (33, 41) soils in unglaciated midlatitude regions and ice marginal zones is potentially an important process that previously has been given little consideration on glacial-interglacial time scales.

Second, extreme polar terrestrial environments are being investigated as analogues of viable extraterrestrial habitats (3, 14). Currently, water on Mars has been observed only as ice in the two polar caps (8). Given that all known bacteria on Earth require liquid water to be metabolically active, some of the most likely potential habitats for microbial life on Mars are the polar environments (36). Moreover, subglacial environments could provide shelter from the harsh conditions at the planet's surface, including large diurnal and seasonal temperature fluctuations and strong UV radiation. Some models suggest that during times of high obliquity on Mars, surficial melting may have occurred in the north polar ice cap (28). Under these conditions, the ice cap may have exhibited polythermal conditions and may have been more dynamic (19), and thus basal melting may have occurred, producing sediment-rich basal ice. Hence, debris-rich ice exposed at the margins of the north polar ice cap or as part of polar layered deposits may provide a record of dormant or possibly extant microbial life that is relatively accessible.

In this study we performed a number of laboratory experiments in which we assessed the diversity and viability of microbes from a high Arctic glaciated environment and examined the effects of microbial activity on biogeochemical processes. In the experiments we also compared microbial population diversity in surficial glacier environments (supraglacial waters and glacier ice, which are characterized by low solute concentrations [<10 µS cm¹] and low sediment concentrations [<0.01 g liter¹]) with microbial population diversity in subglacial environments (subglacial meltwaters and basal ice, which are characterized by high solute concentrations [>100 µS cm¹] and high sediment concentrations [>0.1 g liter¹]).

	MATERIALS AND METHODS

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Field site. The study was performed with samples collected from John Evans Glacier (79°38'N, 74°23'W) on eastern Ellesmere Island, Nunavut, Canada. The local climate is a polar desert, and the mean annual air temperature at the glacier terminus is 14.5°C (4). The glacier has polythermal characteristics; cold (subfreezing) ice at the surface, margins, and terminus of the glacier surrounds a core zone where ice at the glacier bed is at the pressure melting point and basal melting occurs. This core zone begins ca. 7 km upglacier from the glacier snout, where the ice is ca. 400 m thick. Between 4 and 7 km from the glacier snout, temperate ice occurs only at the glacier bed, but in the lowest 4 km of the glacier, where the ice is up to 200 m deep, there is a layer of temperate basal ice up to 20 m thick (L. Copland and M. J. Sharp, submitted for publication). Debris-rich basal ice up to 0.5 m thick is widely exposed at the glacier margin. During the summer melt season, meltwaters which have acquired solutes from subglacial chemical weathering reactions drain from the glacier at a number of locations (35).

Definition of ice and water types. Glacier ice is formed by firnification or superimposed ice generation (refreezing of percolating waters in near-surface snow and firn) at the glacier surface, and it has low solute concentrations (<10 µS cm¹) and low sediment concentrations (<0.01 g liter¹). Supraglacial meltwaters are generated by melting of snow and ice and flow on the surface of the glacier. They are also characterized by low solute and suspended-sediment concentrations. Subglacial waters flow at the bed of the glacier and originate as supraglacial waters which reach the bed through crevasses and moulins and mix with small amounts of groundwater and basal meltwater generated by frictional and geothermal melting of ice at the glacier base. Subglacial waters interact with the rock and sediments that underlie the ice and hence contain high solute concentrations (>100 µS cm¹) and high suspended-sediment concentrations (>0.1 g liter¹). Basal ice contains significant sediment concentrations and forms at the glacier bed by a number of mechanisms, which could include folding (17), regelation of ice into the substrate (18), and basal accretion of supercooled subglacial water (21). This ice provides a record of the geochemical and microbiological conditions at the glacier bed and lies stratigraphically unconformably beneath the glacier ice. At John Evans Glacier the basal ice layers were probably formed by either regelation of ice into the substrate or basal accretion of supercooled water since there is no structural evidence of folding.

Water samples collected in 1996. In the first set of experiments undertaken in 1996 we examined the viability of microbes present in glacial meltwaters having different chemical compositions and suspended-sediment concentrations, and we also examined the relationship between suspended-sediment concentration and microbiological activity. In July 1996, 4-liter samples of subglacial and supraglacial meltwater were collected aseptically from the terminus of John Evans Glacier by using twice-autoclaved plastic carboys (Nalgene) with screw caps fitted with sterile air ports (0.2-µm pore-size Teflon PTFE filters; Nalge). These samples were amended in the field with 10× sterile R2A growth medium lacking agar so that the final concentration was 0.1× R2A medium (1× R2A medium contains [per liter of doubly glass-distilled water] 0.5 g of yeast extract [Difco], 0.5 g of Casamino Acids [Difco], 0.5 g of glucose, 0.5 g of soluble starch [Difco], 0.3 g of K₂HPO₄, 0.3 g of sodium pyrophosphate, 0.25 g of BiTek tryptone [Difco], 0.25 g of Bacto Peptone [Difco], and 24 mg of MgSO₄; adapted from the medium described by Atlas [5]). Samples were stored on ice in the dark at 4°C for up to 1 month in the field, transported to the laboratory (72 h) at 4°C, and then incubated statically in the dark at 4°C. For comparison, other subglacial and supraglacial water samples were incubated without R2A medium. The pH values and dissolved oxygen concentrations of the samples were measured at the time of sampling and 7 weeks later by using an Orion model 290A pH meter fitted with a Ross Sure-flow pH electrode and an Orion dissolved oxygen probe (model 97-08), respectively. H₂S concentrations were determined colorimetrically by using a Hach H₂S test kit.

Ice samples collected in 1997. In the experiments in which we used ice samples collected in 1997 we examined both microbial diversity and rates of biogeochemical activity in thawed samples of glacier ice and basal ice. The chemical and microbiological characteristics of the thawed basal ice also provided information about the nature of subglacial environments. In May 1997, samples of basal ice and glacier ice were collected aseptically from the glacier terminus by using ethanol-flame-sterilized ice axes. The surface few centimeters of ice were scraped away prior to sampling to avoid atmospheric contamination. Samples were then chipped into flame-sterilized metal collection trays and transferred without handling into sterile plastic bags (Whirl-Pak; Nasco Plastics, New Hamburg, Ontario, Canada). Samples remained frozen until they were used in the experiments. Subsequent manipulations were carried out aseptically in a biosafety cabinet with HEPA-filtered airflow.

(i) Anaerobic incubation. Ice was thawed at 4°C in sterile containers and transferred into 168-ml bottles that were fitted with gas-impermeable septa and contained 50-ml portions of half-strength growth media, as follows: nitrate medium (25) and sulfate medium (7) pregassed with sterile 10% CO₂-90% N₂; and R2A medium pregassed with sterile 0.5% CO₂-99.5% N₂. The prepared bottles containing media were precooled on ice prior to inoculation. For glacier ice samples, the inoculum consisted of 50 ml of meltwater, whereas for basal ice 5 g of sediment plus 45 ml of associated meltwater were used. Parallel uninoculated controls containing 100 ml of each growth medium were also prepared. Inoculated, poisoned controls containing 20 mM Na₂MoO₄ and 500 µM HgCl₂ were also included to evaluate abiotic contributions to the aqueous geochemistry. Unamended background controls containing melted glacier ice (100 ml) or basal ice (10 g of sediment plus 90 ml of meltwater) were also prepared. All cultures were sealed with the appropriate sterile headspace gases and incubated statically in the dark at 4°C for 90 days. At 14-day intervals, 0.5-ml samples were withdrawn aseptically through the septa by using fine-gauge needles and syringes. Basal ice samples were clarified by using a 0.45-µm-pore-size filter membrane (Millipore) prior to analysis. The concentrations of Cl, NO₃, and SO₄² in the samples were determined with a Dionex model DX500 ion chromatograph by using a Dionex Ionpac AS4 column and an eluent consisting of 1.7 mM Na₂CO₃ and 1.8 mM NaHCO₃. Headspace concentrations of CO₂ and CH₄ were determined with a Hewlett-Packard model 5890 Series II gas chromatograph equipped with a thermal conductivity detector and with a VarianStar model 3400 or Hewlett-Packard model 5700A gas chromatograph fitted with flame ionization detectors, respectively. The ¹³C values of headspace gases were determined by continuous-flow-isotope ratio mass spectrometry by using a Hewlett-Packard model 5890 Series II gas chromatograph connected to a Finigan Mat 252 mass spectrometer and are reported relative to VPDB.

(ii) Aerobic incubation. Samples consisting of 100 ml of melted glacier ice or 30 ml of melted basal ice were amended with 50 ml of half-strength R2A medium. A parallel uninoculated control containing 50 ml of medium was included. All cultures received portions of a filter-sterilized (pore size, 0.2 µm) solution of sodium [2-¹⁴C]acetate which resulted in final acetate concentrations of 50 µM and 98,000 dpm per flask. The culture flasks were sealed with neoprene stoppers with an aerobic headspace and were incubated at 8°C in the dark with gyratory shaking at 200 rpm for 270 days. At intervals, subsamples of liquid plus headspace were removed aseptically by using a needle and a syringe, acidified, and sparged with N₂ to trap and quantify the evolved ¹⁴CO₂ (11).

Ice samples collected in 1998. In the experiments performed in 1998 and 1999 we recreated in situ subglacial conditions as closely as possible in order to establish whether the microbes were viable and active under such conditions. The rates of microbial respiration were measured for both thawed glacier ice and basal ice at temperatures below and just above 0°C. All manipulations were carried out on ice, and the media used were prechilled to ensure that culture temperatures remained as close to 0°C as possible. Experiments were conducted by using fresh samples of basal ice, which was collected aseptically in July 1998 (as described above) at a site 3 km from the 1997 collection site, and glacier ice collected in 1997, which had been stored frozen and undisturbed in the original sterile bags. Samples were melted at 4°C in sterile containers, and subsamples were transferred with HEPA-filtered airflow to sterile 250-ml bottles fitted with gas-tight septa. Unamended samples consisted of 200 ml of melted glacier ice or 150 g of basal ice sediment plus 75 ml of associated meltwater. Another set of samples was amended by replacing 20 ml of melted ice with 20 ml of R2A medium, which resulted in a final concentration of 0.1× R2A medium. Heat-sterilized controls were also prepared consisting of basal ice containing 100 g of twice-autoclaved sediment plus 28 ml of associated meltwater, glacier ice containing 200 ml of meltwater, and an uninoculated control containing 200 ml of sterile medium. Each culture was supplemented with a filter-sterilized solution of sodium [2-¹⁴C]acetate so that it contained 195,000 dpm and an acetate concentration of 50 to 130 µM depending on the total volume of the sample, and then it was sealed under an aerobic headspace. One set of unamended samples was incubated at 4°C in the dark. The contents of the remaining bottles were frozen by immersion in glycol at 4.8°C; the bottles were shaken every 15 min during freezing to ensure even distribution of any sediment within the ice that was forming. These cultures were incubated in the dark and were submerged in a refrigerated glycol circulator bath (Cole Parmer Polystat) at 4.8°C for 31 days, then at 1.8°C for 35 days, and finally at 0.3°C for 91 days. The bath temperature was monitored by using a thermistor (Campbell Scientific, model 107B) connected to a model CR10 datalogger (Campbell Scientific) and remained within ±0.3°C of the desired temperature. After 66 days the frozen samples were melted at 0.3°C, and the ¹⁴CO₂ that evolved was measured by aseptically removing a subsample of liquid and quantifying the ¹⁴CO₂ (11); sediment-laden samples were first clarified by filtration through a Millipore Millex GS filter unit (pore size, 0.2 µm). The thawed samples were incubated for an additional 91 days at 0.3°C, and subsamples were removed and used for ¹⁴CO₂ analysis at intervals. Samples of the unfrozen (4°C liquid) cultures were obtained after 97, 122, and 157 days and were analyzed to determine the evolved ¹⁴CO₂ content as described above (11). Extraction of all subsamples was performed in a biosafety cabinet, and all cultures were kept on ice during the procedure.

Microscopy. Transmission electron microscopy (TEM) was performed with both cultured and uncultured samples of glacier ice and basal ice in order to examine microbial morphology. Drops of melted ice or amended cultures were placed on Formvar-coated copper grids, briefly dried in vacuo, and then negatively stained with filter-sterilized (pore size, 0.2 µm) 2% phosphotungstic acid and examined with a Philips model 201 electron microscope.

OC concentrations. To examine the availability of organic carbon (OC) in catchment materials, 20 sediment samples were obtained from a variety of soil or sediment environments in the proglacial and ice marginal zones. A subsample of basal ice was also obtained and used for a total OC (TOC) determination. All sediment samples were dried at 105°C for 24 h and allowed to cool in a desiccator prior to weighing. The samples were combusted at 550°C for 16 h and then allowed to cool in a desiccator prior to weighing. The TOC content (expressed as a percentage, by weight) was calculated from 0.4× organic matter content (the weight lost between the drying stage and the combustion stage) (6).

	RESULTS

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Water samples collected in 1996. The initial experiments were primarily qualitative and were intended to determine whether viable microbes were present in subglacial and/or supraglacial waters. After 7 weeks of incubation at 4°C, a 4-liter subglacial sample amended with 0.1× R2A medium had a decreased dissolved oxygen level (1.7 versus 10.9 ppm of O₂) and a decreased pH (pH 6.5 versus 7.8) compared to an unamended sample and contained 0.7 ppm of H₂S (H₂S was not detected in the unamended sample). By contrast, there was no change in the geochemistry of the amended supraglacial sample. When subsamples of R2A medium-amended subglacial waters were incubated with anaerobic nitrate or sulfate medium at 4°C, the media became brown (nitrate medium) or black (sulfate medium) and turbid within 6 weeks. Subsamples of unamended subglacial waters were slower to show positive results (4 months at 4°C), whereas unamended supraglacial waters did not yield positive results within 4 months. Parallel bottles inoculated with the laboratory control medium remained sterile, demonstrating that the anaerobes did not come from the R2A medium. These subculture experiments were repeated several times, and the same results were obtained each time, which confirmed the observation that viable respiring anaerobes were present in subglacial waters. We noted that the appearance of turbid cultures was qualitatively proportional to the amount of mineral sediment associated with the water sample.

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Ice samples collected in 1997. The qualitative results obtained with subglacial and supraglacial water samples described above suggested that the subglacial environment harbored viable populations of both aerobes and anaerobes and thus that direct study of the ice originating from the glacier base was merited. Experiments performed with basal ice showed that aerobic and anaerobic microbial activities were detectable at 8 and 4°C, respectively, if a sample was amended with low levels of organic carbon (i.e., growth medium) or a terminal electron acceptor, such as NO₃ or SO₄². Microbial activity was observed in cultured basal ice but not in cultured glacier ice, again suggesting that there was an association with the sediment. The absence of microbial activity in the glacier ice confirmed that we were using a suitable aseptic technique to prevent contamination of the samples during sampling and analysis.

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View larger version (18K): [in this window] [in a new window]   FIG. 1.   Anaerobic incubation of thawed ice samples at 4°C in the dark. Samples were amended with dilute nitrate medium (5 mM NO3) (A) or dilute sulfate medium (14 mM SO42) (B). Symbols: , basal ice; , glacier ice; , uninoculated medium.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Aerobic incubation of thawed ice samples in the dark. Samples collected in 1997 were supplemented with 98,000 dpm of sodium [2-14C]acetate and incubated at 8°C for 270 days with gyratory shaking at 200 rpm (, basal ice plus dilute R2A medium; , glacier ice plus dilute R2A medium; , dilute R2A medium). Samples collected in 1998 were supplemented with 195,000 dpm of sodium [2-14C]acetate and incubated statically at 4°C for 157 days (, basal ice without R2A medium; , glacier ice without R2A medium).

Ice samples collected in 1998. Whereas the 1997 ice samples were incubated at temperatures higher than those expected in situ and were modestly amended with nutrients, the 1998 ice samples were incubated under conditions as close as possible to those found at the base of the glacier (temperature, ca. 0°C) with minimal organic carbon amendment that was sufficient to detect respiration. The experimental temperature was set at 0.3°C in order to ensure that the entire slurry remained in liquid form (>0°C) due to the tolerances (±0.3°C) of the temperature bath. The experiments were designed to demonstrate not only that viable microbes were present in the basal ice but also that they were active under simulated in situ subglacial conditions. Activity was assessed simply by quantifying aerobic mineralization of radiolabeled acetate to ¹⁴CO₂. [¹⁴C]acetate incorporation into biomass and ¹⁴CO₂ depletion by autotrophy were not assessed, nor was oxygen in the sealed headspace replaced during incubation. Thus, the activity measured should be considered a conservative estimate of aerobic activity.

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Aerobic incubation of 1998 ice samples in the dark at increasing temperatures (4.8, 1.8, and 0.3°C). All samples were supplemented with 195,000 dpm of sodium [2-14C]acetate. Symbols: , basal ice plus dilute R2A medium; , glacier ice plus dilute R2A medium; , basal ice without R2A medium; , glacier ice without R2A medium; , basal ice sterile control; , glacier ice sterile control. Incubation temperatures are also shown.

Microscopy. Microscopic studies revealed that microbes were present in both freshly thawed uncultured basal ice samples and cultured basal ice samples taken in 1998. Typical prokaryotic cell morphologies, including bacilli, cocci, and cells undergoing division, were observed in both cultured and uncultured basal ice samples when TEM was used (Fig. 4). Cells were not exclusively associated with sediment in basal ice samples and were often observed singly rather than in microcolonies or obvious biofilms.

View larger version (73K): [in this window] [in a new window]   FIG. 4.   TEM images of bacteria in meltwater from basal ice samples. Scale bars = 0.5 µm. (A) Coccus associated with sediment from uncultured basal ice immediately after thawing. (B and C) Long thin rod (B) and actively dividing cocci (C) from an aerobic stationary culture incubated at 0.3°C without R2A medium. (D) Short, fat rods with inclusions, associated with sediment from an aerobic culture incubated at 4°C without R2A medium.

Allochthonous OC. Uncultured 1998 basal ice samples had sediment concentrations of 2,000 g liter¹ whereas the glacier ice did not contain visible particulates. The particulate OC concentrations in the basal ice were 0.53 to 0.97 M, compared with 34 µM in the glacier ice. Basal ice had DOC concentrations of 100 µM, compared with 24 µM in the glacier ice. This indicates that there was a much larger source of OC in the basal ice. Sediment samples obtained in the proglacial and ice marginal zones contained cyanobacterial mats, mosses, and plant remains that comprised 0.3 to 4.1% (wt/wt) OC. This confirmed that glacially overridden sediments can provide a source of allochthonous OC that serves as a carbon and energy source in the subglacial environment.

	DISCUSSION

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The 1996 experiments clearly demonstrated that viable microbes are present in both the supraglacial and subglacial environments of a polythermal Arctic glacier. We found that microbial activity increased as the suspended sediment concentration in the water samples increased, which was similar to findings obtained for alpine subglacial environments (35).

The 1997 experiments confirmed that there are culturable microbial populations in the debris-rich basal ice. Moreover, the basal ice harbored numerous types of aerobic and anaerobic bacteria, including heterotrophs, nitrate and sulfate reducers, and methanogens. It is particularly interesting that methanogens were viable in cultures because the samples were not obtained or stored in an anaerobic environment. Moreover, the dissolved oxygen content was close to 100% of the saturation value in the subglacial meltwaters. However, there is considerable variability in the sensitivity of methanogens to oxygen, and viable methanogens have been detected in nominally aerobic environments where anaerobic microenvironments or transient anaerobic conditions occur (42). The presence of methanogens suggests that although the subglacial meltwaters are essentially aerobic, the underlying thawed subglacial sediments contain anaerobic microenvironments which are retained as the basal ice forms.

The 1998 culture experiments showed that aerobic microbial growth can occur in the thawed basal ice under conditions as close to in situ conditions as could be achieved in the laboratory, with minimal amendment of OC and without additional nutrients. Addition of 0.1× R2A growth medium had no effect on the respiration rates in the thawed basal ice samples but resulted in significant increases in the respiration rates in the thawed glacier ice. The latter finding suggests that microbes are present in the glacier ice but are more nutrient and carbon limited than the microbes in the basal ice, probably because of the low solute content of the thawed glacier ice. Hence, the microbes are not necessarily physically associated with sediment particles. Our experiments also revealed no sign of aerobic respiration at subfreezing temperatures; however, it is likely that longer experiments would be required to confirm these observations.

The DOC concentration in the basal ice was at least twice the DOC concentration used for amendment in the culture experiments, and the TOC concentrations were orders of magnitude greater. This confirms that there is sufficient OC available in the subglacial sediments to facilitate subglacial microbial metabolism. The source of the OC in subglacial sediments is most likely permafrozen soils that are overridden by the advancing glacier and then finely ground by subglacial abrasion processes. The organic material in present-day proglacial and ice marginal sediments, which are probably similar to the source material for the subglacial sediments, consists of cyanobacterial mats, plant material, and roots. These types of carbonaceous material are readily biodegradable by microbial activity.

Based on the experiments performed with waters and ice it is evident that there is a clear link between the presence of sediment and the level of microbial activity. The sediment is probably the primary source of the microbes, which are present in the soils and sediments that the glacier overrides. As determined by TEM (Fig. 4A and D), microbes are clearly associated with the sediment, although they may not be physically attached. Additionally, the sediment provides a source of carbon, and weathered sediments produce aqueous nutrients for the subglacial microbial community. Microbes may also be washed into the subglacial environment from the glacier surface; however, it appears from the culture experiments that the combination of microbes, nutrients, and carbon necessary to support viable microbial activity is present only in the subglacial environment. Similarly, in the ice-sediment microbial communities in perennial Antarctic lake ice (30) there is a link between the concentration of sediment in the ice and microbial activity. The sediment provides nutrient-enriched microzones that support the microbial community, and there is evidence that the bacteria are physically attached to the sediment in these microzones. However, the highest concentrations of bacterial cells and DOC are not directly correlated with high sediment concentrations.

The experimental cultures and DOC-TOC results indicate that there are sufficient nutrients and carbon to support aerobic microbial activity in unfrozen basal sediments beneath John Evans Glacier. Therefore, to maintain the aerobic respiration processes in the subglacial environment, a supply of oxygen is clearly required. The potential oxygen sources in the glacier base include oxygenated surficial waters that reach the bed via crevasses or subglacial melting of glacier ice. Assuming that glacier ice contains 0.09 cm³ air of standard composition g of ice¹ (39) and the basal rate of melting is 12 mm year¹ (6 mm year¹ due to geothermal heating and 6 mm year¹ due to friction, assuming that ice slides at a rate of 20 m year¹) (27), basal melting delivers O₂ to the glacier bed at a rate determined by the product of the basal melt rate and the O₂ content of the ice, i.e., 1 mM O₂ m² year¹. If surficial waters reach the bed, the O₂ supply would be significantly higher. If the oxygen supply to the glacier base is completely exhausted, then it is evident that there is a diverse population of anaerobic bacteria in the unfrozen sediments. These anaerobic bacteria are viable at 4°C, and it is highly likely that they would also function at 0.3°C, albeit at lower metabolic rates. Hence, both aerobic and anaerobic microbial activities are tenable processes in unfrozen sediments beneath a high Arctic polythermal glacier. The implications of these findings are relatively wide ranging and are outlined below.

Glacial-interglacial carbon cycling. Microbial populations are found in a range of perennially frozen environments (14, 30, 31, 33), including subglacial environments (20, 29, 35; this study) and subzero marine sediments (34). These environments contain water, nutrients, carbon, and oxygen or a suitable electron acceptor and thus provide the necessary constituents to support microbial activity. Thus, it seems likely that microbial populations were active in the temperate-based sectors of the Pleistocene midlatitude ice sheets, in which water and OC (in overridden boreal soils and peat) were present (32; Copland and Sharp, submitted). Viable aerobic and anaerobic bacteria that are functionally similar to the bacteria cultured from thawed basal ice at John Evans Glacier have been resuscitated from ancient (3-million-year-old) permafrozen boreal soils from Siberia and cultured at 8 to 4°C (14, 33). Hence, reactivation of microbes during sub-ice sheet thawing of permafrozen soils and peat is a tenable process.

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Ice core gases. In this study we found that strictly anaerobic methanogenic bacteria are present in thawed basal ice samples. These bacteria are viable in low-temperature cultures, which suggests that within the unfrozen basal sediments there are anaerobic microenvironments that have the potential to generate methane subglacially. This provides a possible alternative interpretation for the high greenhouse gas load observed in debris-rich basal ice from both the Dye 3 (38) and GRIP (39) ice cores from the Greenland ice sheet (Dye 3, 40,000 parts per million by volume (ppmv) of CO₂; GRIP, 130,000 ppmv of CO₂ and 6,000 ppmv of CH₄). It is thought that the high greenhouse gas load in the basal ice results from incorporation of ground ice into the glacier ice by glacier flow-induced mixing during ice sheet buildup (38, 39). Our results suggest that the gases in these ancient samples may include products of in situ microbial activity at the base of the ice sheet, so that preglacial CH₄ production and subsequent flow mixing may not be required to explain the observed CH₄ values.

Martian polar analogue. In this study we demonstrated that the subglacial environment beneath a polythermal glacier provides a viable habitat for microbial life. There is access to liquid water and sediment (nutrients), and sub-ice microbial communities are protected from diurnal and seasonal temperature fluctuations. Therefore, this environment may provide a model for viable habitats for life on Mars, since similar conditions may exist or may have existed in the basal sediments beneath the Martian north polar ice cap (9).