Results

- Microbial origin of excess methane in glacial ice and implications for life on Mars

Excess Cell Concentrations Correlated with Excess Methane. Fig. 3 Upper shows the concentrations of all cells as a function of depth, determined by scanning and counting Syto-23-stained cells, observed by their fluorescence, and Fig. 3 Lower shows the concentrations of unstained cells detected by their F420 autofluorescence. The number of cells found at a given depth ranged from 4 to 90 for Syto-23 and from 0 to 19 for F420 autofluorescence. The error bars shown in Fig. 3, resulting from application of Poisson statistics, are for an 84% confidence level; the upper limits of 90 cells per ml on F420 fluorescence at depths of 500 to 2,238 m correspond to a null count for a volume of 20 μl. On average, the ratio of concentrations of methanogens to all cells was ≈1 in 300, which is almost an order of magnitude lower than the ratio of ≈1 in 40 found earlier in the GISP2 silty ice (14).

We reiterate that although F420 is present at some level in certain members of all three domains, only in methanogens is its autofluorescence visible. In Fig. 3 Upper we see that, at depths of 4/ml, and we take that as the average microbial concentration resulting from aeolian deposition. In Fig. 3 Lower we see that at depths 5 cells per ml) of Syto-23-stained cells but no methanogens. Because Brook did not measure methane within 3 m of 2,238 m, the null result for methanogens is not significant. Brook measured methane at ≈3,000 m but did not find an excess.

The correspondence of our high concentrations of microbes, and especially of methanogens, with Brook's high methane concentrations at depths of 2,954, 3,018, and 3,036 m strongly supports our hypothesis that methanogenic metabolism accounts for the anomalously high methane in GISP2 ice. The large excess of Syto-23-stained cells at those three depths shows that those layers were also enriched in nonmethanogenic microbes. The absence of excess methane where we saw methanogens at ≈3,000 m suggests that the excess methane and methanogens may have been localized in a layer, probably no thicker than ≈1 m, that did not exactly correspond with the depth of Brook's sample.

To see whether the concentration of cells, including methanogens, was enhanced in the deep, disturbed regions of the GISP2 ice where no excess methane was found, we made measurements not only at 2,954, 3,018, and 3,036 m but also at ≈1 m above and below each of those depths. From Fig. 3 we see that ≈1 m above and below those depths, the concentrations of all cells (Upper) and of methanogens (Lower) drop to low values consistent with background. The sharp drops are consistent with Brook's results: 932 parts per billion by volume (ppbV) at 3,018 m and 632 and 633 ppbV at depths 2 m above and below it.

Metabolic Rates in GISP2 Ice. Price and Sowers (13) recently showed that the average metabolic rates of communities of microbes imprisoned in ice and other solid media can be calculated from the concentrations of trapped gas that they produced during a time t at an absolute temperature T. The metabolic rate R(T), defined as the fractional rate of turnover of carbon per cell per year, is given by

R(T) = Yj(T) / njmjt,

[1]

where Y_j(T) is the concentration of biogenic gas of type j at ice temperature T, with T taken from ref. 19; n_j is the concentration of microbial cells of type j; m_j is the mean carbon mass per cell, with m_j taken to be 19 fg based on measurements of cell size in the silty ice (14); and t is the retention time of the gas in the ice. For depths below ≈2,700 m (including 2,954, 3,018, and 3,036 m) in both GISP2 and GRIP ice, comparisons of δ¹⁸O of O₂ (20, 21) and methane (22), electrical conductivity (23), and stratigraphy (24) have shown that either one or both of the records was sufficiently disturbed that the age vs. depth record does not increase monotonically but falls within the range ≈100 to 250 kyr. Adopting the middle value, we take for retention time, t, the value 180 ± 80 kyr. We used Eq. 1 to estimate metabolic rates for microbial production of CH₄ and CO₂. Fig. 4 shows our results, together with results from Price and Sowers (13), on an Arrhenius plot of rate versus 1/T. In calculating rates for CO₂ production (solid-green triangles), we assumed that cells imaged with Syto-23 fluorescence were mainly anaerobic CO₂ producers such as Fe reducers and sulfate reducers and that the ratio of CO₂ to CH₄ concentrations was 20, as was found in the basal ice at the nearby GRIP (12). Rates for cells imaged in F420 autofluorescence are shown with solid-blue diamonds. The arrows indicate that those two rates might be overestimated if the scanning efficiency for detecting methanogens were 16) or had been in a low-pH environment (16) such as acidic veins (25). The carbon turnover times for the cluster of points reported here are inferred to be at least 10⁵ yr.

The dashed purple line in Fig. 4 is an extrapolation of an Arrhenius line for spontaneous racemization of aspartic acid in microbes from Siberian permafrost, measured at three temperatures from 100 to 145°C (26). The line through the three points in figure 4 of ref. 26 seems to be an exact fit, and no error is quoted. We estimate that the error in racemization rate extrapolated into the interval of 30 to –40°C is less than a factor of two. Rates for racemization of the other amino acids and for depurination of DNA are an order of magnitude lower (27) and can be neglected.