Biological methane production under putative Enceladus-like conditions

Abstract

The detection of silica-rich dust particles, as an indication for ongoing hydrothermal activity, and the presence of water and organic molecules in the plume of Enceladus, have made Saturn's icy moon a hot spot in the search for potential extraterrestrial life. Methanogenic archaea are among the organisms that could potentially thrive under the predicted conditions on Enceladus, considering that both molecular hydrogen (H₂) and methane (CH₄) have been detected in the plume. Here we show that a methanogenic archaeon, Methanothermococcus okinawensis, can produce CH₄ under physicochemical conditions extrapolated for Enceladus. Up to 72% carbon dioxide to CH₄ conversion is reached at 50 bar in the presence of potential inhibitors. Furthermore, kinetic and thermodynamic computations of low-temperature serpentinization indicate that there may be sufficient H₂ gas production to serve as a substrate for CH₄ production on Enceladus. We conclude that some of the CH₄ detected in the plume of Enceladus might, in principle, be produced by methanogens.

Fig. 1

Influence of the different headspace gas compositions on growth of M. marburgensis, M. villosus, and M. okinawensis. The error bars show standard deviations calculated from triplicates. OD curves of a, d, g, j, m M. marburgensis, b, e, h, k, n M. villosus and c, f, i, l, o M. marburgensis for a–c H₂/CO₂, d–f H₂/CO, g–i H₂, j–l Mix 1, and m–o Mix 2. Growth of M. marburgensis was inhibited by the presence of C₂H₄ (see Table 2 for detailed gas composition). Only M. marburgensis seemed to be able to use sodium hydrogen carbonate (supplied in the medium) as C-source in case of a lack of CO₂ (H₂ or H₂/CO as sole gas in the headspace). Both, M. villosus and M. okinawensis showed growth when Mix 1 and Mix 2 were applied to the serum bottle headspace; however, M. villosus exhibited extended lag phases. The dips in the graphs b, c were caused by substrate limitation due to depletion of serum bottle headspace of H₂/CO₂ at high-optical cell densities

Fig. 2

Schematic of the experimental setting and DoE raw data growth curves showing OD measurements. The DoE is based on a central composite design (figure in the upper left corner). NH₄Cl, CH₂O, and CH₃OH were used as factors during the experiment and systematically varied in a multivariate design space (see Supplementary Table 1 for the concrete values). Each of the factors setting was examined in triplicates. The centre point (O) was examined in quintuplicates. The colours of the dots and the letters of the figure in the upper left corner correspond to the growth curves. The line labelled ZC represents the optical density of a corresponding zero control experiment, which was done with the same medium as the experiments labelled with O (central point), but without inoculum. The different colours represent different performances. For better readability, the error bars in this diagram were excluded, which were in a standard deviation range between 0.0009 and 0.1544. According to statistical selection criteria three experiments (one experiment F and two experiments O) were excluded from ANOVA analysis (Supplementary Table 2)

Fig. 3

Growth and turnover rate of M. okinawensis under Enceladus-like conditions at 2 bar. a, c, e Growth curves (OD_578 nm) and b, d, f turnover rates (h⁻¹) as a measure of CH₄ production of M. okinawensis on a, b H₂/CO₂ (4:1), c, d Mix 1 and e, f Mix 2. For detailed composition of gases and media see Table 2 and Supplementary Table 3. I and II (light and dark colours, respectively) denote two independent experiments (each performed in triplicates, error bars = standard deviation). Enceladus-like concentrations were used for NH₄Cl and CH₃OH and mean liquid inhibitor concentrations determined in the DoE were used for CH₂O. The dip in a was caused by substrate limitation due to depletion of serum bottle headspace of H₂/CO₂ at high-optical cell densities

Fig. 4

CH₄ production, MER, and turnover rate of M. okinawensis under Enceladus-like conditions at high pressure. Biological CH₄ production determined by gas chromatography (blue) (Vol.-% h⁻¹) and turnover rates (h⁻¹) (green) and MER·10 (mmol L⁻¹ h⁻¹) (red) measured from headspace gas conversion using M. okinawensis (experiment 1 in light colours, experiment 2 in dark colours) under putative Enceladus-like conditions in a 2.0 L bioreactor (for detailed medium composition, see Supplementary Table 3 and for detailed gas composition see Table 4, n = 2). The positive control experiment contained also the liquid inhibitors but only H₂/CO₂ (4:1) in the headspace. For high-pressure experiments without any inhibitors see Supplementary Fig. 2