Ex Situ Culturing Experiments Revealed Psychrophilic Hydrogentrophic Methanogenesis Being the Potential Dominant Methane-Producing Pathway in Subglacial Sediment in Larsemann Hills, Antarctic

Abstract

It was recognized only recently that subglacial ecosystems support considerable methanogenic activity, thus significantly contributing the global methane production. However, only limited knowledge is available on the physiological characteristics of this kind of methanogenic community because of the technical constraints associated with sampling and cultivation under corresponding environmental conditions. To elucidate methanogenesis beneath the glacial margin in East Antarctic Ice Sheet, we took an integrated approach that included cultivation of microbes associated with the sediment samples in the lab and analysis of mcrA gene therein. After 7 months of incubation, the highest rate of methanogenesis [398 (pmol/day)/gram] was observed at 1°C on a supply of H₂. The rates of methanogenesis were lower on acetate or unamended substrate than on H₂. The rates on these two substrates increased when the temperature was raised. Methanomicrobiales predominated before and after prolonged incubation, regardless whether H₂, acetate, or unamended substrate were the energy source. Therefore, it was inferred that psychrophilic hydrogenotrophic methanogenesis was the primary methane-producing pathway in the subglacial ecosystem we sampled. These findings highlight the effects of temperature and substrate on potential methanogenesis in the subglacial sediment of this area, and may help us for a better estimation on the Antarctica methane production in a changing climate.

Keywords: East Antarctic; climate change; ex situ cultivation; hydrogenotrophic methanogenesis; mcrA gene; subglacial ecosystem.

FIGURE 1

Methane production in subglacial sediment during the incubation in triplicate with different substrates at 1°C (o), 4°C (□), and 12°C (x).

FIGURE 2

Methane production rates in subglacial sediment incubated in triplicate at different temperatures with different substrates.

FIGURE 3

Phylogenetic tree based on translated partial amino acid sequences of the mcrA gene from raw Antarctic subglacial sediment. The sequences from this study and reference sequences were aligned with ClustalW in MEGA 6.0. The tree was constructed with FastTree (Version 2.1.3, ML Model: Jones–Taylor–Thorton) and visualized with MEGA 6.0. The scale bar represents a difference of 0.1 substitutions per site. The mcrA gene sequences marked with black triangles were derived from this study. The number in parentheses indicates the clone proportion.

FIGURE 4

Increase in mcrA gene copy number in subglacial sediment after incubation for 7 months. The mcrA gene copy number in the original sediment was 2.3 × 10⁴ copy/g sediment according to the q-PCR results. (mean ± SEM, triplicate).

FIGURE 5

Methane production rate ((pmol/g)/day) in subglacial sediment and permafrost. For the subglacial sediment, the ex situ incubation temperature and the substrate (H₂: H₂ supplied; Ace: acetate supplied; Raw: no additional substrate supplied) concentrations were obtained from corresponding experiments. For the permafrost sediment, the ex situ data were obtained from corresponding experiments. The in situ data were obtained from the in situ static chambers. The in situ flux values were converted from (mg/m²)/day to (pmol/g)/day according to the depth of active layer and 2 g/cm³ density of soils. Data were derived from this study and references (Supplementary Table S2).