Cultivable microbial community in 2-km-deep, 20-million-year-old subseafloor coalbeds through ~1000 days anaerobic bioreactor cultivation

Abstract

Recent explorations of scientific ocean drilling have revealed the presence of microbial communities persisting in sediments down to ~2.5 km below the ocean floor. However, our knowledge of these microbial populations in the deep subseafloor sedimentary biosphere remains limited. Here, we present a cultivation experiment of 2-km-deep subseafloor microbial communities in 20-million-year-old lignite coalbeds using a continuous-flow bioreactor operating at 40 °C for 1029 days with lignite particles as the major energy source. Chemical monitoring of effluent samples via fluorescence emission-excitation matrices spectroscopy and stable isotope analyses traced the transformation of coalbed-derived organic matter in the dissolved phase. Hereby, the production of acetate and 13C-depleted methane together with the increase and transformation of high molecular weight humics point to an active lignite-degrading methanogenic community present within the bioreactor. Electron microscopy revealed abundant microbial cells growing on the surface of lignite particles. Small subunit rRNA gene sequence analysis revealed that diverse microorganisms grew in the bioreactor (e.g., phyla Proteobacteria, Firmicutes, Chloroflexi, Actinobacteria, Bacteroidetes, Spirochaetes, Tenericutes, Ignavibacteriae, and SBR1093). These results indicate that activation and adaptive growth of 2-km-deep microbes was successfully accomplished using a continuous-flow bioreactor, which lays the groundwork to explore networks of microbial communities of the deep biosphere and their physiologies.

Isolation of an archaeon at the prokaryote-eukaryote interface

“…For iTAG analysis, the universal primer pair 530F/907R, which contained overhang adapters at 5’ ends, was used. The procedures used for library construction, sequencing, and data analysis were described previously 19,46 .…”

Future Opportunities in Scientific Ocean Drilling: Illuminating Planetary Habitability

“…Interestingly, the diversity of microbial communities in these deeply buried coal-associated habitats was found to be very different from commonly observed subseafloor sedimentary microbes. In fact, they resemble anaerobic terrestrial communities living in peat or forest soil and can be cultivated by using down-hanging sponge bioreactors (Inagaki et al, 2015;Imachi et al, 2019). Furthermore, diverse fungal species of both Ascomycota and Basidiomycota isolated from the lignite coal and shale samples resemble terrestrial wood-rotting fungal communities (Liu et al, 2017).…”

Evaluation of reported sediment samples from 20 Ma using a molecular phylogenetic approach: comment on Liu et al. (2017)

“…() and Imachi et al . () would overturn the conventional wisdom on ancient DNA longevity, it is again essential to carefully evaluate the authenticity of their sample dating.…”

“…Recently, microbiological studies involving extraordinarily old samples have been reported (Liu et al, 2017;Imachi et al, 2019). These reports concerned the isolation of fungal communities from deep subseafloor sediments dated to 20 Ma (mega-annum) (Liu et al, 2017) and cultivation of bacteria from 2-km-deep subseafloor microbial communities in lignite coalbeds from 20 Ma (Imachi et al, 2019). These findings are novel in two ways.…”

“…They found that 50-bp fragments can survive much longer than the model predicted, with 0.4%-1.8% of fragments remaining after one million years. Because the studies by Liu et al (2017) and Imachi et al (2019) would overturn the conventional wisdom on ancient DNA longevity, it is again essential to carefully evaluate the authenticity of their sample dating.…”