Accessing the energy-limited and sparsely populated deep biosphere: achievements and ongoing challenges of available technologies

Microbes in marine sediments detected and counted by direct observation of membrane-filtered sediment samples stained with acridine orange. This technique can still be applied to high-biomass (> 10(5) cells/cm(3)) sedimentary habitats, such as organic-rich sediments collected in shallow areas near the seafloor. However, to further explore the nutrients and energy turnover under extremely low energy flux conditions, or in habitats that are close to the lower limit of the biosphere, technological breakthroughs have been required to increase the detection sensitivity for microbial life at densities of a few cells/cm(3) of sediment. These technological developments contributed to increasing fundamental information on microbial life at the fringes of the subseafloor biosphere and led to the discovery of revivable microbes in sediments aged up to 101.5 million years old. More recently, chemical detection methods have revealed the existence of spores in the deep biosphere that are impermeable to conventional DNA stains. Previous applications of molecular biology-based approaches have been limited to relatively higher biomass samples, potentially because the cells surviving in these very low energy flux environments have less integrated genomes. Here, I review the contribution and importance of the technological developments that have been made in the study of microbes from the subseafloor biosphere, recent developments of alternative methods to microscopically detect microbial spores and their application to deep subseafloor sediments, and the challenges associated with applying molecular biological approaches to study low-biomass samples.

Automated summary

Microbes in marine sediments can be detected and counted using membrane-filtered sediment samples stained with acridine orange. Technological breakthroughs are needed to increase the detection sensitivity for microbial life at extremely low energy flux conditions or in habitats close to the lower limit of the biosphere. These technological developments have expanded our knowledge of microbial life in the subseafloor biosphere and led to the discovery of revivable microbes in million-year-old sediments.