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

Abstract 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 collected shallow areas near the seafloor. However, further explore nutrients energy turnover under extremely low flux conditions, or habitats that are close lower limit biosphere, technological breakthroughs have been required increase detection sensitivity for microbial life at densities a few sediment. These developments contributed increasing fundamental information on fringes subseafloor biosphere led discovery revivable microbes aged up 101.5 million years old. More recently, chemical methods revealed existence spores deep impermeable conventional DNA stains. Previous applications molecular biology-based approaches limited relatively higher biomass samples, potentially because cells surviving these very environments less integrated genomes. Here, I review contribution importance made study from recent alternative microscopically detect their application sediments, challenges associated applying biological low-biomass samples.