About 20 years ago, the late Thomas Gold, in his landmark concept paper published in the Proceedings of the National Academy of Sciences of the United States of America, postulated that a "deep, hot biosphere" exists in the crust of the Earth . Gold suggested that this biosphere is as deep as 6 km, and is supported by geological energy sources . The Earth's "deep biosphere", as we know today, should include a variety of subsurface habitats, such as aquifers and sedimentary rocks in the continental regime, and sediments and igneous rock in the marine realm.
The oceanic crust constitutes the most voluminous, continuous aquifer system on the planet, the so-called subsurface ocean. Large areas of volcanic crust are exposed to seawater and serve as the conduits of fluids, heat, solutes, and biological material that are exchanged between the oceans and the lithosphere . Seawater flows through sediments and basalts and actively circulates through faults, fractures, and other permeable conduits in the oceanic crust and underlying mantle. In doing so, it redistributes heat, alters rock, forms secondary mineral deposits, and influences the chemical composition of the oceans and the crust . The rate at which water flows into and out of oceanic crust rivals the flow of all the world's rivers into the oceans. This massive and dynamic plumbing system cycles the entire volume of Earth's ocean water through the subseafloor every 200,000 years .
Oceanic crust is a giant microbial incubator, and hosts an extensive microbial population . Current estimates suggest that ½ to 2/3 of the total biomass of the Earth resides in the deep biosphere . Microorganisms are involved in weathering oceanic crust, in the transformation of minerals, in the exchange of elements between the crust and the ocean, and in the deposition of elements in the crust [8, 9]. Micro-scale reactions mediated by seafloor-dwelling microbial communities exert planetary-scale impacts on biogeochemical cycles. However, given the shallow depth of the magma chambers at a mid-ocean ridge (1.6.4 km below the sea floor for the fast-spreading East Pacific Rise and 3.0.4 km for the slower spreading Mid-Atlantic Ridge), the deep subsurface biosphere is probably limited to older and cooler ridge flanks and the upper few hundred meters of permeable volcanic basalts and metal-sulfide deposits over the ridge .
A major challenge in exploring the deep biosphere is determining cell abundance and viability, i.e., whether detected cells are viable, dormant or dead. Techniques commonly used for detecting subsurface microorganisms include: (1) microscopic cell counts using non-specific fluorescent DNA (RNA) stains (e.g., acridine orange direct count); (2) DNA sequencing; (3) isolation and cultivation; (4) microbial activity inferred from a radiotracer (e.g., 14CH4); (5) intact phospholipid analysis; and (6) microbial processes inferred from pore water geochemistry. All but the phospholipid technique suffer from the shortcoming that a dormant or dead cell can be detected and as a result, a fraction of the total cells measured can be non-living cells . To this end, the phospholipid technique may hold special hope for estimating viable biomass in the deep subsurface environment. Recently, techniques targeting ribosomal RNA such as catalyzed reporter deposition-fluorescence in situ hybridization showed great promise in detecting viable cells in the deep biosphere. For the first time, Schippers et al.  demonstrated the presence of intact microbial membranes and ribosomes in the deep subsurface.
Single-cell genomics are noteworthy because they have opened up a new front in environmental microbiology . Using a method called multiple displacement amplification, a few femtograms (10-15 g) of DNA in a single microbial cell can be amplified to micrograms to be used in sequencing . Thus, single-cell genomics can be extremely useful in exploring the deep biosphere in: (1) characterizing culturable as well as unculturable microorganisms, genotypically and phenotypically (e.g., ); (2) guiding the assemblage of metagenomic sequences (which are contributed to by all of the species and by many cells of each individual species in a sample) to reveal microbial diversity, the core genome of species, evolution and gene transfer, and the linkage between genes and ecology (e.g., ); and (3) understanding mechanisms of metabolically active and dormant microorganisms of microbial populations in the deep subsurface.
Examinations of the deep subseafloor biosphere hosted in rock hold promise for revealing new and remarkable information about unique and remote life on Earth and the possibilities for life on other planetary bodies. Despite recent advances in this young field, many fundamental questions remain to be answered: What is the phylogenetic and physiological diversity and complexity of the deep biosphere? What are the sources and types of energy, metabolic activity and pathways? What are the physical, chemical, and energetic limits of habitability? What are the spatial dimensions of the dark energy environment and the extent of the deep biosphere? Finally, what are the function and dynamics of the deep biosphere. Scientific drilling (e.g., IODP) will continue to provide a major platform for sampling and exploring the deep biosphere.
Jiasong Fang, School of Ocean and Earth Sciences, Tongji University, 1239 Siping Road, Shanghai, China. Li Zhang, State Key Laboratory of Geological Processes and Mineral Resources, College of Earth Sciences, China University of Geosciences, Wuhan, Hubei, China.
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