• Energy Research

Significance Microbial cells are widespread in diverse deep subseafloor environments; however, the viability, growth, and ecophysiology of these low-abundance organisms are poorly understood. Using single-cell–targeted stable isotope probing incubations combined with nanometer-scale secondary ion mass spectrometry, we measured the metabolic activity and generation times of thermally adapted microorganisms within Miocene-aged coal and shale bed samples collected from 2 km below the seafloor during Integrated Ocean Drilling Program Expedition 337. Microorganisms from the shale and coal were capable of metabolizing methylated substrates, including methylamine and methanol, when incubated at their in situ temperature of 45 °C, but had exceedingly slow growth, with biomass generation times ranging from less than a year to hundreds of years as measured by the passive tracer deuterated water.

SignificanceMarine sediment covers 70% of Earth’s surface and harbors as much biomass as seawater. However, the global taxonomic diversity of marine sedimentary communities, and the spatial distribution of that diversity remain unclear. We investigated microbial composition from 40 globally distributed sampling locations, spanning sediment depths of 0.1 to 678 m. Statistical analysis reveals that oxygen presence or absence and organic carbon concentration are key environmental factors for defining taxonomic composition and diversity of marine sedimentary communities. Global marine sedimentary taxonomic richness predicted by species–area relationship models is 7.85 × 103to 6.10 × 105for Archaea and 3.28 × 104to 2.46 × 106for Bacteria as amplicon sequence variants, which is comparable to the richness in seawater and that in topsoil.

Deep drilling into the marine sea floor has uncovered a vast sedimentary ecosystem of microbial cells. Extrapolation of direct counts of stained microbial cells to the total volume of habitable marine subsurface sediments suggests that between 56 Pg (ref. 1) and 303 Pg (ref. 3) of cellular carbon could be stored in this largely unexplored habitat. From recent studies using various culture-independent techniques, no clear picture has yet emerged as to whether Archaea or Bacteria are more abundant in this extensive ecosystem. Here we show that in subsurface sediments buried deeper than 1 m in a wide range of oceanographic settings at least 87% of intact polar membrane lipids, biomarkers for the presence of live cells, are attributable to archaeal membranes, suggesting that Archaea constitute a major fraction of the biomass. Results obtained from modified quantitative polymerase chain reaction and slot-blot hybridization protocols support the lipid-based evidence and indicate that these techniques have previously underestimated archaeal biomass. The lipid concentrations are proportional to those of total organic carbon. On the basis of this relationship, we derived an independent estimate of amounts of cellular carbon in the global marine subsurface biosphere. Our estimate of 90 Pg of cellular carbon is consistent, within an order of magnitude, with previous estimates, and underscores the importance of marine subsurface habitats for global biomass budgets.

m CSF = depth of Core below Sea Floor in meters / m CSF-A: Distance from sea floor to sample within recovered core. This scale allows overlap at core and section boundaries. /m CSF-B: Distance from sea floor to sample within recovered core is compressed, if core recovery > 100%.

Over the past few decades, the subseafloor biosphere has been explored by scientific ocean drilling to depths of about 2.5km below the seafloor. Although organic-rich anaerobic sedimentary habitats in the ocean margins harbor large numbers of microbial cells, microbial populations in ultraoligotrophic aerobic sedimentary habitats in the open ocean gyres are several orders of magnitude less abundant. Despite advances in cultivation-independent molecular ecological techniques, exploring the low-biomass environment remains technologically challenging, especially in the deep subseafloor biosphere. Reviewing the historical background of deep-biosphere analytical methods, the importance of obtaining clean samples and tracing contamination, as well as methods for detecting microbial life, technological aspects of molecular microbiology, and detecting subseafloor metabolic activity will be discussed.

Deep drilling into the marine sea floor has uncovered a vast sedimentary ecosystem of microbial cells (Parkes et al., 1994, doi:10.1038/371410a0; D'Hondt et al., 2004, doi:10.1126/science.1101155). Extrapolation of direct counts of stained microbial cells to the total volume of habitable marine subsurface sediments suggests that between 56 Pg (Parkes et al., 1994, doi:10.1038/371410a0) and 303 Pg (Whitman et al., 1998) of cellular carbon could be stored in this largely unexplored habitat. From recent studies using various culture-independent techniques, no clear picture has yet emerged as to whether Archaea or Bacteria are more abundant in this extensive ecosystem (Schippers et al., doi:10.1038/nature03302; Inagaki et al., doi:10.1073/pnas.0511033103 ; Mauclaire et al., doi:10.1111/j.1472-4677.2004.00035.x; Biddle et al., doi:10.1073/pnas.0600035103). Here we show that in subsurface sediments buried deeper than 1 m in a wide range of oceanographic settings at least 87% of intact polar membrane lipids, biomarkers for the presence of live cells (Biddle et al., doi:10.1073/pnas.0600035103; Sturt et al., 2004, doi:10.1002/rcm.1378), are attributable to archaeal membranes, suggesting that Archaea constitute a major fraction of the biomass. Results obtained from modified quantitative polymerase chain reaction and slot-blot hybridization protocols support the lipid-based evidence and indicate that these techniques have previously underestimated archaeal biomass. The lipid concentrations are proportional to those of total organic carbon. On the basis of this relationship, we derived an independent estimate of amounts of cellular carbon in the global marine subsurface biosphere. Our estimate of 90 Pg of cellular carbon is consistent, within an order of magnitude, with previous estimates, and underscores the importance of marine subsurface habitats for global biomass budgets.