counts at the same depths in sites of ocean margins and upwelling regions8. The presence of dissolved O2, nitrate (NO3−), phosphate (PO4−), and dissolved inorganic carbon (DIC) throughout the sedimentary sequence from the seafloor to the volcanic basement indicates that cell abundance and activity are not limited by availability of electron acceptors or dissolved major inorganic nutrients. From the Redfield stoichiometry of net dissolved O2 reduction to net nitrate production in the sediment, the microbial cells have been inferred to consume oxygen coupled to oxidation of marine organic matter at extremely slow rates8.
Direct evidence of the physiological nature and survival status of microbial cells in this extremely energy-poor setting is previously lacking. A cell must metabolize a certain amount of carbon relative to its own biomass before it can double its size, divide or even sustain a metabolically active state (basal power requirement9). Bioenergetic calculations indicate that the microbes in SPG abyssal clay have access to very little power10. Very low permeability (1.1–2.0 × 10−17 m2 for IODP Site U1365 4H-3 [26.6 meters below seafloor (mbsf)] and 8.9 × 10−18 m2 for IODP Site U1370 [37.5 mbsf], respectively)11, very low estimated pore size of the abyssal clay (~0.02 microns, calculated using above permeability data according to the equation shown in Tanikawa et al.12), and thick porcellanite layers above the oldest sampled horizons appear to preclude cell migration into the sampled sediment. Consequently, the sampled communities have likely been trapped in the sediment since shortly after its deposition. The physiological status and growth potential of these buried communities and, more generally, the fractions of these energy-starved subseafloor microbes that are alive, dormant, or dead, have been essentially unknown.
To document the ecophysiology of the microbes that persist in these sedimentary habitats, we set up microaerobic incubation experiments with stable isotope-labeled substrates as tracers for microbial anabolic activities in mini-plugs of ancient (up to 101.5 Ma) pelagic clay (from IODP Sites U1365 and U1370) and relatively young (up to 13 Ma) but extremely oligotrophic calcareous nannofossil ooze (Site U1368, Supplementary Fig. 1). The results—the low relative abundance of spore formers, the rapid initial increases in incubated cell abundance, the high percentages of active cells in the incubations, and the confining nature of their sedimentary habitat—collectively suggest that microbial communities in oxic subseafloor sediment persist in metabolically active form for at least 101.5 million years.
Results and discussion
Single-cell anabolic activity with 13C-, 15N-labeled substrates
Single cell-targeted ion imaging analysis of 6986 individual cells by NanoSIMS showed that aerobic microbes from every sample of this oligotrophic oxic sediment actively take up isotope-labeled carbon and nitrogen substrates, even from sediment as old as 101.5 Ma13 (U1365 9H-3) (Figs. 1 and 2b, c). The number of cellular regions of interests (ROIs) analyzed per incubation sample ranged from 9 to 210 (Supplementary Data 1). Incorporation of carbon and nitrogen from organic compounds and ammonium was generally seen in the ROIs from all of the microaerobic (~3.3% O2 in the headspace) incubations, with bimodal distribution in the degree of substrate incorporation (Fig. 2b). While initial cell counts were extremely low, ranging from 102 to 3 × 103 cells cm−3 (Fig. 2a)8, the microbes in the oxic sediment responded rapidly to the substrate injection in microaerobic incubations, increasing total numbers by orders of magnitude and taking up labeled carbon and nitrogen within 68 days of incubation.
In contrast to the microaerobic incubations, incubations of ancient (65.5 Ma13) oxic abyssal clay from Site U1370 conducted without adding O2 showed only marginal incorporation of selected substrates with minimal growth of biomass (Supplementary Fig. 2). Although we did not supply additional O2, the oxic sediment contained ~1.5 μM of 