or 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 O2 in its interstitial water7, which introduced 16.4 nmol of O2 in the incubation vial and reached an equilibrated concentration of not more than 14.6 nM in the sediment. Although the slight increase of biomass at the early stage of the U1370 incubations may have been aerobic microbial growth, there was not enough O2 to sustain aerobic growth for the entire period of incubation. Although the sediment contained abundant dissolved nitrate (~46 μM) and sulfate (~27 μM) in its interstitial water7, we did not see intensive growth of biomass in later stages of the incubation. This result suggests that metabolic activity hardly revived and did not persist in the absence of added O2 for microbial communities that have been exposed to dissolved O2 for 65.5 million years. This, in turn, suggests that long-time exposure to dissolved O2 over geological time may extinguish anaerobic microorganisms including anaerobic spore-formers (or at least make them hard to revive).
The microbial metabolic response to microaerobic inoculation conditions was heterogeneous; the extent and rate of labeled isotope incorporation varied considerably from one ROI to another (Fig. 2b, c), and from one labeled substrate to another, similarly to the substrate incorporations seen in anaerobic incubations of deep subseafloor coalbed sediment14. Of the labeled substrates tested, heterotrophic substrates generally supported biomass growth. However, in the case of nannofossil-bearing clay of U1368 2H-5 sediment, the amino acid-supplemented incubation showed biomass increase only at day 21 of incubation and afterward decreased to the level of original cell abundance, indicating the loss of increased biomass at a later stage of incubation. We observed only marginal autotrophic incorporation of bicarbonate (0–23.6 atom%, in average 0.8 atom%, Fig. 2b), consistent with primary reliance on organic matter as carbon source for biomass production.
Rates of substrate uptake and biomass generation
The response to substrate addition, in general, was faster (on ave