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 average, 3.09 times, Table 1) and more extensive for nitrogen incorporation than for carbon incorporation (Fig. 2c). Ammonium, which was added as a nitrogen source in all incubations except for the incubation with 13C and 15N-labeled amino acids, was generally incorporated quickly into cellular biomass. The preferred incorporation of supplemented ammonium over nitrate, which originally existed at ~40 µM in all of the sediment samples6, was predictable given higher energetic costs of reducing nitrate to the oxidation state of N in amino acids15. Although ammonia assimilation requires energy to be incorporated into amino acids15,16, its incorporation was observed even under the condition supplemented with only ammonium without a carbon source. In addition, the high ammonia incorporation and biomass increase during those incubations suggests at least a minor contribution of chemolithoautotrophic ammonia oxidizers17.
The growth rates of microbes were calculated based on the rates of biomass growth and substrate incorporation (Fig. 3a, Table 1, and Supplementary Data 2). The biomass-based specific growth rates were generally higher than the substrate-based biomass generation rates. This difference suggests that the active microbial communities utilized carbon and nitrogen compounds in the sediment other than the supplemented isotope-labeled substrates for their biomass growth. Although the total organic carbon content in the sediment is very low (0.01–0.02 weight percent), it exists in bioavailable form but protected under in situ conditions18. This amount of total organic carbon is enough to sustain the observed growth of microbes without an additional carbon source; it corresponds to potential microbial biomass19 of >1010 cells cm−3, exceeding the original microbial biomass by 7–8 orders of magnitude, and the post-incubation biomass by 4 orders of magnitude.
To document the physiological status of these microorganisms, we calculated the fraction of microbial cells that originally existed in the samples. The calculation