rage, 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 was made by using the observed active ROI fractional ratio (number of active ROI versus total ROI analyzed), increase of biomass, and ROI-based sampling depth (the numerical depth of sampling that the ROI-based analysis covered). The observed “active” ROIs ranged from 18.4 to 100% of the total ROIs analyzed, giving an average of 92%. The minimum original active fractions average 77.7%, ranging from 24.1 to 99.1% (Table 1 and Supplementary Data 2). Even in the sample of oldest sediment (U1365 9H-3, 101.5 Ma13), the revivable heterotrophic population was 39.6–99.1% of the original community. These microaerobic incubations differ notably from the previously published anaerobic incubation14 by rapidly and consistently increasing in biomass after the addition of substrates. The biomass-based doubling time (bTd) of microbes in these microaerobic incubations (Supplementary Data 2) averaged 4.9 days, ranging from 1.4 to 17.8 days. Incubations with the oldest sediment (U1365C 9H-3) showed significantly longer bTd (in average 9.8 days, pairwise t-tests, p < 0.05) than the incubations with younger sediment. The incubations with U1365C 8H-2 (95.4 Ma13) showed slightly longer but statistically insignificant bTd values than the U1368 sediment samples (Table 1).
“Inactive” ROIs, which have no detectable incorporation of supplemented substrates, represent either (i) dormant cells or (ii) dead cells (necromass). Although recycling of biomolecules from necromass is a well-known anabolic strategy in marine sediment20,21, necromass provides only a small fraction of the maintenance power demand for the microbial community in subseafloor sediment22. In our amino-acid-amended incubations, an apparent biomass decrease after an initial increase in the sample of U1368D 2H-5 (Fig. 2a) demonstrates the generation of new biomass and the remineralization after cell death (necromass) at a later incubation stage. The absence of substrate incorporation in the remaining “inactive” cells (accounting for 73.3% of total cells) at the 557-day timepo