 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 timepoint suggested that they should have been inactive throughout the incubation period. Therefore, we infer that these cells comprise living (or at least degradation-resistant) but dormant biomass not revived by the addition of amino acids. This pattern of biomass change was not obvious or absent in the incubations with other substrates.
Comparison of 16S rRNA gene profiles to substrates
We investigated the 16S rRNA gene-based taxonomic composition of microbial cells sorted by fluorescence-activated cell sorting (FACS) from each incubation. Although pilot studies showed the community composition in anoxic sediment outside the SPG23, near-seafloor SPG sediment (<10 cmbsf)24, and SPG surface seawater25, diversity of microbial community in SPG sediment more than a meter below seafloor has not been reported. This absence of community composition information is partially due to the very low cell abundance and the nature of the sediment samples (dominated by fine-grained clay and silt with negligible sand26) that may have adsorbed released DNA from lysed cells during the DNA extraction process. For this study, we separated and sorted cells from the sediment to obtain community taxonomic composition under all of the incubation conditions, including initial sediment samples with very low biomass (~102 cells cm−3) (full dataset can be found in Supplementary Data 2 and detailed methods to eliminate procedural contaminations are found in “Methods” section). The communities of all samples were dominated by bacterial sequences. Dominant bacterial groups included Actinobacteria, Bacteroidetes, Firmicutes, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria (Fig. 3b, c) with a minor fraction of Chloroflexi (0– 2.6%). A small fraction (~7%) of Terrestrial Hot Spring Group of thermophilic Archaea, which was recovered from sediment overlying relatively young (13.5-Ma) basement on the East Pacific Rise flank, was detected in sample U1368D 2H-5 (Supplementary Data 2). The absence of obligately autotroph