int 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 autotrophic taxa and ammonia-oxidizing microorganisms confirmed the marginal incorporation of 13C-labeled bicarbonate and the dominance of heterotrophic ammonia anabolism in the incubations. Also, it should be noted that we found a large fraction (54.9%) of Cyanobacteria in the incubation of U1368 2H-5 supplemented with ammonia at the time point of 557 days. Most of these Cyanobacteria (99.4%) were Chroococcidiopsis, hypolithic cyanobacteria with high tolerance to extreme environmental condition27,28, and minor fraction of non-photosynthetic unclassified Obscuribacterales. The abundant distribution of Chroococcidiopsis in various extreme environments on Earth, including marine environments, are consistent with their survival in this sediment sample. However, the detailed mechanism for Chroococcidiopsis growth under dark incubation condition is unclear.
In general, microbial community composition in the incubations did not closely relate to the specific substrate added (Fig. 3c). This is consistent with the suggestion from NanoSIMS-based results that the active microbial community utilizes native carbon and nitrogen compounds other than the substrates added to the incubation, and additionally suggest that the added substrates just stimulated the activity of microbes for their utilization of native carbon and nitrogen compounds. Rather the community composition showed dependence on incubation time, which suggests that different taxa responded to incubation at different timescales (Fig. 3c).
Abundance of spore formers
The mechanisms that allow microbial survival at extraordinarily low energy fluxes in subseafloor sediment are largely unexplored. Spore formation has been suggested as one of the possible mechanisms for such long-term survival of microorganisms29,30,31. Spore-forming lineages, however, were only a minor constituent of the microbial communities. Firmicutes related to thermophilic spore-forming microorganisms (Thermincola32 and Carboxydocella33) were detected at 0–12% (averaging 0.87 and 0.92%, respectively) thro