ic 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) throughout the samples and incubations. A small fraction (~2.5%) of the populations in incubations of the oldest sediment sample (U1365C 9H-3) is related to the genus Paenibacillus34, mesophilic spore-forming bacteria. This low relative abundance of known spore formers suggests that the revival of spores (germination) was very limited in the incubations.
Sediment samples used in this study were collected by IODP Expedition 329 in the South Pacific Gyre (SPG; Supplementary Fig. 1)7. The samples collected from Site U1365 (23° 51.0377′S 165° 38.6502′W) are zeolitic and/or metalliferous pelagic clays, those from Site U1368 (27° 54.9920′S, 123° 09.6561′W) are calcareous nannofossil oozes, and those from Site U1370 (41° 51.1267′S, 153° 06.3674′W) are black metalliferous clay, containing light yellowish brown clay-bearing nannofossil ooze. The water depths of these sites are respectively 5697, 3739, and 5075 meters below sea level (mbsl) at U1365, U1368, and U1370. Whole-round core samples of U1365C 8H-2 (obtained from 68.9 meters below seafloor [mbsf]), 95.4 Ma), U1365C 9H-3 (74.5 mbsf), U1368D 1H-2 (1.6 mbsf), 1368D 2H-5 (14.7 mbsf), and U1370F 7H-6 (62.9 mbsf) were used for incubation experiments as described in the following section. Those samples were from horizons with no observable coring disturbance by visual core description35. Drilling fluid contamination assessment by chemical tracer revealed minimal drilling contamination of the samples (≤1 cell/g-sediment for U1365C 8H-2 and 1368D 2H-5, below detection to 0 cell/g-sediment for U1365C 9H-3, U1368D 1H-2, and U1370F 7H-6)35. All Expedition 329 data are archived and available online in the IODP database (http://iodp.tamu.edu/tasapps) and archived online in the IODP Expedition 329 Proceedings7. Additional sediment age estimation is available in reports by Dunlea et al.13 and Alvarez Zarikian et al.36.
To identify autotrophic and heterotrophic microbial populations, as well as their potential rates for growth and substrate uptake, sediment samples were incubated with