ughout 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 stable isotope-labeled substrates (13C6-glucose, 13C2-acetate, 13C3-pyruvate, 13C-bicarbonate, 13C-15N-amino acids mix [mixture of 20 Amino Acids], and 15N-ammonium). The incubation experiments were initiated onboard during expedition. Avoiding outer edges of the sediment cores (which are likelier to be contaminated by drill fluid), each 15 cm3 sample was taken from an interior portion of a core with a sterile tip-cut 30 mL syringe, and placed in a 50 cm3 sterile glass vial (Nichidenrika-Glass Co. Ltd.) sealed with a sterile rubber stopper and a screw cap, followed by flushing with 0.22 μm filter-sterilized nitrogen gas and storage at 10 °C. All of the sterilization was done by autoclaving of the materials at 121 °C for 20 min. Given O2 concentrations in interstitial water during Expedition 329 (~70 μM for U1365C 8H-2 and 9H-3, ~150 μM for U1368D 1H-2, ~130 μM for 1368D 2H-5, and ~1.5 μM for U1370F 7H-67), we set O2 concentration in the headspace of vials (other than U1370F 7H-6) at ~3.3% (v/v), which corresponds to the aqueous concentration of oxygen at ~43 μM (assuming salinity of sea water), by adding 0.22 μm filter-sterilized air. The labeled substrates were injected (15 μM of 13C-labeled substrates, and 1.5 μM of 15N-labeled ammonium, dissolved in 50–100 μL of sterile water) onto each subcore sample by syringe and needle and incubated at 10 °C (Supplementary Fig. 3a). All reagents and gas components, including air used for sample preparation, were filtered through 0.22 μm syringe top filter. After setting up the incubations, one of the vials from each set of incubation had no substrate added, and a sediment split from the same sample was fixed by adding equal volume (15 mL) of 4% paraformaldehyde (PFA) in PBS solution for 5 h at 4 °C (time point T0). At each of three time points (T1: ~3 weeks [21 days], T2: 6 weeks [68 days], T3: 18 months [557 days] after starting incubation), vials were opened and sediment samples were fixed with equal volume (15 mL) of 4% PFA in PBS solution for 5 h at 4 °C. Fixed sample