 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 samples were frozen at −80 °C. After storage, they were washed twice with PBS and preserved in PBS/ethanol (1:1 [v/v]) at −20 °C until analysis.
Cell enumeration and selective sorting onto the membrane
To efficiently analyze substrate incorporation into microbial cells with NanoSIMS, cells were separated from their sediment matrix and fluorescence-activated cell sorting (FACS) was conducted to concentrate and purify cells in a small area for analysis (~0.5 mm2, Supplementary Fig. 3b)14,37. Cell separation, enumeration, and FACS were all conducted in the clean-booth and clean-room facilities at the Kochi Institute for Core Sample Research, Japan Agency for Marine-Earth Science and Technology (JAMSTEC).
Cell separation, microscopy, and sorting procedures followed the method of Morono et al.37 with modifications. Eight milliliters of fixed slurry (1/3 [v/v] sediment in ethanol-PBS solution) was mixed with the same volume of 2.5% NaCl solution, followed by centrifugation at 4500 × g for 15 min, after which the supernatant was discarded and the pellet resuspended by adding 2.5% NaCl solution to be 20 mL of sediment slurry. The sediment slurry was added with 2.5 mL each of detergent mix38 (100 mM EDTA, 100 mM sodium pyrophosphate, 1% [v/v] Tween 80) and methanol, then vigorously shaken for 60 min at 500 rpm using a Shake Master (Bio Medical Science, Tokyo, Japan). After shaking, the sediment slurry was sonicated (Bioruptor UCD-250; COSMO BIO) in an ice bath for 20 cycles of 30 s at 200 W on and 30 s off. The processed slurry was then carefully layered onto a manually layered high-density cushion solution consist of (from top) 4 mL of 30% [v/v] Nycodenz, 4 mL of 50% [v/v] nycodentz, 4 mL of 80% [v/v] Nycodentz, and 4 mL of 67% [w/v] of sodium polytungstate. Samples were centrifuged at 4000 × g for 120 min, after which the supernatant, including the high-density layer(s), was carefully removed and transferred to a separate vial. Cells in the supernatant were trapped onto an Anopore Inorganic Membrane (Anodisc, Whatmann, Kent, 