| Sample preparation: | |||
| A 5-cm3 subcore was injected into a sterile 50-mL centrifuge tube containing 10 mL 1:1 ethanol/PBS solution; the suspension was stored at -20°C. | |||
| At select sites: | |||
| 1) A 5-cm3 subcore was fixed in 3.7% formaldehyde in PBS for 1-24 hr. | |||
| 2) The sample was washed twice with PBS (using standard FISH protocols; see Table T11 FISH fixation). | |||
| 3) After washing, the sample was stored at -20°C in a sterile 50-mL centrifuge tube containing 10 mL 1:1 ethanol/PBS. | |||
| The residual round core from which the subcores had been removed was frozen at -80°C for storage. | |||
| Experiments on incorporation of 13C-labeled substrates: | |||
| 1) Sterile bottles with 10 mL of reduced DGH media (see below) and a labeled substrate were sealed in an 80% N2/20% CO2 atmosphere during precruise preparation. In some cases, the headspace also included 20 mL of nonlabeled methane. | |||
| 2) For each sample, 10 mL of 25% sediment slurry was injected into an experiment bottle and incubated at 4°C. | |||
| 3) To terminate incubation, 40 mL of headspace gas was removed (at the rear of the ship to avoid 13C contamination of geological samples), and 20 mL of filtered ethanol was injected into the bottle. Samples were then stored at -20°C. Postcruise, samples will be fixed in 3.7% formaldehyde, washed with filtered PBS buffer, and ultimately stored in a 1:1 ethanol/PBS solution at -20°C. | |||
| Each bottle had one of three possible 13C labeled substrates: glucose, acetate, or methane. Glucose and acetate concentrations were 1 mg/mL of media with half of the substrate 13C labeled, whereas methane concentrations were 20 mL/per serum bottle with one-fourth of the methane 13C labeled. | |||
| DGH media preparation*: | |||
| Component: | NaCl | Concentration: | 10 g/500 mL |
|
|
Na2SO4 |
|
1.5 g/500 mL |
|
|
KH2PO4 |
|
0.1 g/500 mL |
|
|
NH4Cl |
|
0.15 g/500 mL |
|
|
KCl |
|
0.15 g/500 mL |
|
|
CaCl2·H2O |
|
0.1 g/500 mL |
|
|
MgCl2·6H2O |
|
0.15 g/500 mL |
|
|
Wolfe trace elements |
|
|
|
|
0.2% (NH4)2Fe(SO4)2 |
|
0.5 mL/500 mL |
|
|
10% NaOH† |
|
1.5 mL/500 mL |
|
|
ddH20 |
|
500 mL/500 mL |
Notes: DGH media were prepared prior to the cruise. * = 0.25 g NaS was added to reduce the media. The media was dispensed in an anoxic environment with 10 mL added to each 125-mL serum vial. † = media was degassed with N2/CO2, neutralizing the NaOH and resulting in a final pH of 6.8.