To determine potential rates of autotrophic and heterotrophic metabolism within crustal aquifer fluids and deep Atlantic bottom water, fluids were incubated with either 13C-labeled bicarbonate (autotrophy) or 13C-labeled acetate (heterotrophy). For this, 20 mL of freshly sampled fluids were injected into sterile, butyl stoppered Balch tubes using a 60 mL syringe and hypodermic needle using sterile technique. Overpressure was released by insertion of a second hypodermic needle. The tubes were pre-amended with a mix of either unlabeled bicarbonate and 10% 13C-labeled bicarbonate (to a final concentration of 1.8 mM NaHCO3 and 0.2 mM NaH13CO3) or unlabeled acetate and 10% 13C-labeled acetate (to a final concentration of 13.5 uM C2H3NaO2 and 1.5 uM 13C2H3NaO2). All tubes were additionally amended with resarzurin (20 uM final concentration) in order to follow the change in redox potential as a result of oxygen consumption throughout the incubation period. Sterile controls were set up as described above but with an additional filter (0.2 um pore size) inserted between the syringe outlet and the hypodermic needle. Tubes were incubated in the dark at either 5 or 25 degrees C. Incubations were stopped at distinct time intervals by addition of either 0.5 mL of a 1 M NaOH solution (for incubations with bicarbonate) or 5 mL of a 20% zinc acetate solution (for incubations with acetate). Tubes were stored at –20 degrees C until further processing.

For analysis of 13C-labeled biomass, tubes were thawed and the residual pool of bicarbonate or acetate was removed by acidification to a pH of 2 by adding 25% HCl (molecular grade) while stirring and sparging with N2 for at least 30 minutes. The entire volume was then filtered through a pre-combusted glass fiber filter (25mm diameter, 0.7 um particle retention, Whatman, UK). The filters were dried in a desiccator overnight and stored at 5 degrees C until further processing. Filters were weighed into tin capsules and analyzed for 13C/12C ratios with an automated Isotope Cube elemental analyzer (Elementar, Germany) interfaced to a Delta Advantage isotope ratio mass spectrometer (Thermo, Germany). Rates of potential autotrophic metabolism (carbon fixation from 13C-labeled bicarbonate) and heterotrophic metabolism (degradation of 13C-labeled acetate) were calculated from δ13C of the carbon pool on the filters at the start and the end of the incubations (time intervals of not more than 13 days). For this, the transfer of 13C between pools was calculated, according to the following equation:

δBiomass-Final · CBiomass-Final ·VFinal = (CBiomass-Initial · V · δBiomass-Initial) + (CTransferred· V · δLabel)

where δ is the isotopic ratio ((Rsample/ Rstandard –1) · 1000), V is the volume of the incubation (20mL) and C is the concentration of carbon pool. For incubations with 13C-labeled bicarbonate we assumed a concentration of 2.3 mM endogenous dissolved inorganic carbon (DIC) in addition to the added mix of 13C-labeled bicarbonate, thus decreasing the amount of label in the substrate pool. For incubations with 13C-labeled acetate we assumed a concentration of 150 uM endogenous dissolved organic carbon (DOC) in addition to the added 13C-labeled acetate.