Sulfate Reduction 



Sulfate reduction assays were set up using a modification of the 

 technique of Ivanov^ (1964) . Each vial of sediment received approxi- 

 mately 1 pCi of Na„ SO, in 1 ml of anoxic ASW. Samples were mixed and 

 incubated for 2.0 n. The reaction was stopped by the addition of 0.5 

 ml of 2% zinc acetate followed by 0.2 ml of formalin. Samples were 

 assayed and rates determined in the Montana State University lab as 

 described by J^rgensen (1978). H„ S was distilled to traps containing 

 2% zinc acetate. Radioactivity was determined by counting the zinc 

 acetate trap (5 ml) in 10 ml Aquasol (New England Nuclear) on a Beckman 

 LS-100C liquid scintillation counter. Correction for quenching was by 

 the channels ratio method. 



Methane Production 



Methane production was measured by quantifying the increase in 

 methane in the head-space of vials containing sediment. A 0.2 ml gas- 

 eous subsample was removed at desired intervals and analyzed by flame 

 ionization gas chromatography (see below). 



Acetate Metabolism 



..Acetate metabolism was measured by adding approximately 0.5 (JCi of 

 [2- C]-acetate in 1.0 ml of sterile anoxic sulfate-free ASW. Samples 

 were mixed and incubated for 2.0 h unless otherwise stated... The reac- 

 tion was stopped by the addition of 0.2 ml formalin. C0„ and/or 

 CH, were measured in samples of the gas headspace (see below). 



Hydrocarbon Metabolism 



All radiolabelled hydrocarbons except benzene and toluene were di- 

 luted in benzene to the desired activity. The radioisotopes were added 

 to vials and the benzene allowed to evaporate completely before addi- 

 tion of sediment and anaerobic tubing as described above. Anoxic ASW 

 (1.0 ml) was added to each sample to mix sediment and radioisotopes. 

 Radiolabelled benzene and toluene were dissolved in anoxic ASW and 

 added (1.0 ml) after anoxic tubing of sediment. When indicated, sam- 

 ples were incubated in darkness by wrapping with aluminum foil or 

 electricians tape. In one experiment samples contained in anaerobi- 

 cally sealed tubes were incubated within an anaerobic chamber (Gaspak) 

 with a H„ "\/C0 atmosphere. During long term incubations gaseous meta- 

 bolities ( C0„ and/or CH.) were quantified in samples of the gas 

 headspace as described below. After incubation was completed, samples 

 were poisoned by addition of 0.5 ml 10% formalin. Carbon dioxide was 

 reabsorbed by addition of 2 ml 2N NaOH. The sediment was extracted 

 four times by vortex mixing with 6 ml methylene chloride: methanol 

 (9:1) followed by centrifugation to break the emulsion. Solvent frac- 

 tions were removed from beneath the aqueous phase and pooled together 

 with three 6 ml rinses of the original sample vial. Anhydrous sodium 

 sulfate was added to dry the sample. The extract was concentrated to 

 0.1 ml by evaporation and the volume increased by addition of 0.7 ml of 

 hexane. This sample was separated into saturate (f,), aromatic (f ? ) 



163 



