35 14 



H 9 S, and in some cases the generation of CH, within two hours of 



incubation, (Winfrey and Ward, submitted). In long-term incubations no 

 C0„ or CH, was detected in fprmalin-killed controls. In most cases 

 significant amounts of C0„ + CH, were detected in vials incubated 

 aerobically (Table 4). Anaerobic incubation severely reduced the 

 amount of radiolabelled gases formed. However, small amounts of these 

 metabolites were formed from n-hexadecane , n-heptadecane, heptadecene, 

 ring or methyl labelled toluene, and benzene after lengthy anaerobic 

 incubation. The amount of gaseous metabolites formed did not exceed 

 5% of the added radiolabel and reproducibility was poor. Repeated ef- 

 forts by two individuals experienced in cultivation of methanogenic 

 bacteria led to the same observations. Additions of FeCl. or KNO (1 

 ml of 0.5% (w/v) solutions in anoxic ASW replaced the 1 mi addition of 

 anoxic ASW) did not stimulate the formation of C0„ and CH, from 

 n-hexadecane or heptadecene in Aber Wrac'h 5-10 cm sediment. 



The possibility of initial accidental exposure to oxygen during 

 tubing of samples was investigated by late addition of C-toluene 

 which was soluble in water and could be added as an anoxic solution 

 well after any oxygen initially present should have been consumed dur- 

 ing dark incubation. Revsbech, et al, (1980b) have shown that oxygen 

 consumption in intertidal sediments occurs in a matter of minutes fol- 

 lowing, darkening to eliminate photosynthesis... As shown in Figure 6, 

 ring- C-toluene was readily metabolized to CC> when added either at 

 the time of anaerobic tubing or 38 hours after dark anaerobic incuba- 

 tion began. Similar results were found for [methyl- C]-toluene. 



It was conceivable that the radiolabelled gases might have been 

 produced from contaminants rather than from the hydrocarbons them- 

 selves. When an attempt was made tcL recover the added C in, long-term 

 radiolabelling experiments with [1- C] -heptadecane and Jl- C] -hepta- 

 decene, it was noted that the total amount of CO + CH, produced 

 during anaerobic incubations was similar to the level of impurities 

 measured in C-labelled hydrocarbons recovered from formalin controls 

 or from unpurified stocks of added radioisotopes (Table 5). Stock 

 solutions were chromatographically separated into f , f„ ^nd f„ com- 

 ponents which were then tested separately as sources of C-gases in 

 dark anaerobic incubations with anaerobic sediments. The results of 

 such experiments are presented in Eigure 7. The repurified f frac- 

 tions of [1- C] hexadecane and [ 1- , C] -heptadecane were clearly sig- 

 nificant sources for production of CO during dark anaerobic incuba- 

 tions with a slurry of lie Grande oiled 3-6 cm sediment. Increases in 

 CO with time following a lag of 5-15 days also suggested that oxi- 

 dation did not result from any oxygen which might have been introduced 

 accidentally during tubing. Similar results were observed with repuri- 

 fied f of [1- C] -heptadecene. 



A final control was run to test the possibility that slow diffu- 

 sion of oxygen through the vessels containing incubating samples could 

 account for the obsexved metabolism. Darle .anaerobic incubations of 

 repurified f of [1- C] -hexadecane and [1- C] -heptadecane were car- 

 ried out with a slurry of mud from the 3-6 cm interval of Aber Wrac'h 

 sediment. The individual vials were incubated inside an anaerobic 



173 



