processed for -^C incorporation into nucleic acids and protein 

 (Karl 1982). 



During the Pisces V expedition, we continued our initial 

 assessment of CH4 oxidation potential by comparing the rates of 

 CH 4 oxidation in a variety of water samples collected from Pele's 

 Vents. For each water sample that was collected, one subsample 

 was immediately fixed with HgCl2 (as previously mentioned) for 

 measurement of the initial CH4 and CO2 concentrations and a 

 replicate subsample was placed into a sealed glass-stoppered 

 bottle and incubated at 25°C for a period of 30 days, after 

 which the final CH4 and CO2 concentrations were determined. At 

 the end of the experiment, several selected water samples (those 

 which had demonstrated significant CH4 oxidation activity) and 

 the deep-water control were processed for l4 C-CH4 oxidation 

 activity as described previously. 



Adenine incorporation experiments 



Pele's Vent water samples (500 ml) collected on Alvin dives 

 #1799 and #1800, bacterial mats collected on dive #1800 and a 

 deep-water control sample were incubated in polycarbonate bottles 

 at 25°C for 8 and 18 hours in the presence of 100 y Ci ^H-adenine 

 (15 Ci mmol-1; New England Nuclear, Cat. #NET-250). At each 

 time point, 250 ml of the sample was filtered through a Whatman 

 glass fiber filter (GF/F) and the filters were stored frozen for 

 subsequent determination of radioactivity incorporated into RNA, 

 DNA, and protein (Karl 1982). 



Pele's Vent water samples collected on Pisces V dives #28 

 and #29 and a control deep-water sample were analyzed for total 

 rates of microbial RNA and DNA synthesis. Portions (175 ml) of 

 each water sample were incubated with 10 yCi of ^H-adenine (see 

 aforementioned) at 25°C for 16 hours. Following incubation, 

 subsamples were extracted for ATP and were processed for RNA/DNA 

 (Karl and Winn 1984). Rates of RNA and DNA synthesis 

 ( pmol l - -*- hr -1 ) were estimated from the incorporation rates 

 ( nCi hr -1 ) and the direct measurement of the intracellular 

 specific radioactivity of the ATP pool (nCi pmol -1 ) according to 

 the methods described previously (Karl 1981). 



Effects of temperature on bacterial activity 



Portions (25 ml each) of a Pele's Vent water sample 

 collected on Pisces V dive #28 were placed into a series of 16 

 polycarbonate incubation bottles and, in groups of four, were 

 allowed to equilibrate for 1 hour in temperature controlled water 

 baths at 4° 25°, 37° and 60°C. Radiolabeled substrates 

 including, ^H-thymidine (10 yCi; 65 Ci mmol - , New England 

 Nuclear, Cat. #NET-027Z), 3 H-adenine (10 yCi; see previously 

 mentioned for isotope description), ^H-glutamic acid (10 pCi; 

 50 Ci mmol" 1 , New England Nuclear, Cat. #NET-490) and 



197 



