Diversity of Methanogens and other Archaea in Permanently Cold, Anoxic Marine Sediments

Little is known about the mechanism of methanogenic degradation of acetate or the fate of hydrogen and formate in cold marine sediments, or the ability of methanogens to grow and produce methane there. We used cultivation and molecular techniques to examine the microbes that produce methane from these substrates in permanently cold, anoxic marine sediments at Skan Bay, Alaska (57°N, 167°W, depth 65 m), at the Nankai Trough (32°21\u27N, 134°56\u27E, depth 247 m), and at Hydrate Ridge, Oregon (44°35\u27N, 125°10\u27W, depth 800 m). Most probable number (MPN) analyses were conducted for CO2-reducing, methylotrophic, and aceticlastic methanogens from Skan Bay and Hydrate Ridge. At both sites, methanogens were present at low numbers (100 to 103 per gram of sediment). CO2-reducing and methylotrophic methanogens were the most abundant methanogens, and aceticlastic methanogens were not detected. Nine novel strains of methanogens and two strains of syntrophic bacteria were described in this study. Most of these methanogens, strains AK-3, AK-7, AK-8, Nankai-2, and Nankai-3 reduce CO2 with H 2 or formate for energy. Three methanogens that catabolized methylated compounds were isolated: strains AK-4, AK-5, and AK-9. Only one strain of aceticlastic methanogen, strain AK-6, was isolated. These data, together with the MPN enrichment data, support existing geochemical data that most methane produced in marine sediments is the result of CO2-reducing and methylotrophic methanogens. Several strains isolated and described in this study represent previously unrecognized microbial species. Strain AK-5 and AK-9 were described as a new species, Methanococcoides alaskense; strain AK-7 was described as a new species, Methanogenium boonei; the syntrophic bacterium strain AK-B was described as representing a new genus and species, Algorimarina butyrica, and strain Nankai-3 represents the type species of Methanococcus aeolicus. Cultivation-independent molecular analyses were conducted at Skan Bay and Hydrate Ridge to determine archaeal diversity. At both sites, uncultivated organisms dominated the libraries and were related to the Marine Benthic group D (Thermoplasmatales rel.), the putative methane-oxidizing ANME-2 group, and the Marine Benthic group B. Clones related to the ANME-1 methane-oxidizing Archaea and to cultivated methanogens were detected, but these constituted only a minority of the clone libraries