METHANE-CONSUMING MICROBIAL CONSORTIA IDENTIFIED AND STUDIED USING A NOVEL COMBINATION OF FLUORESCENT IN-SITU HYBRIDIZATION AND ION MICROPROBE d13C ANALYSIS

Recently, Hinrichs et al. (1999) found archaeal lipids that were highly depleted in ¹³C in sediments from the Eel River Methane Seep off the coast of California, suggesting an archaeal prokaryote was consuming methane under anoxic conditions. In this investigation, Hinrichs et al. also conducted parallel studies of rRNA genes from the location, revealing several groups of previously unknown archaeal lineages. Additional circumstantial evidence, linking these novel microbial groups to the process of anaerobic methane oxidation, was subsequently provided by fluorescent in-situ hybridization (FISH) studies of methane seep environments by Boetius et al. (2000) and Orphan et al. (in press). The FISH technique revealed microbial aggregates consisting of an archaeal core surrounded by a shell of sulfate-reducing bacteria (SRB).

We utilized a combination of FISH and ion microprobe analysis (House, 2000), to clearly establish that the cells of these microbial consortia predominantly consist of ¹³C-depleted methane-derived carbon. Environmental samples from the Eel River Methane Seep were fixed and deposited on glass slides. The samples were then treated with two different phylogenetic stains – one targeting an archaeal rRNA sequence identified by Hinrichs et al. and one targeting SRB. Aggregates containing the archaeal core surrounded by SRB were located by their florescence - and the carbon isotopic composition of the identified target cells was determined using a method modified from that developed previously for the analysis of microfossils (House, 2000). For comparison, we also studied cells that did not stain for the archaeal rRNA of interest, as well as bacterial and archaeal cells cultivated in the laboratory. The results show that the cell aggregates that contain the archaeal rRNA signature for the species suspected of being responsible for anaerobic methane oxidation are highly depleted in ¹³C (to d ¹³C values of –96.2), whereas all of the other cells investigated are not. This high depletion in ¹³C proves that these archaeal species do consume methane. This novel metabolism may have been an important component of the microbial ecosystem on the Earth during the Archean.