Vertical distribution of methanogens in the anoxic sediment of Rotsee (Switzerland)

Anoxic sediments from Rotsee (Switzerland) were analyzed for the presence a nd diversity of methanogens by using molecular tools and for methanogenic a ctivity by using radiotracer techniques, in addition to the measurement of chemical profiles. After PCR-assisted sequence retrieval of the 16S rRNA ge nes (16S rDNA) from the anoxic sediment of Rotsee, cloning, and sequencing, a phylogenetic analysis identified two clusters of sequences and four sepa rated clones. The sequences in cluster 1 grouped with those of Methanosaeta spp., whereas the sequences in cluster 2 comprised the methanogenic endosy mbiont of Plagiopyla nasuta, Discriminative oligonucleotide probes were con structed against both clusters and two of the separated clones. These probe s were used subsequently for the analysis of indigenous methanogens in a co re of the sediment, in addition to domain-specific probes against members o f the domains Bacteria and Archaea and the fluorescent stain 4',6-diamidino -2-phenylindole (DAPI), by fluorescent in situ hybridization, After DAPI st aining, the highest microbial density was obtained in the upper sediment la yer; this density decreased with depth from (1.01 +/- 0.25) x 10(10) to (2. 62 +/- 0.58) x 10(10) cells per g of sediment (dry weight). This zone corre sponded to that of highest metabolic activity, as indicated by the ammonia, alkalinity, and:pH profiles, whereas the methane profile was constant. Pro bes Eub338 and Arch915 detected on average 16 and 6% of the DAPI-stained ce lls as members of the domains Bacteria and Archaea, respectively. Probe. Ro tcl1 identified on average 4% of the DAPI-stained cells as Methanosaeta spp ., which were present throughout the whole core. In contrast, probe Rotcl2 identified only 0.7% of the DAPI-stained cells as relatives of the methanog enic endosymbiont of P. nasuta, which was present exclusively in the upper 2 cm of the: sediment. Probes Rotp13 and Rotp17 did not detect any cells. T he spatial distribution of the two methanogenic populations corresponded we ll to the methane production rates determined by incubation with either [C- 14]acetate or [C-14]bicarbonate. Methanogenesis from acetate accounted for almost all of the total methane production, which concurs with the predomin ance of acetoclastic Methanosaeta spp. that represented on, average 91% of the archaeal population. Significant hydrogenotrophic methanogenesis was fo und only in the organically enriched upper 2 cm of the sediment, where the probably hydrogenotrophic relatives of the methanogenic endosymbiont of P. nasuta, accounting on average for 7% of the archaeal population, were also detected.