Search

The sub-seafloor biosphere is the largest prokaryotic habitat on Earth but also a habitat with the lowest metabolic rates. Modelled activity rates are very low, indicating that most prokaryotes may be inactive or have extraordinarily slow metabolism. Here we present results from two Pacific Ocean sites, margin and open ocean, both of which have deep, subsurface stimulation of prokaryotic processes associated with geochemical and/or sedimentary interfaces. At 90 m depth in the margin site, stimulation was such that prokaryote numbers were higher (about 13-fold) and activity rates higher than or similar to near-surface values. Analysis of high-molecular-mass DNA confirmed the presence of viable prokaryotes and showed changes in biodiversity with depth that were coupled to geochemistry, including a marked community change at the 90-m interface. At the open ocean site, increases in numbers of prokaryotes at depth were more restricted but also corresponded to increased activity; however, this time they were associated with repeating layers of diatom-rich sediments (about 9 Myr old). These results show that deep sedimentary prokaryotes can have high activity, have changing diversity associated with interfaces and are active over geological timescales.

Culturable bacteria were detected in deep-sea sediment samples collected from the Nankai Trough site 1173 (Ocean Drilling Program, ODP, Leg 190) at 4.15 m below the seafloor with 4791 m of overlying water. In this deep ocean near surface sediment, mainly fermentative heterotrophs, autotrophic acetogens and sulfate-reducing bacteria were enriched by using two different non-selective enrichment culture media. Culturable bacterial population shifts within the deep marine sediment enrichments were monitored by using denaturating gradient gel electrophoresis (DGGE). DGGE analysis revealed a decrease in the number of 16S rRNA gene fragments from high to low carbon concentrations, and from low to high dilution of inoculum, suggesting that fast-growing bacteria were numerically dominant in enrichment culture samples. The dominant 16S rRNA fragments observed in DGGE gels were assigned to the Firmicutes, Proteobacteria (gamma and delta subgroups) and Spirochaeta phyla. Continual sub-culture and purification resulted in two isolates which were phylogenetically identified as members of the genera Acetobacterium and Marinilactibacillus. Our results, which combine enrichment culturing with DGGE analysis, indicated that enrichment cultures derived from inoculum dilution and media with various concentrations of carbon could facilitate the detection and isolation of a greater number of environmentally relevant bacterial species than when using traditional enrichment techniques alone.

Diversity of Bacteria and Archaea was studied in deep marine sediments by PCR amplification and sequence analysis of 16S rRNA and methyl co-enzyme M reductase (mcrA) genes. Samples analysed were from Ocean Drilling Program (ODP) Leg 190 deep subsurface sediments at three sites spanning the Nankai Trough in the Pacific Ocean off Shikoku Island, Japan. DNA was amplified, from three depths at site 1173 (4.15, 98.29 and 193.29 mbsf; metres below the sea floor), and phylogenetic analysis of clone libraries showed a wide variety of uncultured Bacteria and Archaea. Sequences of Bacteria were dominated by an uncultured and deeply branching 'deep sediment group' (53% of sequences). Archaeal 16S rRNA gene sequences were mainly within the uncultured clades of the Crenarchaeota. There was good agreement between sequences obtained independently by cloning and by denaturing gradient gel electrophoresis. These sequences were similar to others retrieved from marine sediment and other anoxic habitats, and so probably represent important indigenous bacteria. The mcrA gene analysis suggested limited methanogen diversity with only three gene clusters identified within the Methanosarcinales and Methanobacteriales. The cultivated members of the Methanobacteriales and some of the Methanosarcinales can use CO2 and H2 for methanogenesis. These substrates also gave the highest rates in 14C-radiotracer estimates of methanogenic activity, with rates comparable to those from other deep marine sediments. Thus, this research demonstrates the importance of the 'deep sediment group' of uncultured Bacteria and links limited diversity of methanogens to the dominance of CO2/H2 based methanogenesis in deep sub-seafloor sediments.