| The microbial populations of the subseafloor may constitute up to a third of the Earth's biomass. The activity and diversity of these subsurface cells has been understudied, and their relevance to the surface world and relationships with each other remain unknown. For my thesis, I examined sediment from Ocean Drilling Program (ODP) Leg 201 to determine both the cultivation-dependent and cultivation-independent microbial diversity in deep sea sediments from the Peru Margin. This work was begun by performing cultivation studies on sediment from ODP Site 1230 and determined that many of the cultivable Bacteria in the near surface sediments were facultative anaerobes. I continued to examine the cultivability of Bacteria to determine if depth, lithology or interstitial water chemistry had an effect on the ability to recover isolates at ODP Site 1229, which had two distinct sulfate/methane transition zones (SMTZ). My results indicated that interstitial water chemistry was the most important factor predicting recovery. Using Site 1228 for comparison, at Site 1229, the cultivation of bacteria was negatively affected within the SMTZs. Sulfate-dependent anaerobic oxidation of methane (AOM) is expected to occur in the SMTZs. The shipboard acridine orange direct cell counts were highest in the SMTZs, presumably because the cells obtained energy from AOM, either directly or indirectly. Fluorescent in-situ hybridization was used to distinguish between members of the archaeal and bacterial domains and found that the archaeal population is highest within these SMTZs, making up to 98% of the hybridized cell count. The carbon isotopic composition of these individual archaeal cells was then analyzed by secondary ion mass spectrometry and showed that they were isotopically heavy (average delta 13C ∼ 20‰). These cells were hypothesized to be isotopically light, since Archaea within seep AOM environments have been shown to uptake isotopically light methane as their biomass. However, upon further inspection of the sediment carbon budget, these deeply buried SMTZ Archaea appear to use methane as an energy source, but not a carbon source, and the cells appear to be heterotrophic. In order to continue the examination of the microorganisms in this sediment, especially the Archaea and their genomic potential, I extracted the total genomic DNA from the sediments at Site 1229 and subjected it to high throughput sequencing. From this metagenome, it was determined that Archaea are indeed the dominant microbial population at depth, using small subunit ribosomal RNA genes as in-silico biomarkers. This thesis describes a multi-faceted approach towards the study of microorganisms in deeply buried sediments. Through these approaches, I have been able to unearth an interesting relationship between Bacteria, Archaea, and the geochemistry of these sediments and uncover clues about a previously unknown and unexpected metabolism of methane oxidation. |
