Enrichment and Cultivation of Microorganisms from Sediment from the Slope of the Peru Trench (ODP Site 1230)

Abstract

The deep biosphere is estimated to hold a significant percentage of the Earth's prokaryotic biomass; however, little is known about the organisms in this environment. Here, we describe investigations of the diversity of microorganisms enriched from surface and subsurface sediment collected during Leg 201 of the Ocean Drilling Program at Site 1230 on the slope of the Peru Trench. This site contains methane hydrates, high levels of organic matter, and high direct cell counts, all of which indicate the potential for thriving microbial populations. To investigate these populations, we examined prokaryotes in samples from seafloor to 258 meters below seafloor (mbsf) using both cultivation and molecular methods. From seafloor samples, we cultivated isolates representing the genera Photobacterium, Shewanella, and Halomonas. The population found in an enrichment cultivated at low temperatures, 0.67 mbsf, contained many cell morphologies and deoxyribonucleic acid (DNA) signatures, but this population, except for a Vibrio sp., was difficult to separate and grow as pure cultures. Most isolates produced extracellular lytic enzymes that were active at low temperatures. Methanogens have been expected to play a large role in the creation of methane hydrates in the sediment; therefore, we also attempted to enrich for psychrophilic methanogens. No methane was found above background levels in anaerobic enrichments incubated for 2 yr, nor was any 16S ribosomal DNA detected following amplification using archaeal primers with DNA extracted from these incubated cultures. These results illus-1 Biddle, J.F., House, C.H., and Brenchley, J.E., 2005. Enrichment and cultivation of microorganisms from sediment from the slope of the Peru Trench (ODP Site 1230).

Biogeographical distribution and diversity of microbes in methane hydrate-bearing deep marine sediments on the Pacific Ocean Margin

“…In a previous study of the Sea of Okhotsk, ␣-and ␥-Proteobacteria dominated in volcanic ash layers, and the genera Halomonas, Marinobacter, and Sulfitobacter were frequently isolated (11). At Site 1230, members of the genera Halomonas, Marinobacter, Shewanella, Photobacterium, and Vibrio were cultivated (14,28). Most cultivable members in deep marine sediments were found to be facultatively anaerobic, heterotrophic, piezophilic (tolerant or adapted to high pressure), or halophilic bacteria (11,14,28,29).…”

Novel Cultivation-Based Approach To Understanding the Miscellaneous Crenarchaeotic Group (MCG) Archaea from Sedimentary Ecosystems

“…Additionally, members of MCG-8 and MCG-4 seem to tolerate oxygen to a certain extent, quite unlike some other anaerobes, which can be killed by oxygen after very short exposure times (e.g., Ͻ2 h in some cases [29]). Interestingly, MCG 16S rRNA gene sequences were recovered from a short-term aerobic culture previously (30), and MCG sequences from fermented seafood (a fermentation process which is not strictly anaerobic) have been reported to coexist with sequences from known aerobic archaea and bacteria (31), which also suggests that some MCG archaea could be tolerant of oxygen. Quite possibly, MCG archaea from the White Oak River basin exhibit oxygen tolerance mechanisms similar to those employed by some sulfatereducing bacteria, which can remain viable after exposure to oxygen (e.g., for a review, see reference 32).…”

Metagenomic signatures of the Peru Margin subseafloor biosphere show a genetically distinct environment

“…Approximately 6 g of product was obtained for the 1-and 16-mbsf samples, and 4 g was obtained for the 32-and 50-mbsf samples. All amplified DNAs were diluted and checked for community composition by intergenic transcribed spacer fingerprinting by using both bacterial and archaeal primer sets as described (3). The overall community composition between samples was similar.…”

“…It has been difficult to determine which microorganisms are responsible for most major geochemical cycles in the subsurface, including those for methane and sulfate (3,4). Recently, a study of the isotopic fractionation of carbon pools in the subsurface led to the suggestion that new metabolisms may be possible in this unique environment, where substrates are restricted and cells may survive on incredibly long time scales (5).…”