Enrichment and cultivation of microorganisms from sediment from the slope of the Peru Trench (ODP Site 1230)

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 illustrate the need for further extensive microbiological studies in order to understand the biogeochemistry of this important subseafloor environment.