Marine sediments constitute a large global carbon reservoir and an environment with numerous microorganisms (Whitman et al., 1998). Although there have been a number of studies on this environment, there are still microbial ecosystems to explore, especially in regions of deep sediment environments. With a few exceptions (Wang et al., 2004), analyses of deep sediment column samples have studied either environmental deoxyribonucleic acid (DNA) (Bidle et al., 1999; Inagaki et al., 2003; Kormas et al., 2003; Newberry et al., 2004) or have cultivated organisms at temperatures higher than those in situ (Bale et al., 1997; Mikucki et al., 2003; Toffin et al., 2004).
Samples from Leg 201 of the Ocean Drilling Program (ODP) presented a unique opportunity for microbiological studies of deep-sea sediments that could be compared with the biogeochemical data. During this cruise, cores were collected from the equatorial Pacific and Peru margin, covering a wide range of marine sediment conditions: organic carbon-rich margin sites to more organic-poor open-ocean sites (D'Hondt, Jørgensen, Miller, et al., 2003). The most organic-rich core from Leg 201 was from Site 1230 on the slope of the Peru Trench (D'Hondt, Jørgensen, Miller, et al., 2003). This site also contained the highest microbial populations, with direct counts of >108 cells/cm3 sediment at the seafloor (D'Hondt, Jørgensen, Miller, et al., 2003). This site is also of special interest because it contains abundant biologically produced methane hydrates throughout the sediment column (D'Hondt, Jørgensen, Miller, et al., 2003).
For these reasons, Site 1230 core serves as an excellent resource for comparing microbial populations at different depths in the same borehole. Although only a small percentage of the total microbial population in an environmental sample can be cultivated, obtaining isolates is an important way to study physiological reactions that may be occurring in situ. In addition, the cultivation of microbes in conjunction with molecular studies of diversity provides a more complete composite of the total population (molecular studies from Leg 201 will be performed by other researchers). With this in mind, we initiated the cultivation of organisms starting with the region of most aerobic heterotrophic activity and abundant carbon source, the seafloor. We were interested in whether psychrophilic organisms could be isolated with extracellular enzymatic activities at seafloor temperatures (2°–6°C). We also examined populations existing in deeper sediments with continued cultivation combined with molecular analyses.
In addition, because of the presence of methane hydrates at Site 1230, we specifically enriched for methanogens that might be within the sediment column. Previous studies have examined archaeal populations of subseafloor sediment (Bidle et al, 1999; Marchesi et al., 2001; Mikucki et al., 2003; Newberry et al., 2004) and have usually not found 16S ribosomal ribonucleic acid (rRNA) genes of methanogens as part of their libraries. Evidence for methanogens has been found using functional gene (mcrA) or more specific 16S rRNA gene primers (Marchesi et al., 2001; Newberry et al. 2004), and recently a mesophilic methanogen species designated Methanoculleus submarinus was isolated from 247 meters below seafloor (mbsf) at the Nankai Trough (Mikucki et al., 2003). The difficulty in finding evidence for methanogens at many sites suggests that they are absent, present in low numbers, or cannot be detected by current methods. Although a few psychrophilic methanogens are in pure culture, none are from a deep marine sediment column (Chong et al., 2002; Franzmann et al., 1997). Additional studies are required to provide information on methanogens in deep sediment columns and clarify their relationship with methane hydrates. Here, we describe our attempts to cultivate methanogens from Site 1230 core samples. We also describe bacterial isolates obtained from seafloor and 0.67 mbsf samples and the studies of mixed populations of Bacteria and Archaea detected by molecular methods in cultures incubated through multiple enrichments at low temperatures.