The deep sea and its sediments represent the largest permanently cold environment on Earth. We incubated cultures at low temperatures (2°–10°C) in order to increase the number of psychrophilic microorganisms and to explore their potential enzyme activities in situ. We isolated and characterized organisms that are phylogenetically related to Photobacterium, Halomonas, Shewanella, and Vibrio species. All isolates are closely related (by >98% 16S rRNA similarity) to previously isolated deep-sea strains, consistent with their being from the core sample rather than contaminants. These genera are also commonly found in deep sediment studies, especially those in the Pacific (Wang et al., 2004). The trend in extracellular degradative enzyme production agrees with previously published results of deep-sea sediment isolates and corresponds to available nutrient sources, suggesting a possible adaptation to this environment or competitive advantage within this ecosystem (Boetius and Lochte, 1994; Luna et al., 2004; Wang et al., 2004). The one Vibrio sp. isolate from the sediment column (0.67 mbsf) adds to the database of other sediment-dwelling microbes isolated from deeper than 0.5 mbsf (Bale et al., 1997; Toffin et al., 2004). Further, the characterization of these isolates increases the numbers of described species for these genera and provides information on their production of cold-active enzymes of possible industrial interest.
Our mixed population studies of cultures from the 0.67-mbsf sample also detected 16S rRNA genes from Halomonas spp. and Shewanella sp., and microscopic examinations suggested the presence of numerous spirochetes and spirilla. This population differs from the Marinilactibacillus and Acetobacter type community described from 4.15 mbsf in the Nankai Trough (Toffin et al., 2004). In other ways, our cultivated mixed population from 0.67 mbsf appears similar to other cultivated populations from below the seafloor (Toffin et al., 2004). Spirochetes have often been found in seafloor and hydrothermal vent environments (Bowman et al., 2000; Campbell and Cary, 2001), and have recently been identified in DNA libraries from the Nankai Trough (Newberry et al., 2004), but their cultivation from deep sediment was a surprise (Toffin et al., 2004). In both studies described here, spirochetes were seen in culture for short periods in media containing yeast extract. It is possible that this provides important nutrients for these organisms. However, because the spirochete population has not been maintained for longer incubations, their growth requirements remain unknown.
The presence of a number of facultative anaerobic microorganisms in our cultivations suggests that the capability for anaerobic growth may allow cell survival after burial by the accumulation of the sediment column over time. However, because facultative organisms grow more rapidly aerobically, especially at low temperatures, we used aerobic cultivation to examine this population. In such an aerobic enrichment culture, it was surprising to find crenarchaeal signatures. Crenarchaea have been shown to be members of the seafloor community (Bowman and McCuaig, 2003; Vetriani et al., 1998, 1999) and have been detected deeper in sediment columns (Bidle et al., 1999). However, to our knowledge, they have not yet been reported as members of a cultivated community from marine sediment. Here we report the existence of a marine benthic Crenarchaeon in a bacteria-dominated enrichment culture at 10°C for more than a week. The conditions needed to prolong the existence of these Crenarchaea in liquid culture are being investigated.
In addition to the numerous crenarchaeal and euryarchaeal sequences retrieved from the subsurface by other researchers during Leg 201 (F. Inagaki, unpubl. data; A. Teske, unpubl. data), methanogens are expected to exist throughout the sediment column. The difficulties, reported here and elsewhere, in detecting subseafloor psychrophilic methanogens are especially puzzling. The light isotopic values of methane in the sediment column (K. Hinrichs et al., unpubl. data) show that the methane is of biological origin. In addition, the dissolved inorganic carbon (DIC) isotopes of Site 1230 sediment (D. Shrag, unpubl. data) show that methanogenesis is occurring within the sediment column, due to the heavy swing of the DIC isotopes in the methane zone (Fig. F5).
Methanogens, however, have been difficult to demonstrate in the marine subsurface where 16S rRNA gene diversity (Bidle et al., 1999; Inagaki et al., 2003; Marchesi et al., 2001) or functional gene diversity (Marchesi et al., 2001; Newberry et al., 2004) studies have encountered patchy and unequally distributed detection of methanogen DNA. Whereas the methanogen population may be limited near the sulfate-rich and oxygenated seafloor, the abundant biogenic methane present suggests that methanogens exist in the subsurface. Based on the assumption that previous inconsistent methanogen detections were the result of their being a minority population, our culturing attempts were designed to measure very small levels of methanogenesis or to encourage growth that could be detected by molecular methods. These enrichments, however, were unsuccessful in increasing numbers of any cold-loving microorganisms, methanogenic or nonmethanogenic, to detectable levels in the time allowed. Because of the long incubation times needed for anaerobic cultivations at low temperatures for seafloor isolates, longer incubations may be required to truly examine anaerobic psychrophilic microorganisms from the deep subsurface.