Cultivations from Leg 201 sediments yielded a wide diversity of cultivable bacteria, predominantly members of two phyla with a Gram-positive cell wall, Firmicutes and Actinobacteria, a range of alpha and gamma-Proteobacteria, and a novel member of the Bacteroidetes phylum that was only distantly related to any other cultured isolate within this phylum (D'Hondt et al., 2004; Biddle et al., this volume; Lee et al., 2005).
The most frequently obtained cultured strains were members of the spore-forming genus Bacillus within the Firmicutes, isolated from open-ocean Sites 1231 and 1226 and Peru margin Sites 1227 and 1229. Actinobacteria were found in Pacific open-ocean Sites 1231, 1225, and 1226 and in Peru margin Site 1227 (D'Hondt et al., 2004). Also, species of the newly described thermophilic genus Thermosediminibacter, within the phylum Firmicutes, were isolated from the upper 10 m of Sites 1227, 1228, and 1230. The isolates grow anaerobically on a variety of sugars (hexoses and pentoses) and other heterotrophic substrates at a temperature optimum of 64°–68°C (Lee et al., 2005). This wide distribution of Gram-positive, spore-forming bacteria in terrestrial soils and marine sediments may be a consequence of broad dispersal of the highly durable resting stages of these bacteria, endospores that are resistant against nutrient depletion, desiccation, salinity and temperature fluctuations, radiation, and changing redox conditions. Spores can remain dormant over extremely long time periods; 34 yr for the revival of a historical bacterial culture of known age (Braun et al., 1981), several 1000 yr for spores from archaeological samples and ancient lake sediments (Gest and Mandelstam, 1987), and 25–40 m.y. for spore-forming bacteria that appear to have been revived from the guts of a bee encased in fossil amber (Cano and Borucki, 1995). Thus, the possibility cannot be ruled out that endospore-forming Gram-positive bacteria have been deposited as spores in deep subsurface sediments and have remained dormant until sample retrieval, resuscitation, and isolation. Thermophilic, spore-forming, Gram-positive, sulfate-reducing bacteria of the genus Desulfotomaculum (Isaksen et al., 1994) and thermophilic, spore-forming, alkane- and aromatic-degrading Geobacillus strains (Marchant et al., 2002) have been found in high numbers in cool marine sediments and cool soils that are too cold for them to grow, indicating effective environmental dispersal.
The most frequently isolated Proteobacteria from Leg 201 sediments include strains closely related to the alpha-Proteobacterium Rhizobium radiobacter and to the gamma-Proteobacterium Vibrio mediterranei (D'Hondt et al., 2004), although R. radiobacter (synonymous with Agrobacterium tumefaciens) is a soil bacterium and plant nodule symbiont, therefore an unlikely inhabitant of deep marine subsurface sediments. R. radiobacter-related strains have been isolated frequently from subseafloor sediments in the Mediterranean and other deep subsurface environments (Suess et al., 2004). By quantitative PCR, as much as 5% of the total bacterial 16S rDNA gene sequences in Mediterranean sediments were shown to be R. radiobacter (Suess et al., 2005). A cultivation survey of surficial and near-surface sediments from Site 1230 yielded gamma-Proteobacteria of the genera Photobacterium, Vibrio, Shewanella, and Halomonas (Biddle et al., this volume). Gamma-Proteobacteria appear to be a major group of frequently cultured subsurface bacteria in Leg 201 samples and other subsurface studies. For example, Vibrio spp. have been isolated from deep Mediterranean sapropel sediments (Suess et al., 2004).
In general, the spectrum of cultured subsurface isolates (Proteobacteria, Actinobacteria, Firmicutes, and the Cytophaga-Flavobacterium-Bacteroides phylum) is narrower than the diverse lineages of bacteria and archaea that were detected using DNA-based molecular methods. The cultured bacteria appear to constitute only a small fraction of the total cell counts in each sample (D'Hondt et al., 2004). Cultivating representatives of the bacterial and archaeal phyla that dominate subsurface clone libraries remains a continuing challenge.