Deep drilling into 45-Ma oceanic basaltic crust in a deepwater (~5000 m) environment provided a promising opportunity to explore the bacterial deep biosphere using a comprehensive multiphase experimental approach.
At Site 1224, 25 samples were taken at various depths, ranging from near-surface sediments to hard rock samples of basaltic basement down to 154 mbsf. Sediment samples from Holes 1224C, 1224D, and 1224E were obtained from different depths ranging from the upper surface layer down to 24.9 mbsf, which was almost the bottom of the sediment coverage at this particular site. The sediment samples consist of light-brown to dark red-brown clay. Five sediment samples were taken over the whole color range near the surface (0.05 mbsf) at 1.45, 3.91, 5.63, 6.28 and 24.93 mbsf.
Ambient seawater samples were collected at 1 m below sea surface, upwind of the JOIDES Resolution, to evaluate the microbial background at Site 1224. The microscopically enumerated total cell counts in the surface water at Site 1224 were 1.4 x 104 cells/mL.
As shown by a comparative determination of total cell counts using the nucleic acid stains SYTO 9 and SYBR Green I and SYTO 62, bacteria were present in all sediment samples taken from Site 1224 down to 24.9 mbsf. As shown for Site 1223, the highest total count was determined in near-surface sediments (Sample 200-1224C-1H-1, 0-5 cm) with 3.8 x 1010 cells per gram of wet sediment. This was followed by a slow decline in total cell counts to 1.1 x 1010 within the sediment layer located at 1.45 mbsf (Sample 200-1224C-1H-1, 145-150 cm). About 4 m downhole (at 5.63 mbsf; Sample 200-1224C-1H-4, 113-118 cm), the number of total cell counts decreased more rapidly by two orders of magnitude to 2.4 x 108 cells per gram of wet sediment. At a depth of 24.93 mbsf (Sample 200-1224E-2R-5, 143-150 cm), bacterial total cell counts slightly increased to 7.3 x 108 cells per gram of wet sediment.
The amount of metabolically active bacteria within the bacterial communities was assessed by fluorescent in situ hybridization (FISH) in two representative sediment samples taken from an upper sediment layer at a depth of 1.45 mbsf (Sample 200-1224C-1H-4, 145-150 cm) and from a depth of 25 mbsf (Sample 200-1224E-2R-5, 143-150 cm). As indicated by fluorescent signals after hybridization with the Bacteria-specific probe EUB338, the amount of metabolically active bacteria ranged in these sediment layers from 41% to 62% of the total cell counts, respectively (Fig. F60). The FISH-based assessment of bacteria with apparent physiological potential was conducted as described in Manz et al. (2001).
Most probable number assays from both sediment samples and rock suspensions from ground tholeiitic basaltic rocks were conducted for both sulfate-reducing bacteria and fermentative bacteria.
Whole-round cores of basement rocks, including glass samples, were collected and transferred within 5 min into an anaerobic chamber. The rock samples were subsequently manually cracked open with sterile chisels, and only pieces from the inner core were used to establish anaerobic and aerobic cultures.
Suspensions obtained from ground rock material were successfully cultivated at various temperatures under both aerobic and anaerobic conditions. Ground rock material obtained from rock samples taken at 27 mbsf (Sample 200-1224D-1R-2, 10-15 cm) and 31.5 mbsf (Sample 200-1224E-3R-4, 82.5-87.5 cm) were cultivated under aerobic conditions in an artificial seawater medium. Preliminary microscopic determination of bacterial colonies gave a strong indication of the presence of Fe2+-oxidizing bacteria (Fig. F61).
The microscopic investigation of a thin section of the massive tholeiitic lava flow unit located at 50.9 mbsf showed unique tubular filamentous structures within CaCO3 cavities, which resembled, with regard to shape and dimension, fungal hyphae including cell walls and dichotomous branches. The cross-sectional dimension of the hyphal network is 5-10 µm with a length ranging from 50 to several hundred micrometers. The hyphae are typically interrupted at irregular intervals by cross walls, or septa, which divide the entire fungal hyphae into single distinctive cells (see arrow in Fig. F62). The diameter of the cavities ranges from 0.5 to 3 mm with a filling matrix of calcite and/or aragonite. In our opinion, these cavities are part of a complex pore-channel system that developed as gas pipes during the cooling process of magma. The net of fungal hyphae shown in Figure F63 filled the entire space spanning from the basalt/carbonate boundary to the center of the cavity.
Pyrite was commonly observed as euhedral crystals within the cavities and outside as framboidal pyrite. Iron oxyhydroxides were never observed adjacent to the cavities' faces. Anaerobic conditions were apparently maintained within the cavities for the remainder of the mineralization process. All cavities where fungi were found were completely filled by secondary phases. At present the question of whether the microbial life reported here grew before, after, or at the same time as the formation of CaCO3 remains open.
A completely different situation is given in fractures and veins where we also detected fungal growth. Here again, the hyphae were detected within a carbonate matrix. Because of the encrustation with iron oxyhydroxide, caused by the contact with seawater, morphological traits of the hyphae are not so well preserved (Fig. F64). This surprising discovery provides strong evidence for eukaryotic life in deep subsurface environments in addition to bacteria.
The number of near-surface bacterial populations shown for Site 1224 is similar to those at Site 1223, with similar overlying water depth. The most abundant bacterial community was seen in the near-surface sediments. This was followed by a slow decrease of the total cell counts down to 1.45 mbsf and a more rapid reduction in cell numbers at a depth of 5.6 mbsf. Interestingly, within sediments obtained from a depth of 25 mbsf, the bacterial cell counts slightly increased again. The application of high-quantum-yield fluorochromes resulted in total cell counts ranging one to two orders of magnitude higher than reported during previous ODP Legs (Cragg et al., 1996).
As a general trend, bacterial population numbers decreased with increasing depth. However, the amount of metabolically active bacteria as revealed for the first time during Leg 200 by FISH, remained remarkably high with 41%-62% of the total cell counts. Both the high total cell counts as well as the amount of bacteria with apparent physiological potential within the sediment layers suggest a higher contribution of sediment bacteria than has been assumed until now. Keeping this in mind, the recently extrapolated contribution of the marine subsurface biosphere, ranging from 10% to 30% of the Earth's living biomass (Parkes et al., 2000; Whitman et al., 1998), might be the minimum contribution of bacterial communities.
The successful cultivation of putative Fe2+-oxidizing bacteria and the microscopic indication of further microbial structures within carbonate-filled cavities in basaltic rocks confirm the presence and even activity of microbial life not only in deep marine sediments, but also in old and cold oceanic crust far away from any deep-sea volcanic vent. Both Mn(II) and Fe(II) diffuse upward from reduced sediments and are deposited as oxide layers in the presence of low oxygen contents. Although Fe(II) oxidation is very rapid at neutral pH, filamentous bacteria such as Gallionella ferruginea living at the oxic/anoxic interface are known to drive iron oxidation as a mode of energy conservation in freshwater environments. The successful cultivation of aerobic bacteria capable of ferrous iron oxidation in a marine system would be an important contribution to our understanding of the complex biogeochemical iron cycle in marine environments. The phylogenetic affiliation of the obtained cultures will be further investigated by shore-based DNA extraction and characterization, electron microscopy, and biomarker analysis. The detection of fungi within a massive tholeiitic lava flow unit describes eukaryotic life in such a deep-sea environment for the first time. Along with the description of microfossils (fossilized microorganisms) as suggested by McKinley and Stevens (2000), the microbiological characterization of their modern equivalents will result in a more profound knowledge of the biogeochemical processes of the deep biosphere.
In summary, these results constitute further evidence for continuing microbiological activity in the deep subseafloor biosphere environment. The presence of fossilized iron-encrusted bacteria and fungi at interfaces may therefore serve as an indicator of anoxic to dysaerobic conditions in various paleo(micro)environments. Thereby, Leg 200 significantly contributes to the elucidation of the unique but almost unknown deep subseafloor biosphere environment. The secrets that microorganisms associated with the deep-marine component of our biosphere might reveal about the origin and limits of life on Earth are still unspoken and remain to be reported in further investigations.