In all of the sedimentary environments sampled during Leg 201, sedimentary properties and, by inference, past oceanographic conditions affect the current rates and sediment depths of several principal microbial activities. This point is most broadly demonstrated by the general correspondence between each site's geographic location (open ocean, equatorial upwelling, or coastal upwelling) and its current cell concentrations, rates of net general activity, and rates of net specific activities (Figs. F5, F7, F8, F9).
At individual sites, the depths at which specific microbial activities are most pronounced are commonly directly related to sediment properties. These properties in turn were largely determined by oceanographic conditions at the time of the sediment's original deposition. For example, correspondences between dissolved iron concentration and magnetic susceptibility suggest that current subseafloor rates and foci of microbial iron reduction may be ultimately controlled by the availability of oxidized iron in mineral form. This relationship is beautifully illustrated at Site 1225, where concentration of dissolved iron closely follows magnetic susceptibility (Fig. F13). At that site, peak Fe concentration is present in lithologic Subunit IC, where magnetic susceptibility is highest. These sediments were deposited during the middlelate Miocene "carbonate crash" of the equatorial Pacific (Farrell et al., 1995). Leg 201 magnetic reversal records suggest that the magnetic susceptibility of these sediments dates to the time of their original deposition. The overlying interval of near-zero magnetic susceptibility and very low dissolved Fe concentration was deposited during the late Miocene "biogenic bloom" of the Indo-Pacific tropical ocean (Farrell et al., 1995).
Relationships between dissolved manganese concentration, lithology, and physical properties similarly suggest that current rates and stratigraphic foci of microbial Mn reduction are contingent on oceanographic history. Such a relationship is seen at Site 1226, where peak dissolved Mn concentration is present in the oldest sediments (Fig. F14). This sedimentary interval exhibits the lowest natural gamma radiation and, as shown by Leg 138 shipboard studies, the lowest average total organic carbon (TOC) concentration of this 16-m.y. sedimentary sequence (Shipboard Scientific Party, 1992a). This interval of low gamma radiation and low TOC was deposited at very slow rates over 8 m.y. (Shipboard Scientific Party, 1992a). The overlying interval of higher gamma radiation, higher TOC, and much lower dissolved Mn concentration was deposited during the late Miocene biogenic bloom (Shipboard Scientific Party, 1992a). These relationships suggest that manganese reducers now active in these sediments are utilizing oxidized manganese that accumulated during an interval of unusually low carbon accumulation, from 8 to 16 m.y. ago.
Profiles of magnetic susceptibility and concentrations of dissolved Fe, Mn, and sulfide suggest that current iron and manganese reducing activity may be similarly contingent on depositional history at the Peru shelf sites. For example, at Site 1229 peak dissolved Fe concentration and low dissolved sulfide concentration coincide with intervals of relatively high magnetic susceptibility at 87 mbsf and below 123 mbsf (Fig. F15). These magnetic susceptibility occur in intervals of terrigenous-dominated sediments deposited during sea level lowstands. The peak Fe concentration at 87 mbsf is within the well-developed methanogenic zone at this site (Fig. F16). The peak concentration below 123 mbsf lies within the underlying sulfate-reducing zone. In short, higher Mn and Fe concentrations are consistently associated with relatively high-susceptibility intervals at both open-ocean and ocean-margin sites. This association illustrates one way in which current rates and stratigraphic depths of an individual microbial process may depend on past ocean history.
At the Peru shelf sites, the lithologic context of anaerobic methane oxidation (AMO) zones suggests another way that past ocean history may affect current microbial activity. At these sites, AMO and related processes predominantly occur in narrow subsurface zones that are associated with thin sedimentary intervals characterized by high grain density (Fig. F17), accompanied by high natural gamma radiation (NGR), high resistivity, and low porosity. These thin low-porosity intervals are unusually rich in terrigenous sediment and are interpreted to have been deposited during sea level lowstands. This lithologic association of AMO zones with high-density, low-porosity lowstand sediments provides intriguing evidence that on the Peru shelf the position of AMO zones is pinned within the sediment column by lithologic properties and, by extension, depositional history. More detailed determination of the extent to which physical and compositional properties control subseafloor biogeochemical zonations in this region will require further investigation.
A different aspect of the Peru shelf records provides evidence that geologically recent oceanographic changes may also affect the activity of subseafloor organisms at shallow burial depths. At Sites 1228 and 1229, a brief positive excursion in alkalinity, DIC, ammonium, and sulfide coincides with a brief negative excursion in dissolved sulfate at 23 mbsf (Fig. F18). This near-surface pore water anomaly indicates that the steady-state diffusion of biologically active chemicals past the upper sediment column was disrupted by late Quaternary environmental change and has not yet fully recovered. There are least three possible explanations of this anomaly. It may result from ongoing activity in a microbial "hot spot" at this shallow sediment depth, it may be a chemical relic of past microbial activity and is now relaxing back to a diffusional steady state, or it may be due to the establishment of an oxygen minimum at this water depth, causing the extinction of a bioirrigating benthos and a stimulation of sulfate reduction. The first and third of these explanations imply that this anomaly is a fingerprint of current microbial activity.
Chemical profiles of the Leg 201 sites provide consistent evidence of microbial influence on their sedimentary environment. This evidence is most clearly expressed at the ocean-margin sites, where microbial activity is highest. At these sites, the precipitation and dissolution of a number of minerals, including pyrite, barite, dolomite, and apatite, are catalyzed by microbial activities.
Dissolved barium, sulfate, and methane profiles provide a particularly clear illustration of the interplay between microbial activity and authigenic mineral formation and dissolution at these sites (1227, 1228, 1229, and 1230). The dissolved Ba2+ profiles of these sites are broadly similar to their CH4 profiles and are inversely related to their dissolved SO42 profiles (Fig. F19). The inverse relationship between SO42 and Ba2+ is inferred to be controlled by the solubility product of BaSO4 (barite) and the in situ activity of sulfate-reducing bacteria. Within the zone of AMO, dissolved SO42 concentration declines toward 0 mM, barite dissolves, and the dissolved Ba2+ concentration rises. Diffusion of the dissolved Ba2+ past the AMO zone is suspected to sustain modern barite formation in the zone of dissolved SO42. At sites with particularly high dissolved Ba2+ concentration (such as Site 1230), a significant fraction of the SO42 diffusing toward the AMO zone may participate in this cycle of barite precipitation and dissolution before finally being reduced by microbial activity.
A broad range of microbiological studies was initiated during Leg 201 in order to study the diversity and population size of subsurface microbial communities and to analyze the pathways and rates of their metabolic activities. These studies will not yield immediate results, partly because of the expected slow growth of deep subsurface bacteria and partly because the research requires special analytical equipment available only in shore-based laboratories.
A very large number of cultivation experiments and viable counts based on MPN methods were among the studies initiated on board. These cultivation experiments target a broad physiological spectrum of heterotrophic and autotrophic microorganisms that utilize diverse electron acceptors and donors in their energy metabolism. Some experiments target chemoautotrophs using different electron donor/acceptor combinations. Others focus on heterotrophs using different electron acceptors for respiration. Others focus on methanogenic and acetogenic organisms, fermenting, or spore-forming organisms. Still others target microbes adapted to different monomeric or polymeric carbon sources, different temperature adaptations (psychrophilic, mesophilic, or thermophilic), and/or different pH, salinity, and pressure requirements. Some cultivation experiments focus on consortia depending on syntrophic degradation of organic substrates. Gradient cultures were initiated to screen for different substrate concentration requirements. Radiotracer MPN cultivations were inoculated for detection of minimal growth.
A wide range of other microbiological studies will be conducted postcruise. An extensive sampling scheme was developed for postcruise studies of microbial populations using culture-independent molecular analyses. Molecular techniques based on 16S rRNA (ribosomal ribonucleic acid) gene sequence information will be used to analyze microbial diversity and function. Sequence libraries for populations of different sediment depths and geochemical interfaces are expected to demonstrate differences related to the special conditions for life in the deep subsurface. Quantitative analyses of the microbial communities will be done using, for example, FISH and real-time polymerase chain reaction (PCR). Secondary ion mass spectrometry (SIMS) will be used to analyze the stable carbon isotope composition of individual cells or cell clusters that are identified with nonspecific fluorescence stains or FISH probes. The potential metabolic activity of dominant members of these communities will be analyzed by sequencing of functional genes for key enzymes, such as dissimilatory sulfite reductase and adenosine-5'-phosphosulfate (APS) reductase for microbial sulfate reduction, coenzyme-M methyl reductase for methanogenesis, and formyl tetrahydrofolate synthase for acetogenesis. Finally, the carbon isotopic signature of biomarkers will be analyzed in order to identify the carbon substrate of the dominant microbial populations.
It was an important goal of Leg 201 to identify and quantify the dominant microbial processes in the deep subsurface. To identify gross rates of microbial processes and rates that involve intermediate metabolites, experiments using radioactive tracers were conducted on the ship. Such use of radiotracers increases the sensitivity of process rate measurements by several orders of magnitude. The following microbial processes were analyzed at all relevant sites with samples from many depths and geochemical zones: sulfate reduction (35S), methanogenesis (14C) and acetogenesis (14C), anaerobic methane oxidation (14C), acetate (14C) and hydrogen (3H) turnover, and bacterial growth, thymidine incorporation (14C). Most of these experiments were terminated by the end of the cruise. Their activities will be measured in shore-based laboratories shortly after the cruise. The results of these experiments will be compared to net rates obtained by modeling of pore water chemical gradients.
The study of deep subsurface microbial communities is highly dependent on rigorous contamination control. Contamination tests are necessary to assess the extent to which bacteria from the surface environment may have reached the microbiological sediment samples at any point. Contaminating bacteria may be introduced from drilling fluid during the coring operation, they may penetrate into cores from their contaminated periphery during the wireline trip, or they may be introduced during the subsequent sectioning and subsampling of sediment. Consequently, refinement of routine methods for testing and avoiding potential contamination was an important part of the microbiological work during Leg 201.
During the drilling operation at all sites, perfluorocarbon tracer (PFT) was fed in trace quantities into the seawater pumped through the drill string (Smith et al., 2000a, 2000b). This practice ensured that the core liner was bathed in a dissolved tracer that could later be analyzed at high sensitivity in retrieved cores and microbiological samples. The quantity of PFT detected in a sample provided an estimate of the maximal amount of seawater introduced into that sample. The PFT provides an upper-bound estimate of bacterial contamination, as it may penetrate by molecular diffusion through pore space unavailable to bacteria. To further evaluate the extent to which contaminating bacteria may have penetrated into a sample, fluorescent beads of bacterial size were applied as an additional contamination tracer. A suspension of >1011 0.5-µm-sized beads was released in the core cutting shoe at the critical moment of impact against the sediment. Microscopic counts of beads subsequently indicated whether samples used for microbiology had become "infected." This test may be more realistic with respect to the possibility of bacterium-sized particles penetrating a sediment core. However, it is rather qualitative because the beads are not uniformly delivered to all surfaces of the core. Taken together, the two approaches provide a critical test on which to base confidence in the noncontaminated nature of microbiological samples.
A summary of the extensive contamination tests for all Leg 201 sites is given in Figure F20, which shows PFT and bead concentrations observed in selected samples. The two contamination indicators are positively correlated, thus improving confidence in the prediction of potential contamination by their combined use. The subset of samples presented in Figure F20 was chosen to reflect the wide range of tracer results observed during Leg 201. Out of the 117 PFT tests conducted, 90% had <0.1 µL potential seawater contamination and 26% had no detectable contamination (<0.01 µL potential seawater contamination). The centers of the cores, which were used for microbiological sampling, were compared to the periphery in contact with the core liner. The results show that core centers are much less contaminated than core peripheries. Advanced piston corer (APC) cores generally show less potential contamination than the "biscuits" of sediment retrieved in extended core barrel (XCB) cores. The large scatter in these data shows that potential contamination is highly irregular from sample to sample. Consequently, contamination tests must be conducted routinely on all samples used for microbiology. It is not sufficient to rely on general experience with a specific coring/sampling technique or sediment type.
Samples specifically used for isolations and viable bacterial counts during Leg 201 were generally proven by both PFT and bead tests to have very low or undetectable contamination. Through the extensive experience gained during the leg, our confidence has strengthened that, with rigorous contamination controls and aseptic sampling techniques, deep subsurface samples can routinely be obtained without the introduction of microorganisms from the surface environment.
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