Igneous samples from Hole 801C selected for microbiological studies represent a range of environments within the volcanic basement, including massive basalt, fractured basalt, and different types of alteration and vein filling. Core liners were split and core samples were chosen for microbiological analyses as well as for fluorescent microsphere and perfluorocarbon tracer (PFT) analyses. Table T15 gives a core-by-core list of samples collected for each type of analysis. Thin sections of volcanic glass were examined for "microbial" and chemical alteration patterns. Drilling fluid, surface seawater, and borehole water were collected to enumerate and characterize background levels of microbes potentially introduced during the drilling process. Sediment samples collected from Core 185-801D-1W were used to test sampling protocols and tracer techniques in preparation for sediment sampling at Site 1149.
Details of Hole 801C samples used for microbiological analyses are shown in Tables T16 and T17 and complete petrographic descriptions of the six samples used for the full suite of microbiological analyses are given in "Sample Descriptions of Rocks Used in Microbial Cultures". The analyses included cultivation at 1 atm; enumeration of cells by epifluorescence microscopy; observation of cells with scanning electron microscopy (SEM); DNA extraction and community characterization; in situ hybridization; and maintenance at in situ pressure. An additional 11 samples were maintained at in situ pressure with no additional culturing medium. Additional culturing experiences were started on the ship using anaerobic media (see "Microbiology" in the "Explanatory Notes" chapter). Cultures started from Sample 185-801C-23R-1 (Piece 10, 119-125 cm) were incubated in 25° or 30°C incubators, within 3°C of in situ temperature (Table T16) and at in situ pressure of 620 atm. The pressure cultures were incubated at 25°C for one month, then transferred to 1 atm before the end of the leg. Cultures will be analyzed onshore for products of metabolism and for the presence of cells by microscopy. Shore-based analyses of preserved rock samples will include enumeration of cells, SEM, DNA extraction, in situ hybridization, and cultivation at in situ pressure.
Tracer tests were carried out as a part of Leg 185 microbiology studies to determine whether it is possible to collect microbiological samples without contamination introduced during drilling or sample handling. Eight fluorescent microsphere and two PFT tests were conducted on cores from Hole 801C. Methods used for these tests and results can be found in "Methods for Quantifying Potential Microbial Contamination during Deep Ocean Coring" (Smith et al., 2000) and in Smith et al. (in press). Fluorescent microspheres were not observed in the crushed interior rock used for the microbiological analyses, even though they were detected on the outside of the cores (see Table T18). PFT was detected in the cores, indicating that 0.01-0.03 µL drill water/g of rock had penetrated the rock. (see Table T19 and Figs. F74, F75). Based on the abundance of cells in surface seawater at Site 801 (see below), this volume of drill water would translate into 3-13 cells/g of rock.
This sample was taken from lithologic Unit 37 (Sequence IV), which consists of pillow basalts and interpillow material (Fig. F76). The basalt is fine grained to glassy and is aphyric. In Figure F76 the right side of Piece 8 and the left side of Piece 9 are basalt, and the lower left (greenish) of Piece 8 and the right side of Piece 9 are the interpillow material. The vertical white zone in the center of Piece 8 that separates the basalt from the interpillow material is mostly calcite. A thin section was made from a pillow margin and interpillow material from 10 cm lower in the core (Sample 185-801C-14R-3, 120-122 cm), which is the same material as found in Piece 9. The thin section contained altered volcanic glass, calcite, celadonite, and chalcedony that have filled open spaces. The glass contained some olivine that has been altered to iron oxides and altered laths of plagioclase (Fig. F77). The sample that was crushed and was used to inoculate cultures was mostly interpillow material similar to the right half of Piece 9 in Figure F76.
This section of the core contains pillow basalts with interpillow material composed of fractured basalt, glass, and secondary minerals. Some pillow basalts enclose breccia made of basalt clasts cemented with calcite (Fig. F78). The clasts are typically 0.5-1 cm across and some have been oxidized, giving them their red color. This sample also has secondary sulfides. The presence of both secondary sulfides and oxidized basalt clasts indicates that conditions have evolved from oxidizing to reducing. The rock also has irregular rounded voids that are filled with calcite (Fig. F78). A photomicrograph of a thin section from Sample 185-801C-16R-5, 107-110 cm (Fig. F79), shows a contact between glass and clay and calcite infilling of a void space that is ~20 cm deeper in the hole than the biological sample. The glass is no longer fresh and has dark halos around crystals and voids and along the contact between the clay and the glass.
This basalt flow or pillow lava is lithologic Subunit 44 of Unit 50 (Sequence IV). The core is part of geochemical Unit 13. Along with Pieces 8 and 9, it makes up a complete lava flow, with grain size increasing toward the middle of this subunit. Piece 10 has two veins that are filled with more than 90% carbonate and 1% or 2% sulfide, and the remainder is clay (Fig. F80). A thin section of this sample shows that ~10% of the original rock has been replaced with secondary clay and calcite, and some minerals also contain secondary sulfide. This rock appears to be the least altered of the rocks used for microbiological studies. A thin section from Sample 185-801C-23R-1, 27-28 cm, is from ~1 m shallower in the hole and is from lithologic Unit 50, but it appears to be from rock very similar to that used for the biological samples. The groundmass is composed of plagioclase, pyroxene, and opaque cubes of titanomagnetite. Approximately 1% of the vesicles are filled with clay. Figure F81 shows that the plagioclase and pyroxene appear fresh and that there is some secondary clay between grains of groundmass and in vesicles.
This sample contains volcanic glass, interpillow material, and pillow rims (Fig. F82). The top of Piece 5 (as shown in Fig. F80) is missing in the archive photo because it was removed as soon as the core arrived on deck in order to minimize biological contamination. The basalt of Piece 5 is relatively fresh. A thin section from Piece 2 (Fig. F83) shows that the interpillow material is made of calcite, clay, chert, recrystallized radiolarians, and iron oxyhydroxide. Some fresh glass is also present.
Core 37R contains most of the thickest lava flow from Hole 801C, and this piece is from near the top of the flow. The basalt is similar to others at this site in terms of chemical composition, and it contains olivine phenocrysts (<1% of the rock) that have been completely altered to clay. Many of the veins in this unit are filled with clay and some are filled with calcite. The vein shown in Figure F84, which was crushed for microbiological studies, was composed primarily of calcite. Other veins in this piece contain abundant saponite clay and minor iron oxides. Of the three thin sections taken from Section 37R-1, one was from the interval 27-29 cm and corresponds to the location of this sample. The thin section shows that the vein is filled carbonate with <5% iron oxides. Outward from the vein in the groundmass of the basalt are three parallel zones of different secondary mineralogy. The first is a 2-mm-wide zone where the groundmass has been stained red with iron oxides, magnetite is more abundant than in the groundmass or the other zones, and sulfides are absent. The next zone is ~1.5 mm wide, is free of red staining, and has low magnetite abundance and no sulfides. The third zone is ~0.5 mm wide and has ~10% secondary sulfides and no red iron oxides (Fig. F85).
The final sample used for inoculating cultures was Piece 3 from the last unit (Unit 60) and the last core (Core 52M) collected at this site (see the "Core Descriptions" contents list). The veins in this section are primarily filled with smectite clay, and the vein used for this study was ~1 mm thick and appeared to be completely filled with clay and sulfide. Based on thin section 52M-1, 43-45 cm, the rock is relatively fresh and contains ~5% saponite replacing interstitial material in the groundmass. The fresh groundmass is plagioclase, augite, and opaque oxides. There is a small amount of secondary pyrite. A thin section from Unit 60, but from Core 50M, has similar mineralogy and also contains a vein filled with pyrite and chalcedony (Fig. F86). This suggests that fluids in these fractures were reducing compared to fluids at higher levels that oxidized the surrounding groundmass (see Fig. F77).
The extent of the subsurface biosphere in the oceanic crust is of major importance to understanding chemical fluxes on Earth. Evidence of microbial activity in basalt glass from the ocean basins has been reported previously (Thorseth et al., 1992; Furnes et al., 1996; Giovannoni et al., 1996; Fisk et al., 1998; Torsvik et al., 1998; Furnes and Staudigel, 1999). The evidence includes concentrations of carbon, DNA, phosphorus, and microbe-sized particles in areas where the glass is being altered to clay. Associated with chemical and visual clues of microbes are a variety of alteration fronts in glass (i.e., densely pitted or intermittently distributed tunnels) that do not appear to be caused solely by abiological chemical activity. Microbes are also known to weather a number of igneous minerals, but evidence for this in deep-sea basalts has not yet been reported. A survey of the occurrence of alteration textures in volcanic glasses from surface and subsurface basalts indicates that the microbial alteration phenomenon is widespread in the oceanic crust (Fisk et al., 1998). Basalt glass with ages of 3-145 Ma appear to be affected by microbial alteration. These basalts were collected from the ocean floor and from depths >1500 mbsf. Only glasses from high-temperature (>140°C) environments appear not to have microbial alteration textures. Other factors that might prevent microbes from living in the oceanic crust are the absence of metabolic substrates, low fluid flux, or excessive pressure.
Hole 801C is important for determining if old, stable crust can support microbial life and for potentially extending the known depth of the deep biosphere in the oceans; therefore, volcanic glass from Hole 801C was studied for evidence of microbial activity. The location of fresh glass from flow margins, pillow margins, and hyaloclastites are reported in Table T5. Thin sections were made from nine of these samples and examined microscopically. Six of the thin sections come from a 50-m interval in Core 129-801C-12R and Cores 185-801C-13R to 17R between 588 and 637 mbsf (Table T17). One thin section is from Core 185-801C-32R at 767 mbsf, and the last two thin sections are from Cores 42R and 48R in a 40-m interval (881-920 mbsf). In two cases, where little glass remained in the core, the thin sections failed to intersect the glass margin. Of the seven thin sections that had glass, four contained the style of alteration similar to the microbial alteration seen in younger and shallower oceanic crust. These styles of alteration are documented in Figure F87. Additional glasses will be examined in shore-based studies.
The four samples shown in Figure F87 all have the characteristic alteration patterns associated with microbial activity. It is possible that these are fossil textures and that nothing was living in the rocks at the time they were drilled. If the textures in Figure F87 are the result of microbial activity, however, we hypothesize that life was present and that fluid was circulating though the crust when the basalts were collected. This is because as the chemical front progresses, it recrystallizes the glass to clay and destroys the microbial alteration textures. Thus, for microbial alteration to be observed in the basalts, it must continually occur ahead of the chemical alteration front.
Additional evidence of the presence of microbes in these samples may come from chemical mapping of thin sections for DNA, carbon, and phosphorus. Confirmation that the patterns of tunnels and alteration observed in Hole 801C are the consequence of microbial activity will require culturing microbes, microbial experiments using glass substrates, and molecular analysis of microbial DNA.
In addition to the irregular alteration that could be caused by the localized activity of microbes, there is alteration in Hole 801C that does not have the attributes of microbial alteration. Chemical alteration acting along the edge of a fracture would be expected to produce smooth alteration fronts in an isotropic substrate such as glass. This is observed in three samples from Hole 801C (Fig. F88).
The absence of microbial alteration in three of the seven thin sections that contain glass from Hole 801C is unusual in our experience of examining glass in seafloor basalts. Temperatures in the hole are between ~21° and 33°C, based on the extrapolation of the temperature gradient measured by Larson et al. (1993) for the upper 90 m of basement; thus, temperature should not prevent microbial growth. High pressure may prevent some microbes from living at great depth, but presently this effect has not been thoroughly investigated. Fluid flow, however, may be restricted by the filling of fractures and veins with secondary minerals. This could limit the supply of metabolic substrates required for microbial growth. Fluid flow might be directly related to the porosity of the formation. Porosity (measured at atmospheric pressure) of rocks near the glassy samples in Table T17 ranges from 2.2% to 15.6% (see "Index Properties"), but there does not appear to be any correlation of porosity with the occurrence of microbial alteration. Other proxies for fluid flow might be the width of veins, the volume of veins, or the number of veins per meter of core (see Fig. F40), but these also do not appear to correlate with the presence of microbial textures in the glass. Shore-based studies of alteration mineralogy may help us understand the causes of the absence and presence of microbial textures.
Microbial alteration occurs in the basalts near the top and the bottom of Hole 801C, but presently we are not sure what controls the distribution of organisms. The absence of organisms at intermediate levels of Hole 801C suggests that this environment is less habitable than other deep-sea, subsurface environments.
Surface- and drill-water samples were analyzed to evaluate the background cell levels and microbial population composition introduced into the formation during drilling. Drill water is surface seawater, but it was interesting to study both samples since additions to the drill water, such as mud and grease, could affect the microbial population.
A sample of water from the drill pipe was collected when it was opened to retrieve the core barrel for Core 185-801C-32R. This sample contained 2.5 × 105 cells/mL (±6 × 104 cells/mL). The surface seawater sample from the launch (see "Water" in "Microbiology" in the "Explanatory Notes" chapter) contained 4.2 × 105 cells/mL (±2.1 × 105 cells/mL). These numbers are typical for surface waters and are not statistically different. Separate aliquots of the surface seawater sample were incubated at downhole pressure (620 atm) and temperature (30°C) and inoculated into anaerobic growth media. Cultures will be started under pressure on shore and will be compared to those started on board. Both water samples were preserved for shore-based DNA extraction and community analysis.
The conditions in the borehole were investigated with two lowerings of the WSTP tool (Barnes, 1988). The undisturbed water in Hole 801C could be an excellent in situ microbial culture, and we wished to sample it before it was disturbed by drilling. Our first attempt to sample formation water (WSTP1) at 490 m was not successful, but the temperature was determined (Fig. F89; Table T20). The second attempt (WSTP2) at 540 m was successful both in collecting a sample and in determining the water temperature. Approximately 100 mL of gas and 310 mL of water was withdrawn from the relief valve in the overflow reservoir with syringes to avoid contamination with air or other potential contaminants. The sample coil was taken to the microbiology lab, and 23 mL of water was withdrawn in the anaerobic chamber for culturing of cells and DNA analysis. Sample coil fluids and overflow gas and water were split for shipboard and shore-based chemical analyses (Table T21). Samples of 17 mL were stored in a pressure vessel for later culturing experiments.
Temperatures at the ocean floor (mudline) were 1.7°C on both deployments of the WSTP (Fig. F89), which is consistent with bottom-water temperature in this part of the Pacific (Dietrich, 1963). Because the mudline temperatures were taken inside the drill string, the string appears to have cooled to the bottom temperature. Inside the hole, the drill string also appears to have started to approach ambient temperatures. As the WSTP was lowered on the cable from the mudline to the drill bit, it recorded an increase in temperature inside the drill string. In the case of WSTP1 the increase was from 1.7° to 7°C at a depth of 460 mbsf and for WSTP2 the temperature increased from 1.7° to ~10°C at 510 mbsf. The tool was then latched into the drill bit, the temperature probe extended ~50 cm below the drill bit, and the temperature was recorded in the undisturbed open hole. Next, the whole drill string was lowered ~30 m to depths of 490 and 540 mbsf for deployments WSTP1 and WSTP2, respectively. While the string was being lowered, the temperature increased ~8°C in WSTP1 and ~12°C in WSTP2. This suggests that there was a relatively large temperature gradient in this part of the hole. The final temperature we recorded at 490 mbsf was 15.5°C, and that measured at 540 mbsf was 22.3°C (Table T20; Fig. F90). The highest recorded temperatures at these depths were chosen as the ambient temperatures (Fig. F90). These highest temperatures occurred shortly after the probe reached its maximum depth. The probe was left in place for 10 min and during this time the temperature decreased slightly, which we attribute to the pumping action of the drill string in the hole. Even though sea conditions were calm, the probe was only 50 cm below the end of the drill string, and small motions of the drill string could have mixed cool water down the hole.
During Leg 144, temperatures within Hole 801C were measured (Larson et al., 1993) from the bottom of the casing (480 mbsf) to 570 mbsf, and within this interval the temperature gradient was linear and can be calculated using the equation
T °C = 8.17 + 0.0268 depth (mbsf).The temperature measured in the undisturbed hole at 540 mbsf during Leg 144 (22.6°C) is nearly identical to the temperature we measured at this depth during Leg 185 (22.3°C). However, the temperature measured during Leg 144 at 490 mbsf is 6°C more than our measurement (15.5°C). The difference in the two measured temperatures at 490 m may be caused by the introduction of cooler water from shallower in the hole by the lowering of the drill string. Alternatively, the lower temperature may be due to surface seawater being drawn into the formation between 490 and 540 mbsf.
The major hydrothermal zone in this hole extends from 512 to 528 mbsf. During Leg 144 permeability was estimated using a packer (Larson et al., 1993), and it was hypothesized that the most permeable region of the hole was located within the hydrothermal zone. Negative temperature anomalies and low resistivity between 515 and 530 mbsf from the downhole logs measured after drilling during Leg 185 (see "Temperature") indicates that this zone is highly permeable. If the 6°C temperature anomaly of WSTP1 is due to the gradual drawdown of surface water into the hydrothermal region, then this is a relatively recent phenomenon, as there was no evidence of drawdown in the temperature survey of Larson et al. (1993). This leads to two possibilities. The anomalously cool water at 490 mbsf in Hole 801C is caused by the mixing of water within the cased hole during the deployment of WSTP1 or to renewed drawdown of water in the hole, possibly stimulated by the packer tests.
Water was analyzed chemically to determine the origin of the water collected with WSTP2. Results indicate that the collected water was normal bottom water mixed with some distilled water that came from the coil (Table T20). High levels of Li are attributed to the grease used in the WSTP.
Sterilization of sampling equipment is critical in obtaining uncompromised microbiological samples. Prior to deployment, the WSTP samplers were flushed with 1 ppm chlorine dioxide followed by 0.2-µm-filtered Nanopure water. This water was not sterile because it was pumped into the WSTP from nonsterile carboys with an inline pump that also was not sterile. The total abundance of microbial cells in the Nanopure water before and after it passed through the WSTP was determined by epifluorescence microscopy. Results are shown in Table T22. The first time the WSTP tools were flushed, there were slightly higher levels of microbes in the outgoing wash water as compared to the ingoing water, indicating that cells were flushed out of the WSTP. The final wash before sampling showed no significant difference between ingoing and outgoing water. Residual levels of cells in the WSTP tools were 103 cells/mL (Table T22). After flushing, the sample coil was filled with 0.2-µm-filtered Nanopure water, and the sample reservoir was emptied and filled with nitrogen. The tool was stored at room temperature for at least 12 hr before use. Although WSTP1 failed to collect a water sample, the Nanopure water remaining in the coil was collected and cells were enumerated in this water (Table T22). Results showed that the cells in the coil had undergone four generations of cell growth, with an approximate generation time of 3 hr. The second WSTP tool was successful in collecting water from 540 mbsf. Total cell numbers in the WSTP reservoir were 9600 (± 6000) cells/mL (Table T22). Assuming cell growth occurred at the same rate as in WSTP 1 after the sampler was sealed, 30%-60% of these cells are likely to be contaminants. This is an approximate estimate, but it shows that there was considerable contamination in the WSTP. Major modifications would be required if the WSTP is to be used for microbiological sampling in the future. Ideally, a 0.2-µm filter that withstands the 1500-psi pump pressure would be placed in line after the pump for flushing and pressure testing the sampler. Alternative types of water samplers that are easier to sterilize should be investigated if water sampling for microbial investigations is to be a part of future legs.
A single push core was taken in Hole 801D. The rationale for taking this core was to test sampling protocols and tracer techniques in preparation for eventual sediment sampling at Site 1149. Samples to determine the total abundance of microorganisms and for community characterization were taken from whole rounds from each of the six sections of Core 185-801D-1W. Samples for abundance (0.5 cm3) were preserved in formalin (2% final concentration), and samples for microbial community analysis were frozen in liquid nitrogen and stored at -70°C. In addition to the microbiological samples, a contamination test using fluorescent microspheres was conducted in Hole 801D. Smear slides were prepared from whole rounds to examine the presence of microspheres (see "Methods for Quantifying Potential Microbial Contamination during Deep Ocean Coring" [Smith et al., 2000] and Smith et al., in press). Microspheres were observed in both the interior and exterior of every section, suggesting that the core was disturbed during the drilling process (see "Lithostratigraphy"). This is corroborated by pore-water chemistry data (see "Interstitial Water Chemistry and Headspace Gas"). Disturbance of the core was expected given that this wash core was taken from the mudline to 19.3 mbsf; thus, there was a large amount of solid and aqueous advection during coring. The disturbance and contamination were also enhanced by the high porosities of the sediments. The methods for sampling the spheres were modified for Site 1149 to provide quantitative data for estimating the amount of contamination in sediment samples.
Igneous rock samples were collected from Hole 801C for microbiological analyses. The core interiors were isolated in anaerobic conditions, used to inoculate microbial cultures, and examined microscopically. Samples were preserved for shore-based analyses, which will include enumeration of cells, SEM, DNA extraction, in situ hybridization, and cultivation at in situ pressure.
The primary microbiological objective for Leg 185 was to determine the types and abundance of microbes in unconsolidated sediments, sedimentary rocks, and igneous rocks of the oceanic crust. This goal could only be achieved if uncontaminated samples were obtained. Contamination tests were carried out to determine the extent of contamination introduced into the samples by the drilling and sampling processes. Results suggest that microbial contamination of samples used for microbiology is unlikely, although some drilling fluid did penetrate the rock (see Figs. F74, F75, and Table T19).
Observation of thin sections from Hole 801C showed signs of both microbial and chemical alteration in basaltic glass. Four of the seven thin sections that contained glass showed microbial alteration patterns.
Surface, drilling, and borehole water samples were collected to determine background microbial populations that could be introduced into the formation during drilling. These samples will be evaluated in parallel with rock samples. A water sample was collected of the water in Hole 801C before drilling. The water was determined to be bottom water. A good microbiological sample was not obtained because it was not possible to sterilize the sampler. Water temperatures were measured at two depths in Hole 801C.
A sediment push core was collected in Hole 801D. Samples from this core were used to test microbiology and tracer methods in advance of sediment coring at Site 1149.