Samples were collected prior to curation from Holes 1188A and 1188F for shipboard studies (direct microscopic enumeration and micromorphological descriptions, ATP analysis for biomass activities, and enrichment cultures). Corresponding samples will be used for shore-based studies (aerobic and anaerobic cultivation, biochemical and molecular typing, microscopic determination of the role of microorganisms in mineralization and alteration, and potential bioactive molecules search).
Following the methods described in "Microbiology" in the "Explanatory Notes" chapter, samples were stained with 4,6-diamindino-2-phenylindole (DAPI), and the results of direct bacterial counts are shown in Table T19. The DAPI dye binds with DNA; hence, these bacterial counts represent both the dead (provided that the DNA is intact) and the living microbial population. The uppermost core enumerated (Sample 193-1188A-5R-1, 17-21 cm; 33.7 mbsf) contains 1.5 × 107 cells/cm3, and the bacterial population decreased to 5.8 × 105 cells/cm3 in the next lower sample (Sample 193-1188A-7R-1, 55-58 cm; 48.75 mbsf). No bacteria were detected by this method in cores from deeper in Hole 1188A nor from any of the samples from Hole 1188F (Table T19). The detection limit of the direct count procedure is ~1 × 105 cells/cm3, and it is possible that a more sparse bacterial population than this exists below this detection limit. A major drawback of this direct count procedure is the difficulty in distinguishing bacterial cells from mineral particles that fluoresce.
The lack of microbial habitation observed with increasing depths is most likely caused by temperature limitations. Although the temperature in Hole 1188A was not measured, it is plausible that a progressive increase in temperature occurs with increasing depth in this hydrothermal system. The temperature measurements immediately after drilling, at various depth intervals in Hole 1188F, range from ~80°-100°C (see "Downhole Measurements"). A temperature of 313°C measured at 360 mbsf in Hole 1188F 8 days after drilling indicates that the geothermal gradient at Snowcap hydrothermal site is very steep, limiting the vertical extent of any potential microbial habitat to the shallowest part of the basement.
ATP was measured to determine the biomass activity using the luciferin-luciferase method, and the results are shown in Table T19. ATP was detected only in the uppermost cores (Samples 193-1188A-5R-1, 17-21 cm [33.77 mbsf], and 7R-1, 55-58 cm [48.75 mbsf]). All samples collected from depths below 60 mbsf in Hole 1188A showed no indications of biomass activity. However, note that the detection limit of this analytical procedure is 0.5 pg/cm3 (1 × 104 cells/cm3), and it therefore remains possible that microorganisms are present at deeper levels in amounts below the detection limit of this procedure. Because of an unavailability of reagents, ATP was not measured in samples from Hole 1188F. The results from the ATP analysis corroborate the bacterial enumeration data (Table T19); hence, we are confident that we have established a variation of biomass with depth in Hole 1188A that restricts detectable biomass to the uppermost 50 to 60 mbsf.
Enrichment cultivation experiments were performed to improve the yield of microorganisms in the samples. The cultivation experiments were conducted at varying temperatures and oxygen partial pressures (Table T20). Bacterial growth was determined by comparing culture medium inoculated with core samples with the uninoculated medium, where turbidity in the medium indicates growth. In cultures where it was difficult to make an assessment based on visual inspection, ATP analysis was used to test for growth.
Results from these experiments (after 1 week incubation time) are shown in Table T20. In the aerobic experiments, growth was observed at both 4°C (Sample 193-1188A-2R-1, 0-4 cm [9.60 mbsf]) and 25°C (Samples 2R-1, 0-4 cm [9.60 mbsf], and 5R-1, 17-21 cm [33.77 mbsf]). No growth was observed in any other sample. In the anaerobic cultures, growth was observed at 25°C (Samples 193-1188A-2R-1, 0-4 cm [9.60 mbsf], through 7R-1, 55-58 cm [48.75 mbsf]), and at 60°/90°C (Samples 2R-1, 0-4 cm [9.60 mbsf], and 5R-1, 17-21 cm [33.77 mbsf]). Bacteria isolated from these enriched cultures will be used in shore-based studies.
Although no bacteria were detected by direct microscopic count in any of the samples from Hole 1188F, enrichment cultivation experiments were conducted to improve the yield (and also to verify the presence) of any microorganism present at these deeper depths. In the aerobic cultures, bacterial growth was observed in both samples (Samples 193-1188F-1Z-4, 60-65 cm [222.23 mbsf], and 3Z-2, 70-75 cm [224.75 mbsf]) at 25°C. No bacteria were observed in any other cultures (Table T20). In the anaerobic cultures, bacterial growth observed was in media inoculated with Sample 193-1188F-1Z-4, 60-65 cm (222.23 mbsf), at 25°C. No bacterial growth was observed in any other sample (Table T20). In view of the high temperatures recorded at these depths (~100°C at depths >200 mbsf), the microbial mass enriched at 25°C cultures (both aerobic and anaerobic) is most likely caused by contamination from the seawater used as drilling fluid.
Optical and epifluorescence microscopic techniques were used to examine the interactions between microorganisms and minerals, particularly the micromorphology, size, chemical composition, and structure of minerals associated with the microorganisms. Such information is essential in establishing the biological habitat and the role of microbes in the mineralization and alteration processes in the PACMANUS hydrothermal system.
The uppermost cores (Samples 193-1188A-2R-1, 0-4 cm [9.6 mbsf], and 5R-1, 17-21 cm [33.77 mbsf]) consist of brown translucent fragments of dacitic volcanic glass (Fig. F115). The textures of these fragments range from aphanitic to microlitic (Fig. F115). When stained with DAPI, bacteria were located on the surfaces and along the edges of these fragments. Some microcrystalline domains exhibit ~20-µm spots of stray fluorescence, indicating either the presence of bacterial colonies or, more likely, autofluorescent minerals.
Although no bacteria were detected in core samples obtained from depths >50 mbsf (Table T19), Figure F116 shows the micromorphological observations that may suggest possible bacterial habitation at these depths (Samples 193-1188A-7R-1, 55-58 cm [48.75 mbsf], to 21R-1, 105-114 cm [184.15 mbsf]). The exopolymer clusters (clusters of material of inorganic and organic origin that contain fragments with bacterial morphology and/or are stained by DAPI), flaky particles composed of volcanic glass fragments, and mineralized bacterial particles (chains of particles that have bacterial morphology and/or are stained by DAPI) are shown in Figure F116.
Fresh plagioclase phenocrysts and magnetite embedded in volcanic glass are common in core materials from Hole 1188A, but no bacterial habitation was associated with these minerals. Samples 193-1188F-1Z-4, 115-130 cm (222.23 mbsf), to 26Z-1, 62-65 cm (304.53 mbsf), from Hole 1188F were composed mostly of quartz, plagioclase, pyrite, anhydrite (Fig. F117A), and volcanic glass. No bacteria were apparent within these mineral particles. Assemblages of clay minerals and plagioclase fragments also showed no definitive bacterial mass (Fig. F117B).
Further shore-based studies using high-resolution microscopic techniques will be needed to verify the bacterial habitation within these samples.
The chemical tracer test was conducted while coring with the RCB (Core 193-1188A-11R) at this site. To estimate the amount of fluid intrusion into the recovered cores, a chemical tracer was deployed as previously described (Smith et al., 2000). Perfluoro(methylcyclohexane) was used as the perfluorcarbon tracer (PFT). Calibrations of the gas chromatograph (HP 5890) with standard solutions yielded a slope of 4.05 × 1012 area units/g of PFT. The detection limit of the samples was 0.01 µL of drilling fluid. The tracer was detected on the outer edge of the core, indicating a successful delivery. Estimates of drilling fluid intrusion in this sample range from below detection to 0.5 µL/g. The drilling fluid intrusion into the center of the core was below the detection limit.