Two samples were collected prior to curation from Hole 1191A for shipboard studies (direct microscopic enumeration, adenosine triphosphate [ATP] analysis, micromorphological descriptions, and enrichment cultures). Corresponding samples were collected for shore-based studies (aerobic and anaerobic culturing, biochemical and molecular typing, and microscopic determination of the role of microbes in mineralization and alteration).
Following methods described in "Microbiology" in the "Explanatory Notes" chapter, samples were stained with 4,6-diamindino-2-phenylindole (DAPI), and the direct bacterial counts are shown in Table T6. Bacteria were found in both samples. The bacterial mass decreased from 1.4 × 107 cells/cm3 in Sample 193-1191A-2R-1, 115-130 cm (10.55 mbsf), to 5.8 × 106 cells/cm3 in Sample 193-1191A-3R-1, 62-65 cm (15.32 mbsf). ATP was detected in both samples, the amounts being approximately the same in both samples (Table T6), therefore, indicating similar biomass activities at these depths. The detection limits of total bacterial counts and ATP analysis are 1.0 × 105 cells/cm3 and 0.5 pg/cm3, respectively. These results show that a large and active bacterial population exists to the bottom of the hole at a depth of 15 mbsf.
Enrichment cultivation of microbes was conducted to improve the yield of microorganisms in the core samples. The cultivation cultures were conducted at varying temperatures and oxygen partial pressures (Table T7). Bacterial growth was routinely determined by comparing inoculated cultures with the uninoculated medium, where turbidity indicates bacterial growth. In cultures where it was difficult to make an assessment based on a visual observation, ATP analysis was conducted to confirm growth. Results of these experiments (after 1 week of incubation) are shown in Table T7. In the aerobic cultures, growth was observed at 4°C (Sample 193-1191A-2R-1, 115-130 cm [10.55 mbsf]) and 25°C (Samples 193-1191A-2R-1, 115-130 cm [10.55 mbsf], and 3R-1, 62-65 cm [15.32 mbsf]). No growth was observed with any of the samples at 60°C. In the anaerobic cultures, growth was observed in both samples at all the incubation temperatures. These observations suggest that a large diversity of aerobic and anaerobic (thermophilic, extreme thermophilic, and hyperthermophilic) bacteria exist to 15 mbsf and are, therefore, consistent with the direct count and ATP results.
Optical and epifluorescence microscopic techniques were used to characterize the interactions between microorganisms and minerals, in particular the micromorphology, size, chemical composition, and structure of minerals associated with the microorganisms. These observations are essential for establishing the biological habitat and the role of microbes in the mineralization and alteration processes in this hydrothermal system.
Samples 193-1191A-2R-1, 115-130 cm (10.55 mbsf), and 3R-1, 62-65 cm (15.32 mbsf), consist mainly of transparent and translucent fragments of dacitic volcanic glass with embedded magnetite (Fig. F12A). Some fragments contained spherical opaque grains of magnetite embedded in glass (Fig. F12B) and lath-shaped plagioclase microlites (Fig. F12B). Sparse bacterial populations were located on the surface of these fragments, including a few bacterial colonies (Fig. F12C).