MICROBIOLOGY

Samples were collected prior to curation from Holes 1189A and 1189B for shipboard studies (direct bacterial count, adenosine triphosphate (ATP) analysis for biomass activities, micromorphological descriptions, and enrichment cultivation). Corresponding samples will be used for shore-based studies, including aerobic and anaerobic culturing, biochemical and molecular typing, microscopic determination of the role of microorganisms in mineralization and alteration, and search for potential bioactive molecules.

Following the 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 Tables T14 and T15. The DAPI dye binds to DNA, hence these bacterial counts represent both the dead (provided that the DNA is intact) and the living microbial population. In Hole 1189A, bacteria were found in the three uppermost samples, and the amount of microbial mass decreases with increasing depth (Table T14). The uppermost core enumerated (Sample 193-1189A-2R-1, 35-38 cm; 9.70 mbsf) contains 1.3 × 10⁷ cells/cm³, the population decreases to 4.6 × 10⁵ cells/cm³ in the next sample (Sample 193-1189A-5R-1, 33-35 cm; 39.13 mbsf), and to 9.8 × 10⁵ cells/cm³ (Sample 193-1189A-6R-1, 18-21 cm; 48.6 mbsf). No bacteria were detected in samples from greater depths. The results of the bacterial enumeration from Hole 1189B are listed in Table T15. Bacteria were detected only in the uppermost core counted (Sample 193-1189B-1R, 0-4 cm; 31.0 mbsf) at a population of 3.1 × 10⁶ cells/cm³. No bacteria were detected in samples from greater depths. The detection limit of the direct count procedure is ~1 × 10⁵ cells/cm³. It is, therefore, possible that a more sparing microbial population exists below this detection limit. As noted previously (see "Microbiology" in the "Site 1188" chapter), the major limitation with this direct count procedure is the difficulty in distinguishing bacterial cells from mineral particles that fluoresce.

The microbial mass profile observed at this site is similar to that in Site 1188, where bacterial mass was detected down to ~50 mbsf. These results demonstrate that microogranisms may exist in the subseafloor at sites of active venting of high-temperature hydrothermal fluid; therefore, they may play a role in mineralization and alteration processes.

ATP was measured using the luciferin-luciferase method to determine the biomass activities, and the results of the analysis of samples from Hole 1189A are shown in Table T14. ATP was detected only in the two uppermost samples (Samples 193-1189A-2R, 35-38 cm [9.7 mbsf with 18.2 pg/cm³] and 193-1189A-5R, 33-35 cm [39.13 mbsf with 4.8 pg/cm³]). All samples collected from depths >40 mbsf showed no evidence of biomass activity. ATP was not measured in samples from Hole 1189B because of an unavailability of the reagents required to do these analyses.

The bacterial count data suggest bacterial existence as deep as ~50 mbsf (Table T14). The absence of microbial activities below this depth suggests an unsuitable environment for existence and/or bacterial mass with much reduced activity. However, note that the detection limit of this analytical procedure is 0.5 pg/cm³ (1 × 10⁴ cells/cm³). Therefore, it remains possible that microorganisms are present at deeper levels but in sparse amounts.

Enrichment cultivation experiments were performed to improve the yield of microorganisms in the samples. These experiments were conducted at varying temperatures and oxygen partial pressures for a period of 1 week. 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 verify growth.

The results of the enrichment cultivation experiments with samples from Hole 1189A are shown in Table T16. In the aerobic experiments, growth was observed at 4°C (Sample 193-1189A-2R-1, 35-38 cm, 9.70 mbsf) and 25°C (Samples 193-1189A-2R-1, 35-38 cm [9.70 mbsf]; 5R-1, 33-35 cm [39.13 mbsf]; and 6R-1,18-21 cm [48.6 mbsf]). No bacterial growth was observed in samples incubated at 60°C. In the anaerobic experiments, bacterial growth was observed at 25°C (Samples 193-1189A-2R-1, 35-38 cm [9.70 mbsf]; 5R-1, 33-35 cm [39.13 mbsf]; 6R-1, 18-21 cm [48.60 mbsf]; and 8R-1, 39-41 cm [68.39 mbsf]), 60°C (Samples 193-1189A-5R-1, 33-35 cm [39.13 mbsf]; 6R-1, 18-21 cm [48.60 mbsf]; and 8R-1, 39-41 cm [68.39 mbsf]) and 90°C (Samples 193-1189A-2R-1, 35-38 cm [9.70 mbsf]; 5R-1, 33-35 cm [39.13 mbsf]; and 8R-1, 39-41 cm [68.39 mbsf]). Enrichment cultivation experiments with samples from Hole 1189B were conducted only in anaerobic conditions and results are shown in Table T17. Microbial growth was observed only in samples at depths between 80 and 100 mbsf (Samples 193-1189B-6R-1, 7-19 cm [79.07 mbsf], and 8R-1, 0.0-4.0 cm [118.10 mbsf]) at both 60° and 90°C, and as deep as ~130 mbsf (Samples 193-1189B-10R-1, 20-24 cm [118.1 mbsf], and 11R-1, 66-76 cm [128.94 mbsf]) at 90°C. No growth was observed in the samples from the uppermost 70 mbsf (Samples 193-1189B-1R-1, 0-4 cm [31.0 mbsf] through 5R-1, 14-18 cm [69.8 mbsf]) or from depths >140 mbsf (Samples 193-1189B-12R-1, 130-133 cm [140.01 mbsf] through 16R-1, 25-28 cm [175.5 mbsf]). A negative control experiment (surface seawater used instead of core) reveals no microbial growth, suggesting that microbial growth is not caused by contamination from seawater.

The distribution of anaerobic high-temperature microbial populations differs among the two holes at Site 1189. In Hole 1189A, microbial mass was detected in the uppermost 70 mbsf. In contrast, microbial mass was established at depths between 80 and 130 mbsf in Hole 1189B. Furthermore, the bacterial growths in the anaerobic (and high temperature) cultures were detected in samples from greater depths (80-130 mbsf) in Hole 1189B compared to <35 mbsf in holes from Site 1188. These data suggest differences in nutrient supplies and temperature profiles between the two holes at Site 1189 and more so, between the Snowcap hydrothermal site (Site 1188) and Roman Ruins (Site 1189) hydrothermal sites. These observations probably reflect a large biological diversity that likely exists within this hydrothermal system.

A cultivation experiment was conducted to study the effects of different types of seawater and core, at different growth conditions, on the microbial habitation. Seawater was used in the aerobic experiments, whereas sterilized seawater and artificial seawater were used in the anaerobic experiments. These seawater media were inoculated with ~5 g of core (Sample 193-1189A-5R-1, 33-35 cm; 39.13 mbsf) and incubated at 25° and 60°C in the dark for a period of 4 weeks (Table T18). Corresponding control experiments (without core) were also conducted. Sample 193-1189A-5R-1, 33-35 cm (39.13 mbsf), is a completely altered hydrothermal breccia with flow-laminated volcanic clasts and hosts an active bacterial population (Table T14). The descriptions of the cloudy materials at the bottom of the culture bottle, the estimations of the microbial growth, and the pH of the medium after 4 weeks of incubation are presented in Figure F119.

Microscopic observations reveal that the cloudy materials formed at the bottom of the culture bottles are composed of a suspension of clay minerals (most probably mixed-layer chlorite-smectite clay, smectite, and illite as identified by XRD in the core used). This suspension was present in cultures of natural seawater, artificial seawater, and to lesser extent in sterilized seawater. The fact that no such clay suspension was observed in the aerobic culture at 25°C suggests that formation of this suspension is temperature dependent. In contrast, under anaerobic conditions, formation of such a suspension appears to be dependent on the type of seawater.

In the aerobic cultures, bacterial mass exists within the suspension of clay minerals (Fig. F120A) and forms exopolymeric clusters (Fig. F120B, F120C). The bacterial population can be clearly distinguished from fluorescent minerals. Small numbers of similar bacterial populations were also found in the control culture. These observations imply that natural seawater microbes are able to exist in such clay mineral assemblages, under aerobic conditions at temperatures 60°C. In the anaerobic cultures, bacterial populations exist within the clay mineral suspensions in both the sterilized seawater and the artificial seawater at 25°C, and to a lesser extent at 60°C (Fig. F119). These bacteria are morphologically distinct from those observed in aerobic cultures. The clay mineral suspension and bacterial habitation within these suspensions are shown in Fig. F121. Bacteria were not detected in the control cultures. These observations mean that bacteria within the core materials are able to exist at temperatures 60°C within the clay mineral suspensions.

The microbial habitation is also dependent on pH, and because the core contains sulfides (e.g., pyrite) and sulfates (e.g., anhydrite), dissolutions of these minerals could alter the pH of the medium. Likewise, changes in pH can result from microbial activity (e.g., demineralization, production of organic acids). As shown in Table T18 and Figure F119, the pH of natural and sterilized seawater cultures decreased, whereas that of the synthetic seawater increased after the 4 weeks of incubation. In both cases, however, the changes in the pH were small (0.2-0.5 pH units). Likewise, changes in pH (of similar magnitudes) were also observed in the control cultures. These observations, therefore, suggest that mineral dissolution and/or bacterial activities do not significantly alter the pH of the media at temperatures 60°C.

The results of these experiments suggest that clay-sulfide-sulfate assemblages may be the primary habitats for microbial populations within the PACMANUS hydrothermal field and that seawater is the main source of nutrients and microorganisms, although nutrient concentrations may be affected by fluid-rock interaction.

Optical and epifluorescence microscopic techniques were used to characterize 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 this hydrothermal system.

The three uppermost samples examined show definite microbial habitation. Sample 193-1189A-2R-1, 35-38 cm (9.70 mbsf) consists of brown translucent fragments (volcanic glass with abundant fine-grained inclusions of silica, clay, and magnetite) and translucent materials (broken plagioclase phenocrysts). Staining with DAPI reveals that bacteria are located exclusively on the brown translucent volcanic glass. Samples 193-1189A-5R-1, 33-35 cm (33.13 mbsf), and 193-1189A-6R-1, 18-21 cm (48.6 mbsf), are completely altered hydrothermal breccias with flow-laminated volcanic clasts. They contain transparent blocky crystals of anhydrite and flaky brown translucent clusters of altered volcanic glass, clay, and pyrite (Fig. F122A). Bacterial staining with DAPI indicates the presence of bacteria on the surface of the flaky clusters (Fig. F122B), whereas the anhydrite crystals did not show any bacterial habitation (Fig. F122C).

The direct bacterial counts for Hole 1189A show bacterial habitation as deep as ~50 mbsf (Sample 193-1189A-6R-1, 18-21 cm; 48.6 mbsf). However, as noted previously, bacterial mass may exist below the detection limit of this procedure. Staining with DAPI revealed mineralized bacteria in Samples 193-1189A-8R-1, 39-41 cm (68.39 mbsf), and 193-1189A-12R-1, 41-50 cm (106.91 mbsf) (in chains of three to six bacteria), and bacterial secretion-like material (exopolymeric clusters, ~300 µm in size) in Sample 193-1189A-12R-1, 41-40 cm (106.91 mbsf) (Fig. F123). In view of the difficulties in distinguishing bacteria and minerals alluded to previously, further shore-based high-resolution microscopic studies will be conducted to verify the bacterial habitation profiles of Holes 1189A and 1189B.

Sample 193-1189A-10R-1, 103-115 cm (88.33 mbsf), is a completely altered hydrothermal breccia that is similar to Samples 193-1189A-5R-1, 33-35 cm (39.13 mbsf), and 193-1189A-6R-1, 18-21 cm (48.60 mbsf). However, no bacteria were located in this sample perhaps implying depth and temperature restrictions to microbial habitation.