The primary microbiology objective for Leg 193 is to establish the nature, extent, and habitat control of microbial activity within the PACMANUS (Papua New Guinea-Australia-Canada-Manus) hydrothermal system. Furthermore, we aim to interpret the differences encountered in the diversity and biomass in terms of the nutrient supplies and environmental habitat in the context of the geochemical and hydrological understanding of the total hydrothermal system.
To achieve these objectives, samples from the core of the hydrothermal system were collected for both shipboard and shore-based studies. Shipboard studies included
Shore-based studies will include
Interpretations of shipboard and shore-based results are complicated by the possibility of contamination of samples with microbes from seawater, drilling equipment, and postcollection processing of samples. To minimize the problems of contamination, special handling, sampling, and sample treatment protocols were established. Further, to confirm suitability of the core material for microbiological research, contamination assays were conducted to quantify the intrusion of drill water using perfluorocarbon and fluorescent microsphere tracer procedures.
Whole-round cores or large fragments of the core were collected in the core splitting room immediately after the core liner was split (rotary core barrel [RCB] core) or cores were removed from barrels (ADCB core). The cores were only handled with latex gloves washed with 70% ethanol. Following the selection of the appropriate piece and photography by handheld digital camera, the whole-round sample was transferred to the fume hood in the chemistry lab where the outside of the sample was flamed (sterilized) using a torch. Subsequently, the sample was transferred into the anaerobic chamber, equilibrated with an atmosphere containing a mixture of N2 (90%), CO2 (5%), and H2 (5%) usually within 20-30 min of the core arriving on deck. The outer surface of the whole-round core was split off using a hydraulic rock trimmer to minimize transfer of drilling-induced contamination from the outer sample surfaces to the interior of the sample. The remaining parts of the sample (all the outer parts) were returned to the core laboratory for curation.
The pieces selected for shore-based microscopic observations were placed in sterile opaque vessels or vials. Other samples for shore-based studies were fixed in 4% paraformaldehyde or mixed with anaerobic or aerobic media and stored at 4°C or at -70°C, or they were fixed in 4% paraformaldehyde (10% phosphate buffer saline [PBS]) and refrigerated for 2-24 hr, subsequently rinsed with PBS and stored in 1:1 ethanol-PBS at -20°C. Samples for postcruise biochemistry and molecular biology studies were stored in sterile bags at -80°C.
To determine potential levels of contamination of samples during drilling, two tracer test systems were used—perfluorocarbon tracer (PFT) and fluorescent microspheres (Smith et al., 2000). Tests were conducted on an average of two to three times for each hole.
The abundance of subsurface microorganisms was determined by direct fluorescence microscopic counting after 4,6-diamindino-2-phenylindole (DAPI) staining (see below). Such observations can indicate the extent of the deep biosphere. However, it is not possible to determine the activity of the microorganisms from the direct counts, because fluorescent stains can still bind to intact dead cells. For direct counting, a crushed rock sample (1-2 cm3) was diluted in 10 mL of filtered-sterilized (0.2 µm) 2% formaldehyde in artificial seawater (FFSW). This solution was vortexed vigorously, and 1 mL was removed with a wide-bore pipet tip, diluted in 10 mL of FFSW, and agitated in a ultrasonic cleaner for 10 min at moderate power. Samples were mixed thoroughly before removing an aliquot for filtration (on preblacked polycarbonate filters—Isopore by Millipore—with a pore size of 0.2 µm and a diameter of 25 mm). The filters were stained with a filtered (0.2 µm) DAPI (0.1 mg/mL final concentration) or with SYBR 1 (Bioprobes). The cells on the filters were examined with a Zeiss fluorescence microscope at 400×-2000× magnification (100× Plan-NEOFLUAR objective) using epifluorescence illumination (100-W Hg bulb) with ultraviolet and blue filter set for DAPI and SYBR 1, respectively. Cells were enumerated and normalized to the volume filtered.
The ATP concentration extracted from subsurface core samples was measured. The ATP is a biomolecule that serves as a proxy for living biomass and for the source of metabolic energy indicating subsurface bioactivity. The assays were performed in a luminometer (AF-70) using the luciferin-luciferase analysis method (TOA Co., Japan). Crushed rock (1-2 cm3) was diluted with 10 mL of 0.5-M trichloroacetic acid solution (TCA; 0.25-M Na2HPO4) and agitated in an ultrasonic cleaner for 2 min at moderate power. Then, 100 mL of the slurry was transferred to a new vial containing 9.9 mL of N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) buffer. The diluted sample was transferred again to a new vial and an ATP releasing agent (100 µL) and the luciferin-luciferase solution (100 µL) were added to the vial and mixed. Light production was immediately quantified in the luminometer. Blanks were determined using 100 µL of sterile distilled water, and standards were analyzed using a purified ATP solution. Note, however, that the detection limit of this method is 1.0 fmol ATP/cm3 (equivalent to 104 cells/mL).
Microorganisms were cultured at different thermal and redox conditions. Some microorganisms are believed to be capable of growing at temperatures >100°C, but there is only limited information to address this hypothesis and pressure cells were not available on board. Subsurface samples were incubated at different temperatures using culture media for anaerobic and aerobic bacteria to try to enrich viable microbial populations from the core samples. The media used was of seawater composition (Table T10), and culturing was conducted in air-tight 100-mL serum bottles. The anaerobic media were reduced with cysteine or sodium sulfide, whereas sulfate or bicarbonate was added as an electron acceptor. Electron donors were organic carbon sources such as yeast extract and peptone. Approximately 1 cm3 of crushed rocks or rock chips were added to the aerobic and anaerobic culture media, respectively. The bottles were incubated at 4°, 25°, 60°, and 90°C. Growth was verified by the accumulation of microbial cells or by metabolic products as seen by increased turbidity.