MICROBIOLOGY

The primary microbiological objective at Site 1200 was to estimate the microbial community structure and phylogenetic diversity from core samples derived from a natural window (i.e., South Chamorro Seamount) into the ultradeep subsurface biosphere. The approach was to use molecular biological techniques to assess the degree of commonality and uniqueness among different microbial taxa originating from these communities, by taking advantage of small-subunit ribosomal deoxyribonucleic acids (SSU rDNAs) as discriminators for categorizing multiple microbial populations (community structure) and as descriptors of a single microbial population's ancestry (phylogeny). We also planned to correlate these molecular phylogeny results with variations in pore water geochemistry to interpret the metabolic potential of the most dominant microbial populations. Our goal during these comparisons was to test our hypothesis that these microbial taxa are the residual survivors of ultradeep subsurface conditions and mud volcanism mass transport encountered in the Mariana subduction system.

To achieve these objectives, samples from RCB and APC cores from Site 1200 were collected for both shipboard and shore-based studies.

Shipboard studies and preservation procedures included

1. Determination of the abundance and distribution of microorganisms by direct counting fluorescence microscopy, along with morphological characterizations by light microscopy;
2. The preservation of subsamples for molecular biological analyses by quick freezing with liquid nitrogen and then maintenance at -70°C until transport to shore-based laboratories on dry ice;
3. The preservation of subsamples in 2.5% glutaraldehyde for scanning electron microscopy (SEM) and, potentially, transmission electron microscopy (TEM) preparations, stored at -20°C;
4. The preservation of subsamples for fluorescent in situ hybridization probing, first for 2-3 hr at a final concentration of 3.0% (w/v) paraformaldehyde (in phosphate buffered saline [PBS], 10-mM sodium phosphate, 138-mM NaCl [pH = 7.4]), washed twice with 1 x PBS, and finally, stored in 1x PBS:ethanol solution (1:1) at -20°C;
5. The aseptic cryopreservation of subsamples placed in 30% glycerol solution and quick freezing in liquid nitrogen for use with multiple enrichment culture procedures; and
6. The preservation of subsamples in serum vials stored at 4°C under nitrogen-flushed headspace for use in enrichment cultures under various conditions (thermal and aerobic/anaerobic).

Sampling and preservation was conducted in order to carry out the following shore-based studies:

1. Molecular biological assays, including amplified ribosomal DNA restriction analysis as a means of determining the initial community structure from both the bacterial and archaeal components of the microbial community (Moyer, 2001);
2. Sequencing of the SSU rDNAs from the most dominant community members to allow for a molecular phylogeny analysis so that evolutionary relationships and potential physiological characteristics can be inferred (Moyer, 2001);
3. High-throughput community tracking with terminal-restriction fragment length polymorphism to examine the spacial distribution of predetermined marker populations across an array of core samples (Marsh et al., 2000);
4. Fluorescent in situ hybridization and/or other staining procedures to determine the role of microorganisms in these respective communities (Ravenschlag et al., 2001);
5. SEM techniques and/or TEM studies to further examine sediment-microbial interaction processes;
6. Lipid extraction and analysis to determine total viable biomass, community structure, and metabolic status using phospholipid fatty acid analysis; and
7. Anaerobic and aerobic enrichment culturing of targeted populations after the preliminary interpretation of initial molecular biological assays.

Interpretation of shipboard and shore-based results is 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.

Whole-round cores or large fragments of the core were collected immediately after the core reached the catwalk and after the core liner was split. Whole rounds were broken away from the rest of the core to minimize the potential for cross contamination by cutting. The cores were handled only with nitrile gloves. Following selection of an appropriate piece, the whole-round sample was transferred to the laminar flow hood in the microbiology lab, where the outside of the sample was stripped away using a flamed (sterilized) spatula. Subsequently, the subsamples were processed and preserved, usually within 20-30 min of the core arriving on deck.

To confirm the suitability of the core material for microbiological research, contamination assays were conducted to quantify the intrusion of drill water using in situ pore water chemistry (e.g., chlorinity) (see "Geochemistry"), and fluorescent microspheres were used as particulate tracers (Smith et al., 2000a, 2000b). These tests were carried out on either the same or an adjacent 10-cm whole-round sample.

The abundance of subsurface microorganisms was determined by direct fluorescence microscopic counting after acridine orange staining. Such observations give a first-order approximation of the extent of the deep subsurface biosphere. For direct counting, small sediment samples (0.1-0.2 cm³) were diluted in 1.0 mL of filtered (0.2 µm), sterilized PBS. After this solution was vortexed vigorously, 50 µL was removed with a wide-bore aerosol-resistant pipet tip and diluted in 1.0 mL of PBS. Samples were mixed thoroughly before removing an aliquot for filtration on preblacked polycarbonate filters (Isopore, Millipore; pore size = 0.2 µm; diameter = 25 mm). The filters were stained with a filtered (0.2 µm) acridine orange (0.025% [w/v] final concentration in PBS). The cells on the filters were examined with a Zeiss fluorescence microscope at 400-2000x magnification (100x Plan-Neofluar objective) using epifluorescence illumination (100-W Hg bulb) with ultraviolet and blue filters set for acridine orange. Cells were enumerated and normalized to the volume filtered.