Recent progress in exploring the deep subseafloor biosphere has revealed that microbial populations are consistently present in core samples recovered from the deep oceanic subsurface (Parkes et al., 1994; Wellsbury et al., 1997). Calculations suggest that the subsurface biosphere, extending deep into the crust (Fisk et al., 1998; Furnes and Staudigel, 1999; Furnes et al., 1999), is the largest reservoir of biomass on Earth (Whitman et al., 1998). However, the structure, diversity, and function of subsurface microbial communities remains poorly understood.
The primary shipboard microbiology objective was to obtain sediment and oceanic basalt samples while ensuring the use of sterile techniques at all times of sampling, sample handling, and storage. Little-altered microcystalline to fine-grained gabbro recovered at Site 1253 was different from the heavily veined and altered thin basalt flows and pillow basalts expected. However, rock samples were taken from the lower part of Subunit 4B for the purposes stated below. Potential contamination will be monitored using chemical tracers at Site 1253 and particulate tracer tests at all shipboard sites. We will begin extraction of deoxyribonucleic acid (DNA) from sediment and rock samples for genetic analyses to quantify bacterial abundance and diversity. We will relate these molecular-based microbial identifications to fluid and solid chemistry in the sediments and to alteration textures and chemical maps of altered crust and analyze the isotopic carbon fractionation of the glass postcruise. This information will allow for better assessment of the microbial community in the deep biosphere, as well as the role that microorganisms play in the alteration of basalts. It will also provide us with more accurate estimates of the microbial role in important geochemical processes such as the evolution of crustal composition and the cycling of elements in the ocean.
Syringe plugs were too difficult to obtain from cut core sections on the catwalk because the sediments were very indurated, especially at Site 1253. Instead, a 5-cm3 whole round was selected for microbiological sampling adjacent to interstitial water whole rounds and it was rapidly transported to the microbiology laboratory for paring and sampling. A sterile spatula was used to free ~0.5 cm3 of sediment from the most central part of the core. This 0.5-cm3 sample was scraped into a sterile 2-mL Eppendorf tube containing 1.5-mL 2% formalin solution to fix cells for postcruise cell counting. A ~5-cm3 plug was excised using a 5-cm3 autoclaved, cut-off syringe; this specimen was expelled from the syringe immediately into a sterile, 15-mL Falcon tube, capped tightly, and frozen at -80°C for adenosine triphosphate quantification as a means of calibrating cell counts. In the same fashion, a second 5-cm3 plug was taken, sealed likewise in a sterile Falcon tube, and frozen at -80°C for postcruise microbial community assessment. Samples of drilling fluid were also obtained, caught in 50-mL sterile Falcon tubes as the fluid ran out of the top of the core liner on the catwalk; drilling fluid samples were immediately frozen at -80°C and will be processed similarly to the sediment samples postcruise. Sampling for contamination tests is described below. Taking microbiological samples where interstitial fluid and squeeze cake geochemical analyses are planned increases the future compatibility of geochemical and microbiological results.
Upon core retrieval, samples of drilling fluids were taken in sterile Falcon tubes to determine the microbial populations that naturally occur in these fluids ("background contamination"), then the liner was cut open without disturbing the rock fragments. Pieces indicating some degree of low-temperature alteration were picked with sterile forceps, placed on sterile foil, and immediately transferred from the splitting room to the anaerobic glove box and put on ice to maintain core temperature at or below the in situ temperature.
Working within the glove box, samples were rinsed of unwanted debris with an N2-flushed marine salts solution (23.5 g NaCl and 10.8 mg MgCl2·6H2O per liter). An initial core segment was cut using a hydraulic press under aseptic and anoxic conditions, and any unused portions were immediately returned to the core. All materials, including core cutters, storage vials, and so on, were sterilized by autoclave or ethanol. Digital images were taken of the core piece prior to and after initial microbial sampling. Images were also taken of the sample used for further microbial work. The images serve as a record for complete core descriptions. The sampled core section was then crushed into several pieces to be used for different investigations. Crushing was done by wrapping the rock in sterile aluminum foil and breaking it into several pieces with a sterile chisel and hammer or mortar and pestle.
A glove bag (Coy, Grass Lake Michigan) containing a nitrogen atmosphere with 5% CO2 and >2% H2 was used for anaerobic handling of the core. Sections were brought into the glove bag and insulated on blue ice, and work was performed quickly to minimize warming. Hydrogen is present to combine with residual oxygen in a reaction catalyzed by palladium pellets maintained within the bag. Several hours before each use, the bag was flushed with a gas mixture. As an additional precaution to minimize oxygen contamination, tools and glassware to be used for manipulation and storage of samples for strict anaerobe work were stored within the glove bag.
Although cores were processed as quickly and as carefully as possible, shipboard handling should not be simply accepted as aseptic. We recommend that investigators receiving samples treat them as potentially contaminated and subsample accordingly whenever possible.
The crushed rock samples were partitioned into five different parts. One portion was put aside for future fluorescent in situ hybridization analyses. Rock fragments were fixed in 4% paraformaldehyde (Table T4) in sterile Falcon tubes for 4-24 hr. The samples were then washed and stored in a 1:1 mixture of phosphate buffered saline:100% ethanol at -20°C. A second portion was set aside for shore-based microscopic observations. The samples were placed in 10- or 50-mL sterile Falcon tubes depending on sample size. They were then fixed in 2% glutaraldehyde for 4-24 hr before being washed and stored in 0.25% glutaraldehyde at 4°C. A third sample was stored in sterile sample bags at -80°C for shore-based chemical analyses. A fourth sample was immediately stored in sterile 2-mL Eppendorf tubes at -80°C for future DNA extractions and genetic analyses. Finally, a fifth sample was set aside and put on ice to inoculate growth media.
The greatest challenge for subsurface microbiological investigations is verification that observed populations and activities are authigenic and not the result of introduced contaminants. Chemical (perfluorocarbon) and particulate (latex microsphere) tracers were used during microbiological coring to check for the potential intrusion of drill water and confirm the suitability of the core material for microbiological research. The presence or absence of these two tracers also acts as a quality assurance check on core handling methods. These tracer techniques were used during several legs and are described in ODP Technical Note 28 (Smith et al., 2000a). Samples of the drilling fluids were also collected upon core retrieval to determine the "background contamination." Note, however, that these methods were developed for APC coring methods. In the absence of other protocols, Leg 205 scientists used these approaches for RCB coring, with limited success.
Perfluorocarbon tracer (PFT) was continuously fed to the drill water at a concentration close to the limit of solubility (1 µg/g) and well above the detection limit for gas chromatographic analysis (1 pg/g). Samples for PFT analysis were taken from all cores intended for microbiological studies. In the sediments, 3-cm3 syringe subcores were taken from the interior (to monitor intrusion) and exterior (to verify delivery) of a freshly broken core or biscuit surface, extruded into headspace vials, and sealed with Teflon septa. Upon recovery of Subunit 4B, the core liner was split to observe and sample the core. To verify delivery of the PFT, the interior of the liner was wiped with a sterile cotton ball, which was placed in a headspace vial and sealed with a Teflon septum. A small rock fragment was taken from the interior of the core (to monitor tracer intrusion) and stored in a similar fashion as the cotton swab. Air samples were occasionally taken on the catwalk and in the glove box to monitor the background (blank) level of PFT. Samples were analyzed by GC. To avoid high background contamination levels of PFT in the laboratories, PFT containers should be opened on the helideck.
Latex fluorescent microspheres (Polysciences, Inc., Warrington, Pennsylvania; 0.5-µm diameter; YG) were used as a particulate tracer complementary to the volatile PFT. A 2-mL aliquot of microsphere stock (2.69% solids) was diluted with 40 mL of distilled water, sonicated for 2 min, and heat-sealed into a 4-oz Whirl-Pak bag. The bag was then attached with thread to the core catcher, a slight modification from procedures as described in Smith et al. (2000a, 2000b). The bag was positioned to rupture upon impact of the core tube with bottom sediments or rock, mixing the microspheres with seawater and coating the outside of the core as it is pushed into the liner.
During core processing, subsamples of sediments were collected from outer and inner layers for microscopic examination. Weighed samples were mixed with saturated sodium chloride solution and mixed thoroughly to extract microspheres. The slurry was then centrifuged to separate the liquid phase (Marathon 21K; 3 min; 2000 rotations per minute). The uppermost 10 mL of supernatant was filtered onto a black polycarbonate filter (0.2-µm pore size; Millipore), and the filter was mounted on a clean slide for microscopic examination. The usable filter area was ~1.98 x 108 µm2 as determined by the inner diameter of the filtration tower (15.64 mm). Microspheres in slide preparations were counted using a Zeiss Axioplan fluorescence microscope equipped with the Zeiss number 9 filter set (BP 450-490; LP 520), and the number of spheres observed was used to quantify contamination in spheres per gram (sediments) or milliliter (gabbro) of sample. The 100x objective was used for detecting cells, where the area for one entire field of view was ~3.14 x 104 µm2 (the diameter for one field of view = 200 µm). A total of 50 fields of view were analyzed for each sample.
Given the RCB coring during Leg 205, an additional method may be used. Intact biscuits, pared aseptically shipboard, were taken contiguous with interstitial water whole rounds. At Sites 1254 and 1255, elevated sulfate concentrations are indicative of seawater contamination of biscuit interiors (see "Inorganic Geochemistry" in the "Site 1254" chapter and "Inorganic Geochemistry" in the "Site 1255" chapter).
Samples of the exterior gabbro core for microsphere analyses were taken by washing the core sample with sterile N2-flushed marine salts solution and collecting the fluids in a syringe to be transferred to Falcon tubes for storage. Interior gabbro samples were crushed with a steel mortar and pestle, mixed with the marine salts solution, and collected in Eppendorf tubes. Prior to filtering, the samples were vortexed and allowed ~5 min each for settling of the larger particulates. A 5-mL aliquot was taken from each sample and filtered. Samples were visualized and counted as stated previously. Given the indurated and often fractured nature of the igneous rock, these methods are not optimal; all igneous microbiology samples are assumed to have been in contact with seawater.
To enrich microorganisms, different media, which were prepared prior to the cruise and are described in Table T5, were inoculated with a slurry from the cold gabbro subsamples set aside for culturing work. The slurry was prepared by first crushing the rock fragment into very small pieces (<2 mm) by wrapping the pieces in sterile foil and smashing with a hammer or with a mortar and pestle. Next the finely crushed pieces were placed into sterile beakers with anoxic (N2-flushed) minimal salts solution used for initial rinsing of the core. The beakers were shaken and vortexed to remove microorganisms attached to the rock fragments. Sterile syringes flushed with nitrogen were used to transfer the slurry into capped 5-mL serum vials or into gradient tubes. A sterile needle was used each time to inoculate the various media.
Genetic analysis of gabbro samples will allow more detailed identification of microorganisms inhabiting the samples. Following collection of samples, DNA will be extracted following the protocol in Table T6. DNA will then be purified from the samples, following the protocols of Edwards et al. (2000). The 16S ribosomal ribonucleic acid genes will be amplified by polymerase chain reaction using universal primers. Cloning and sequencing (University of Maine sequencing facility) will take place on shore. Determining the phylogenetic relationships of the microbial community will provide more information regarding whether organisms obtained from the samples are contamination artifacts (phylogenies similar those of organisms commonly found in surficial environments or drilling fluids) or if they truly live in gabbro (phylogenies match those of organisms found in subsurface environments).