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An analysis of the microbial populations present in the subsurface biosphere at Atlantis Massif was undertaken during Expeditions 304 and 305. The goal for these microbiological analyses is to characterize the endolithic microbes associated with the oceanic core complex via culture-dependent and culture-independent methodologies. Throughout each expedition, an effort was made to establish a culture collection of microbes that interact with the various lithologies recovered. Upon completion of both expeditions, molecular analyses will be carried out on cultures, as well as on rock samples, to identify the microbes present in the various igneous rock samples from Atlantis Massif.

Sampling procedures

Upon arrival in the core laboratory, a section of the core was evaluated as a potential microbiological sample by visual inspection with minimal handling using ethanol-rinsed gloves. The core segment of interest was putatively identified, photographed, and placed in a sterile plastic bag. The core was then transferred to an anaerobic hood, where all subsequent manipulations were performed.


To determine the extent of contamination from drilling fluid, 0.5 µm fluorescent microspheres (Expeditions 304 and 305) and perfluorcarbon tracer (PFT; Expedition 304 only) were deployed with each core barrel intended for microbiological sampling (for details, see Smith et al., 2000). PFT was metered into the drilling fluid during coring operations, and samples were collected from the outside of the core to ensure delivery of the tracer and from the inside of the core to determine the extent of PFT infiltration into the sample. To determine whether the PFT reached the core, the surface of the whole-rock piece (~10 cm) was washed with filtered seawater. This rinse water was then placed into a vial and immediately sealed. Subsamples from the exterior and interior of each rock sample were crushed, placed into a vial, and immediately sealed.

Fluorescent beads were deployed in a plastic bag wedged into the core catcher that likely ruptured as the first cored material entered the core barrel. Beads on the outside of the core indicate dispersion of the microspheres on the external portion of the core; beads detected on the interior of the core indicate the presence of permeable pathways in excess of 0.5 µm in width, suggesting that contamination of the interior of the core may have occurred.

Each core collected was rinsed, and this rinse water was then assessed via microscopy (Zeiss Axiophot) to ensure that microspheres were dispersed. Contamination of the interior of the core was determined by the presence or absence of microspheres.

To further constrain the degree of contamination of cores, water samples were obtained using a sterile Water Sampling Temperature Probe (WSTP). The WSTP water collection device was initially flushed with distilled water and then sterilized with a (10% v/v) bleach solution that remained in the tool for 0.5 h. The tool was then rinsed with nanopure water. Approximately 5 mL of seawater was collected and immediately frozen at –80°C. A comparison of seawater microorganisms present in the water samples to the microbes identified via a molecular analysis in the interior portions of core samples will determine the extent of contamination by seawater microorganisms.

Cultivation experiments

Expedition 304

Anaerobic cultivation experiments targeted a broad physiological spectrum of chemoautotrophic and chemoorganotrophic microorganisms that may utilize energy sources from the deep oceanic crust. To verify that microspheres were dispersed during drilling operations, the exterior of the core (~10 cm) was washed with 50 mL of filtered seawater. Seawater remaining on the exterior of the rock surface was removed with paper. The core sample, which was wrapped in plastic, was split by means of a hydraulic rock splitter. The interior portions of the core were then crushed using a sterile mortar and pestle, and ~1 cm3 of sample was used as the innoculum. Each culture bottle contained 10 mL of media to enrich for chemoautotrophic microbes. Following inoculation culture, vessels were sealed with butyl rubber stoppers.

For sediment samples, ~0.5 mL of sediment was used as the innoculum in three different types of anaerobic media: methanogen enrichment media, sulfate-reducing bacteria media, and media to enrich for organic-matter-utilizing microbes. The culture bottles for methanogen enrichment media and sulfate-reducing bacteria media were incubated at 5°, 37°, 65°, and 85°C. The culture bottles for the enrichment of organic-matter-utilizing microbes were incubated at 37° and 65°C. Cell counts of in situ cell densities as well as determination of cell densities in cultures were performed with 4′,6-diamidino-2′-phenylindole-dihydrochloride, a nucleic acid stain.

Molecular studies based on 16S ribosomal ribonucleic acid gene sequence information, including polymerase chain reaction amplification, will be part of a postcruise research program. In addition, onshore investigations will address the potential metabolic activity of microbial populations and microscale observations of sulfide, phosphate, graphite, and stable isotope compositions to determine the impact of biomineralization.

Expedition 305

In the anaerobic hood, the core was first rinsed with 300 mL of nanopure water to collect a sample from the exterior of the core to confirm that microspheres were dispersed during drilling. The core sample was then broken into two sections using a hydraulic rock splitter with sterile blades. One section of the core sample was immediately frozen at –80°C for shore-based molecular analysis. The exterior portions of the remaining core section were removed, and interior pieces, manipulated with sterile forceps, were crushed using a sterile mortar and pestle.

Approximately 2 cm3 of sample was used as the innoculum in four different types of anaerobic media: artificial seawater media amended with nitrate, artificial seawater amended with lactate and overpressured with hydrogen, natural seawater with no amendments, and methanogen enrichment media. Cultures were established in anaerobic culture tubes, and all media, except for methanogen enrichment media, contained 5 g of sterile fine-fraction olivine derived from crushed dunite. Samples from the depths of 400–800 mbsf were incubated at 20° and 30°C. The sample from 400 mbsf was also incubated at 50°C. Samples from the depths of 900–1100 mbsf were incubated at 30° and 60°C. Finally, samples from the depths of 1200–1400 mbsf were incubated at 60° and 90°C.

Approximately 5 cm3 of inner core material from several samples was placed in 25 mL of sterile artificial seawater media. A 50 µL aliquot of this was spread onto marine agar 2216, R2A agar, artificial seawater, and natural seawater agar plates. Plates were incubated at 30°C and monitored for colony formation.

Following inoculations, 6 mL of paraformaldhyde (4% w/v) and 600 µL of sodium pyrophosphate (0.1M) were added to the remaining interior core material, which was then agitated for 3 h on a vortexer to detach cells from rock material. After vortexing, samples were stored for 12 h at 4°C and subsequently stained with a 0.2 µm filter-sterilized acridine orange (0.01% w/v) solution that absorbs wavelengths of 440–480 nm. Samples were then filtered onto a 0.2 µm filter and examined using epifluorescence microscopy and brightfield illumination to determine in situ cell densities.

Samples inoculated with core material were monitored for growth via change in liquid media turbidity. An increase in turbidity of liquid media is a potential indicator for an increase in cell density. Cultures that appeared more turbid with time were subsampled (240 µL/sample) and fixed with 50 µL of 0.2 µm filter-sterilized 10% paraformaldahyde. Samples were then stained with 15 µL of acridine orange and examined using epifluorescence microscopy.