The primary microbiology objective for Leg 209 was to determine the diversity and potential metabolic pathways of microbes in peridotites. To do this, peridotites were collected from the cores for cultivation experiments, onshore characterization of deoxyribonucleic acid (DNA) within the rocks, and examination of extant and fossil microbial activity by various microscope techniques.
Interpretation of results is complicated by the possibility of contamination of samples with microbes from the seawater (surface: used as drilling fluid, and bottom), the ship and drilling equipment, and from postcollection processing of samples. To determine the extent of microbial contamination introduced during drilling, surface and bottom seawater were sampled to characterize DNA and downhole tracer tests were conducted. Postcollection contamination was minimized by using an established sample handling protocol.
Whole-round core samples were removed from the split core liner in the core splitting room. The cores were handled with latex gloves washed with 70% ethanol. Drilling-induced contamination of the outer surface was minimized by quickly flaming the outer surface with a propane torch. After the sterilization, the samples were placed in nitrogen-flushed plastic bags (Zip-Lock) and taken immediately to the anaerobic chamber in the microbiology laboratory. Samples for enrichment cultures were kept moist with sterile artificial seawater and cool with ice.
Pieces of core, kept moist with 1 mL of artificial seawater, were broken with a hydraulic rock splitter and then gently crushed into smaller grains in a sterile steel mortar. Additional pieces of core were used whole as described below.
Fourteen types of bacterial culture media—all microaerophilic—were used to cultivate viable microbial populations from the rock samples. The media were based on the composition of seawater, and the cultures were contained in airtight Balch tubes. Nitrate, Mn(IV), Fe(III), S(IV), dimethylsulfoxide, and fumarate were added as electron acceptors, and the solutions were buffered to pH 7.5 and 9.5. Approximately 1 g of crushed rock was added to 20 mL of each type of culture medium. Additionally, each culture tube contained ~5 g of sterile olivine with Fe(II) as the electron donor. Mercuric chloride was added to one of each media type to inhibit all biological reactions within the tube. Uninoculated negative controls were incubated with each medium as well. These controls allow for differentiation of abiotic and biotic reactions in the growth media. Samples were incubated at 4° and 15°C in nitrogen-filled containers. Growth is confirmed by the accumulation of metabolic products and microbial cells.
Hydrothermal clay cultures were set up with the 14 enriched media listed above. Approximately 1 g of clay from the core interior was added to 15 mL of each type of culture medium. Similarly, mercuric chloride was added to one of each media type to inhibit all biological reactions within the tube. Samples were incubated at 4° and 15°C. Growth is confirmed by the accumulation of metabolic products and microbial cells.
A small fragment of core (>1 g) from the interior of the larger microbiology core segment was placed in the bottom of a 50-mL tube and overlain with 25 mL of R2A medium that had been precooled to ~48°C. Additionally, another fragment of core was inoculated into marine broth supplemented with 5% tryptic soy broth. The tubes were capped and incubated at room temperature in the dark for the duration of the leg. Sample tubes were monitored daily for growth. If growth occurred, broth media was inoculated by sterile loop, incubated at room temperature for 48 hr, and archived in glycerol at –70°C. Molecular techniques will be utilized during shore-based analysis of isolates and/or core fragments.
Whole-rock pieces split from the cores adjacent to the pieces used for culture experiments were preserved for shore-based DNA extraction. Rocks were frozen at –80°C in sterile sample bags and transported frozen to Oregon State University.
Excess pieces crushed for culture experiments and not used were preserved for examination by scanning electron microscopy. Rock chips were preserved in 1% glutaraldehyde solution and maintained at 4°C.
One-third of each sample of hard rock collected for microbiology was placed in a glass jar that had been baked at 550°C for 12 hr. The glass jars were sealed and stored at 4°C for postcruise total organic carbon analysis.
Sediment samples were collected using sterile 50-mL tubes. Sample weight was calculated by subtracting collection tube weight.
Ten milliliters of autoclaved and filter sterilized artificial seawater (ASW) was added to separate tubes for direct counts and enrichment cultures. Tubes were vortexed by hand until the mud pellets were in solution. Tubes were then allowed to sit at room temperature until settling occurred and clear ASW was visible. Aliquots of the ASW were then used for direct count analysis and culturing by spread plate technique using R2A. Direct counts were reported as counts per milliliter of cleared ASW following mixing and settling. Cultures results were reported as colony forming units per gram of mud after adjusting for the added ASW.
Surface seawater samples were collected using a sterile flask lowered from the bow of the ship, to avoid the ship's wastewater and cooling water. One liter of seawater was filtered through a 0.2-µm vacuum filtration rig from each drill site. The filter was preserved with a 20% sterile glycerol and 1x phosphate buffered saline (PBS) solution, then was frozen at –80°C. The filters were used for shore-based DNA analysis.
Bottom water samples were taken at the drill site with the water sampling temperature probe (WSTP). Prior to deployment, the WSTP was flushed with 10% bleach to sterilize the equipment and then flushed with filtered (0.2-µm pore size) distilled water. The coil and overflow tank of the WSTP contained filtered distilled water at the time of deployment. Water from the coil reservoir was extracted with forced air flow. The water samples from both the coil and overflow reservoirs were filtered and the filters were preserved with a 20% sterile glycerol and 1x PBS solution and frozen for DNA extraction on shore at Oregon State University.
Two types of tracer tests were conducted to determine the level of contamination of samples recovered by drilling: perfluorocarbon tracer (PFT) and fluorescent microspheres. Detailed methods for these tests are described in "Materials and Methods" in "Methods for Quantifying Potential Microbial Contamination During Deep Ocean Coring" (Smith et al., 2000).
Perfluoro(methylcyclohexane) was used as a liquid tracer within the borehole circulation fluid (seawater). The PFT was not diluted prior to introduction into the circulation fluid. The PFT was injected into the drilling fluid automatically and achieved a final concentration of 1 mg/L. The tracer was pumped early enough to reach the bit before the coring began and maintained a steady concentration throughout the drill string.
Once the cores arrived on deck, several small pieces were placed into headspace vials and sealed to confirm that the PFT reached the core. Next, the PFT was flamed off the surface of the core with a propane torch to prevent spreading the PFT to the interior while breaking up the core. The exterior rock was chipped away, and pieces of the interior were crushed and sealed in headspace vials. The headspace vials and gas-tight syringes were heated to 70°C. Five milliliters of headspace gas was injected into a Hewlett-Packard 5890 GC. Volume of drilling fluid in the core was calculated based on PFT concentration in the rocks and the concentration in the drilling fluid.
Yellow-green fluorescent carboxylate microspheres, 0.518 µm (±0.021 µm) in diameter, were used as a particulate tracer. The microspheres emit bright green fluorescence when observed under epifluorescence microscopy using a blue filter set. To achieve an approximate concentration of 1010 microspheres/mL, 2 mL of the microsphere suspension (2.86% solids in deionized water) was diluted to 40 mL with nanopure water. The diluted solution was placed in an ultrasonic bath to disrupt aggregates. The diluted solution was then poured into a plastic bag, excess air was expelled, and the bag was heat sealed and wedged into a recess in the top of an auxiliary core catcher insert supported by the core catcher fingers. Core entering the barrel ruptured the bag, dispersing the microspheres into the core barrel.
Pieces of rock close to the top of the core were selected to maximize the probability of finding the microspheres. The surface of the rock was washed with distilled water, and aliquots of the wash water were filtered onto polycarbonate filters. Rock thin sections were then prepared with no special precautions. A Zeiss Axiophot epifluorescence microscope with a 100-W mercury lamp, a blue filter set, and a 100x Plan-Neofluar oil-immersion objective was used to check for presence of microspheres.
The primary objectives of atmospheric microbiology studies during Leg 209 were to determine what types of microorganisms and pollutants were being transported across the Atlantic in African dust clouds and how nutrients in these clouds impact primary productivity in the tropical mid-Atlantic. To do this, both membrane filtration and liquid impingement were used to collect volumes of air on a daily basis. The air samples were then used to isolate and identify culturable and nonculturable bacteria, fungi, and viruses from the atmosphere. Contamination from shipboard air was minimized by collecting atmospheric samples on the windward side of the ship.
Atmospheric condition values were obtained using National Aeronautics and Space Administration (NASA) SeaWiFS global images and a photo manipulation software package (Ulead Photo Impact) for the purpose of graphing atmospheric conditions in relation to microbiology data. SeaWiFS images were opened in the software package and a 3° latitude x 3° longitude box was superimposed on the sample site (~15°N, 45°W). Image pixel tone for that area was then calculated using an image tone mapper. The value was calculated based on the pixel tone sum graphed by the software's tone mapper (bell-shaped curves within a 5 pixel x 5 pixel box). Baseline values were obtained by taking 10-tone map measurements of clear, light cloud cover, heavy cloud cover, light dust, and heavy dust conditions using a series of SeaWiFS images (20 May–23 May 2003). Baseline measurement ranges were
See Figure F11 for a graph of baseline values.
A portable membrane filtration unit was used to take samples on the windward side of the ship (Fig. F12). Presterilized filter housings containing 47-mm-diameter, 0.2-µm pore size filter membranes were used to collect all air samples (Fisher Scientific, Atlanta, Georgia, 09-74030G) in duplicate. The filter housings were removed from their respective sterile bags and placed on an analytical filter manifold. The lids were removed, and vacuum was applied using a vacuum pump for a set period of time. Air flow rates through the filters were 6.5 L/min for 40 min per sample. To control for handling contamination, an additional filter housing was removed from its bag, placed on the manifold, and allowed to sit for 1 min without removing the lid or turning on the vacuum. Filter housings were then removed from the manifold; the lids were sealed with parafilm and replaced in their respective bags for transport to the ship laboratory. R2A agar (Fisher Scientific, Atlanta, Georgia, DF1826-17-1) was used for microbial culture-based analysis. The sample filters were placed on R2A agar plates, sample side up, incubated in the dark at room temperature (~21°C), and monitored for growth over a 48-hr period. Fungal and bacterial colonies were separated by isolation streaking onto fresh plates of R2A. Once isolated, colonies were grown overnight at room temperature in tryptic soy broth (Fisher Scientific, Atlanta, Georgia, DF0370-17-3). The following day, 1 L of each culture was transferred to a sterile cryogenic storage tube containing 200 µL of sterile glycerol and stored at –80°C for shore-based identification using DNA sequencing of each isolate 16S (bacterial) or 18S (fungal) ribosomal DNA (rDNA) sequences.
A sterile liquid impinger vial containing 30 mL of autoclaved 0.5-M tris buffer (pH = 7.5) and a LaMotte (Chestertown, Maryland) air sampling pump was used to collect samples (Fig. F13). The assembled impinger unit was set to pump 1.5 L/min and allowed to run for 8 hr. After the sample collection was complete, the unit was transported to the laboratory for processing. Five milliliters of sample (in duplicate) was used for onboard microbial direct count assay using 0.02-µm pore size glass fiber filters, SYBR gold nucleic acid stain (Molecular Probes, Inc.), and epifluorescent microscopy. Another 5 mL of sample was preserved with 0.02-µm filtered formaldehyde and stored in a refrigerator at 4°C for shore-based analysis of the viral community using transmission electron microscopy. The remaining volume of sample was transferred to a sterile 15-mL tube and frozen at –80°C for shore-based analysis of bacterial community DNA using the polymerase chain reaction coupled with denaturing gradient gel electrophoresis and pathogen screening.
Surface water samples were collected each day at 1400 hr by lowering a sterile 50-mL tube from the bow of the JOIDES Resolution to prevent shipborne contamination. Ten milliliters of the sample (in duplicate) was filtered through a 0.02-µm glass filter using a sterile glass filter funnel. After filtration, the microorganisms on the filter surface were stained with SYBR gold nucleic acid stain. A negative control was used to account for microbial contamination of staining reagents, handling, and glassware. Ten microscope fields per sample were counted, and the averages of the field counts in the duplicate samples were used to determine total numbers of bacteria and viruses per milliliter of sample, via epifluorescent microscopy. Direct counts were also performed on aliquots of bottom water samples.
Samples for the analysis of metals and chemical pollutants were collected using a roofed four-place air sample unit (Fig. F14). The unit was placed at the crown of the ship drill tower, and samples were collected over three 1-week periods. The unit was turned off to prevent aerosol contamination during certain phases of drilling operations. Air sampling was based on conventional methods comparable to the U.S. Environmental Protection Agency's method TO-4A and related methods for semivolatile organic compounds, or IO-3, methods for determining inorganic species in suspended particulate matter. Four sampler cartridges were deployed for contaminant analyses: one for current-use pesticides, polyaromatic hydrocarbons, and other organic chemicals; one for antibiotics and pharmaceuticals; one for dioxins; and one for trace metals. Sampling trains for organic compounds consisted of a glass-fiber filter followed by two polyurethane foam plugs in series contained in a Teflon cartridge. Total air concentrations were determined by combined analysis of vapor and particle phases to achieve low detection levels. Quartz fiber filters were used to collect atmospheric particles for trace metal analysis. Field equipment blank samples were collected at each site for each sample type. Air pump flow rates through each filter type were recorded at the beginning and end of each sampling period.