The primary microbiology objective for Leg 187 was to determine the diversity and habitat of microbes in igneous rocks of different ages and to identify and quantify microbes participating in the alteration of the basalt. To achieve these objectives, samples of pillow lavas and other lava flows were collected from the cores for cultivation experiments, for later onshore characterization of DNA contained within them, and for examination of extant and fossil microbial activity by electron microscope techniques. Some sediment samples were also collected for cultivation and DNA analysis. The shore-based studies will be performed at the University of Bergen, Norway, and at Japan Marine Science and Technology Center (high-pressure cultivation).
Interpretation of results is complicated by the possibility of contamination of samples with microbes from the seawater (used as drilling fluid), the ship and drilling equipment, and from postcollection processing of samples. To determine the extent of microbial contamination during drilling, surface seawater was sampled to characterize DNA, and downhole tracer tests were conducted. A sample handling protocol was established to minimize postcollection contamination.
Whole-round cores were collected on the catwalk through the ends of unsplit core liners or in the core splitting room immediately after the core liner was split. The cores were handled only with latex gloves washed with 70% ethanol. Two approaches to minimize drilling-induced contamination of the outer rock sample surfaces were followed. The outer surface was either quickly flamed with an acetylene torch, or it was split off using a hydraulic rock trimmer. Flaming proved to be the simplest and fastest method, which minimized the time the rock samples were exposed to oxygen. After the sterilization, the samples were split into pieces of suitable size. Samples for enrichment cultures were placed in sterile plastic bags inside cold glass flasks containing 5 mL of seawater to keep them moist; these samples were immediately refrigerated (~5°C). Samples for anaerobic enrichment were placed in nitrogen-flushed flasks. The samples were contained in anaerobic and cold conditions within 20-30 min of the core arriving on deck.
Pieces of core, kept moist with 1 mL of anaerobic or aerobic seawater, were gently crushed into smaller grains in a sterile percussion mortar. For anaerobic enrichment this was done inside an anaerobic glove bag (Instruments for Research and Industry, model S30-20) flushed with a mixture of N2 (90%), H2 (5%), and CO2 (5%). Additional pieces of core were used whole as described below.
Fourteen types of bacterial culture media? anaerobic and 3 aerobic—were used to try to enrich viable microbial populations from the rock samples. The media were based on the composition of seawater and were contained in airtight 10-mL serum bottles. The anaerobic media were reduced with sulfide, and Mn(IV), Fe(III), sulfate, or bicarbonate were added as electron acceptors. For the aerobic media, oxygen was the electron acceptor. Electron donors were organic carbon sources (formate, acetate, lactate, succinate, glucose, yeast extract, trimethylamine, and peptone), hydrogen, and methane. Approximately 1 g (0.25-0.5 cm3) of crushed rock was added to each type of culture medium.
In addition, 4-5 g of crushed rock was added to two types of microcosm in 250-mL flasks filled with 200 mL anaerobic seawater. One contained Mn(II) as an added electron acceptor and the other contained Fe(II) and sulfate. Chitin, pectin, and acetate were added to both types as carbon sources. This setup is designed to establish a gradient from anaerobic conditions at the bottom of the flask to aerobic conditions at the top, with a micro-aerobic intersection in between. Enrichment of bacteria participating in the manganese-, iron-, and sulfur-redox cycles was the aim of these microcosm experiments, where the elements will be reduced in the anaerobic zone and oxidized in the aerobic zone of the flask. All samples were incubated at ~5°C. Growth is confirmed by the accumulation of metabolic products and of microbial cells. Finally, 5-10 g of sample was immediately frozen and stored at -70°C for high-pressure cultivation.
Three approaches were used for cell recovery from the rocks for later DNA analysis. First, a phosphate-buffered saline (PBS) solution of NaH2PO4 (2 mM), Na2HPO4 (8 mM), and NaCl (130 mM), which had a pH of 7.2, was applied to wash cells off fracture surfaces in the samples. This potential cell suspension was immediately frozen at -70°C.
Second, samples were crushed in a mortar filled with PBS. After some settling, the PBS was collected and centrifuged at 4000 rpm for 20 min. The supernatant was then discarded, and the potential cell pellet was frozen at -70°C. These samples were frozen for shore-based analysis.
Third, pieces of rock (1-2 cm3) were fixed with 70% ethanol in sterile 15-mL Falcon plastic tubes for onshore in situ hybridization.
Samples from both glassy margins and crystalline interiors of lava flows and pillows were collected for shore-based examination of extant and fossil microbial activity by electron microscope techniques. Some of the samples were subsamples of those collected for microbial cultivation and DNA extraction. The samples were either air dried or preserved in artificial seawater containing 2% glutaraldehyde.
Sediment samples taken from the interiors of the cores using sterile spatulas and spoons were immediately placed in sterile plastic bags inside cold, anaerobic flasks. The sediment samples (~1 g) were inoculated inside the anaerobic glove bag using the same 10 anaerobic and 3 aerobic media as described for the igneous rock samples. No microcosms were prepared with sediment inoculum.
For shore-based DNA analysis, sediment samples were fixed in 70% ethanol in sterile 15-mL Falcon plastic tubes and stored at 5°C.
Surface seawater samples were collected using a flask lowered from the bow of the ship to avoid wastewater and cooling water from the ship. One aliquot of the seawater samples was immediately fixed by adding ethanol to a final concentration of 40%. The ethanol-fixed seawater was later filtered for enumeration of microbes. A second aliquot was centrifuged (4000 rpm, 20 min) to separate seawater supernatant from a residual cell pellet. The cell pellet was washed with PBS and then frozen at -70°C. The cell pellet will be used for shore-based DNA analysis.
To determine potential levels of contamination of samples during drilling, we used yellow-green fluorescent carboxylate microspheres (Polysciences, Inc.) 0.518 µm (±0.021 µm) in diameter as a particulate tracer. These microspheres show 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 distilled water. This diluted solution was placed in an ultrasonic bath to disrupt aggregates. The diluted solution was 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. The first core to enter the barrel ruptures the bag, dispersing the microspheres into the core barrel.
After splitting the core liner, 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. For sediment cores, smear slides were made from both the outer surface and interior of the core using nonfluorescent immersion oil (Olympus). A Zeiss Axiophot epifluorescence microscope with a 100W mercury lamp, a blue filter set, and a 100× Plan-Neofluar oil-immersion objective was used to check for presence of microspheres.