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doi:10.2204/iodp.proc.336.104.2012

Microbiology

Microbiological hard rock and sediment samples were collected from every interval cored with the RCB in Hole U1382A. Roughly 11% (3.8 m total) of core recovered from Hole U1382A was taken as whole-round samples from the core splitting room and dedicated to microbiological analysis. The 46 hard rock microbiology samples and 2 sediment samples span a range of lithologic units, alteration states, and presence of chilled margins, and some contain at least one vein or fracture. Table T10 provides a description of the collected samples, and Appendix Figures AF1–AF47 provide photographs of the whole-round samples taken (see the “Appendix”). Additionally, a few background contamination samples were collected for shore-based DNA analysis, including recovered plastic bags that held the fluorescent microsphere solutions in the core catcher, samples of the drilling mud from the mud pumps, scrapings of drilling mud from a recovered drill bit, and surface seawater injected into the drill pipe.

In accordance with protocols described in “Microbiology” in the “Methods” chapter (Expedition 336 Scientists, 2012), samples were selected in the core splitting room as quickly as possible after core recovery, following initial discussion with petrologists on sample representation and photographing the sample before it was removed from the core liner. When sample volume permitted, samples were preserved for shore-based DNA and RNA analysis, shore-based FISH and cell counting analysis, ship-based culturing and enrichment, shore-based isotopic analysis, and ship-based fluorescent microsphere analysis. Microspheres were used during all coring operations to help evaluate core contamination. Microsphere density was evaluated in all samples following iterative washing of the exterior of the whole-round samples with sterile seawater. Table T10 summarizes the results of the microsphere contamination survey, listing whether microspheres were detected in the first or last of 3–4 total washes with sterilized seawater. Of the 46 hard rock samples collected from Hole U1382A, 14 had microspheres in the first wash but none in the final wash, 26 did not have any detectable microspheres in any wash, and 6 had microspheres in all washes. In the latter case, only one microsphere was observed on the filter in the final wash. For all samples with observed microspheres, the microspheres were always in an abundance of <10. Of the two sediment samples collected, neither had microspheres on the interior of the core, and only one had detectable microspheres in the exterior.

Several enrichment and cultivation experiments were started with rock samples collected from Hole U1382A (see “Microbiology” in the “Methods” chapter [Expedition 336 Scientists, 2012]). Carbon-fixation incubations were initiated with one sample of massive oxidized basalt from Sample 336-U1382A-3R-4-MBIOA and one sample of a glassy pillow basalt from Sample 336-U1382A-7R-2-MBIOB. Enrichments for carbon- and nitrogen-cycling (carbon fixation, methane oxidation, and nitrate reduction) microorganisms were conducted with basalt from Samples 336-U1382A-2R-1-MBIOC, 3R-1-MBIOA, 3R-3-MBIOA, 3R-4-MBIOA, 3R-4-MBIOB, 5R-2-MBIOF, 6R-1-MBIOC, 8R-4-MBIOD, and 11R-1-MBIOA. Enrichments for methanogens, sulfate reducers, sulfide oxidizers, and nitrate-reducing iron-oxidizing bacteria were conducted with rocks from several horizons: Samples 336-U1382A-3R-4-MBIOA, 5R-2-MBIOF, 7R-2-MBIOB, 8R-2-MBIOF, 8R-3-MBIOG, and 10R-1-MBIOA. Enrichments for heterotrophic metabolisms were conducted with materials from Samples 336-U1382A-3R-4-MBIOA, 7R-2-MBIOB, 8R-2-MBIOF, and 8R-3-MBIOG.

Deep UV (<250 nm) native fluorescence scanning of hard rock materials with the Deep Exploration Biosphere Investigative portable tool (DEBI-pt) did not yield usable data for most of the Hole U1382A materials because of technical issues associated with operating in a shipboard environment with a new instrument. The heave experienced by the ship translated to rocking of the sample, which was corrected by using a heavier sample stage that was more stable. A second issue was encountered when part of the x-axis motor control failed, requiring a new component to be fabricated in the shipboard machine shop. Following these modifications, one sample from Hole U1382A was successfully scanned to assess the distribution of microorganisms on the surface of the sample (Fig. F31). Scanning of a subsample of Sample 336-U1382A-10R-3-MBIOD (Fig. AF43) indicated that microorganisms were heterogeneously distributed across the surface. Based on published fluorescence cross sections for bacteria (Seaver et al., 1998), the bacterial biomass for this sample is estimated to be ~10⁴ cells/cm². Signal intensities indicative of bacteria were generally associated with patches of iron oxides. DEBI-pt scanning also identified regions of the sample in which aluminosilicates contained higher quantities of calcium and potassium, suggesting alteration.

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