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doi:10.2204/iodp.proc.327.102.2011

Microbiology

The primary microbiology objective for Expedition 327 was to determine the microbial community composition harbored in the buried basaltic oceanic crust at Sites U1362 (prospectus Site SR-2) and U1363 (prospectus Sites GRB-1, GRB-2, and GRB-3) on the Juan de Fuca Ridge flank. These studies expand on previous work in this region at nearby Site U1301 during IODP Expedition 301 and postexpedition in Holes 1026B, U1301A, and U1301B (Cowen et al., 2003; Engelen et al., 2008; Fisher et al., 2005; Lever et al., 2006; Nakagawa et al., 2006; Orcutt et al., 2011; Wheat et al., 2010). The secondary microbiology objective of this expedition was to collect sediment from Grizzly Bare outcrop (Site U1363) to examine the influence of presumed seawater recharge on sediment microbiology and to compare these findings with microbiological results obtained from sediments drilled during Expedition 301 (Engelen et al., 2008; Lever et al., 2010).

The microbiology strategy for Expedition 327 was to

• Collect deep basaltic crust from Hole U1362A,

• Collect upper oceanic crust from drill bit samples (when available),

• Collect sediment and upper basement from Grizzly Bare outcrop (Site U1363),

• Collect microbiological tracer samples during the 24 h pumping test in Hole U1362B, and

• Deploy new microbial colonization experiments downhole in the subseafloor borehole observatories (“CORKs”) in Holes U1362A and U1362B.

This section focuses on the shipboard methods used for rock, sediment, and pore water sample collection and handling for microbiological analyses; CORK-related experiments are described in Fisher, Wheat, et al.; tracer experiment–related microbial activities are briefly described here and in more detail in Fisher, Cowen, et al. Briefly, samples of oceanic crust collected for microbiology were subsampled for environmental DNA extraction and analysis, cell counts, fluorescent in situ hybridization (FISH) studies, and contamination tests. Sediment samples were also collected for DNA extraction and analysis, cell counts, FISH, and contamination tests, and additional samples were collected to examine the potential for microbial dehalogenation metabolic activities.

Core handling and sampling

To examine potential contamination of hard rock and sediment core samples, slurries of yellow-green fluorescent microspheres (Fluoresbrite Carboxylate Microspheres; Polysciences, Inc., 15700) were sealed in plastic bags and placed inside the core catcher prior to deployment of the core barrel according to standard protocol (Smith et al., 2000a, 2000b). Perfluorocarbon tracer (PFT) contamination checks were not conducted during this expedition.

Hard rock cores

Hard rock samples for microbiology originated from RCB coring in Hole U1362A and APC/XCB coring at Grizzly Bare outcrop Site U1363. Priority was given to large (>10 cm in length) intact pieces or samples with interesting lithology. Nominally one sample was collected per section. Immediately following delivery of core on deck, rocks were exposed for subsampling in the core splitting room by either splitting the recovered core liner or shaking the recovered rocks into another split core liner (which, because of frequent splitting blade breakage, was much faster than trying to split the recovered core liner). Rocks for microbiological sampling were identified immediately, photographed in place, and then collected using combusted aluminum foil for transport to the microbiology laboratory. All sample handlers wore gloves to reduce contamination. In the laboratory, rock pieces were transferred to a flame-sterilized rock processing box (Fig. F8) and broken into smaller pieces using flame-sterilized chisels and forceps. Subsampling was done as rapidly as possible (5–15 min) to minimize oxygen exposure and cell degradation. Care was taken to separate outer layers with higher potential for contamination. Rock fragments from the outer and inner layers were prepared and preserved for later microbiological analyses using one of the following methods:

1. Fixed in cold 3.7% [w/v] paraformaldehyde in 1× phosphate-buffered saline (PBS; 150 mM NaCl + 10 mM sodium phosphate, pH 7.2) for cell counts and FISH.

2. Transferred to a sterile plastic tube, covered with 3% formaldehyde, and then stored at 4°C for 4–6 h before filtration onto a 0.1 µm mesh black polycarbonate membrane.

3. Transferred to sterile sample bags and frozen immediately at –80°C.

4. Sprayed with ethanol and flamed to remove outer layers of contamination from the rock surface and then allowed to cool before being transferred to sterile sample bags and frozen at –80°C.

5. Covered with lysis buffer before being stored at –80°C.

6. Mixed with distilled water for microsphere contamination checks.

Leftover rock material was washed in deionized water, dried, and returned to shore-based laboratories for use as substrate in future colonization experiments.

Sediment cores

Sediments for microbiological analysis were collected as whole-round or syringe samples. APC and XCB cores were cut on the catwalk using sterilized tools (autoclaved spatulas and ethanol-cleaned and combusted foil-wrapped end caps), and all handlers wore gloves to minimize contamination. Prior to whole-round sample collection, cores were visually inspected for gas voids, cracks, or drilling disturbance. In Hole U1363B, roughly two microbiological sample sets were collected per sediment core throughout the entire sediment column. Samples were typically collected near the base of the core and also in Section 3, except near the sediment/basalt interface, where sampling frequency was higher. For each sample set in Hole U1363B,

1. Three to four whole-round samples (for shore-based DNA analysis, halogenated organic matter characterization, and incubation experiments) were taken next to an interstitial water sample, immediately capped with ethanol-cleaned end caps, and bagged for sample storage.

   1. Whole-round samples for shore-based DNA extraction and analysis and shore-based analysis of halogenated organics were immediately transferred to a sterile sample bag and frozen at –80°C.
   2. Whole-round samples for shore-based dehalogenation enrichment studies were capped, transferred into sealable gas-tight bags, and then impulse-sealed with anaerobic gas packs (BD BBL GasPak Plus) and stored cold (4°C).

2. Three syringe samples were collected (two interior and one exterior) from the whole-round core with sterile cut-end syringes for headspace analysis (one interior) and shore-based cell count and microsphere contamination analyses (one interior and one exterior). Samples for cell counts and microsphere contamination analyses were mixed with cold 3.7% [w/v] formaldehyde in 1× PBS (pH 7.4) for fixation.

Because of compressed coring schedules at the end of the cruise, microbiological sediment sample sets were compressed in Holes U1363C–U1363G to include only whole-round samples for shore-based DNA analysis and syringe samples for headspace and microsphere contamination checks (as described above) to allow for higher sampling frequency.

A subsample of the above cores was further sampled for parallel molecular diversity and organic geochemistry analyses. The outer perimeter and tops and bottoms of whole-round samples were removed with sterile scalpels that had also been rinsed with a methanol-methylchloride (1:1 v/v) solution. The remaining sample was then split into subsamples using a methanol-methylchloride–rinsed spatula for the following:

1. DNA extraction: subsamples were transferred to sterile 50 mL tubes, covered with lysis buffer, and frozen at –80°C.

2. Microscopy and FISH: samples were placed in similar 50 mL tubes, covered with 3.7% formaldehyde solution, and refrigerated at 4°C for several hours before being frozen at –80°C.

3. Lipid biomarker analyses: subsamples were transferred to a combusted glass jar, covered with a polytetrafluoroethylene-lined cap, and frozen at –80°C.

Splits of sediment pore water also were collected for microbial and organic analyses (see “Pore water geochemistry”). Whole-round samples were processed in a N₂-purged glove bag where their perimeters, tops, and bottoms were removed with clean spatulas. The remaining whole-round samples were placed in stainless steel press assemblies before being pressed under as much as 25,000 psi. However, for these samples the first 11 mL of pore water was collected into a sterile syringe under low pressure: 1 mL from this syringe was transferred directly to a 2 mL cryovial, and the remaining 10 mL was filtered through a 0.1 µm mesh Supor filter. Then, 90 µL of 37% formaldehyde was added to the cryovial, which was then stored at 4°C for several hours before being frozen at –80°C. The Supor filter was placed at the bottom of a sterile 15 mL tube, covered in lysis buffer, and frozen at –80°C. Other pore water aliquots were distributed among combusted glass scintillation vials for shore-based analysis of dissolved organic carbon, amino acids, and low molecular weight organic acids.

Hole U1362B tracer injection experiment microbiological sampling

A 24 h pumping and tracer injection experiment was performed in Hole U1362B as part of a large-scale, long-term multi-CORK tracer transport project. The rationale and injection and monitoring methods for this experiment are described elsewhere (Fisher, Cowen, et al.). The tracers used include (1) fluorescent microspheres of different sizes (0.5 and 1.0 µm) and surface charges (neutral or carboxyl) intended as stable proxies for microorganisms and (2) fluorescently stained (4′,6-diamindino-2-phenylindole [DAPI]) microorganisms concentrated from 63 µm strained surface seawater. Fluid sampling at the rig floor during the 24 h tracer injection was conducted to monitor the injection profile of fluorescent microspheres and DAPI-stained bacteria; these samples were then preserved for shore-based microscopy analysis for comparison with future colonization experiments to be recovered from the Hole U1362B CORK.

Storage and shipment conditions

All samples for shore-based DNA extraction and analysis and shore-based halogenated organic analysis were stored and shipped at –80°C, whereas samples for shore-based cell counts, FISH, and dehalogenation enrichment studies were stored and shipped cold (4°C).

Analytical methods

Cell counts and FISH

Samples for cell counts and FISH were fixed in cold 3.7% [w/v] formaldehyde in 1× PBS. After 1–4 h of fixation, a subsample was removed and washed twice with cold 1× PBS before being stored in 1:1 (v/v) 1× PBS:ethanol. This washed sample will be used for shore-based FISH analyses, whereas the unwashed sample will be used for cell counting with either SYBR Green I or acridine orange fluorescent dyes using previously described methods (Morono et al., 2009). On the basis of results from DNA extraction and analysis, microbial groups of interest will be investigated using group-specific FISH primers according to published protocols (Biddle et al., 2006).

DNA extraction and analysis

DNA will be extracted in shore-based laboratories using a variety of methods, depending on sample type. Genes of interest, including the 16S rRNA gene, as well as functional genes, will be amplified using the DNA extracts and polymerase chain reaction (PCR). PCR amplicons will then be cloned and sequenced to generate clone libraries following published protocols (Lever et al., 2010; Orcutt et al., 2011).

Dehalogenation analysis

Halogenated organics will be extracted from frozen sediments in a shore-based laboratory following published protocols (Covaci et al., 2007). Concentrations of compounds of interest will be quantified using a combination of coupled gas chromatography mass spectrometry, liquid chromatography, and gas chromatography. The potential for dehalogenation activities will be examined using sediment slurry enrichment incubations with compounds of interest in a shore-based laboratory, similar to previously used protocols (Futagami et al., 2009). The identity of potential dehalogenating microorganisms in sediment will be examined using group-specific 16S rRNA gene and functional gene analysis of DNA extracts in a shore-based laboratory.

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