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doi:10.2204/iodp.proc.347.102.2015

Microbiology

All sampling for microbiology was completed during the offshore phase of Expedition 347. At four sites (M0059, M0060, M0063, and M0065), at least one separate hole was drilled to sample for microbiology, biogeochemistry, geochemistry, and detailed interstitial water chemistry. Cores from these holes were subsampled in a specialized microbiology containerized laboratory within 1–2 h of recovery onto the deck. Only cores taken by piston corer were used for microbiology, as other types of coring retrieved sediment were too disturbed and contaminated with drilling fluid.

As soon as a core arrived on deck, it was curated into 1.5 m sections (Sections 1 and 2 with a short Section 3 if recovery was >91%). Sediment samples were immediately taken from the freshly exposed lower end of Section 1 using cut-off syringes; two 5 cm³ syringe samples were taken from every core for methane analyses conducted. Three 5 cm³ syringe samples were taken for PFC contamination tests from all cores shallower than 30 mbsf and every second core deeper than 30 mbsf. The positions of the three PFC samples at the exposed core end were (A) in the center (“interior”), (B) halfway between the center and the periphery (“halfway”), and (C) at the periphery, against the inner wall of the core liner (“exterior”). Three 5 cm³ samples were also taken, on a limited number of occasions, with sterilized cut-off syringes in the same A-B-C pattern for later DNA analyses of potentially contaminating microorganisms.

The core sections were then taken immediately to core curation for labeling and entering into the DIS. From there, the sections were run through the Fast-track MSCL without waiting for equilibration to room temperature (as per the usual procedure). As soon as logging was complete (<25 min), each section was transferred to the core reception container where subsampling for microbiology and biogeochemistry took place.

Subsampling was undertaken using cut-off syringes, scooping out using a sterile spatula, or by taking whole-round core segments, predominantly of 5 or 10 cm lengths. The short core segments remaining following microbiology sampling from this hole were capped. If they were longer than 15 cm, they were allowed to equilibriate and run through the normal slow-track MSCL. All remaining core was then stored together with the other core sections in the refrigerated container at +4°C.

Perfluorocarbon contamination tracer

PFC tracer was injected into the stream of drilling fluid during the coring of each microbiology hole to serve as a tracer for sediment core contamination with microbes from drilling fluid. Similar methodologies have been applied during previous ocean drilling expeditions (Smith et al., 2000a, 2000b; Lever, 2006). The PFC tracer used was perfluoromethylcyclohexane (C₇F₁₄) (Smith et al., 2000b). The PFC was pumped undiluted directly into the main stream of drilling fluid (seawater or drilling mud) at a rate of 0.1 mL/min (0.18 g/min) using a high-performance liquid chromatography (HPLC) pump (Alltech Model 301). The target concentration of PFC in the drilling fluid was 1 mg PFC/L, which is the maximum solubility of perfluoromethylcyclohexane in water (Smith et al., 2000b).

The three 5 cm³ syringe samples taken for PFC analyses from the freshly exposed lower end of Section 1 were immediately extruded into 20 mL headspace vials, prefilled with 5 mL Milli-Q water, and capped with a butyl rubber septum and an aluminum crimp seal. The vials were kept upside down at +4°C until later analysis in the shore-based laboratory within 1–2 weeks. A 10 mL liquid sample was also taken in a headspace vial for PFC analysis of seawater (D) collected from the top end of each whole piston core immediately after the core arrived on the cutting table (“liner fluid”). Finally, a sample was also taken of the drilling fluid (E) being used directly at the drill derrick, which was immediately subsampled into a 20 mL headspace vial. “D” and “E” provided controls for the detailed PFC analysis.

Analyses of PFC samples were performed at the Center for Geomicrobiology at Aarhus University in Denmark. All measurements were carried out using a gas chromatograph with an electron capture detector (Agilent 7820A GC), following the technical specifications described previously (Smith et al., 2000b) and subsequently used during other ocean drilling expeditions (e.g., Smith et al., 2000a; Lever et al., 2006). However, to ensure full PFC recovery from sediments, all headspace vials containing samples were preincubated for ≥2 h at 80°C. During this time, headspace vials were rotated horizontally using a rotisserie in a hybridization oven (Problot 12 hybridization system, Labnet International, USA). Preliminary tests conducted during IODP Expedition 337 and at the Center for Geomicrobiology at Aarhus University had shown this modified preincubation technique to be necessary for full PFC extraction into headspace (M.A. Lever, pers. comm., 2013).

Microbiology sampling

Sites M0059, M0060, and M0065 had shallow water depths of <100 m. Because of the relatively shallow water and the time required for processing the samples, it was necessary to moderate the pace of piston coring in the uppermost part of the hole where sampling density was greatest. This worked effectively and helped minimize time between the core arriving on deck and being sampled. Between 20 and 30 mbsf, the microbiology sampling became faster as fewer samples were requested, and the normal rate of coring was resumed.

To manage the large and complex sampling program, a detailed sampling scheme was produced for each new site. This scheme was not only available in tabular form but was also prepared graphically for each core run with a corresponding estimated depth interval. As coring proceeded and the core run lengths were modified to account for any sediment expansion, the real depths sampled increasingly deviated from the predicted depths, yet the depth indication remained instructive as a guideline for execution of the sampling program. The graphical sampling scheme included two pages for each core number, one page for the initial syringe sampling scheme (e.g., Fig. F19), and one page for the cutting of whole-round cores (WRCs; e.g., Fig. F20). To improve the overview and avoid mistakes, an easily recognizable color coding scheme was used for each individual sample request and their sample requirements (Fig. F21). The sampling schematics were displayed where cutting and sample distribution took place and also in the cooled microbiology container where further sample preparation and bagging was done.

The sections from the microbiology hole were taken to the core reception container immediately after MSCL logging for further sampling and data entry of samples. All persons handling the samples wore nitrile gloves to minimize contamination of microbiology or biomarker samples. The endcaps on core sections were removed, and subsamples were taken using cut-off sterile syringes from the exposed core ends according to the sampling scheme. The condition of each section was assessed in case there were disturbed parts of the core, which were then avoided when sampling. This assessment was also guided by previous analysis of the occurrence of cracks, bubbles, or free liquid surrounding the core in the paleoenvironment cores recovered from the hole drilled immediately prior to the microbiological hole. These observations were useful for the later evaluation of potential contamination of core samples. Syringe samples were taken from both the bottom of Section 1 and the top and bottom of Section 2. No samples were taken from the top of Section 1, which had been exposed to drilling fluid, or from the short (~30 cm) Section 3/core catcher material.

After initial syringe sampling, core sections were cut into consecutive short whole-round segments with a hacksaw according to the sampling plan. A black line was first made with a marker pen around the core liner where the cut was to be made, using as a guide a 5 cm short piece of split core liner that could be clipped around the core section being worked on. During sawing, care was taken to cut only the liner where possible, while rotating the core to minimize contamination of the central part through smearing by the hacksaw blade. A sterilized wire was then used to cut the WRCs, which were subsequently sealed with standard endcaps. Concurrent with the subsampling, the data for each sample was entered into the DIS.

Immediately after sampling in the core reception container, the syringe samples and WRCs were carried to the microbiology container, where further treatment was carried out. The microbiology container was kept at +12°C to avoid the ambient temperature rising to a level that could adversely affect heat-sensitive cells. The in situ temperature of the cored sediments varied from the annual mean water temperature of approximately +9°C at the southwestern sites to about +5°C in the Landsort Deep. The ambient air temperatures ranged mostly from +4°C to about +12°C and thus largely resembled the in situ temperature of cored sediments.

Counting of microbial cells

Total counts of microbial cells were carried out as the only offshore standard IODP microbiology analysis. Further counts were done during the onshore sampling party. Remaining counts will be done after the OSP at the Kochi Core Repository (Japan) and Cardiff University (Wales, UK); however, these will be conducted as postcruise research.

Direct counts were carried out on fluorescently labeled cells using three different methods: (1) acridine orange direct counts and fluorescence microscopy, (2) SYBR green DNA stain and flow cytometry, or (3) SYBR green DNA stain and fluorescence microscopy.

Acridine orange direct counts (AODC)

Samples were taken with a sterile 5 cm³ cut-off plastic syringe from the core end. A 1 cm³ plug was extruded into a sterile serum vial containing 9 mL of 2% (v/v) filter-sterilized (0.2 µm) formaldehyde in 2% (w/v) NaCl. The vial was capped, crimped, and shaken vigorously using a vortex mixer to disperse the sediment plug.

Numbers of total prokaryotic cells and dividing or divided cells were determined using acridine orange as a fluorochrome dye with epifluorescence microscopy (Fry, 1988). Fixed samples were mixed thoroughly, and a 5–20 µL subsample was added to 10 mL of 2% (v/v) filter-sterilized (0.1 µm) formaldehyde in 2% NaCl. Acridine orange (50 µL of a 1 g/L filter-sterilized [0.1 µM] stock solution) was added, and the sample was incubated for 3 min. Stained cells and sediment were filtered down on a 0.2 µm black polycarbonate membrane (Osmonics, USA). Excess dye was flushed from the membrane by rinsing with a further 10 mL aliquot of 2% (v/v) filter-sterilized formaldehyde in 2% NaCl, and the membrane was mounted for microscopic analysis in a minimum of paraffin oil under a coverslip. Mounted membranes were viewed under incident illumination with an Olympus BX53 microscope fitted with a 100 W mercury vapor lamp, a wide-band interference filter set for blue excitation, a 100× (numerical aperture = 1.3) Plan Neofluar objective lens, and 10× oculars. Prokaryote-shaped fluorescing objects were enumerated, with the numbers of cells on particles doubled in the final calculation to account for masking by sediment grains.

The detection limit for prokaryotic cells by this procedure is ~1 × 10⁵ cells/cm³ (Cragg, 1994).

The percentage of cells involved in division has been suggested as an indication of growth, although the assessment of dividing cells has never had a standardized approach in the literature. Dividing cells were defined operationally as those having clear invagination. A divided cell is operationally defined as a visually separated pair of cells of identical morphology. The percentage of cells involved in division is then calculated as follows:

Cell counts by flow cytometry

A 1 cm³ sample of sediment was taken from every 1.5 m core section by a cut-off syringe with sterilized tip and immediately mixed with 9 mL of fixation solution (4% formaldehyde in 2.5% NaCl). This 10% slurry was dispersed by vortex mixer and then stored at +4°C. A 40 µL sample of the slurry was diluted 1250 times in pH 8.0 tris-EDTA (TE) buffer. The diluted slurry was sonicated for 10 min to separate sediment particles and cells. The sample was then filtered through a 40 µm cell strainer (BD Falcon) to remove large particles. An aliquot of 2 µL of SYBR green I (Invitrogen) fluorescent stain was mixed with 50 µL of the diluted sample and incubated at room temperature for 10 min. A total of 950 µL of TE buffer was added to the stained sample. The final sample volume was thus 1002 µL, with a total final dilution of 1:25,000.

For cell counting on board the Greatship Manisha we used an Accuri C6 flow cytometer that is designed for fieldwork and may be operated on a research vessel. We customized the excitation filters of the Accuri C6 to 530/30 and 530/43 nm to detect SYBR green. A formaldehyde-fixed Escherichia coli suspension was used as a standard to calibrate the instrument. A 100 µL aliquot of each sample was used to analyze cell abundance. Because of ship motion, the volumetric pump system of the Accuri C6 was not accurate. We therefore estimated the sample volume by two independent methods: (1) by calculating the volume based on the E. coli calibration sample and (2) by calculating the volume based on a known number of added fluorescent beads that passed through the flow cell together with the sample.

Microscopic counts using SYBR green

The flow-cytometric counts were compared with direct cell counts of the same samples in the fluorescence microscope. Membrane-based counting was carried out at JAMSTEC in Kochi, Japan.

The slurry was first diluted with 2.5% NaCl and sonicated for 40 min using an ultrasonic homogenizer (Bioruptor UCW-310, Cosmo Bio Co.,Tokyo, Japan). The sample was treated with 1% HF for 20 min at room temperature after sonication. A 50 µL aliquot from the HF treated sample was filtered through a 0.22 µm pore size black polycarbonate membrane. About 5 mL of filtered (0.22 µm) 2.5% NaCl solution was placed into the filter tower prior to the addition of the supernatant to ensure even distribution of cells on the filter. The membrane was then washed with 5 mL of TE buffer, and roughly 2 × 10⁸ fluorescent microsphere beads (Fluoresbrite Bright Blue Carboxylate Microsphere [BB beads], 0.5 µm, Polysciences, PA, USA) were added for use in focus adjustment (Morono et al., 2009). A quarter of the membrane was cut and stained with SYBR green at room temperature in the dark for 10 min followed by washing with 50 mM tris-EDTA (TE) buffer. The membrane was sealed on a glass slide for microscopic cell counting. Microscopic fluorescence image acquisition (at 525/36 nm [center wavelength/bandwidth] and 605/52 nm × 490 nm excitation) was performed automatically using a fluorescence microscope system equipped with an automatic slide shifter (Morono and Inagaki, 2010). The resulting images were analyzed using the macro of Metamorph software (Molecular Devices, Sunnyvale, CA, USA) to discriminatively enumerate microbial cells on the membrane.

Onshore microbiology analyses

Apart from the total cell counts, all other analyses of the microbiology samples were conducted onshore as postcruise research. To give an overview of the large microbiological and biogeochemical research program on this expedition, we provide the following overview of samples. Requests are arranged according to type of analysis.

Individual samples were treated differently on board the ship. Many were kept at –80°C and transferred as frozen samples to the requesting laboratories. Some frozen syringe samples that depended on having intact cells upon thawing were frozen on board the ship in a Cells Alive System (CAS) freezer (casfresh.trustpass.alibaba.com) provided by JAMSTEC. The CAS freezer exposes the samples to a high-frequency alternating magnetic field that allows the sample to be cooled to –10°C without freezing (Iwasaka et al., 2011). When the magnetic field is switched off, the samples rapidly freeze without formation of destructive ice crystals. Samples were subsequently transferred to –80°C storage.

Samples containing live bacteria for later experiments were kept at +4°C in the ESO refrigerated container until they could be transferred to onshore laboratories. Most of those samples were sensitive to oxygen and needed to be kept in an anoxic atmosphere. They were therefore placed in gas-tight plastic bags and flushed with di-nitrogen gas before the bags were sealed with a heat sealer. The plastic bags available as supplied by ESO were made from multilaminated aluminum tube foil consisting of ortho-phthalaldehyde/polyethylene/aluminum/polyethylene layers with thicknesses of 15/15/12/75 µm (Gruber-Folien GmbH & Co, Straubing, Germany). Other varieties of gas-tight bags were supplied by individual laboratories requesting samples. Some bags were also supplied with an oxygen scrubber (Oxoid AnaeroGen) which develops CO₂, but no H₂, when taking up O₂ from the air.

Because of the nature of mission-specific platforms, laboratory space was not available to undertake experiments with live microorganisms offshore. To avoid deterioration of these samples due to prolonged storage on board the ship, the samples were offloaded from the vessel and sent to the respective laboratories as soon as possible after each microbiology hole had been cored. For further information on this process, see the “Operations” section in each site chapter. Frozen microbiology samples were offloaded halfway through the expedition during a port call to Nynäshamn on the Swedish Baltic Sea coast and at the end of the Expedition in Kiel, Germany. Distribution of both cooled and frozen samples to the requesting laboratories was organized jointly by ESO and the Center for Geomicrobiology at Aarhus University.

Samples taken for onshore research encompassed the following areas:

• Total cell enumeration;
• Phylogenetic and functional genetic diversity;
• Extraction of DNA for metagenomic analysis, functional genes of predominant metabolism, genes diagnostic of low-energy subsistence, and metagenomic data to support single-cell genomes;
• Intracellular vs. extracellular DNA extraction to distinguish living vs. ancient DNA, as well as real-time polymerase chain reaction (qPCR) of marker genes of the two DNA pools, including 16S rRNA genes of bacteria and archaea and 18S rRNA genes of animals and plants;
• Bacterial and archaeal 16S rRNA and eukaryotic 18S rRNA genes, as well as quantification of specific bacterial groups (including JS1) using qPCR;
• Sorting of single cells, amplification and sequencing of single-cell genomes; focus on archaea; qPCR of functional genes; and catalyzed reporter deposition–fluorescence in situ hybridization (CARD-FISH) of specific groups;
• Metatranscriptomics and identification of active microbial communities by RNA, as well as focus on S and Fe metabolism;
• Cultivation of fermenters and iron/manganese/sulfate reducers, qPCR of functional genes (dsr, apr, mcrA, and cbbL) and of 16S rRNA genes of specific groups (including JS1 and Chloroflexi, Geobacteraceae, Archaea, and Bacteria), and CARD-FISH of Bacteria and Archaea; and
• Endospores of thermophilic bacteria, quantification by most probable number (MPN) technique, and cultivation of eukaryotic spores.
• Virus;
• Viral abundance, viral diversity by PCR/pulsed-field gel electrophoresis (PFGE) or by metagenomics, and viral production/lysogeny; and
• Prophages in bacterial isolates and potential viral production in sediment by ³H-thymidine incorporation.
• Microbial activity in live samples;
• Sulfate reduction rate experiments using ³⁵SO₄^2–, modeling of rates, and calculation of cell-specific rates;
• ¹⁴C-tracer experiments with methane oxidation, acetogenesis, and methanogenesis from diverse substrates; cultivation and DNA extraction; and stable isotope probing (SIP); and
• Combined analyses of microbial activity and diversity with focus on sapropels and varved clays.
• Methanogenic degradation of complex organic molecules; cultivation; and SIP and δ¹³C isotope signatures;
• Biomarkers of microbial activity;
• Analyses of living microbial biomass, microbial necromass, and bacterial endospores from bacterial signature molecules (total amino acids, D/L-amino acids, muramic acid, and dipicolinic acid); and intact polar lipids.
• Microbial substrates; and
• Analyses of low molecular weight organic acids (volatile fatty acids [VFA])
• Contamination tests;
• Measurement of PFC contamination tracer at sediment depths used for microbiology and analyses done in Aarhus and results distributed to laboratories receiving microbiology samples

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