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doi:10.2204/iodp.proc.331.102.2011

Microbiology

Total prokaryotic cell counts

The abundance of microbial cells in sediments was evaluated using SYBR Green I as a fluorochrome dye with epifluorescence microscopy (Weinbauer et al., 1998). Approximately 0.5 cm³ of sediment and/or deposit sample was taken with a sterilized spatula. In a clean area of the laboratory, samples were immediately fixed with 4% paraformaldehyde in a phosphate-buffered saline (PBS) buffer and stored at 4°C for 3 h. Fixed samples were diluted with 1× PBS and sonicated for 20 s with an ultrasonic homogenizer UH-50 (SMT, Tokyo, Japan). Microbial cells were directly stained with SYBR Green I (1/2000 [v/v]) at room temperature for 15 min. Cells were then filtered onto a 0.2 μm pore-size membrane filter (GTBP, Millipore). Excess dye was flushed from the membrane by rinsing with a further aliquant of Milli-Q water, and the membrane was mounted for microscopic analysis under glycerol/PBS (1:1) or glycerol using a coverslip. Microbial cells were enumerated with a fluorescence microscope (Axioplan 2, Zeiss) equipped with the fluorescent filter set of 470/20 nm (center wavelength/bandwidth) for excitation and 515 nm for emission.

The lower detection limit to detect one cell depends on the volume put on the filter, the total number of fields, the number of fields of view, and the desired probability to find one cell (Kallmeyer, 2008); the equation is given by

where

• n = number of cells required,

• T_fov = microscopic factor (total fields of view),

• C_fov = counted fields, and

• (1 – p) = probability to detect one cell.

The average number of counted fields was 100 fields per sample. To find the optimal balance between the covering of the slide with sediment and detection limit, we varied the volume on the filter and the number of fields slightly during the expedition. Nevertheless, the lower detection limit varied between and 0.65 × 10⁶ and 4.4 × 10⁶ cells/mL sediment because of the limited volume that can be put on the filter by this method (Table T7). In some cases, actual cell counts were less than the theoretical detection limit, as the probability to detect one cell was empirically reduced.

Cultivation of thermophiles

Growth of members of Thermococcales (e.g., Thermococcus), Aquificales (e.g., Persephonella), thermophilic Epsilonproteobacteria (e.g., Nitratiruptor), and thermophilic and hydrogenotrophic methanogens (e.g., Methenothemococcus and Methanocaldococcus) from core samples was used as a biological probe of hydrothermal fluid input in subseafloor environments. Cultivation of Thermococcales, Aquificales, and thermophilic Epsilonproteobacteria was carried out for all regular microbiology samples, whereas enrichment of methanogens was only examined for samples where growth of Thermococcales, Aquificales, and/or thermophilic Epsilonproteobacteria was observed. Media for cultivation of Thermococcales, Aquificales, thermophilic Epsilonproteobacteria, and thermophilic and hydrogenotrophic methanogens were the MJYPS media (Takai et al., 2000), MMJHS media (Takai et al., 2003), and MMJ media (Takai et al., 2002), respectively. N₂ was used as the headspace gas for the MJYPS media, and a mixture of 80% H₂ and 20% CO₂ (2 atm) was used for the MMJHS and MMJ media. Incubation temperatures for the MJYPS, MMJHS, and MMJ media were 80°C, 55°C, and 55°C and 70°C, respectively.

Additional cultivation of Thermococcus was conducted using the same method as described in Holden et al. (2001) with YPS media in 10 mL Balch tubes using headspace flushed with argon at both 80°C and 90°C.

Cultivation of sulfate reducers

Sediment slurries from a variety of layers were cultivated to isolate sulfate-utilizing bacteria and to determine if they were capable of using methyl sulfides as a substrate. Artificial seawater (Lyimo et al., 2002) (15 mL) with 0.1 g/L of sodium sulfide was placed in gas-tight 50 mL tubes and autoclaved under 80% N₂ and 20% CO₂ (0.5 atm). Substrates for sulfate reducers were added later, prior to inoculation. A total of four types of media were prepared:

• Two types of substrate mixture with/without 25 mM bromoethanesulfonic acid (BES), which is an inhibitor of methanogenic archaea;

• A mixture of dimethyl sulfide (DMS) and methyl mercaptan (MeSH) at concentrations of 10 µM and 11 µM, respectively, following Lyimo et al. (2009); and

• A mixture of lactate and acetate (1.25 µM each).

Sediment slurry for each section was inoculated in these four types of media, and tubes were incubated at temperatures similar to downhole conditions.

Cultivation of iron-oxidizing bacteria

Sediment slurry was also used for the onboard cultivation of iron-oxidizing bacteria (FeOB). We used essentially the same protocol as described in Emerson and Floyd (2005), using two types of media:

1. A modified artificial seawater (ASW) media with 5 mM MES (pH 6.5), minerals, and zero valence iron (Fe⁰) and

2. A modified ASW media with 5 mM sodium nitrate, 5 mM MES (pH 6.5), minerals, and Fe⁰.

The first media (without sodium nitrate) is referred to as ASW media A. The second media (with sodium nitrate) is referred to as ASW media B. All samples inoculated in ASW media A were incubated at room temperature in a microaerophilic environment using a CampyPak microaerophilic system in an anaerobic (i.e., gas-tight) jar. All samples inoculated in ASW media B were incubated at room temperature in an anaerobic environment using a GasPak anaerobic system in an anaerobic jar. Samples were incubated for 3–6 days, after which growth was assessed using epifluorescence microscopy with either a SYTO 13 or SYBR Green I nucleic acid stain. For successful samples, between 0.02 and 0.5 mL was also fixed with one drop 50% glutaraldehyde overnight, washed with sterile water, and dried at room temperature before being imaged using the onboard SEM and analyzed by X-ray EDS. Subsamples of successful enrichments were stored both with and without 10% glycerol in ASW media A; both were subsequently fast frozen in liquid nitrogen and stored at –80°C for future shore-based analyses.

Contamination test

Fluorescent microspheres were used for contamination assessment of each HPCS core from which microbiology samples were taken following standard IODP procedures (Smith et al., 2000). Perfluorocarbon tracer (PFT) (perfluoromethylcyclohexane; MW = 350.6; Matrix Scientific, USA) was also applied for contamination assessment as a chemical tracer for all coring systems. When drill mud components were solved in surface seawater, PFT was added to the drill mud tank. The concentration of PFT in the drilling fluid was monitored after PFT was added (Table T8). In addition, potential maximum PFT concentrations in the drill hole were monitored from adhered mud on a cored rock surface from Site C0016 (see Table T6 in Expedition 331 Scientists, 2011b). Quantification of PFT in core samples and original drilling mud fluids was carried out following standard IODP procedures (Smith et al., 2000) and using an Agilent Technologies model 6890N gas chromatograph (GC) with an electron capture detector (ECD) (Agilent Technologies). The GC was equipped with an HP-PLOT Al₂O₃ “M” deactivation column (length = 30 m; inside diameter = 0.53 mm; coating thickness = 15 µm). Helium and nitrogen were used as the carrier gas and make-up gas, respectively.

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