MICROBIOLOGY AND INORGANIC GEOCHEMISTRY

In response to organic matter deposition and the relative concentrations of dissolved oxygen, bacteria catalyze many redox reactions in sediments. These reactions influence the pore-water profiles of many compounds, such as nitrate, ammonia, phosphate, dissolved and particulate phases of manganese and iron, sulfate, and methane. Therefore, whereas pore-water chemistry is important for understanding sediment chemistry and diagenesis, pore-water chemistry profiles also reveal the habitats of many different types of aerobic and anaerobic bacteria (e.g., nitrifying and denitrifying bacteria, sulfate-reducing and sulfide-oxidizing bacteria, and methane-oxidizing and methanogenic bacteria).

The first core of Hole 1179B was designated specifically for microbiological and geochemical sampling at closer depth intervals near the sediment/water interface. In this core, samples were collected for sedimentary and pore-water analyses at the following depths: 0.1, 0.14, 0.25, 0.50, 0.75, 1, 4, 5, 6, and 7 mbsf. In subsequent cores, sediments and pore waters were collected from the following depths: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, and 250 mbsf. All of these samples were collected with APC coring.

Five- to ten-centimeter whole-round core (WRC) samples were collected for analysis of interstitial water (IW). Prior to squeezing the WRC for IW, ~3 cm³ of sediment was collected for carbonate and carbon, nitrogen, and sulfur (CNS) analyses. In addition, the squeeze cakes left over from pore-water collection were reserved for coulometric determination of carbonate, CNS analysis, and inductively coupled plasma-atomic emission spectroscopy (ICP-AES) analyses of Fe and Mn.

IWs were retrieved by applying the minimum pressure necessary to the sediment and gradually increasing it up to a maximum pressure of 205 MPa (30,000 psi) using a hydraulic press (Manheim and Sayles, 1974). Before squeezing, the sediment was immediately extruded from the whole-round core liner, the surface was carefully scraped to remove potentially contaminated exteriors, and the cleaned sediment was placed into a titanium squeezer atop a filter previously rinsed with high purity water to remove processing acids. Interstitial water was collected in two splits. An unfiltered sample was immediately treated with zinc acetate at 12.5 mg/10 mL of sample and frozen for subsequent shore-based ³⁴S analysis of the sulfate. The other IW split used for routine chemical analyses was passed through a filter into a plastic syringe attached to the bottom of the squeezer assembly and filtered through a 0.45-µm polycarbonate filter. Samples were stored in plastic vials pending shipboard analyses. Archived aliquots for future shore-based analyses were placed in acid-washed plastic tubes and heat-sealed glass ampules.

IW samples were routinely analyzed for salinity as total dissolved solids with a Goldberg optical handheld refractometer (Reichart), for pH and alkalinity by Gran titration with a Brinkmann pH electrode and a Metrohm autotitrator, for dissolved chloride by titration, and for phosphate, nitrate, and ammonium by spectrophotometric methods with a Milton Roy Spectronic 301 spectrophotometer, following the analytical techniques described by Gieskes et al. (1991). International Association of Physical Sciences Organizations (IAPSO) standard seawater was used for calibrating most techniques. The reproducibility of these analyses, expressed as 1- standard deviations of the means of multiple determinations of IAPSO standard seawater or of a standard, are as follows: alkalinity, <1.5%; chloride, <0.2%; and phosphate, nitrate, and ammonium, 4%.

Potassium, calcium, magnesium, sodium, chloride, and sulfate were analyzed by ion chromatography using the Dionex DX100 ion chromatograph. The reproducibility of these analyses, expressed as 1- standard deviations of the means of multiple determinations of IAPSO standard seawater, are as follows: potassium, <2%; calcium, ~2%; magnesium, ~5%; and sulfate, ~1%. For sodium and chloride, the precision with the ion chromatograph is ~1% for Cl and <2% for Na but the accuracy for chloride by ion chromatography is lower than that obtained by titration.

Fe, Mn, B, Li, and Sr concentrations in pore waters were determined by ICP-AES following the general procedure outlined by Murray et al. (2000). Chemical data for all interstitial waters are reported in molar units.

Inorganic carbon (IC) of sediment samples was determined using a Coulometrics 5011 carbon dioxide coulometer equipped with a System 140 carbonate carbon analyzer. A known mass, ranging from 18 to 20 mg of freeze-dried (dedicated carbonate samples) ground sediment was reacted in a 2-N HCl solution. The liberated CO₂ was titrated in a monoethanolamine solution with a colorimetric indicator; the change in light transmittance was monitored with a photodetection cell. The percentage of carbonate was calculated from the IC percentage content assuming that all carbonate is present as calcium carbonate:

CaCO₃ = IC × 100/12.

The precision of these analyses, expressed as 1- standard deviations of the means of multiple determinations of a pure carbonate standard, is <1%.

Sediment residues were retained after acid digestion from carbonate analysis and washed several times with deionized water for subsequent CNS analysis. The purpose of this experimental procedure was to investigate whether these samples more accurately reflect concentrations of particulate (nonsulfate) sulfur.

Total nitrogen, carbon, and sulfur in sediment samples were determined using a Carlo Erba Model NA1500 NCS analyzer. Mixtures of vanadium pentoxide and crushed freeze-dried samples (~5 mg) were combusted in an oxygen atmosphere at 1000°C, converting total (organic and inorganic) carbon to CO₂, sulfur to SO₂, and nitrogen to NO₂. The NO₂ was reduced to N₂ using copper. The gases were then separated by gas chromatography and measured with a thermal conductivity detector. The precision of these analyses, expressed as 1- standard deviations, is 2%-3%. Total organic carbon (TOC) was calculated by difference between total carbon (TC) from the NCS analyzer and IC from the coulometer:

TOC = TC - IC.

In addition, organic carbon was also measured directly from the acidified residues remaining from carbonate analysis.

In addition to the WRC samples taken for chemical analyses (above), WRC samples were also taken for microbiological analyses. These included 2-cm WRCs for postcruise lipid analyses, which were collected just below the intervals taken for IWs. These WRCs were immediately collected upon retrieval of the core on the catwalk before the end caps were sealed with acetone, placed in heat-sealed polyethylene bags, and frozen in the -86°C freezer. In addition, four WRCs were collected for microbial incubation experiments. A 15-cm whole-round sediment sample was collected at 5 mbsf from Hole 1179B, and 10-cm whole rounds were collected at 30, 100, and 200 mbsf from Holes 1179B and 1179C. These WRCs were also collected on the catwalk prior to acetone application, capped, sealed with insulation tape, and immediately transferred to the anaerobic chamber for additional processing. In the case of the 30-mbsf sample, where there was a 3-hr delay between collection and processing, the capped and sealed sample was instead stored in the refrigerator until processing.

The anaerobic chamber had been prepared ahead of time by placing two large sheets of aluminum foil, one above the other, on the floor of the chamber. Both sheets were swabbed inside the chamber with 70% ethanol. Following this procedure, the gas inside the working and portal chambers was exchanged once with a mixture of gas containing 90% N₂, 5% H₂, and 5% CO₂. Autoclaved scalpels and spatulas had also been placed inside the chamber for processing of the core.

The capped WRC was placed inside the chamber. The outside liner was swabbed once with alcohol before extruding the core onto an unused and fresh portion of the aluminum foil. The core liner and caps were then removed from the working part of the chamber. In order to remove possible contaminants from the core, the ends were cut off and the sides were scraped with the autoclaved spatulas and scalpels. Between each scrape, the tools were swabbed with 70% ethanol and heated with an electric platinum wire inside the chamber. Once the entire core was prepared, the discarded sediment was wrapped inside the upper sheet of foil and removed from the working chamber. The lower sheet of foil was swabbed once more with 70% ethanol before the scraped core was transferred to this sheet for further processing.

Incubation experiments were designed to observe sulfate-reducing and methanogenic bacterial activity. From each WRC, ~50 replicate 20-mL serum vials were prepared, each containing 5-cm³ subsamples of the sediment. The serum vials and butyl-rubber stoppers had been autoclaved in advance. The subsamples were collected from the core with 5-mL presterile plastic syringes from which the luer end had been removed with a sterile scalpel blade. The 5- and 30-mbsf WRC subsamples could be pulled into the syringe by suction. In the 100- and 200-mbsf cores, where water porosity was noticeably lower, sediments could not be collected with suction. Instead, the plunger of the syringe was first withdrawn and the syringe barrel was immersed repeatedly into the core until it was filled. Either 6 or 16 mL of sterile filtered (0.2 µm) seawater (SW) was added to the different vials and subsequently crimp sealed with standard rubber and aluminum cap stoppers. The vials with 6 mL of SW added contained 10 mL of gas headspace that could be subsequently measured for changes in methane concentration with time from 1-mL subsamples of the gas headspace. Methane measurements were made onboard the ship using a Hewlett Packard gas chromatograph with a HayeSep Q column and flame ionization detector. Replicates of these vials were collected soon after setup in the anaerobic chamber and were immediately placed in the -86°C freezer to serve as the first "T-0" time point. The vials with 16 mL of sterile SW added had no gas headspace and were designed specifically for measuring sulfate reduction by the appearance of sulfide using an Orion ion-specific electrode. Because of slow response time of the electrode when used with sediment slurries, the sulfide measurements will be completed after the cruise from frozen samples.

For comparative purposes, the samples collected at 5, 30, and 100 mbsf were all incubated at 7°C. In situ temperatures, as determined with an Adara temperature probe during coring, varied from ~2°C at the sediment/water interface to 8.2°C at 100 mbsf. Temperatures were not measured below 150 mbsf, where a temperature of ~11.5°C was measured. By extrapolation, a temperature of ~18°C was estimated for 200 mbsf. Therefore, incubation experiments from this depth were made at 20°C, as this was the lower limit of the only remaining and available incubator on board the ship.

To confirm the suitability of the core material for microbiological research, contamination assays were conducted to quantify the intrusion of drill water using the chemical tracer techniques that utilize perfluorocarbon tracers (PFTs) as described in ODP Technical Note 28 (Smith et al., 2000). This was completed on Cores 191-1179B-5H and 191-1179C-21H. Each time, the second, fourth, and sixth sections of the core were sampled for subsequent PFT analysis. Samples for PFT analysis were taken from the cores using the sampling protocol described in ODP Technical Note 28 (Smith et al., 2000). Unfortunately, an attempt to conduct PFT tests during XCB coring on Core 191-1179C-27H coincided with the contact of the chert layer, during which <1 m of sediment was collected, thus precluding PFT measurements.