Instrument Type: Inductively Coupled Plasma Optical Emission Spectrometer

Once on deck, OsmoSamplers were sectioned. Individual coils of Teflon tubing (each ~300 m long) were removed, sealed, and labeled. Each coil was then sectioned into 1.2 m lengths and the fluid expelled into acid washed (60C for 14 hours in 10%HCl) and rinsed (18 mega ohm water) micro centrifuge tubes. Centrifuge tubes were labeled and packaged for shipment.

In the laboratory aliquots from these centrifuge tubes were diluted before being analyzed using an inductively coupled plasma optical emission spectrometer (ICPOES). In addition the fresh water reservoir for each of the OsmoSampler pumps was analyzed for salinity to asses if the samplers over pumped and if so, by how much.

Additional descriptions for the recovered samplers can be found at Edwards et al. (2012) and Wheat et al. (2011).

Location: North Atlantic, western flank of the mid-Atlantic Ridge 22.75589 N 46.08125 W

Methodology:

Prior to the extraction, we freeze-dried, ground and sieved sediment samples to less than 125 μm (Ruttenberg 1992). For a given sample, we weighed four sample replicates (2 g) and placed each in 250 mL HDPE bottles. Sodium dithionite (F.W. 147.12 g/mol; 7.4 g) was added to each sample split, followed by 200 mL of citrate-bicarbonate solution (pH 7.6). This step produces effervescence, so the solution should be added slowly to the sample. We shook samples for 8 h and then centrifuged them at 3,700 rpm for 15 min. We filtered the supernatants with a 0.4 μm polycarbonate filter. We took 20 mL aliquots from the filtrate for each sample split for MRP and total P analyses, and kept them refrigerated until analysis within 24 h. We added 200 mL of ultrapure water to the solid residue for each sample split as a wash step after the above reductive step, shook samples for 2 h, and then centrifuged them at 3,700 rpm for 15 min. We filtered the supernatants with 0.4 μm polycarbonate filters and set aside 20 mL of filtrate from each sample split for MRP and total P analyses. We then extracted the solid sample residues in 200 mL of sodium acetate buffer (pH 4.0) for 6 h. At the end of this extraction step, we centrifuged the bottles at 3,700 rpm for 15 min, filtered the supernatants with 0.4 μm polycarbonate filters and took a 20 mL aliquot of filtrate from each sample split for MRP and total P analyses. We added 200 mL of ultrapure water to the solid residue for each sample split as a wash step, shook samples for 2 h, and then centrifuged them at 3,700 rpm for 15 min.  We filtered the supernatants with 0.4 μm polycarbonate filters and set aside 20 mL of filtrate from each sample split for MRP and total P analyses. We repeated the water rinse step, and collected aliquots for MRP and total P analyses as in the previous steps. The concentrations of  TP were determined as described below.

Solid sediment sample residues following the pretreatment described above were transferred to two 50 mL centrifuge tubes (2 sample replicates combined per tube). We added 20 mL of 0.25 M NaOH + 0.05 M Na2EDTA solution to each tube, vortexed until all sediment was resuspended and then shook samples for 6 h at room temperature (Cade-Menun et al. 2005). We used a solid to solution ratio of 1:5 for this step to minimize the amount of freeze-dried material that will need to be dissolved for the 31P NMR experiments. Large amounts of salts from the NaOH-EDTA concentrated in NMR samples lead to higher viscosity and increase line broadening on NMR spectra (Cade-Menun and Liu 2014). We chose an extraction time of 6 h to improve total P recovery while limiting the degradation of natural P compounds in the sample. At the end of the extraction, samples were centrifuged at 3,700 rpm for 15 min and supernatants decanted into 50 mL centrifuge tubes. We collected a 500 μL aliquot from each sample, which we diluted with 4.5 mL of ultrapure water. These were refrigerated until analysis for total P content on the ICP-OES. The sample residues and supernatants were frozen on a slant to maximize the exposed surface area during the lyophilization step; this was done immediately after the removal of the 500 μL aliquot. Once completely frozen, the uncapped tubes containing supernatants and residues were freeze-dried over the course of 48 h. Each tube was covered with parafilm with small holes from a tack to minimize contamination. Freeze-dried supernatants from identical sample splits were combined and dissolved in 500 μL each of ultrapure water, D2O, NaOH-EDTA and 10 M NaOH prior to 31P NMR analysis.  The D2O is required as signal lock in the spectrometer (Cade-Menun and Liu 2014). Sample pH was maintained at a pH > 12 to optimize peak separation (Cade-Menun 2005; Cade-Menun and Liu 2014). Sample pH was assessed with a glass electrode, and verified with pH paper to account for the alkaline error caused by the high salt content of our samples (Covington 1985).

Freeze-dried sample residues were ashed in crucibles at 550oC for 2 h and then extracted in 25 mL of 0.5 M sulfuric acid for 16 h (Olsen and Sommers 1982; Cade-Menun and Lavkulich 1997). We centrifuged samples at 3,700 rpm for 15 min, filtered supernatants with 0.4 μm polycarbonate filters, and measured P content on an ICP-OES. 

Total P concentrations in sediment extracts were measured using inductively coupled plasma optical emission spectroscopy (ICP-OES). Standards were prepared with the same solutions as those used for the extraction procedure in order to minimize matrix effects on P measurements. Sediment extracts and standards (0 μM, 3.2 μM, 32 μM and 320 μM) were diluted to lower salt content to prevent salt buildup on the nebulizer (1:20 dilution for step 1, 1:10 for steps 2 – 4). Concentration data from both wavelengths (213 nm and 214 nm) were averaged to obtain extract concentrations for each sample. The detection limit for P on this instrument for both wavelengths is 0.4 μM. The MRP concentrations were measured on a QuikChem 8000 automated ion analyzer. Standards were prepared with the same solutions used for the extraction step to minimize matrix effects on P measurements. Sediment extracts and standards (0 – 30 μM PO4) were diluted ten-fold to prevent matrix interference with color development. The detection limit for P on this instrument is 0.2 μM. We derived MUP concentrations by subtracting MRP from total P concentrations.

X-ray diffraction (XRD)

Powdered samples were examined for mineral presence using a Rigaku Ultima III X-ray Diffraction (XRD) System at the Institute of Imaging and Analytical Technologies (i2AT) at Mississippi State University. These analyses were conducted at 40 kV and 40 mA.

Elemental analysis of experimental aragonite crystals and experimental fluids with ICP-MS

Crystals were dissolved and solutions were analyzed by ICP-MS. Experimental fluids were also analyzed with ICP-MS, as well. Analyses were conducted on an Agilent 7900 quadrupole ICP-MS equipped with a glass nebulizer at the University of Rochester (Rochester, NY). Prior to analysis, fluid samples were diluted with 2% trace metal grade HNO3 (Fisher) in the volume proportion of 1:100. Aragonite crystals were dissolved in HNO3 in the mass proportion of 1:1000. Experimental fluid samples were analyzed using a 5-point calibration curve (one blank and four standards). Seawater standards were prepared by diluting a seawater certificate reference material (CRM-SW; High-Purity Standards) to a final concentration of ~300, 200, 150, and 80 ppm. Experimental crystals were analyzed using a 5-point calibration curve (one blank and four standards). Calcium carbonate standards were prepared by dissolving and diluting the original MACS-3 powder to a final concentration of ~111, 55, 27, and 11 ppm. Because S concentration was not reported in the certificate of analysis provided by the USGS, we added a small amount of sulfur single element standard (Inorganic Ventures) to a final concentration of ~1.5, 0.75, 0.375, and 0.150 ppm, in each MACS-3 standard we prepared. Samples, blanks, and standards were also spiked with 2 ppb indium, which was used as the internal standard to check for possible instrumental drift during each analytical session.

The instrument was tuned at the beginning of each analytical session (two analytical sessions in two consecutive days). 24Mg, 34S, 43Ca, 88Sr, 138Ba, and 238U were analyzed.  Integration times were the following: 0.3 s for 24Mg; 0.12 s for 43Ca, 88Sr, and 138Ba; 0.99 s for 238U. The calibration curve was run before analyzing each set of samples. For each element analyzed, the correlation among standards was 0.9923 or better. Elements were identified using three peaks. Each analysis included three replicates, with 100 sweeps/replicate. The carrier gas (argon) flow was set at 1.15 L/min. In addition, we used helium flow of 4.2 milliliters per minute in the collision reaction cell in the ICP-MS to minimize oxide interference. At the end of each tuning, oxides and doubly-charged ion interferences were below 0.8% and 1.4%, respectively. Data were computed automatically during the run using the Agilent Mass Hunter 4.1 workstation software v. C.01.01.

Results

XRD spectra identified the presence of aragonite and monohydrocalcite (Table 2). Only aragonite crystallized at intermediate pressure (110 bars). Calculations of fluid carbonate chemistry were conducted with a CO2SYS spreadsheet (Lewis and Wallace, 1998)  and presented in Table 1. There, measured pH and total alkalinity (TA) were used to calculate the concentration of CO32-, which together with Ca2+ is necessary to calculate fluid saturation states with respect to aragonite (Omega-Ar). Precipitation started at saturation state, which exceeds that of artificial seawater (ASW) by the factor of ~25. Over the course of the experiments, Omega-Ar decreased back to 2, i.e. Omega-Ar value of ASW prior to the addition of Na2CO3. Table 2 contains E/Ca of experimental products (solids and liquids) as well as the Doener-Hoskins apparent partition coefficients between solid and fluid (KE). Fluid composition changed during individual experiments, and therefore, values of KE were calculated using the Doener-Hoskins relationship:

       KE= log(1+ mE^aragonite /mE^fluid) / log(1+ mCa^aragonite /mCa^fluid)    (1)

where m^aragonite is the total number of moles of element (i.e., Mg, S, Sr, Ba, or U) or Ca in the final precipitate, and m^fluid is the total number of moles of element or Ca in the final fluid (Doerner and Hoskins, 1925).

Software products used:
XRD: Jade and Microsoft Excel
ICP-MS: Agilent Mass Hunter 4.1 workstation software v. C.01.01 and Microsoft Excel

Push cores were collected by the ROV Jason II on dive J2-773 on 11-April-2014.

Pore waters were extracted in a cold room at 4C using acid-rinsed, washed and dried rhizomes. The first 2 ml of fluid was discarded, then the remaining fluid was filtered through a 0.45 micron membrane and stored in hot acid (60C) washed high-density polyethylene bottles. Fluids were analyzed by electrode (pH), titration (chlorinate, calcium, and alkalinity), colorimetric (nitrate), inductively coupled optical emission spectroscopy (ICPOES), and inductively coupled mass spectroscopy (ICPMS). Precision was generally 0.2% for chlorinate and calcium titrations, 2% for alkalinity titrations, 0.01  for pH, 5% for nitrate determinations, 2% for ICPOES measurements and 5% for ICPMS measurements. All measurements were above detection limits with the exception of Mn (0.1 umol/kg) and Fe (0.01 mol/kg).

Careful consideration of these data should examine the potential of sampling artifacts for many of the solutes. We believe, for example, the Ca difference from bottom seawater values is an artifact of sample handling, resulting from the movement of sediment with carbonate present to the ship where the sediment was handled. Other solutes are likewise probably affected.

Samples were taken from the following locations and depths:
Kilo Moana (~2600 m water depth),
ABE (~2100 m water depth),
Tui Malila (~1875 m water depth), and
Mariner (~1900 m water depth) hydrothermal vent fields

The East Lau Spreading Center (ELSC) and Valu Fa Ridge (VFR) comprise a ridge segment in the southwest Pacific Ocean where rapid transitions in the underlying mantle chemistry manifest themselves as gradients in seafloor rock geochemistry. The investigators studied the geology and microbial diversity of three silicate rock samples and three inactive sulfide chimney samples collected, from north to south, at the vent fields Kilo Moana, ABE, Tui Malila, and Mariner. This is the first study of microbial populations on basaltic andesite, which was sampled at Mariner vent field. Silicate rock geochemistry exhibits clear latitudinal trends that are mirrored by changes in bacterial community composition. Alphaproteobacteria, Epsilonproteobacteria, and Bacteroidetes are most common on a silicate collected from Kilo Moana and their proportions decrease linearly on silicates collected further south. Conversely, a silicate from Mariner vent field hosts high proportions of a unique lineage of Chloroflexi unrelated (<90% sequence similarity) to previously recovered environmental clones or isolates, which decrease at ABE and are absent at Kilo Moana. The exteriors of inactive sulfide structures are dominated by lineages of sulfur oxidizing Alphaproteobacteria, Gammaproteobacteria, and Epsilonproteobacteria, while the interior of one chimney is dominated by putative sulfur-reducing Deltaproteobacteria. A comparison of bacterial communities on inactive sulfides from this and previous studies reveals the presence of a clade of uncultured Bacteroidetes exclusive to sulfidic environments, and a high degree of heterogeneity in bacterial community composition from one sulfide structure to another. In light of the heterogeneous nature of bacterial communities observed here and in previous studies of both active and inactive hydrothermal sulfide structures, the presence of numerous niches may be detected on these structures in the future by finer scale sampling and analysis.

Gravity core sampling took place at Dorado Outcrop off the west coast of Costa Rica in the Pacific Ocean during the R/V Atlantis cruise  AT26-24.  For more information about operations of this cruise see the cruise page which contains a link to the cruise report (AT26-24). 

Phosphate, N+N, silicate, and NH4, were all run at Oregon State University using standard colorimetric techniques.  Dissolved inorganic carbon (DIC) was run at Oregon State University using coulometry.  Remaining elements were analyzed using Inductively Coupled Plasma Optical Emission Spectrophotometer (ICPOES).  Comparison of Si measured via the two techniques shows an offset with the colorimetric results being ~7 uM higher than the ICP-OES results.

Push core sampling took place at Dorado Outcrop off the west coast of Costa Rica in the Pacific Ocean during the R/V Atlantis cruise  AT26-24.  For more information about operations of this cruise see the cruise page which contains a link to the cruise report (AT26-24). 

Phosphate, N+N, silicate, and NH4, were all run at Oregon State University using standard colorimetric techniques.  Dissolved inorganic carbon (DIC) was run at Oregon State University using coulometry.  Remaining elements were analyzed using Inductively Coupled Plasma Optical Emission Spectrophotometer (ICPOES).  Comparison of Si measured via the two techniques shows an offset with the colorimetric results being ~7 uM higher than the ICP-OES results.

Push core sampling took place at Dorado Outcrop off the west coast of Costa Rica in the Pacific Ocean during the R/V Atlantis cruise  AT26-24.  For more information about operations of this cruise see the cruise page which contains a link to the cruise report (AT26-24). 

Total inorganic carbon (TIC) was measured using coulometry and CaCO3 is calculated from this value. Organic carbon (OC) and nitrogen (N) were measured on a Perkin Elmer Series II 2400 CHNS/O analyzer. The Fe, Cr, Cu, Mn, and Zn represent results from dithionite extractions (see Roy et al., 2012, Continental Shelf Research, 54, 67-79). Metals were analyzed by Inductively Coupled Plasma Optical Emission Spectrophotometer (ICPOES) at the University of Akron. d13C was measured on a Carlo Erba NA-1500 elemental analyzer connected to a DeltaPlus XL mass spectrometer at Oregon State University.

Gravity core sampling took place at Dorado Outcrop off the west coast of Costa Rica in the Pacific Ocean during the R/V Atlantis cruise  AT26-24.  For more information about operations of this cruise see the cruise page which contains a link to the cruise report (AT26-24). 

Total inorganic carbon (TIC) was measured using coulometry and CaCO3 is calculated from this value. Organic carbon (OC) and nitrogen (N) were measured on a Perkin Elmer Series II 2400 CHNS/O analyzer. The Fe, Cr, Cu, Mn, and Zn represent results from dithionite extractions (see Roy et al., 2012, Continental Shelf Research, 54, 67-79). Metals were analyzed by Inductively Coupled Plasma Optical Emission Spectrophotometer (ICPOES) at the University of Akron. d13C was measured on a Carlo Erba NA-1500 elemental analyzer connected to a DeltaPlus XL mass spectrometer at Oregon State University.

Syringe samples of hydrothermal water were collected during Alvin dives.

The Alvin dives were conducted off the west coast of Costa Rica in the Pacific Ocean at Dorado Outcrop during the R/V Atlantis cruise AT26-24.  For more information about operations of this cruise see the cruise pages which contain links to cruise reports (AT26-24)

Chemical Measurements:

All fluids were measured using established standard protocols. At sea, alkalinity was determined potentiometrically via titration with 0.1N hydrochloric acid; a pH measurement is conducted as part of this procedure. An Inductively Coupled Plasma Optical Emission Spectrophotometer (ICPOES) was used to measure Na, K, Sr, Li, Mg, Ca, and S with a 1:200 dilution and Sr, Li, Fe, Mn, and B with a 1:25 dilution in 1% nitric acid. An inductively coupled plasma mass spectrometry (ICP-MS) was used to analyze Rb, Cs, Ba, Mo, V and U with a 1:75 dilution in 3% nitric acid. Nutrients (silicate, phosphate, total nitrogen and nitrate) were measured at Oregon State University using standard colorimetric, automated, segmented flow procedures. Chlorinity was determined potentiometrically via titration with silver nitrate. Carbon analyses were conducted at Oregon State University