Dataset: Stable carbon isotopes of seafloor basalts
Deployment: MSM20-5

Acquisition methods are described in the following publication:
Orcutt, B.N., Sylvan, J.B., Rogers, D.R., Delaney, J., Lee, R.W., & Girguis, P.R. 2015. Carbon fixation by basalt-hosted microbial communities. Front. Microbiol. doi:10.3389/fmicb.2015.00904

In summary (excerpted from above):
Basalt samples used in this study were collected from three different crustal formation areas. One glassy, seafloor-exposed basalt came from the ASHES vent field in the Axial Seamount volcano caldera on the Juan de Fuca Ridge off the western coast of North America. The sample was collected in 2009 with the Alvin submersible during dive AD-4527 on RV Atlantis cruise AT15-51 (Sample JdF2009). The basalt piece had a thick (up to 1 cm depth) glassy rim overlying moderately to sparsely vesicular cryptocrystalline groundmass. A film of iron oxide discoloration was evident in the contact between the glass and groundmass. Two altered, seafloor-exposed basalts were collected from the Loihi Seamount off the coast of the big island of Hawai’i in October 2009 by ROV Jason-II during R/V Kilo Moana cruise KM0923: one from "Marker 2" in Pele's Pit on the summit of the Loihi Seamount, and one from the Ula Nui vent field at the base of the Loihi Seamount. Sample Ula Nui, collected on dive J2-477, was glassy, highly vesicular, and friable, contained ∼3 mm olivine phenocrysts, and displayed visible iron oxide staining. Sample Marker 2, collected on dive J2-481 from an area away from diffuse venting, was pillow basalt with an altered rind Finally, two seafloor-exposed rocks were collected from outcrops surrounding North Pond in April 2012 by ROV Jason-II on dives J2-626 and J2-627 during the MSM20-5 cruise on the R/V Maria S. Merian. The J2-626-R1 rock was a breccia of mm- to cm-sized clasts of aphanitic basalt in a greenish-gray matrix, while the J2-627-R3 rock was a highly serpentinized harzburgite (25–30% orthopyroxene surrounded by completely serpentinized olivine and with chrysotile-filled veins.

Upon retrieval of the samples, all seafloor-exposed rocks and surrounding water from the plastic sampling containers were immediately transferred to glass jars and placed at 4 degrees C until processing. Basalts were then transferred to ethanol- and flame-sterilized steel processing trays and subsampled with ethanol- and flame-sterilized chisels and tweezers. The experiments utilized the glassy rims of the basalts, which were removed, broken into smaller pieces (<1 cm diameter) and transferred to sterile glass serum vials (30–100 ml volume, depending on experiment) containing 0.2-mm-mesh filter-sterilized oxic bottom seawater. All samples were maintained at 4 degrees C.

Basalt fragments (5–20 cm3) were transferred to sterile and baked glass serum vials (to remove organics, vials had been heated to 500 degrees C for 2 h), which were filled to overflowing with sterile oxic seawater then sealed with autoclaved butyl rubber septa and aluminum crimp seals. Multiple replicate bottles were prepared for each sample to enable as many time series as possible with the limited sample volume, including a no-tracer-addition control.

Time series samples were injected with a small volume of 0.2-um filter-sterilized 13C-bicarbonate-labeled solution (in sterile filtered seawater) to achieve the following starting concentrations: JdF2009 incubations received a final concentration of 0.75 mM 13C-labeled bicarbonate against a background of seawater bicarbonate (∼2 mM, or, 27% 13C label); the Ula Nui and Marker 2 rock incubations received a final concentration of 2.7 mM 13C-labeled bicarbonate in background bottom seawater (57% 13C label); and the seafloor-exposed North Pond basalt incubations contained a final concentration of 4.5 mM 13C-labeled bicarbonate in surface seawater (69% 13C label). Vials were incubated in the dark at 4 degrees C until sampling. At each time point, the vials were opened and rock fragments were transferred to sterile plastic centrifuge tubes and frozen for shore-based DNA and organic carbon extraction and analysis. For the JdF2009, Marker 2, and Ula Nui samples, time series were stopped after 1 h, 1 day, and 1 week of incubation. The North Pond samples were incubated for 2-weeks, 2-months, and 4-months intervals. Final concentrations of dissolved inorganic carbon were not measured, as rates of carbon consumption were presumed to be significantly slow compared to the bulk pool size.

The carbon content and stable carbon isotopic composition of biofilms on the incubated basalts were determined by IRMS analysis of subsamples of the basalts that had been stored frozen. Samples were analyzed using a Costech elemental analyzer in line with a Micromass Isoprime continuous flow stable isotope mass spectrometer. Results are presented in the standard δ notation, where isotopic ratios (R) are expressed in per mil (‰) differences relative to the conventional standard, the PeeDee Belemnite limestone. Although the instrument precision was established as 0.3‰, rates of change were conservatively assumed to be robust if there was more than a 2‰ difference between samples; values less than 2‰ difference are reported as below the detection limit (BDL).