Dataset: Jason-II sample log AT26-10
Deployment: AT26-10

The taxonomic composition of the microbial community present in fluid samples and shipboard incubations conducted in gas-tight isobaric samplers (IGT) was investigated by sequencing 16S rRNA amplicons using 454 technology. Further, samples from shipboard incubations were taken to obtain cell counts, activity measurements using nano-scale secondary ion mass spectrometry (nanoSIMS), to determine bulk 13C-incoproration into biomass, and measure pH and the concentration of select chemicals (nitrate, ammonium, sulfide, hydrogen, methane, oxygen). On select samples obtained with the large volume pump (LVP), metagenomic, metaproteomic, and lipid biomarker analyses will be performed.

Samples were collected at several sites at the 9ºN deep-sea hydrothermal vent field on the East Pacific Rise.  They included ROV Jason-II deployments J2-758, J2-759, J2-760, J2-761, J2-762. DNA was extracted following established protocols. We were able to successfully sequence 16S rRNA amplicons for Bacteria and Archaea from a total of 17 shipbaord incubations, 10 LVP samples, and one basalt rock sample. Sequence data are currently being analyzed and will be deposited in GenBank prior to publication and will be made available to the scientific community. From the incubations, the following analyses have been completed: total cell numbers, NanoSIMS analyses, 13C-bulk organic carbon analysis, and chemical measurements. These data are currently being prepared for a manuscript and data will be publically released with the publication. Metagenomic sequencing, metaproteomic analyses, and lipid biomarker analysis of the LVP samples are currently underway. Data will be made available to the scientific community once the data processing is complete and data are published. This is expected to be the case in the first half of 2016.

16S rRNA amplicons for Bacteria and Archaea were generated using 454 sequence technology. Obtained sequences are currently being analyzed using the QIIME pipeline. The reads are being dereplicated, denoised, screened for chimeric sequences and taxonomically classified using the RDP and GreenGenes databases. Multivariate ordination techniques are being used to discriminate among samples with similar community structures. Total sulfide was determined by combining a 2mL sample with sulfide antioxidant buffer and measuring voltage with a sulfide-selective electrode. The electrode was calibrated daily with a serial dilution of a standard sodium sulfide solution. To account for oxidation of the sulfide solution, the solution was titrated daily with lead nitrate to determine the actual sulfide concentration. pH was measured using an electrode, which was calibrated daily. Methane and hydrogen were determined by quantitative headspace extraction of a known volume of sample and measured on a GC-FID (for methane and concentrations of hydrogen > 5μM) or GC-TCD (for concentrations of hydrogen <5μM). Oxygen was determined by passing hydrothermal fluid through a specially designed flow-through cell fitted with a commercially available oxygen optode (Pts3, Presens, Germany). Fluorescence lifetime decay was measured every second using a computerized system corrected for temperature effects (Fibox 3, Presens, Germany), and the most stable final oxygen value was used. The oxygen optode spot was calibrated once with oxygen-free water (treated with sodium dithionate) and air-saturated water to make a two-point calibration. Prior to measurements, the optode flow-through cell was flushed with N2-purged FBSW to remove air bubbles and connected to the IGT sample valve while both were dripping liquid to avoid entrainment of air bubbles in the sample chamber. For subsequent nutrient analysis, fluids were filtered through a 0.2µm GTTP membrane and stored frozen at -20oC. Total nitrate+nitrite was determined by conversion to NO and chemiluminescent determination using the NoxBox instrument (Teledyne, San Diego CA, USA) following the original protocol (Garside 1982). Similarly, filtered samples were analyzed shipboard for total ammonium+ammonia by flow injection as previously described. All standards were pure chemicals made up in distilled water. All analytes, with the exception of oxygen, were measured in analytical duplicates. Cells were prepared for counting by preserving 1.5mL of fluids with 40µL of borate-buffered formalin, and subsequently adding 200µL of 0.1% acridine orange solution. The fixed and stained sample was then filtered under gentle vacuum onto a black 0.2µm polycarbonate filter, and enumerated aboard the ship by fluorescence microscopy. 10 grids were counted per sample and extrapolated using the area filtered to determine cell concentrations. All counts were done in analytical duplicates.