|Created||June 3, 2015|
|Modified||February 10, 2017|
|State||Final no updates expected|
|Brief Description||sample log from Jason-II dives for Sievert|
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.
General term for a laboratory instrument used for deciphering the order of bases in a strand of DNA. Sanger sequencers detect fluorescence from different dyes that are used to identify the A, C, G, and T extension reactions. Contemporary or Pyrosequencer methods are based on detecting the activity of DNA polymerase (a DNA synthesizing enzyme) with another chemoluminescent enzyme. Essentially, the method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step.
Fibox 3/Pts3, Presens, Germany
For nitrate+nitrite measurement. Teledyne, San Diego CA, USA
The chemiluminescence method for gas analysis of oxides of nitrogen relies on the measurement of light produced by the gas-phase titration of nitric oxide and ozone. A chemiluminescence analyzer can measure the concentration of NO/NO2/NOX.
One example is the Teledyne Model T200: http://www.teledyne-api.com/products/T200.asp
unique sample identification or number; any combination of alpha numeric characters; precise definition is file dependent
latitude, in decimal degrees, North is positive, negative denotes South; Reported in some datasets as degrees, minutes
longitude, in decimal degrees, East is positive, negative denotes West; Reported in some datsets as degrees, minutes
Observation/sample depth below the sea surface. Units often reported as: meters, feet.
When used in a JGOFS/GLOBEC dataset the depth is a best estimate; usually but not always calculated from pressure; calculated either from CTD pressure using Fofonoff and Millard (1982; UNESCO Tech Paper #44) algorithm adjusted for 1980 equation of state for seawater (EOS80) or simply equivalent to nominal depth as recorded during sampling if CTD pressure was unavailable.
unique sample identification or number; any combination of alpha numeric characters; precise definition is file dependent
collection method: IGT=Isobaric Gastight Sampler ; LVP=Large Volume Pump
|Stefan M. Sievert||Woods Hole Oceanographic Institution (WHOI)||✓|
|Craig Taylor||Woods Hole Oceanographic Institution (WHOI)|
|Jeffrey S. Seewald||Woods Hole Oceanographic Institution (WHOI)|
|Nancy Copley||Woods Hole Oceanographic Institution (WHOI BCO-DMO)|
|Project Title||An Integrated Study of Energy Metabolism, Carbon Fixation, and Colonization Mechanisms in Chemosynthetic Microbial Communities at Deep-Sea Vents|
|Acronym||Microbial Communities at Deep-Sea Vents|
|Created||June 11, 2012|
|Modified||June 11, 2012|
Deep-sea hydrothermal vents, first discovered in 1977, are poster child ecosystems where microbial chemosynthesis rather than photosynthesis is the primary source of organic carbon. Significant gaps remain in our understanding of the underlying microbiology and biogeochemistry of these fascinating ecosystems. Missing are the identification of specific microorganisms mediating critical reactions in various geothermal systems, metabolic pathways used by the microbes, rates of the catalyzed reactions, amounts of organic carbon being produced, and the larger role of these ecosystems in global biogeochemical cycles. To fill these gaps, the investigators will conduct a 3-year interdisciplinary, international hypothesis-driven research program to understand microbial processes and their quantitative importance at deep-sea vents. Specifically, the investigators will address the following objectives: 1. Determine key relationships between the taxonomic, genetic and functional diversity, as well as the mechanisms of energy and carbon transfer, in deep-sea hydrothermal vent microbial communities. 2. Identify the predominant metabolic pathways and thus the main energy sources driving chemoautotrophic production in high and low temperature diffuse flow vents. 3. Determine energy conservation efficiency and rates of aerobic and anaerobic chemosynthetic primary productivity in high and low temperature diffuse flow vents. 4. Determine gene expression patterns in diffuse-flow vent microbial communities during attachment to substrates and the development of biofilms.
Integration: To address these objectives and to characterize the complexity of microbially-catalyzed processes at deep-sea vents at a qualitatively new level, we will pursue an integrated approach that couples an assessment of taxonomic diversity using cultivation-dependent and -independent approaches with methodologies that address genetic diversity, including a) metagenomics (genetic potential and diversity of community), b) single cell genomics (genetic potential and diversity of uncultured single cells), c) meta-transcriptomics and -proteomics (identification and function of active community members, realized potential of the community). To assess function and response to the environment, these approaches will be combined with 1) measurement of in situ rates of chemoautotrophic production, 2) geochemical characterization of microbial habitats, and 3) shipboard incubations under simulated in situ conditions (hypothesis testing under controlled physicochemical conditions). Network approaches and mathematical simulation will be used to reconstruct the metabolic network of the natural communities. A 3-day long project meeting towards the end of the second year will take place in Woods Hole. This Data Integration and Synthesis meeting will allow for progress reports and presentations from each PI, postdoc, and/or student, with the aim of synthesizing data generated to facilitate the preparation of manuscripts.
Intellectual Merit. Combining the community expression profile with diversity and metagenomic analyses as well as process and habitat characterization will be unique to hydrothermal vent microbiology. The approach will provide new insights into the functioning of deep-sea vent microbial communities and the constraints regulating the interactions between the microbes and their abiotic and biotic environment, ultimately enabling us to put these systems into a quantitative framework and thus a larger global context.
Broader Impacts. This is an interdisciplinary and collaborative effort between 4 US and 4 foreign institutions, creating unique opportunities for networking and fostering international collaborations. This will also benefit the involved students (2 graduate, several undergraduate) and 2 postdoctoral associates. This project will directly contribute to many educational and public outreach activities of the involved PIs, including the WHOI Dive & Discover program; single cell genomics workshops and Cafe Scientifique (Bigelow); REU (WHOI, Bigelow, CIW); COSEE and RIOS (Rutgers), and others. The proposed research fits with the focus of a number of multidisciplinary and international initiatives, in which PIs are active members (SCOR working group on Hydrothermal energy and the ocean carbon cycle, http://www.scorint. org/Working_Groups/wg135.htm; Deep Carbon Observatory at CIW, https://dco.gl.ciw.edu/; Global Biogeochemical Flux (GBF) component of the Ocean Observatories Initiative (OOI), http://www.whoi.edu/GBF-OOI/page.do?pid=41475)
|Stefan M. Sievert||Woods Hole Oceanographic Institution (WHOI)||Lead Principal Investigator|
|Costantino Vetriani||Rutgers University||Principal Investigator|
|Dionysis I. Foustoukos||Carnegie Institution for Science (CIS)||Principal Investigator|
|Ramunas Stepanauskas||Bigelow Laboratory for Ocean Sciences||Principal Investigator|
|Craig Taylor||Woods Hole Oceanographic Institution (WHOI)||Co-Principal Investigator|
|Jeffrey S. Seewald||Woods Hole Oceanographic Institution (WHOI)||Co-Principal Investigator|
|Nadine Le Bris||Laboratoire d'Écogéochimie des Environnements Benthiques (LECOB)||International Collaborator|
|Niculina Musat||Max Planck Institute for Marine Microbiology (MPI)||International Collaborator|
|Thomas Schweder||University of Greifswald||International Collaborator|
|Fengping Wang||Shanghai Jiao Tong University (SJTU)||International Collaborator|