Award: OCE-1136608

This effort is linked to an NSF-DOB project that investigates microbial processes and their contribution to the evolution of ecological habitats at deep-sea hydrothermal vents. The lead principal investigator of this interdisciplinary and collaborative project is Sievert (WHOI) accompanied with several principal investigators including Foustoukos (CIW), Seewald (WHOI), Stepanauskas (Bigelow), Taylor (WHOI) and Vetriani (Rutgers University). The primary objectives of this program are: i) determine the relationship between taxonomic, genetic and functional diversity, ii) identify predominant metabolic pathways, iii) constrain the rates of chemosynthetic primary productivity and iv) investigate gene expression patterns associated with biofilm development. The CIW team was responsible for the shipboard continuous culturing of vent fluids collected from Crab Spa and Tica hot springs during the AT26-10 expedition at 9 oN EPR. This was accomplished by utilizing our high-pressure bioreactor and deploying isobaric gas-tight samplers (IGTs) to collect hydrothermal vent fluids at the diffuse flow sites (Seewald). Experiments were designed to study the cycling to N through the metabolic processes of denitrification and dissimilatory nitrate reduction to ammonia (DNRA) under in-situ deep-sea vent temperature and pressure conditions. To our knowledge, this is the first time that microorganisms from deep-sea vents have been sampled, transferred and cultures under in-situ pressure and temperature conditions. We studied the evolution of nitrate reducing microorganisms at mesophilic (30 oC) and thermophilic (50 oC) conditions at pressures ranging from 5 to 250 bar. Vent fluids (16 IGTs) were delivered in the bioreactor and homogeneously mixed with aqueous media solution enriched in dissolved nitrate, hydrogen and 13C labeled bicarbonate to facilitate the growth of nitrate reducing microorganisms. Experiments simulated near-seafloor hydrothermal fluid-seawater mixing environments. Two distinct sets of experiments were lasted for 356 and 100 hours. In short, experimental results constrained the function and metabolic rates of the denitrifying microbial communities in the Crab Spa fluids, while DNRA metabolic pathways were identified for the populations residing in the moderate temperature vent fluids (50 oC) of the Alvinella colony at Tica. Data will help us evaluate the tradeoffs between growth efficiency and reaction kinetics during chemolithoautotrophic NO3- reduction, and their contribution to the N and C cycles at deep-sea hydrothermal vents. During the course of the experiments we monitored the growth of deep-sea microbial communities by measuring the concentrations of dissolved aqueous species directly involved in nitrate based metabolism, such as NO3-, NH4+, H2 and H2S. We also monitored cell densities by utilizing an epi-fluorescence microscope. Dissolved gas and NH4+ concentrations were attained by gas and ion chromatography (Seewald, Sylva). Subsamples were also collected for a number of offshore analysis to determine: i) the 15N/14N isotope composition of NO3-,/NH4+ and constrain kinetic isotope effects associated with denitrification/DNRA (CIW), ii) to study the rates of autotrophic carbon fixation following the microbial assimilation of 13C-labelled bicarbonate by NanoSIMS (Musat, UFZ) and EA-IRMS (CIW), iii) to perform single cell genomics on the microbial populations grown in the bioreactor (Ramunas) and (iv) to isolate and characterize novel microogranisms from the communities developed in the high-pressure bioreactor (Perez-Rodriguez, CIW and Vetriani). Data have been released to the BCO-DMO depository: http://www.bco-dmo.org/dataset/628993 and http://www.bco-dmo.org/dataset/529026. This award supported the development of procedures and techniques to conduct high-pressure culturing of microbial communities sampled and transferred under seafloor pressures. The CIW high-pressure bioreactor allows for experimentation of micro...