Dataset: Incubation in diffuse flow vent fluids - Crab Spa
Deployment: AT26-10

From AT26-10 cruise report (01/29/2014):
DOB: An Integrated Study of Energy Metabolism, Carbon Fixation, and Colonization Mechanisms in Chemosynthetic Microbial Communities at Deep-Sea Vents
Cruise Report by the CIW research team: Dr. Ileana Perez-Rodriguez, Mr. Matt Rawls and Dr. Dionysis I. Foustoukos
The CIW team was responsible for the shipboard continuous culturing incubations of vent fluids collected from Crab Spa and Tica hot springs during the AT26-10 expedition at 9oN EPR by utilizing our high-pressure bioreactor (Fig. 1). This was accomplished through a collaborative effort with Jeff Seewald and Sean Sylva (WHOI), who deployed isobaric gas-tight samplers (IGTs) to collect hydrothermal vent fluids at the diffuse flow sites. 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.

We studied the evolution of nitrate reducing microorganisms at mesophilic (30oC) and thermophilic (50oC) 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 (Fig. 2). The 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 (60oC) of the Alvinella colony at Tica.

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 (Sievert, WHOI). Dissolved gas and NH4+ concentrations were attained by gas and ion chromatography (Seewald - Sylva, WHOI). 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 (Perez-Rodriguez, CIW), ii) to study the rates of autotrophic carbon fixation by NanoSIMS (Musat, UFZ), iii) to perform single cell genomics on the microbial populations grown in the bioreactor (Ramunas, Bigelow) and (iv) to isolate and characterize novel microogranisms from the communities cultured in our experiments (Perez-Rodriguez, CIW and Vetriani, Rutgers).