Identification of Chemoautotrophic Microorganisms from a Diffuse Flow Hydrothermal Vent

At deep-sea hydrothermal vents chemolithoautotrophic microbes mediate the transfer of geothermal chemical energy to higher trophic levels. To better understand these underlying processes and the organisms catalyzing them, this research used DNA Stable Isotope Probing (SIP) combined with Catalyzed Activated Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH) to identify the microorganisms chemoautotrophically supporting the food web at a diffuse flow hydrothermal vent. Both anaerobic and aerobic shipboard incubations containing various augmented electron donor and acceptor species showed that Epsilonproteobacteria were the dominant chemoautotrophs with greater than 70% of the cells counted within the first 24 hours. 1C DNA SIP identified unique organisms not previously characterized from low temperature diffuse flow venting: green sulfur bacteria (Chlorobi-like organisms) possibly utilizing photoautotrophy, aerobic Lutibacter litoralis-like organisms growing under anaerobic conditions, and Epsilonproteobacterial Thioreductor sp. at temperatures above maximum known tolerances. This research illustrates both the promise and pitfalls of the SIP technique applied to hydrothermal systems, concluding that timing of the incubation experiments is the critical step in eliminating undesired 1 C labeling. These results set the stage for a more thorough future examination of diffuse flow microorganisms by presenting interesting questions that second generation experiments could be designed to answer. History and Background: Hydrothermal Vent Systems Deep-sea hydrothermal vents were first discovered 32 years ago on the spreading center of the Galapagos ridge (Lonsdale, 1977, Corliss et al., 1979). They are commonly located along mid-ocean ridges where oceanic plates are spreading apart due to an underlying magma chamber in the Earth's mantle. At these locations, cold, deep-sea water is penetrating the highly porous, newly formed oceanic crust, and through seawater-rock interactions at depth and at high temperature and pressure it is being transformed into a highly reduced, superheated, and acidic fluid, also referred to as hydrothermal fluid. The hydrothermal fluid that is highly enriched in reduced chemical species, such as H2, H2S, Fe2 +, travels back to the seafloor where it is emitted into the deep ocean in basically two modes: as focused flow or diffuse flow. At focused flow, fluids are emitted undiluted at high temperatures (to 400*C) through so-called black smoker chimneys. At diffuse-flow vents, hydrothermal fluid mixes in the subseafloor with various amounts of oxygenated seawater and exits the seafloor at lower temperatures, often between 10*C to 60C. Over the years, hydrothermal vent science has focused mainly on studying the hot focused black smoker venting, and less attention has been paid to diffuse-flow vents. However, it is the mixing zones in the upper crust that have the potential to harbor abundant microbial life due to the simultaneous presence of oxidized and reduced chemical species creating chemical disequilibria that can be harnessed by a diverse array of metabolically versatile microorganisms. At present information on the identity of the microbes living in this vast system and their activities and contribution to overall biomass production at vents is limited. This study addresses the diversity and identity of chemoautotrophic microbial communities associated with diffuse