Billy Brazelton

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The Lost City Hydrothermal Field (LCHF) is a novel type of hydrothermal system on 1.5 million year-old ultramafic oceanic crust near the mid-Atlantic ridge. Fluid circulation is primarily driven by exothermic serpentinization reactions - not magmatic heating as in typical black smoker systems. The highly reducing, high-pH fluids emanate from large (up to 60m tall) carbonate structures and contain significant amounts of hydrogen, methane, and organic compounds. We have performed a set of microbiological and molecular analyses on samples collected from the LCHF during an expedition to the site in April-May 2003. Diverse communities of bacteria are prevalent within fluids as well as on the carbonate structures, as evidenced by microscopy and 16S rDNA clone libraries. Fluid and rock samples were inoculated into a wide range of enrichment culture medias; culture tubes with headspaces of H2/CO2 or CH4 were particularly successful in supporting growth. Functional gene analysis by PCR amplification from environmental samples and cultures will be conducted to characterize possible microbial metabolisms involved in the geochemistry of the system. The discovery of the LCHF suggests that other off-axis, geochemically-driven, volcanic-independent systems may be present on the seafloors of Earth as well as other planets. Furthermore, because this type of system requires only the formation of oceanic crust and not necessarily modern tectonic activity, it may represent a likely analog for habitats widespread on the early Earth. Molecular and Organismal Characterization of Microbial Communities at the Lost City Hydrothermal Field William J. Brazelton, Matthew O. Schrenk, Sheryl A. Bolton, and John A. Baross University of Washington, School of Oceanography, Center for Astrobiology and Early Evolution Abstract 3864-1524 3864-1537 3881-1408 2001 Active Carbonates 2001 Inactive n=26 n=50 n=54 n=53 n=35 2003 Active Carbonates ANME-2 Methano- sarcinales ANME-1 Methanomicrobiales LCHF 16S rDNA Clone Libraries Enrichment Cultures Environmental Cell Counts Bacterial 16S rDNA was amplified from rock samples collected from the LCHF. (Construction of Archaeal 16S rDNA libraries is in progress.) A summary of the closest BLAST hits to the sequenced clones is below: Proteobacteria Alpha: Afipia (methylotroph) and other heterotrophs Beta: Ralstonia & Burkholderia (pathogens) Gamma: Thiomicrospira (sulfur oxidizer) Marinomonas protea (Antarctic) Methylomonas rubra (methylotroph) Epsilon: Sulfurospirillum (sulfur reducer) Firmicutes Desulfotomaculum (sulfate-reducer) Deep-sea Clostridia Thermoterrabacterium (thermophilic Fe-reducer, H2-oxidizer) CFB Saprospira grandis Zobellia (mesophilic aerobe) Thermotoga Thermosipho (vent hyperthermophile) The distribution of bacterial groups in six bacterial 16S rDNA clone libraries constructed from LCHF rock samples is shown below. Each of the libraries contains a unique bacterial community distribution, suggesting large variations in microbial diversity among microenvironments on the same structure. (Samples 3864-1524 and 3864-1537 were both collected from Marker 2; 3881-1408 is from Marker 3 (large vent at top of structure).) We designed a high-pH, carbonate-buffered basal medium for culturing microbes from LCHF. A large array of enrichment culture media was prepared in order to target microbial groups that may play a role at LCHF: methanogens, methanotrophs, sulfate-reducing bacteria, H2-oxidizers, and Fe-reducers. Culture tubes containing headspaces of H2/CO2 or CH4 or both were more successful at permitting growth than other media types. Of approximately 380 inoculated tubes, 41 were observed to contain growing cultures. Of those 41, 36 were inoculated from rock samples, and only 5 were inoculated from fluids. Three of the positive enrichment cultures contained an Archaea 16S rDNA sequence with high identity to a phylotype that completely dominates Archaea 16S rDNA clone libraries constructed from environmental samples (data not shown). A phylogenetic tree showing the relationships among the phylotype from the environmental clone libraries (LC1022a-1 and LC1149a-56), the enrichment culture (LC206), and other related species is at right. This data shows that a community of Methanosarcina-related organisms present in our enrichment cultures is prevalent on active vent structures at LCHF, possibly playing a role in methane generation or oxidation. Acknowledgements The crew and science party of Atlantis cruise 7-32, chief scientist Deborah Kelley, and the Baross Lab Hydrothermal fluid samples and carbonate rock samples collected from the LCHF were filtered onto 0.2um membranes. DAPI-stained cells were counted using the most probable number (MPN) technique. Rock samples tend to contain many more cells than fluids from the same location, and fluids do not appear to have more cells than deep sea background waters. However, Marker 3 (large, central vent) fluids appear to be slightly enriched, and Marker 2 (a diffusely venting flange) is somewhat depleted of cells relative to background. The Lost City Hydrothermal Field The LCHF is a peridotite-hosted system, driven by serpentinization reactions that occur when mantle rocks are uplifted and exposed to seawater. As mantle rocks are uplifted and exposed to water, the peridotite is altered to serpentinite, releasing hydrogen, methane, organics, and energy. This process does not depend on tectonic activity, and it is possible that Lost City represents a new class of biological habitat capable of thriving on geochemical reactions independent of solar or tectonic energy. n=32 LC1149B LC1022B LC1231B Number of positive enrichment cultures incubated at 50, 70, or 90°C Summary Rock samples from the LCHF contained significantly more cells than fluid samples from the same structure. Bacterial 16S rDNA clone libraries indicate a wide diversity of bacteria associated with the carbonate structures. A Methanosarcina-related organism from the carbonate structures has been cultivated in enrichment culture. References Kelley, DS et al. An off-axis hydrothermal vent field near the Mid-Atlantic Ridge at 30 degrees N.Nature 2001 Jul 12;412(6843):145-9. Schrenk, MO et al. Incidence and diversity of microorganisms within the walls of an active deep-sea sulfide chimney.Appl. Env. Microbiol. 2003 Jun;69(6):3580-92. Fruh-Green, GL et al. 30,000 years of hydrothermal activity at the lost city vent field. Science. 2003 Jul 25;301(5632):495-8. Incubation Temperature *rock samples * * * * * * * * * *