|Created||November 17, 2015|
|Modified||August 19, 2016|
|State||Final no updates expected|
Assessments of bacterial and archaeal diversity from three distinct hydrothermal vents in the Middle Valley vent field along the Juan de Fuca Ridge.
Sampling and Analytical Methodology: Once on board ship, samples were directly transferred to sterile anaerobic seawater and handled/processed using appropriate sterile microbiological techniques. DNA was extracted from this crushed deposit sample with a protocol modified from (Santelli et al. 2008). Subsamples were washed with 0.1 N HCl, followed by two rinses with a sterile solution containing 10 mM Tris (pH 8.0) and 50 mM EDTA. A known mass of material was added to PowerSoil beadbeating tubes(MoBio Laboratories, Carlsbad CA), incubated at 70 degrees C for 10 minutes, and then amended with 200 ng of poly-A. Subsamples were subjected to beadbeating, followed by three cycles of freeze-thaw steps to further lyse cells. Nucleic acids were extracted using hot phenol (60 degrees C for 3 min.), followed by two chloroform:isoamyl separations and precipitated with ethanol. DNA was resuspended in TE (pH 8.0) and quantified using the Qubit™ fluorometer (Life Technologies, Grand Island, NY).
Data Processing: DNA samples were sequenced using 454 pyrotag methods similar to those described previously (Dowd et al. 2008). All samples were sequenced at the Research and Testing Laboratory (Lubbock, TX) using a 454FLX instrument (Roche Inc.) with Titanium™ reagents. The resulting bacterial and archaeal 16S rRNA (bacterial V1-V3 and archaeal V3-V4 of the 16S rRNA genes; primers are shown in Table 1) as well as drsB sequences were analyzed via Mothur (Schloss et al. 2009). Sequences were trimmed, quality checked, aligned to the SILVA-compatible alignment database reference alignment (dsrB gene datasets were aligned to a dsrB gene database generated from the Ribosomal Database Project (RDP)), analyzed for chimeras, classified against the Greengenes99 database and clustered in to OTUs. Rarefaction curves were used to examine the number of OTUs as a function of sampling depth. Alpha diversity was assessed by generating values from the Chao1 richness estimators and the inverse Simpson diversity index.
unique sample identification or number; any combination of alpha numeric characters; precise definition is file dependent
|Peter R. Girguis||Harvard University|
|Kiana L. Frank||Harvard University|
|Shannon Rauch||University of Hawaii at Manoa (SOEST)||✓|
BCO-DMO Project Info
|Project Title||Characterizing the distribution and rates of microbial sulfate reduction at Middle Valley hydrothermal vents|
|Acronym||Middle Valley Vents|
|Created||November 17, 2015|
|Modified||November 19, 2015|
This project characterizes rates of microbially mediated sulfate reduction from three distinct hydrothermal vents in the Middle Valley vent field along the Juan de Fuca Ridge, as well as assessments of bacterial and archaeal diversity, estimates of total biomass and the abundance of functional genes related to sulfate reduction, and in situ geochemistry. Maximum rates of sulfate reduction occurred at 90°C in all three deposits. Pyrosequencing and functional gene abundance data reveal differences in both biomass and community composition among sites, including differences in the abundance of known sulfate reducing bacteria. The abundance of sequences for Thermodesulfovibro-like organisms and higher sulfate reduction rates at elevated temperatures, suggests that Thermodesulfovibro-like organisms may play a role in sulfate reduction in warmer environments. The rates of sulfate reduction observed suggest that – within anaerobic niches of hydrothermal deposits – heterotrophic sulfate reduction may be quite common and might contribute substantially to secondary productivity, underscoring the potential role of this process in both sulfur and carbon cycling at vents.
This project was funded, in part, by a C-DEBI Graduate Student Fellowship.