Award: OCE-1405237

The detection and characterization of active microbial populations within subsurface crustal basalts represents an expansion of EarthÆs biosphere and provides a new environment to test the limits of life. Over the past several decades, microbial populations have been qualitatively and quantitatively characterized in marine sediments from near shore locations to gyre centers, and from the sediment surface to two kilometers below the surface. Recent exploration of the crustal material has targeted exposed basalts and free-living, interstitial water populations. Limited access to subsurface basalt samples has inhibited biological characterization of microbial communities within this unique, isolated, potentially habitable environment. Basalt contains redox active iron and additional metals available as energy sources for microbial populations (Canfield et al., 2006). Basalt has been shown to geochemically oxidize over millions of years, thus remaining biologically redox active (Bach and Edwards, 2004). Basalt is also porous allowing fluid exchange and providing single-celled life with dissolved metabolites and nutrients required for cell maintenance and growth. Initial cultivation-based and in situ analysis of subsurface basalt has produced some structural identification of populations that have the potential to alter the crust. The study reported here represents the first to directly sample and identify metabolically active microbial populations within subsurface crustal material. Drilling during Integrated Ocean Drilling Program Expedition 336 in the æNorth PondÆ sediment pond feature on the western flank of the Mid-Atlantic Ridge (Expedition 336 Scientists, 2012. IODP Preliminary Report 336) provided basalt samples from 144-219 meters below seafloor. Samples were collected using standard biological sampling protocols to prevent operational contamination. Each sample passed drilling contamination quality control standards for microbial analysis. Descriptions of each sample were recorded shipboard and then visually confirmed prior to RNA extraction. The samples from drill site 1382 were aphyric with glassy margins (7R-2B) and a sedimentary breccia with basalt clasts and a rust color (8R-4D). Site 1383 samples included two highly altered phyric basalts (10R-1B and 11R-1B) and a basalt glass with rust alterations (20R-1A). Total RNA was extracted from all samples and multiple controls following methods described in Mills et al. (2012). The targeted 16S rRNA V1-V3 region was sequenced using 454 pyrosequencing and analyzed following a standardized Mills Lab work flow. Sequencing efforts produced 2539 sequences; with each sample library containing 124 to 1323 sequences phylogenetically related to 4 to 10 phyla. These lineages were not observed in any of the control samples, thus represent metabolically active populations only found within the crustal material. Distinct communities were observed in each of the five samples with few lineages shared. Analysis of these lineages suggested both anaerobic and aerobic metabolisms with populations capable of utilizing the available carbonate present within the basalt samples. Additional characteristics suggested metabolisms capable of taking advantage of substrates within the basalt. As seen in the overlying sediments, many lineages were capable of using nitrogen species in redox reactions, supporting the importance of this pathway in energy-limited environments. This analysis will support further targeted approaches for enhanced molecular characterizations and culture-based analysis to enhance understanding of the metabolic processes and potential biomarkers available for detection of life within basalts on Earth and potentially in the Martian subsurface. Last Modified: 12/14/2015 Submitted by: Heath Mills