Gas seeps emanating from Yanartaş (Chimera), Turkey, have been documented for thousands of years. Active serpentinization produces hydrogen and a range of carbon gases that may provide fuel for life. Here we report a newly discovered, ephemeral fluid seep emanating from a small gas vent at Yanartaş. Fluids and biofilms were sampled at the source and points downstream. We describe site conditions, and provide microbiological data in the form of enrichment cultures, Scanning electron microscopy (SEM), carbon and nitrogen isotopic composition of solids, and PCR screens of nitrogen cycle genes. Source fluids are pH 11.95, with a Ca:Mg of ~200, and sediments under the ignited gas seep measure 60°C. Collectively, these data suggest the fluid is the product of active serpentinization at depth. Source sediments are primarily calcite and alteration products (chlorite and montmorillonite). Downstream, biofilms are mixed with montmorillonite. SEM shows biofilms distributed homogeneously with carbonates. Organic carbon accounts for 60% of the total carbon at the source, decreasing downstream to <15% as inorganic carbon precipitates. δ13C ratios of the organic carbon fraction of solids are depleted (−25 to −28‰) relative to the carbonates (−11 to −20‰). We conclude that heterotrophic processes are dominant throughout the surface ecosystem, and carbon fixation may be key down channel. δ15N ratios ~3‰, and absence of nifH in extracted DNA suggest that nitrogen fixation is not occurring in sediments. However, the presence of narG and nirS at most locations and in enrichments indicates genomic potential for nitrate and nitrite reduction. This small seep with shallow run-off is likely ephemeral, but abundant preserved microterracettes in the outflow and the surrounding area suggest it has been present for some time. This site and others like it present an opportunity for investigations of preserved deep biosphere signatures, and subsurface-surface interactions.

A b s t r a c tThe microbiology of caves is an important topic for better understanding subsurface biosphere diversity. The diversity and taxonomic composition of bacterial communities associated with cave walls of the Oylat Cave was studied first time by molecular cloning based on Sanger/pyrosequencing approach. Results showed an average of 1,822 operational taxonomic units per sample. Clones analyzed from Oylat Cave were found to belong to 10 common phyla within the domain Bacteria. Proteobacteria dominated the phyla, followed by Actino bacteria, Acidobacteria and Nitrospirae. Shannon diversity index was between to 3.76 and 5.35. The robust analysis conducted for this study demon strated high bacterial diversity on cave rock wall surfaces. This copy is for personal use only -distribution prohibited.Gulecal-Pektas Y. 70After collection, the samples were frozen on dry ice on site, and stored at -20°C upon return to the laboratory (Groth et al., 1999). Environmental DNA was extracted from samples using Fast DNA Spin kit for soil (MP biomedicals, Solon, OH USA).Clone library construction and Sanger sequence analysis. Total genomic DNA was used as a template for 16S rRNA PCR amplification using bacteria 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') universal pri mers (Weisburg et al., 1991). Each 20 µl reaction mixture contained: l µl environmental DNA template, 2.25 mM MgCl 2 , 2 µl GeneAmp 10X PCR Buffer II (Applied Biosystems, Foster City, CA, USA), 100 µM dNTPs (SigmaAldrich, Saint Louis, MO, USA), 0.2 µM each primer, 2.5 U AmpliTaq Gold DNA Polymerase (Applied Biosystems, Carlsbad, CA, USA). Thermal cycling was as follows: initial denaturation 5 min at 94°C, 25 cycles of 94°C for 1 min, hybridization at 50°C for 25 s and elongation at 72°C for 2 min followed by a final elongation at 72°C for 20 min. PCR products were purified using a QIAquick kit (QIAGEN, Valencia, CA, USA), and were cloned into Escherichia coli hosts using the TOPO TA Cloning kit with the pCR 2.1 Vector (Invitrogen Corporation, Carlsbad, CA, USA). Plasmid DNA was extracted and purified using the Ultra Clean Standard Mini Plasmid Prep Kit (MoBio Laboratories). Cloning products were sequenced by TUBITAK MAM DNA Services Facility at Gebze, Turkey, using standard M13 primers.Partial sequences were assembled with CodonCode Aligner v.1.2.4 (CodonCode, USA) and manually checked. Assembled sequences were checked for chimera by Bellerophon server (Huber et al., 2004) and Chimera_Check v 2.7 (Cole et al., 2005). Sample sequences were aligned by BioEdit (Ibis Biosciences, Carlsbad, CA, USA). Phylogenetic analysis was performed in PAUP (Sinauer Associates, Sunderland, MA) using parsimony, neighbor-joining, and maximum likelihood analyses. The 16S rRNA gene sequences were submitted to the NCBI Gen Bank database under accession numbers JQ065958-JQ065959 and JQ219081-JQ219137.454 pyrosequencing and sequence analysis. For the pyrosequencing, the V6 region of the 16S rRNA gene was amplified using PCR with a bacterial primer set 967f...

No abstract