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Each analysis required 45 min in a dual mobile phase gradient separation on a 250-mm C18 5 μm HS reversed phase column (Alltech Assoc.) so as many as 30 samples could be analyzed daily. Light and dark COC, as described previously (Cuhel et al., 2002). The third and hottest water sample, Stevenson Island 72, comes from a deep trench east of Stevenson Island in the central portion of the lake, where small, well-developed hydrothermal vents coalesce along northwest-trending deep fissures that reach maximally 133 m depth, the deepest point in the lake (Morgan et al., 2003b).
Analytical equipment was transported to Yellowstone National Park and set up as a field laboratory at the National Park Service Lake Station.
Freshly collected samples for stable analytes were filtered through 0.2-μm filters (Supor-200, Pall Corp.) and aliquoted for the different analyses.
Near-complete 16S r RNA genes were PCR-amplified with bacterial primers 8F (5); these primers were successful in recovering extremely diverse bacterial communities, including members of novel phyla (Teske et al., 2002). Phylogenetic ecology of the freshwater Actinobacteria ac I lineage.
PCR products were cloned using the TOPO XL PCR cloning kit (Invitrogen Corporation, Carlsbad, CA, USA) following the manufacturers instructions, and sequenced at the sequencing center of the University of North Carolina with primers M13F (5).
Total DIC was analyzed by the Teflon-membrane flow injection method of Hall and Aller (1992), in which the sipper tube was inserted through the three-way valve and the syringe plunger used to prevent formation of headspace during injection.
For deep samples from Stevenson Island, unavoidable degassing effects were reduced by shaking the syringe thoroughly just before sipping, as the method measures all forms of CO including dissolved gas.
Direct observations by SCUBA and ROV have revealed a wide range of hydrothermal features, including large hydrothermal chimneys; gas fumaroles; seepage of hot, shimmering water; and sulfur-oxidizing microbial mats growing around hot water seeps and vents (Remsen et al., 2002).
Examination of an ancient vent chimney revealed internal conduit structures with metal sulfide precipitates, indicating long-term hydrothermal activity (Cuhel et al., 2004).
The fifth sample from the West Thumb Basin, West Thumb 98, represents a cooler, low activity water sample from the West Thumb area.
In situ temperatures measured by ROV, and chemistry of the syringe-sampled waters are shown in Table 1.