Dataset: Thermophile abundance - MPN
Deployment: RB1403

In August 2012, September and October 2013, August 2014, and August 2015, 7-40°C diffuse hydrothermal fluids were collected from 10 vent sites at 1515-1716 m depths from Axial Seamount on the Juan de Fuca Ridge. The fluid samples were drawn into 650 ml Tedlar plastic bags with polyethylene valves within rigid housings using the NOAA Hydrothermal Fluid and Particle Sampler. The sampler pumped vent fluid through a titanium nozzle and recorded the temperature of the fluid within the intake nozzle once every second during pumping. Samples were collected using the research submarines Jason II and ROPOS. Background seawater was collected by shipboard hydrocasts at 1500 m depth directly over the caldera (25 m above the bottom) and 3 km west of the summit with 10 L Niskin bottles. The hydrothermal fluid and background seawater samples were divided for cultivation-dependent Most Probable Number (MPN) concentration estimates of thermophiles and hyperthermophiles (100 ml), microcosm incubations (400 ml), and total cell counts (40 ml). All operations at sea occurred on the research vessels Marcus G. Langseth, Thomas G. Thompson, Falkor, and Ronald H. Brown.

Most Probable Number (MPN) Cell Estimates: Three-tube MPN analyses were used by adding 3.3, 0.33, and 0.03 ml of the hydrothermal fluid samples in triplicate to the methanogen, autotrophic sulfur reducer, and heterotroph media. After inoculation, the tubes were incubated at 80°C and 55°C for up to 7 days. Growth in the tubes was confirmed using phase-contrast light microscopy. Growth of methanogens and H2-producing heterotrophs was verified by analyzing all of the tubes for CH4 and H2, respectively, in the headspace using gas chromatography. Total and H2-producing heterotroph cell concentration estimates were scored and reported separately based on tubes that had cells versus those with H2. In order to estimate the concentration of non-methanogenic autotrophs in the autotrophic sulfur medium, the estimated number of methanogens in the autotrophic sulfur medium MPN tubes was subtracted from the estimated concentration of total cells.

Full methodology