Dataset: Thermophile growth
Deployment: TN327

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 (dives J820-J826) 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.

For each sample site, 25 ml of hydrothermal fluid or background seawater was added without exposure to air to each of 16 sealed 60 ml serum bottles that had been pre-flushed with either H₂:CO₂ (80%:20%) or N₂:CO₂ (80%:20%), depending on the headspace composition used for incubation (see table in full methods pdf). The bottles were divided into four sets of four bottles with a pair of bottles from each set incubated at 55°C and 80°C for up to a week or until visibly turbid. Three of the four sets of microcosms (sets A-C) were incubated each of the four study years. Set A was flushed and filled with 200 kPa of H₂:CO₂ yielding an estimated aqueous H₂ concentration of 1.2 mM at their incubation temperatures based on calculations using the geochemical prediction software Geochemist’s Workbench. Sets B and C were flushed and filled with 200 kPa of N₂:CO₂, and half of these bottles (set B) were given 1 ml of H₂:CO₂ in exchange for 1 ml of N₂:CO₂ to produce an estimated aqueous H₂ concentration of 20 mM at their incubation temperatures. In 2012 and 2013, the remaining four serum bottles (set D) were amended with 4.7 mM NH₄Cl (2012 only) or 47 mM NH₄Cl (2013 only) and flushed and filled with 200 kPa of H₂:CO₂ to test for growth stimulation by ammonium. The NH₄Cl concentration was based on that added to our defined methanogen growth medium (see below). In 2014 and 2015, the remaining four serum bottles (set E) were amended with 0.5% (wt vol^-1) tryptone plus 0.01% (wt vol^-1) yeast extract and flushed and filled with 200 kPa of N₂:CO₂ to test for H₂ syntrophy. All samples were reduced with 0.025% (wt vol^-1) each of cysteine-HCl and Na₂S•9H₂O. Growth of methanogens was determined by analyzing for CH₄ in the headspace using gas chromatography once the cells in the bottle had reached stationary growth phase. In 2015, an aliquot of the 80°C and 55°C tryptone/no H₂ samples (set E) that showed CH₄ production were filtered onto 0.2-µm pore size nucleopore filters prestained with Irgalan black (Sterlitech, Kent, WA, USA), stained with acridine orange, and examined using epifluorescence microscopy. In 2015, the 80°C and 55°C tryptone/no H₂ samples from the Marker 113 vent site were also separately filtered through Sterivex GP 0.22 µm sterile filter units (Millipore, Billerica, MA, USA) and frozen at -80°C until analyzed. In2015, 10 ml of hydrothermal fluid was added to sealed Balch tubes without exposure to air, amended separately with 0.1% (wt vol^-1) sodium formate and 0.5% (wt vol^-1) sodium acetate, flushed and filled with 200 kPa N₂:CO₂, and incubated in duplicate at 80°C and55°C for up to seven days to determine if these substrates can support methanogenesis at high temperatures.

Total cell counts in the original hydrothermal fluids were done by preserving in duplicate 18 ml of hydrothermal fluid with 1.8 ml of 37% formaldehyde. Samples were stored at 4°C for less than a month prior to counting by epifluorescence microscopy as described above.

Full methodology