During Alvin Dive 4905, May 8, 2017, 5 major samples were collected at the diffuse-flow vent Crab Spa. The fluid from these samplers was used in an on-deck Vent-SID incubation. The purpose of the incubation was to simulate a sea-floor incubation of the Vent-SID. Upon arrival of majors on the ship, we transferred fluid from the majors into N2 flushed 1L Restek bags. The bags were stored at 4˚C and taken out as needed for setting up a new Vent-SID incubation. In total, six incubations were conducted, all at 25(+/-2)˚C.

Microbial cell counts: Samples for cell numbers were fixed with formaldehyde (1% final concentration) and then counted on the board the ship after staining with acridine orange by fluorescence microscopy as described in McNichol et al. (2016).

Carbon fixation rates: 13C-labeled bicarbonate was added to the incubation chambers to assess chemoautotrophic production. At the beginning and the end of the incubation, fluids were filtered onto pre combusted GFF filters, which were frozen until analysis back in the shore lab. Gas chromatography combustion (Fisons 1108 Elemental Analyzer equipped with a Costech "Zero Blank" sample carousel) coupled to an isotope ratio mass spectrometer (GC-IRMS) (Finnigan-MAT Conflo-II interface attached to a DeltaPlus Isotope Ratio Mass Spectrometer) was used to measure the incorporation of 13C-labelled bicarbonate into biomass during the incubations to determine chemoautotrophic production.

Nitrate concentrations:  Vent fluid from the Crab Spa site was incubated on the deck of the ship in the Vent-SID reaction chamber, in the dark at a temperature of 25(+/- 2)°C. Incubations consisted of a vent fluid with added NO3- (10 µmol/L), NO2- (1 µmol/L) and H13CO3- (0.7 mmol/L) and time-point samples were taken for nitrate+nitrite measurements at 0, 3, and 6 hours. Six incubations were conducted that varied in combinations of 15NO3-/14NO3 or 15NO2-/14NO2-, keeping the total concentration of added NO3- and NO2- constant across incubations. 

Nitrate concentrations were measured in time-point samples utilizing the vanadium(III) reduction of nitrate and nitrite method described by Braman and Hendrix, 1989. Samples were injected into a heated Vanadium acid solution whereby NO3- and NO2- were reduced to NOx gases. The NOx then passed into a Teledyne T200 NOx analyzer where a photodetector measured the light produced from the chemiluminescent reaction of NOx and instrument generated ozone.

After initial collection, vent fluid samples were stored in 15 mL conical tubes at -20C until analyzed on the Teledyne NOx analyzer. Once thawed, samples were injected using Hamilton 1700 series gastight syringes and Nitrate concentrations were calculated alongside standards generated from a Ricca Nitrate Nitrogen Standard (CAT#5459-16, 1000 ppm N, 4427 ppm NO3). The series of standards ranged from 1 uM to 50 uM and were made up by diluting the Ricca standard in Milli-Q water.

Peak Simple Version 4.49 was used to generate chromatographs from the photodetector and raw data was further processed in Microsoft Excel.