Dataset: Chemoautotrophic assimilation of inorganic carbon within the ETNP OMZ R/V Atlantis AT50-08 from the Eastern Tropical Pacific Minimum Oxygen Zone from February to March 2023 (MicroPro project)

1L whole water samples were collected on deck under nitrogen pressure at the rosette. A three-volume overflow was performed for each sample to minimize oxygen contamination. Bottles were sealed with deoxygenated butyl rubber septa after confirming all bubbles were excluded. A He gas headspace was added in the shipboard lab no more than 15 minutes after collection. To quantify community chemoautotrophic activity, triplicate incubations with ¹⁴C-bicarbonate were prepared in 16 mL Hungate tubes with degassed butyl rubber septa under nitrogen pressure. Chilled N2-purged ¹⁴C-bicarbonate in an alkaline brine (pH 9.5; S = 60 on the practical salinity scale) was injected (0.92 µCi Mex1, 0.54 µCi Mex2) into the bottom before sealing (Tuttle and Jannasch 1973a). One depth per cast was selected for stimulation experiments with alternative electron donors. Selected incubations were spiked with ¹⁴C-bicarbonate and one of the following: NH₄⁺, DMSP, H₂S or S₂O₃. Incubations were conducted in a dark ice chest for 18-24 hours and terminated by filtration onto 25 mm nitrocellulose 0.22 µm filters (25 mm diameter Osmonics). Selected non-amended incubations were size fractionated into a “particle-associated” fraction (>2.7 µm) and a free-living size fraction (0.22 – 2.7 µm) by sequential filtration through stacked 25 mm Swinnex filter holders. Unassimilated ¹⁴C-bicarbonate was purged from the filters in a saturated HCl atmosphere enclosure for >1h in a fume hood. Filters were then dried, suspended in scintillation cocktail (Hionic-Fluor) and radioassayed. Table 1 summarizes sample types. Data were corrected for isotopic fractionation (multiplied by 1.06) and for nonbiological sorption by use of control samples processed immediately after introduction of the radiotracer (T₀ samples). If blanks were higher than T₀ samples, blanks were used instead. Three T₀ sample sets were made, one for standard+stimulation experiments, one for the free-living size fraction and one for the particle-associated size fraction. Rates of dark ¹⁴C-assimilation were normalized to total nanomoles of carbon per day via equation detailed under 'Data Processing' section.

Sample code	Tracer/Amendments	Experiment type	Notes
standard	14C-bicarbonate	Standard
S2O3	14C-bicarbonate and S2O3	Alternative electron donor amendment	Target stimulant concentration: 10uM. For all samples prepared Feb.19 or later, target concentration is 5uM
DMSP	14C-bicarbonate and DMSP	Alternative electron donor amendment	Target stimulant concentration: 100nM. For all samples prepared Feb.19 or later, target concentration is 50nM
NH4+	14C-bicarbonate and NH4+	Alternative electron donor amendment	Target stimulant concentration: 10uM. For all samples prepared Feb.19 or later, target concentration is 5uM
H2S	14C-bicarbonate and H2S	Alternative electron donor amendment	Target stimulant concentration: 10uM. For all samples prepared Feb.19 or later, target concentration is 5uM
0.2_SF	14C-bicarbonate	Size fractionation
2.7_SF	14C-bicarbonate	Size fractionation