Dataset: In situ pCO2 manipulation experiments 2010-2011
Deployment: KM1016

Rates of primary production were assessed using the 14C-bicarbonate incorporation technique. Rates of bacterial production were assessed using incorporation of 3H-leucine. Whole seawater samples from six discrete depths (5, 25, 45, 75, 100, and 125 m) were collected into duplicate acid-washed 20 L carboys. Control carboys were unamended; 43 mL of 1.0 N HCl and 4 mmol sodium bicarbonate were added to a treatment carboy at each depth, to increase the pCO2 to ~750 µatm, while minimizing changes to total alkalinity. Water from control and treatment carboys were then each subsampled into acid washed 500 mL polycarbonate bottles, with triplicate bottles per depth and treatment. To each bottle, was then added ~1.85 MBq 14C-bicarbonate. Water from each depth and treatment was also added to acid-cleaned 40 mL polycarbonate centrifuge tubes, each tube was then inoculated with 3H-leucine to a final concentration of 20 nmol L-1. For each depth and treatment, there was a dark (in a opaque cloth bag) and light incubation. Time zero blanks were immediately subsampled from each tube, by aliquoting 1.5 mL of seawater into 2 mL microcentrifuge tubes each containing 100 µL of 100% TCA. Following addition of radioactive substrates, the bottles and tubes were affixed to a free-drifting array and incubated in situ at the original depth of sample collection from dawn to dusk.

Upon recovery of the array, the total radioactivity added to each primary production sample bottle was determined by subsampling 250 µL aliquots of seawater into scintillation vials containing 500 µL of β-phenylethylamine. 400 mL from each 500 mL sample bottle was filtered at low vacuum (<50 mm Hg) onto 25 mm diameter, 10 µm porosity polycarbonate membrane filters. The filtrate was collected and filtered onto 25 mm diameter 2 µm porosity polycarbonate membrane filters. 100 mL of that filtrate was then filtered onto 25 mm diameter 0.2 µm porosity polycarbonate membrane filters.  Filters were stored frozen in 20 mL scintillation vials until analysis. Analysis consisted of acidification via addition of 1 mL of 2 N hydrochloric acid, and passively venting at least 24 hours in a fume hood to remove all inorganic 14C. Addition of 10 mL Ultima Gold LLT liquid scintillation cocktail and counting on a Perkin Elmer 2600 liquid scintillation counter completed the primary production analysis.

Upon recovery of the array, triplicate 1.5 mL subsamples were removed from each polycarbonate tube for bacterial production rate measurements, and aliquoted into 2 mL microcentrifuge tubes containing 100 µL of 100% TCA. The microcentrifuge tubes were frozen (-20°C) for subsequent processing, following the procedures described in Smith and Azam 1992.

Samples for the determination of dissolved inorganic carbon and total alkalinity were collected from each carboy and analyzed according to the protocols of the Hawaii Ocean Time-series (Dore et al. 2009; Winn et al. 1998). DIC and TA samples were collected into precombusted 300 mL borosilicate bottles. Care was taken to avoid introduction of air bubbles into samples during filling; bottles were allowed to overflow three times during filling. Once filled, samples were immediately fixed with 100 µL of a saturated solution of mercuric chloride; bottles were capped with a grease seal, and stored in the dark for later analysis.

Samples for measurement of fluorometric chlorophyll a were collected according to the protocols of the Hawaii Ocean Time-series; analysis was performed following Letelier et al. (1996).