Dataset: Experimental pCO2 manipulation experiments 2010-2012
Deployment: KM1015

Rates of primary production were assessed using the 14C-bicarbonate incorporation technique. Rates of bacterial production were assessed using incorporation of 3H-leucine. Whole near-surface seawater was collected into acid-washed 20 L carboys. Control carboys were bubbled with air; treatment carboys were bubbled with a mixture of air and CO2, to increase the pCO2 to either ~750 or 1100 µatm. Sampling of each time point was conducted before dawn, experiments lasted between 2 and 5 days. Water from each carboy was subsampled into acid washed 500 mL polycarbonate bottles for primary production rate measurements. To each bottle, was then added ~1.85 MBq 14C-bicarbonate. Water from each carboy was also collected in an opaque polyethylene amber bottles and then subsampled into six 1.5 mL microcentrifuge tubes for bacterial production rate measurements. Each tube was then inoculated with 3H-leucine to a final concentration of 20 nmol L-1. Three tubes from each carboy were incubated in the dark (in a opaque cloth bag) and three in the light. Time zero blanks were immediately subsampled from each amber bottle, 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 primary production bottles and bacterial production tubes were placed in shaded (~50% irradiance) surface seawater-cooled incubators for the duration of the photoperiod.

After sunset, 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. 100 mL from each 500 mL sample bottle was filtered at low vacuum (<50 mm Hg) onto 25 mm diameter, 0.2 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.

After sunset, 100 µL of 100% TCA was added to each microcentrifuge tube. 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).