Physiological Methods

Photosynthesis, respiration, and calcification measurements were performed on each fragment using 300 mL temperature-controlled respirometry chambers filled with seawater from the treatment aquaria that was continuously stirred with a magnetic stir bar.  The chambers were used to assess the rates of respiration (Rd) in the dark and rates of photosynthesis (Pn) and calcification in the light.  Light was supplied by a series of blue and red LEDs with adjustable intensity (150 uMol quanta m-2 sec-1).  Water samples were taken from each chamber prior to a cycle and also at the end of both dark and light incubations (60 minutes each) for measurements of pHT (pH on the total scale) and total alkalinity (AT) as described in Martin and Gattuso (2009).  

Calcification - Total alkalinity (TA) values were measured using an automatic potentiometric titrator (Metrohm 807 Titrando, Riverview, FL) to the second end point of a 15.3-g accurately weighed seawater sample.  Total alkalinity values were then computed using the Gran equation (DOE, 1994) with pH values lower than 3.9 for creating the Gran plot. The pH electrodes (Metrohm 807 Titrando) were calibrated daily as described above.  The acid titrant concentration was 0.05N HCl (JT Baker, Phillipsburg, NJ).  Alkalinity was calculated using the first derivative of the curve for the evaluation of the exact end point.  Standards for total seawater alkalinity and provided by Dickson were run daily (Dickson, 2007).  The differences between duplicate samples and standards were less than 5 uEq kg-1 (for calibration of the titrator, differences were measured between triplicate samples).  Water samples were analyzed immediately or stored in darkness at 4C and processed within 24 hours of collection.  

Dickson AG, Sabine CL, and Christian JR (2007) Guide to best practices for ocean CO2 measurements: PICES Special Publication. 3, 191 p.

Martin S and Gattuso J-P (2009) Response of Mediterranean coralline algae to ocean acidification and elevated temperature. Glob Change Biol 15:2089-2100. 
Marubini F and Thake B (1999) Bicarbonate addition promotes coral growth. Limnol and Oceanogr 44: 716-720.

Riebesell U, Fabry VJ, Hansson L, and Gattuso JP (2010) Guide to best practices for ocean acidification research and data reporting. European Commission, European Research Area. Brussels. 258 p