Experiment Description:
The experiment was conducted onboard the RV Seward Johnson II, from June 20 to July 4, 2005, with water collected at 57° 58’ N, 15° 32’W. Four treatments were used with 6 replicates each: (1) 12°C and 390 ppm CO₂ (LTLC), (2) 12°C and 690 ppm CO₂ (LTHC), (3) 16°C and 390 ppm CO₂ (HTLC), and (4) 16°C and 690 ppm CO₂(HTHC). Sea surface temperature at this location was 12°C at the time of water collection. Experiments were run using a seawater continuous culture system, termed an ‘Ecostat’ (Hutchins et al. 2003, Hare et al. 2005, 2007). Briefly, whole seawater was collected from 5 to 10 m depth using a trace-metal-clean, towed-intake Teflon pump system (Hutchins et al. 2003), prefiltered through 200 μm Nitex mesh to remove mesozooplankton and incubated in twenty-four 2.7 l trace-metal-clean, clear polycarbonate bottles. Bottles were placed in racks in a temperature-controlled deck incubator with recirculating water and shaded to 30 percent of surface irradiance (I0) using a neutral-density shade screen. Temperatures in the 16°C incubator were gradually increased over a period of 24 h to avoid heat-shocking the plankton. Bottles were bubbled with either air or a commercially prepared air/CO2 mixture with 750 ppm CO2 using an inflow tube through the cap and an airstone to maximize gas transfer to the liquid phase. The gases used for bubbling were filtered through a 0.2 μm HEPA filter to avoid contamination of experimental bottles by trace metals (Hare et al. 2005). The system was run in batch mode for 3 days prior to turning on the pumps, in order to stimulate phytoplankton growth and prevent wash-out of slower growing species. After this batch growth period, whole seawater in each incubation bottle was slowly diluted at a continuous rate using seawater collected at the initial site. This seawater medium was filtered through a 0.2 μm inline capsule filter initially, then re-filtered through a second 0.2 μm inline capsule filter immediately prior to use as a diluent. The medium was stored in trace-metal-clean, 50 l carboys in the dark. Initial in situ nutrient concentrations were low (0.32 umol nitrate l^–1, 0.12 umol phosphate l^–1, 0.7 umol silicate l^–1), so the medium and the whole water in the incubation bottles were amended with 5 and 0.31 umol l^–1 (final concentration) of nitrate and phosphate. The dilution rate of 0.5 d^–1 was controlled in each incubation bottle using a peristaltic pump and calibrated daily to ensure constant flow rate. This flow rate constituted a 50 percent dilution of the experimental bottle volume daily. Incubation bottles were mixed by inverting the rack 120° every 5 to 15 min using a compressed-air-driven system. Diluted seawater flowed out of the incubation bottles at a continuous rate and into 2.7 l polycarbonate bottles stored in the dark, which were used as outflow collection vessels. Seawater carbonate system measurements were performed as described in Feng et al. (2009).

References:
Feng, Y., C.E. Hare, K. Leblanc, G.R. DiTullio, P.A. Lee, S.W. Wilhelm, J. Sun, J.M. Rose, N. Nemcek, I. Benner, and D.A. Hutchins. 2009. The effects of increased pCO2 and temperature on the North Atlantic Spring Bloom: I. The phytoplankton community and biogeochemical response. Marine Ecology Progress Series 388: 13-25.

Hare, C.E., G.R. DiTullio, C.G. Trick, S.W. Wilhelm, K.W. Bruland, E.L. Rue, and D.A. Hutchins. 2005. Phytoplankton community structure changes following simulated upwelled iron inputs in the Peru upwelling region. Aquatic Microbial Ecology 38: 269-282.

Hare, C.E., K. Leblanc, G.R. DiTullio, R.M. Kudela, Y. Zhang, P.A. Lee, S.F. Riseman, and D.A. Hutchins. 2007. Consequences of increased temperature and CO2 for phytoplankton community structure in the Bering Sea. Marine Ecology Progress Series 352: 9-16.

Hutchins, D.A., F. Pustizzi, C.E. Hare, and G.R. DiTullio. 2003. A shipboard natural community continuous culture system for ecologically relevant low-level nutrient enrichment experiments. Limnology and Oceanography: Methods 1: 82-91.

Related files and references:
Rose, J.M., Y. Feng, C.J. Gobler, R. Gutierrez, C.E. Hare, K. Leblanc, and D.A. Hutchins. 2009. The effects of increased pCO2 and temperature on the North Atlantic Spring Bloom. II. Microzooplankton abundance and grazing. Marine Ecology Progress Series 388: 27-40.

Additional parameters measured during these experiments are described in: Feng, Y., C.E. Hare, K. Leblanc, G.R. DiTullio, P.A. Lee, S.W. Wilhelm, J. Sun, J.M. Rose, N. Nemcek, I. Benner, and D.A. Hutchins. 2009. The effects of increased pCO2 and temperature on the North Atlantic Spring Bloom: I. The phytoplankton community and biogeochemical response. Marine Ecology Progress Series 388: 13-25.

Lee, P.A., J.R. Rudisill, A.R. Neeley, D.A. Hutchins, Y. Feng, C.E. Hare, K. Leblanc, J.M. Rose, S.W. Wilhelm, J.M. Rowe, and G.R. DiTullio. 2009. The effects of increased pCO2 and temperature on the North Atlantic Spring Bloom: III. Dimethylsulfoniopropionate. Marine Ecology Progress Series 388: 41-49.