Initial pCO2 incubation experiment

A mixed natural dinoflagellate bloom dominated by Lingulodinium polyedrum, Prorocentrum micans, Alexandrium sp. and Gonyaulax sp. at a total cell density of ~700 cells per ml was collected off Venice Beach, California in September, 2009. This large regional bloom extended throughout the Southern California Bight region. Samples were collected near shore for both the initial incubations and all experimental dilution water used throughout the 12-month experiment.

The experiment was incubated in the laboratory at 18 degrees C under 90 photons per meters squared per second of cool white fluorescent illumination on a 14-h light: 10-h dark cycle. Triplicate sterilized 1 L polycarbonate bottles were gently bubbled (60 bubbles per minute) using commercially prepared air/CO2 mixtures (230 ppm, 433 ppm and 765 ppm, Praxair Gas). Preliminary experiments verified that growth rates of cultures bubbled at this rate were not significantly different from those of unbubbled cultures (data not shown), and these methods have been employed for other CO2 experiments (Hutchins et al. 2007; Fu et al. 2007), including dinoflagellate studies (Fu et al. 2008; 2010). Filtered seawater was amended with L1/20 nutrient, vitamin and trace metal concentrations (Guillard and Hargraves 1993), except NH4Cl+ was substituted for NaNO3- and silicate was omitted. Nutrient concentrations at the Redfield ratio (by atoms) of 16 N: 1 P (Redfield 1958) were added initially to the incubation bottles, and replenished once at the one week dilution (below).

The CO2-amended treatments were maintained in active growth using semi-continuous culture methods (Tatters et al. 2012). Each bottle was diluted to the original time-zero in vivo chlorophyll a fluorescence value after one week with nutrient-amended filtered seawater. Aliquots were removed initially, and after one and two weeks for examination of carbonate buffer system parameters and community structure using microscopic cell counts. Samples for cell counts were obtained at the 1 week time point (after dilution), and after the 2 week incubation, in order to calculate acclimated growth rates (1-2 week rates) and final abundances of all species.

Clonal culture isolations

Three individual cells representing the four dominant genera were isolated from each incubation bottle at the end of the two-week incubation of the natural community, and maintained in long-term culture (52 weeks) at the pCO2 from which they were obtained under conditions of temperature, light, nutrients, CO2 bubbling, etc., identical to the 2-week natural community experiment. Cultures were maintained in exponential phase using autoclave-sterilized enriched seawater growth medium with semi-continuous weekly dilutions based on specific growth rates within each bottle, calculated as in Tatters et al. (2012). The approximate number of generations during this time period was: L. polyedrum (48-62), P. micans (58-71), Alexandrium sp. (34-38) and Gonyaulax sp. (75-126).

Artificial community competition experiments

The conditioned clonal cultures were recombined into artificial communities after 4, 8 and 12 months in the same relative proportions and cell densities as the original natural bloom assemblage. The 8- and 12-month experiments used triplicate communities of all four species, but due to logistical limitations, the 4-month experiment used only L. polyedrum, P. micans, and Alexandrium sp. in duplicate communities. Because Gonyaulax sp. was not included in this preliminary 4-month experiment and replication was different, it is not fully comparable to the other experiments. The dinoflagellates in the artificial community trials were allowed to compete under identical experimental conditions of light, temperature, nutrient availability, and pCO2 for the same time period and diluted exactly as in the original natural bloom incubation. Samples were collected for cell counts and carbonate system parameters (Table 1) in all experiments.

The initial bloom counts, per mL, are as follows:

Lingulodinium: 464
Prorocentrum: 50.5
Alexandrium: 20.5
Gonyaulax: 27

Three samples (A, B, C) were taken for each partial pressure of CO2. The partial pressures of CO2 were targeted at 190, 390, and 750 ppm but the biology can interfere a bit with the actual values achieved.

Literature Cited

Fu, F.-X., M. E. Warner, Y. Zhang, Y. Feng, and D. A. Hutchins. 2007. Effects of increased temperature and CO2 on photosynthesis, growth and elemental ratios of marine Synechococcus and Prochlorococcus (Cyanobacteria). J. Phycol. 43:485-496.

Fu, F.-X., A. R. Place, N. S. Garcia, and D. A. Hutchins. 2010. CO2 and phosphate availability control the toxicity of the harmful bloom dinoflagellate Karlodinium veneficum. Aquat. Mcrob. Ecol. 59: 55-65.

Fu, F.-X., Y. Zhang, M. E. Warner, Y. Feng, and D. A. Hutchins. 2008. A comparison of future increased CO2 and temperature effects on sympatric Heterosigma akashiwo and Prorocentrum minimum. Harmful Algae. 7:76-90

Hutchins, D. A., F.-X. Fu, Y. Zhang, M. E. Warner, Y. Feng, K. Portune, P. W. Bernhardt, and M. R. Mulholland. 2007. CO2 control of Trichodesmium N2 fixation, photosynthesis, growth rates, and elemental ratios: Implications for past, present and future ocean biogeochemistry. Limnol. Oceanogr. 52:1293-1304.

Redfield, A. C. 1958. The biological control of chemical factors in the environment. Am. Sci. 46:205-222.

Tatters, A. O., F.-X. Fu, and D. A. Hutchins. 2012. High CO2 and silicate limitation synergistically increase the toxicity of Pseudonitzschia fraudulenta. PLoS ONE 7(2):e32116.doi:10.1371/journal.pone.0032116.