Technical Comments

Response to Comment on "Phytoplankton Calcification in a High-CO2 World"

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Science  05 Dec 2008:
Vol. 322, Issue 5907, pp. 1466
DOI: 10.1126/science.1161501

Abstract

Recently reported increasing calcification rates and primary productivity in the coccolithophore Emiliania huxleyi were obtained by equilibrating seawater with mixtures of carbon dioxide in air. The noted discrepancy with previously reported decreasing calcification is likely due to the previously less realistic simulation of bicarbonate due to addition of acid or base to obtain simulated future CO2 partial pressure conditions.

Riebesell et al. (1) highlight our report of increased calcification and productivity of the coccolithofore Emiliania huxleyi at high CO2 partial pressures (Pco2) (2), discuss discrepancies between our data (2, 3) and those of others (4, 5) and speculate on the shortcomings of our experimental protocol. However, Riebesell et al. ignore the problem we noted (2) in the design of previous experiments (3): adding acid or base when aiming for simulation of the future high-CO2 ocean. Here, we address their critiques (1) and provide a further analysis of what we believe is the central issue of this debate.

The first issue raised by Riebesell et al. (1) concerns potential drifts in the carbonate system caused by differences in cell concentrations. The nature of these drifts is undocumented experimentally, and it is unclear how this would change the main conclusion of our work (2). We followed established semicontinuous culturing protocols and maintained cells at densities several-fold lower than those reported in recent studies specifically measuring the effect of carbonate chemistry on E. huxleyi physiology [e.g., (6)]. More important, the cells were not nutrient-limited and were growing exponentially (i.e., not exhibiting lag-phase), and therefore we expect a negligible effect, if any, on the observed responses.

Regarding the issue of potential nutrient limitation in precultures, nutrient stress in our cultures is not supported by our observations: Cells had high growth rates (0.53 to 0.76 d–1), and the minimum ambient nutrient concentrations during measurements (35 to 84 μM nitrate and 2 to 5 μM phosphate) (7) were at least an order of magnitude higher than reported limiting values (8). Even using the lowest N:P ratios reported for blooms (N:P <10) (9), the nitrate and phosphate availabilities (35 μM and 0.9 μM, respectively) in our cultures are well above the measured cell quotas in E. huxleyi under N- or P-limitation (8). When E. huxleyi cells were grown with ambient N:P = 1:1 (N-limited) and 300:1 (P-limited), the cellular N:P ratios varied between 12:1 and 41:1 (8). Applying the lowest cellular ratios, our experiments are nutrient-replete. A problem with Riebesell et al.'s argument (1) is their suggestion that the observed increase in cell size was a result of nutrient limitation. This is not supported by evidence showing that E. huxleyi cell size decreases under nitrate limitation (10) and increasing Pco2 (11), whereas phosphate limitation induces an increase in size (10).

Riebesell et al. also point out the potential for non–steady-state growth in our preconditioned cells. We monitored the growth of semicontinuous cultures of exponentially growing nutrient-replete cells for nine generations, bubbled with the constant air-CO2 mixtures, before harvesting was conducted during the final subculturing. Impacts of changes in the ocean carbon chemistry on physiology and cell size should, however, not be surprising because cell size is sensitive to many factors impacting on phytoplankton physiology, often within one or two cell divisions (12).

Riebesell et al. further question the value of expressing physiological rates on a per cell basis. We contend that any understanding of cellular responses requires cell-specific measurements and that this is key to understanding the eco-physiological responses of calcifying phytoplankton to increasing Pco2. It also allows our results to be compared with the rich historical primary literature [e.g., (3)]. Biogeochemically, the biomass-normalized rate is most likely more valuable, as discussed in (2), showing that the range of Pco2 enhances both photosynthesis and calcification.

Experiments conducted at various dissolved inorganic carbon (DIC) conditions, that is, different relative proportions of CO2, bicarbonate (HCO3), and carbonate ions (CO32–) (35, 13) are, in principle, valid for unraveling the physiology of E. huxleyi. However, great care must be taken when extrapolating these results to E. huxleyi performance in the future high-CO2 ocean. In these experiments, the manipulation of the carbonate system is central to the data interpretation. Because of the long residence time of alkalinity in the ocean, we can assume constant alkalinity until the end of the century (14, 15). The increasing atmospheric Pco2 will yield an increase of total DIC, CO2(aqueous), and HCO3, a decrease of CO32– and pH: ocean acidification. Upon adding acid/base to seawater to mimic future high Pco2/past ocean, one inevitably causes a decrease/increase in alkalinity and a lesser increase/decrease of bicarbonate as compared to their predicted stability and stronger increase/decrease, respectively, in the future high-CO2/past ocean (Fig. 1). Thus, from comparing the initial conditions (Fig. 1) of the simulation by bubbling CO2 in air (2) with the previous acid/base perturbations (3), one realizes that CO2 bubbling correctly mimics the constant alkalinity accompanied by major changes in bicarbonate ion of the future high-CO2 ocean. In contrast, the acid/base treatment (3) shows major perturbation of alkalinity and too small changes of bicarbonate ions. Any conceivable relationship (2, 13, 16) between the rate of calcification and bicarbonate ions in the future high-CO2 ocean will be obscured by the acid/base treatment (3).

Fig. 1.

Upon CO2 in air bubbling (2) of seawater, the initial alkalinity [(ALK) 10–6 mol L–1] (filled circles) remains constant, and the initial bicarbonate [10–6 mol L–1] (filled triangles) changes strongly versus the desired Pco2 [10–6 atm] to best simulate the corresponding initial conditions of a bloom of E. huxleyi in the future high-CO2 ocean. In contrast, by acid/base manipulation (3), the initial alkalinity (open circles) changes strongly, whereas the initial bicarbonate has a far lesser change versus the desired Pco2 than the predicted (14, 15) future high-CO2 ocean. For latter acid/base treatments, the initial values of alkalinity and dissolved inorganic carbon were provided by U. Riebesell, from which the initial bicarbonate values were calculated. Initial conditions are for the subsequent growth experiments at light intensity of 150 10–6 mol m–2 s–1 and T = 19°C and T = 15°C after (2) and after figure 2 in (3), respectively.

Finally, Riebesell et al. (1) encourage efforts to better understand the response of marine organisms to ocean acidification, as do we. Contrary to their assertion of the methodological shortcomings of our study, we have justified our reasoning and approach and demonstrated that a previous report (3) has confounded the issue by using an approach (acid/base manipulation of seawater) that is not appropriate for predicting the calcification response of E. huxleyi in a future high-CO2 ocean.

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