The coccolithophores are microalgae belonging to the class Prymesiophyceae in the division Haptophyta and the most common species of coccolithophore globally is Emiliania huxleyi. Uniquely, the coccolithophores synthesize calcium carbonate scales (coccoliths) within vesicles in the cell, which are then extruded to the cell surface. Emiliania huxleyi frequently forms huge blooms (up to 8 million km2; Moore et al., 2012) in coastal and open marine systems and coccolithophores account for c. 50% of the global ocean calcium carbonate production and export to the deep ocean. In view of the observed and predicted future drop in oceanic pH (ocean acidification) that is a consequence of increasing atmospheric CO2 levels, there is a great deal of interest, but conflicting reports in the literature (Riebesell et al., 2000; Iglesias-Rodriguez et al., 2008), about the consequences of ocean acidification for the growth and calcification of this important group of microalgae. The paper by Bach et al., in this issue of New Phytologist (pp. 121–134) presents a series of elegant experiments, using a variety of approaches, that makes significant headway towards answering these questions.
‘ … the presence of a CCM suggests that E. huxleyi growth will not be stimulated by elevated CO2 as global climate change progresses …’
A number of biological functions for coccoliths have been proposed (see Raven & Crawfurd, 2012) and include ballasting, protection against viruses and grazers and photoprotection. One additional function/consequence of calcification is the generation of H+, which can drive the intracellular formation of CO2 (Eqn )
This CO2 production from calcification has been thought by some to act as a form of CO2 concentrating mechanism (CCM; see Reinfelder, 2011 for a recent review). The central enzyme of CO2 assimilation, ribulose bis-phosphate carboxylase oxygenase (Rubisco) in microalgae shows a relatively low affinity for CO2 and consequently would be severely constrained were cells to rely on diffusive entry of CO2 at present day levels. However, most microalgae and cyanobacteria have evolved mechanisms, based on active transport of CO2 and/or , that accumulate CO2 at the active site of Rubisco thereby increasing rates of carbon fixation (Giordano et al., 2005).
There has been considerable debate in the literature about whether E. huxleyi and other coccolithophores possess a CCM. Early work suggested that CCMs were absent in E. huxleyi (Nimer et al., 1992), but recent studies suggest that this organism does possess active CCMs (Rost et al., 2007; Reinfelder, 2011). The occurrence of a CCM is supported by data on the in vitro CO2 half-saturation value of Rubisco (Shiraiwa et al., 2004; Boller et al., 2011) and the Rubisco content (Losh et al., 2013; Raven, 2013) of E. huxleyi. The Rubisco half-saturation value of at least 70 mmol m−3 is at least five times the half-saturation value for CO2 of intact E. huxleyi cells grown in air-equilibrated seawater (Bach et al.), while the CO2 saturated Rubisco activity is just sufficient to account for the rate of light-saturated photosynthesis in seawater (Losh et al., 2013). At least five times as much Rubisco per E. huxleyi cell would be needed to explain the in vitro CO2 affinity in terms of diffusive entry of CO2 and setting of the in vivo maximum photosynthetic rate by the capacity of the thylakoid reactions needed to regenerate ribulose-1,5-bisphosphate, a co-substrate of Rubisco. However, due to high rates of leakage of CO2 from the cell, the efficiency of CCMs in this group is thought to be very low (Rost et al., 2006). Recent work from our laboratory indicates a highly active CCM in cultures of Southern Hemisphere strains of E. huxleyi (Stojkovic et al., 2013) and in another coccolithophore Gephryocapsa oceanica (Larsen, 2013). Nonetheless the complexities of inorganic carbon use (CO2 or or both) and the potential role of calcification in supplying CO2 internally have made resolving the details of these processes and likely responses to ocean acidification elusive.
Future global change predictions are for a rise in CO2 from the current 394 ppm to 1000 ppm (corresponding to dissolved CO2 in the oceans at 15°C of 39 mmol m−3, Beardall et al. (1998) by the end of this century. Concomitant with this is a predicted drop in oceanic pH from 8.1 (8.2 in pre-industrial times) to 7.7 which will mean that dissolved inorganic carbon (DIC) equilibria (Eqn ) will be shifted, such that concentrations will approximately halve and concentrations increase by < 10%, despite the much larger increase in CO2 levels.
Bach et al. address a number of these critical issues in carbon acquisition and usage in E. huxleyi, such as whether or not E. huxleyi has a CCM, whether CO2 or pH per se influence growth and calcification under ocean acidification, and whether photosynthesis and calcification are inextricably linked.
Thus by changing pH, CO2 and total DIC concentrations in a multifactorial experimental design, the authors were able to separate effects of CO2 and concentrations and pH on growth rates, organic matter production through photosynthesis and on calcification. To tie all of this together, the authors also carried out quantitative reverse-transcriptase PCR (qRT-PCR) on 15 target genes related to carbon acquisition (various carbonic anhydrases, Rubisco, anion exchange like transporter), Ca2+ transporters and H+ pumps (see table 1 of Bach et al. for the list of genes investigated).
In essence the experiments reported by Bach et al. indicate that many processes such as organic carbon production rates are decreased at CO2 and bicarbonate concentrations below thresholds of 10 mmol m−3 and 2000 mmol m−3, respectively, and concentrations above this had little impact. Perhaps not surprisingly, lower CO2 concentrations also resulted in lower carbon/nitrogen (C:N) ratios in the cells. Only pH values below 7.74 also reduced particulate organic carbon (POC) and growth rates, indicating that these processes were relatively insensitive to pH.
Calcification rates were strongly inhibited at concentrations below present day values but showed no effect of higher concentration and were independent of CO2 or concentrations, reinforcing the current understanding that calcification is supported by transport. When pH was low (7.74), higher concentrations of were needed to support calcification than at higher pH (8.34), suggesting that providing sufficient is available, calcification may continue, despite the impacts of high H+ (low pH) reported by Bach et al. (2011). Interestingly, at lower DIC levels, POC production decreased less than did particulate inorganic carbon production (PIC; i.e. calcification). This perhaps ties in well with observations by Stojkovic et al. (2013) that, in calcifying strains of E. huxleyi, CO2 is the main source of carbon for photosynthesis, but in noncalcifying strains more is available to participate in carbon fixation into organic matter. Bach et al. report that as calcification rates dropped at low DIC, so were genes associated with CCMs up-regulated, strongly supporting the idea that calcification does not play a role as a CCM. No doubt calcification can produce CO2, which may be then used in photosynthesis, but this is not an obligate link and at low DIC levels cells invest in CCMs to support organic carbon production and growth at the expense of calcification. Interestingly, CCM related genes were only up-regulated at DIC values below 1000 mmol m−3, that is, half present day air equilibrium levels. This induction of CCM activity at low DIC was related to CO2 and concentrations but did not respond directly to pH.
What does all this mean to the functioning of coccolithophores such as E. huxleyi in the real world and under increasing ocean acidification in a higher CO2 ocean? This paper by Bach et al. clearly establishes a number of relevant features of the biology of E. huxleyi. First, they provide additional evidence for a CCM and show convincingly that this is not driven by calcification (which does not rule out use in photosynthesis of CO2 produced by calcification, simply that there is no obligatory role for this process in coccolithophore CCMs). Second, the work suggests that under low DIC levels, is preferentially channeled into production of organic matter rather than into calcification but that rising CO2 and bicarbonate above present day levels does not affect PIC levels at either pH 7.74 or pH 8.34 and third, the authors show that induction of genes associated with calcification, CCMs and other physiological processes are responsive only to CO2 and concentrations well below those of the present day and what might be expected by the end of the century. Induction of CCMs at low DIC could still be relevant as E. huxleyi can form extensive and very dense blooms in which the available CO2 can drop to half air equilibrium values or less. However, the presence of a CCM suggests that E. huxleyi growth will not be stimulated by elevated CO2 as global climate change progresses, as has been postulated previously.
This paper, in solving some of the enigmas in E. huxleyi biology, neatly illustrates the power of combining carefully constructed physiological experiments with molecular approaches. May we see more of its like in the future!