Plant growth depends on CO2 concentration (reviewed by Urban 2003), which is expected to increase from current levels of about 400 ppm to between 700 and 1000 ppm during the next century (Watson et al. 2001). Oceanic primary production constitutes about 46% of the total primary production on earth (Field et al. 1998), and experiments examining how C uptake by microalgae responds to rising CO2 are needed to understand how oceanic primary production will change in the future. The ocean–atmosphere flux is partially controlled by a biological pump, by which dying phytoplankton sink C into deep ocean sediments. Mathematical simulations have estimated that pre-industrial levels of CO2 would have been as high as 460 ppm without the operation of such a pump (Sarmiento & Toggweiler 1984), whereas pre-industrial atmospheric CO2 levels were around 280 ppm (Etheridge et al. 1996). The discrepancy between a model ocean lacking biologically mediated C fixation and an ocean with biological C fixation suggests that biotic sequestration of C plays an important role in regulating atmospheric CO2 levels. It has been suggested that increases in CO2 may lead to an increase in algal biomass, which would in turn lead to more CO2 being removed from the atmosphere by these algae. In addition to these ecological responses to rising CO2, microalgal communities may also adapt to the increased supply of CO2, resulting in unknown long-term changes to C uptake and cycling in oceans.
Many microalgal species respond to CO2 limitation by the induction of a C concentrating mechanism (CCM) (Badger et al. 1998; Sültemeyer 1998; Badger and Spalding 2000) The CCM is an inducible system that enables microalgae to respond to extracellular changes in inorganic C, and occurs in most microalgal species studied to date (Colman et al. 2002). The CCM elevates CO2 concentration in the vicinity of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco), the main carboxylating enzyme in C fixation, when C is scarce (Moroney & Somanchi 1999). Conversely, CCM expression decreases at higher levels of CO2, resulting in lower affinity C uptake when inorganic C is abundant (Bozzo & Colman 2000). This presumably allows algae to avoid paying the cost of unnecessary enzyme production and active transport.
Over the long term, changes in CO2 levels have the potential to affect two different processes connected to phytoplankton growth: C uptake and C fixation. Most predictions of phytoplankton responses to CO2 enrichment make the tacit assumption that a functional CCM will continue to exist in phytoplankton and that their basic physiology will remain more or less unchanged as CO2 rises. If prolonged growth at elevated CO2 can result in drastic changes to the CCM or to C fixation, considerable uncertainty would be introduced into predictions about the outcome of competition between populations, as well as estimates of global C pool dynamics.
The presence of a CCM in microalgae buffers the C fixation machinery from changes in extracellular inorganic C concentrations. In some cases, growth and photosynthesis rates are nonetheless stimulated by elevated concentrations of CO2 within the range of expected global increases (Hein & Sand-Jensen 1997). In other cases, primary productivity of species or species assemblages of phytoplankton are more or less insensitive to extracellular increases in inorganic C (Tortell & Morel 2002). Even in replicate lines descending from isogenic ancestors, both increases in growth and insensitivity to CO2 enrichment have been reported (Collins & Bell 2004). This variability in responses over all time scales makes it difficult to make general predictions about how phytoplankton populations may respond to elevated CO2, even when other complicating factors are ignored, such as changes in other nutrients or competition between species.
Variability in the responses of phytoplankton to CO2 enrichment has led to considerable debate over the role that C uptake by phytoplankton plays in the sequestration of anthropogenic CO2. This has inspired several experiments designed to evaluate the presence and magnitude of a biological C pump (Honda 2003; Buesseler et al. 2004; Coale et al. 2004). For anthropogenic CO2 to be ‘sunk’ by phytoplankton, it must stimulate increases in net primary productivity, either by increasing rates of net C uptake or by increasing population sizes. Although the response of phytoplankton to changes in CO2 remains controversial, estimates of increases in primary production of 15–19% in response to reasonable increases in CO2 have been observed in natural populations in unenriched seawater from the central Atlantic Ocean (Hein & Sand-Jensen 1997).
We previously used a microalgal model system, Chlamydomonas reinhardtii, to evaluate changes in fitness caused by the spread of novel mutations over 1000 generations in response to increasing CO2 (Collins & Bell 2004). We found that there was no direct response to selection at high CO2, whereas many of the high selected lines had lowered fitness at ambient CO2. The lines also had lower maximum population densities at high CO2, despite normal or increased rates of photosynthesis. From these characters, we suggested that this negative correlated response was caused by the accumulation of conditionally deleterious mutations in the CCM, though this hypothesis was not directly tested. Here, we measure rates and affinities of CO2 uptake by mass spectrometry to evaluate the hypothesis that prolonged growth at elevated CO2 can result in the degradation of high-affinity CO2 uptake. A mass spectrometer was used to distinguish between the following hypotheses: that high selected lines were unable to take up C, that high selected lines were leaking C or that high selected lines were unable to fix C at ambient levels of CO2, even though it was actively being taken up. Because changes in CO2 uptake have the potential to affect larger-scale ecological processes, we then use these results to explore the possible effect of evolutionary change on net C uptake by algal populations.