Carbon acquisition of marine phytoplankton: Effect of photoperiod length

We investigated the carbon acquisition of three marine microalgae, Skeletonema costatum, Phaeocystis globosa, and Emiliania huxleyi in response to different light regimes. Rates of photosynthetic O2 evolution and CO2 and HCO uptake were measured by membrane inlet mass spectrometry in cells acclimated to cycles of 16 : 8 light : 3 dark (LD; h : h) and 12 : 12 LD and were compared with those obtained under continuous light. In addition, cellular leakage was estimated for different photoperiods and ambient CO2 concentrations during growth. Maximum rates of photosynthesis more or less doubled under LD cycles compared with continuous light. In S. costatum and E. huxleyi, a remarkably higher contribution of HCO to the overall carbon uptake was observed under LD cycles. In 3 contrast, P. globosa did not change its CO2 : HCO uptake ratio in response to daylength. Half saturation concen3 trations (K1/2) for O2 evolution and inorganic carbon (Ci) uptake were also influenced by the photoperiod. Under LD cycles K1/2 values for photosynthesis in S. costatum and P. globosa were similar or higher compared with continuous light, whereas they were much lower in E. huxleyi. With the exception of CO2 uptake in E. huxleyi and P. globosa, affinities for Ci decreased under the LD cycles. Cellular leakage was highest for E. huxleyi and lowest for S. costatum and generally decreased with increasing CO2 concentration. Although this study confirms speciesspecific differences in the CO2-concentrating mechanisms (CCMs), the effect of daylength on CO2 and HCO uptake 3 has hitherto not been described. We put forward the idea that variations in light condition influence the cellular carbon demand, thereby imposing a stronger control on CCM regulation than the naturally occurring changes in CO2 supply. Inorganic carbon acquisition has been suggested to play an important role in marine phytoplankton ecology and evolution (e.g., Badger et al. 1998; Tortell 2000; Giordano et al. 2005). Despite the relatively high concentrations of dissolved inorganic carbon in the ocean, phytoplankton cells have to invest considerable resources in carbon acquisition to allow for high rates of photosynthesis. This situation is mainly caused by the ‘‘imperfection’’ of their primary carboxylating enzyme, ribulose-1,5-bisphosphate carboxylase/ oxygenase (RubisCO), which is characterized by a low affinity for its substrate CO2, a slow maximum specific turnover rate, and susceptibility to a competing reaction with O2. To avoid the risk of carbon limitation, most microalgae have thus developed different mechanisms that enhance the intracellular CO2 concentration at the site of carboxylation (Badger et al. 1998). These CO2-concentrating mechanisms (CCMs) involve active uptake of CO2 or HCO or both, as 3 1 Corresponding author ([email protected]). Acknowledgments We thank Steffen Burkhardt, Gabi Amoroso, Dominik Müller, and Christoph Thyssen for technical support and laboratory assistance and two anonymous reviewers for their constructive comments on the manuscript. This work was supported by the German Science Foundation (TH74412) and the German–Israeli Cooperation in Marine Sciences, which is funded by the German Federal Ministry of Education and Research. well as the enzyme carbonic anhydrase (CA), which accelerates the otherwise slow conversion rate between HCO3 and CO2. Recent data suggest the possibility that a C4-like pathway might operate, together with active HCO uptake, 3 in diatoms (Reinfelder et al. 2000, 2004). This involves the formation of oxalacetate and malate by phosphoenolpyrovate carboxylase, which has the advantage over RubisCO of a high affinity for its carbon source HCO along with insen3 sitivity to O2. Phytoplankton species differ in efficiency and regulation of their carbon acquisition (e.g., Burkhardt et al. 2001; Rost et al. 2003; Giordano et al. 2005). Species relying on diffusive CO2 uptake or those with inefficient CCMs are CO2 sensitive in their photosynthesis, whereas species with highly efficient CCMs are rate saturated even under low ambient CO2 concentrations. The capability of regulation allows phytoplankton to adjust CCM efficiency to their actual need, thereby optimizing the allocation of resources. Understanding the factors influencing CCM efficiency could help to elucidate the role of carbon acquisition in phytoplankton ecology. As one of these factors, CO2 supply has early on been identified and subsequently used to investigate the properties of CCMs by comparing incubations at different CO2 concentrations. While most studies compare unnaturally high with ambient CO2 levels, implying distinct repression or induction of the CCM, current findings indicate that under the natural range of CO2 concentrations, there is a fine-scale 13 Effect of daylength on C acquisition tuning in the degree to which the CCM is expressed (Berman-Frank et al. 1998; Burkhardt et al. 2001; Rost et al. 2003). Photon flux density (PFD) also influences CCM efficiency in microalgae. Increasing light limitation yielded a decrease in dissolved inorganic carbon affinities, which has been ascribed to the effect of energy supply on active carbon uptake (Beardall 1991; Berman-Frank et al. 1998). The effect of daylength has been studied on different aspects of algal physiology, including photosynthesis. Nielsen (1997) investigated the influence of daylength on the photosynthesis of Emiliania huxleyi, finding a threefold higher chlorophyll a (Chl a)-specific maximum photosynthetic rate when cells were grown under a cycle of 12 : 12 light : dark (LD) compared with continuous light. Moreover, some microalgae are able to keep their carbon-specific growth rate more or less constant independent of daylength (Price et al. 1998; Burkhardt et al. 1999; Rost et al. 2002), which requires that rates of carbon fixation increase with decreasing photoperiod length. When carbon-specific growth rates are normalized for the duration of the photoperiod, an almost twofold increase from continuous light to 12 : 12 LD for Skeletonema costatum (Burkhardt et al. 1999) and about 1.5fold increase from continuous light to 16 : 8 LD for E. huxleyi (Rost et al. 2002) was observed. Mortain-Bertrand et al. (1987a) reported a stimulation of photosynthesis in cycling light for S. costatum compared with continuous light. Further indication for the effect of photoperiod on carbon acquisition stems from experiments on carbon isotope fractionation («p). Species such as S. costatum and E. huxleyi showed significantly lower «P values under LD cycles compared with continuous light (Burkhardt et al. 1999; Rost et al. 2002). On the basis of this pattern of isotope fractionation, as well as the daylength-dependent changes in the carbon fixation rate, Rost et al. (2002) postulated that daylength influences the regulation of carbon acquisition in E. huxleyi and other microalgae. In this study, we investigated inorganic carbon acquisition of three dominant bloom-forming species, S. costatum, Phaeocystis globosa, and E. huxleyi, in response to changes in photoperiod length. We examined O2 evolution under steady-state photosynthesis and quantified CO2 and HCO3 uptake rates as well as cellular leakage (CO2 efflux : Ci uptake) with the use of membrane inlet mass spectrometry. Material and methods Culture conditions and sampling—S. costatum, P. globosa (both strains collected in the North Sea and maintained in stock culture for several years), and a calcifying strain of E. huxleyi (B92/11) were grown at 158C in 0.2-mm filtered seawater (salinity 32), which was enriched according to f/2 medium (Guillard and Ryther 1962). Batch cultures were grown in 1-liter glass tubes with 360 parts per million by volume (ppmv) CO2 and an incident PFD of 180 mmol photons m22 s21. Cells were acclimated for at least 4 d to LD cycles of 16 : 8 and 12 : 12. Cells were harvested by centrifugation 4 to 7 h after the beginning of the photoperiod to allow photosynthesis and CCM activity to be fully induced (Marcus et al. 1986). A subsample of the culture was used for potentiometric pH measurements. Cultures in which the pH had shifted significantly from that of a cell-free control (pH drift .0.05) were excluded from further measurements. To concentrate the cells for the measurements, 800 ml of the culture were centrifuged at 500–1,000 3 g at 158C for 4 min. Subsequently, cells were washed in CO2-free f/2 medium buffered with 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES, 50 mmol L21, pH 8.0). Samples for Chl a determination were taken after the measurements by centrifuging 2 ml of the cell suspension (4,500 3 g, 4 min). Chl a was subsequently extracted in 1 ml of methanol (1 h in darkness, at 48C) and determined spectrophotometrically at 652 and 665 nm. Chl a concentrations in the culture ranged from 5 to 35 mg L21 at the time of sampling. The carbonate system was determined according to Burkhardt et al. (2001). Determination of net photosynthesis and inorganic carbon fluxes—To investigate inorganic carbon (Ci) fluxes during steady-state photosynthesis, a quadropole membrane inlet mass spectrometer (MSD 5970; Hewlett Packard) was used. Net photosynthesis was measured by monitoring the O2 concentration over consecutive LD intervals with increasing Ci concentrations. Simultaneous measurements of the CO2 concentration enabled us to determine the CO2 uptake and HCO uptake kinetics according to equations by Badger et 3 al. (1994). This method has been applied in several studies of cyanobacteria and freshwater microalgae (Palmqvist et al. 1994; Tchernov et al. 1997; Sültemeyer et al. 1998) and recently also of marine phytoplankton (Burkhardt et al. 2001; Rost et al. 2003). Cellular leakage was estimated from the CO2 efflux observed right after turning off the light (Badger et al. 1994). In this study, we largely followed the protocol described by Burkhardt et al. (2001). All measurements were performed in f/2 medium buffered with 50 mmol L21 HEPES (pH 8.0) at 158C. Dextran-bound sulfonamide (DBS), a membrane-impermeable inhibitor of external CA, was added to the cuvette to a final concentration of 100 mmol L21. Chl a concentration ranged between 1 and 3 mg ml21. Light and dark intervals during the assay lasted 6 and 7 min, respectively. The incident photon flux density was 300 mmol m22 s21. For further details on the method and calculation, we refer to Badger et al. (1994) and Burkhardt et al. (2001).