Independence of nutrient limitation and carbon dioxide impacts on the Southern Ocean coccolithophore Emiliania huxleyi
Calcification of the cosmopolitan coccolithophore species Emiliania huxleyi was investigated in relation to the cell division cycle with the use of batch cultures. With a 12 : 12 h light : dark cycle, the population was synchronised to undergo division as a cohort, simultaneously passing through the G1 (assimilation), S (DNA replication), and G2+M (cell division and mitosis) phases. Cell division was followed with the use of quantitative DNA staining and flow cytometry. Simultaneously, carbon-14 (14C) assimilation in organic and inorganic carbon as well as cell abundance, size, and organic nitrogen content were measured at 2-h intervals. In additional experiments, changes in calcification and cell cycle stages were investigated in nitrogen-, phosphorus-, and lightlimited cultures. Calcification occurred only during the G1 cell cycle phase, as seen by the very tight correlation between the percentage of cells in G1 and calcification during the dark period. When growth was limited by nitrogen, cells decreased in size, remained in the G1 phase, and showed a moderate increase in the cell-specific calcite content. Limitation of growth by phosphorus, however, caused a significant increase in cell size and a dramatic increase in cellular calcite. Light limitation, by slowing the growth rate, prolonged the time cells spent in the G1 phase with a corresponding increase in the cellular calcite content. These results help explain the differing responses of coccolithophorid growth to nitrogen, phosphorus, and light limitation. Coccolithophores are unicellular photosynthetic algae that produce platelets of calcium carbonate, called coccoliths, that surround the cells. They are the dominant planktonic calcifiers in the present ocean and are responsible for up to 80% of global oceanic calcification (Deuser and Ross 1989; Fabry 1989) of 0.8–1.4 Pg of CaCO3-C y21 (Feely et al. 2004). The cosmopolitan species Emiliania huxleyi in particular forms huge seasonal blooms that extend over .100,000 km2 (Brown and Yoder 1994), making it an important player in the marine environment. Calcification plays a substantial role in the marine carbon cycle in that formation and export of calcium carbonate reduce alkalinity in the surface ocean and cause a net release of CO2 to the atmosphere, counteracting the CO2 drawdown by photosynthesis. Calcification thus decreases the efficiency with which the oceans’ ‘‘biological pump’’ takes up atmospheric CO2 (Antia et al. 2001). Hence, variations in the ratio of calcification to photosynthesis (C : P) and ratio of particulate inorganic carbon to particulate organic carbon (PIC : POC) leaving the surface ocean are important in determining the efficiency of biogenic carbon sequestration by the ocean. Additionally, because coccolith formation is negatively affected by decreasing seawater pH, changes in the abundance of calcifiers is expected because of ongoing ocean acidification (Riebesell 2004; Delille et al. 2005) with unknown effects on marine ecosystems. Although there is no consensus as to why coccolithophores calcify (Harris 1994; Young 1994; Bratbak et al. 1996), several factors that influence the rate of calcification, as well as the ratio of calcification to photosynthesis, have been identified. In numerous controlled laboratory experiments and mesocosm and field studies, changes in calcification and the PIC : POC ratios were seen to change with dependence on light, nutrient availability, growth rate, and strain diversity (summarized in Paasche 2002). Elevated bulk calcite production and cell-specific calcium carbonate quota are found particularly under high light conditions and when phosphorus rather than nitrogen limits growth (Paasche and Brubak 1994; Riegmann et al. 2000; Zondervan 2007). Higher cell–calcite quotients also result from nitrogen limitation, but to a lesser extent (Paasche 1998). The physiological reasons underlying these observations are unclear. Calcification is energy consuming, fueled by photosynthesis in the light and respiration in the dark (Sekino and Shiraiwa 1996). Though E. huxleyi calcifies primarily during the light phase of the diel cycle, cells that have been decalcified by acidification can build coccoliths when incubated in the dark, albeit at a much slower rate (Sekino and Shiraiwa 1996). In his comprehensive review of the coccolithophore E. huxleyi, Paasche (2002) speculated that calcification is linked to the cell division cycle, with calcification being primarily a G1 (gap 1, assimilation) process and thus reduced in the dark when dividing cells pass through the S (DNA synthesis) and G2+M (gap 2 [cell division] + mitosis) phases of division. This argument is supported by the observation that the coccolith vesicle is not present during nuclear division and is reconstituted after mitosis (van Emburg 1989). If calcification is related to the G1 phase of the cell cycle, processes arresting cells in G1, such as nutrient limitation, would cause an increase in bulk calcification. 1 Corresponding author (firstname.lastname@example.org). Acknowledgments We thank H. Schäfer for his help with the DNA staining protocol, D. Hümmer and K. Straube for help during sampling, K. Nachtigall for PON measurements, and R. Surberg for measuring the Ca samples. We also thank two anonymous reviewers for their helpful comments. Limnol. Oceanogr., 53(2), 2008, 506–512 E 2008, by the American Society of Limnology and Oceanography, Inc.