Utilization of dissolved inorganic carbon (DIC) and the response of the marine flagellate Isochrysis galbana to carbon or nitrogen stress

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1 New Phytol. (1999), 144, Utilization of dissolved inorganic carbon (DIC) and the response of the marine flagellate Isochrysis galbana to carbon or nitrogen stress D. R. CLARK*, M. J. MERRETT AND K. J. FLYNN Swansea Algal Physiology Research Unit, School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea SA1 8PP, UK Received 15 February 1999; accepted 21 July 1999 SUMMARY The growth of the marine flagellate Isochrysis galbana was followed in batch cultures at four concentrations of dissolved inorganic carbon (DIC), from C- and N-replete lag phase into C- andor N-deplete stationary phase. Organic buffers were omitted from the growth medium, and culture ph was maintained at by the addition of acid or alkali. The responses of the flagellate to N stress included an increase in the C:N ratio, and decreases in the ratios of glutamine (Gln): glutamate (Glu) and Chl a:c, and the cell Chl a quota. Conversely, the responses to C stress included a decrease in the C: N ratio, and increases in the ratios of Gln:Glu and Chl a: C, and the cell Chl a quota. The relationship between carbon-specific growth rate (C-µ), and the concentration of extracellular DIC, [DIC] ext, exhibited Michaelis Menten type kinetics with a half saturation constant, K G(DIC), of 81 µm. Comparative studies of the diatom Phaeodactylum tricornutum showed similar results, although the value of K G(DIC) was lower at 3 µm. Key words: carbon, nitrogen, stress, DIC, carbonic anhydrase, Gln: Glu, nutritional status. INTRODUCTION Over the past 2 million yr, increases in the atmospheric CO concentration have been correlated with increases in the export of organic carbon from surface waters to the marine sediments (Mix, 1989; Lyle et al., 1992). Photosynthetic CO fixation by marine phytoplankton decreases the concentration of CO in the surface waters, increasing the ocean atmosphere interface concentration gradient, and enhancing atmospheric CO exchange with dissolved inorganic carbon (DIC) in surface waters. Intense biological activity may lead to significant CO undersaturation, and may potentially limit marine diatom growth rates (Riebesell et al., 1993). However, it is often assumed that the availability of DIC is non-rate-limiting to the growth of marine phytoplankton compared with other essential nutrients such as N, P, Fe (Walsh, 1991; Falkowski & Wilson, 1992) and Si in the case of diatom productivity (Egge & Jacobsen, 1997). Although elevated biological activity may lead to significant *Author for correspondence ( bbclarkswansea.ac.uk). decreases in sea-surface DIC concentrations (Codispoti et al., 1982), the potential for DIC limitation of phytoplankton growth rates has not been clarified. In laboratory studies, steps are often taken to ensure that DIC does not become limiting, although there is a lack of data relating growth to DIC availability. Steady-state growth in continuous culture has been used to derive numerous relationships (Herzig & Falkowski, 1989; Heath et al., 199) and subsequently used, for example, to determine the biomass of a natural population from Chl a determinations, despite recognized uncertainties between these variables (Fogg, 1975). The marine environment is a dynamic system. Parameterization of cellular variables (growth rate, cell C and N quotas, photosynthetic pigments) is required for mathematical models of phytoplankton growth to enable the prediction of nutrient utilization and C fixation under non-steady-state conditions. The day-averaged carbon specific growth rate (C-µ) is the summation of all cellular processes, and may be more ecologically relevant in mathematical models capable of predicting C sequestration by marine phytoplankton than a consideration of the short-

2 464 D. R. Clark et al. term variation in a single process with DIC availability (e.g. photosynthesis; Caperon & Smith, 1978; Turpin et al., 1985). The aim of this study was to investigate the kinetics of DIC utilization in relation to C-µ and the extracellular DIC concentration, [DIC] ext. Using four initial DIC concentrations, the growth of the marine flagellate Isochrysis galbana was followed through two consecutive growth cycles from C- and N-replete lag and exponential phase into C- andor N-deplete stationary phase. This allowed an estimation of the concentration of DIC at which halfmaximum C-µ was attained (K G(DIC) ), and the theoretical maximum rate of C-µ (V max ). The parameters K G(DIC) and V max, when considered together, may have implications for species succession. In addition the responses to C andor N stress could be compared. MATERIALS AND METHODS Axenic cultures of Isochrysis galbana (Parke) (CCAP 9271) were grown at 181C in artificial seawater (Harrison et al., 198). No organic buffer was added, and ph was adjusted to Bicarbonate was added at.25,.5, 1. and 2. mm. Phosphate and nitrite were added at 32 and 5 µm, respectively. Nitrite was used for rapidity and ease of analysis. Either C or N would then become the yield-limiting nutrient. All media were filter-sterilized through 47 mm diameter,.2 µm pore Durapore filters (Millipore). Cultures were grown in 3-l conical flasks sealed with a gas-tight silicon bung through which three PTFE tubes passed. The first of the three tubes was attached via silicon tube to a length of glass tubing which extended into the culture medium. Samples were removed through this tube after the flask contents had been mixed by swirling. CO -free air replaced the volumes of media removed for sampling and entered the culture vessel via the second tube; this air first passed through a Dreschel bottle containing 1 N NaOH (thus removing CO ), and then through a second Dreschel bottle containing unbuffered HPLC-grade water. The third tube allowed for the manual correction of culture medium ph to by the addition of small volumes of freshly made weak acid or alkali (.25 M HCl or NaOH) via an Acrodisc filter (Gelman, Ann Arbor, MI, USA). Cultures were grown at a photon flux density of 2 µmol m s (measured within water-filled flasks using Biospherical Instruments QSL 1 meter (Biospherical Instruments, San Diego, CA, USA) with a 4 π detector) in a 12:12 h light: dark cycle supplied by cool white fluorescent tubes. Experimental cultures were inoculated from cultures grown under identical conditions, batches being started with a 5% (vv) inoculation from the previous batch once this had attained stationary phase (as judged by cell counts). Cell numbers and biovolume were measured in triplicate (Elzone 282PC particle analyser (Partical Data, Luxenbourg)) and nitrite in duplicate at, 6 and 12 h into the light phase on each day. Additional samples were collected by filtration under low pressure (75 mm Hg) onto pre-ashed, 13 mm Gelman AE filters at 6 h into the light phase and subsequently used for amino-acid, pigment and elemental C and N analysis. Nitrite was determined by diazotizing with sulphanilamide and coupling with N-(1-napthyl)- ethylenediamine (Parsons et al., 1984) before being measured by a Cecil Instruments 6 series (Cecil Instruments, Cambridge, UK), double-beam spectrophotometer at 543 nm. The DIC concentration was determined in cell-free medium by the Gran titration technique described by Butler (1982). Intracellular amino acids were extracted and analysed by HPLC according to the method described by Flynn & Flynn (1992). Pigments were extracted using N,N-dimethylformamide (Inskeep & Bloom, 1985) and incubated for 4 h at 4C before estimating by spectrophotometry (Jeffery & Humphrey, 1975). Particulate organic carbon (POC) and particulate organic nitrogen (PON) were measured using Europa Scientific Ropoprep and Tracer-mass instruments (Europa, Crewe, UK) using isoleucine as the standard. Carbon-specific growth rates (C-µ) were determined from biovolume (BV). Biovolume was linearly related to POC by Eqn 1: POC (µg ml ).22BV (nl ml ) (r.99, df 45) Eqn 1 Carbon-specific growth could therefore be determined from the rate of BV-specific growth, for which more frequent, triplicate values were taken than for POC. The coefficient of variation for replicate BV determinations is also much lower (5%) than for replicate POC measurements. Only those values for C-µ corresponding to the N-replete exponential phase were used in the construction of the C-µ: [DIC] ext relationship. C-µ was determined using Eqn 2: C-µ (ln(bv )ln(bv ))(t t ) Eqn 2 (BV and BV are the biovolume values at times t and t, respectively). The half-saturation constant for C-specific growth (K G(DIC) ) and the theoretical maximum rate of C-specific growth (V max ) were determined using an iterative fit procedure (Biosoft s FigP (Biosoft, Durham, NC, USA)) from Eqn 3: C-µ V max ([DIC]([DIC]K G(DIC) )) Eqn 3 Using Microsoft Excel, an analysis of variation between the means of cell counts and BV measurements for consecutive growth cycles was performed, which showed that these were not significantly

3 C and N stress in Isochrysis galbana 465 Cell numbers ( 1 6 ml 1 ) Extracellular DIC (mm) Biovolume (nl ml 1 ) Extracellular DIN (µm) Time (d) Time (d) Fig. 1. Changes in cell density and biovolume during the growth of Isochrysis galbana at dissolved inorganic carbon (DIC) concentrations of.25 (closed squares),.5 (open squares), 1. (closed circles) and 2. mm (open circles). All cultures were supplied with 5 µm NO at 18C with a photon flux density of 2 µmol m s. For each DIC treatment the results presented are from the second of two consecutive growth cycles, which were not significantly different (P.5). different (P.5). A similar set of experiments was conducted using the diatom Phaeodactylum tricornutum (Bohlin) (CCAP 1526) for comparisons between C kinetics data only. RESULTS Growth of I. galbana was followed from nutrientreplete lag phase into C-stressed (.25 mm DIC), C N-stressed (.5 and 1. mm DIC), and N- stressed (2. mm DIC) stationary-phase cells. Growth was followed in terms of cell numbers and BV (Fig. 1), with simultaneous measurements of DIC and DIN depletion (Fig. 2). Generally, cell division occurred during the dark phase while BV increased during the light phase. Table 1 shows selected parameters determined during the stationary phase of the growth cycles. The value of C-µ could be related to [DIC] ext by a rectangular hyperbolic curve. C-µ increased rapidly up to a [DIC] ext of.5 mm, after which C-µ approached saturation, increasing by only 11% as [DIC] ext reached 2. mm (Fig. 3). This trend was Fig. 2. Changes in extracellular dissolved inorganic carbon (DIC) and dissolved inorganic nitrogen (DIN) during the growth of Isochrysis galbana at DIC concentrations of.25 (closed squares),.5 (open squares), 1. (closed circles) and 2. mm (open circles). Nutritional status of I. galbana cultures during stationary phase varied from N-stressed (2. mm DIC cultures), through C Nstressed (1. and.5 mm DIC cultures), to C-stressed cultures (.25 mm DIC cultures). also demonstrated by the diatom P. tricornutum grown under identical conditions. For the diatom, C-µ increased by 5% as [DIC] ext increased from.5 to 2. mm. Table 2 gives estimates of K G(DIC) and V max determined for both species. Phaeodactylum tricornutum attained the higher affinity for DIC for C-specific growth, which was almost three times greater than that of the flagellate. The V max attained by the diatom was almost twice that of the flagellate. Changes in the nutritional status of I. galbana were indicated by changes in the C:N ratio (Fig. 4a), and the ratio of intracellular glutamine (Gln) to glutamate (Glu) (Fig. 4b). Nitrogen stress was most significant in the 2. mm DIC culture, with an increase in the C: N ratio noticeable beyond day 3 of growth. Concurrently, cellular concentrations of Glu increased in these cultures, resulting in a decrease in the Gln:Glu ratio to.1 by day 3, indicative of N stress. Nitrogen stress was less significant in the 1. and.5 mm DIC cultures with slower changes in the C: N ratio, and with final stationary phase values of 9.7 and 8.7, respectively. A decrease and subsequent

4 466 D. R. Clark et al. Table 1. Selected parameters determined for Isochrysis galbana during the stationary phase of growth, with 95% confidence limits Culture (mm DIC) Cell C quota (pg cell ) Cell N quota (pg cell ) Cell Chl a quota (ng cell ) Chl a:c ratio (1) DIC, dissolved inorganic carbon. 1. Phaeodactylum tricornutum C µ (d 1 ) Isochrysis galbana Extracellular DIC (mm) Fig. 3. The relationship between the C-specific growth rate (C-µ) and the extracellular dissolved inorganic carbon ([DIC] ext ) concentration for Isochrysis galbana (closed squares) and Phaeodactylum tricornutum (open squares). Both species exhibited Michaelis Menten type-c kinetics. The diatom P. tricornutum attained an affinity for DIC (K G(DIC) )of32 µm which was almost three times greater than that of the flagellate (K G(DIC) 81 µm). Further, P. tricornutum attained a theoretical maximum rate of C-µ (V max ) of.79.7 d, exceeding that of the flagellate by almost twice (V max.48.1 d ). Gln : Glu ratio C : N ratio Time (d) Table 2. The half saturation constant for C-specific growth (K G(DIC) ) and the theoretical maximum rate of the C-specific growth (V max ) for Isochrysis galbana and Phaeodactylum tricornutum, with 95% confidence limits Parameter I. galbana P. tricornutum K G(DIC) (µm) V max (d ) increase in the Gln:Glu ratio of the 1. mm DIC culture indicated that these cultures became transiently N stressed at the level of the internal aminoacid pool, while the Gln:Glu ratio of the.5 mm DIC cultures fluctuated around a value of.3, consistent with a balance of C and N for amino-acid synthesis. Cellular concentrations of glutamine increased towards the end of the growth cycle in the.25 mm DIC cultures, leading to the highest Fig. 4. Changes in the C: N mass ratio and the ratio of intracellular glutamine (Gln) to glutamate (Glu) as indicators of nutritional status during the non-steady-state growth of the marine flagellate Isochrysis galbana at dissolved inorganic carbon (DIC) concentrations of.25 (closed squares),.5 (open squares), 1. (closed circles) and 2. mm (open circles). Gln:Glu ratio of approx..7, indicative of C stress. The.25 mm DIC culture also attained the lowest C:N ratio of all cultures at the end of the growth cycle; N stress was not detected. In all cultures of I. galbana, the concentration of total internal free amino acids reached a maximum during the first 2 d of the growth cycle, with internal amino acid N (InAA-N) representing a maximum of 18% of the total cell N. As cultures entered exponential growth, the concentration of the internal amino-acid pool decreased. In both C- and N- deplete stationary phase cultures, InAA-N represented 4.2.7% of the total cell N.

5 C and N stress in Isochrysis galbana 467 Chl a : C ratio ( 1) Chl a quota (ng cell 1 ) Time (d) Fig. 5. Changes in cell Chl a quota and the Chl a:c ratio during the non-steady-state growth of Isochrysis galbana into C-stressed (.25 mm dissolved inorganic carbon (DIC), closed squares), C N-stressed (.5 mm DIC, open squares, and 1. mm, closed circles), and N-stressed stationary phase (2. mm DIC, open circles). With increasing C stress, Chl a synthesis was stimulated (.25 mm DIC cultures) whereas decreases in the cell Chl a quota and Chl a:c ratio resulted from N stress (2. mm DIC cultures). Chl a per ml of culture increased in the 1. and 2. mm DIC cultures and appeared to reach a plateau with the onset of N-deplete growth. Upon entering N-deplete growth, the cell Chl a quota of the 1. and 2. mm DIC cultures decreased by 35 and 32%, respectively, of those attained during nutrientreplete growth (Fig. 5a). By contrast, Chl a per ml culture increased in the.25 and.5 mm DIC cultures throughout the growth cycle, consistent with C rather than N stress. The cell Chl a quota of the C-stressed.25 mm DIC cultures was greater than those attained during C- and N-replete growth by a factor of 2. The variation in cell Chl a quota due to nutrient stress was reflected in the Chl a:c ratio of the I. galbana cells (Fig. 5b). Thus, as cultures entered nutrient-deplete growth, increased N stress resulted in a decrease in the Chl a:c ratio, while increased C stress resulted in an increase in the Chl a:c ratio. Correlations between extractable pigments and PON or POC revealed that only two of these relationships were independent of nutritional status. POC (µg ml 1 ) POC (µg ml 1 ) PON (r = 96) POC (r = 93) Chl a (µg ml 1 ) PON (r = 77) POC (r = 97) Carotenoid (µg ml 1 ) Fig. 6. Correlation between particulate organic carbon (POC), particulate organic nitrogen (PON), and extractable Chl a or carotenoids for Isochrysis galbana during non-steady-state growth. When all the data were collated including those points corresponding to C- and N-stressed cultures, correlations between Chl a and PON, and between carotenoids and POC, were highly significant. Correlations between Chl a and POC, and between carotenoids and PON, were affected to a greater extent by C andor N stress. Extractable Chl a was related to PON (Fig. 6a), described by Eqn 4: N(µg ml ) 9.9Chl a (µg ml ) (r.96, df 45) Eqn 4 Similarly, carotenoids were related to POC (Fig. 6b) by Eqn 5: C(µg ml ) carotenoids (µg ml ) (r.97, df 45) Eqn 5 The correlation between Chl a and POC (Fig. 6a), or between carotenoid and PON (Fig. 6b), was found to be linear only during nutrient-replete growth. However, the correlation coefficient deteriorated with the inclusion of data from nutrient-stressed cells. DISCUSSION Two related processes associated with photosynthetic CO fixation by many algae are the internal accumulation of DIC and the alkalization of the PON (µg ml 1 ) PON (µg ml 1 )

6 468 D. R. Clark et al. surrounding medium (Shiraiwa et al., 1993). The latter results in changes in the medium s ph which affect the growth of phytoplankton in a number of ways: for example by changing the distribution of DIC forms and therefore the availability of CO and HCO, and by altering the availability of trace metals (Fe, Zn, Mn, Cu) and other essential nutrients. At the extremes, ph may have direct physiological effects. Alkalization of the medium during algal growth in culture is usually prevented by the use of medium containing organic buffers. A set of preliminary investigations was conducted to test the buffering capacity of a range of organic buffers including Tricine, Trizma, Hepes and Bistrispropane. The results demonstrated that even at greatly elevated concentrations, the use of organic buffers with appropriate Pka values failed to maintain the ph of the medium to within The ph consistently increased with time as POC increased. Further, for Nanaochloropsis oculata (CCAP 8491), elevated concentrations of organic buffer led to a decreased cell yield, whereas investigations with I. galbana and P. tricornutum suggested that these species may be able to use organic buffers as a DOCDON source. Manual adjustments of the culture ph circumvented the need for organic buffers which also interfere in the determination of [DIC] by acid titration methods. The Gran titration method employed in these investigations, which is widely accepted and routinely used at sea (Dyrssen, 1965; Bradshaw et al., 1981), was found to have a higher degree of accuracy and reproducibility by comparison with single-point titration methods. For P. tricornutum and I. galbana the relationship between [DIC] ext and C-µ exhibited Michaelis Menten type kinetics. With these species, C-µ approached saturation at [DIC].5 mm. Low DIC availability usually results in the expression of CO concentrating mechanisms (CCM) (Badger et al., 198; Colman, 1991). As a component of CCM, extracellular carbonic anhydrase (CA ext ) activity has been shown to represent 31% of the total CA activity in I. galbana (Burns & Beardall, 1987), whereas CA ext activity in P. tricornutum has been shown to increase below a [DIC] of.7 mm (Iglesias- Rodriguez & Merrett, 1996). Further, in low-co - adapted I. galbana, Rubisco, which may be the primary rate-limiting step of photosynthetic C metabolism (Woodrow & Berry, 1988), represents 23% of the total cell protein (Falkowski et al., 1989; Herzig & Falkowski, 1989) which is three to nine times greater than that of a range of low-co - adapted micro-algae (Raven, 1991). Such cellular acclimatizations in response to low [DIC] ext will be contributory factors enabling both species of phytoplankton to attain high rates of C-µ (i.e. 89% of those attained at 2. mm DIC) even when grown at [DIC] ext of 25% of normal atmosphere-equilibrated [DIC]. While the results of the present investigation give no indication of the form of DIC used (CO or HCO ), under the experimental regimes employed the results would suggest that, for I. galbana and P. tricornutum, DIC rate limitation of growth may be of significance only when [DIC] ext declines to.5 mm. In the present study, P. tricornutum attained a K G(DIC) which was about one-third that of the flagellate, whereas the theoretical V max was 2 times higher. Such differences are potential sources of competitive selection for organisms growing in natural populations. The results suggest that under the same conditions of growth (i.e. of light and nutrient availability) at any level of DIC availability, the rate of C-µ achieved by P. tricornutum would enable its succession over I. galbana. Further investigations with species from other algal groups are required to determine whether Michaelis Menten type [DIC] ext :C-µ kinetics are typical of algal physiology. Cell size (surface area to volume ratio), motility (affecting the thickness of the boundary layer) and the presence or absence of a CCMCA activity may significantly affect the kinetics of DIC utilization, and need to be considered in this regard. The decline of total amino acids per cell, and amino acid N as a percentage of total cell N from the peak values shown during N-sufficient growth, show a trend which is reflected in the decline of individual proteins such as those constituting the photosynthetic apparatus (Falkowski et al., 1989). Nitrogen limitation may therefore affect protein synthesis at the level of translation, by lowering the internal amino-acid pool concentration. As a biomarker which may respond rapidly to changes in nutritional status, the Gln: Glu ratio (Flynn et al., 1989) may be expected to be of use in the determination of nutritional status because of the primary role of these amino acids in the assimilation of N following uptake (Syrett, 1981). The changes in the internal aminoacid pool of algae, cyanobacteria and yeast during N deprivation and nutrient spiking have demonstrated that the ratio could be used as a sensitive monitor of N status (Flynn et al., 1989). Amino-acid synthesis depends as much on the supply of C as on that of N. The Gln: Glu ratio may increase as a result of decreased levels of α-oxoglutarate relative to Gln synthesis, resulting in an increase in Gln (indicative of a surplus of N relative to C), andor as a result of decreased levels of Glu (due to the removal of Glu for the synthesis of other amino acids indicative of a continuance of amino-acid synthesis). A Gln: Glu ratio of.5 is suggested to indicate an N-replete population (Flynn et al., 1989). The lowest Gln:Glu ratio of c..5 in the 2. mm DIC cultures was a result of very low Gln concentrations relative to Glu, indicative of N stress. Concurrently, the cell N quota of the 2. mm DIC cultures remained stable, while the cell C quota increased by 1%, resulting in an increase in the

7 C and N stress in Isochrysis galbana 469 C: N ratio as cultures entered stationary phase. Upon depletion of the DIN source, the Gln:Glu ratio in the 1. mm DIC cultures declined to.1 before recovering slightly. This event has been noted by Davidson et al. (1992) and has been ascribed to N recycling. During the recovery period in the current study, the cell Chl a quota decreased by 35% to a level similar to that of the significantly N- stressed 2. mm DIC cultures. Concurrently, cellular concentrations of Glu decreased, indicative of a continuance of amino-acid synthesis. Catabolism of photosynthetic apparatus may have supplied N during N-deprived growth for amino-acid synthesis, thereby relieving N stress. The transient increase and subsequent decrease in N stress due to cellular acclimatization, as demonstrated at the level of the internal amino-acid pool, was not reflected in the C:N ratio of these cultures. While the Gln: Glu ratio of the.5 mm DIC cultures fluctuated c..3 throughout the growth cycle, the Gln: Glu ratio of the.25 mm DIC cultures increased to.67 at the end of the growth cycle. This increase was because of an increase in cellular Gln concentrations, indicative of a limiting C supply as seen also in C-limited yeast (Flynn & Hipkin, 199). There was a concurrent twofold increase in the cell Chl a quotas. These results suggest that C stress stimulates Chl a synthesis, increasing the photosynthetic capacity to fix CO in response to an inadequate supply of C skeletons for amino-acid synthesis. Ecologists usually infer the distribution of phytoplankton biomass from the distribution of Chl a. However, the correlation between Chl a and POC is dependent on nutritional status, and becomes less significant during N-deprived growth. In this study, Chl a:c ratios were highest in C-stressed cultures, and decreased with increased N stress. The high degree of variability in the Chl a:c ratio (by a factor of 2) of I. galbana due to nutrient deprivation (as also noted by Flynn et al., 1993) indicated that Chl a measurements could not be used to reliably estimate phytoplankton biomass. In addition, dinoflagellates generally have lower Chl a :C ratios than diatoms (Tang, 1996), and the use of Chl a measurements to estimate the biomass of a mixed population of unknown nutrient history is debatable. The relationship between carotenoid and PON was also found to be dependent on CN status in contrast to the Chl a: PON and carotenoid: POC ratios. The latter has also been demonstrated for ammonium-limited I. galbana under continuous light, and a light dark cycle (Davidson et al., 1992), suggesting that for this species extractable carotenoid may be used as a measure of population biomass. In conclusion, the responses of I. galbana to C stress included a decrease in the C: N ratio and increases in the Gln : Glu ratio, Chl a:c ratio and the cell Chl a quota. The responses to N stress were opposite to those for C stress. However, the most significant result is that even during intense phytoplankton blooms in which total [DIC] may decrease to as low as 1.75 mm (Codispoti et al., 1982), the results suggest that for the species investigated a DIC rate limitation of phytoplankton growth rate may be of minor consequence in the marine environment. ACKNOWLEDGEMENTS The authors wish to acknowledge the support of the NERC via a studentship award to D.R.C. REFERENCES Badger MR, Kaplan A, Berry JA Internal inorganic carbon pool of Chlamydomonas reinhardtii. Evidence for a carbon dioxide concentrating mechanism. Plant Physiology 66: Bradshaw AL, Brewer PG, Shafer DK, Williams RT Measurements of total carbon dioxide and alkalinity by potentiometric titration in the GEOSECS program. Earth and Planetary Science Letters 55: Burns BD, Beardall J Utilisation of inorganic carbon by marine microalgae. Experimental Marine Biology and Ecology 17: Butler NB Carbon dioxide equilibria and their applications. Wokingham, UK: Addison-Wesley, Caperon J, Smith DF Photosynthetic rates of marine algae as a function of inorganic carbon concentration. Limnology and Oceanography 23: Colman JR The molecular and biochemical analysis of CO -concentrating mechanisms in cyanobacteria and microalgae. Plant, Cell and Environment 14: Codispoti LA, Friederich GE, Iverson RL, Hood DW Temporal changes in the inorganic carbon system of the southeastern Bering Sea during spring 198. Nature 296: Davidson K, Flynn KJ, Cunningham A Non-steady state ammonium-limited growth of the marine phytoflagellate Isochrysis galbana Parke. New Phytologist 122: Dyrssen D A Gran titration of sea water on board Sagitta. Acta Chemica Scandinavica 19: Egge JK, Jacobsen A Influence of silicate on particulate carbon production in phytoplankton. Marine Ecology Progress Series 147: Falkowski PG, Sukenik A, Herzig R Nitrogen limitation in Isochrysis galbana (Haptophyceae). II. Relative abundance of chloroplast proteins. Journal of Phycology 25: Falkowski PG, Wilson C Phytoplankton productivity in the North Pacific Ocean since 199 and implications for absorption of anthropogenic carbon dioxide. Nature 358: Flynn KJ, Dickson DMJ, Al-Amoudi OA The ratio of glutamine: glutamate in microalgae: a biomarker for nitrogen status for use at natural cell densities. Journal of Plankton Research 11: Flynn KJ, Flynn K Non-protein free amines in microalgae: consequences for the measurement of intracellular amino acids and of the glutamineglutamate ratio. Marine Ecology Progress Series 89: Flynn KJ, Hipkin CR Changes in intracellular amino acids and glutamine: glutamate during N-deprivation and feeding in Candida nitraphila. New Phytologist 114: Flynn KJ, Zapata M, Garrido JL, Opik H, Hipkin CR Changes in carbon and nitrogen physiology during ammonium and nitrate nutrition and nitrogen starvation in Isochrysis galbana. European Journal of Phycology 28: Fogg GE Primary productivity. In: Riley JP, Skirrow G, eds. Chemical oceanography, Vol. 2, 2nd edn. London, UK: Academic Press, Harrison PJ, Waters RE, Taylor FJR A broad spectrum artificial seawater for coastal and open ocean phytoplankton. Journal of Plankton Research 3:

8 47 D. R. Clark et al. Heath MR, Richardson K, Kioboe T Optical assessment of phytoplankton nutrient depletion. Journal of Plankton Research 12: Herzig R, Falkowski PG Nitrogen limitation in Isochrysis galbana (Haptophyceae). I. Photosynthetic energy conversion and growth efficiencies. Journal of Phycology 25: Iglesias-Rodriguez MD, Merrett MJ Dissolved inorganic carbon utilisation and the development of extracellular carbonic anhydrase by the marine diatom Phaeodactylum tricornutum. New Phytologist 135: Inskeep WP, Bloom PR Extinction coefficients of chlorophyll a and b in N,N-dimethylformamide and 8% acetone. Plant Physiology 77: Jeffery SW, Humphrey GF New spectrophotometric equations for determining chlorophylls a, b, c and c2 in higher plants, algae and natural phytoplankton. Biochemical Physiology 167: Lyle MW, Prahl FG, Sparrow MA Upwelling and productivity changes inferred from a temperature record in the central Equatorial Pacific. Nature 355: Mix AC Influence of productivity variations on long-term atmospheric CO. Nature 337: Parsons R, Maita Y, Lalli CM A manual of chemical and biological methods for seawater analysis. Oxford, UK: Pergamon, Raven JA Physiology of inorganic carbon acquisition and implications for resource use efficiency by marine phytoplankton relation to increased CO and temperature. Plant, Cell and Environment 14: Riebesell U, Wolf-Gladrow DA, Smetacek V Carbon dioxide limitation of marine phytoplankton growth rates. Nature 361: Shiraiwa Y, Goyal A, Tolbert NE Alkalisation of the medium by unicellular green algae during uptake of dissolved inorganic carbon. Plant and Cell Physiology 34: Syrett PJ Nitrogen metabolism of microalgae. Canadian Bulletin of Fisheries and Aquatic Science 21: Tang E Why do dinoflagellates have lower growth rates? Journal of Phycology 32: Turpin DH, Miller AG, Parslow JJ, Elrif IR, Canvin DT Predicting the kinetics of dissolved inorganic carbon limited growth from the short-term kinetics of photosynthesis in Synechococcus leopoliensis (Cyanophyta). Journal of Phycology 21: Walsh J Importance of continental shelf margins in the marine biogeochemical cycling of carbon and nitrogen. Nature 35: Woodrow IE, Berry JA Enzymatic regulation of photosynthetic CO fixation in C plants. Annual Review of Plant Physiology and Molecular Biology 39: