CO2 Availability Affects Elemental Composition (C:N:P) of the Marine Diatom Skeletonema Costa Tum

MARINE ECOLOGY PROGRESS SERIES I Published August28 Mar EcoI Prog Ser

CO2 availability affects elemental composition (C:N:P) of the marine diatom Skeletonema costa tum

Steffen Burkhardt*, Ulf Riebesell

Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, D-27570 Bremerhaven, Germany

ABSTRACT. The effect of variable CO, concenti-ations on the elemental composition (C N:P) of marine diatoms was investigated in 2 strains of Skeletonema costatum (Grev.)Cleve Five or 6 concentrations of dissolved molecular carbon dioxlde [CO, (aq)],ranging from 0.5 to 39 pm01 I-', were applied in dilute batch cultures. In both strains, elemental ratios were clearly dependent on [CO.(aq)]. With decreasing CO2 concentrations, a decline in C.P and N P and an increase in C:N was observed. The close correlation between C:P or N:P and [CO, (aq)]corresponded to a ca 45 to 65?<, decrease in elemental ratios from highest (230 pm01 I-') to lowest (ca 1 pm01 I-')COz concentrations. C:N at low [COz (aq)]was up to 24 % higher than at high [CO, (aq)].To date, the elemental composition of marine phytoplankton has been considered to be independent of CO, availability. If dependency of the C:N:P ratio on [CO2(aq)] proves to be a general phenomenon in marine phytoplankton, changes in the elemental composition may be expected in response to the currently observed increase in partial pressure of atmospher~cCOz.

KEY WORDS: Redfield ratio - CO, - Phytoplankton Manne diatoms Cell stoichiometry

INTRODUCTION become a limiting factor in the growth of marine plants in the sea' due to the high concentrations of inorganic In the 1930s, Alfred C. Redfield developed the con- carbon compared to phosphorus and nitrogen. cept of a constant elemental composition of planktonic The high concentration of total dissolved inorganic biomass in the oceans (Redfield 1934, Redfield et al. carbon (DIC) in the oceans constitutes a large reservoir 1963). Since then the Redfield ratio of C:N:P = 106:16:1 for the build-up of algal biomass. However, utilization (by atoms) in marine plankton has become a corner- of inorganic carbon by phytoplankton depends on the stone in biogeochemical and ecophysiological studies, chemical form of DIC (molecular CO2, HCO;, or CO:-) in spite of minor revisions in the proportions of these present in seawater Pathways of inorganic carbon elements (e.g. Takahashi et al. 1985, Anderson & acquisition in marme microalgae still represent a con- Sarmiento 1994, Anderson 1995).Its application ranges troversial issue owing to the limited and sometimes from the use as biological indicator of in situ nutrient contradictory information available. Cellular uptake of limitation of phytoplankton (e.g. Perry 1976, Sakshaug DIC by passive diffusion of CO2 versus active transport & Holm-Hansen 1977, Hecky et al. 1993) to modelling of CO2 and/or HCOj, interconversion of CO, and global carbon flux (e.g Heinze et al. 1991). Redfield et HCO;, by extracellular and/or intracellular carbonic al. (1963) proposed that since N:P ratio of available nu- anhydrase, and lntracellular accumulation of CO2 by trients closely resembled planktonic composition, both an inorganic carbon concentrating mechanism are elements would be potentially limiting to phytoplank- discussed as factors controlling carbon availability in ton growth. With regard to inorganic carbon supply to marine microalgae (for review see Raven 1991, Raven microalgae they stated that 'clearly carbon does not et al. 1993). Studies of stable carbon isotope fractionation by marine microalgae show that diffusive uptake of dissolved molecular CO2, which represents only

0 Inter-Research 1997 Resale of fullarticle not permitted Mar Ecol Prog Ser 155: 67-76. 1997

0.5 to 1% of the total DIC, can satisfy cellular carbon grown in f/2-enriched (Guillard & Ryther 1962) aged demand for the observed growth rates over a wide 0.2 pm filtered natural seawater. Nutrient enrichment range of (CO2 (aq)] (Degens et al. 1968, Gleitz et al in SK B cultures was modified to nitrate, silicate, and 1996, H. Kukert & U. Riebesell unpubl.). A different phosphate concentrations of 100, 100, and 6.25 pm01 mode of carbon acquisition might be employed by I-', respectively. The diatoms were incubated in a means of either active CO2 transport or utilization of Rumed 1200 light-thermostat at 15°C with a light-dark the external HCOi pool below a critical concentration cycle of 18 h:6 h and an incident photon flux density of of molecular CO2 (Sharkey & Berry 1985, Gleitz et al. 150 pm01 photons m-' S-'. 1996, Laws et al. in press). Phytoplankton relying on Experimental carbonate system. An intrinsic prop- diffusive transport of CO2 as carbon source can be sub- erty of the carbonate system is the interdependency of ject to transport limitation (Gavis & Ferguson 1975). its components. In order to vary [CO2(aq)], at least 1 of CO2 availability, therefore, can affect carbon uptake the parameters DIC, alkalinity, and pH needs to be and fixation by algal cells (Riebesell et al. 1993) and changed. Concentration of molecular CO2 in ocean thereby may also influence the elemental composition surface water depends on the physical parameters of the organic matter produced. atmospheric pC02, air-sea gas exchange, mixing In this study, 2 strains of the marine diatom Skele- events, temperature, salinity, and the biological tonema costaturn were used in dilute batch culture processes photosynthesis, respiration and calcite pro- experiments to evaluate the potentlal effect of variable duction. Thus, either DIC concentration or, in the case [CO2 (dq)] on cell stoichiometry. To date, the Redfield of calcite production, alkalinity are affected. Whenever ratio has been considered to be independent of arnbi- DIC concentration has been altered, changes in [COz ent CO2 concentrations. Variation of elemental ratlos (aq)]are accompanied by changes in pH. in response to changes in [CO2 (aq)] would provide In routine experiments, concentrations of DIC within first evidence that the chemical composition of marjne treatments were approximately the same (DIC const) diatoms can be sensitive to changes in atmospheric (Table I). Five concen.trations of dissolved molecular partial pressure of CO2 (pCOz). carbon dioxide [CO2 (aq)],ranging from 1.5 to 32 pm01 I-', were adjusted by keeping pH fixed at values between 7.88 and 9.03. The respective pH was MATERIAL AND METHODS achieved by addition of 1 N HC1 or 1 N NaOH to the culture medium. Further gas exchange was prevented Diatom cultures. Two strains of Skeletonenla costa- by closing the incubation vessels tightly without head- turn, SK A and SK B, were used in laboratory experi- space immediately after pH adjustment to ensure con- ments. SK A was collected in the North Sea by vertical stancy of pH and [CO2(aq)]. In experiments with SK A net tows and maintained In stock culture for several (DIC const), triplicate bottles were incubated at each of years. SK B was provided by the Biologische Anstalt the 5 CO, (aq) levels. Because of reasonable agree- Helgoland, where cells were isolated from North Sea ment in elemental ratios between replicates, incuba- phytoplankton net sa.mples i.n February 1996. Mean tion of only 1 bottle at each [CO2(aq)] was considered. cell diameter of SK B cells (12.9 pm) exceeded that of sufficient in further experiments. SK A (4 7 pm) by a factor of 3. Dilute batch cultures To evaluate potential pH effects on cell physiology, were pre-adapted to experimental conditions for at which, might be reflected In the chemical composition least 5 d. Experiments were designed to permit at least of the cells, control experiments with SK A were per- 9 cell divisions under the specific conditions of the formed at identical pH of 8.21 to 8.23 (pH const) in respective treatment. To avoid large pH drift and cor- 6 parallel treatments of different [CO, (aq)].Unlike responding cha~y~sin DIC speciation, cultures were variation in [COl (aq)] under natural conditions, con- inoculated at low cell concentrations (SK A: ca 4 X centration of molecular CO2 in this treatment was ma- to4 cells I-';SK B: cd 1 X 104 cells 1-l) and harvested at nipulated by alteration of both DIC concentration a.nd ca 2.5 X 107 cells 1-' (SK A) or 6 X 106 cells I-' (SK B), alkalinity (Table 1).To achieve lower CO2 (aq)concen- which corresponds to ca 30 pm01 1-' inorganic carbon, trations at constant pH, 1 N HC1 was first added to the 5 pm01 I-' nitrate a.nd 0.5 pm01 1-' phosphate asslmi- culture medium, which resulted In a decrease in alka- lated during the experiment. Bacterial biomass never linity and supersaturation of CO, (aq) relative to at- exceeded 1 'Yu of algal biomass in the cultures and its mospheric pC02. [CO2 (aq)] was then decreased by contribution to elemental ratios was therefore c0nsi.d- bubbling with alr. Because of the open system, DIC de- ered negligible. creases until [<:02 (aq)] approaches equilibrium with Experiments were performed in 2.4 1 borosilicate the atmosphere, accompanied by an increase in pH. In glass bottles, tightly closed with PBT-lined screw caps the following step, pH was readjusted to initial values to avoid air bubbles in the culture vessel. SK A was by addition of 1 N NaOH. The incubation vessels were Burkhardt & Riebesell: CO, effect on C:N:P (Redfield ratio) 69

Table 1 Skeletonema costatum. Experimental carbonate sys- on a Carlo Erba 1500 CHN analyzer after removal of tem. Two strains (SK A. SK B) were incubated at either con- inorganic C with 0.1 N HC1. Triplicate filters for deter- stant concentrations of dissolved inorganic carbon (DIC const) mination of total particulate phosphorus (TPP) were or at constant pH (pH const). pH and concentrations of dissolved molecular carbon dioxide ICO, (aq)]were calculated placed directly in 10 m1 borosilicate tubes with teflon- from DIC, total alkalinity (tAlk),temperature (15"C), salinity lined screw caps and stored frozen at -25°C. Upon (32 psu) and concentrations of phosphate (6.25 pm01 1.') and analysis, TPP filters were digested in a 1 %, potassium silicate (100 pn~olI-') persulfate solution in an autoclave at 1213(: for 1 h. Subsequently, TPP was measured spectrophotometri- DIC tAlk cally by the ammonium-molybdate method (Strickland (mmol l '1 (mecl I-') & Parsons 1972) as orthophosphate in l cm cells at

SK A 7.89 2.11 2.24 885 nm C:N.P was calculated from molar concentra- DLC const 8.28 2.13 2.45 t~onsof P0C:PON:TPP. 8.59 2.14 2.71 Cell size, cell concentration and growth rates. Cells 8.85 2.14 3.01 were enumerated on 20 m1 samples preserved in 9.03 2.14 3.26 Lugol's solution under an inverted microscope. At least SK B 7.88 2.14 2.25 400 cells were counted in duplicate for calculation of DIC const 8.12 2.16 2.37 8.51 2.16 2.63 cell concentrations. For determination of cell size, at 8.72 2.17 2.85 least 25 individual cells were chosen at random in the 8.93 2.17 3.12 microscopic sample. Cell counts before and after the SK A 8.22 5.94 6.51 experiment served as an estimate of growth rates at pH const 8.23 2.17 2.47 the various CO2 (aq) concentrations. To avoid distur- 8.21 0.76 0.95 8.22 0.48 0.65 bance of the closed system, no further subsamples 8.22 0.17 0.32 were taken from the incubation vessels during the 8.21 0.07 0.21 experiment. Growth rates, however, \yere compared to rates obtained from subsequent cell counts twice a day over a 3 d period in pre-cultures. These values were then sealed with screw caps immediately to prevent also used to define the duration of the experiment (72 h further exchange with atmospheric CO2. To achieve at [CO2(aq)] 2 2 pm01 l.', 96 h at [CO2(aq)] i 2 pm01 l') higher [CO2 (aq)]at constant pH, Na2C0, was added which permitted 9 cell divisions prior to sampling and pH then readjusted to initial values with 1 N HC1. Growth rates (p)were calculated according to the [CO2 (aq)]before and after the experiments was cal- equation p = ln(hrl/N,,)/t,where No and N,are cell con- culated from DIC, total alkalinity (tAlk),temperature, centrations at the beginning and the end of the exper- sallnity and concentrations of phosphate and silicate, ~mentand t equals duration of incubation in days. assunling dissociation constants according to Mehr- bach et al. (1973). pH was measured with a micro- processor pH meter (WTW pH 3000) using a combined RESULTS AgCl/KCl electrode, calibrated with NBS buffer solu- tions. In addition, pH was calculated from DIC and Elemental ratios tAlk. The difference between measured and calculated pH was, on average, less than 0.05 pH units, tAlk Both strains of Skeletonerna costaturn (SKA and SK B) was calc.ulated from linear Gran-plots (Gran 1952) showed decreases in C:P and N:P, but increases in C:N, after potentiometric titration of duplicate 100 m1 sam- towards lowest [CO2(aq)] (Figs. 1 & 2). Minimum C:P ples with 0.05 N HC1 (Bradshaw et al. 1981, Brewer et and N:P ratios were respectively only 41 to 55% and al. 1986). DIC samples were stored at 4OC in 300 m1 35 to 49 % of maximum values over the range of CO2 (aq) borosilicate glass bottles without headspace after addi- concentrations (>30 to <2 pm01 I-') tested (Figs. lb. c & tion of 1 m1 of 50Y0 saturated HgC12 solution. DIC was 2b, c). The effect of [CO, (aq)]on C:N was also signifi- measured in triplicate by coulometric titration (UIC cant, but less pronounced, with C:N increasing by up to coulometer) in an automated gas extraction system 24% from highest to lowest CO2 (aq) concentrations (Johnson et al. 1993). (Figs. la & 2a). Results from 3 independent incubations C:N:P measurements. Samples for determination of of SK A at each of 5 experimental CO2concentrations are C:N:P were filtered on precombusted (500°C, 12 h) shown in Fig. 1. Elemental ratios of the other strain, SK B, GF/C glass fiber filters (Whatman), rinsed with 3 X 5 m1 were similar with C:P and N:P ratios being slightly lower 0.17 M Na2S0, solution, and stored at -25OC until than in SK A. In spite of differences in cell diameter by a analysis. Analysis of particulate organic carbon (POC) factor of 3, both strains showed similar va~lationin C:N:P and nitrogen (PON) was performed on triplicate filters ratios with changing [CO?(aq)] (Fig. 1). Mar Ecol Prog Ser 155: 67-76, 1997

SK A. DIC const SK A, pH const A SK B. DIC const

CO, [pmolI"]

Fig. 1 Skeletonema costatum. Vanation in (a) C:N, (b) C:P, Fig. 2. Skeletonema costatum. Variation in (a] C:N, (b) C:P, and (c) N:P ratios of SK A (0) and SK B (A)at constant DIC in and (C] N:P ratios of SK A at constant pH ~n relation to relation to [CO2 (aq)].Each data point represents the mean [CO, (aq)l value of triplicate measurements of C, N, and P. C:P and N:P curves are derived from a least-squares fit using the Michaelis-Menten equation (see Table 2). The C:N curve was experiment with SK A, in which pH was kept con- calculated as C:N = C:P/N:P stant at all CO, levels. C:N:P ratios varied by the same order of magnitude as in experiments with constant DIC and variable pH. Although absolute values If [CO2 (aq)] of ocean surface water varies in in Fig. 2' differ somewhat from Fig 1, identical trends response to biological activity or, over longer time in elemental ratios were taken as indication that vari- scales, by air-equilibration in response to increasing ability in elemental ratios was caused by changes pC02, alkalinity remains unaffected whereas DIC in supply of CO2 rather than pH effects on cell physi- and pH change. As mentioned above, only 1 of the ology. parameters DIC, alkalinity, and pH can be kept con- To describe COp-dependentvariation in C:P and N:P, stant when manipulating seawater. In the experimen- it was assumed that elemental ratios (E) approach tal carbonate system, DIC was kept constant when maximum values (E')at saturating CO2 levels. There- changing [CO2 (aq)]. Corresponding changes in pH fore, the Michaelis-Menten equation E = E' [CO, (aq)]/ in this system were of the same magnitude as under (KE + [COz (aq)]),where KE is the half-saturation con- natural conditions. If the observed variation in C:N:P stant, was used for an iterative, non-linear least- were caused by changes in pH and not CO2 concen- squares fit to C:P and N:P measurements. C:N curves tration, one would expect no variation in elemental were calculated as C:N = C:P/N:P (Figs. 1 & 2). Calcu- ratios of Skeletonema costatum at variable [CO2 (aq)] lated constants and their standard deviations for all but constant pH. Fig 2 shows data from the control treatments are listed in Table 2. Burkhardt & Riebesell: CO2 effect on C:N:P (Redfield ratio) 7 1

Table 2. Skeletonerna costaturn. Results from a least-squares Table 3. Skeletonerna costatum. Cell quota for C, N, P and fit to CO2-dependent variation in C:P and N:P. Maximum ele- average growth rates (p) of SK A, grown at different CO2 con- mentdl ratios E' and half-saturation constants KE were calcu- centrations [CO:, (aq)]at constant DIC lated according to the Michaelis-Menten equation for C:P and N:P molar ratios of 2 clones (SK A, SK B). Standard deviations are given in parentheses [for further explanations see text) [CO, (aq)] C/cell N/cell P/cell P (~lmol1') (pg C) (W NI (pg P) (d-')

Clone Treatment E' KE (1.1mol 1.') 30 6 17 70 3.54 0.58 2.1 12 0 14 49 2.84 0.58 2.1 SK A DIC const (C-P)'= 77.47 (0.51) KC,. = 1.14 (0.04) 5 4 14.98 2.77 0.65 2.0 (NP)' = 13.40 (0.57) KN,P= 1.80 (0.31) 2 7 12.29 2.05 0.61 1.9 15 12 03 2.09 0.65 1.6 SK B DIC const (C.P)'= 76.86 (0.02) KC p = 2.46 (0.01) (N:P)'= 13 06 (1 16) = 3.55 (1.10) SK A pH const (C:P)'= 82 01 (0 74) KC,= 0.90 (0.04) (hT P)' = 12.50 (0 78) KNp = 1.13 (0.31) growth as indicated by a decrease in growth rate of SK A from 2.1 d-' at high [COz (aq)]to 1.6 d-' at the lowest [CO2 (aq)] (Table 3). These results concur with other Cell quota and growth rates studies in which growth limitation of marine diatoms was observed at low CO2 concentrations (Riebesell et Table 3 summarizes cellular carbon, phosphorus, al. 1993, Chen & Durbin 1994). and nitrogen contents (cell quota) for SK A at constant DIC. Most of the variation in C:P ratios was accounted for by decreasing amounts of organic carbon per cell, Inorganic carbon acquisition whereas P/cell was largely unaffected by changes in COz concentrations. N/cell was also variable, but to a The interpretation of CO2 effects on algal physiology greater degree than C/cell, which resulted in high C:N is complicated by limited knowledge of mechanisms and low N:P at low [CO2 (aq)]. Growth rates deter- and pathways of inorganic carbon acquisition by mined for the different treatments (Table 3) equalled marine phytoplankton. RUBISCO (ribulose 1,5-bispho- those obtained from repeated cell counts in pre-cul- sphate carboxylase-oxygenase) is the primary enzyme tures (data not shown), indicating that the cells grew in of photosynthetic carbon fixation in microalgae (Raven the experimental growth phase throughout the experi- 1996) and uses molecular CO2 as substrate. Several ments. Growth rate decreased at CO2 concentrations mechanisms which involve active transport of inor- below ca 2 pm01 I-' to a minimum value of 1.6 d-l. At ganic carbon through the plasma membrane or the higher CO2 levels, growth rates were largely unaf- chloroplast envelope have been proposed to actively fected by variation in [COz (aq)] and SK A grew at increase CO2 concentration at the site of carboxylation maximum rates of 2.1 d-l. (Badger et al. 1980, Badger & Andrews 1982, Badger & Price 1992) Such an inorganic carbon concentrating mechanism might be coordinated with the enzymatic DISCUSSION activity of extracellular carbonic anhydrase which would increase external CO, concentration by catalyz- Response to variable CO2 concentration ing dehydration of HC0: (Aizawa & Miyachi 1986, Badger & Price 1994). If changes in external [CO2(aq)] This study provides the first evidence that the C:N:P affected CO2 availability at the carboxylation site of elemental ratios of a common marine diatom can be RUBISCO or the energy budget of an algal cell, corre- affected by the availability of CO2 (aq). As indicated by sponding shifts in metabolic pathways might be identical trends at constant pH in the control experi- reflected in variable elemental composition. The ment, pH effects on algal physiology did not seem to extent to which marine microalgae can make use of the have accounted for the observed changes in elemental large HC03 pool in seawater and whether they have ratios. Under controlled conditions with sufficient light adopted a common strategy to accommodate variations and excess nutrients, variation in CO2 concentrations in [CO2(aq)] is not known yet. appeared to be the cause of changes in chemical com- In this study, C:N:P ratios of Skeletonema costatum position of the cells in this study. Differences in cell showed consistent increases or decreases with de- size between the 2 strains were not reflected in the creasing [CO2(aq)] (Figs. l & 2) over the range of CO2 CO2 dependency of C:N:P ratios. concentrations (ca 5 to 25 pm01 I-') typically encoun- CO2 availability not only affected elemental ratios of tered in the ocean's surface waters (Goericke & Fry Skeletonema costatum but also limited its rate of 1994). At the lowest experimental [COz (aq)] of ap- 72 Mar Ecol Prog Ser 155: 67-76. 1997

proximately 1 to 2 pm01 I-', however, trends in C:N in appears to lack storage polyphosphates (Sakshaug & all treatments and in C:P and N:P of SK B were re- Holm-Hansen 1977). C:P ratios of approximately 40 at versed. It has been suggested that a different mode of [CO, (aq)]12 pm01 l'were similar to lowest C:Pratios inorganic carbon acquisition might be induced below a of S. cos fa tun^ in experiments by Sakshaug & Holm- criticdl level of CO2 supply in marine phytoplankton Hansen (1977)and might thus represent minimum val- (Raven 1991, H~ngaet al. 1994, Laws et al. in press) ues which can be maintained in S. costatum irrespec- Below a CO2 threshold concentration of approximately tive of the kind of environmental factor affecting the 2 pm01 1-', S. costatum might have responded by chemical composition of the cells. changing its inorganic carbon acquisition, pathway to Whereas a decrease in C:P ratios with decreasing compensate for limited CO2supply. Such physiological [CO, (aq)]fits the classical perception of intracellular adaptation should be accompanied by changes in the shortage of a limiting nutrient, a concomitant increase biochemical composition and might have accounted in C:N appears to vlolate this concept. In a comparison for reversal of observed trends in changes in elemental of P-limited and N-limited chemostat cultures of the ratios. Another possible response to insufficient COz marine diatom Thalassiosira pseudonana, Perry (1976) supply could be enhancement of carbon fixation by P- observed Increases in C:N ratios in response to nutri- carboxylases. While RUBISCO is considered 'the sole ent exhaustion irrespective of the kind of limiting carboxylase involved in converting exogenous inor- nutrient. Similar observations of high C:N during N or ganic C into at least 95 O/oof cellular organic C' (Raven P starvation were made in other studies (e.g. Sakshaug 1996), Descolas-Gros & Fontugne (1985) found p-car- & Holm-Hansen 1977, Goldman et al. 1979, Laws & boxylation to Increase in batch cultu.res of S. costatum Bannister 1980). Harrison et al. (1977) found highly at the end of the exponential growth phase. Whether elevated ratios of C:N = 13.2 in silicate-starved and such shifts in enzymatic pathways also apply to growth C:N = 14.8 in ammonium-starved Skeletonerna costa- at low [CO2(aq)] has not been Investigated yet. turn.Thus, In contrast to C:P ratios, elevated C:N might serve as a general indicator of unfavorable environmental conditions irrespective of the nutrient (CO2in Factors affecting C:N:P this study) in shortest supply. The C:N ratio can also be affected by light availabil- Compared to variability of C:P or C:N ratios mea- ity. Over a range in photon flux density of 4.4 to sured under severe P or N limitation, the magnitude of 209 pm01 photons m-2 SS', C:N of the marine diatom change caused by CO2 availability can be considered Thalassiosira fluviatilis varied to an extent (molar C:N moderate. Elemental ratios found at high [CO2(ay)] in = 5.1 to 7.1) similar to CO2-related changes in C:N in this study were close to values reported by Sakshaug & this study (Laws & Bannister 1980) Llght-limited Holm-Hansen (1977) for Skeletonema costatum under growth, however, resulted in minimum C:N values at nutrient-replete conditions during exponential growth the lowest growth rates, contrary to maximum C:N (C:N 7.4, C:P 90, N:P 12; continuous culture, N:P of during nutrient-limited growth. medium 20). In their study, however. C:P covered a Likewise, iron starvation might affect the biochemi- wide range from lowest C:P = 39 during exponential cal composition. Compared to exponentially growing growth (N:P of medium = 1.2) to C:P > 900 during star- cells, Sakshaug & Holm-Hansen (1977) observed a vation in P-deficient cells (N:P of medium = 310 and decrease in C:P and N:P in Fe-deficient cultures of 830).Large increases in C:N and C:P under Nor P lim- Skeletonema costatum similar in magnitude to varia- itation appear, to a great extent, to be the result of tion of these ratios in the present study. For C:N they accumulation of P-1,3 glucan, which is considered a found no systematic trend due to the large scatter of common reserve polysaccharide in marine diatoms values. On the other hand, C:N:P rati.os of the chryso- (Myklestad 1974, 1977). It can be speculated that, in phyte Pavlova (Monochrysis) lutheri (Sakshaug & contrast to an overflow production of carbon under N- Holm-Hansen 1977), of the diatom Phaeodactylum tri- or P-limiting conditions, variation in elemental rati.os of cornutum (Greene et al. 1991) and of natural phyto- S, costaturn in response to changes in [CO2(aq)] might plankton communities from the Southern Ocean (van result from differences in the allocation of assimilated Leeuwe et al. 1997) appeared to be largely unaffected carbon to intracellular carbon pools. More detailed by Fe starvation. analysis of carbohydrate, protein, and lipid composition, however, is required to test this hypothesis. As shown in Table 3, P content per cell rem<~inedrel- Ecological significance of CO2-related C:N:P variation atively constant, whereas amounts of intracellular organic carbon decreased at low [CO2(aq)]. Regard- While variation in the elemental composition of less of the nutrient status, Skeletonema costatum marine diatoms in response to changes in [CO2(aq)] is Burkhardt & Riebesell: CO2 effect on C:N:P (Redfield ratio) 73

interesting from a physiological point of view, its eco- Biogeochernical relevance logical significance depends on the degree to which such changes In C:N:P develop and persist under nat- Over geological time scales, atmospheric pCOz ural conditions. Drastic and rapid changes in C:N and increased from a glacial minimum of 180 ppmv to pre- C:P ratios are known to occur in microalgae during industrial levels of 280 ppmv (e.g. Delmas et al. 1980, nutrient exhaustion (Banse 1974, Sakshaug & Holm- Neftel et al. 1982). At a constant temperature, [CO, Hansen 1977), which occurs upon termination of algal (aq)]in air-equilibrated seawater would show a corre- blooms. Such large deviations from Redfield stoichio- sponding increase (Broecker et al. 1979). It needs to be metry easily override the comparatively small differ- considered, however, that over geological time scales ences in elemental ratios related to variable CO2 or not only [COz (aq)] but also other factors such as light supply. nitrate, phosphate, iron and light availability are likely Above a certain threshold concentration, however, to have varied significantly. Furthermore, shifts in spe- elemental ratios of marine phytoplankton seem to be cies composit~onamong the major taxonomic groups of largely unaffected by concentrations of potentially lim- marine microalgae might have occurred over such iting nutrients. C:N ratios of surface layer particulate time scales. All these factors are known to influence organic matter are often similar to or less than the Red- the average elemental composition of marine phyto- field ratio even at nitrate concentrations below 2 pm01 plankton, which complicates evaluation of potential 1-' (see Sambrotto et al. 1993 for reference). Further changes in the Redfield ratio due to increases in [CO, examples of little variation in C:N at low nitrate con- (as)] centrations are given by Sakshaug & Olsen (1986) for Unlike the above-mentioned long-term variability, blooms dominated by Skeletonema costatum or the burning of fossil fuel has led to an increase in atmos- coccolithophorid Emlliania huxleyi and by Dortch, et pheric pCO, on a much shorter time scale. Within al. (1985)for mixed algal assemblages. Highly efficient about 200 yr, atmospheric pC02 increased from uptake mechanisms for both nitrate (Eppley et al. 1969, 280 ppmv to present levels of 360 ppmv and is pre- Harrison et al. 1996) and phosphate (Perry 1976) may dicted to exceed 600 ppmv by the year 2100 in 'busi- provide the means to compensate for deficiency in ness as usual' emission scenarios (Wigley et al. 1996). nutrient supply. Assuming total alkalinity = 2.3 meq I-', salinity = Particulate organic matter close to the Redfield ratio 35 psu and an increase in surface seawater tempera- below the euphotic layer (Copin-Montegut & Copin- ture from 10 to 11°C, an increase in atmospheric pC02 Montegut 1983) provldes further evidence that the to 600 ppmv would raise [CO2(aq)] in air-equilibrated major portion of biogenlc sinking flux is not character- seawater from pre-industrial 12.3 pm01 l'to 25.5 pm01 ized by the extremely high C:N ratios observed upon 1-l. As a result of this increase in [CO2 (aq)],C:P of nutrient exhaustion Phytoplankton spring blooms, as Skeletonema costatum would be expected to increase they occur in high latitude ecosystems, are character- by 4.6% (from 70.9 to 74.2), C:N to decrease by 2.3% ized by high rates of export production because (from 6.07 to 5.93) according to Table 2 (SK A, DIC autotrophic production and heterotrophic consump- const). tion are largely uncoupled (Bienfang & Ziemann The rapid increase in today's pC02 and the associ- 1992). As long as nutrient supply does not drop below ated increase in [CO2 (aq)]of surface seawater repre- a critical level, CO2-related changes in the Redheld sents a uni-directional trend of a single environmental ratio are of similar magmtude as variability caused by factor which is not accon~paniedby other factors and other factors contributing to stoichiometry of sinking has a potential influence on elemental composition of particles. marine phytoplankton. Systematic changes in the ele- In the subarctic North Pacific, the equatorial Pacific mental composition of marine phytoplankton in or the Southern Ocean, where high nitrate levels per- reponse to the rapid increase in [COz (aq)]would thus sist throughout the year but phytoplankton stocks are be superimposed on a background scatter due to sto- low, iron limitation of phytoplankton growth and bio- chastic variability of other factors affecting the Red- mass represents an important factor controlling pri- field ratio. Changes in C:P are of particular importance mary productivity (Martin & Fitzwater 1988, de Baar et because phosphate is the nutrient whlch ultimately al. 1995, Coale et al. 1996). Since the effect of iron star- controls marine phytoplankton biomass (Redfield vation on elemental composition of phytoplankton is 1958, Broecker 1982, van Cappellen & Ingall 1996) and moderate or negligible (Sakshaug & Holm-Hansen therefore C:P is commonly used in global biogeochem- 1977, Greene et al. 1991, van Leeuwe et al. 1997) and ical models to quantify inorganic carbon fixation (e.g. nitrate and phosphate are in ample supply, CO2- Broecker et al. 1985, Six & Maier-Reimer 1996). related effects on plankton stoichiometry should be Several recent studies recognize deviations from noticable. Redfield stoichiometry and caution against calculation Mar Ecol Prog Ser 155: 67-76, 1997

of organic carbon export based on a constant Redfield that, in spite of the much higher CO2 (aq) and lower ratio (e.g Fanning 1992, Sambrotto et al. 1993, Banse HCOi concentrations in freshwater, limnetic micro- 1994).According to Maier-Reimer (1996),'the assump- algae might show a similar stoichiometric response to tion of a constant Redfield ratio (for global modelling variation in [COz [aq)]as marine phytoplankton. This studies) represents an efficient mechanism to reduce observation is of particular interest because freshwater the number of free parameters and, thus, to increase and marine phytoplankton might employ different the efficiency of any model pred~ction'.Deviations modes of inorganic carbon acquisition in adaptation to from Redfield ratio due to intra- and interspecific vari- their different environments. ability of cell stoichiometry 'seem to compensate each In conclusion, results from this study demonstrate other in the large-scale chemical distribution in the clear correlation of C:N:P elemental ratios with con- deep sea' (Maier-Reimer 1996). If changes in elemen- centrations of CO, (aq) in 2 strains of the marine ta.1 composition, however, are caused by factors which diatom Skeletonema costatum. The physiological exhibit systematic trends, deviations from the Redfleld mechanisms responsible for the observed trends and ratio will no longer be compensated by random vari- the question whether this variation in Redfield ratios ability. Knowledge of the magnitude by which relevant represents a common feature in marine phytoplankton factors affect the elemental composition of planktonic need to be elucidated. organisms, therefore, provides the framework for improved models of large-scale processes. Acknowledgements.\Ve thank A. Dauelsberg who performed CO2-dependentstoichiometry of marine microalgae all DIC measurements, H. Halliger (Biologische Anstalt Hel- goland, Sylt) for supplying strain SK B, and H. Schwarz for has not yet been considered a factor contributing to technical assistance in C:N measurements. V. Smetacek and variability of the Redfield ratio. If the observed correla- M Pahlow provided helpful comments on earlier versions of tion of elemental ratios with [CO2(aq)] proves to be a the manuscript. This is publication no. 1286 of the Alfred general trend in marine phytoplankton, changes in the Wegener Institute for Polar and Marine Research. Redfield ratio may be expected in response to the currently observed increase in atmospheric pC02.Depen- LITERATURE CITED dency of elemental ratios on CO2 concentration would Aizawa K, Miyachi S (1986) Carbonic anhydrase and CO2 affect the efficiency of the 'biological carbon pump' concentrating mechanisms in microalgae and cyanobacte- (Volk & Hoffert 1985)due to variation in the amount of ria. FEMS Microbiol Rev 39:215-233 carbon fixed relative to other inorganic nutrients. Anderson LA (1995) On the hydrogen and oxygen content of marine phytoplankton. 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This art~clewas subnlitted to the editor iklanuscript received. December 23, 1996 Revised version accepted: July 8, 1997